Linker compounds and antibody-drug conjugates thereof, method for preparing same, and use thereof

Abstract

Provided are a linker compound having a hydrophilic side chain, an antibody-drug conjugate formed by the linker compound and an antibody, and a pharmaceutical use of the conjugate.

Description

A class of linker compounds and their antibody-drug conjugates, their preparation methods and uses Technical Field

[0001] This invention relates to linker compounds with hydrophilic side chains, antibody-drug conjugates formed by the linker compounds and antibodies, and the pharmaceutical uses of the conjugates. Background Technology

[0002] Drug conjugates (ADCs) are a class of innovative targeted therapies that enhance the specificity and therapeutic efficacy of small molecule drugs by binding them to monoclonal antibodies, peptides, nucleic acid aptamers, or oligonucleotides. The core technology of ADCs comprises three key components: the targeting moiety, the small molecule payload, and the linker. The targeting moiety is responsible for targeting by recognizing and binding to specific therapeutic targets, while the small molecule payload typically possesses potent physiological activity. The design of the linker is crucial to the success of ADCs; it needs to remain stable in the bloodstream to avoid premature drug release, while simultaneously enabling rapid release of the effective active ingredient upon reaching the target cells.

[0003] Linkers can be classified into cleavable and non-cleavable types. Cleavable linker drug conjugates (DDCs) utilize the difference between the tumor microenvironment and the normal physiological environment to release small molecule payloads, while non-cleavable linker DDCs release the small molecule payload and linker residues in the lysosomes after ADC internalization. Currently, existing linkers have drawbacks such as weak hydrophilicity, poor stability, and complex structure. In particular, the hydrophilicity issue affects the overall pharmaceutical properties, pharmacokinetic (DMPK) properties, efficacy, and safety of the conjugated drug.

[0004] Therefore, the technical problem that this invention needs to solve is to optimize the linker design in order to improve the efficacy and safety of existing conjugated drugs and better meet clinical needs. Summary of the Invention

[0005] This invention provides a method for preparing linker compounds, antibody-drug conjugates, and intermediates thereof, as well as their uses. The linker compounds of this invention enable the conjugated drugs to have wide applications in the biomedical field, treating various diseases such as those affecting the immune system, hematologic system, tumors, or metabolism. The main technical advantages of this invention are: the novel linker designed allows for the conjugation of highly hydrophobic loads, such as camptothecin and sea haretoxin, with antibodies, peptides, nucleic acid aptamers, or oligonucleotides using specific chemical or biological methods, resulting in conjugates with high hydrophilicity and stability. When using the linker used in this invention to conjugate small molecule loads with target regions, compared to traditional conjugate linkers, less aggregation occurs during the conjugation process, and the resulting conjugated drugs have higher DAR values. Furthermore, the inventors have found that the conjugated drugs obtained from the linker compounds of this invention exhibit high plasma stability and lower hematologic toxicity while maintaining higher antitumor activity.

[0006] Linker compounds

[0007] A first aspect of the invention relates to a linker compound, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker compound having the structure shown in Formula I.

[0008] RG-L 3 -L 2 -L 1 -D (I)

[0009] in

[0010] RG stands for reactive group;

[0011] L 3 C is an optional replacement 1-6 Alkylene-CO, C 2-6 imide-CO, C 2-6 acetylenoid-CO, C 1-6 Alkylene-C 3-8 Cycloalkylene-CO, C 1-6 Alkylene-C 3-8 Cycloalkyl-C 1-6 Heteroalkyl-CO or C 1-6 Heteroalkyl-CO;

[0012] L 2 It consists of divalent polypeptide residues composed of 2 to 5 amino acids;

[0013] L 1 It is a divalent spacer group; and

[0014] D stands for cytotoxin.

[0015] Each occurrence of a "-" is an independent single key;

[0016] The divalent polypeptide residue L 2 and / or the divalent linker L 1 Hydrophilic side chains (H) SC (hydrophilic side chain) replaces;

[0017] The hydrophilic side chain H SC Each is selected independently from:

[0018] a)-(CH2) t -SO3M, where t is an integer of 1, 2, 3, 4, 5 or 6, and M is H or an optionally substituted alkyl group (preferably C). 1-4 Alkyl groups or alkali metal ions;

[0019] b) Where t is an integer of 1, 2, 3, 4, 5 or 6;

[0020] c)-C(=O)-(CH2) t4 -P(=O)(OM1)2, where t4 is an integer of 1, 2, 3, 4, 5 or 6, and M1 is selected from H or alkali metal ions;

[0021] d)-Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H , where t0 is an integer of 1, 2, 3, 4, 5, or 6, and n is any integer from 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), and / or

[0022] e)–(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, where T is

[0023] Wherein Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or absent, wherein R is H or an optionally substituted alkyl group, preferably R is H or C. 1-4Alkyl; p1, p2, p3, q1, q2 and q3 are each independently an integer of 1, 2, 3, 4, 5 or 6, q4 is an integer of 0, 1, 2, 3, 4, 5 or 6, and s is an integer of 1, 2, 3 or 4;

[0024] Y H for Or -C(=O)-(CH2) t4 -P(=O)(OM1)2, where t1, t2, t3, t4 are each independently an integer of 1, 2, 3, 4, 5, or 6, and M and M1 are independently H or optionally substituted alkyl groups (preferably C). 1-4 Alkyl groups or alkali metal ions.

[0025] RG

[0026] In some embodiments, RG refers to an active functional group capable of specifically reacting with thiol (-SH) or amino (-NH2) groups in the side chains of antibody amino acids, or a leaving group that can be removed during coupling as a linker. Specifically, the active functional group includes, but is not limited to: maleimide groups that can react with thiol groups, and N-hydroxysuccinimide esters (NHS esters) that can react with amino groups; the removable group includes functional groups with good leaving ability such as halogens, halogenated groups (such as iodoacetyl groups), and methanesulfonyl groups.

[0027] In some implementations, RG is halogen,

[0028] L 3

[0029] In some implementations, L 3 C 1-6 Alkylene-CO, C 2-6 acetylenoid-CO, C 1-3 Alkylene-C 3-6 Cycloalkylene-CO, C 1-3 Alkylene-C 3-6 Cycloalkyl-C 1-6 Heteroalkyl-CO or C 1-6 Heteroalkyl-CO, wherein each heteroalkyl group independently contains a -C(=O)NH- or a -CH2CH2-O- heteroatom group, for example, containing one -C(=O)NH- and / or 2-12 -CH2CH2-O- heteroatom groups.

[0030] In some implementations, L 3 for Where n1 is an integer from 2 to 12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0031] RG-L 3

[0032] In some implementations, RG-L 3 for Where n1 is an integer from 2 to 12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, preferably n1 is 4.

[0033] L2

[0034] In some implementations, L 2 H by one or more hydrophilic side chains SC replace.

[0035] In some implementations, L 2 The expression is X1-X2-X3-X4-X5, where X1 and L 3 The linkage, wherein X1 and X2 are each independently an amino acid residue, and X3, X4, and X5 are each independently an amino acid residue or are absent; wherein at least one of X1, X2, X3, X4, and X5 is a Lys or Val residue. In some embodiments, X1 is Lys or Val; X2 is Val, Gly, or Ala; X3 is Ala, Gly, Cit, or absent; X4 is Phe or absent; and X5 is Gly or absent. In some embodiments, X1 is Lys. In some embodiments, X1 is Lys, wherein the Lys has an amino group with a long-chain amino side chain (i.e., -(CH2)4-NH2) substituted with 1-3 hydrophilic side chains H. sc .

[0036] In some implementations, L 2 Lys-Val-Ala Lys-Val-Cit Or Lys-Gly-Gly-Phe-Gly Each Lys contains a long-chain amino side chain (i.e., -(CH2)4-NH2) with 1-3 hydrophilic side chains H. sc .

[0037] In some implementations, H SC for

[0038] a)-(CH2) t-SO3M, where t is an integer of 1, 2, 3, 4, 5 or 6, and M is H or an optionally substituted alkyl or alkali metal ion;

[0039] b) Where t is an integer of 1, 2, 3, 4, 5 or 6;

[0040] c)-C(=O)-(CH2) t4 -P(=O)(OM1)2, where t4 is an integer of 1, 2, 3, 4, 5 or 6, and M1 is selected from H or alkali metal ions;

[0041] d)-Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H Where t0 is an integer of 1, 2, 3, 4, 5, or 6, and n is any integer from 1 to 11.

[0042] e)–(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, where T is

[0043] Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or not present, where R is H or an alkyl group that is optionally substituted; p1, p2, p3, q1, q2 and q3 are each independently an integer of 1, 2, 3, 4, 5 or 6, q4 is an integer of 0, 1, 2, 3, 4, 5 or 6, and s is an integer of 1, 2, 3 or 4;

[0044] Y H for Or -C(=O)-(CH2) t4 -P(=O)(OM1)2, where t1, t2, t3, t4 are each independently an integer of 1, 2, 3, 4, 5 or 6, and M and M1 are independently H, an optionally substituted alkyl or alkali metal ion.

[0045] In some implementations, Hsc is The variables are defined as above; or preferably t1, t2 and t3 are 3, where t0 is an integer of 1, 2, 3, 4, 5 or 6, Y1 is -C(=O)-, -N(CH3)-C(=O)- or -CH2-N(CH3)-C(=O)-, Y2 is non-existent, and other variables are defined as above.

[0046] In some implementations, H SC for

[0047] In some implementations, L 2 for

[0048] In some implementations, L 2 No hydrophilic side chain H SC Replacement. In some implementations, L 2 Val-Ala

[0049] L 1

[0050] In some implementations, L 1 for Or C 1-6 Heteroalkyl groups.

[0051] In some implementations, L 1 Unaffected by hydrophilic side chain H SC Replacement. In some implementations, L 1 for

[0052] In some implementations, L 1 H by one or more hydrophilic side chains SC Replacement. In some implementations, L 1 H by a hydrophilic side chain SC Replacement. In some implementations, L 1 for Hsc is a hydrophilic side chain. In some implementations, H... SC For -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H Where t0 is an integer of 1, 2, 3, 4, 5, or 6, and n is an integer from 1 to 11, Y H for Or -C(=O)-(CH2) t4-P(=O)(OM1)2; where t1, t2, t3, t4 are each independently an integer of 1, 2, 3, 4, 5 or 6, M and M1 are independently H, optionally substituted alkyl or alkali metal ions; Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or not present, where R is H or optionally substituted alkyl.

[0053] In some implementations, H SC For –(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, where T is

[0054] Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or not present, where R is H or an alkyl group that is optionally substituted; p1, p2, p3, q1, q2 and q3 are each independently an integer of 1, 2, 3, 4, 5 or 6, q4 is an integer of 0, 1, 2, 3, 4, 5 or 6, and s is an integer of 1, 2, 3 or 4.

[0055] In some implementations, H SC for

[0056] In some implementations, L 1 for

[0057] Cytotoxin D

[0058] In some embodiments, the cytotoxicant is selected from topoisomerase I inhibitors such as camptothecin compounds and their derivatives (e.g., eczemacon, delutecan (DXd), 14-aminocamptothecin, 9-aminocamptothecin, belotecine, etc.), topoisomerase II inhibitors such as doxorubicin compounds and their derivatives (e.g., daunorubicin), maytansine compounds and their derivatives, calichimycin compounds and their derivatives, docarmicin compounds and their derivatives (e.g., duocarmycin derivatives), and pyrrolobenzodiazepine compounds and their derivatives (e.g., PBD) as DNA alkylating agents, tubulin inhibitors such as salamander toxin compounds and their derivatives (e.g., MMAE and its derivatives or MMAF and its derivatives), immunosuppressants such as rapamycin and its derivatives, and other small molecule compounds with cytotoxic activity. In some embodiments, the cytotoxicant is selected from eczemacon, delutecan, MMAE, and rapamycin.

[0059] In some implementations, the compound of formula (I) is

[0060] Where H SC It is -C(=O)-(CH2) t4 -P(=O)(OH)2;-Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H , where t0 and t4 are each independent integers of 1, 2, 3, 4, 5, or 6, and n is an integer from 1 to 11; –(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, where T is

[0061] Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or not present, where R is H or an alkyl group that is optionally substituted; p1, p2, p3, q1, q2 and q3 are each independently an integer of 1, 2, 3, 4, 5 or 6, q4 is an integer of 0, 1, 2, 3, 4, 5 or 6, and s is an integer of 1, 2, 3 or 4;

[0062] YH for Or -C(=O)-(CH2) t4 -P(=O)(OH)2, where t1, t2, t3 and t4 are each independently an integer of 1, 2, 3, 4, 5 or 6, M is H, an optionally substituted alkyl or alkali metal ion, X2, X3, X4 and X5 are each independently an amino acid residue or are not present, and other variables are as defined for formula (I).

[0063] In some implementations, the compound of formula (I) is in

[0064] H SC For -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H Where t0 is an integer of 1, 2, 3, 4, 5 or 6, n is an integer of 1-11, and Y H for Or -C(=O)-(CH2) t4 -P(=O)(OH)2; where t1, t2, t3, and t4 are each independently an integer of 1, 2, 3, 4, 5, or 6, and M is H, an optionally substituted alkyl group, or an alkali metal ion; Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2-, or absent, where R is H or an optionally substituted alkyl group; or

[0065] H SC For –(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, where T is

[0066] Wherein Y1 and Y2 are independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or absent, wherein R is H or an optionally substituted alkyl group (preferably C). 1-4Alkyl); p1, p2, p3, q1, q2 and q3 are each independently an integer of 1, 2, 3, 4, 5 or 6, q4 is an integer of 0, 1, 2, 3, 4, 5 or 6, and s is an integer of 1, 2, 3 or 4;

[0067] Other variables are as defined in equation (I).

[0068] In some implementations, the compound of formula (I) is

[0069] Targeted drug conjugates

[0070] The second aspect of the invention relates to a targeted drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the targeted drug conjugate is formed by conjugating a linker compound (e.g., of formula (I), (II), or (III)) of the first aspect of the invention with a targeting moiety.

[0071] In some embodiments, the targeted drug conjugate has the structure shown in formula (IV).

[0072] TX-[RGR-L 3 -L 2 -L 1 -D] m (IV)

[0073] in

[0074] TX is the target region, selected from antibodies or their antigen-binding fragments, small molecule ligands, peptides, and proteins;

[0075] m is any value between 2 and 10 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6...). 4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.5, 9, 9.5, 10, or such as 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8);

[0076] Where RGR is the residue resulting from the reaction of RG (active group) and TX; other variables RG, L 3 L 2 L 1 D is as defined above in equation (I), equation (II) or equation (III).

[0077] There are no particular limitations on the antibodies or antigen-binding fragments thereof that can be used in this invention, and they can be selected from monoclonal antibodies (such as mouse antibodies, rabbit antibodies, phage-displayed antibodies, yeast-displayed antibodies, chimeric antibodies, humanized antibodies, fully human antibodies), bispecific antibodies, multispecific antibodies, nanobodies (VHH, Variable Domain of Heavy Chain Antibody), Fab fragments, Fab' fragments, and F(ab'). 2 Fragments, Fd, Fv, dAb, complementarity-determining region fragments, single-chain variable fragments (scFv), scFV-Fc bivalent molecules, single-domain antibodies (sdAb), proantibodies, and antibody fusion proteins.

[0078] In some preferred embodiments, the targeting portion targets one or more of the following antigens: aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancer), CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA242 (colorectal cancer), placental alkaline phosphatase (cancer), prostate-specific antigen (prostate), prostate acid phosphatase (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma), CD3ε (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma), CD2... CD2 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (chronic lymphocytic leukemia)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumor multiple myeloma), CD50 (leukemia, systemic lupus erythematosus). CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (cancer), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer and lung cancer), DLL4 (Δ-like protein-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hematologic oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epidermal cell adhesion molecule, bladder cancer, head and neck cancer, colon cancer, non-Hodgkin's lymphoma, prostate cancer and ovarian cancer), ERBB2 (epidermal growth factor receptor 2);Lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (a cell surface antigen glycolipid, melanoma), GD3 specific antigen (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (non-Hodgkin lymphoma), HLA-DRB (non-Hodgkin lymphoma, B-cell leukemia), human chorionic gonadotropin (cancer), IGF1R (insulin-like growth factor 1 receptor, solid tumors, hematologic malignancies), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castells disease, IL6-dependent tumors), integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, used for various cancers) MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE-4 (cancer), anti-transferrin receptor (cancer), p97 (melanoma), MS4A1 (Membrane-crossing 4-domain subfamily A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer, and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (Membrane-crossing 4-domain subfamily A, small cell lung cancer, non-Hodgkin's lymphoma), ribosomal protein, neuroproto-oncogene product (cancer), P21 (cancer), anti-(N-acetylneuraminic acid, breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate), ROBO4, TAG 72 (tumor-associated glycoprotein 72, acute myeloid leukemia, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, non-Hodgkin's lymphoma, other cancers, rheumatoid arthritis, and systemic lupus erythematosus), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1, lymphoma, non-Hodgkin's lymphoma, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, or VEGF-2 (CD309) (various cancers).

[0079] In some implementations, the targeting portion targets one or more of the following antigens: various differentiation clusters. differentiations, CD) (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, C D30, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD 49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD7 1. CD72, CD74, CD79 (CD79a, CD79b), CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, C D95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, C D138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166 , CD168, CD184, CDw186, CD195, CD202(a, b), CD209, CD235a, CD271, CD303, CD304); Annexin A1, Trop-2, STEAP-1, STEAP-2, DLL-3, DLL-4, Claudin 18.2. Nucleolin, tissue factor, endothelin (CD105), ROBO4, aminopeptidase N, Δ-like protein 4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, B7-H4, WT1, MUC1, LMP2, HPV E6 / E7, EGFRvIII, HER-2 / neu, specific antigen, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, PD-1, PD-L1, MelanA / MART1, Ras mutation, gp100, p53 mutation, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation Breakpoints), BCMA, FGFR2, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, sugar-rich GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, spermin 17, LCK, high molecular weight polypeptide, AKAP-4, SSX2, XAGE 1, B7H3, leguminous proteinase, Tie 2. Page 4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, Fos-related antigen 1.

[0080] As is generally known in the art, given that the RG has been determined, those skilled in the art can determine the RGR after the coupling reaction. For example, the coupling between a maleimide (Mal) group and a mercapto (-SH) group forms a stable thioether bond, and the coupling between an azide group (-N3) and a terminal alkyne via copper-catalyzed azide-alkyne cycloaddition (CuAAC) forms a triazole bond.

[0081] In some preferred embodiments, the targeted drug conjugate is selected from the following formulas (IV-1-1) to (IV-1-3), (IV-2-1) to (IV-2-3), and (IV-3):

[0082] The value of m can be any value from 2 to 10, preferably any value from 3.5 to 8.5; preferably 3.5 to 4.5 or 7.5 to 8.5.

[0083] Each of the Hsc1 is independently selected from

[0084] The Hsc2 mentioned is selected from

[0085] In some implementations, Hsc1 is In some implementations, Hsc1 is

[0086] In some implementations, Hsc2 is... In some implementations, Hsc2 is... In a preferred embodiment, the targeted drug conjugate has the following structure:

[0087] The Hsc1 mentioned is In some preferred embodiments, the Hsc1 is preferably...

[0088] Antibody-drug conjugates

[0089] Preferably, the targeting portion is an antibody or its antigen-binding fragment. This invention relates to an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody-drug conjugate is formed by conjugating an antibody with a linker compound (e.g., formula (I), (II), or (III)) as described in the first aspect of this invention.

[0090] In some embodiments, the antibody-drug conjugate has the structure shown in formula (V).

[0091] Ab-[RGR-L 3 -L 2 -L 1 -D] m (V)

[0092] in

[0093] Ab represents an antibody or its antigen-binding fragment;

[0094] m is any value between 2 and 10 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6...). 4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.5, 9, 9.5, 10, or such as 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8);

[0095] Where RGR is the residue resulting from the reaction of RG (active group) and Ab; other variables RG, L 3 L 2 L 1 D is as defined above in equation (I), equation (II) or equation (III).

