Antibody drug conjugate

Abstract

According to the doxamine compound antibody drug conjugate and the application and the preparation method thereof, hydrophilic 1ink is introduced to be connected with the doxamine compound, and the antibody drug conjugate with good stability, hydrophilicity and good pharmacokinetic properties is obtained.

Description

An antibody-drug conjugate

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to a prior application, filed with the State Intellectual Property Office of China on April 27, 2023, with patent number 2023104680075, entitled “An Antibody Drug Conjugate.” The entire text of that prior application is incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology and medicine, and particularly relates to an antibody-drug conjugate, a preparation method and application thereof. Background Art

[0004] Antibody-drug conjugates or antibody-drug conjugates (ADCs) are one of the biggest hotspots in the current research and development of anti-tumor drugs. As of now, there are 14 antibody-drug conjugates on the market. They are: Mylotarg, approved in 2000 for the treatment of CD33-positive acute myeloid leukemia (AML); Adcetris, approved in 2011 for Hodgkin's lymphoma; Kadcyla, approved in 2013 for Her2-positive breast cancer; Besponsa, approved in 2017 for B-cell lymphocytic leukemia; Lumoxiti, approved in 2018 for relapsed or refractory capillary leukemia; Polivy, approved in 2019 for relapsed or refractory diffuse large B-cell lymphoma. Padcev for urothelial carcinoma, approved in 2019; Enhertu for HER2-positive breast cancer, approved in 2019; Trodelvy for triple-negative breast cancer, approved in 2020; Akalux, a photoimmunotherapy drug for head and neck cancer, approved in 2020; Zynlonta for large B-cell lymphoma, approved in 2021; Adixit for HER2-positive gastric cancer, approved in 2021; Tivdak for cervical cancer, approved in 2022; and Elahere for ovarian cancer, approved in 2022. It's worth noting that Mylotarg was withdrawn from the market in 2010 due to its high toxicity, but was re-launched in 2017 after adjusting its clinical dosage. Meanwhile, Blenrep received accelerated approval for multiple myeloma in 2020, but was withdrawn from the market due to unsatisfactory subsequent clinical results.

[0005] Antibody-drug conjugates consist of three parts: antibodies, linkers, and bioactive molecules (drugs). Among them, antibodies are closely related to disease targets. Linkers and drugs determine the effectiveness and safety of ADCs and are the most core parts. Among the ADC drugs that have been launched on the market, most use the technology developed by Seattle Genetics decades ago, which uses microtubule inhibitors as active molecules, coupled with tissue protease hydrolysis linkers, and conjugated to antibody cysteine ​​through maleimide. The ADCs obtained by this technology are often highly toxic, highly hydrophobic, and have poor pharmacokinetic properties. Although the ADCs obtained by Daiichi Sankyo using a hydrophilic tetrapeptide linker have been greatly improved, they are still quite different from the properties of naked antibodies. Trodelvy uses a lysine linker containing polyethylene glycol and a salt-forming lysine. Although the resulting ADC has good hydrophilicity, it has poor stability. Literature reports that a linker containing polycreatine can greatly improve the hydrophilicity of ADCs. However, this linker is only suitable for active molecules containing secondary amine structures, such as MMAE. For active molecules with primary amine structures such as Exatecan, the carbonamide bond formed by this linker is less stable.

[0006] Dolidomide compounds have been used as clinical therapeutics in immunomodulation and anti-tumor settings for many years. For example, thalidomide has long been used for leprosy and various skin diseases, such as discoid lupus erythematosus, subacute cutaneous lupus erythematosus, and Behçet's syndrome. Pomalidomide and lenalidomide are commonly used in the treatment of multiple myeloma. Recent studies have shown that this class of dolidomide molecules works like a "molecular glue," achieving therapeutic effects by degrading their target proteins. Marketed small molecules of dolidomide suffer from significant toxicity and suboptimal efficacy, such as thalidomide, which was the culprit in the famous "thalidomide incident." A growing number of studies are targeting the structural modification of these dolidomide compounds. For example, Orum Therapeutics has engineered Smol006, a compound in this class, and combined it with antibody-conjugated delivery technology to create the antibody-drug conjugates ORM-5029 and ORM-6151. These antibody-drug conjugates have demonstrated excellent anti-tumor activity and a favorable safety profile. However, during our research, we discovered that Smol006's activity was only moderate, making it insufficient as the active moiety in an ADC for tumors with low receptor expression. The inventors described in patent application PCT / CN2024 / 076883 a novel amide-based molecular glue derivative with superior anti-tumor efficacy and improved safety. Therefore, there is an urgent need to develop ADCs based on amide compounds with improved activity.

[0007] Summary of the Invention

[0008] To solve the problems existing in the prior art, the present invention provides an antibody drug conjugate having a structure of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels: A-(LD) m (I)

[0009] wherein A is a targeting ligand selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment, a small molecule ligand, or a polypeptide;

[0010] L is a linker moiety, one end of which is connected to ligand A and the other end is connected to the bioactive molecule D;

[0011] D is a biologically active molecule containing an amino group or a tautomer, mesomer, racemate, enantiomer, diastereomer, or isotope-labeled substance thereof, and is covalently linked to the linker portion L through the amino group in its molecular structure;

[0012] m represents the molar ratio of cytotoxic drug molecules to antibody A (also known as DAR, i.e. drug-antibody coupling ratio).

[0013] m is an integer or decimal from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0014] When m is a decimal, it refers to the average number of linker-drug molecules (LD) conjugated per antibody unit (A).

[0015] In some embodiments, A is selected from an antibody or antigen-binding fragment thereof that targets HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, dll3, TROP2, B7-H3, c-Met, CD20, CD22, CD30, CD33, CD123, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis Y, VEGFR, VEDF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, or Mesothelin.

[0016] In some embodiments, A can be modified, such as by changing, adding, or removing one or more amino acids.

[0017] In some embodiments A is selected from antibodies or antigen-binding fragments thereof targeting Her2, BCMA, CD33, CD123.

[0018] In some embodiments, the CDR combinations of the heavy chain variable region VH and light chain variable region of antibody A are as follows, in the order of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3:

[0019] CDR Combination I:

[0020] CDR Portfolio II:

[0021] CDR combination III:

[0022] CDR Portfolio IV:

[0023] CDR Portfolio V:

[0024] CDR Portfolio VI:

[0025] CDR Combination VII:

[0026] CDR Combination VIII:

[0027] CDR Portfolio IX:

[0028] CDR Portfolio X:

[0029] Also preferably, A is selected from trastuzumab (Trastuzumab, heavy chain and light chain combination SEQ ID NO.1-2), 007 (Pertuzumab, heavy chain and light chain combination SEQ ID NO.3-4), 014 (Gemtuzumab, heavy chain and light chain combination SEQ ID NO.5-6), 018 (CD33AB, heavy chain and light chain combination SEQ ID NO.7-8), 019 (heavy chain and light chain combination SEQ ID NO.9-10), 020 (heavy chain and light chain combination SEQ ID NO.11-12), 021 (heavy chain and light chain combination SEQ ID NO.13-14), 022 (heavy chain and light chain combination SEQ ID NO.15-16), 023 (heavy chain and light chain combination SEQ ID NO.17-18), 008 (heavy chain and light chain combination SEQ ID NO.19-20) or a biosimilar thereof.

[0030] In some embodiments, the linker moiety -L- is represented by the formula:

[0031] -L1-L2-L3-L4-,

[0032] Among them, L1 is selected from (or its open-loop form ), or Where * indicates connection to the sulfhydryl group of A (such as a monoclonal antibody), and ** indicates connection to L2;

[0033] L2 is a spacer; preferably, it is selected from -L 2a -C(O)-、-L 2a -L 2b -C(O)-、-L 2a -NH-C(O)-、-L 2a -L 2b -NH-C(O)-、-L 2a -L 2b -C(O)-NH-、-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-、-L 2a -C(O)-NH-L 2b -C(O)-NH-、-L 2a -NH-C(O)-L 2b -NH-C(O)-、-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)-, where L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C6-C 14 Arylene-, -C6-C 14arylene-C1-C8 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C8 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C6 L is substituted by one or more substituents of alkyl, 1-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl are selected from one or more of N, O or S; 2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, 2-8 membered heteroalkyl, C6-C 14Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more of N, O or S;

[0034] L3 is a polypeptide sequence selected from peptide residues consisting of 2-8 (e.g., 2, 3, 4, 5, 6, 7, 8) natural or unnatural amino acids, wherein any of the amino acids is further substituted with one or more substituents selected from C1-C6 alkyl, 2-6 atom heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl, or C3-C8 cycloalkyl;

[0035] L4 is a self-cleaving fragment,

[0036] Further preferably, L4 is a self-cleaving fragment modified with a hydrophilic group,

[0037] The self-cleaving fragment is selected from:

[0038] or

[0039] Where * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group of the bioactive molecule D, and X is absent or *** indicates connection with carbon atoms, **** indicates connection with oxygen atoms, and arrows indicate the modification sites of hydrophilic groups;

[0040] Further preferably, the self-cleaving fragment is selected from the following structures before modification with the hydrophilic group:

[0041] Wherein Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, and n is an integer from 0 to 6;

[0042] The hydrophilic group has at least one azide group and comprises a polyethylene glycol group, a polynatural amino acid or non-natural amino acid group, a monosaccharide, an oligosaccharide or a polysaccharide or a combination of the above groups;

[0043] Preferably, L4 is selected from:

[0044] or

[0045] Where * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D;

[0046] R 2 is a hydrophilic segment, preferably, selected from a linear or branched heteroalkyl group comprising 4-50 (preferably 4-24, more preferably 6-24, most preferably 8-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) -OCH2CH2- structural units, a 24, more preferably 8-24, most preferably 10-24, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) protein amino acids (such as glycine) or non-protein amino acids (such as sarcosine), a linear or branched heteroalkyl group containing a monosaccharide, oligosaccharide or polysaccharide; X is absent or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is a C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) -OCH2CH2- structural units, and n is an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, 6);

[0047] When A is selected from antibodies targeting CD33 or CD123, L4 may also be:

[0048] Where * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino or hydroxyl group of the bioactive molecule D, and X is absent or *** indicates bonding to a carbon atom, and **** indicates bonding to an oxygen atom.

[0049] In some embodiments, R 2 Selected from the following structures:

[0050] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); a is selected from a bond, a C1-C3 alkylene group, such as methylene, ethylene, n-propylene, isopropylene; R b Selected from C1-C3 alkyl, such as methyl, ethyl, n-propyl, isopropyl.