[0096] As is generally known in the art, given that the RG has been determined, those skilled in the art can determine the RGR after the coupling reaction. For example, the coupling between a maleimide (Mal) group and a mercapto (-SH) group forms a stable thioether bond, and the coupling between an azide group (-N3) and a terminal alkyne via copper-catalyzed azide-alkyne cycloaddition (CuAAC) forms a triazole bond.

[0097] In this invention, the drug-to-antibody ratio (DAR) refers to the number of drug molecules conjugated to the antibody (e.g., m in formula (IV)). The number of drug molecules contained in the antibody-drug conjugates described in this application can be an integer or a decimal. Whether integer or decimal, it refers to the average number of drug molecules conjugated to each antibody. "m is any value between 2 and 8" means that m can be any integer selected from 2 to 8 (including the endpoints 2 and 8), or any decimal selected from 2 to 8, such as 2.3, 3.9, 4.0, and 4.2. Furthermore, those skilled in the art will understand that even using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches may not be exactly the same; for example, they may fluctuate within a range of no more than 0.5.

[0098] Drug-antibody ratio (DAR) can be verified using conventional analytical methods such as mass spectrometry, ELISA, HIC, and HPLC. The quantitative distribution of the ADC in the m-axis can also be determined. In some cases, the separation, purification, and verification of homogeneous ADCs with a certain m-value from ADCs carrying other drug loads can be achieved using methods such as reversed-phase HPLC or electrophoresis.

[0099] In this invention, the linker compound is covalently coupled to the antibody Ab. The coupling methods include, but are not limited to, for example, coupling maleimide with the thiol group in the antibody to form a -S-succinimide structure, or forming a structure by reacting the -NH2 group of lysine with a group containing NHS; or exposing the -NH2 or -SH by removing the removable group and then coupling.

[0100] There are no particular limitations on the antibodies or antigen-binding fragments thereof that can be used in this invention, and they can be selected from monoclonal antibodies (such as mouse antibodies, rabbit antibodies, phage-displayed antibodies, yeast-displayed antibodies, chimeric antibodies, humanized antibodies, fully human antibodies), bispecific antibodies, multispecific antibodies, nanobodies (VHH, Variable Domain of Heavy Chain Antibody), Fab fragments, Fab' fragments, and F(ab'). 2 Fragments, Fd, Fv, dAb, complementarity-determining region fragments, single-chain variable fragments (scFv), scFV-Fc bivalent molecules, single-domain antibodies (sdAb), proantibodies, and antibody fusion proteins.

[0101] Examples of antibodies include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti-CA-125), Abciximab (anti-CD41 (integrin alpha-IIb), Adalimumab (anti-TNF-α), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-α), Afutuzumab (anti-CD20), and Alacizumab. pegol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti-CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin (CD62L)), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-β-amyloid), Basiliximab (Simulect, anti-CD52). 25 (IL-2 receptor α chain)), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11 (eosinophil chemokine-1)), Besilesomab (Scintimun, anti-CEA-associated antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II β chain), Bivatuzumab (anti-CD44v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30) TNFRSF8), Briakinumab (anti-IL-12, IL-23), Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM),Anti-CD3), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimzia anti-TNF-α), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (IL-2 receptor α chain)), Daratumumab (anti-CD38 (cyclic ADP ribohydrolase)), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphocyte lymphoma). (Lymphoma cells), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD11a)), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2 / neu, CD3), Etaracizumab (Abegrin, anti-integrin αvβ3), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec,Anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80), Gantenerumab (anti-β-amyloid), Gavilimomab (anti-CD147 (basement membrane protein)), Gemtuz umab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α), Gomiliximab (anti-CD23 (IgE receptor)), anti-HLA-DR antibody, Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-α), I Intetumumab (anti-CD51), Inolimomab (anti-CD25 (IL-2 receptor α chain)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF-β2), Lexatumumab (anti-TRAIL-R2), L ibivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab (anti-CD23 (IgE receptor)), Mapatumumab (anti-TRAIL-R1), Maslimomab (anti-T cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF-β1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2,Anti-GD3 gangliosides), Morolimumab (anti-Rh factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin α4), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-Rα), Omalizumab (Xolair, anti-IgE) Fc segment), Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-HER2 / neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-(N-acetylneuraminic acid (NeuGc, NGNA)-ganglioside GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab,Anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-α), Rovelizumab (LeukArrest, anti-CD11, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart)MI95 (anti-CD33), Solanezumab (anti-β-amyloidosis) (Pyrophylloid protein), Sonepizumab (anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myosin), Sulesomab (LeukoScan, anti-NCA-90 (granulocyte antigen)), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin αIIbβ3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, anti-aggregation factor A), Telimomab, Tenatumomab (anti-tenascin) C) Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, anti-IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, anti-HER2 / neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus), Urtoxazumab (anti-E. coli), Ustekinumab (Stelara, anti-IL-12),IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab (anti-integrin α4β7), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1)), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-angiotensin αvβ3), Volociximab (anti-integrin α5β1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, anti-EGFR), Zanolimu mab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basement membrane protein)), Zolimomab (anti-CD5), Etanercept, Alefacept, Abatacept, Rilonacept (Arcalyst), 14F7 (anti-IRP-2 (ferrregular protein 2)), 14G2a (anti-GD2 ganglioside, from the National Cancer Institute, used for melanoma and solid tumors), J591 (anti-PSMA, used at Weill Cornell Medical College for prostate cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma-associated antigen), Sorin Radiofarmaci SRL (Milan, Italy) for melanoma, COL-1 (anti-CEACAM3, CGM1, from the National Cancer Institute, used for colorectal and gastric cancer), CYT-356 (for prostate cancer), HNK20 (OraVax for respiratory syncytial virus), ImmuRAIT (Immunomedics for non-Hodgkin's lymphoma), Lym-1 (anti-HLA-DR10, Peregrine Pharm for cancer), MAK-195F (anti-TNF (tumor necrosis factor; TNFA)).TNF-alpha (TNFSF2), from Abbott / Knoll, used for septic shock; MEDI-500, T10B9, anti-CD3; TRαβ (T cell receptor α / β complex), from MedImmune, used for graft-versus-host disease; RING SCAN (anti-TAG). 72 (Tumor-associated glycoprotein 72), from Neoprobe for breast, colon, and rectal cancer; Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTD1 (tumor-associated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2), anti-KSA; KS1 / 4 antigen; M4S; tumor antigen 17-1A; CD326, from NeoRx for colon, ovarian, prostate, and non-Hodgkin's lymphoma); anti-Trop-2 humanized antibody hRS7; LymphoCide (Immunomedics, New Jersey); Smart ID10 (Protein Design Labs); Oncolym (Techniclone, California); Allomune (BioTransplant, California); anti-VEGF (Genentech, California); CEAcide (Immunomedics, New Jersey); IMC-1C11 (ImClone) Systems) and Cetuximab (ImClone). ,

[0102] In some embodiments, the antibody or antigen-binding fragment targets one or more of the following antigens: aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancer), CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA242 (colorectal cancer), placental alkaline phosphatase (cancer), prostate-specific antigen (prostate), prostate acid phosphatase (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma), CD3ε (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma), CD2... CD2 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (chronic lymphocytic leukemia)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumor multiple myeloma), CD50 (leukemia, systemic lupus erythematosus). CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (cancer), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer and lung cancer), DLL4 (Δ-like protein-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hematologic oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epidermal cell adhesion molecule, bladder cancer, head and neck cancer, colon cancer, non-Hodgkin's lymphoma, prostate cancer and ovarian cancer), ERBB2 (epidermal growth factor receptor 2);Lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (a cell surface antigen glycolipid, melanoma), GD3 specific antigen (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (non-Hodgkin lymphoma), HLA-DRB (non-Hodgkin lymphoma, B-cell leukemia), human chorionic gonadotropin (cancer), IGF1R (insulin-like growth factor 1 receptor, solid tumors, hematologic malignancies), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castells disease, IL6-dependent tumors), integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, used for various cancers) MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE-4 (cancer), anti-transferrin receptor (cancer), p97 (melanoma), MS4A1 (Membrane-crossing 4-domain subfamily A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer, and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (Membrane-crossing 4-domain subfamily A, small cell lung cancer, non-Hodgkin's lymphoma), ribosomal protein, neuroproto-oncogene product (cancer), P21 (cancer), anti-(N-acetylneuraminic acid, breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate), ROBO4, TAG 72 (tumor-associated glycoprotein 72, acute myeloid leukemia, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, non-Hodgkin's lymphoma, other cancers, rheumatoid arthritis, and systemic lupus erythematosus), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1, lymphoma, non-Hodgkin's lymphoma, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, or VEGF-2 (CD309) (various cancers).

[0103] In some embodiments, the antibody or antigen-binding fragment targets one or more of the following antigens: various differentiation clusters. differentiations, CD) (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, C D30, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD 49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD7 1. CD72, CD74, CD79 (CD79a, CD79b), CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, C D95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, C D138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166 , CD168, CD184, CDw186, CD195, CD202(a, b), CD209, CD235a, CD271, CD303, CD304); Annexin A1, Trop-2, STEAP-1, STEAP-2, DLL-3, DLL-4, Claudin 18.2. Nucleolin, tissue factor, endothelin (CD105), ROBO4, aminopeptidase N, Δ-like protein 4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, B7-H4, WT1, MUC1, LMP2, HPV E6 / E7, EGFRvIII, HER-2 / neu, specific antigen, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, PD-1, PD-L1, MelanA / MART1, Ras mutation, gp100, p53 mutation, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation Breakpoints), BCMA, FGFR2, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, sugar-rich GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, spermin 17, LCK, high molecular weight polypeptide, AKAP-4, SSX2, XAGE 1, B7H3, leguminous proteinase, Tie 2. Page 4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, Fos-related antigen 1.

[0104] In some embodiments, the antibody-drug conjugate is selected from the following formulas (V-1-1)-(V-1-3), (V-2-1)-(V-2-3), and (V-3):

[0105] Ab represents the antibody or its antigen-binding fragment; m is any value from 2 to 10, preferably any value from 3.5 to 8.5; preferably 3.5 to 4.5 or 7.5 to 8.5; -S- represents a thiol residue in the antibody or its antigen-binding fragment, through which the linker compound is coupled to the antibody or its antigen-binding fragment.

[0106] The Hsc1 mentioned is selected from

[0107] The Hsc2 mentioned is selected from

[0108] In some implementations, Hsc1 is In a preferred embodiment, Hsc1 is

[0109] In some implementations, Hsc2 is... In some implementations, Hsc2 is...

[0110] In a preferred embodiment, the antibody-drug conjugate has the following structure:

[0111] The Hsc1 mentioned is In some preferred embodiments, the Hsc1 is preferably...

[0112] In some specific embodiments, the antibody-drug conjugate has the following structure:

[0113] In some embodiments, the Ab is an anti-EGFR antibody or its antigen-binding fragment, wherein the anti-EGFR antibody or its antigen-binding fragment comprises light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; wherein the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are the amino acid sequences shown in SEQ ID NO:5, 6, 7, 8, 9, and 10, respectively.

[0114] In some embodiments, the antibody is an anti-EGFR antibody or its antigen-binding fragment, the anti-EGFR antibody comprising a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region being SEQ ID NO:3; and the amino acid sequence of the heavy chain variable region being SEQ ID NO:4.

[0115] In some embodiments, the antibody is an anti-EGFR antibody or its antigen-binding fragment, the anti-EGFR antibody comprising a light chain and a heavy chain, the amino acid sequence of the light chain being SEQ ID NO:1; and the amino acid sequence of the heavy chain being SEQ ID NO:2.

[0116] In some implementations, the antibody is JMT101.

[0117] In some embodiments, the antibody is an anti-HER2 antibody or its antigen-binding fragment, the anti-HER antibody comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is SEQ ID NO:11; and the amino acid sequence of the heavy chain is SEQ ID NO:12.

[0118] In some implementations, the anti-HER2 antibody is Trastuzumab.

[0119] In some embodiments, the antibody is an anti-CD20 antibody or its antigen-binding fragment, the anti-CD20 antibody comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is SEQ ID NO:13 and the amino acid sequence of the heavy chain is SEQ ID NO:14.

[0120] In some embodiments, the anti-CD20 antibody is rituximab.

[0121] Information on some sequences involved in this invention is provided in the table below.

[0122] In some embodiments, an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, is involved, said antibody-drug conjugate having a structure represented by formula (VI-A1), (VI-3A), (VI-10A), or (VI-15A).

[0123] The Ab is an anti-EGFR antibody or its antigen-binding fragment, and the anti-EGFR antibody or its antigen-binding fragment includes light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0124] The light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are the amino acid sequences shown in SEQ ID NO:5, 6, 7, 8, 9, and 10, respectively.

[0125] The value of m can be any value between 3.5 and 4.5.

[0126] Preferably, in some embodiments, the value of m is about 4.0.

[0127] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is SEQ ID NO:3; and the amino acid sequence of the heavy chain variable region is SEQ ID NO:4.

[0128] In some embodiments, the anti-EGFR antibody or its antigen-binding fragment comprises a light chain and / or a heavy chain, wherein the amino acid sequence of the light chain is SEQ ID NO:1 and the amino acid sequence of the heavy chain is SEQ ID NO:2.

[0129] Some embodiments relate to the use of antibody-drug conjugates with the structure shown in formula (VI-A1), (VI-3A), (VI-10A) or (VI-15A), or pharmaceutically acceptable salts, solvates or solvates of said salts, for the treatment or prevention of tumors.

[0130] In some embodiments, the use of antibody-drug conjugates with structures shown in formula (VI-A1), (VI-3A), (VI-10A), or (VI-15A) in the preparation of medicaments for treating tumors is involved.

[0131] The cancers mentioned are selected from lung cancer, nasopharyngeal carcinoma, head and neck cancer, breast cancer, colorectal cancer, colon cancer, rectal cancer, stomach cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, and pancreatic cancer.

[0132] The patients with the lung cancer were resistant to osimertinib and / or erlotinib.

[0133] The lung cancer in question is an EGFR-mutated lung cancer.

[0134] The lung cancer is lung cancer containing the Del19 / T790M / C797S mutation or lung cancer containing the T790M / L858R mutation.

[0135] The lung cancer mentioned is lung adenocarcinoma.

[0136] The lung cancer is either lung cancer with the Del19 / T790M / C797S mutation or lung adenocarcinoma with the T790M / L858R mutation.

[0137] Pharmaceutical Compositions and Pharmaceutical Uses

[0138] A third aspect of the invention provides a pharmaceutical composition comprising the aforementioned linker compound, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned targeted drug conjugate (including antibody drug conjugate), or a pharmaceutically acceptable salt, solvate, or solvate of said salt; and at least one pharmaceutical excipient.

[0139] A fourth aspect of the invention provides the use of the aforementioned linker compound, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors.

[0140] A fifth aspect of the invention provides a method for treating and / or preventing tumors, comprising: administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned linker compound, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0141] In this invention, "tumor" refers to a lesion formed by excessive proliferation of cells in a localized area of ​​an organ or tissue, including hematologic malignancies and solid tumors, including benign and malignant tumors. In some embodiments, the tumor is selected from tumors that are positive for or highly express the following markers: HER2, EGFR, and CD20.

[0142] Some embodiments involve an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof.

[0143] The antibody-drug conjugate has the structure shown in formula (a), formula (b), or formula (c).

[0144] The Ab is an anti-EGFR antibody or its antigen-binding fragment, and the anti-EGFR antibody or its antigen-binding fragment includes light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3.

[0145] The light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are the amino acid sequences shown in SEQ ID NO:5, 6, 7, 8, 9, and 10, respectively.

[0146] The n is an integer between 3 and 11; preferably 3, 5, 7, 9, 11; most preferably 11.

[0147] The value of m is any value between 3.5 and 4.5. Preferably, the value of m is approximately 4.0.

[0148] Wherein, -S- represents a thiol residue in the antibody or its antigen-binding fragment, and the linker compound is coupled to the antibody or its antigen-binding fragment through this thiol residue.

[0149] In some embodiments, the antibody-drug conjugate has the structure shown in formula (d), formula (e), or formula (f):

[0150] The present invention provides the use of the above-mentioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of tumors.

[0151] The present invention also provides a method for treating and / or preventing tumors, comprising: administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned pharmaceutical composition thereof.

[0152] In this invention, "tumor" refers to a lesion formed by excessive proliferation of cells in a localized area of ​​an organ or tissue, including hematologic malignancies and solid tumors, including benign and malignant tumors. In some embodiments, the tumor is selected from tumors that are positive for or highly express epidermal growth factor receptor (EGFR).

[0153] Some embodiments relate to the use of antibody-drug conjugates with the structure shown in formula (a), (b), (c), (d), (e), or (f), or pharmaceutically acceptable salts, solvates, or solvates of said salts for the treatment or prevention of tumors.

[0154] Some embodiments involve the use of antibody-drug conjugates with the structure shown in formula (a), (b), (c), (d), (e), or (f), or pharmaceutically acceptable salts, solvates, or solvates of said salts, in the preparation of medicaments for treating or preventing tumors.

[0155] The tumors mentioned are selected from lung cancer, nasopharyngeal carcinoma, head and neck cancer, breast cancer, colorectal cancer, colon cancer, rectal cancer, stomach cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, and pancreatic cancer.

[0156] The lung cancer mentioned is lung cancer that is resistant to osimertinib and / or erlotinib.

[0157] The lung cancer in question is an EGFR-mutated lung cancer.

[0158] The lung cancer is lung cancer containing the Del19 / T790M / C797S mutation or lung cancer containing the T790M / L858R mutation.

[0159] The lung cancer mentioned is lung adenocarcinoma.

[0160] The lung cancer is either lung cancer with the Del19 / T790M / C797S mutation or lung adenocarcinoma with the T790M / L858R mutation. Attached Figure Description

[0161] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein:

[0162] Figures 1a and 1b show the HIC and SEC spectra of the antibody-drug conjugate MAB802-A1(B1), respectively.

[0163] Figures 2a and 2b show the HIC and SEC spectra of the antibody-drug conjugate MAB802-A12(B13), respectively.

[0164] Figures 3a and 3b show the HIC and SEC spectra of the antibody-drug conjugate MAB802-A7(B20), respectively.

[0165] Figures 4a and 4b show the HIC and SEC spectra of the antibody-drug conjugate MAB802-A4(B4), respectively.

[0166] Figure 5 shows the plasma stability test results of antibody-drug conjugates B1 and B50. Detailed Implementation

[0167] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0168] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this invention are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0169] definition

[0170] In this invention, unless otherwise stated, any numerical range should be understood to include any value within the range or any subrange.

[0171] In this invention, unless otherwise stated, RG, L3, L2, L1, and D are read from left to right. The definitions of L3, L2, and L1 are also expressed from left to right. Indicates the connection location.

[0172] In this invention, the terms "coupler," "compound," and "linker compound" refer to all stereoisomers, geometric isomers, tautomers, and isotopes including the described structure. Unless otherwise stated, compounds identified by name or structure as a particular tautomer form in this invention include other tautomer forms.

[0173] Some compounds exist as tautomers. These tautomers exist in equilibrium. For example, a compound containing an amide group may be in equilibrium with a tautomer containing an imino acid. Regardless of which tautomer is shown, and regardless of the nature of the equilibrium between the tautomers, those skilled in the art will understand that these compounds include both amide and imino acid forms. Therefore, compounds containing an amide group are considered to include their imino acid tautomers. Similarly, compounds containing imino acids are considered to include their amide tautomers.

[0174] Any molecular formula or structure given in this invention is also intended to represent both the unlabeled and isotopically labeled forms of the compound. Isotopically labeled compounds have the structures described by the formulas listed in this invention, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that may be incorporated into the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example, but not limited to, isotopes of these elements. 2 H (deuterium, D) 3 H (tritium) 11 C 13 C 14 C 15 N、 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I. The various isotope-labeled compounds disclosed herein, such as those labeled with radioactive isotopes (e.g., 3 H, 13 C and 14 C) Labelled compounds can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or for the treatment of patients with radiotherapy.