[0051] In some embodiments, R 2 Selected from the following structures:

[0052] In some embodiments, Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 C1-C6 alkylene or -C1-C6 alkylene-(OCH2CH2) o -, wherein o is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0053] In some embodiments, Y is C1-C3 alkylene or -R 3 -C(O)-, where R 3 C1-C3 alkylene or -C1-C3 alkylene-(OCH2CH2) o -, wherein o is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0054] In some embodiments, Y is methylene, ethylene, n-propylene, isopropylene, or -R 3 -C(O)-, where R 3 Methylene -(OCH2CH2) o -, ethylene-(OCH2CH2) o -, n-propylene-(OCH2CH2) o -, isopropylidene-(OCH2CH2) o -, wherein o is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0055] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0056] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2a Selected from -C1-C6 alkylene-, -C1-C6 alkylene-C3-C6 cycloalkylene-, -C6-C 10 Arylene-, -C6-C 10 arylene-C1-C6 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C6 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C3 L is substituted by one or more substituents of alkyl, 1-3 atoms of heteroalkyl, C1-C3 alkoxy, hydroxy, amino, carboxyl or C3-C6 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl are selected from one or more of N, O or S; 2b41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C1-C3 haloalkyl, 2-6 membered heteroalkyl, C6-C 10 Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical are each optionally substituted by one or more substituents independently selected from C1-C3 alkyl, 2-3 atoms of heteroalkyl, C1-C3 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclic radical, heteroaryl, heteroalkylene are selected from one or more of N, O or S.

[0057] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2aSelected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, -C1-C3 alkylene-cyclohexylene-, -phenylene-, -phenylene-C1-C3 alkylene-, -5-6 membered heteroaryl-C1-C3 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched heteroalkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms in a straight or branched chain heteroalkylene-3-6 membered heteroalkylene cycloalkyl, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, and heterocyclylene are each optionally substituted by one or more substituents independently selected from halogen, methyl, ethyl, n-propyl, isopropyl, 1-3 atoms of heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, hydroxy, amino, carboxyl or cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, wherein the heteroalkylene, heterocyclylene, and heteroalkylene contain 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, and heteroalkylene are selected from one or more of N, O, and S; 2b is selected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, a straight or branched chain heteroalkylene radical of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, R 1Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-6 membered heterocyclyl, -CF3, -CF2CF3, 2-3 atoms of heteroalkyl, phenyl, 5-6 membered heteroaryl, said alkyl, heteroalkyl, phenyl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from methyl, ethyl, n-propyl, isopropyl, 2-3 atoms of heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, amino or carboxyl, said heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of said heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more of N, O or S.

[0058] In some embodiments, L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene, C6-C 14 Arylene-C1-C8 alkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms of straight or branched heteroalkylene, the alkylene, cycloalkylene, arylene, heteroalkylene are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heteroalkylene, heteroarylene are selected from one or more of N, O or S; L 2b is selected from -C1-C8 alkylene-, a straight or branched chain heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms, R 1 Selected from hydrogen, C1-C6 alkyl, C2-C8 heteroalkyl, C6-C 14 Aryl, the alkyl, heteroalkyl, and aryl groups are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl group contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkyl and heteroalkylene groups are selected from one or more of N, O or S.

[0059] In some embodiments, L 2a Selected from -CH2CH2CH2CH2CH2-, -CH2CH2(OCH2CH2) p -、-(OCH2CH2) p-, -CH2(OCH2CH2) p -、-CH2CH2(OCH2CH2) p CH2-, -CH2CH2(OCH2CH2) p CH2CH2-、-CH2(OCH2CH2) p CH2CH2-, wherein p is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L 2b Selected from -CH2OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2-, -CH2O-, -CH2CH2O-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -(OCH2CH2) q -、-CH2CH2(OCH2CH2) q -, -CH2(OCH2CH2) q -、-CH2CH2(OCH2CH2) q CH2-, -CH2CH2(OCH2CH2) q CH2CH2-、-CH2(OCH2CH2) q CH2CH2-, R 1 Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, wherein q is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0060] In some embodiments, L2 is selected from the following structures:

[0061] Wherein, w is selected from an integer of 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection with L1, **

[0062] Indicates connection with L3.

[0063] In some embodiments, L3 is selected from 2-8 peptide residues formed by amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (such as C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, isopropyl substituted lysine), etc. For example -ValCit-;-CitVal-;-AlaAla-;-AlaCit-;-CitAla-;-AsnCit-;-CitAsn-;-CitCit-;-ValGlu-; itAsp--AlaVal--ValAla--PheAla--AlaPhe--PheLys--LysPhe--ValLys--LysVal--AlaLys--LysAla--PheCit--CitPhe--LeuCit--CitLeu--IleCi t-;-CitIle-;-PheArg-;-ArgPhe-;-CitTrp-;-TrpCit-;-AlaAlaAla-;-PhePheLys-;-LysPhePhe-;-DPhePheLys-; heLeuGly- -GlyLeuPheGly- -GluValCit- -AlaLeuAlaLeu- -GlyGlyGly- -GlyGlyGlyGly- -GlyPheValGly- -GlyValPheGly- -GlyGlyPheGly- -GlyGlyValGly-.

[0064] In some embodiments, L3 is selected from the following structures:

[0065] * indicates connection with L2, and ** indicates connection with L4.

[0066] In some embodiments, L4 is selected from:

[0067] wherein v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D.

[0068] In some embodiments, L4 is selected from:

[0069] wherein r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the bioactive molecule D.

[0070] In some embodiments, L is selected from the following structures:

[0071] wherein v and w are each independently selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); * indicates linkage to the sulfhydryl group of antibody A; ** indicates linkage to the amino group of biologically active molecule D; and r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0072] In some embodiments, L is selected from the following structures:

[0073] wherein v and w are each independently selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); * indicates linkage to the sulfhydryl group of antibody A; ** indicates linkage to the amino group of biologically active molecule D; and r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0074] In some embodiments, L is selected from the following structures:

[0075] Where * indicates connection with the sulfhydryl group of antibody A, and ** indicates connection with the amino group of bioactive molecule D.

[0076] In some embodiments, the bioactive molecule D is selected from camptothecin derivatives, eribulin, and molecular glues.

[0077] In some embodiments, the bioactive molecule D is selected from a molecular glue compound having a structure shown in formula (A-1):

[0078] wherein RA is selected from an optionally substituted 3-16 membered heterocyclic group, wherein the heteroatom of the heterocyclic group is selected from nitrogen; m1 and n1 are each independently selected from an integer of 0-6, such as 0, 1, 2, 3, 4, 5, 6.

[0079] In some embodiments of the present invention, RA is selected from the group consisting of optionally substituted 3-8 membered monocyclic heterocyclyl, 6-14 membered spirocyclic heterocyclyl, 5-14 membered fused ring heterocyclyl, 5-14 membered bridged ring heterocyclyl, wherein the heteroatom of the heterocyclyl is selected from nitrogen.

[0080] In some embodiments of the present invention, RA is selected from optionally substituted 5-6 membered monocyclic heterocyclyl.

[0081] In some embodiments of the present invention, RA is selected from the group consisting of optionally substituted azetidinyl, pyrrolidinyl, piperidinyl,

[0082] In some embodiments of the present invention, RA is selected from the group consisting of the optionally substituted substituents:

[0083] In some embodiments of the present invention, the optionally substituted substituent is selected from H, C 1-3 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl.

[0084] In some embodiments of the present invention, the compound of the structure represented by formula (A-1) has the structure represented by formula (A-2) or (A-3):

[0085] wherein m1 and n1 are independently selected from integers of 0-6, such as 0, 1, 2, 3, 4, 5, 6.

[0086] In some embodiments of the present invention, the compound of formula (A-1) has a structure represented by formula (A-4), (A-5) or (A-6):

[0087] In some embodiments of the present invention, the structure represented by formula (A-1) is connected to L via the nitrogen atom on the amino group, and more preferably via the nitrogen atom on RA.

[0088] For example:

[0089] The wavy line shows the connection site between the bioactive molecules D and L.

[0090] In some embodiments, the biologically active molecule D is selected from the group consisting of compounds of the formula:

[0091] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0092] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0093] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v and w are each independently selected from an integer of 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0094] In some embodiments, the antibody drug conjugate of Formula I and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled products are selected from the following structures:

[0095] Preferably, the antibody A of structures 9-11 in the above table targets CD33 or CD123, further preferably 018, 019, 020, 021, 022 or 023.

[0096] In some embodiments, the present invention provides use of an antibody-drug conjugate of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products in the preparation of drugs for treating cancer. The cancers include liver cancer, kidney cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), stomach cancer, esophageal cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood tumors or multifocal glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma), cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

[0097] In some embodiments, the present invention provides an antibody drug conjugate of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products for use in treating cancer. The cancers include liver cancer, kidney cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), stomach cancer, esophageal cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood tumors or multifocal glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma), cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

[0098] In some embodiments, the cancer is a tumor in which tumor cells express HER2 (HER2+), CD33 (CD33+), and CD123 (CD123+).

[0099] In some embodiments, the present invention provides antibody drug conjugates of formula (I) structure and tautomers, mesomorphs, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels thereof for the treatment of cancer. The cancer is preferably a cancer associated with abnormal expression of HER2, CD33, and CD123. Cancers associated with abnormal expression of CD33 or CD123 include leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia). Cancers associated with abnormal expression of HER2 include lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), breast cancer, ovarian cancer, endometrial cancer, gastric cancer, and prostate cancer. Cancers associated with abnormal expression of BCMA include lymphoma (e.g., multiple myeloma).

[0100] In some embodiments, the present invention provides a pharmaceutical composition comprising an antibody drug conjugate having a structure of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels and one or more pharmaceutically acceptable excipients.

[0101] Another aspect of the present invention provides a linker-drug compound represented by formula (II) or its tautomers, mesomers, racemates, enantiomers, diastereomers, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products: L'-D (II)

[0102] Wherein L' is a linker moiety;

[0103] D is an amino-containing bioactive molecule of formula (I) or a pharmaceutically acceptable salt thereof, which is covalently linked to the linker portion L' via the amine group in its molecular structure;

[0104] In some embodiments, the linker moiety L' is represented by the formula:

[0105] L1'-L2-L3-L4-

[0106] Wherein, L1' is selected from or Among them, ** indicates connection with L2;

[0107] L2, L3, L4, and D are defined the same as in formula (I).