[0175] This invention also includes “deuterated analogues” of the compounds described herein, wherein one to n hydrogen atoms bonded to carbon atoms are replaced with deuterium, where n is the number of hydrogen atoms in the molecule. These compounds exhibit greater tolerance to metabolism and can therefore be used to extend the half-life of the compounds described herein when administered to mammals, particularly humans. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci. 5(12):524-527 (1984). These compounds can be synthesized by methods known in the art, for example using starting materials with one or more hydrogen atoms replaced by deuterium.

[0176] The deuterium-labeled or substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, involving distribution, metabolism, and excretion (ADME). Substitution with a heavier isotope (such as deuterium) may provide certain therapeutic advantages, such as prolonged in vivo half-life, reduced dose requirement, and / or improved therapeutic index due to enhanced metabolic stability. 18F-labeled compounds can be used in PET or SPECT studies. The isotope-labeled compounds and their prodrugs disclosed herein can generally be prepared by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents through the procedures disclosed in Scheme I or in the examples and preparations described below. In this context, deuterium is considered as a substituent in the compounds described herein.

[0177] The concentration of such heavier isotopes (particularly deuterium) in the compounds disclosed herein can be defined by isotope enrichment factors. In the compounds disclosed herein, any atom not specifically designated as a particular isotope represents any stable isotope of that atom. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen," that position is understood to have a naturally occurring isotopic composition of hydrogen. Therefore, in the compounds disclosed herein, any atom specifically designated as deuterium (D) represents deuterium.

[0178] prefix "C" u-v "" indicates that the following group contains u to v carbon atoms. For example, "C 1-6 "Alkyl" indicates that an alkyl group contains 1 to 6 carbon atoms.

[0179] "Halogens" refer to fluorine, chlorine, bromine, and iodine.

[0180] "alkyl" refers to a substituted or unsubstituted straight-chain or branched saturated aliphatic hydrocarbon group, including but not limited to alkyl groups with 1 to 20 carbon atoms, alkyl groups with 1 to 8 carbon atoms, alkyl groups with 1 to 6 carbon atoms, and alkyl groups with 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and their various branched isomers. The alkyl group used in this application is defined in accordance with this definition. An alkyl group can be monovalent, divalent, trivalent, or tetravalent.

[0181] "Alkylene" refers to substituted or unsubstituted straight-chain and branched divalent saturated hydrocarbon groups, including -(CH2). v - (v is an integer from 1 to 10), examples of alkylene include, but are not limited to, methylene, ethylene, propylene, and butylene.

[0182] "Alkenyl" refers to a substituted or unsubstituted straight-chain and branched unsaturated hydrocarbon group having at least one, typically one, two, or three, carbon-carbon double bonds. The main chain has, but is not limited to, 2 to 10, 2 to 6, or 2 to 4 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, and 2-methyl -1-Butenyl, 2-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 1-octenyl, 3-octenyl, 1-nonenyl, 3-nonenyl, 1-decenyl, 4-decenyl, 1,3-butadiene, 1,3-pentadiene, 1,4-pentadiene, and 1,4-hexadiene, etc. The alkenyl groups appearing in this application are defined in accordance with this definition. Alkenyl groups can be monovalent, divalent, trivalent, or tetravalent. "Alkenylene" refers to a divalent alkenyl group.

[0183] "Alynyl" refers to a substituted or unsubstituted straight-chain and branched monovalent unsaturated hydrocarbon group having at least one, typically one, two, or three, carbon-carbon triple bonds. The main chain comprises 2 to 10 carbon atoms, including but not limited to groups with 2 to 6 carbon atoms or 2 to 4 carbon atoms on the main chain. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3... -Pentynyl, 4-pentynyl, 1-methyl-1-butynyl, 2-methyl-1-butynyl, 2-methyl-3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-1-pentynyl, 2-methyl-1-pentynyl, 1-hepynyl, 2-hepynyl, 3-hepynyl, 4-hepynyl, 1-octyynyl, 3-octyynyl, 1-nonynyl, 3-nonynyl, 1-decynyl, 4-decynyl, etc. The ynyl group can be monovalent, divalent, trivalent, or tetravalent. "Idemynyl" refers to a divalent ynyl group.

[0184] The term "cycloalkyl" refers to a substituted or unsubstituted saturated carbocyclic hydrocarbon group, typically having 3 to 12 carbon atoms. Cycloalkyl groups include monocyclic C16 groups. 3-8 For example, single-cycle C 3-6 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.; cycloalkyl groups also include bicyclic cycloalkyl groups, such as bicyclic C 6-11 Cycloalkyl groups, such as bicyclo[3.3.0]octyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.0]hexyl, or bicyclo[3.1.1]heptyl, etc. Cycloalkyl groups appearing in this application are defined as described above. Cycloalkyl groups can be monovalent, divalent, trivalent, or tetravalent.

[0185] "Cycloalkylene" refers to divalent cycloalkylene.

[0186] A "heteroalkylene" chain contains at least one carbon atom and at least one heteroatom group. "Heteroalkylene" includes straight-chain or branched saturated chains having carbon atoms and heteroatoms. For example, one, two, or three or more carbon atoms can be independently replaced by the same or different heteroatom groups. Heteroatom groups include, but are not limited to, -NR-, -C(=O)NR-, -NRC(=O)-, -CH2CH2-O-, -O-, -S-, -S(O)-, -S(O)2-, etc., where R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl. "Heteroalkylene" refers to a divalent heteroalkyl group. Examples of heteroalkyl groups include, but are not limited to: -CH2OCH2, -CH(CH3)OCH2, -CH2CH2OCH2, -OCH2, -CH(CH3)O, -CH2CH2O, -CH2CH2OCH2CH2OCH2, -CH2CH2OCH2CH2O, -CH2SCH2, -CH(CH3)SCH2, -CH2CH2SCH2, -CH2CH2SCH2CH2SCH2, -SCH2, -CH(CH3)S, -CH2 CH2S, -CH2CH2SCH2CH2S, -CH2S(O)2CH2, -CH(CH3)S(O)2CH2, -CH2CH2S(O)2CH2, -CH2CH2S(O)2CH2CH2OCH2, -CH2NRCH2, -CH(CH3)NRCH2, -CH2CH2NRCH2, -CH2CH2NRCH2CH2NRCH2, etc., wherein each R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl. Heteroalkylene groups contain 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

[0187] "Aryl" or "aromatic ring" refers to a substituted or unsubstituted aromatic hydrocarbon group having a monocyclic or fused ring, wherein the number of ring atoms in the aromatic ring includes, but is not limited to, 6 to 18, 6 to 12, or 6 to 10 carbon atoms. Aryl groups include phenyl and naphthyl groups. In some embodiments, the aryl group is phenyl.

[0188] "Heteroaryl" or "heteroary ring" refers to a substituted or unsubstituted aromatic hydrocarbon group containing 1 to 5 heteroatoms or a group containing heteroatoms (including but not limited to N, O, or S(=O)n, where n is 0, 1, or 2). In some embodiments, heteroaryl refers to a 5-14 member containing a cyclic carbon atom and 1-4 cyclic heteroatoms, such as a 5- or 6-membered monocyclic ring or an 8- to 12-membered bicyclic ring.

[0189] "Substituted" refers to one or more hydrogen atoms in a group, for example, up to five, preferably one to three hydrogen atoms, which are independently substituted by a corresponding number of substituents, such as halogens, alkyl groups, alkenyl groups, alkynyl groups, heteroalkyl groups, alkoxy groups, alkylthio groups, hydroxyl groups, mercapto groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclic groups, aryl groups, heteroaryl groups, oxo groups, haloalkyl groups, hydroxyalkyl groups, and -OR groups. a -SR a NR a R b -C(=O)-R a -OC(=O)-R a -C(=O)-OR a The substituents are replaced by R, where R is a substituent. a and R b Each can be independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl.

[0190] In this invention, the term "pharmaceutically acceptable salt" refers to a salt formed by an acidic functional group present in the coupling compound provided by this invention and a suitable inorganic or organic cation (base), or a salt formed by a basic functional group present in the coupling compound provided by this invention and a suitable inorganic or organic anion (acid).

[0191] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic substance with a suitable acid providing a pharmaceutically acceptable anion, or by reacting an adequate amount of an acid with a suitable base providing a pharmaceutically acceptable cation.

[0192] In this invention, solvates refer to these forms of the antibody-drug conjugates of the invention: solid or liquid complexes formed by coordination with solvent molecules. Hydrates are a specific form of solvate having coordinated water molecules. Hydrates are preferred solvates in this invention.

[0193] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be obvious to those skilled in the art according to the disclosure of the present invention. As described in Remington's Pharmaceutical Sciences, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed thereto at the doses and concentrations used.

[0194] In this invention, pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical formulation. Besides acting as a formulator, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified into natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0195] In this invention, the pharmaceutical composition can be formulated into various suitable dosage forms according to the route of administration.

[0196] The term "treatment" as used in this invention generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. The term "treatment" as used in this invention covers any treatment of a patient's disease, including: (a) preventing the occurrence of a disease or symptoms in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., preventing its progression; or (c) alleviating the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0197] In this invention, "subject" refers to a vertebrate. In some embodiments, vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, mammal refers to a human.

[0198] In this invention, "effective amount" refers to the amount that effectively achieves the desired therapeutic or preventative effect at the necessary dose and time. The "therapeutic effective amount" of the substance / molecule of this invention may vary depending on factors such as an individual's disease state, age, sex, weight, and the substance / molecule's ability to elicit the desired response in the individual. Therapeutic effective amount also encompasses the amount in which the beneficial therapeutic effect of the substance / molecule outweighs any toxic or harmful consequences. "Preventative effective amount" refers to the amount that effectively achieves the desired preventative effect at the necessary dose and time. Typically, but not necessarily, the preventative effective amount will be lower than the therapeutic effective amount because the preventative dose is administered to the subject before the onset of the disease or in its early stages. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; shrink the tumor volume; inhibit (i.e., slow down, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent.

[0199] In this invention, the 20 common amino acids and their abbreviations follow their usual usage. See Immunology-ASynthesis (2nd edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0200] In this invention, polypeptide residues, such as Lys-Val-Ala, represent a linking fragment composed of Lys, Val and Ala sequentially linked to form a peptide bond. Those skilled in the art will understand that it does not limit the specific stereo configuration of Lys, Val and Ala, such as L-type or D-type.

[0201] In this invention, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of identical polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). The light chains of antibodies can be classified into two types: κ and λ. The heavy chains can be classified into five types: μ, δ, γ, α, or ε. Based on the heavy chain, antibodies can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids. The heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the C1q component of the complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0202] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0203] Furthermore, during humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described in this invention. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs are usually limited to one or two.

[0204] In some embodiments, the drug module is coupled to the antibody in a coupling reaction with less than the theoretical maximum. Generally, antibodies do not contain many free and reactive cysteine ​​thiols that can link to the drug module; in fact, most cysteine ​​thiols in antibodies exist as disulfide bridges. In some embodiments, the antibody can be reduced under partially or completely reducing conditions with reducing agents such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiols.

[0205] The abbreviations used in this application have the following meanings:

[0206] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0207] Nuclear magnetic resonance (NMR) 1 The H NMR measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0208] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0209] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0210] Mass spectrometry (MS) measurements were performed using a Waters (ESI) mass spectrometer, model Waters 2998. Detector: QDA.

[0211] The HPLC determination was performed using an Agilent 1260 Infinity.

[0212] Detailed Implementation

[0213] Intermediate synthesis:

[0214] Synthesis of Compound 1

[0215] Step 1: Synthesis of Compound 1-1

[0216] At 25°C, compound SM-1 (1000 mg, 2.436 mmol) was dissolved in a mixed solution of dichloromethane (10 mL) and methanol (10 mL). Then, p-aminobenzyl alcohol (450 mg, 3.654 mmol) was added and stirred until completely dissolved. EEDQ (903.68 mg, 3.654 mmol) was added and stirred until completely dissolved. A large amount of solid gradually precipitated as the reaction proceeded. The reaction was continued at 25°C for 5 hours. LC-MS confirmed the end of the reaction. Then, n-heptane (30 mL) was added to the reaction solution and stirred for 2 hours. The solid was obtained by filtration. Vacuum drying yielded compound 1-1 (900 mg, 70.3%). ESI-MS (m / z): 516 [M+H] + .

[0217] Step 2: Synthesis of Compounds 1-2

[0218] Compound 1-1 (800 mg, 1.552 mmol) was dissolved in DMF (4 mL) at 25 °C, followed by the addition of 4-nitrophenyl carbonate (613.60 mg, 2.017 mmol). After complete dissolution, DIPEA was added. The reaction was maintained at 25 °C for 2 h. HPLC analysis confirmed the end of the reaction. A 1:1 mixture of ethyl acetate (EA) and methyl tert-butyl ether (MTBE) (20 mL) was added to the reaction mixture, and the mixture was stirred and slurried for 2 h. The solid was then filtered and dried in a vacuum oven to obtain compound 1-2 (993 mg, 93%). ESI-MS: m / z 681.7 [M+H] + .

[0219] Step 3: Synthesis of compounds 1-3

[0220] Compounds 1-2 (280 mg, 0.414 mmol) were dissolved in DMF (4 mL) at 25 °C, followed by the addition of eczema mesylate (200 mg, 0.376 mmol), DIPEA (97.26 mg, 0.753 mmol), and HOBT (20.32 mg, 0.150 mmol). The reaction was maintained at 25 °C for 3 h, and the reaction was confirmed to be complete by HPLC. No further processing was performed, and the solution was directly used for the next reaction.

[0221] Step 4: Synthesis of Compound 1

[0222] Diethylamine (1 mL) was added to the reaction solution of compounds 1-3 at 25 °C, and the reaction was maintained at 25 °C for 3 h. The reaction was confirmed to be complete by HPLC. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 80% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound 1 (240 mg, two-step yield 84.5%). ESI-MS: m / z 756 [M+H] + .

[0223] Synthesis of Compound 2

[0224] Synthesis of compound SP-Val-Ala-OH

[0225] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (800 mg, 2.982 mmol) was dissolved in 3 mL of DMF. DIC (413.9 mg, 3.280 mmol) and HOSu (377.5 mg, 3.280 mmol) were added with stirring at 20 °C. After the addition was complete, the reaction was allowed to proceed at 20 °C for 4.5 h. The reaction was confirmed to be complete by LC-MS. Then, H-Val-Ala-OH (561.3 mg, 2.982 mmol) and DIPEA (493 μL, 2.982 mmol) were added to the reaction solution. The reaction was continued at 20 °C for 15 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 55% B gradient over 40 min; detector, UV). The collected fraction was concentrated and freeze-dried to obtain SP-Val-Ala-OH (571 mg, 43.7%), ESI-MS: m / z 439.24 [M+H). + .

[0226] Step 1: Synthesis of Compound 2-2

[0227] Compound 2-1 (5000 mg, 27.913 mmol) was dissolved in 42 mL of dichloromethane at 25 °C. Then, 30 mL of ethanol and monomethylamine hydrochloride (4523.3 mg, 66.991 mmol) were added, followed by slow dropwise addition of DIPEA (11.534 mL, 69.782 mmol). The reaction was carried out at 25 °C for 40 h. LC-MS confirmed the reaction was complete. The reaction solution was filtered, and the filter cake was washed twice with MTBE (10 mL * 2). The resulting solid was dried under vacuum at 40 °C to give compound 2-2 (5227.2 mg, 89.1%). ESI-MS: m / z 211.03 [M + H]. + .

[0228] Step 2: Synthesis of Compounds 2-3

[0229] Compound 2-2 (5000 mg, 23.788 mmol) was dissolved in 70 mL of tetrahydrofuran under nitrogen protection. The mixture was cooled to 0-5 °C and stirred. A 2.0 M borane dimethyl sulfide tetrahydrofuran solution (30 mL, 59.470 mmol) was slowly added dropwise. The mixture was then refluxed at 70 °C for 4.5 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was cooled to 0-10 °C, and methanol (16.7 mL, 411.532 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 1 h. The temperature was then controlled at 0-5 °C, and 11 mL of 4 M HCl in methanol solution was added dropwise. After the addition was complete, the temperature was raised to 65 °C, and the reaction was continued for 8 h. The reaction solution was then cooled to 0-5 °C, stirred for 1 h, and filtered. The filter cake was washed twice with dichloromethane (10 mL * 2). The obtained solid was dried under vacuum at 30 °C to give compound 2-3 (3586.3 mg, 76.8%). ESI-MS: m / z 197.05 [M+H] + .

[0230] Step 3: Synthesis of compounds 2-4

[0231] Compound 2-3 (3100 mg, 15.799 mmol) was dissolved in 13 mL of tetrahydrofuran under nitrogen protection. Di-tert-butyl dicarbonate (4356 μL, 18.959 mmol) was added with stirring at 25 °C, followed by the dropwise addition of triethylamine (1933 μL, 31.599 mmol). After the addition was complete, the reaction mixture was reacted at 25 °C for 17 h. LC-MS confirmed the end of the reaction. The reaction solution was then cooled to 20 °C, and 23 mL of purified water was slowly added dropwise. After stirring for 0.5 h, the mixture was extracted three times with ethyl acetate (20 mL * 3). The ethyl acetate solutions were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness to obtain a solid, which was then dried under vacuum at 35 °C to give compound 2-4 (4188 mg, 89.5%). ESI-MS: m / z 297.12 [M+H] + .

[0232] Step 4: Synthesis of compounds 2-5

[0233] Compound 2-4 (3000 mg, 10.12 mmol) was dissolved in 60 mL of methanol under nitrogen protection. 600 mg of 10% Pd / C was added with stirring at 25 °C, followed by ammonium formate (6384.3 mg, 101.24 mmol). The reaction was carried out at 25 °C for 6 h. After LC-MS confirmation, diatomaceous earth was added to filter the reaction solution. The filtrate was concentrated to dryness, and then 50 mL of dichloromethane and 50 mL of purified water were added. The mixture was stirred, extracted, and separated. 120 mL of MTBE was slowly added dropwise to the resulting dichloromethane phase to precipitate a solid. The filtered solid was dried under vacuum at 35 °C to obtain compound 2-5 (1671 mg, 61.9%). ESI-MS: m / z 267.15 [M+H] + .

[0234] Step 5: Synthesis of compounds 2-6

[0235] Compound SP-Val-Ala-OH (197.6 mg, 0.451 mmol) was dissolved in 3 mL of DMF. Compound 2-5 (120.0 mg, 0.451 mmol), DIC (85.3 mg, 0.676 mmol), and HOAT (92.0 mg, 0.676 mmol) were added under stirring at 20 °C. The reaction was carried out at 20 °C for 5 h. LC-MS was used to confirm the end of the reaction. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound 2-6 (199 mg, 64.3%). ESI-MS: m / z 687.59 [M+H] + .

[0236] Step Six: Synthesis of Compounds 2-7

[0237] Compound 2-6 (99 mg, 0.144 mmol) was dissolved in DMF (3 mL), and 4-nitrophenyl carbonate (131.5 mg, 0.433 mmol) and DIPEA (71.5 μL, 0.433 mmol) were added with stirring at 20 °C. The reaction was carried out at 20 °C for 10 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 50% B to 55% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound 2-7 (112 mg, 91.3%). ESI-MS: m / z 852.87 [M+H] + .

[0238] Step 7: Synthesis of compounds 2-8

[0239] Compound 2-7 (62 mg, 0.073 mmol) was dissolved in 2 mL of DMF. Exatecan mesylate (61.9 mg, 0.116 mmol) and DIPEA (30.1 μL, 0.182 mmol) were added with stirring at 15 °C, and the reaction was carried out at 15 °C for 20 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 40% B to 75% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to give compound 2-8 (68 mg, 81.4%). ESI-MS: m / z 1149.59 [M+H] + .

[0240] Step 8: Synthesis of Compound 2

[0241] Compound 2-8 (68 mg, 0.059 mmol) was added to 3 mL of dichloromethane, and 450 μL of trifluoroacetic acid was added with stirring at 25 °C. The reaction was carried out at 25 °C for 2 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 40% B to 75% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound 2 (49 mg, 78.9%). ESI-MS: m / z 1049.55 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ10.12(s,1H),9.13(s,2H),8.26(d,J=6.7Hz,1H),8.11(d,J=8.7 Hz,1H),7.94(t,J=4.3Hz,2H),7.80(d,J=10.9Hz,1H),7.51(s,2H),7.34(s,1H),5.46(s, 2H),5.36-5.17(m,5H),4.42(t,J=6.4Hz,1H),4.25(s,3H),3.59(s,10H),2.69(t,J=5.2 Hz,3H),2.47-2.28(m,6H),1.94-1.77(m,4H),1.33(d,J=7.1Hz,3H),0.95-0.82(m,10H).

[0242] Synthesis of Compound 3A and Compound 3B

[0243] Step 1: Synthesis of Compound 3-2

[0244] Compound 3-1 (1.7 g, 2.074 mmol) and Boc-Lys-OMe (600 mg, 2.305 mmol) were dissolved in 20 mL of DMF, and PyAOP (1.8 g, 3.475 mmol) and DIPEA (1.14 mL, 6.914 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 50% B to 90% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to give compound 3-2 (2.1 g, 79.98%), ESI-MS: m / z 491.97 [(M-100) / 2+H] + .

[0245] Step 2: Synthesis of Compound 3-3

[0246] Compound 3-2 (2.1 g, 1.940 mmol) was dissolved in 8 mL of DMF, and diethylamine (2 mL) was added. The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to give compound 3-3 (800 mg, 44.83%), ESI-MS: m / z 860.5 [M+H]. + .

[0247] Step 3: Synthesis of Compound 3A and Compound 3B

[0248] Compound 3-3 (200 mg, 0.233 mmol) and 1,3-propanesulfonyl lactone (0.4 mL, 4.651 mmol) were dissolved in 2 mL of methanol, and DIPEA (770 μL, 4.651 mmol) was added. The mixture was stirred and refluxed at 85 °C for 3 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 15% B to 45% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to give compound 3A (125 mg, 39.47%), ESI-MS: m / z 613.7 [M / 2+H] + Compound 3B (120 mg, 42.02%), ESI-MS: m / z 552.7 [M / 2+H] + .

[0249] Synthesis of Compound 4

[0250] Step 1: Synthesis of Compound 4-2

[0251] Compound 4-1 (2000 mg, 2.381 mmol) was dissolved in DMF (5 mL) at 25 °C, and benzyl alcohol (463.47 mg, 4.286 mmol) was added until completely dissolved. DIC (540.87 mg, 4.286 mmol) and DMAP (122.17 mg, 0.119 mmol) were added, and the reaction was carried out at 25 °C for 2 h. The reaction was confirmed by LC-MS, and the reaction solution was filtered. The filtrate was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 70% B gradient for 25 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound 4-2 (1149 mg, 51.7%), ESI-MS: m / z 931.1 [M+H]. + .

[0252] Step 2: Synthesis of Compound 4

[0253] Compound 4-2 (1400 mg, 1.505 mmol) was dissolved in DCM (3 mL) at 25 °C, and morphine (1 mL) was added until completely dissolved. The reaction was carried out at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The solution was concentrated to dryness, dissolved in DMF, and purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 25% B gradient for 25 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound 4 (880 mg, 82.5%), ESI-MS: m / z 708.9 [M+H]. + .

[0254] Synthesis of Compound 5

[0255] Step 1: Synthesis of Compound 5-2

[0256] Compound 5-1 (500 mg, 0.831 mmol) was dissolved in DMF (2 mL) at 25 °C, followed by the addition of 4-nitrophenyl carbonate (300 mg, 0.997 mmol). After complete dissolution, DIPEA (412 μL, 2.493 mmol) was added. The reaction was maintained at 25 °C for 16 h, and the reaction was confirmed to be complete by HPLC. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 80% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound 5-2 (210 mg, 31.31%). ESI-MS: m / z 767 [M+H] + .

[0257] Step 2: Synthesis of Compound 5

[0258] Compound 5-2 (210 mg, 0.273 mmol) was dissolved in DMF (5 mL) at 25 °C, followed by the addition of ixotecan mesylate (190 mg, 0.356 mmol) and DIPEA (136 μL, 0.822 mmol). The reaction was maintained at 25 °C for 2 h, and HPLC was used to confirm the end of the reaction. Diethylamine (150 μL) was then added to the reaction solution, and the reaction was maintained at 25 °C for 1 h. HPLC was used to confirm the end of the reaction. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 60% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound 5 (120 mg, 49.5%). ESI-MS: m / z 841 [M+H] + .

[0259] Example A1: Synthesis of Compound A1

[0260] Step 1: Synthesis of compound A1-01

[0261] Compound 3A (125 mg, 0.102 mmol) was dissolved in 5 mL of water, and 1 mL of TFA was added at 25 °C. The mixture was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated and freeze-dried to obtain compound A1-01 (105 mg, 82.89%), ESI-MS: m / z 563.3 [M / 2+H] + .

[0262] Step 2: Synthesis of compound A1-02

[0263] Compound A1-01 (105 mg, 0.093 mmol) was dissolved in 6 mL of 50% methanol / water, and NaOH (11.2 mg, 0.28 mmol) was added at 25 °C. The reaction was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 45% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound A1-02 (80 mg, 73.78%), ESI-MS: m / z 557.0 [M / 2+H]. + .

[0264] Step 3: Synthesis of compound A1-03

[0265] 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (7 mg, 0.026 mmol) was dissolved in 2 mL of DMF, and DIC (5 μL, 0.029 mmol) and HOSu (3.3 mg, 0.029 mmol) were added. After stirring at 25 °C for 1 h, A1-02 (25 mg, 0.022 mmol) and DIPEA (13 μL, 0.078 mmol) were added. The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 15% B to 55% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A1-03 (25 mg, 77.58%), ESI-MS: m / z 681.9 [M / 2+H] + .

[0266] Step 4: Synthesis of Compound A1

[0267] Compound A1-03 (25 mg, 0.018 mmol) and compound 1 (13.8 mg, 0.018 mmol) were dissolved in 1 mL of DMF. PyAOP (14.3 mg, 0.028 mmol) and DIPEA (9 μL, 0.055 mmol) were added at 25 °C. The mixture was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 15% B to 55% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A1 (11 mg, 28.55%). ESI-MS: m / z 1050.5 [M / 2+H] + . 1H NMR(400MHz,DMSO-d6)δ10.00(s,1H),9.13-9.09(m,2H),8.19-8.18(m,1H), 8.09-8.06(m,2H),7.82-7.78(m,2H),7.72-7.67(m,1H),7.63-7.60(m,2H), 7.40-7.37(m,2H),7.33(s,1H),5.47(s,2H),5.33-5.29(m,3H),5.09(s,2H) ,4.42-4.38(m,1H),4.30-4.28(m,1H),4.23-4.16(m,1H),3.73-3.68(m,1H) ,3.58-3.48(m,65H),3.42(s,3H),3.29-3.18(m,1H),3.17-3.10(m,1H),3.0 6-2.98(m,2H),2.41-2.36(m,3H),2.35-2.28(m,5H),2.23-2.21(m,2H),2.0 2-1.95(m,7H),1.89-1.78(m,4H),1.69-1.61(m,1H),1.58-1.48(m,1H),1.4 1-1.34(m,2H),1.32(d,J=6.8Hz,3H),1.28-1.25(m,2H),0.92-0.83(m,9H).

[0268] Example A2: Synthesis of Compound A2

[0269] Step 1: Synthesis of compound A2-1

[0270] Compound 4 (45 mg, 0.053 mmol) was dissolved in DMF (1 mL) at 25 °C. 3-phosphonopropionic acid (163.23 mg, 1.060 mmol) was added. HATU (483.45 mg, 1.271 mmol) and DIPEA (493.00 mg, 3.814 mmol) were added and completely dissolved. The reaction was allowed to proceed for 2 h, and HPLC analysis confirmed the end of the reaction. The mixture was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 30% B gradient for 20 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A2-1 (20 mg, 49%), ESI-MS: m / z 844.9 [M+H]. + .

[0271] Step 2: Synthesis of compound A2-2

[0272] Compound A2-1 (20 mg, 0.022 mmol) was added to ACN (8 mL) at 25 °C, followed by TFA (4 mL), and dissolved completely. The reaction was carried out at 70 °C for 4 h, and the reaction was confirmed to be complete by HPLC. The solution was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 30% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A2-2 (15 mg, 70%), ESI-MS: m / z 754.7 [M+H]. + .

[0273] Step 3: Synthesis of compound A2

[0274] At 25°C, compound A2-2 (8 mg, 0.010 mmol) was dissolved in DMF (1 mL), and compound 2 (10 mg, 0.009 mmol) was added until completely dissolved. HATU (20 mg, 0.053 mmol) and DIPEA (78.2 mg, 0.605 mmol) were added. The reaction was carried out at 25°C for 1 h, and the reaction was confirmed to be complete by LC-MS. The solution was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 60% B gradient for 25 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A2 (12 mg, 67.1%).

[0275] Example A3: Synthesis of Compound A3

[0276] Step 1: Synthesis of compound A3-1

[0277] Compound 4 (100 mg, 0.141 mmol) was dissolved in ACN (8 mL) at 25 °C, and 1,3-propanesulfonyl lactone (966.28 mg, 7.911 mmol) was added. DIPEA (1095.56 mg, 8.476 mmol) was added and the mixture was completely dissolved. The reaction was carried out at 85 °C for 5 h. LC-MS confirmed the end of the reaction. The mixture was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 30% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A3-1 (50 mg, 32.9%), ESI-MS: m / z 1075.3 [M+H]. + .

[0278] Step 2: Synthesis of compound A3-2

[0279] Compound A3-1 (50 mg, 0.047 mmol) was added to ACN (8 mL) at 25 °C, followed by TFA (4 mL), and dissolved completely. The reaction was carried out at 70 °C for 4 h, and the reaction was confirmed to be complete by HPLC. The solution was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 20% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A3-2 (30 mg, 65.5%), ESI-MS: m / z 985.1 [M+H]. + .

[0280] Step 3: Synthesis of compound A3

[0281] At 25°C, compound A3-2 (10 mg, 0.010 mmol) was dissolved in DMF (1 mL), and compound 2 (10 mg, 0.009 mmol) was added until completely dissolved. HATU (4 mg, 0.011 mmol) and DIPEA (5 mg, 0.035 mmol) were added. The reaction was carried out at 25°C for 2 h, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative high-performance liquid chromatography (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 60% B gradient for 25 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A3 (4 mg, 22%). ESI-MS: m / z 1008.1 [M / 2+H] + .

[0282] Example A5: Synthesis of Compound A5

[0283] Step 1: Synthesis of compound A5-02

[0284] Compound A5-01 (50 mg, 0.118 mmol) and 1,3-propanesulfonyl lactone (103 μL, 1.178 mmol) were dissolved in 2 mL of methanol, and DIPEA (195 μL, 1.178 mmol) was added. The reaction was stirred at 25 °C for 3 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A5-02 (20 mg, 21.23%), ESI-MS: m / z 791.22 [M+H]. + .

[0285] Step 2: Synthesis of compound A5-03

[0286] Compound A5-02 (20 mg, 0.025 mmol) was dissolved in 2 mL of 3N hydrochloric acid aqueous solution and stirred at 25 °C for 6 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was diluted with 20 mL of water and lyophilized to obtain compound A5-03 (15 mg, 79.92%). ESI-MS: m / z 735.19 [M+H] + .

[0287] Step 3: Synthesis of compound A5-04

[0288] Compound A5-03 (15 mg, 0.020 mmol) and compound 1 (15.4 mg, 0.020 mmol) were dissolved in 2 mL of DMF, and PyAOP (16 mg, 0.031 mmol) and DIPEA (10 μL, 0.061 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A5-04 (15 mg, 44.47%), ESI-MS: m / z 736.43 [M / 2+H] + .

[0289] Step 4: Synthesis of compound A5-05

[0290] Compound A5-04 (15 mg, 0.010 mmol) was dissolved in 2 mL of DMF, and diethylamine (0.5 mL) was added. The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A5-05 (10 mg, 77.71%), ESI-MS: m / z 625.30 [M / 2+H]. + .

[0291] Step 5: Synthesis of Compound A5

[0292] Compound A5-05 (10 mg, 0.008 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (2.1 mg, 0.008 mmol) were dissolved in 2 mL of DMF, and HATU (4.5 mg, 0.012 mmol) and DIPEA (4 μL, 0.024 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to give compound A5 (5 mg, 69.44%), ESI-MS: m / z 750.53 [M / 2+H] + .

[0293] Example A6: Synthesis of Compound A6

[0294] Step 1: Synthesis of A6

[0295] Compound A5-05 (4 mg, 0.003 mmol) and succinimide 6-(maleimide)hexanoate (1.2 mg, 0.004 mmol) were dissolved in 1 mL of DMF and 2 μL of DIPEA (0.012 mmol). The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to give compound A6 (3.8 mg, 78.17%). ESI-MS: m / z 722.2 [M / 2+H] + .

[0296] Example A7: Synthesis of Compound A7

[0297] Step 1: Synthesis of compound A7-02

[0298] Compound A7-01 (89 mg, 0.209 mmol) and Boc-PEG were used. 12-COOH (150 mg, 0.209 mmol) was dissolved in 2 mL of DMF, and PyAOP (163 mg, 0.313 mmol) and DIPEA (104 μL, 0.627 mmol) were added. The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 55% B to 95% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to give compound A7-02 (150 mg, 61.93%), ESI-MS: m / z 484.96 [(M-100-56)+H] + .

[0299] Step 2: Synthesis of compound A7-03

[0300] Compound A7-02 (150 mg, 0.133 mmol) was dissolved in 5 mL of methanol, and 1 mL of 3N hydrochloric acid was added. The reaction was stirred at 60 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A7-03 (100 mg, 72.73%), ESI-MS: m / z 492.0 [M / 2+H] + .

[0301] Step 3: Synthesis of A7-04

[0302] A7-03 (100 mg, 0.102 mmol) and 1,3-propanesulfonyl lactone (89 μL, 1.018 mmol) were dissolved in 5 mL of methanol, and DIPEA (168 μL, 1.018 mmol) was added. The reaction mixture was stirred at 25 °C for 16 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A7-04 (30 mg, 23.74%), ESI-MS: m / z 614.0 [M / 2+H] + .

[0303] Step 4: Synthesis of compound A7-05

[0304] Compound A7-04 (30 mg, 0.024 mmol) was dissolved in 2 mL of 50% tetrahydrofuran / water, and NaOH (2 mg, 0.049 mmol) was added at 0 °C. The reaction was stirred at 0 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A7-05 (15 mg, 48.05%), ESI-MS: m / z 606.5 [M / 2+H]. + .

[0305] Step 5: Synthesis of compound A7-06

[0306] Compound A7-05 (10 mg, 0.008 mmol) and compound 1 (9.4 mg, 0.012 mmol) were dissolved in 1 mL of DMF. PyAOP (6.46 mg, 0.012 mmol) and DIPEA (4 μL, 0.025 mmol) were added at 25 °C. The mixture was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A7-06 (12 mg, 70.9%). ESI-MS: m / z 650.8 [M / 3+H] + .

[0307] Step Six: Synthesis of A7-07

[0308] Compound A7-06 (12 mg, 0.006 mmol) was dissolved in 2 mL of DMF, and 0.5 mL of diethylamine was added at 25 °C. The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A7-07 (7 mg, 65.19%), ESI-MS: m / z 864.5 [M / 2+H]. + .

[0309] Step 7: Synthesis of Compound A7

[0310] Compound A7-07 (7 mg, 0.004 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylacrylic acid (1.6 mg, 0.006 mmol) were dissolved in 2 mL of DMF, and HATU (2.3 mg, 0.006 mmol) and DIPEA (2 μL, 0.012 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A7 (5.5 mg, 68.15%). ESI-MS: m / z 989.5 [M / 2+H] + .

[0311] Example A9: Synthesis of Compound A9

[0312] Step 1: Synthesis of compound A9-01

[0313] Compound 3B (120 mg, 0.108 mmol) was dissolved in 4 mL of 50% methanol / water, and NaOH (13.0 mg, 0.326 mmol) was added at 25 °C. The reaction was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 15% B to 55% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound A9-01 (100 mg, 76.92%), ESI-MS: m / z 1091.0 [M+H]. + .

[0314] Step 2: Synthesis of compound A9-02

[0315] Compound A9-01 (100 mg, 0.092 mmol) and compound 1 (69 mg, 0.092 mmol) were dissolved in 3 mL of DMF. PyAOP (57.4 mg, 0.110 mmol) and DIPEA (46 μL, 0.275 mmol) were added at 25 °C. The reaction mixture was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A9-02 (60 mg, 34.72%). ESI-MS: m / z 914.1 [M / 2+H] + .

[0316] Step 3: Synthesis of compound A9-03

[0317] A9-02 (60 mg, 0.033 mmol) was dissolved in 4.5 mL of water, and 0.5 mL of TFA was added at 25 °C. The reaction was stirred at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25% B to 65% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A9-03 (30 mg, 50.78%), ESI-MS: m / z 863.2 [M / 2+H]. + .

[0318] Step 4: Synthesis of Compound A9

[0319] A9-03 (15 mg, 0.009 mmol) and succinimide 6-(maleimide)hexanoate (2.7 mg, 0.009 mmol) were dissolved in 1 mL of DMF and DIPEA (4.5 μL, 0.027 mmol). The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to give compound A9 (5 mg, 29.07%). ESI-MS: m / z 960.6 [M / 2+H] + .

[0320] Example A10: Synthesis of Compound A10

[0321] Step 1: Synthesis of A10-01

[0322] Compound A1-02 (50 mg, 0.045 mmol) and succinimide 6-(maleimide)hexanoate (27.7 mg, 0.090 mmol) were dissolved in 1 mL of DMF, and DIPEA (22 μL, 0.135 mmol) was added. The reaction was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 15% B to 55% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound A10-01 (40 mg, 67.08%), ESI-MS: m / z 653.2 [M / 2+H]. + .

[0323] Step 2: Synthesis of compound A10

[0324] Compound A10-01 (25 mg, 0.019 mmol) and compound 1 (14.5 mg, 0.019 mmol) were dissolved in 2 mL of DMF, and PyAOP (15.0 mg, 0.028 mmol) and DIPEA (9.2 μL, 0.056 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A10 (21 mg, 53.28%). ESI-MS: m / z 1022.0 [M / 2+H] + . 1 H NMR (400MHz, DMSO-d6) δ9.99 (s, 1H), 8.17 (d, J = 6.4Hz, 1H), 8.08 (d, J = 6.0Hz, 1 H),7.96(d,J=8.0Hz,1H),7.81-7.78(m,2H),7.65-7.60(m,3H),7.38(d,J=8.4H z,2H),7.33(s,1H),7.01(s,2H),5.46(s,2H),5.35-5.27(m,3H),5.09(s,2H),4 .41-4.37(m,1H),4.28-4.20(m,3H),3.89-3.85(m,2H),3.58-3.45(m,60H),3.3 9-3.36(m,2H),3.29-3.23(m,1H),3.18-3.10(m,1H),3.08-2.99(m,2H),2.50- 2.49(m,2H),2.39(s,3H),2.31-2.28(m,2H),2.24-2.19(m,2H),2.17-2.10(m,2 H),1.99-1.90(m,7H),1.87-1.83(m,2H),1.68-1.58(m,1H),1.51-1.46(m,5H), 1.38-1.35(m,2H),1.31(d,J=7.2Hz,3H),1.25-1.18(m,4H),0.91-0.83(m,9H).

[0325] Example A11: Synthesis of compound A11

[0326] Step 1: Synthesis of compound A11-1

[0327] Compound 1 (50 mg, 0.066 mmol) was dissolved in DMF (1 mL) at 25 °C, and N-α-Fmoc-N-ε-Boc-L-lysine (31.04 mg, 0.066 mmol) was added until completely dissolved. HBTU (27.63 mg, 0.073 mmol) and DIPEA (11.13 mg, 0.086 mmol) were added, and the reaction was carried out at 25 °C for 30 min. The reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 30% B to 90% B gradient; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A11-1 (50 mg, 59.4%), ESI-MS: m / z 1206.3 [M+H]. + .