[0108] L2 is a spacer; preferably, it is selected from -L 2a -C(O)-、-L 2a -L 2b -C(O)-、-L 2a -NH-C(O)-、-L 2a -C(O)-NH-、-L 2a -L 2b -NH-C(O)-、-L 2a -L 2b -C(O)-NH-、-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-、-L 2a -C(O)-NH-L 2b -C(O)-NH-、-L 2a -NH-C(O)-L 2b -NH-C(O)-、-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)-, where L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C6-C 14 Arylene-, -C6-C 14arylene-C1-C8 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C8 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C6 L is substituted by one or more substituents of alkyl, 1-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl are selected from one or more of N, O or S; 2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, 2-8 membered heteroalkyl, C6-C 14Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more of N, O or S;

[0109] L3 is a polypeptide sequence selected from peptide residues consisting of 2-8 (e.g., 2, 3, 4, 5, 6, 7, 8) natural or unnatural amino acids, wherein any of the amino acids is further substituted with one or more substituents selected from C1-C6 alkyl, 2-6 atom heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl, or C3-C8 cycloalkyl;

[0110] L4 is a self-cleaving fragment modified with a hydrophilic group,

[0111] The self-cleaving fragment is selected from:

[0112] or

[0113] Where * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group of the bioactive molecule D, and X is absent or *** indicates connection with carbon atoms, **** indicates connection with oxygen atoms, and arrows indicate the modification sites of hydrophilic groups;

[0114] Further preferably, the self-cleaving fragment is selected from the following structures before modification with the hydrophilic group:

[0115] or

[0116] Wherein Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 -OCH2CH2- structural units, and n is an integer from 0 to 6;

[0117] The hydrophilic group has at least one azide group and comprises a polyethylene glycol group, a polynatural amino acid or non-natural amino acid group, a monosaccharide, an oligosaccharide or a polysaccharide or a combination of the above groups;

[0118] Preferably, L4 is selected from:

[0119] or

[0120] Where * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D;

[0121] R 2 47,48,49,50) (OCH2CH2) structural units, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) natural or unnatural amino acid peptide chain, a linear or branched heteroalkyl group containing a monosaccharide, oligosaccharide or polysaccharide; X is absent or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is a C1-C6 alkylene or -R 3 -C(O)-, where R 3 It is a C1-C6 alkylene group or a heteroalkylene group containing 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) -OCH2CH2- structural units, and n is an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, 6).

[0122] In some embodiments, R 2 Selected from the following structures:

[0123] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); a is selected from a bond, a C1-C3 alkylene group, such as methylene, ethylene, n-propylene, isopropylene; R bSelected from C1-C3 alkyl, such as methyl, ethyl, n-propyl, isopropyl.

[0124] In some embodiments, R 2 Selected from the following structures:

[0125] In some embodiments, Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 C1-C6 alkylene or -C1-C6 alkylene-(OCH2CH2) o -, wherein o is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0126] In some embodiments, Y is C1-C3 alkylene or -R 3 -C(O)-, where R 3 C1-C3 alkylene or -C1-C3 alkylene-(OCH2CH2) o -, wherein o is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0127] In some embodiments, Y is methylene, ethylene, n-propylene, isopropylene, or -R 3 -C(O)-, where R 3 Methylene -(OCH2CH2) o -, ethylene-(OCH2CH2) o -, n-propylene-(OCH2CH2) o -, isopropylidene-(OCH2CH2) o -, wherein o is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0128] In some embodiments, Y is selected from -CH2-(OCH2CH2)4C(O)-.

[0129] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2a Selected from -C1-C6 alkylene-, -C1-C6 alkylene-C3-C6 cycloalkylene-, -C6-C10 Arylene-, -C6-C 10 arylene-C1-C6 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C6 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, a straight or branched chain heteroalkylene group, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, C1-C3 L is substituted by one or more substituents of alkyl, 1-3 atoms of heteroalkyl, C1-C3 alkoxy, hydroxy, amino, carboxyl or C3-C6 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl are selected from one or more of N, O or S; 2b 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched chain heteroalkylene, 1 Selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C1-C3 haloalkyl, 2-6 membered heteroalkyl, C6-C 10Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical are each optionally substituted by one or more substituents independently selected from C1-C3 alkyl, 2-3 atoms of heteroalkyl, C1-C3 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclic radical, heteroaryl, heteroalkylene are selected from one or more of N, O or S.

[0130] In some embodiments, L2 is selected from -L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-, where L 2aSelected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, -C1-C3 alkylene-cyclohexylene-, -phenylene-, -phenylene-C1-C3 alkylene-, -5-6 membered heteroaryl-C1-C3 alkylene-, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, straight or branched heteroalkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 47,48,49,50) atoms, wherein the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene is independently selected from halogen, methyl, ethyl , n-propyl, isopropyl, 1-3 atoms of heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, hydroxy, amino, carboxyl or cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl one or more substituents, the heteroalkylene, heterocyclylene, heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroalkylene, heterocyclylene, heteroarylene, heteroalkyl heteroatoms are selected from one or more of N, O or S; L 2b is selected from methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, -CH2(CH3)CH2CH2CH2CH2-, a straight or branched chain heteroalkylene radical of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, R 1selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 3-6 membered heterocyclyl, -CF3, -CF2CF3, 2-3 atoms of heteroalkyl, phenyl, 5-6 membered heteroaryl, said alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from methyl, ethyl, n-propyl, isopropyl, 2-3 atoms of heteroalkyl, methoxy, ethoxy, n-propoxy, isopropoxy, amino or carboxyl, said heteroalkyl, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, said heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene heteroatoms are selected from one or more of N, O or S.

[0131] In some embodiments, L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene, C6-C 14 Arylene-C1-C8 alkylene, 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms of straight or branched heteroalkylene, the alkylene, cycloalkylene, arylene, heteroalkylene are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heteroalkylene contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene, heteroarylene heteroatoms are selected from one or more of N, O or S; L 2b is selected from -C1-C8 alkylene-, a straight or branched chain heteroalkylene of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) atoms, R 1 Selected from hydrogen, C1-C6 alkyl, C2-C8 heteroalkyl, C6-C 14 Aryl, the alkyl, heteroalkyl, aryl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more of N, O or S. In some embodiments, L 2a Selected from -CH2CH2CH2CH2CH2-, -CH2CH2(OCH2CH2) p -、-(OCH2CH2) p -, -CH2(OCH2CH2)p -、-CH2CH2(OCH2CH2) p CH2-, -CH2CH2(OCH2CH2) p CH2CH2-、-CH2(OCH2CH2) p CH2CH2-, wherein p is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L 2b Selected from -CH2OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2-, -CH2O-, -CH2CH2O-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -(OCH2CH2) q -、-CH2CH2(OCH2CH2) q -, -CH2(OCH2CH2) q -、-CH2CH2(OCH2CH2) q CH2-, -CH2CH2(OCH2CH2) q CH2CH2-、-CH2(OCH2CH2) q CH2CH2-, R 1 Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, wherein q is an integer from 1 to 12 (preferably 2-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0132] In some embodiments, L2 is selected from the following structures:

[0133] Wherein, w is selected from an integer of 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection with L1, and ** indicates connection with L3.

[0134] In some embodiments, L3 is selected from 2-8 peptide residues formed by amino acids selected from phenylalanine (F), glycine (G), valine (V), citrulline (C), glutamic acid (E), alanine (A), lysine (K), C1-C6 alkyl substituted lysine (such as C1-C3 alkyl substituted lysine, such as methyl substituted lysine, ethyl substituted lysine, n-propyl substituted lysine, isopropyl substituted lysine), etc.

[0135] In some embodiments, L3 is selected from the following structures:

[0136] * indicates connection with L2, and ** indicates connection with L4.

[0137] In some embodiments, L4 is selected from:

[0138] , wherein v is selected from an integer of 1-12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), * indicates connection to the carboxyl group of L3 via an amide bond, and ** indicates connection to the amino group of the bioactive molecule D.

[0139] In some embodiments, L4 is selected from:

[0140] wherein r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v is selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). * indicates linkage to the carboxyl group of L3 via an amide bond, and ** indicates linkage to the amino group of the bioactive molecule D.

[0141] In some embodiments, L' is selected from the following structures:

[0142] wherein v and w are each independently selected from an integer of 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); ** represents linkage to the amino group of the biologically active molecule D; and r, s, t, and u are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

[0143] In some embodiments, L' is selected from the following structures:

[0144] Where ** indicates connection with the amino group of the biologically active molecule D.

[0145] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope labels, wherein the biologically active molecule D is selected from camptothecin derivatives, eribulin, and molecular glues.

[0146] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein the biologically active molecule D is selected from formula (A-1) to formula (A-6) described in the first aspect;

[0147] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein the biologically active molecule D is selected from the compounds of the following formula:

[0148] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0149] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below:

[0150] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and v and w are each independently selected from an integer of 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0151] In some embodiments, the linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products thereof has a structure as shown below: Beneficial effects

[0152] The present invention provides an antibody-drug conjugate based on a molecular glue with good activity. In addition, by introducing a hydrophilic linker into the ADC, most of the connections with the above-mentioned active molecules can be met, so that the obtained ADC has excellent efficacy while having good stability and hydrophilicity as well as good pharmacokinetic properties.

[0153] Abbreviations and definitions

[0154] Unless otherwise indicated, the following terms used in this application have the following meanings.

[0155] When a trade name is used in this application, unless the context indicates otherwise, the trade name includes the product formulation, generic drug, and active pharmaceutical ingredient of the trade name product.

[0156] The term "antibody-drug conjugate" or "antibody-drug conjugate" (ADC) refers to a targeting ligand such as an antibody (eg, monoclonal antibody) or antibody fragment linked to a biologically active molecule through a stable chemical linker compound.

[0157] The term "linker-drug compound" or "linker-drug compound" refers to the partial structure of an antibody-drug conjugate consisting of a linker compound and a biologically active compound.

[0158] The term "linker" refers to a chemical structure fragment, represented by L, that is linked to an antibody at one end and to a cytotoxic drug at the other. It is formed by linking a "linker compound" to the antibody and a bioactive compound, respectively. In certain embodiments of the present invention, a portion of the linker compound is first linked to the antibody, and another portion is linked to the bioactive compound. The "linker" is then formed through a chemical reaction between the different portions of the linker. The linker herein comprises a portion that connects to the antibody, a spacer, a polypeptide sequence, and a self-cleaving fragment.

[0159] The linker-drug compound of the present invention is linked to the antibody via conventional coupling methods in the art, including lysine coupling, inter-light and heavy chain reductive disulfide bond coupling, and directional coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6: 15-17; McCombs JR, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 2015, 17: 339-351). The present invention preferably couples the antibody via inter-light and heavy chain reductive disulfide bond coupling, i.e., one or more inter-light and heavy chain disulfide bond sites (two sites between the heavy chain and two sites between the heavy and light chains) are reduced to form a thiol group (sulfur atom of a cysteine ​​residue) for coupling.

[0160] In the present invention, the connecting portion of the linker L and the antibody is represented by L1, which is formed by the reaction of the L1' group in the linker compound with the antibody, for example or Wherein, * indicates connection with the sulfhydryl group of the antibody, and ** indicates connection with the spacer.

[0161] The term "spacer" refers to the linker L and the connecting portion of the antibody and the polypeptide sequence, which can be any divalent organic group, such as a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; said C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof and optionally interrupted by carbonyl, O, S, or N atoms; the C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group may be optionally replaced by C 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6 Alkyl substituted; preferably, as defined for L2.

[0162] The term "polypeptide sequence" is well known in the art and is selected from a divalent peptide group comprising 2 to 8 optionally substituted natural or non-natural amino acid residues, L-form or D-form, each of which is the same or different and is independently selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle) or analogs of the above amino acids.