[0328] Step 2: Synthesis of compound A11-2

[0329] Compound A11-1 (50 mg, 0.041 mmol) was dissolved in DMF (1 mL) at 25 °C, and diethylamine (0.3 mL) was added until complete dissolution. The reaction was allowed to proceed at 25 °C for 1 h, and HPLC analysis confirmed the end of the reaction. The mixture was then purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 30% B to 90% B gradient; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A11-2 (37 mg, 81.6%). ESI-MS: m / z 984.05 [M+H] + .

[0330] Step 3: Synthesis of A11-3

[0331] Compound A11-2 (37 mg, 0.034 mmol) was dissolved in DMF (1 mL) at 25 °C, and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (9.09 mg, 0.034 mmol) was added until completely dissolved. HBTU (15.24 mg, 0.041 mmol) and DIPEA (6.57 mg, 0.051 mmol) were added, and the mixture was reacted at 25 °C for 1 h. The reaction was confirmed to be complete by HPLC. The mixture was purified by preparative HPLC (column: C18; mobile phase: column, C18; mobile phase A: water (0.05% TFA), B: ACN; 30% B to 90% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A11-3 (33 mg, 77.4%), ESI-MS: m / z 1234.2 [M+H]. + .

[0332] Step 4: Synthesis of compound A11-4

[0333] Compound A11-3 (33 mg, 0.027 mmol) was added to DCM (4 mL) at 25 °C, followed by TFA (1 mL), and dissolved completely. The reaction was allowed to proceed at 25 °C for 1 h, and HPLC analysis confirmed the end of the reaction. The solution was concentrated to dryness, and then dissolved and concentrated three times more by adding DCM. A mixture of DCM and MeOH was then added to dissolve the compound, followed by extraction with water. The organic phase was retained and concentrated to dryness. Compound A11-4 (23 mg, 71.4%) was obtained. ESI-MS: m / z 1134.3 [M+H] + .

[0334] Step 5: Synthesis of compound A11

[0335] Compound A11-4 (18 mg, 0.016 mmol) was dissolved in DMF (1 mL) at 25 °C, and 3-phosphonopropionic acid (25 mg, 0.162 mmol) was added until completely dissolved. HATU (1.96 mg, 0.237 mmol) and DIPEA (25 mg, 0.193 mmol) were added. The reaction was carried out at 40 °C for 5 h, and the reaction was confirmed to be complete by HPLC. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 90% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A11 (9 mg, 44.6%), ESI-MS: m / z 635.66 [M / 2+H]. + . 1H NMR (400MHz, DMSO-d6) δ10.10(s,1H),9.13(s,2H),8.20(d,J=6.8Hz,1H),8.09(d,J=8.9Hz,2H),7.91(s,1H),7.77(dd,J=22 .8,10.0Hz,2H),7.62(d,J=8.3Hz,2H),7.41-7.31(m,4H),6.54(s,1H),5.47(s,2H),5.31(s,4H),5.09(s,2H),4.46-4.38(m, 1H),4.34-4.26(m,1H),3.42(s,3H),3.15(s,1H),3.01(t,J=6.2Hz,2H),2.57(t,J=6.6Hz,2H),2.40(s,3H),2.28(dt,J=17.6 ,8.6Hz,2H),2.00(q,J=7.1Hz,1H),1.96-1.79(m,3H),1.79-1.65(m,2H),1.41-1.21(m,14H),0.88(dt,J=14.4,7.1Hz,11H).

[0336] Example A12: Synthesis of compound A12

[0337] Step 1: Synthesis of compound A12-1

[0338] Compound 1 (50 mg, 0.066 mmol) was dissolved in DMF (1 mL) at 25 °C, and N-α-Boc-N-ε-Fmoc-L-lysine was added until completely dissolved. HBTU (30.15 mg, 0.079 mmol) and DIPEA (12.84 mg, 0.099 mmol) were added, and the reaction was carried out at 25 °C for 1 h. The reaction was confirmed to be complete by HPLC. After preparative HPLC purification (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 30% B to 90% B gradient; detection wavelength: 220 nm), the preparative solution was freeze-dried to obtain compound A12-1 (50 mg, 62.3%), ESI-MS: m / z 1206.3 [M+H] + .

[0339] Step 2: Synthesis of compound A12-2

[0340] At 25°C, A12-1 (50 mg, 0.041 mmol) was dissolved in DMF (1 mL), and diethylamine (0.2 mL) was added until complete dissolution. The reaction was allowed to proceed at 25°C for 1 h, and HPLC analysis confirmed the end of the reaction. The solution was purified by preparative HPLC (column, C18; mobile phase: A: water (0.05% TFA), B: ACN; 35 min 20% B to 90% B gradient; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A12-2 (50 mg, 62.3%), ESI-MS: m / z 984.05 [M+H]. + .

[0341] Step 3: Synthesis of compound A12-3

[0342] At 25°C, compound A12-2 (50 mg, 0.051 mmol) was dissolved in DMF (1 mL), and Fmoc-PEG was added. 12 -COOH (51.41 mg, 0.061 mmol) was completely dissolved. HATU (29 mg, 0.077 mmol) and DIPEA (10 mg, 0.077 mmol) were added. The reaction was carried out at 25 °C for 1 h, and the reaction was confirmed to be complete by HPLC. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 90% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A12-3 (20 mg, 21.7%), ESI-MS: m / z 903.5 [M / 2+H]. + .

[0343] Step 4: Synthesis of compound A12-4

[0344] Compound A12-3 (20 mg, 0.011 mmol) was dissolved in DMF (1 mL) at 25 °C, and morpholine (0.2 mL) was added until completely dissolved. The reaction was allowed to proceed at 25 °C for 1 h, and HPLC analysis confirmed the end of the reaction. The mixture was then purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 90% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A12-4 (10 mg, 57.4%), ESI-MS: m / z 792.5 [M / 2+H]. + .

[0345] Step 5: Synthesis of A12-5

[0346] Compound A12-4 (20 mg, 0.013 mmol) was dissolved in DMF (1 mL) at 25 °C, and 3-phosphonopropionic acid (30 mg, 0.195 mmol) was added until completely dissolved. HATU (90 mg, 0.237 mmol) and DIPEA (93.84 mg, 0.726 mmol) were added. The reaction was carried out at 40 °C for 5 h, and the reaction was confirmed to be complete by HPLC. The solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 90% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A12-5 (16 mg, 73.6%). ESI-MS: m / z 860.5 [M / 2+H] + .

[0347] Step Six: Synthesis of A12-6

[0348] Compound A12-5 (16 mg, 0.013 mmol) was added to DCM (6 mL) and TFA (1 mL) at 25 °C and dissolved completely. The reaction was allowed to proceed at 25 °C for 2 h, and HPLC analysis confirmed the end of the reaction. The solution was concentrated to dryness, and the process was repeated three times with the addition of DCM to achieve complete dissolution and concentration. Compound A12-6 (12 mg, 79%) was obtained. ESI-MS: m / z 810.4 [M / 2+H] + .

[0349] Step 7: Synthesis of Compound A12

[0350] Compound A12-6 (20 mg, 0.013 mmol) was dissolved in DMF (1 mL) at 25 °C, and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (4 mg, 0.195 mmol) was added until completely dissolved. HBTU (12 mg, 0.032 mmol) and DIEA (7.82 mg, 0.061 mmol) were added. The reaction was carried out at 20 °C for 1 h, and the reaction was confirmed to be complete by HPLC. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 90% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A12 (6 mg, 43.30%), ESI-MS: m / z 935.5 [M / 2+H]. + .

[0351] Example A14: Synthesis of compound A14

[0352] Step 1: Synthesis of compound A14-2

[0353] Compound A14-1 (50 mg, 0.188 mmol) was dissolved in DMF (2 mL) at 25 °C, and Boc-piperazine (105.05 mg, 0.564 mmol) was added until completely dissolved. HATU (214.32 mg, 0.564 mmol) and DIPEA (145.80 mg, 1.128 mmol) were added, and the reaction was carried out at 25 °C for 2 h. The reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25 min 20% B to 70% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A14-2 (100 mg, 88.5%), ESI-MS: m / z 603.5 [M+H]. + .

[0354] Step 2: Synthesis of compound A14-3

[0355] Compound A14-2 (100 mg, 0.166 mmol) was dissolved in DCM (3 mL) at 25 °C, and TFA (1 mL) was added until complete dissolution. The reaction was carried out at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. After preparative HPLC purification (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 25% B gradient for 30 min; detection wavelength: 210 nm), the preparative solution was freeze-dried to obtain compound A14-3 (50 mg, 74.96%), ESI-MS: m / z 403.5 [M+H] + .

[0356] Step 3: Synthesis of compound A14-4

[0357] At 25°C, compound A14-3 (50 mg, 0.124 mmol) was dissolved in DMF (1 mL), and DIPEA (64.11 mg, 0.496 mmol) was added until completely dissolved. The solution was then cooled to -20°C in a dry ice ethanol bath, and this solution was labeled as solution 1. 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (22.10 mg, 0.124 mmol) was added to DMF (1 mL), followed by HATU (70.68 mg, 0.186 mmol). The reaction was carried out at 25°C for 20 min, and this solution was labeled as solution 2. Solution 2 was then added dropwise to solution 1, and the reaction was maintained at -20°C. The reaction was confirmed to be complete by LC-MS detection; after preparative HPLC purification (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 5% B to 25% B gradient; detection wavelength: 210 nm), the preparative solution was freeze-dried to obtain compound A14-4 (38 mg, 55.0%), ESI-MS: m / z 563.6 [M+H] + .

[0358] Step 4: Synthesis of compound A14-5

[0359] Compound A14-4 (38 mg, 0.067 mmol) was added to DMF (2 mL) at 25 °C, followed by 3,6,9-trioxaundecanoic acid (59.55 mg, 0.268 mmol), until completely dissolved. The mixture was then cooled to 0 °C. HATU (70.68 mg, 0.186 mmol) and DIPEA (64.11 mg, 0.496 mmol) were added. The reaction was maintained at 0 °C for 1 h, and HPLC analysis confirmed the end of the reaction. The mixture was purified by preparative HPLC (column, C18; mobile phase: A: water (0.05% TFA), B: ACN; 5% B to 25% B gradient for 30 min; detection wavelength: 210 nm). The solution was freeze-dried to obtain compound A14-5 (30.8 mg, 60.0%), ESI-MS: m / z 767.8 [M+H]. + .

[0360] Step 5: Synthesis of compound A14

[0361] At 25°C, compound A14-5 (16 mg, 0.021 mmol) was dissolved in DMF (1 mL), and compound 2 (15 mg, 0.014 mmol) was added until completely dissolved. HATU (7.98 mg, 0.021 mmol) and DIPEA (7.24 mg, 0.056 mmol) were added. The reaction was carried out at 25°C for 1 h, and the reaction was confirmed to be complete by LC-MS. The solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 25% B gradient for 30 min; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A14 (8 mg, 36%). ESI-MS: m / z 899.6 [M / 2+H] + .

[0362] Example A15: Synthesis of Compound A15

[0363] Step 1: Synthesis of A15-2

[0364] Compound A15-1 (100 mg, 0.561 mmol) was dissolved in DMF (2 mL) at 25 °C, and Boc-piperazine (313.66 mg, 1.684 mmol) was added until completely dissolved. HATU (640.35 mg, 1.684 mmol) and DIPEA (435.33 mg, 3.368 mmol) were added, and the reaction was carried out at 25 °C for 2 h. The reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25 min 20% B to 70% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A15-2 (230 mg, 79.6%), ESI-MS: m / z 515.5 [M+H]. + .

[0365] Step 2: Synthesis of compound A15-3

[0366] Compound A15-2 (230 mg, 0.447 mmol) was dissolved in DCM (3 mL) at 25 °C, and TFA (1 mL) was added until complete dissolution. The reaction was carried out at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The solution was concentrated to dryness, dissolved in DMF, and purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25 min 20% B to 70% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A15-3 (120 mg, 85.4%), ESI-MS: m / z 315.4 [M+H]. + .

[0367] Step 3: Synthesis of compound A15-4

[0368] At 25°C, compound A15-3 (120 mg, 0.382 mmol) was dissolved in DMF (1 mL), and DIPEA (246.67 mg, 1.908 mmol) was added until completely dissolved. The solution was then cooled to -20°C in a dry ice ethanol bath, and this solution was labeled as solution 1. Compound A15-1 (271.98 mg, 1.527 mmol) was added to DMF (1 mL), and HATU (290.27 mg, 0.763 mmol) was added. The reaction was carried out at 25°C for 20 min, and this solution was labeled as solution 2. Solution 2 was then added to solution 1, and the reaction was carried out at -20°C. The reaction was confirmed to be complete by LC-MS detection; after preparative HPLC purification (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 25 min 5% B to 25% B gradient; detection wavelength: 210 nm), the preparative solution was freeze-dried to obtain compound A15-4 (130 mg, 53.67%), ESI-MS: m / z 635.6 [M+H] +.

[0369] Step 4: Synthesis of compound A15-5

[0370] Compound A15-4 (130 mg, 0.205 mmol) was added to DMF (2 mL) at 25 °C, followed by Boc-piperazine (38.15 mg, 0.205 mmol), and dissolved completely. The mixture was then cooled to -10 °C. HATU (77.89 mg, 0.205 mmol) and DIPEA (79.43 mg, 0.615 mmol) were added. The reaction was maintained at -10 °C for 1 h, and the reaction was confirmed to be complete by LCMS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 5% B to 35% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A15-5 (75 mg, 45.60%), ESI-MS: m / z 803.8 [M+H]. + .

[0371] Step 5: Synthesis of compound A15-6

[0372] Compound A15-5 (75 mg, 0.093 mmol) was dissolved in DCM (3 mL) at 25 °C, and TFA (1 mL) was added until complete dissolution. The reaction was carried out at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The solution was concentrated to dryness, dissolved in DMF, and purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 5% B to 25% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A15-6 (60 mg, 91.4%), ESI-MS: m / z 703.5 [M+H]. + .

[0373] Step Six: Synthesis of A15-7

[0374] Compound A15-6 (60 mg, 0.085 mmol) was added to DMF (1 mL) at 25 °C, followed by 2-(2-methoxyethoxy)acetic acid (11.45 mg, 0.085 mmol) until completely dissolved. The mixture was then cooled to -10 °C. HATU (32.46 mg, 0.085 mmol) and DIPEA (33.11 mg, 0.256 mmol) were added. The reaction was maintained at -10 °C for 1 h, and the reaction was confirmed to be complete by LCMS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 5% B to 35% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A15-7 (32 mg, 45.70%). ESI-MS: m / z 835 [M+H] + .

[0375] Step 7: Synthesis of Compound A15

[0376] Compound A15-7 (8.20 mg, 0.010 mmol) was dissolved in DMF (1 mL) at 25 °C, and compound 2 (10 mg, 0.009 mmol) was added until completely dissolved. HATU (7.98 mg, 0.021 mmol) and DIPEA (7.24 mg, 0.056 mmol) were added. The reaction was carried out at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 35 min 30% B to 90% B gradient; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A15 (5 mg, 30.2%), ESI-MS: m / z 925.5 [M / 2+H]. + .

[0377] Example A16: Synthesis of Compound A16

[0378] Step 1: Synthesis of compound A16-2

[0379] Compound A16-1 (50 mg, 0.225 mmol) was dissolved in DMF (2 mL) at 25 °C, and Boc-piperazine (125.55 mg, 0.675 mmol) was added until completely dissolved. HATU (256.50 mg, 0.675 mmol) and DIPEA (158.33 mg, 1.225 mmol) were added, and the reaction was carried out at 25 °C for 2 h. The reaction was confirmed to be complete by LC-MS. The solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 70% B gradient for 35 min; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A16-2 (93 mg, 74.4%), ESI-MS: m / z 559.6 [M+H]. + .

[0380] Step 2: Synthesis of compound A16-3

[0381] Compound A16-2 (93 mg, 0.166 mmol) was dissolved in DCM (3 mL) at 25 °C, and TFA (1 mL) was added until completely dissolved. The reaction was carried out at 25 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The solution was concentrated to dryness, dissolved in DMF, and purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% B to 25% B gradient for 30 min; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A16-3 (50.5 mg, 85%), ESI-MS: m / z 359.4 [M+H]. + .

[0382] Step 3: Synthesis of compound A16-4

[0383] At 25°C, compound A16-3 (50 mg, 0.139 mmol) was dissolved in DMF (1 mL), and DIPEA (64.11 mg, 0.496 mmol) was added until completely dissolved. The solution was then cooled to -20°C in a dry ice ethanol bath, and this solution was labeled as solution 1. 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (24.70 mg, 0.139 mmol) was added to DMF (1 mL), followed by HATU (70.68 mg, 0.186 mmol). The reaction was carried out at 25°C for 20 min, and this solution was labeled as solution 2. Solution 2 was then added dropwise to solution 1, and the reaction was maintained at -20°C. The reaction was confirmed to be complete by LC-MS detection; after preparative HPLC purification (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30 min 5% to 25% ACN gradient; detection wavelength: 210 nm), the preparative solution was freeze-dried to obtain compound A16-4 (36 mg, 50.0%), ESI-MS: m / z 519.6 [M+H]+ .

[0384] Step 4: Synthesis of compound A16-5

[0385] Compound A16-4 (36 mg, 0.069 mmol) was added to DMF (2 mL) at 25 °C, followed by 3,6,9-trioxaundecanoic acid (60.38 mg, 0.272 mmol), until completely dissolved. The mixture was then cooled to 0 °C. HATU (70.68 mg, 0.186 mmol) and DIPEA (64.11 mg, 0.496 mmol) were added. The reaction was maintained at 0 °C for 1 h, and the reaction was confirmed to be complete by HPLC. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 5% to 25% ACN gradient for 30 min; detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A16-5 (20 mg, 40.8%), ESI-MS: m / z 359.4 [M+H]. + .

[0386] Step 5: Synthesis of Compound A16

[0387] At 25°C, compound A16-5 (16 mg, 0.021 mmol) was dissolved in DMF (1 mL), and compound 2 (10 mg, 0.009 mmol) was added until completely dissolved. HATU (7.98 mg, 0.021 mmol) and DIPEA (7.24 mg, 0.056 mmol) were added. The reaction was carried out at 25°C for 1 h, and the reaction was confirmed to be complete by LC-MS. The solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 35 min 30% B to 90% B gradient, detection wavelength: 210 nm). The preparative solution was freeze-dried to obtain compound A16 (5 mg, 31.7%), ESI-MS: m / z 877.5 [M / 2+H]. + .

[0388] The following compounds were synthesized in a similar manner:

[0389] In some implementations, the compound of formula (I) is

[0390] Comparative Example C1: Synthesis of Compound A4

[0391] Step 1: Synthesis of Compound A4

[0392] Compound 1 (11.3 mg, 0.015 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (4 mg, 0.015 mmol) were dissolved in 2 mL of DMF, and HATU (8.5 mg, 0.022 mmol) and DIPEA (5 μL, 0.030 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A4 (8 mg, 48.1%), ESI-MS: m / z 1005.3 [M+H]. + . 1 H NMR (400MHz, DMSO-d6) δ9.96(s,1H),9.13(s,2H),8.22(d,J=6.8Hz,1H),8.07(d,J=7.2Hz,1H),7.94(d,J=8.4Hz,1H),7.7 9(d,J=10.8Hz,1H),7.61(d,J=8.4Hz,2H),7.38(d,J=8.4Hz,2H),7.32(s,1H),6.55(brs,1H),5.46(s,2H),5.33-5.29(m, 3H),5.09(s,2H),4.42-4.38(m,1H),4.25-4.21(m,1H),3.42(s,3H),3.30-3.22(m,1H),3.18-3.12(m,1H),2.59-2.54(m, 2H),2.44-2.35(m,5H),2.26-2.17(m,2H),2.02-1.97(m,1H),1.91-1.84(m,4H),1.32(d,J=7.2Hz,3H),0.91-0.87(m,9H).

[0393] Comparative Example C2: Synthesis of Compound A53

[0394] Step 1: Synthesis of compound A53-2

[0395] Compound A53-1 (1.0 g, 1.191 mmol) and Boc-Lys-OtBu (430 mg, 1.429 mmol) were dissolved in 20 mL of DMF, and PyAOP (0.93 g, 1.786 mmol) and DIPEA (0.59 mL, 3.572 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was used directly in the next step without purification.

[0396] Step 2: Synthesis of compound A53-3

[0397] Diethylamine (0.15 mL) was added to the reaction solution of compound A53-2 at 25 °C, and the reaction was maintained at 25 °C for 1 h. HPLC detection confirmed the end of the reaction. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 70% B gradient for 30 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A53-3 (880 mg, 44.69%). ESI-MS: m / z 902 [M+H] + .

[0398] Step 3: Synthesis of compound A53-4

[0399] Compound A53-3 (125 mg, 0.202 mmol) and D-glucose (1.0 g, 5.542 mmol) were dissolved in 10 mL MeOH, and NaCNBH3 (343 mg, 5.542 mmol) was added. The mixture was stirred at 60 °C for 8 h, and the reaction was confirmed to be complete by LC-MS. After filtration, the filtrate was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 60% B gradient for 40 min; detection wavelength: 220 nm). The preparative solution was freeze-dried to obtain compound A53-4 (400 mg, 52.79%), ESI-MS: m / z 1231 [M+H]. + .

[0400] Step 4: Synthesis of compound A53-5

[0401] Compound A53-4 (400 mg, 0.325 mmol) was dissolved in 20 mL of water, and 2 mL of TFA was added at 25 °C. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated and freeze-dried to obtain compound A53-5 (220 mg, 50.40%), ESI-MS: m / z 538 [M / 2+H]. + .

[0402] Step 5: Synthesis of compound A53-6

[0403] A53-5 (210 mg, 0.195 mmol) and succinimide 6-(maleimide)hexanoate (90.4 mg, 0.293 mmol) were dissolved in 5 mL of DMF, and DIPEA (97 μL, 0.586 mmol) was added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 10% B to 50% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to give compound A53-6 (120 mg, 46.49%), ESI-MS: m / z 634.5 [M / 2+H]. + .

[0404] Step Six: Synthesis of Compound A53

[0405] A53-6 (30 mg, 0.024 mmol) and compound 5 (24 mg, 0.028 mmol) were dissolved in 2 mL of DMF, and HATU (18 mg, 0.047 mmol) and DIPEA (16 μL, 0.095 mmol) were added. The reaction mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 30% B to 70% B gradient over 40 min; detector, UV 254 nm). The collected fraction was concentrated and freeze-dried to obtain compound A53 (30 mg, 58.50%), ESI-MS: m / z 697 [M / 3+H]. + . 1H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.13(d,J=7.2Hz,1H),8.08(d,J=7.2Hz,1H),7.9 8(d,J=8.0Hz,1H),7.84-7.78(m,2H),7.68(d,J=8.0Hz,1H),7.62(d,J=8.0Hz,2H),7.37 (d,J=8.0Hz,2H),7.33(s,1H),7.01(s,2H),6.54(s,1H),6.05-6.01(m,1H),5.51-5.45 (m,5H),5.34-5.27(m,3H),5.09(s,2H),4.84-4.80(m,2H),4.63-4.61(m,4H),4.47-4.4 3(m,3H),4.27-4.18(m,2H),4.04-3.94(m,2H),3.98-3.81(m,2H),3.69-3.67(m,2H),3 .60-3.42(m,61H),3.39-3.38(m,4H),3.07-2.92(m,4H),2.39(s,3H),2.32(t,J=8.0Hz, 2H),2.22-2.18(m,2H),2.13-2.09(m,2H),2.04-1.98(m,2H),1.91-1.85(m,2H),1.73-1 .57(m,2H),1.49-1.45(m,6H),1.40-1.33(m,3H),1.33-1.29(m,4H),0.93-0.82(m,9H).

[0406] Comparative Example C3: Synthesis of Compound A47

[0407] Step 1: Synthesis of A47-2

[0408] Fmoc-Val-OH (340 mg, 1.00 mmol) was dissolved in 5 mL of THF. Dicyclohexylcarbodiimide (DCC, 310 mg, 1.50 mmol) and HOSu (172.64 mg, 1.50 mmol) were added under stirring at 20 °C. After addition, the reaction was allowed to proceed at 20 °C for 4.5 h, and the reaction was confirmed to be complete by LC-MS. Then, L-glutamic acid-5-tert-butyl ester (223.56 mg, 1.10 mmol) and DIPEA (193.86 mg, 1.50 mmol) were added to the reaction solution, and the reaction was continued at 20 °C for 2 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated to dryness, dissolved in DMF, and purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 90% B gradient over 40 min; detector, UV). The collected fraction was concentrated and freeze-dried to obtain compound A47-2 (450 mg, 85.78%), ESI-MS: m / z 525.5 (220 nm).

[0409] [M+H]+.

[0410] Step 2: Synthesis of A47-3

[0411] Compound A47-2 (450 mg, 0.86 mmol) was dissolved in 10 mL of DCM, and 10 mL of MeOH was added. The mixture was stirred until dissolved. p-Aminobenzyl alcohol (160.1 mg, 1.30 mmol) and EEDQ (321.48 mg, 1.30 mmol) were added under stirring at 20 °C. After the addition was complete, the mixture was reacted at 20 °C for 18 h. The reaction was confirmed to be complete by LC-MS. The reaction solution was concentrated to dryness, dissolved in DMF, and purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 90% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound A47-3 (500 mg, 92.3%). ESI-MS: m / z 630.3 [M+H] + .

[0412] Step 3: Synthesis of A47-4

[0413] Compound A47-3 (500 mg, 0.79 mmol) was dissolved in 5 mL of DMF and stirred until dissolved. 4-Nitrophenyl carbonate (240.3 mg, 1.20 mmol) and DIPEA (323.10 mg, 2.50 mmol) were added under stirring at 20 °C. After addition, the reaction was carried out at 20 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 90% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound A47-4 (580 mg, 92.3%). ESI-MS: m / z 795.1 [M+H] + .

[0414] Step 4: Synthesis of A47-5

[0415] Compound A47-4 (580 mg, 0.73 mmol) was dissolved in 5 mL of DMF and stirred until dissolved. Icenotecan mesylate (388 mg, 0.73 mmol) and DIPEA (284 mg, 2.20 mmol) were added under stirring at 20 °C. After addition, the reaction was carried out at 20 °C for 2 h, and the reaction was confirmed to be complete by LC-MS. Diethylamine (1 mL) was then added, and the reaction was continued for another 2 h, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 20% B to 90% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain compound A47-5 (500 mg, 80.1%). ESI-MS: m / z 869 [M+H] + .

[0416] Step 5: Synthesis of A47-6

[0417] Compound A47-5 (500 mg, 0.58 mmol) was dissolved in 5 mL of DMF and stirred until dissolved. N-[15-[(2,5-dioxo-1-pyrrolidinyl)oxy]-15-oxo-3,6,9,12-tetraoxapentadecan-1-yl]-2,5-dihydro-2,5-dioxo-1H-pyrrolidinamide (297.81 mg, 0.58 mmol) and DIPEA (112.44 mg, 0.87 mmol) were added under stirring at 20 °C. After the addition was complete, the reaction was carried out at 20 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 10% B to 60% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to give compound A47-6 (546.7 mg, 75.2%). ESI-MS: m / z 634.5 [M / 2+H] + .

[0418] Step Six: Synthesis of A47-7

[0419] Compound A47-6 (540 mg, 0.43 mmol) was dissolved in 5 mL of DCM and stirred until dissolved. TFA (1.0 mL) was added with stirring at 20 °C, and the reaction was carried out at 20 °C for 2.0 hr. The reaction was confirmed to be complete by LC-MS. The solution was concentrated to dryness to give compound A47-7 (358 mg, 70%). ESI-MS: m / z 1211 [M+H] + .

[0420] Step 7: Synthesis of A47

[0421] Compound A47-7 (100 mg, 0.08 mmol) was dissolved in 3 mL of DMF and stirred until dissolved. 3-((2-aminoethyl)dimethylamino)propane-1-sulfonic acid inner salt (25 mg, 0.12 mmol), PyAOP (62.5 mg, 0.12 mmol), and DIPEA (20.70 mg, 0.16 mmol) were added under stirring at 20 °C. After addition, the reaction was carried out at 20 °C for 1 h, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by preparative HPLC (column, C18; mobile phase, A: water (0.05% TFA), B: ACN; 10% B to 60% B gradient over 40 min; detector, UV 220 nm). The collected fraction was concentrated and freeze-dried to obtain a yellow solid A47 (54 mg, 49.2%). ESI-MS: 702 m / z [M / 2+H] + .

[0422] Preparation of antibody-drug conjugates

[0423] Example B1: Preparation and characterization of antibody-drug conjugate B1 (MAB802-A1)

[0424] Take MAB802 antibody (HER2), adjust the antibody pH to approximately 7.2 with Tris solution, detect the protein concentration using Nanodrop, weigh the net antibody solution, and calculate the total protein content. Add 10 mM TCEP solution to the antibody, mix well, and place on a 3D shaker. React at room temperature for at least 120 minutes, continuously mixing to completely reduce the disulfide bonds between antibody chains.

[0425] Add an excess of DMSO solution of compound A1 to the reduced antibody solution. The ratio of A1 to MAB802 antibody is 1:2:1. Mix well and place on a shaker at room temperature for at least 30 minutes, mixing continuously. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and place on a 3D shaker at room temperature for at least 20 minutes, mixing continuously.

[0426] The conjugate product was purified using 30kD ultrafiltration centrifuge tubes and displaced into a storage medium (10mM Histidine, pH 5.8) at a displacement factor greater than 1000-fold. It was then filtered through a 0.22μm sterile filter to obtain the antibody-drug conjugate MAB802-A1, which was stored at 4°C.

[0427] The protein concentration of the antibody-drug conjugate was determined using the UV / BCA method, the DAR value was detected using hydrophobic interaction chromatography (HIC), and the purity was determined using size exclusion chromatography (SEC). The concentration of antibody-drug conjugate B1 (MAB802-A1) was 4.51 mg / ml; the DAR value was 7.9; and the Mono% was 98.96%. The HIC chromatogram is shown in Figure 1a, and the SEC chromatogram is shown in Figure 1b. The analytical results are shown in Table 2.

[0428] Table 1: HIC and SEC analysis methods used in the examples

[0429] Example B2: Preparation and characterization of antibody-drug conjugate B2 (JMT101-A1)

[0430] Following the preparation method of Example B1 above, the linker drug conjugate A1 was conjugated with JMT101 antibody (EGFR), with a feed ratio of A1 to JMT101 antibody of 12 / 1. The antibody-drug conjugate JMT101-A1 was thus prepared.

[0431] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as those used in Example B1. The concentration of antibody-drug conjugate B2 was determined to be 5.34 mg / ml; the DAR value was 7.8; and the Mono% was 97.37%.

[0432] Example B3: Preparation and characterization of antibody-drug conjugate B3 (MAB801-A1)

[0433] Following the preparation method of Example B1 above, the linker drug conjugate A1 was conjugated with the MAB801 antibody (CD20), with a feed ratio of A1 to MAB801 antibody of 12 / 1. The antibody-drug conjugate MAB801-A1 was thus prepared.

[0434] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B3 was determined to be 5.76 mg / ml; the DAR value was 7.8; and the Mono% was 99.31%.

[0435] Table 2: Results of Examples B1, B2 and B3

[0436] Example B4: Preparation and characterization of antibody-drug conjugate B4 (MAB802-A9)

[0437] Referring to the method in Example B1, the linker drug conjugate A9 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B4 (MAB802-A9) was thus prepared.

[0438] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B4 was determined to be 3.26 mg / ml; the DAR value was 8.0; and the Mono% was 98.55%.

[0439] Example B5: Preparation and characterization of antibody-drug conjugate B5 (JMT101-A9)

[0440] Referring to the method in Example B1, the linker drug conjugate A9 was conjugated with the JMT101 antibody at a ratio of 12 / 1. Antibody-drug conjugate B5 (JMT101-A9) was thus prepared.

[0441] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B5 was determined to be 2.92 mg / ml; the DAR value was 8.0; and the Mono% was 98.45%.

[0442] Example B6: Preparation and characterization of antibody-drug conjugate B6 (MAB801-A9)

[0443] Referring to the method in Example B1, the linker drug conjugate A9 was conjugated with antibody MAB801, with a feed ratio of A9 to MAB801 antibody of 12 / 1. Antibody-drug conjugate B6 (MAB801-A9) was thus prepared.

[0444] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B6 was determined to be 2.81 mg / ml; the DAR value was 8.0; and the Mono% was 99.14%.

[0445] Example B7: Preparation and characterization of antibody-drug conjugate B7 (MAB802-A10)

[0446] Referring to the method in Example B1, the linker drug conjugate A10 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B7 (MAB802-A10) was thus prepared.

[0447] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B7 was determined to be 0.78 mg / ml; the DAR value was 8.0; and the Mono% was 99.73%.

[0448] Example B8: Preparation and characterization of antibody-drug conjugate B8 (JMT101-A10)

[0449] Referring to the method in Example B1, the linker drug conjugate A10 was conjugated with the JMT101 antibody at a ratio of 12 / 1. Antibody-drug conjugate B8 (JMT101-A10) was thus prepared.

[0450] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B8 was determined to be 2.89 mg / ml; the DAR value was 8.0; and the Mono% was 98.34%.

[0451] Example B9: Preparation and characterization of antibody-drug conjugate B9 (MAB801-A10)

[0452] Referring to the method in Example B1, the linker drug conjugate A10 was conjugated with antibody MAB801, with a feed ratio of A10 to MAB801 antibody of 12 / 1. Antibody-drug conjugate B9 (MAB801-A10) was thus prepared.

[0453] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B9 was determined to be 3.18 mg / ml; the DAR value was 8.0; and the Mono% was 99.2%.

[0454] Example B10: Preparation and characterization of antibody-drug conjugate B10 (MAB802-A11)

[0455] Referring to the method in Example B1, the linker drug conjugate A11 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B10 (MAB802-A11) was thus prepared.

[0456] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B10 was determined to be 5.43 mg / ml; the DAR value was 7.6; and the Mono% was 95.52%.

[0457] Example B11: Preparation and characterization of antibody-drug conjugate B11 (JMT101-A11)

[0458] Referring to the method in Example B1, the linker drug conjugate A11 was conjugated with the JMT101 antibody at a ratio of 12 / 1. This yielded antibody-drug conjugate B11 (JMT101-A11).

[0459] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B11 was determined to be 4.62 mg / ml; the DAR value was 7.8; and the Mono% was 92.84%.

[0460] Example B12: Preparation and characterization of antibody-drug conjugate B12 (MAB801-A11)

[0461] Referring to the method in Example B1, the linker drug conjugate A11 was conjugated with the MAB801 antibody at a ratio of 12 / 1. Antibody-drug conjugate B12 (MAB801-A11) was then prepared.

[0462] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B12 was determined to be 5.07 mg / ml; the DAR value was 7.7; and the Mono% was 96.44%.

[0463] Example B13: Preparation and characterization of antibody-drug conjugate B13 (MAB802-A12)

[0464] Referring to the method in Example B1, the linker drug conjugate A12 was conjugated with the MAB802 antibody at a ratio of 12 / 1. The antibody-drug conjugate MAB802-A12 was thus prepared.

[0465] The protein concentration of the antibody-drug conjugate was determined using the UV / BCA method, the DAR value was measured using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B13 was determined to be 5.71 mg / ml; the DAR value was 7.9, and the Mono% was 98.72%. The HIC chromatogram is shown in Figure 2a, and the SEC chromatogram is shown in Figure 2b.

[0466] Example B14: Preparation and characterization of antibody-drug conjugate B14 (JMT101-A12)

[0467] Referring to the method in Example B1, the linker drug conjugate A12 was conjugated with the JMT101 antibody at a ratio of 12 / 1. Antibody-drug conjugate B14 (JMT101-A12) was then prepared.

[0468] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B14 was determined to be 4.55 mg / ml; the DAR value was 7.9, and the Mono% was 97.37%.

[0469] Example B15: Preparation and characterization of antibody-drug conjugate B15 (MAB801-A12)

[0470] Referring to the method in Example B1, the linker drug conjugate A12 was conjugated with the MAB801 antibody at a ratio of 12 / 1. This yielded antibody-drug conjugate B15 (MAB801-A12).

[0471] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B15 was determined to be 4.39 mg / ml; the DAR value was 8.0, and the Mono% was 99.09%.

[0472] Example B16: Preparation and characterization of antibody-drug conjugate B16 (MAB802-A2)

[0473] Referring to the method in Example B1, the linker drug conjugate A2 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B16 (MAB802-A2) was thus prepared.

[0474] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B16 was determined to be 5.17 mg / ml; the DAR value was 7.8, and the Mono% was 97.61%.

[0475] Example B17: Preparation and characterization of antibody-drug conjugate B17 (JMT101-A2)

[0476] Referring to the method in Example B1, the linker drug conjugate A2 was conjugated with the JMT101 antibody at a ratio of 12 / 1. Antibody-drug conjugate B17 (JMT101-A2) was then prepared.

[0477] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B17 was determined to be 5.05 mg / ml; the DAR value was 7.8, and the Mono% was 97.68%.

[0478] Example B18: Preparation and characterization of antibody-drug conjugate B18 (MAB801-A2)

[0479] Referring to the method in Example B1, the linker drug conjugate A2 was conjugated with the MAB801 antibody at a ratio of 12 / 1. Antibody-drug conjugate B18 (MAB801-A2) was thus prepared.

[0480] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B18 was determined to be 5.22 mg / ml; the DAR value was 7.9, and the Mono% was 97.18%.

[0481] Example B19: Preparation and Characterization of Antibody-Drug Conjugate B19 (MAB802-A5)

[0482] Referring to the method in Example B1, the linker drug conjugate A5 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B19 (MAB802-A5) was thus prepared.

[0483] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B19 was determined to be 1.52 mg / ml; the DAR value was 7.8, and the Mono% was 99.78%.

[0484] Example B20: Preparation and characterization of antibody-drug conjugate B20 (MAB802-A7)

[0485] Referring to the method in Example B1, the linker drug conjugate A7 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B20 (MAB802-A7) was thus prepared.

[0486] The protein concentration of the antibody-drug conjugate was determined using the UV / BCA method, the DAR value was measured using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B20 was determined to be 5.24 mg / ml; the DAR value was 7.8, and the Mono% was 98.76%. The HIC chromatogram is shown in Figure 3a, and the SEC chromatogram is shown in Figure 3b.

[0487] Example B21: Preparation and characterization of antibody-drug conjugate B21 (MAB802-A14)

[0488] Referring to the method in Example B1, the linker drug conjugate A14 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B21 (MAB802-A14) was thus prepared.

[0489] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B21 was determined to be 8.94 mg / ml; the DAR value was 7.9, and the Mono% was 98.69%.

[0490] Example B22: Preparation and characterization of antibody-drug conjugate B22 (MAB802-A15)

[0491] Referring to the method in Example B1, the linker drug conjugate A15 was conjugated with the MAB802 antibody at a ratio of 12 / 1. This yielded antibody-drug conjugate B22 (MAB802-A15).

[0492] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B22 was determined to be 4.92 mg / ml; the DAR value was 7.9, and the Mono% was 99.02%.

[0493] Example B23: Preparation and characterization of antibody-drug conjugate B23 (MAB802-A16)

[0494] Referring to the method in Example B1, the linker drug conjugate A16 was conjugated with the MAB802 antibody at a ratio of 12 / 1. This yielded antibody-drug conjugate B23 (MAB802-A16).

[0495] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B23 was determined to be 3.41 mg / ml; the DAR value was 7.9, and the Mono% was 98.89%.

[0496] Example B31: Preparation and characterization of antibody-drug conjugate B31 (MAB802-A18)

[0497] Referring to the method in Example B1, the linker drug conjugate A18 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B31 (MAB802-A18) was thus prepared.

[0498] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B31 was determined to be 4.8 mg / ml; the DAR value was 6.9, and the Mono% was 95.50%.

[0499] Example B32: Preparation and characterization of antibody-drug conjugate B32 (MAB802-A20)

[0500] Referring to the method in Example B1, the linker drug conjugate A20 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B32 (MAB802-A20) was thus prepared.