[0163] The term "self-cleaving fragment" is well known in the art, such as the p-aminobenzyl carbonate fragment commonly used in the art. In a specific embodiment of the present invention, the "self-cleaving fragment" of the linker of the present invention comprises a modification of the "hydrophilic fragment".

[0164] The term "hydrophilic segment" is well known in the art, and the hydrophilic segment may comprise a polyethylene glycol group, such as a polyethylene glycol group with a methoxy terminal group, or further connected to other hydrophilic segments via polyethylene glycol groups. The hydrophilic segment may also comprise a polyamino acid segment, such as polyglycine or polysarcosine, which may be combined with a polyethylene glycol group. The hydrophilic segment may also be a monosaccharide, a disaccharide or an oligosaccharide, which may be a sugar in the form of a chain molecule or a cyclic molecule, and may comprise a glycosamine, a sugar acid or a phosphate sugar. Preferably, the sugar group is combined with a polyethylene glycol segment or a polyamino acid segment; further preferably, at least two sugar groups are introduced via a multivalent linking group, such as a linking group based on aspartic acid, glutamic acid or lysine. It may also comprise a polycarboxylic acid group, a polysulfonic acid group or a chelating group, etc.; the structure of the polycarboxylic acid group may be as follows:

[0165] The structure of the polysulfonic acid group is as follows:

[0166] The chelating group may be, for example, a DOTA group or a NOTA group.

[0167] The terms "solvate" and "solvate" mean a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain circumstances, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of isolation. The solvent molecules in the solvate may exist in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0168] The term "stereoisomer" refers to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0169] The term "tautomer" refers to isomers with different energies that are interconvertible across a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include tautomers that interconvert via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include tautomers that interconvert via reorganization of bonding electrons.

[0170] The term "plurality" in "one or more" includes two or more (e.g., 3, 4, 5, etc.);

[0171] The term "heteroatom" refers to nitrogen, oxygen, sulfur, and halogen atoms.

[0172] The term "C m -C n " means that the moiety has an integer number of carbon atoms ranging from m to n. For example, "C1-C3" means that the group can have 1 carbon atom, 2 carbon atoms or 3 carbon atoms.

[0173] The term "-(CH2) n -" means that the part has n -CH2- connected groups. For example, when the number of a connecting group is 0, such as "-(CH)0-", it means that the connecting group is a covalent bond.

[0174] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C 1-10 Alkyl), further preferably containing 1 to 8 carbon atoms (C 1-8 Alkyl), more preferably containing 1-6 carbon atoms (ie C 1-6 Alkyl). For example, "C 1-6 The term "alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl and the like.

[0175] The term "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined above, i.e., containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5 or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, and the like.

[0176] The term "halogen" or "halo" refers to F, Cl, Br, I.

[0177] The term "haloalkyl" refers to that one, two or more hydrogen atoms or all hydrogen atoms are replaced by halogen in the alkyl as defined above. Representative examples of haloalkyl include CCl , CHCl , CH Cl, CF , CHF , CH F, CBr , CHBr , CH Br, CI , CHI , CH I, CH CF , CF CF Deng.

[0178] The term "aryl" refers to an aromatic carbocyclic ring system containing 6-16 carbon atoms, or 6-14 carbon atoms, or 6-12 carbon atoms, or 6-10 carbon atoms, preferably 6-10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, or pyrenyl. The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing 5-14 members, or preferably 5-10 members, or preferably 5-8 members, more preferably 5-6 members, wherein 1, 2, 3 or more of the ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N, or S, and the number of heteroatoms being preferably 1, 2, or 3. Examples of heteroaryl groups include, but are not limited to, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, benzopyridinyl, benzopyrimidinyl, benzo pyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl, etc.

[0179] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocycle, a bridged ring, or a spirocycle. Preferably, it comprises 3-12 carbon atoms (i.e., C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), further preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, etc.

[0180] The term "-alkyl-" or "alkylene" refers to a saturated straight or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight or branched alkylene group having 1 to 8 carbon atoms.

[0181] The term "heterocyclic radical" refers to a non-aromatic ring that is fully saturated or partially undersaturated (non-completely unsaturated heteroaromatic) and can exist as a monocycle, a bridged ring or a spirocycle. Unless otherwise indicated, the heterocycle is typically a 3 to 7 ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms, but excluding -OO-, -OS- or -SS- moieties) independently selected from sulphur, oxygen and / or nitrogen. Non-limiting examples of heterocyclic radicals include but are not limited to oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl etc.

[0182] The term "heteroalkyl" by itself or in combination with another term refers to a stable linear or branched alkyl radical or combinations thereof, consisting of a certain number of carbon atoms and at least one heteroatom. The number of carbon atoms can be 1-50 (preferably 1-20, more preferably 1-12, and most preferably 1-8), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. The heteroalkyl group may optionally contain one, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from N, O, and S (or interpreted as optionally heteroatoms inserted into the alkyl group, optionally C-C bonds and C-H bonds). The heteroatoms O, N, and S may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0183] Unless otherwise stated, the definition of a term herein also applies to the group containing that term, for example the definition of C1-6 alkyl also applies to C1-6 alkyloxy.

[0184] In various parts of the present invention, linking substituents are described. When the structure clearly requires a linking group, the Markush variables listed for the group should be understood as linking groups. For example, if the structure requires a linking group and the Markush group definition for the variable lists "heteroalkyl", "aryl", etc., it should be understood that the "heteroalkyl" and "aryl" represent the heteroalkylene group or arylene group connected, respectively. And it should also be understood that when the Markush variables listed for the group are understood as linking groups, whether they are defined as "alkylene" or "heteroalkylene", the definition scope of the two is the same. Therefore, for example, the definitions of the terms "alkylene", "cycloalkylene", "arylene", "heteroalkylene", "heteroarylene", and "heterocyclylene" are equivalent or refer to the "alkyl", "cycloalkyl", "aryl", "heteroalkyl", "heteroaryl", and "heterocyclyl" as defined above.

[0185] The term "derivative" refers to a compound formed by replacing an atom or an atomic group in a parent compound molecule with another atom or an atomic group, which is called a derivative of the parent compound.

[0186] The phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a drug-linker or an antibody-linker-drug conjugate). The compound may contain at least one amino, imino, hydroxyl or carboxyl group and may therefore form an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobromides, hydroiodides, nitrates, bisulfates, phosphates, acid phosphates (-H2PO4) salts, phosphites, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, ammonium salts, calcium salts, and the like. In addition, pharmaceutically acceptable salts have more than one charged atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter atoms. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter atoms.

[0187] The term "amino acid" refers to naturally occurring amino acids or non-naturally occurring amino acids, represented by NH2-C(R'R")-C(=O)OH, wherein R' and R" are each independently hydrogen, optionally substituted straight chain, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl, or R" and the N-terminal nitrogen atom can be combined to form a heterocycle, for example, proline.

[0188] The term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and / or one hydroxyl group is removed from the carboxyl terminus of an amino acid, for example -NH-C(R'R")-C(O)-.

[0189] The term "peptide" refers to a short chain of amino acid monomers connected by peptide (amide) bonds.

[0190] The term "tumor" refers to a new organism formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in its clonal abnormal proliferation.

[0191] The term "linker" refers to a chemical structure fragment that is connected to an antibody at one end and to a cytotoxic drug at the other end, and is represented by L.

[0192] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule, represented by A. It encompasses intact polyclonal antibodies, intact 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, biparatopic antibodies, etc.), multivalent antibodies (e.g., trivalent, tetravalent, etc. antibodies having three, four, or more antigen-binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibody exhibits the desired biological activity.

[0193] CDR disclosed herein may also include variants. Typically, the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Therefore, a "variant CDR" has a specific identity and shares a biological function with a parent or reference CDR of the present invention, including but not limited to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR. For example, a "variant CDR" may be a sequence that contains 1, 2, 3 or 4 amino acid changes compared to a parent or reference CDR of the present invention and shares or improves the biological function, specificity and / or activity of the parent CDR. BRIEF DESCRIPTION OF THE DRAWINGS

[0194] Figure 1: Total Flux [p / s] detection of experimental mice in Example 5 of the present invention

[0195] Figure 2: Tumor growth curve of experimental mice in Example 5 of the present invention DETAILED DESCRIPTION

[0196] The present application is further illustrated by examples, but these examples are not intended to limit the scope of this application. The test methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, ratios, proportions, or parts are by weight.

[0197] Abbreviation Description

[0198] Table 1

[0199] Example 1: Synthesis of Compounds 8-11

[0200] Compound 1 (50 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The temperature was raised to 70°C and the reaction was refluxed for 6 hours. After cooling, white crystals precipitated. The remaining solid was filtered and washed with methanol (20 mL x 3) to obtain a white solid 2 (50 g, 100% yield). MS (ESI): (M+H) + Calculated value 266.1, experimental value 266.2.

[0201] Compound 2 (50 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol) and ethanol (400 mL) were added to a reaction flask and stirred at room temperature to dissolve. After slowly adding carbon disulfide (17 g, 283 mmol), the temperature was raised to 100°C and the reaction was refluxed for 5 hours to complete. After removing the solvent under reduced pressure, water (50 mL) was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 3 (54 g, yield 93%). MS (ESI): (M+H) + Calculated value 308.1, experimental value 308.2.

[0202] Compound 3 (5 g, 16.3 mmol), triethylamine (2 g, 19.6 mmol), and tetrahydrofuran (30 mL) were added to the reaction flask. Methyl iodide (2.5 g, 17.9 mmol) was then added to the reaction solution. The reaction was stirred at 25°C for 1.5 hours until completion. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 4 (3.4 g, 65% yield). MS (ESI): (M+H) + Calculated value 322.1, experimental value 322.2.

[0203] Compound 4 (3.4 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 5, which was directly used for the next reaction.

[0204] The crude product 5, diglycolic anhydride 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25°C for 1.5 hours. The solvent was removed under reduced pressure, and ether was added to the residue. After filtration, the residue was washed with water and ether to obtain compound 7 (3.5 g, yield 99%). MS (ESI): (M+H)+ Calculated value 338.0, experimental value 338.2.

[0205] Compound 7 (3.5 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 8 (2.3 g, yield 60%). MS (ESI): (M+H) + Calculated value 370.0, experimental value 370.1.

[0206] Table 2

[0207] The synthesis of compounds 9-11 refers to the synthetic route of compound 8.

[0208] Example 2: Synthesis of Compound 14

[0209] Tert-butyl glycolate (2.57 g, 19.5 mmol) was slowly added dropwise to a solution of chlorosulfonyl isocyanate (2.75 g, 19.5 mmol) in dichloromethane (40 mL) at 0°C, and the temperature was restored to 25°C for reaction for 1 hour. A solution of compound 5 (5 g, 19.5 mmol) in dichloromethane (10 mL) was added dropwise to the above reaction system. After 6 hours of reaction, water (40 mL) was added and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 12 (7.68 g, yield 86%). MS (ESI): (M+H) + Calculated value 459.1, experimental value 459.0.

[0210] Compound 12 (7.68 g, 16.8 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (4.2 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 13, which was directly used for the next reaction.

[0211] The crude product 13 and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (4 g, 25.2 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 14 (3.28 g, 45% yield). MS (ESI): (M+H) + Calculated value 435.0, experimental value 435.1.

[0212] Example 3: Synthesis of Compound 18

[0213] The synthesis of compound 18 followed the synthesis method of compound 14.

[0214] Example 4: Synthesis of Compounds 20, 22, and 37

[0215] p-Bromobenzaldehyde (7.97 g, 43 mmol), sodium azide (5.57 g, 86 mmol), cuprous iodide (817 mg, 4.3 mmol), L-proline (1.48 g, 12.9 mmol), sodium hydroxide (515 mg, 12.9 mmol), ethanol (70 mL), and water (20 mL) were added to a reaction flask and reacted at 95°C for 24 hours. After the reaction, the mixture was cooled, water was added, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 19 (5.5 g, 87% yield). MS (ESI): (M+H) + Calculated value 148.0, experimental value 148.1.

[0216] Zinc powder (7.3 g, 113 mmol) and tetrahydrofuran (50 mL) were added to a reaction flask, and a solution of propyne bromide (14.9 g, 125.5 mmol) in tetrahydrofuran (30 mL) was added dropwise at 10°C. The mixture was reacted at 10°C for 2 hours, and a solution of 19 (2.75 g, 18.7 mmol) in tetrahydrofuran (30 mL) was slowly added dropwise, and stirred for another 2 hours. The reaction solution was poured into a 1N HCl aqueous solution at 0°C and extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 20 (3.15 g, 90% yield). MS (ESI): (M+H) + Calculated value 188.0, experimental value 188.1.

[0217] Compound 20 (1 g, 5.3 mmol) and dichloromethane (5 mL) were added to a reaction flask, and dichlorothionyl (1.9 g, 16 mmol) was added dropwise at 0°C. After the addition, the temperature was restored to 25°C and the reaction was continued for 2 hours. The solvent and the remaining dichlorothionyl were removed under reduced pressure, and potassium thioacetate (906 mg, 8 mmol) and N,N-dimethylformamide (10 mL) were added. The reaction was continued at 25°C for 12 hours. Water was added and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Tetrahydrofuran (10 mL) and potassium hydroxide (594 mg, 10.6 mmol) solution were added, the reaction was continued at 25°C for 2 hours, water was added, extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 21 (612 mg, yield 56%). MS(ESI):(M+H) + Calculated value 204.0, experimental value 204.1.

[0218] Cyclohexene oxide (1.47 g, 15 mmol) and 40% trimethylbenzylammonium hydroxide (2.5 g, 6 mmol) were added to a reaction flask. Compound 21 (612 mg, 3 mmol) was dissolved in N,N-dimethylformamide (6 mL) and added dropwise to the above reaction system. The mixture was reacted at 25°C for 3 hours. Water was added and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 22 (813 mg, 90% yield). MS (ESI): (M+H) + Calculated value 302.1, experimental value 302.1.

[0219] Trimethylsilyl cyanide (3.86 g, 39 mmol) was added to tetrahydrofuran (20 mL), and tetrabutylammonium fluoride (35.7 mL, 1 M) was added dropwise at 0°C. After reacting for 1 hour, a solution of compound 30 (10 g, 32.5 mmol) in acetonitrile (30 mL) was added. The mixture was reacted at 80°C for 1 hour. After cooling, the solvent was removed, and the residue was purified by silica gel chromatography to obtain compound 31 (5.4 g, 66% yield). MS (ESI): (M+H) + Calculated value 254.0, experimental value 254.1.

[0220] Compound 31 (5.4 g, 21.5 mmol) was added to tetrahydrofuran, and a borane tetrahydrofuran solution (21.5 mL, 1 M) was added dropwise at 0°C. The mixture was then reacted at 25°C for 16 hours. Methanol was added to quench the reaction, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 32 (4.1 g, 74% yield). MS (ESI): (M+H) + Calculated value 258.0, experimental value 258.1.

[0221] Compound 32 (4.1 g, 15.9 mmol) and triethylamine (3.2 g, 31.8 mmol) were added to dichloromethane (30 mL), and trifluoroacetic anhydride (5 g, 23.8 mmol) was added dropwise at 0°C. The mixture was then reacted at 25°C for 1 hour, diluted with water, and extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 33 (5.6 g, 60% yield). MS (ESI): (M+H) + Calculated value 354.0, experimental value 354.1.

[0222] Compound 33 (5.6 g, 9.5 mmol) and paraformaldehyde (0.37 g, 12.4 mmol) were added to sulfuric acid and reacted at 25°C for 1 hour. The reaction solution was poured into ice water and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 34 (2.77 g, yield 80%). MS (ESI): (M+H) + Calculated value 366.0, experimental value 366.1.

[0223] Compound 34 (2.77 g, 7.6 mmol) was added to tetrahydrofuran, and diisobutylaluminum hydride was added dropwise at 0°C. The mixture was then reacted at 25°C for 2 hours. Water was added to quench the reaction, and the filtrate was filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 35 (1.65 g, 90% yield). MS (ESI): (M+H) + Calculated value 242.0, experimental value 242.1.

[0224] Compound 35 (1.65 g, 6.8 mmol), sodium azide (3.5 g, 54.4 mmol), cuprous iodide (130 mg, 0.68 mmol), L-proline (0.4 g, 2 mmol), and sodium hydroxide (80 mg, 2 mmol) were added to ethanol (20 mL) and water (10 mL), and the mixture was reacted at 95°C for 24 hours. After cooling, water was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 36 (0.97 g, 70% yield). MS (ESI): (M+H) + Calculated value 205.1, experimental value 205.1.

[0225] Compound 36 (0.97 g, 4.8 mmol), compound 28 (1.8 g, 5.04 mmol), and triethylamine (0.76 g, 7.2 mmol) were added to tetrahydrofuran (10 mL) and reacted at 25°C for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 37 (1.7 g, 80% yield). MS (ESI): (M+H) + Calculated value 447.2, experimental value 447.1.

[0226] Example 5: Synthesis of Compound 45

[0227] Compound 38 (10 g, 50 mmol) was dissolved in tetrahydrofuran (120 mL), and sodium hydride (4 g, 100 mmol) was added at 0°C and stirred for 0.5 hours. Allyl bromide (12.1 g, 100 mmol) was then added and allowed to react for 2 hours. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted with ethyl acetate (10 mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 39 (4.65 g, 66% yield). MS (ESI): (M+H) + Calculated value 142.2, experimental value 142.3.

[0228] Compound 39 (4.65 g, 33 mmol) was added to tetrahydrofuran (100 mL) and a tetrahydrofuran solution of 9-borabicyclo[3.3.1]nonane (80 mL, 0.5 M) was added. The mixture was reacted at 80° C. for 2 hours, cooled to room temperature, and the reaction solution was directly used for the next reaction.

[0229] To the reaction mixture of compound 40, N,N-dimethylformamide (100 mL) and an aqueous solution of potassium phosphate (8.7 g, 41 mmol) were added, stirred for 10 minutes, and 3-chloro-4-bromoaniline (6.8 g, 33 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (2.4 g, 3.3 mmol) were added. The reaction was carried out at 90°C for 3 hours, cooled to room temperature, and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 41 (3.7 g, 42% yield). MS (ESI): (M+H) + Calculated value 269.1, experimental value 269.1.

[0230] Compound 41 (3.7 g, 13.8 mmol) was added to dichloromethane (90 mL), and diisopropylethylamine (3.5 g, 27 mmol) and phenyl chloroformate (2.5 g, 16 mmol) were added to the solution in sequence. The mixture was reacted at 25°C for 0.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 42 (4.66 g, yield 89%). MS (ESI): (M+H) + Calculated value 389.2, actual value 389.2.

[0231] Compound 42 (4.66 g, 12 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of compound 43 (4.08 g, 13.2 mmol). The solid was dispersed in the solution by ultrasound, and diisopropylethylamine (48 mmol) was added. The reaction was carried out at 50°C for 3 hours. The mixture was cooled to room temperature, and water was added. The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 44 (6.4 g, yield 94%). MS (ESI): (M+H) + Calculated value 568.2, experimental value 568.4.

[0232] Compound 44 (1.6 g, 2.8 mmol) was added to dichloromethane (19 mL), and a solution of hydrogen chloride in ethyl acetate (11 mL, 4 M) was added dropwise at 0°C. The reaction was continued for 2 hours. The solvent was removed under reduced pressure and the mixture was dried in vacuo to obtain compound 45 (1.6 g, 94% yield). MS (ESI): (M+H) + Calculated value 568.2, experimental value 568.3.

[0233] Example 6: Synthesis of Compounds 46-49

[0234] Table 3

[0235] The synthesis of compounds 46-49 followed the synthetic route of compound 45.

[0236] Example 7: Synthesis of Compounds 52 and 55

[0237] Compound 50 (1.1 g, 1.06 mmol, obtained from solid-phase peptide synthesis) and diethylamine (3.89 g, 53.24 mmol) were dissolved in dichloromethane (5 mL). The reaction solution was purged with N2 three times and then reacted at 25°C for 1 hour. After complete conversion of the starting material, the solvent was removed under reduced pressure, and low-boiling point compounds were removed under high vacuum. Compound 51 (3.15 g, 1.6 mmol) and N,N-dimethylformamide (4 mL) were then added and stirred. Triethylamine (3.23 g, 3.2 mmol) was added dropwise at 25°C. After 6 hours of reaction, no starting material remained as determined by LCMS. Excess triethylamine was removed under reduced pressure, and the residue was directly separated by reverse phase chromatography to obtain compound 52 (810 mg, 10% yield). MS (ESI): (M+H) + Calculated value 812.4, experimental value 812.3.

[0238] Compound 53 (1.2 g, 1.36 mmol, synthesized according to the synthetic route reported in patent CN 111757757 A) and compound 54 (447 mg, 1.36 mmol, obtained from solid-phase peptide synthesis) were added to N,N-dimethylformamide (40 mL), followed by the addition of 1-hydroxybenzotriazole (40 mg, 0.26 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (285 mg, 1.46 mmol). The reaction was stirred at 25°C for 16 hours. After terminating the reaction, the solvent was removed under reduced pressure, and the residue was separated by reverse phase chromatography to obtain compound 55 (820 mg, 50% yield). MS (ESI): (M+H) + Calculated value 1208.5, experimental value 1208.6.

[0239] Example 8:

[0240] Compound 45 (0.1 mmol) was dissolved in DMF (2 mL), and Linker-04 (0.11 mmol) was added to the solution. DIPEA (0.3 mmol) was added dropwise and the mixture was stirred. The reaction progress was monitored by TLC and LCMS. When the reaction was complete, ethyl acetate and saturated sodium chloride solution were added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound LD02 (0.085 mmol, 85%). LC-MS (ESI): (M+H)+ Calculated value 1080.4, experimental value 1080.9.