[0501] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B32 was determined to be 5.3 mg / ml; the DAR value was 8.0, and the Mono% was 97.25%.

[0502] Example B35: Preparation and characterization of antibody-drug conjugate B35 (MAB802-A38)

[0503] Referring to the method in Example B1, the linker drug conjugate A38 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B35 (MAB802-A38) was thus prepared.

[0504] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B35 was determined to be 4.3 mg / ml; the DAR value was 7.6, and the Mono% was 98.96%.

[0505] Example B51: Preparation and characterization of antibody-drug conjugate B51 (MAB802-A26)

[0506] Referring to the method in Example B1, the linker drug conjugate A26 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B51 (MAB802-A26) was thus prepared.

[0507] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B51 was determined to be 3.5 mg / ml; the DAR value was 5.2, and the Mono% was 98.65%.

[0508] Table 3: Characterization of Results for Examples B4-B23, B31-B32, B35, and B51

[0509] Example B29: Preparation and characterization of antibody-drug conjugate B29 (JMT101-A3 DAR4)

[0510] Referring to the method in Example B1, the linker drug conjugate A3 was conjugated with the JMT101 antibody at a ratio of 6:1. Antibody-drug conjugate B29 (JMT101-A3 DAR4) was then prepared.

[0511] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B29 was determined to be 3.5 mg / ml; the DAR value was 4.3, and the Mono% was 97.80%.

[0512] Example B30: Preparation and characterization of antibody-drug conjugate B30 (JMT101-A1 DAR4)

[0513] Take JMT101 antibody (EGFR antibody), add an appropriate amount of Tris-EDTA solution, adjust the antibody pH to 7.6 with Tris solution, detect the protein concentration using Nanodrop, weigh the net antibody solution, and calculate the total protein content. Add 10 mM TCEP solution to the antibody to make the TCEP to antibody molar ratio 2.6, mix well, and place on a 3D shaker to react at room temperature for at least 120 min.

[0514] Add solution A1 (dissolved in DMSO) to the reduced antibody solution. The ratio of A1 to JMT101 antibody is 6:1. Mix well and place on a shaker. React at room temperature for at least 90 minutes, mixing continuously. After the reaction is complete, add excess N-acetylcysteine ​​solution to the reaction solution. Place on a 3D shaker and react at room temperature for at least 20 minutes, mixing continuously.

[0515] The conjugate product was purified using 30kD ultrafiltration centrifuge tubes. Excess small molecules were adsorbed with activated carbon, filtered, and then displaced into a storage medium (10mM Histidine, pH 5.8) at a displacement factor greater than 1000-fold. It was then filtered through a 0.22μm sterile filter to obtain antibody-drug conjugate B30 (JMT101-A1 DAR4), which was stored at 4°C.

[0516] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B30 was determined to be 3.4 mg / ml; the DAR value was 4.0, and the Mono% was 97.60%.

[0517] Example B33: Preparation and characterization of antibody-drug conjugate B33 (JMT101-A10 DAR4)

[0518] Referring to the method in Example B1, the linker drug conjugate A10 was conjugated with the JMT101 antibody at a ratio of 6:1. Antibody-drug conjugate B33 (JMT101-A10 DAR4) was then prepared.

[0519] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B33 was determined to be 3.0 mg / ml; the DAR value was 4.0, and the Mono% was 97.90%.

[0520] Example B34: Preparation and characterization of antibody-drug conjugate B34 (JMT101-A15 DAR4)

[0521] Referring to the method in Example B1, the linker drug conjugate A15 was conjugated with the JMT101 antibody at a ratio of 6:1. Antibody-drug conjugate B34 (JMT101-A15 DAR4) was then prepared.

[0522] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B34 was determined to be 3.2 mg / ml; the DAR value was 4.2, and the Mono% was 97.90%.

[0523] Table 4: Results Characterization of Examples B29-B30 and B33-B34

[0524] Example B28: Preparation and characterization of antibody-drug conjugate B28 (MAB802-A3)

[0525] Take MAB802 antibody (HER2), add an appropriate amount of Tris-EDTA solution, adjust the antibody pH to approximately 7.5 with Tris solution, detect the protein concentration using Nanodrop, weigh the net antibody solution, and calculate the total protein content. Add 10 mM TCEP solution to the antibody, mix well, and place on a 3D shaker. React at room temperature for at least 120 minutes, continuously mixing to completely reduce the disulfide bonds between antibody chains.

[0526] Add excess A3 solution (dissolved in DMSO) to the reduced antibody solution. The ratio of A3 to MAB802 antibody is 12 / 1. Mix well and place on a shaker at room temperature for at least 90 minutes, mixing continuously. After the reaction is complete, add excess N-acetylcysteine ​​solution to the reaction mixture and place on a 3D shaker at room temperature for at least 20 minutes, mixing continuously.

[0527] The conjugate product was purified using 30kD ultrafiltration centrifuge tubes. Excess small molecules were adsorbed with activated carbon, filtered, and then displaced into a storage solution (10mM Histidine, pH 5.8) at a displacement factor greater than 1000-fold. It was then filtered through a 0.22μm sterile filter to obtain the antibody-drug conjugate MAB802-A3, which was stored at 4°C.

[0528] The protein concentration of the antibody-drug conjugate was determined using the Nanodrop method, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods are shown in Table 5 below. The concentration of antibody-drug conjugate B28 was determined to be 11.8 mg / ml; the DAR value was 7.7, and the Mono% was 98.4%.

[0529] Table 5: Nanodrop, MASS, and SEC detection and analysis methods used in the examples

[0530] Example B36: Preparation and characterization of antibody-drug conjugate B36 (MAB802-A39)

[0531] Following the preparation method of Example B28 above, linker drug conjugate A39 was conjugated with MAB802 antibody (HER2), with a feed ratio of A39 to MAB802 antibody of 12 / 1. Antibody-drug conjugate B36 (MAB802-A39) was thus prepared.

[0532] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B36 was determined to be 9.6 mg / ml; the DAR value was 8.0, and the Mono% was 98.8%.

[0533] Example B37: Preparation and characterization of antibody-drug conjugate B37 (MAB802-A40)

[0534] Following the preparation method of Example B28 above, the linker drug conjugate A40 was conjugated with the MAB802 antibody (HER2), with a feed ratio of A40 to MAB802 antibody of 12 / 1. Antibody-drug conjugate B37 (MAB802-A40) was thus prepared.

[0535] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B37 was determined to be 14.7 mg / ml; the DAR value was 8.0, and the Mono% was 99.0%.

[0536] Example B40: Preparation and characterization of antibody-drug conjugate B40 (MAB802-A43)

[0537] Following the preparation method of Example B28 above, the linker drug conjugate A43 was conjugated with the MAB802 antibody (HER2), with a feed ratio of A43 to MAB802 antibody of 12 / 1. Antibody-drug conjugate B40 (MAB802-A43) was thus prepared.

[0538] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B40 was determined to be 6.7 mg / ml; the DAR value was 8.0, and the Mono% was 97.1%.

[0539] Example B41: Preparation and characterization of antibody-drug conjugate B41 (MAB802-A44)

[0540] Following the preparation method of Example B28 above, linker drug conjugate A44 was conjugated with MAB802 antibody (HER2), with a feed ratio of A44 to MAB802 antibody of 12 / 1. Antibody-drug conjugate B41 (MAB802-A44) was thus prepared.

[0541] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B41 was determined to be 7.8 mg / ml; the DAR value was 8.0, and the Mono% was 99.5%.

[0542] Example B42: Preparation and characterization of antibody-drug conjugate B42 (MAB802-A45)

[0543] Following the preparation method of Example B28 above, the linker drug conjugate A45 was conjugated with the MAB802 antibody (HER2), with a feed ratio of A45 to MAB802 antibody of 12 / 1. This yielded antibody-drug conjugate B42 (MAB802-A45).

[0544] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B42 was determined to be 11.3 mg / ml; the DAR value was 8.0, and the Mono% was 99.2%.

[0545] Table 6: Results Characterization of Antibody-Drug Conjugates B28, B36-37, and B40-B42

[0546] Control Example D1: Preparation and Characterization of Antibody-Drug Conjugate B24 (MAB802-A4)

[0547] Following the method in Example B1, the linker drug conjugate A4 was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B24 (MAB802-A4) was prepared. The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, DAR was determined using HIC, and purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B24 was determined to be 4.77 mg / ml; the DAR value was 0.9, and the Mono% was 66.73%. The HIC chromatogram is shown in Figure 4a, and the SEC chromatogram is shown in Figure 4b. The analytical results are shown in Table 7.

[0548] Table 7: Results Characterization of Antibody-Drug Conjugate B24

[0549] Control Example D2: Preparation and characterization of antibody-drug conjugate B25 (MAB802-GGFG-Dxd)

[0550] Referring to the method in Example B1, the linker drug conjugate MC-GGFG-Dxd (purchased from Haoyuan Biotechnology) was conjugated with the MAB802 antibody at a ratio of 12 / 1. Antibody-drug conjugate B25 (MAB802-GGFG-Dxd) was thus prepared.

[0551] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B25 was determined to be 3.14 mg / ml; the DAR value was 7.6, and the Mono% was 94.69%. The analytical results are shown in Table 8.

[0552] Control Example D3: Preparation and characterization of antibody-drug conjugate B26 (JMT101-GGFG-Dxd)

[0553] Referring to the method in Example B1, the linker drug conjugate MC-GGFG-Dxd (purchased from Haoyuan Biotechnology) was conjugated with the JMT101 antibody at a ratio of 12 / 1. Antibody-drug conjugate B26 (JMT101-GGFG-Dxd) was thus prepared.

[0554] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B26 was determined to be 3.27 mg / ml; the DAR value was 7.9, and the Mono% was 97.29%. The analytical results are shown in Table 8.

[0555] Control Example D4: Preparation and characterization of antibody-drug conjugate B27 (MAB801-GGFG-Dxd)

[0556] Referring to the method in Example B1, the linker drug conjugate MC-GGFG-Dxd (purchased from Haoyuan Biotechnology) was conjugated with the MAB801 antibody at a ratio of 12 / 1. Antibody-drug conjugate B27 (MAB801-GGFG-Dxd) was thus prepared.

[0557] The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, the DAR value was determined using HIC, and the purity was determined using SEC. The HIC and SEC analytical methods were the same as in Example B1. The concentration of antibody-drug conjugate B27 was determined to be 3.26 mg / ml; the DAR value was 7.8, and the Mono% was 98.14%. The analytical results are shown in Table 8.

[0558] Table 8: Results Characterization of Antibody-Drug Conjugates B25, B26, and B27

[0559] Control Example D5: Preparation and Characterization of Antibody-Drug Conjugate B44 (MAB802-A47)

[0560] Following the preparation method of Example B28 above, the linker drug conjugate A47 was conjugated with the MAB802 antibody (HER2), with a feed ratio of A47 to MAB802 antibody of 12 / 1. This yielded antibody-drug conjugate B44 (MAB802-A47).

[0561] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B44 was determined to be 9.5 mg / ml; the DAR value was 8.0, and the Mono% was 96.7%.

[0562] Control Example D6: Preparation and Characterization of Antibody-Drug Conjugate B50 (MAB802-A53)

[0563] Following the preparation method of Example B28 above, the linker drug conjugate A53 was conjugated with the MAB802 antibody (HER2), with a feed ratio of A53 to MAB802 antibody of 12 / 1. Antibody-drug conjugate B50 (MAB802-A53) was thus prepared.

[0564] The protein concentration of the antibody-drug conjugate was detected using Nanodrop, the DAR value was determined using MASS, and the purity was determined using SEC. The MASS and SEC analytical methods were the same as in Example B28. The concentration of antibody-drug conjugate B50 was determined to be 7.44 mg / ml; the DAR value was 8.0, and the Mono% was 96.8%.

[0565] Table 9: Results Characterization of Antibody-Drug Conjugate B50

[0566] Example E1: In vitro plasma stability of ADC

[0567] B30 (JMT101-A1 DAR4.0), B29 (JMT101-A3 DAR4.3), B33 (JMT101-A10 DAR4.0), and B26 (JMT101-GGFG-Dxd DAR8, control) were incubated with 1% BSA-PBS, cynomolgus monkey plasma, and human plasma, respectively, at a concentration of 100 μg / mL. Samples were collected after incubation at 37°C for 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 7 d, 10 d, 14 d, 17 d, and 21 d.

[0568] B1 (MAB802-A1), B4 (MAB802-A9), B10 (MAB802-A11), B28 (MAB802-A3), B23 (MAB802-A16), B22 (MAB802-A15), B32 (MAB802-A20), B31 (MAB802-A18), B35 (MAB802-A38), and B25 (MAB802-GGFG-Dxd, control) were incubated with 1% BSA-PBS, cynomolgus monkey plasma, and human plasma, respectively. Samples were collected after incubation at 37°C for 0 h, 1 d, 3 d, 7 d, 13 d, and 21 d.

[0569] B1 (MAB802-A1, DAR7.9), B44 (MAB802-A47, DAR8.0, Control 1), and B50 (MAB802-A53, DAR7.9, Control 2) were incubated with cynomolgus monkey and human plasma at 37°C, respectively. A 1% BSA-PBS system was used as a negative control. Incubation samples were collected at 0h, 24h, 72h, and 168h, respectively. The concentration of shed payload in plasma samples was detected by LC-MS / MS.

[0570] The results are shown in Tables 10, 11 and 12.

[0571] Table 10: In vitro plasma stability of JMT101 antibody-drug conjugates B30, B29, B33 and B26 (control)

[0572] *BLQ: Below the quantitative threshold

[0573] The results showed that after incubation at 37°C for different times, the JMT101 antibody-drug conjugate generated free payloads in 1% BSA-PBS, cynomolgus monkey plasma, and human plasma. After 21 days of incubation, the stability of the ADC from strongest to weakest was B30(JMT101-A1)≈B29(JMT101-A3)>B33(JMT101-A10)>B26(JMT101-GGFG-Dxd).

[0574] Table 11: In vitro plasma stability of MAB802 antibody-drug conjugate

[0575] After incubation at 37°C for different times, MAB802-ADC generated free payloads in 1% BSA-PBS, cynomolgus monkey plasma, and human plasma. After 21 days of incubation, the MAB802-ADC of this invention exhibited superior stability compared to MAB802-GGFG-Dxd.

[0576] Table 12: In vitro plasma stability of MAB802 antibody ADC

[0577] The results showed that after incubation at 37℃ for different times, B1, B44, and B50 all produced free eccentrica in 1% BSA-PBS, cynomolgus monkey plasma, and human plasma, with the release of eccentrica gradually increasing with prolonged incubation time. In the PBS negative control group, there was no significant difference in the release of eccentrica from B1, B44, and B50. In plasma from different species, the release of eccentrica from B44 and B50 was significantly higher than that from B1, indicating that B1 has better stability in the circulatory system than B44 and B50.

[0578] Example E2: Determination of the cytotoxic activity of ADC compounds against tumor cells

[0579] AU565 (human breast cancer, ATCC, a HER2-highly expressing cell line) and NCI-N87 (human gastric cancer, Institute of Cell Biology, Chinese Academy of Sciences), cells in logarithmic growth phase were digested with trypsin, collected, centrifuged, and resuspended in complete culture medium to a cell density of 4.44 x 10⁻⁶ cells / mL. 4 / ml, add 90μL of cells to each well of a 96-well plate, ensuring 4000 cells per well. Incubate the plate overnight in an incubator; then prepare working solutions of 10X series concentrations: the 21 ADCs are prepared up to 10uM, and diluted 5-fold in sequence; add 10uL of the above working solution of 10X concentration to each well of the corresponding cell well; incubate the 96-well plate in a cell culture incubator for 6 days; after the culture, add 10μL of PrestoBlue (Invitrogen, Cat: A13262) detection reagent to each well, and incubate the cells in an incubator for 1h; set the excitation light to 560nm and the emission light to 590nm, and read the plate using a microplate reader (Molecular Devices, SpectraMax M5); finally, use GraphPad Prism 10.4.0 (621) software to fit and obtain IC50. 50 Values. The results are shown in Table 13.

[0580] Table 13: Killing activity of MAB802 antibody ADC against HER2-overexpressing cells

[0581] The results showed that the ADCs (i.e., the target compounds) conjugated with MAB802 antibody (anti-HER2 antibody) all had significant killing effects on HER2-overexpressing tumor cell lines, while the ADCs (B3, B12, B15, B18, and B27) conjugated with MAB801 antibody (anti-CD20 antibody) had significantly lower killing activity against tumor cells than the target compounds.

[0582] Example E3: Determination of the cytotoxic activity of ADC compounds against tumor cells

[0583] MDA-MB-468 (human breast cancer, Institute of Cell Biology, Chinese Academy of Sciences, an EGFR-highly expressing cell line) and DiFi (human colorectal cancer, Medicilon, an EGFR-highly expressing cell line) cells in logarithmic growth phase were digested with trypsin. Cells were collected, centrifuged, counted, and resuspended in complete culture medium to a density of 4.44 x 10⁻⁶ cells / mL. 4 / ml, add 90μL of cells to each well of a 96-well plate, ensuring 4000 cells per well. Incubate the plate overnight in an incubator; then prepare working solutions of 10X series concentrations: the 16 ADCs are prepared up to 10uM, and diluted 5-fold in sequence; add 10uL of the above working solution of 10X concentration to each well of the corresponding cell well; incubate the 96-well plate in a cell culture incubator for 6 days; after the culture, add 10μL of PrestoBlue (Invitrogen, Cat: A13262) detection reagent to each well, and incubate the cells in an incubator for 1h; set the excitation light to 560nm and the emission light to 590nm, and read the plate with a microplate reader (Molecular Devices, SpectraMax M5); finally, use GraphPad Prism 10.4.0 (621) software to fit and obtain IC50. 50 Values. See Table 14 for experimental results.

[0584] Table 14: Killing activity of JMT101 antibody ADC against EGFR-overexpressing cells

[0585] The results showed that the ADCs (i.e., the target compounds) conjugated with JMT101 antibody (anti-EGFR antibody) all had significant killing effects on EGFR-overexpressing tumor cell lines, while the ADCs (B3, B6, B9, B12, B15, B18, and B27) conjugated with MAB801 antibody (anti-CD20 antibody) had significantly lower killing activity against tumor cells than the target compounds.

[0586] Example E4: Determination of the cytotoxic activity of ADC compounds against tumor cells

[0587] AU565 (human breast cancer, ATCC, a Her2-highly expressing cell line) and BXPC-3 (human orthotopic pancreatic adenocarcinoma cells, Institute of Cell Biology, Chinese Academy of Sciences, a Her2-lowly expressing cell line) cells in logarithmic growth phase were digested with trypsin, collected, centrifuged, and resuspended in complete culture medium to a cell density of 2.67 x 10⁻⁶ cells / mL. 4 / ml, add 150μL of cells to each well of a 96-well plate, ensuring 4000 cells per well. Incubate the plate overnight. Then prepare working solution at 4X sequence concentration: prepare 10uM for each of the 16 ADCs, and dilute 5-fold sequentially. Add 50uL of the above working solution at 4X concentration to each well. Incubate the 96-well plate in a cell culture incubator for 6 days. After incubation, add 20μL of PrestoBlue (Invitrogen, Cat: A13262) detection reagent to each well and incubate the cells for 1 hour. Set the excitation light to 560nm and the emission light to 590nm, and read the plate using a microplate reader (Molecular Devices, SpectraMax M5). Finally, use GraphPad Prism 10.4.0 (621) software to obtain the IC50 value. The results are shown in Table 15.

[0588] Table 15: Killing activity of ADCs against Her2 high / low expression cells

[0589] The results showed that the ADCs (target compounds) conjugated with MAB802 antibody (anti-Her2 antibody) all had significant killing effects on the HER2-overexpressing tumor cell line (AU565), while the killing activity against the HER2-low-expressing tumor cell line (BXPC-3) was significantly lower than that against the overexpressing cell line, which indirectly verified the importance of antigen expression level to tumor killing activity.