[0241] Example 9:

[0242] Compound 57 (0.1 mmol) was dissolved in DMF (2 mL), and Linker-04 (0.11 mmol) was added to the solution. DIPEA (0.3 mmol) was added dropwise and stirred. The reaction progress was monitored by TLC and LCMS. When the reaction was complete, ethyl acetate and saturated sodium chloride solution were added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound LD06 (0.083 mmol, 83%). LC-MS (ESI): (M+H) + Calculated value 1052.4, experimental value 1052.7.

[0243] Example 10: Synthesis of Compound LD38

[0244] At 0°C, 1-(4-aminophenyl)-3-butyn-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and anhydrous DCM (90 mL) were added to a reaction flask. After 3 hours of reaction, the solvent was removed under reduced pressure, and the residue was slurried in 500 mL of MTBE to obtain compound 56 (9007 mg, 64% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 455.2, experimental value 455.2.

[0245] At 0°C, 56 (4000 mg, 8.8 mmol, dr = 1:1) and 100 mL of THF were added to a reaction flask. DBU (1337 mg, 8.8 mmol) was slowly added with stirring. After half an hour of reaction, the mixture was allowed to cool to room temperature. After TLC monitoring, the starting material disappeared, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 57 (1776 mg, 87% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 233.1, experimental value 233.2.

[0246] Compound 57 (1624 mg, 7 mmol, dr = 1:1), Fmoc-Val-OSu (362 mg, 8.4 mmol, CASRN: 688585-20-8), and N,N-dimethylformamide (150 mL) were added to a reaction flask, stirred, and DIEA (1158 uL, 7 mmol) was slowly added dropwise. The reaction was carried out at 25°C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added for slurrying to precipitate a white solid. This reaction was repeated three times to obtain compound 58 (dr = 1:1). MS (ESI): (M+H) + Calculated value 554.3, experimental value 554.4.

[0247] Compound 58 (1303 mg, 2.4 mmol, dr = 1:1), p-nitrophenyl chloroformate (964.8 mg, 4.8 mmol), and THF (120 mL) were added to a reaction flask, stirred, and Py (382.8 uL, 4.8 mmol) was added dropwise. The reaction was continued at 65°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to obtain compound 59 (1430 mg, 83% yield, dr = 1:1). MS (ESI): (M+Na) + Calculated value 741.3, experimental value 741.5.

[0248] Compound 59 (3.72 g, 5.174 mmol), compound 45 (2.6 g, 4.311 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask, stirred, and DIPEA (1.668 g, 2.254 mL, 12.933 mmol) was slowly added dropwise. The mixture was reacted at 25°C for 2 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 60 (3.86 g, 79.52% yield). MS (ESI): (M+H) + Calculated value 1147.5, experimental value 1147.8.

[0249] Compound 60 (3.85 g, 3.419 mmol) and N,N-dimethylformamide (50 mL) were added to a reaction flask, and DBU (417.3 mg, 409.5 μL, 2.735 mmol) was added dropwise. The reaction was allowed to react at 0°C for 0.5 hours. After the reaction was complete, methanesulfonic acid (590.8 mg, 399 μL, 6.154 mmol) was added to neutralize the reaction system, and the pH was measured to be approximately 7. The solvent was removed under reduced pressure, and the residue was slurried sequentially with MTBE and DCM. The solid was collected by filtration and dried to obtain compound 61 (2.728 g, 78.22% yield). MS (ESI): (M+H) + Calculated value 925.4, experimental value 925.8.

[0250] Compound 61 (1.15 g, 1.125 mmol), compound 8 (622 mg, 1.687 mmol) and HOBT (30 mg, 0.225 mmol) were dissolved in N,N-dimethylformamide (22.5 mL), EDC (262 mg, 298 μL, 1.687 mmol) was added, and the mixture was reacted at 25°C for 2 hours. After the reaction was completed, 110 mL of deionized water was placed in an ice bath, and the reaction solution was slowly added dropwise to deionized water with stirring to precipitate a white solid. The filter cake was filtered and washed with deionized water and n-hexane, and the solid was collected and dried to obtain an off-white solid compound 62 (1.256 g, yield 87.44%). MS (ESI): (M+H) + Calculated value 1276.4, experimental value 1276.8.

[0251] Compound 62 (896.3 mg, 0.7025 mmol), N3-24PEG-OMe (860.9 mg, 0.7728 mmol), sodium ascorbate (27.8 mg, 0.1405 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (61.0 mg, 0.1405 mmol), and cuprous bromide (20.1 mg, 0.1405 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and tert-butanol (40 mL) and acetonitrile (40 mL) were added. The reaction was allowed to react at 60°C for 18 hours. After completion of the reaction as monitored by LCMS, the solid was removed by filtration, the solvent was removed under reduced pressure, the solution was diluted with water (20 mL), and acidified to a pH close to 5 with formic acid. After purification by reverse-phase chromatography, the solvent was removed under reduced pressure, and the product was lyophilized to afford compound LD38 (703.4 mg, 41.91% yield). MS (ESI): (M+2H) 2+ Calculated value 1195.5, experimental value 1195.9.

[0252] Example 11: Synthesis of Compound LD39-54

[0253] Table 4

[0254] The synthetic routes of compounds LD39-45 refer to the synthesis of compound LD38.

[0255] The structures of Con1-5, Pep1-5, Trig1-3, HDP1-3, and Pay8-12 are as follows:

[0256] The nitrogen end was connected to Fmoc.

[0257] Among them, the # position is connected to -N3, the ## position is connected to -OH, and the ### position is connected to alkynyl.

[0258] Example 12: General method for ADC sample preparation and DAR determination

[0259] Antibodies with known sequences can be obtained by protein expression methods well known to those skilled in the art, and then the fermentation broth can be subjected to affinity chromatography, ion chromatography and other steps to obtain a sample with higher purity.

[0260] The antibody sample was diluted to approximately 10 mg / mL in an appropriate buffer (the same as the sample buffer). An appropriate amount of the reducing agent, TCEP, was added, with the equivalents adjusted based on the target DAR (e.g., 8-10 equivalents for a target DAR of 8). The pH was then adjusted to 7-7.4 with Tris buffer and reduced at room temperature for 1-1.5 hours. The antibody reduction intermediates were monitored by CE-SDS. Once the antibody was fully reduced, an appropriate amount of saturated citric acid solution was added to adjust the pH to approximately 6.5. An excess of the linker-drug solution in DMSO was then added to achieve a linker-drug equivalent ratio of 15-20 times that of the antibody. The coupling reaction was carried out at room temperature for approximately 30 minutes. After the coupling was complete, the reaction solution was filtered and then ultrafiltered using a centrifugal concentrator to remove excess linker-drug and other small molecule impurities. After purification, the resulting sample was subjected to DAR determination by mass spectrometry. Data statistics for all ADC samples are shown in the table below.

[0261] Table 5

[0262] The amino acid sequence of the antibody used for conjugation is as follows:

[0263] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0264] VHCDR1 is provided as SEQ ID NO. 21: GFNIKDTYIH

[0265] VHCDR2 is provided as SEQ ID NO. 22: RIYPTNGYTRYADSVKG

[0266] VHCDR3 is provided as SEQ ID NO. 23: WGGDGFYAMDY>Light chain (SEQ ID NO. 2)

[0267] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0268] VLCDR1 is provided as SEQ ID NO. 24: RASQDVNTAVA

[0269] VLCDR2 is provided as SEQ ID NO. 25: SASFLYS

[0270] VLCDR3 is provided as SEQ ID NO. 26: QQHYTTPPT

[0271] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0272] VHCDR1 is provided as SEQ ID NO. 27: GFTFTDYTMD

[0273] VHCDR2 is provided as SEQ ID NO. 28: DVNPNSGGSIYNQRFKG

[0274] VHCDR3 is provided as SEQ ID NO. 29: NLGPSFYFDY

[0275] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0276] VLCDR1 is provided as SEQ ID NO. 30: KASQDVSIGVA

[0277] VLCDR2 is provided as SEQ ID NO. 31: SASYRYT

[0278] VLCDR3 is provided as SEQ ID NO. 32: QQYYIYPYT

[0279] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0280] VHCDR1 is provided as SEQ ID NO. 33: DSNIH

[0281] VHCDR2 is provided as SEQ ID NO. 34: YIYPYNGGTDYNQKFKN

[0282] VHCDR3 is provided as SEQ ID NO. 35: GNPWLAY

[0283] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0284] VLCDR1 is provided as SEQ ID NO. 36: RASESLDNYGIRFLT

[0285] VLCDR2 is provided as SEQ ID NO. 37: AASNQGSG

[0286] VLCDR3 is provided as SEQ ID NO. 38: QQTKEVPWS

[0287] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0288] VHCDR1 is provided as SEQ ID NO. 39: DSNIH

[0289] VHCDR2 is provided as SEQ ID NO. 40: YIYPYNGGTDYNQKFKN

[0290] VHCDR3 is provided as SEQ ID NO. 41: GNPWLAY

[0291] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0292] VLCDR1 is provided as SEQ ID NO. 42: RASESLDNYGIRFLT

[0293] VLCDR2 is provided as SEQ ID NO. 43: AASNQGSG

[0294] VLCDR3 is provided as SEQ ID NO. 44: QQTKEVPWS

[0295] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0296] VHCDR1 is provided as SEQ ID NO. 45: SYYIH

[0297] VHCDR2 is provided as SEQ ID NO. 46: VIYPGNDDISYNQKFQG

[0298] VHCDR3 is provided as SEQ ID NO. 47: EVRLRYFDV

[0299] KSFNRGEC

[0300] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0301] VLCDR1 is provided as SEQ ID NO. 48: KSSQSVFFSSSQKNYLA

[0302] VLCDR2 is provided as SEQ ID NO. 49: WASTRES

[0303] VLCDR3 is provided as SEQ ID NO. 50: HQYLSSRT

[0304] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0305] VHCDR1 is provided as SEQ ID NO. 51: NYDIN

[0306] VHCDR2 is provided as SEQ ID NO. 52: WIYPGDGSTKYNEKFKA

[0307] VHCDR3 is provided as SEQ ID NO. 53: GYEDAMDY

[0308] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0309] VLCDR1 is provided as SEQ ID NO. 54: KASQDINSYLS

[0310] VLCDR2 is provided as SEQ ID NO. 55: RANRLVD

[0311] VLCDR3 is provided as SEQ ID NO. 56: LQYDEFPLT

[0312] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0313] VHCDR1 is provided as SEQ ID NO. 57: DYNMH

[0314] VHCDR2 is provided as SEQ ID NO. 58: YIYPYNGGTGYNQKFK

[0315] VHCDR3 is provided as SEQ ID NO. 59: GRPAMDY

[0316] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0317] VLCDR1 is provided as SEQ ID NO. 60: RASESVDNYGISFMN

[0318] VLCDR2 is provided as SEQ ID NO. 61: AASNQGS

[0319] VLCDR3 is provided as SEQ ID NO. 62: QQSKEVPWT

[0320] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0321] VHCDR1 is provided as SEQ ID NO. 63: SYYIH

[0322] VHCDR2 is provided as SEQ ID NO. 64: VIYPGNDDISYNQKFQG

[0323] VHCDR3 is provided as SEQ ID NO. 65: EVRLRYFDV

[0324] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0325] VLCDR1 is provided as SEQ ID NO. 66: KSSQSVFFSSSQKNYLA

[0326] VLCDR2 is provided as SEQ ID NO. 67: WASTRES

[0327] VLCDR3 is provided as SEQ ID NO. 68: HQYLSSRT

[0328] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (e.g.

[0329] As defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987):

[0330] VHCDR1 is provided as SEQ ID NO. 69: SSIMH

[0331] VHCDR2 is provided as SEQ ID NO. 70: YIKPYNDGTKYNEKFKG

[0332] VHCDR3 is provided as SEQ ID NO. 71: EGGNDYYDTMDY

[0333] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0334] VLCDR1 is provided as SEQ ID NO. 72: RASQDINSYLS

[0335] VLCDR2 is provided as SEQ ID NO. 73: RVNRLVD

[0336] VLCDR3 is provided as SEQ ID NO. 74: LQYDAFPYT

[0337] The heavy chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0338] VLCDR1 is provided as SEQ ID NO. 75: NYWMH

[0339] VLCDR2 is provided as SEQ ID NO. 76: ATYRGHSDTYYNQKFKG

[0340] VLCDR3 is provided as SEQ ID NO. 77: GAIYDGYDVLDN

[0341] The light chain variable region (VH) of the antigen binding protein may include the following CDRs or variants of these CDRs (as defined by Kabat (Kabat et al.: Sequences of proteins of Immunological Interest NIH, 1987)):

[0342] VLCDR1 is provided as SEQ ID NO. 78: SASQDISNYLN

[0343] VLCDR2 is provided as SEQ ID NO. 79: YTSNLHS

[0344] VLCDR3 is provided as SEQ ID NO. 80: QQYRKLPWT

[0345] Example 13 Preparation of Control ADC Samples

[0346] ORM-5029 was obtained by the method of reference document WO 2021 / 198965 A1. ORM-6151 (using 018) was obtained by the method of reference document WO 2022 / 254376 A1 as a control example of the pay8-pay11 series ADC, and the DAR was determined to be 3.5 by LC-MS. In addition, low DAR samples of ADC38-ADC44 were obtained under the same coupling conditions, and were respectively recorded as ADC38-4 to ADC44-4. The DARs determined by LC-MS were also 3.5±0.1. In particular, when the linker-drug used in ORM-5029 was coupled to the antibody, due to the high polymer ratio, it was not possible to successfully obtain qualified ADC samples with high Dar values ​​(>7). Enhertu is a commercially available product.

[0347] Effect Example 1: In vitro activity test of ADC samples

[0348] 1.1 SK-BR-3 cells with high HER2 expression were selected as test cells. The cells were cultured according to the general method and then seeded into 96-well plates at an appropriate density. After observing that the cells were in normal condition, the test sample was added. The concentration gradient was set from 0 to 100 nM. After culturing for 144 hours, the cell viability was determined using the CTG method to calculate the IC 50 See Table 6 below:

[0349] Table 6 Note: A in the table represents IC 50 Value <15pM, B represents IC 50 The value is between 15pM and 25pM, and C represents IC 50 >25pM.

[0350] From the activity test results, it can be seen that the ADC in the present invention has significantly improved activity compared with the same type ADC ORM-5029, and also has significantly improved activity compared with Enhertu with the same target.

[0351] In vitro cytotoxicity against other HER2-positive cells

[0352] BT-474 and NCI-N87 cells with high HER2 expression and T-47D cells with low HER2 expression were selected as test cells. The cells were cultured according to the general method and then seeded into 96-well plates at an appropriate density. After the cells were observed to be normal, the test sample was added with a concentration gradient of 0-100 nM. After culturing for 144 hours, the cell viability was determined using the CTG method to calculate the IC 50 value.

[0353] Table 7: BT-474

[0354] Table 8: NCI-N87

[0355] Table 9: T-47D

[0356] From the activity test results, it can be seen that the ADC of the present invention has excellent in vitro killing activity against various cells.

[0357] 1.2 Select NCI-H929 cells with high BCMA expression as test cells. Culture the cells according to general methods and then seed them into 96-well plates at an appropriate density. After observing that the cells are in normal condition, add the test sample. Set the concentration gradient from 0 to 100 nM. After culturing for 144 hours, use the CTG method to measure cell viability and calculate the IC 50 value.

[0358] Table 10

[0359] From the activity test results, it can be seen that the ADCs in the present invention all have good activity.

[0360] 1.3 MV-4-11 cells with high CD33 expression were selected as test cells. The cells were cultured according to the general method and then seeded into 96-well plates at an appropriate density. After observing that the cells were in normal condition, the test sample was added. The concentration gradient was set from 0 to 100 nM. After culturing for 144 hours, the cell viability was determined using the CTG method to calculate the IC 50 value.

[0361] Table 11: Cellular activity of ADC samples

[0362] From the activity test results, it can be seen that the ADC of the present invention has excellent activity in this type of cells, and most samples have significantly improved activity compared to the control substance ORM-6151.

[0363] Effect Example 2: In vivo activity test of ADC samples

[0364] JIMT-1 cells with low to moderate HER2 expression were inoculated into NUNU mice. After tumor formation, mice with tumor volumes of approximately 150 cubic millimeters were selected and divided into different groups of 5 mice each. Different drugs were administered via tail vein injection at pre-set doses. Tumor size and mouse weight were regularly observed and measured after administration. The final tumor size and mouse weight were measured at the end of the experiment, and the tumor inhibition rate was calculated. The test results are shown in Table 12 below:

[0365] Table 12: In vivo efficacy of non-hydrophilic linker ADCs

[0366] Among them, T-ADC is prepared according to the method of document WO 2021 / 198965 A1, and the following linker drug compound is first prepared:

[0367] Then conjugate with the antibody to obtain the target ADC:

[0368] Table 13: In vivo efficacy of hydrophilic linker ADCs

[0369] The in vivo efficacy results in Table 12 show that, for ADCs using non-hydrophilic linkers, T-ADC significantly outperforms ORM-5029 and slightly underperforms Enhertu, using the same linker. The in vivo efficacy results in Table 13 show that ADCs using hydrophilic linkers are more effective than Enhertu. Combining the results in Tables 12 and 13, we can conclude that ADCs using hydrophilic linkers are significantly more effective than those using non-hydrophilic linkers.

[0370] Effect Example 3: In vivo activity test of ADC samples

[0371] HCC-1954 cells were inoculated into NUNU mice. After tumor formation, mice with tumors approximately 100 cubic millimeters were selected and divided into different groups, with 5 mice in each group. Different drugs were administered via tail vein injection at pre-set doses. Tumor size and mouse weight were regularly observed and measured after administration. Tumor size and mouse weight were measured at the end of the experiment, and the tumor inhibition rate was calculated. The test results are shown in Table 14 below:

[0372] Table 14: In vivo efficacy of hydrophilic linker ADCs

[0373] From the in vivo efficacy results in Table 14, it can be seen that ADC using a hydrophilic linker has a good effect.

[0374] Effect Example 4: In vivo activity test of ADC samples

[0375] MV-4-11 cells were inoculated into NOD SCID mice. After tumor formation, mice with tumor volumes of approximately 100 cubic millimeters were selected and divided into different groups of 6 mice each. Different drugs were administered via tail vein injection at pre-set doses. Tumor volumes and mouse body weights were regularly observed and measured after administration. The results are shown in Table 15 below:

[0376] Table 15: In vivo efficacy of hydrophilic linker ADCs

[0377] From the in vivo efficacy results in Table 15, it can be seen that ADCs using hydrophilic linkers have excellent therapeutic effects. Some samples have significant advantages over the Yangshen therapy for this indication and can achieve complete cure.

[0378] Effect Example 5: In vivo activity test of ADC samples

[0379] MV-4-11-luc cells were selected and inoculated into NCG mice via tail vein administration. The exposure time was fixed. When the Total Flux[p / s] in the mice was detected to be around 5E6, the mice were randomly divided into different groups, with 6 mice in each group. Different drugs were administered through tail vein injection according to the pre-set dose. After administration, the weight of the mice and the Total Flux[p / s] in the mice were observed and measured regularly (Figures 1 and 2). The final fluorescence intensity and mouse weight were measured at the end of the experiment, and the tumor inhibition rate was calculated. The test results are shown in Table 16 below:

[0380] Table 16: In vivo efficacy of hydrophilic linker ADCs

[0381] As can be seen from the in vivo efficacy results in Table 16, ADCs using hydrophilic linkers have excellent therapeutic effects, achieving complete cure without rebound, and have significant advantages over the reference substance ORM-6151.

[0382] Effect Example 6: In vivo PK property evaluation of the antibody-drug conjugate of the present invention

[0383] T-ADC-2 was prepared according to the method of WO 2021 / 198965 A1, and the following linker drug compound was first prepared:

[0384] Then it is coupled with the antibody to obtain the target T-ADC-2:

[0385] The pharmacokinetic behavior of the compounds ADC38-4, ADC38, T-ADC-2, and Enhertu in rats following a single intravenous injection of 6 mg / kg was investigated. Rats were randomly assigned to three groups, each containing three male rats. The ADC compounds were injected via the tail vein at a dose of 6 mg / kg. The protocol is shown in Table 17. Animals were fasted for 2 hours prior to dosing, but were not allowed to drink water. Dosing solution was dispensed based on animal weight on the day of the experiment.

[0386] Table 17: In vivo PK experimental plan for hydrophilic linker ADC

[0387] Before dosing and at 0.5 h, 18 h, 24 h, 48 h, 96 h, and 168 h after dosing, retroorbital bleeding was performed, and blood was drawn into citrate tubes and processed into plasma. The antibody-drug conjugate content in the blood samples was determined using enzyme-linked immunosorbent assay, and pharmacokinetic parameters were calculated using a non-compartmental model. The content of the shed small molecule in the blood samples was determined using LC-MS / MS, and pharmacokinetic parameters were calculated. The results are shown in Table 18.

[0388] After converting the areas under the curves of the antibody-drug conjugate and the shed small molecule into the same units, the small molecule shedding rate was calculated based on the sample DAR value. The results are shown in Table 18.