[0590] Example E5: In vivo efficacy test of anti-EGFR antibody ADC against human lung adenocarcinoma PC9 (Del19 / T790M / C797S, osimertinib resistant) CDX model

[0591] The experiment used NOD / SCID mice (5–10 weeks old, 20 mice). A human lung adenocarcinoma PC9-Del19 / T790M / C797S osimertinib-resistant CDX model was used to investigate the in vivo antitumor effects of the two ADC drugs. The experiments were conducted according to the drug dosages and groupings in Table 16.

[0592] Table 16: Grouping and Dosage of Animals in the In Vivo Efficacy Model of Human Lung Adenocarcinoma PC9 (Del19 / T790M / C797S) CDX Model Note: IV: intravenous administration; IV / 1 time: once-daily administration.

[0593] This experiment used human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib resistant) cells to construct a mouse tumor transplantation model, and the tumor volume was increased to 150 mm². 3Animals were grouped according to tumor volume according to Table 16. They were given a single intravenous dose of 10 mL / kg. The solvent control group was given 5% glucose. The experiment ended after 14 days of administration. The tumor inhibition effects of the two ADC drugs were compared.

[0594] At the experimental endpoint, compared with the solvent group, both B30 and B26 (JMT101-GGFG-Dxd) inhibited tumor volume, and the tumor-suppressing effect showed a certain dose-dependent effect. At high doses, B30 showed the most significant tumor-suppressing effect, with a tumor inhibition rate as high as 78.2%, indicating that the ADC prepared using the drug linker of this invention still has a significant inhibitory effect on osimertinib-resistant tumors compared with the ADC prepared by GGFG-Dxd.

[0595] Table 17: Tumor volume after ADC administration in human lung adenocarcinoma PC9 (Del19 / T790M / C797S) CDX model

[0596] TGI = (1-T i / C i )×100; T i and C i These represent the average tumor volume in the treatment group and the solvent group at a specific time point.

[0597] Example E6: In vivo efficacy test of anti-EGFR antibody ADC against human lung adenocarcinoma NCI-H1975 (T790M / L858R, erlotinib resistant) CDX model

[0598] The experiment used BALB / c nude mice (6-7 weeks old, 40 mice). A human lung adenocarcinoma NCI-H1975-T790M / L858R erlotinib-resistant CDX model was used to investigate the in vivo antitumor effects of four ADC drugs. The experiments were conducted according to the drug dosages and groupings in Table 18.

[0599] Table 18: Animal grouping and dosage in in vivo efficacy test of human lung adenocarcinoma NCI-H1975 (T790M / L858R) CDX model Note: IV: intravenous administration; IV / 1 time: once-daily administration.

[0600] This experiment used human lung adenocarcinoma NCI-H1975-T790M / L858R erlotinib-resistant cells to construct a mouse tumor transplantation model, and the tumor volume was increased to 150 mm². 3 Animals were grouped according to tumor volume according to Table 19. They were given a single intravenous dose of 10 mL / kg. The solvent control group was given physiological saline. The experiment was completed after 14 days of administration. The tumor inhibition effects of the four ADC drugs were compared.

[0601] At the experimental endpoint, compared with the solvent group, B30, B29, B34, and B26 (JMT101-GGFG-Dxd, positive control) all inhibited tumor volume. At a dose of 3 mg / kg, each ADC showed significant tumor-suppressive effects. The consistent effect was B30 > B29 > B34 > B26 (positive control).

[0602] Table 19: Tumor parameters of each group 14 days after ADC administration

[0603] TGI = (1-T i / C i )×100; T i and C i These represent the average tumor volume in the treatment group and the solvent group at a specific time point. Example E7: In vivo efficacy test of anti-EGFR antibody ADC against a human lung adenocarcinoma HCC827 CDX model.

[0604] The experiment used BALB / c nude mice (6-7 weeks old, 30 mice). A human lung adenocarcinoma HCC827 CDX model was used to investigate the in vivo antitumor effects of five ADC drugs. The experiments were conducted according to the drug dosages and groupings in Table 20.

[0605] Table 20: Animal grouping and dosage in in vivo efficacy test of human lung adenocarcinoma HCC827 CDX model Note: IV: intravenous administration; IV / 1 time: once-daily administration.

[0606] In this experiment, human lung adenocarcinoma HCC827 cells were used to construct a mouse tumor transplantation model. The tumor volume was increased to 150 mm². 3 Animals were grouped according to tumor volume according to Table 20. They were given a single intravenous dose of 10 mL / kg. The solvent control group was given physiological saline. The experiment was completed after 32 days of administration. The tumor inhibition effects of the five ADC drugs were compared.

[0607] At the experimental endpoint, compared with the solvent group, all ADCs significantly inhibited tumor volume, with tumor inhibition rates exceeding 95%. In the treatment group, tumors began to shrink in all mice during weeks 2-3 of the experiment and continued to shrink until the end of the experiment.

[0608] Table 21: Tumor volume after ADC administration in human lung adenocarcinoma HCC827 CDX model

[0609] TGI = (1-T i / C i )×100; T i and C i These represent the average tumor volume in the treatment group and the solvent group at a specific time point.

[0610] Example E8: In vivo efficacy test of anti-EGFR antibody ADC against human lung adenocarcinoma PC-9CDX model

[0611] The experiment used BALB / c nude mice (6-8 weeks old, 30 mice). A human lung adenocarcinoma PC-9CDX model was used to investigate the in vivo antitumor effects of 45 ADC drugs. The experiments were conducted according to the drug dosages and groupings in Table 22.

[0612] Table 22: Animal grouping and dosage in in vivo efficacy test of PC-9CDX model of human lung adenocarcinoma Note: IV: intravenous administration; IV / 1 time: once-daily administration.

[0613] This experiment used human lung adenocarcinoma PC-9 cells to construct a mouse tumor transplantation model, and the tumor volume was increased to 150 mm². 3 Animals were grouped according to tumor volume according to Table 22. They were given a single intravenous dose of 10 mL / kg. The solvent control group was given physiological saline. The experiment ended after 28 days of administration. The tumor inhibition effects of the four ADC drugs were compared.

[0614] At the experimental endpoint, compared with the solvent group, all ADCs significantly inhibited the volume of the transplanted tumor, and their tumor inhibition effect was better than that of the control group B26 (JMT101-GGFG-Dxd).

[0615] Table 23: Tumor volume in human lung adenocarcinoma PC-9CDX model after ADC administration

[0616] TGI = (1-Ti / Ci) × 100; Ti and Ci are the average tumor volumes of the treatment group and the solvent group at a certain time point, respectively.

[0617] Example E9: In vitro hematologic toxicity assay of anti-EGFR antibody ADC (HemaTox) TM Erythroid Medium)

[0618] Collect CD34 cells (Milstone) and use HemaTox TM Cells were resuspended in Erythroid Medium and seeded at 2000 cells / 200 μL into 96-well plates. ADCs of 5000, 1000, 200, 40, 8, 1.6, 0.32, 0.064, and 0.013 nM were added to the 96-well plates and incubated for 7 days. CD71 and CD235a antibodies were added for staining. CD71 was detected using FACS. + CD235a +The expression was then performed; finally, the IC was obtained by fitting a four-parameter logistic (4PL) nonlinear regression model using Xfit software (5.5.0.5). 50 Values. See Table 24 for experimental results.

[0619] Table 24: Killing activity of ADCs against Erythroid cells

[0620] B30, B29, and B26 all exhibited certain killing effects on CD34 and induced differentiated Erythroid cells. However, the killing effects of B30 and B29 were weaker than those of the positive control B26, indicating that B30 and B29 had a weaker bone marrow suppression effect on Erythroid cells than B26.

[0621] Example E10: In vitro hematologic toxicity assay of anti-EGFR antibody ADC (HemaTox) TM Myeloid Medium cells)

[0622] Collect CD34 cells (Miao Shun) and use HemaTox TM Cells were resuspended in Myeloid Medium and seeded at 1000 cells / 200 μL into 96-well plates. ADCs of 5000, 1666.667, 555.556, 185.185, 61.728, 20.576, 6.859, 2.286, and 0.762 nM were added to the 96-well plates and incubated for 7 days. CD13 and CD15 antibodies were added for staining. CD13 was detected using FACS. + CD15 + The expression was then performed; finally, the IC was obtained by fitting a four-parameter logistic (4PL) nonlinear regression model using Xfit software (5.5.0.5). 50 Values; experimental results are shown in Table 25.

[0623] Table 25: Killing activity of ADCs against Myeloid cells

[0624] B30, B29, B33, and B26 (control) all exhibited some killing effects on CD34 cells and induced differentiated Myeloid cells, with B33 showing the strongest killing effect, possibly due to its unstable linker. Among the stable linker ADCs, B30 and B29 showed weaker killing effects than B26 (control), indicating that B30 and B29 had a weaker effect on Myeloid-induced myelosuppression than B26 (control).

Claims

1. A linker compound, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, having the structure of Formula I, ###0001### Formula I wherein RG-L 3 -L 2 -L 1 -D (I) RG is an active group; D is a cytotoxin, L 3 C1-C6-alkylene-CO, C 1-6 C1-C6-alkylene-CO, C 2-6 C1-C6-alkylene-CO, C 2-6 C1-C6-alkylene-CO, C 1-6 C1-C6-alkylene-CO, C 3-8 C1-C6-alkylene-CO, C 1-6 C1-C6-alkylene-CO, C 3-8 C1-C6-alkylene-CO, C 1-6 C1-C6-alkylene-CO, C 1-6 C1-C6-alkylene-CO, C L 2 is a divalent polypeptide residue consisting of 2 to 5 amino acids; L 1 is a divalent spacer group; and each occurrence of "-" is independently a single bond; wherein t is an integer of 1, 2, 3, 4, 5, or 6; wherein the divalent polypeptide residue L 2 and / or the divalent linking group L 1 is substituted by one or more hydrophilic side chains; said hydrophilic side chain H SC selected from the group consisting of: a) -(CH2) t -S03M, wherein t is an integer of 1, 2, 3, 4, 5 or 6, and M is H or an optionally substituted alkyl or alkali metal ion; b) 8. The linker compound of claim 7, wherein X1 is Lys or Val; X2 is Val, Gly, or Ala; X3 is Ala, Gly, Cit, or absent; X4 is Phe or absent; and X5 is Gly or absent. c) -C(=O)-(CH2) t4 - P(=O)(OM1)2, wherein t4 is an integer of 1, 2, 3, 4, 5 or 6; each M1 is independently selected from H or an alkali metal ion; d) -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H wherein t0 is an integer of 1, 2, 3, 4, 5 or 6, wherein n is any integer from 1 to 11, and / or e) -(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, wherein T is wherein Y1and Y2are each independently -C(=0)-, -N(R)-C(=0)-, -CH2-N(R)-C(=0)-, -C(=0)NR-, -C(=0)0-, -OC(=0)-, -OC(=0)NR-, -0-, -NR-, -S-, S(=0)2-, or absent, wherein R is H or optionally substituted alkyl (preferably C 1-4 alkyl); each of p1, p2, p3, q1, q2, and q3is independently an integer of 1, 2, 3, 4, 5, or 6, q4is an integer of 0, 1, 2, 3, 4, 5, or 6, and s is an integer of 1, 2, 3, or 4. Y H for or -C(=O)-(CH2) t4 -P(=O)(OM1)2, wherein each of t1, t2, t3, and t4 is independently an integer of 1, 2, 3, 4, 5, or 6, and M and M1are independently H, optionally substituted alkyl, or an alkali metal ion.

2. The linker compound of claim 1, wherein RG is halogen, 3. The linker compound of claim 1, wherein L 3 is C 1-6 alkylene-CO, C 2-6 alkynylene-CO, C 1-3 alkylene-C 3-6 cycloalkylene-CO, C 1-3 alkylene-C 3-6 cycloalkylene-C 1-6 heteroalkylene-CO or C 1-6 heteroalkylene-CO, wherein each heteroalkylene independently contains a -C(=O)NH- or -CH2CH2-O- heteroatom group.

4. The linker compound of claim 1, wherein L 3 is wherein n1 is an integer from 2 to 12, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

5. The linker compound of any one of claims 1-4, wherein R is 3 is wherein n1 is 4.

6. The linker compound of any one of claims 1-5, wherein L 2 is substituted with one or more hydrophilic side chains H SC .

7. The linker compound of claim 6, wherein L 2 is X1-X2-X3-X4-X5, wherein X1 is attached to L 3 and wherein each of X1 and X2 is independently an amino acid residue, each of X3, X4, and X5 is independently an amino acid residue or is absent.

9. The linker compound of any one of claims 6-7, wherein X1 is Lys. wherein t is an integer of 1, 2, 3, 4, or 5; 10. The linker compound of claim 9, wherein X1 is Lys, the amino group of Lys bearing a long chain amino side chain (i.e., -(CH2)4-NH2) is substituted with 1-3 hydrophilic side chains H sc .

11. The linker compound of claim 9, wherein L 2 is Lys-Val-Ala Lys-Val-Cit or Lys-Gly-Gly-Phe-Gly wherein the amino group of each Lys bearing a long chain amino side chain (i.e., -(CH2)4-NH2) is substituted with 1-3 hydrophilic side chains H sc .

12. The linker compound of any one of claims 6-11, wherein H SC is a) -(CH2) t -S03M, wherein t is an integer of 1, 2, 3, 4, 5 or 6, and M is H or an optionally substituted alkyl or alkali metal ion; b) wherein Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2-, or absent, wherein R is H or optionally substituted alkyl; pi, p2, p3, qi, q2, and q3 are each independently an integer of 1, 2, 3, 4, 5, or c) -C(=0)-(CH2) t4 -P(=0)(OM1)2; t4 is an integer of 1, 2, 3, 4, 5 or 6; M1 is H or an optionally substituted alkyl or an alkali metal ion; d) -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H wherein t0 is an integer of 1, 2, 3, 4, 5 or 6, n = an integer from 1 to 11, e) -(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, wherein T is wherein Y1 and Y2 are each independently -C(=O)-, -N(R) -C(=O)-, -CH2-N(R)-C(=O)-, -C(=0)NR-, -C(=O)O-, -OC(=O)-, -OC(=0)NR-, -O-, -NR-, -S-, S(=O)2-, or Y H for or -C(=O)-(CH2) t4 -P(=O)(OM1)2, wherein each of t1, t2, t3, and t4 is independently an integer of 1, 2, 3, 4, 5, or 6, and M and M1are independently H, optionally substituted alkyl, or an alkali metal ion.

13. The linker compound of claim 12, wherein Hsc is wherein the variables are as defined above; or preferably ti, t2 and t3 are 3, Y1 is -C(=0)-, -N(CH3)-C(=0)- or -CH2-N(CH3)-C(=0)-, Y2 is absent, and the other variables are as defined above.

14. The linker compound of claim 12, wherein H SC is 15. The linker compound of claim 12, wherein L 2 is 16. The linker compound of claim 1, wherein L 2 is not substituted by a hydrophilic side chain H SC substituted.

17. The linker compound of claim 1, wherein L 2 is Val-Ala 18. The linker compound of claim 1, wherein L 1 is or C 1-6 heteroalkylene.

19. The linker compound of any one of claims 1-18, wherein L 1 is not a hydrophilic side chain H SC substituted.

20. The linker compound of claim 20, wherein L 1 is 21. The linker compound of any one of claims 1-5, wherein L 1 is substituted with one or more hydrophilic side chains H SC .

22. The linker compound of claim 21, wherein L 1 is substituted with one hydrophilic side chain H SC substituted.

23. The linker compound of claim 22, wherein L 1 is wherein Hsc is a hydrophilic side chain.

24. The linker compound of claim 23, wherein H SC is -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H wherein t0 is an integer of 1, 2, 3, 4, 5 or 6, n is an integer of 1-11, Y H is or -C(=O)-(CH2) t4 -P(=O)(OM1)2; wherein ti, t2, t3 and t4 are each independently an integer of 1, 2, 3, 4, 5 or 6, M is H or an alkali metal ion, M1 is H or an alkali metal ion; Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or absent, wherein R is H or optionally substituted alkyl.

25. The linker compound of claim 23, wherein H SC is -(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, wherein T is 28. The linker compound of claim 1, wherein the cytotoxin is a camptothecin, a doxorubicin, a maytansinoid, a dolastatin, a rapamycin (preferably exatecan, deruxitcan, MMAE, or rapamycin.

26. The linker compound of claim 22, wherein H SC is 27. The linker compound of claim 22, wherein L 1 is wherein Y1 and Y2 are each independently -C(=O)-, -N(R )-C(=O)-, -CH2-N(R)-C(=O)-, -C(= O)NR-, -C(=O)O-, -OC(=O)-, -OC(= O)NR-, -O-, -NR-, -S-, S(=O)2-, or - absent, wherein R is H or optionally substituted alkyl; pi, p2, p3 qi, q2, and q3 are each independently an integer of 1, 2, 3, 4, 5, or 6, q4 is an integer of 0, 1, 2, 3, 4, 5, or 6, and s is an integer of 1, 2, 3, or 4.

29. The linker compound of claim 1, wherein the compound of formula (I) is ###00010### wherein H SC -C(=O)-(CH2) t4 -P(=O)(OH)2; -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H wherein t0and t4are each independently an integer of 1, 2, 3, 4, 5, or 6, n = an integer of 1-11; -(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, wherein T is ​ Y H for or -C(=O)-(CH2) t4 -P(=O)(OH)2, wherein each of t1, t2, t3, and t4 is independently an integer of 1, 2, 3, 4, 5, or 6, M is H, optionally substituted alkyl, or an alkali metal ion, each of X2, X3, X4, and X5 is independently an amino acid residue or is absent, and the other variables are as defined for formula (I).

30. The linker compound of claim 1, wherein the compound of formula (I) is ###0007### wherein H SC is -Y1-(CH2) t0 -(OCH2CH2) n -OCH2CH2-Y2-Y H wherein t0 is an integer of 1, 2, 3, 4, 5 or 6, n is an integer of 1-11, Y H is or -C(=O)-(CH2) t4 -P(=O)(OH)2; wherein ti, t2, t3 and t4 are each independently an integer of 1, 2, 3, 4, 5 or 6, M is H, optionally substituted alkyl, optionally substituted aryl or optionally substituted heteroaryl or an alkali metal ion; Y1 and Y2 are each independently -C(=O)-, -N(R)-C(=O)-, -CH2-N(R)-C(=O)-, -C(=O)NR-, -C(=O)O-, -OC(=O)-, -OC(=O)NR-, -O-, -NR-, -S-, S(=O)2- or absent, wherein R is H or optionally substituted alkyl; or H SC is -(CH2) q1 -Y1-(T) s -(CH2) q2 -(OCH2CH2) q3 -O-(CH2) q4 CH3, wherein T is wherein Y1and Y2are each independently -C(=0)-, -N(R)-C(=0)-, -CH2-N(R)-C(=0)-, -C(=0)NR-, -C(=0)0-, -OC(=0)-, -OC(=0)NR-, -0-, -NR-, -S-, S(=0)2-, or absent, wherein R is H or optionally substituted alkyl; pi, p2, p3, qi, q2, and q3are each independently an integer of 1, 2, 3, 4, 5, or 6, q4is an integer of 0, 1, 2, 3, 4, 5, or 6, s is an integer of 1, 2, 3, or 4; The other variables are as defined for formula (I).

31. The linker compound of claim 1, which is 32. An antibody drug conjugate comprising the linker compound of claim 1, said antibody drug conjugate having a structure represented by formula (IV), Ab-[RGR-L 3 -L 2 -L 1 -D] m (IV) wherein Ab is an antibody or an antigen binding fragment thereof; m is any numerical value between 2 and 8 (such as 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, or such as 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, or 7.5-8); wherein RGRis the residue after the reaction of RG (active group) and Ab; The other variables are as defined above in claim 1.

33. A pharmaceutical composition comprising the linker compound of any one of claims 1-31, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the antibody drug conjugate of claim 32, or a pharmaceutically acceptable salt, solvate, or solvate of the 34. A method of treating and / or preventing a tumor comprising: administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of the linker compound of any one of claims 1-31, or a pharmaceutically acceptable, solvate, or solvate of the salt thereof, or the antibody drug conjugates of claim 32, or a pharmaceutically acceptable, solvate, or solvate of the salt.

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