[0389] Table 18 Small molecule shedding rate in hydrophilic ADC rat PK model

[0390] Comparing the PK parameters of ADC38 and T-ADC-2 reveals that, despite having the same high DAR value, ADC38 exhibits a significantly higher AUC than T-ADC-2. Comparing ADC38 with ADC38-4 reveals that increasing the DAR value from 4 to 8 does not significantly reduce the ADC's AUC. This suggests that the introduction of a hydrophilic side chain can significantly improve the metabolic stability of ADCs with high DAR values. Comparing the small molecule shedding rates of ADC38 and T-ADC-2 reveals that the linker technology of this invention and the hydrophilic side chain can improve the in vivo stability of the ADC linker.

Claims

1. An antibody drug conjugate having the structure of formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: A-(LD) m (I) wherein A is a targeting ligand selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment, a small molecule ligand, a polypeptide; L is a linker part, one end of which is connected to the ligand A and the other end is connected to the bioactive molecule D; D is an amino-containing biologically active molecule or its tautomer, mesomer, racemate, enantiomer, diastereomer, isotope-labeled substance, which is covalently linked to the linker portion L through the amino group in its molecular structure; m is an integer or decimal from 1 to 12; The linker moiety -L- is represented by the formula: -L1-L2-L3-L4-, in, L1 is selected from or Wherein * indicates connection with the sulfhydryl group of A (such as monoclonal antibody), and ** indicates connection with L2; L2 is a spacer selected from -L 2a -C(O)-、-L 2a -L 2b -C(O)-、-L 2a -NH-C(O)-, -L 2a -L 2b -NH-C(O)-, -L 2a -L 2b -C(O)-NH-, -L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-、-L 2a -C(O)-NH-L 2b -C(O)-NH-, -L 2a -NH-C(O)-L 2b -NH-C(O)-, -L 2a -NR 1 -SO2-NH-C(O)-OL 2b -C(O)-or-L 2a -NR 1 -SO2-NH-C(O)-OL 2b -NH-C(O)-, where L 2a Selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C6-C 14 Arylene-, -C6-C 14 Arylene-C1-C8 alkylene-, -5-6 membered heteroarylene-, -5-6 membered heteroarylene-C1-C8 alkylene-, 1-50 straight chain or branched heteroalkylene, 1-50 straight chain or branched heteroalkylene-3-8 membered heterocyclylene, the alkylene, cycloalkylene, arylene, heteroalkylene, heteroarylene, heterocyclylene are each optionally substituted by one or more substituents independently selected from halogen, C1-C6 alkyl, 1-6 heteroalkylene atoms, C1-C6 alkoxy, hydroxyl, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene, heterocyclylene, heteroalkylene contains 1-12 heteroatoms, and the heteroatoms of the heteroalkylene, heterocyclylene, heteroarylene, heteroalkylene are selected from one or more of N, O or S; L 2b is selected from -C1-C8 alkylene-, a straight chain or branched chain heteroalkylene of 1 to 50 atoms, R 1 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C1-C6 haloalkyl, 2-8 membered heteroalkyl, C6-C 14 Aryl, 5-6 membered heteroaryl, the alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, amino or carboxyl, the heteroalkyl, heteroalkylene contains 1-12 heteroatoms, the heteroatoms of the heteroalkyl, heterocyclyl, heteroaryl, heteroalkylene are selected from one or more of N, O or S; L3 is a polypeptide sequence selected from peptide residues consisting of 2-8 natural or unnatural amino acids, wherein the optional amino acids are further substituted by one or more substituents selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxyl, amino, carboxyl or C3-C8 cycloalkyl; L4 is a self-cleaving fragment, More preferably, L4 is a self-cleaving fragment modified with a hydrophilic group, The self-cleaving fragment is selected from: or Wherein, * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group of the bioactive molecule D, and X is absent or *** indicates connection with carbon atoms, **** indicates connection with oxygen atoms, and arrows indicate the modification sites of hydrophilic groups; Further preferably, the self-cleaving fragment is selected from the following structures before modification with the hydrophilic group: or Wherein Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1 to 8 -OCH2CH2- structural units, and n is an integer from 0 to 6; The hydrophilic group has at least one azide group and comprises a polyethylene glycol group, a polynatural amino acid or a non-natural amino acid group, a monosaccharide, an oligosaccharide or a polysaccharide or a combination of the above groups; Preferably, L4 is selected from: or Wherein * indicates connection with the carboxyl group of L3 by an amide bond, and ** indicates connection with the amino group of the bioactive molecule D; R 2 is a hydrophilic fragment selected from a linear or branched heteroalkyl group containing 4 to 50 -OCH2CH2- structural units, a peptide chain containing 4 to 50 protein amino acids or non-protein amino acids, a linear or branched heteroalkyl group containing a monosaccharide, an oligosaccharide or a polysaccharide; X is absent or *** indicates connection with a carbon atom, **** indicates connection with an oxygen atom; Y is C1-C6 alkylene or -R 3 -C(O)-, where R 3 is a C1-C6 alkylene group or a heteroalkylene group containing 1 to 8 -OCH2CH2- structural units, and n is an integer from 0 to 6; When A is selected from antibodies targeting CD33 or CD123, L4 may also be: Wherein, * indicates that it is connected to the carboxyl group of L3 by an amide bond, ** indicates that it is connected to the amino group or hydroxyl group of the biologically active molecule D, and X is absent or is *** indicates connection with carbon atom, **** indicates connection with oxygen atom; The bioactive molecule D is selected from a molecular glue compound, and the molecular glue compound has a structure as shown in the following formula (A-1): wherein RA is selected from an optionally substituted 3-16 membered heterocyclic group, wherein the heteroatom of the heterocyclic group is selected from nitrogen; and m1 and n1 are each independently selected from an integer of 0-6.

2. The antibody drug conjugate having the structure of formula (I) according to claim 1 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope labels, wherein A is selected from the group consisting of targeting HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, dll3, TROP2, B7-H3, c-Met, CD20, CD22, CD30, CD33, CD123, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis Antibodies or antigen-binding fragments thereof to Y, VEGFR, VEDF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4 or Mesothelin.

3. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 2, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L2 is selected from the following structures: in, w is an integer selected from 1-12, * indicates connection with L1, and ** indicates connection with L3.

4. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 3, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L3 is selected from the following structures: Among them, * indicates connection with L2, and ** indicates connection with L4.

5. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 4, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L4 is selected from the following structures: in, v is an integer selected from 1-12, * indicates connection with the carboxyl group of L3 via an amide bond, and ** indicates connection with the amino group of the biologically active molecule D.

6. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 5, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein R 2 Select from the following structures: in, r, s, t, and u are each independently selected from an integer of 1 to 50; R a is selected from a bond, a C1-C3 alkylene group, such as methylene, ethylene, n-propylene, isopropylene; R b Selected from C1-C3 alkyl, such as methyl, ethyl, n-propyl, isopropyl.

7. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 6, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L is selected from the following structures: Wherein, * indicates connection with the sulfhydryl group of antibody A, and ** indicates connection with the amino group of biologically active molecule D.

8. An antibody drug conjugate having a structure of formula (I) as described in any one of claims 1 to 7, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein: The molecular glue compound has a structure as shown in formula (A-1), wherein RA is selected from the following optionally substituted substituents: a 3-8 membered monocyclic heterocyclic group, a 6-14 membered spirocyclic heterocyclic group, a 5-14 membered condensed ring heterocyclic group, a 5-14 membered bridged ring heterocyclic group, wherein the heteroatom of the heterocyclic group is selected from nitrogen; Preferably, RA is selected from an optionally substituted 5-6 membered monocyclic heterocyclic group; Preferably, the optionally substituted substituent is selected from H, C 1-3 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl; Preferably, the compound of the structure shown in formula (A-1) has the structure shown in formula (A-2) or (A-3): Wherein m1 and n1 are each independently selected from integers of 0-6; Preferably, the compound of the structure shown in formula (A-1) has the structure shown in formula (A-4), (A-5) or (A-6): Preferably, the bioactive molecule D is selected from the compounds of the following formula:

9. An antibody-drug conjugate having a structure of formula (I) according to any one of claims 1 to 8, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is selected from antibodies or antigen-binding fragments thereof targeting Her2, BCMA, CD33, CD123; Preferably, in the order of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3, the CDR combinations of the heavy chain variable region VH and the light chain variable region of the antibody or antigen-binding fragment thereof are as follows: CDR combination I: SEQ ID NO. 21-26 CDR combination II: SEQ ID NO. 27-32 CDR combination III: SEQ ID NO.33-38 CDR combination IV: SEQ ID NO.39-44 CDR combination V: SEQ ID NO. 45-50 CDR combination VI: SEQ ID NO. 51-56 CDR combination VII: SEQ ID NO. 57-62 CDR combination VIII: SEQ ID NO. 63-68 CDR combination IX: SEQ ID NO. 69-74 CDR combination X: SEQ ID NO. 75-80; Also preferably, A is selected from trastuzumab (Trastuzumab, heavy chain and light chain combination SEQ ID NO.1-2), 007 (Pertuzumab, heavy chain and light chain combination SEQ ID NO.3-4), 014 (Gemtuzumab, heavy chain and light chain combination SEQ ID NO.5-6), 018 (CD33AB, heavy chain and light chain combination SEQ ID NO.7-8), 019 (heavy chain and light chain combination SEQ ID NO.9-10), 020 (heavy chain and light chain combination SEQ ID NO.11-12), 021 (heavy chain and light chain combination SEQ ID NO.13-14), 022 (heavy chain and light chain combination SEQ ID NO.15-16), 023 (heavy chain and light chain combination SEQ ID NO.17-18), 008 (heavy chain and light chain combination SEQ ID NO.19-20) or a biosimilar thereof.

10. An antibody drug conjugate having a structure of formula (I) according to any one of claims 1 to 9, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, which are selected from the following structures: Preferably, antibody A of structures 9-11 in the above table targets CD33 or CD123, further preferably 018, 019, 020, 021, 022 or 023.

11. A pharmaceutical composition comprising an antibody-drug conjugate having a structure of formula (I) as claimed in any one of claims 1 to 10, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products, and one or more pharmaceutically acceptable excipients.

12. Use of an antibody drug conjugate having a structure of formula (I) as claimed in any one of claims 1 to 10, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products, or the pharmaceutical composition as claimed in claim 11 in the preparation of a drug for treating cancer.

13. The use of claim 12, wherein the cancer comprises liver cancer, kidney cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), gastric cancer, esophageal cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood tumors or multiform gliomas, lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma or recurrent anaplastic large cell lymphoma), cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, etc.

14. A linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: L'-D (II) Wherein L' is a linker moiety; D is an amino-containing biologically active molecule or a pharmaceutically acceptable salt thereof, which is covalently linked to the linker portion L' via the amine group in its molecular structure; L' is represented by the following formula: L1'-L2-L3-L4- in, L1' is selected from or Among them, ** indicates connection with L2; The definitions of L2, L3, L4, and D are the same as those of claims 1-10.

15. A linker-drug compound represented by formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer, and pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, the structure of which is shown below:

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