Antibody drug conjugate as well as pharmaceutical composition and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to an antibody drug conjugate, and a pharmaceutical composition and application thereof. Specifically, the invention provides an antibody drug conjugate as shown in formula (I), a drug-linker for preparing the antibody drug conjugate, and an application of the antibody drug conjugate in tumor resistance. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a class of antibody-drug conjugates, their pharmaceutical compositions and applications. Background Art

[0002] Antibody-drug conjugates (ADCs) couple antibodies and small molecule cytotoxic drugs through specific linkers. Antibodies act as carriers to transport small molecule drugs into target cells, thereby reducing systemic exposure and improving safety. They are a hot topic in the research and development of targeted tumor therapy. Traditional ADCs use lysine residues of antibodies or cysteine ​​residues obtained by opening interchain disulfide bonds for conjugation. The resulting ADCs are heterogeneous mixtures with different drug loadings and different connection positions, which are likely to affect the pharmacokinetics, stability, tolerance and efficacy of the drug. The new generation of ADC conjugation technology has turned to site-specific conjugation technology with precise drug loading and controlled connection sites. The ADCs obtained by this technology have a controllable drug-antibody ratio (DAR), high uniformity, better stability and pharmacokinetic properties, and are also more suitable for large-scale production of ADCs. In recent years, a variety of site-specific conjugation methods have been developed to provide precise modification of specific amino acid residues or sugar residues.

[0003] With the development of ADC technology, immunostimulating antibody conjugates (ISACs) have been developed based on ADCs. ISACs are formed by coupling immunostimulators or immunomodulators such as TLR7 and / or TLR8 agonists to antibodies through cleavable / non-cleavable linkers. Immunostimulating antibody conjugates combine the precise targeting of antibodies with the immune activation and long-lasting immune memory effects of immunostimulants, achieving the safety of systemic administration and triggering anti-tumor immune responses.

[0004] In the research of anti-tumor treatment, there is a more obvious development trend from single therapy to combination therapy. From the perspective of clinical needs, ideal combination therapy can improve clinical efficacy, reduce drug toxicity, and delay or avoid the development of drug resistance. Combination therapy is the combination of two existing drugs, such as the combination of cytotoxic drugs and immunostimulants, and the combination of two active ingredients to form a new single drug.

[0005] With the help of antibody targeting and site-specific coupling technology, the bifunctional immunostimulatory antibody drug conjugate (iADC) obtained by coupling cytotoxic drugs and immunostimulatory agents to antibodies can simultaneously enter tumor cells and immune cells in the tumor microenvironment, exerting the dual effects of tumor killing by cytotoxic drugs and local immune activation by immunostimulatory agents, thereby improving the efficacy of antibody drug conjugates in treating cancer. Summary of the invention

[0006] In the first aspect, the present application provides an antibody drug conjugate as shown in formula (I):

[0007]

[0008]

[0009] in,

[0010] Ab is the targeting group;

[0011] Aa is an amino acid fragment;

[0012] L 1 To connect Aa and D 1 The linker of

[0013] L 2 To connect Aa and D 2 The linker of

[0014] D 1 It is a cytotoxic drug fragment;

[0015] D 2 It is a TLR (Toll-like receptor) agonist fragment;

[0016] m is selected from 1 to 5;

[0017] M is selected from the following structures:

[0018]

[0019] The 1 position of M is connected to Ab, and the 2 position is connected to Aa;

[0020] A is unsubstituted or substituted with one or more R 1 A substituted 3-20 membered heterocyclic subunit, wherein the heterocyclic subunit contains at least one nitrogen atom;

[0021] R 1 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6Haloalkyl and oxo;

[0022] a are each independently selected from 1-6 (e.g., 1, 2, 3, 4, 5 or 6);

[0023] b are each independently selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0024] c is each independently selected from 0-6 (eg, 0, 1, 2, 3, 4, 5 or 6).

[0025] In some embodiments, A is optionally substituted with one or more R 1 substituted 5-6 membered heterocyclic subunit or optionally substituted by one or more R 1 Substituted 18-20 membered heterocyclic subunits.

[0026] In some embodiments, the heterocyclic ring contains 1-2 nitrogen atoms.

[0027] Optionally, the heterocyclic ring further contains 1, 2, 3 or 4 other kinds of heteroatoms (eg, oxygen atoms or sulfur atoms).

[0028] In some embodiments, the heterocycle is selected from piperidine, piperazine, 1,4,10,13-tetraoxo-7,16-diaza-cyclooctadecane.

[0029] In some embodiments, A is selected from:

[0030]

[0031] In some embodiments, the 2' position is close to the 1 position in M.

[0032] In some embodiments, a is 1. In some embodiments, a is 2.

[0033] In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 8.

[0034] In some embodiments, c is 1. In some embodiments, c is 2.

[0035] In some embodiments, M is selected from the following structures:

[0036]

[0037] wherein each b is independently selected from 1-10, and the 1 position of M is connected to Ab, and the 2 position of M is connected to Aa.

[0038] In some embodiments, M is selected from the following structures:

[0039]

[0040]

[0041] Among them, the 1 position of M is connected to Ab, and the 2 position is connected to Aa.

[0042] In some embodiments, Aa is selected from the following structures:

[0043]

[0044] Among them, position 3 is connected to M, and position 4 is connected to L 1 and L 2 One of them is connected, and position 5 is connected to L 1 and L 2 Preferably, the 3 position is connected to M and the 4 position is connected to L 1 Connected, 5 position and L 2 connected.

[0045] In some embodiments, Aa is selected from the following structures:

[0046] In some embodiments, L 1 The invention relates to a method for preparing a cleavable linker comprising: selecting a non-cleavable linker and a cleavable linker, wherein the cleavable linker can be cleaved by acid or by an enzyme present in a pathological environment, wherein the enzyme is selected from a protease, a phosphatase, a pyrophosphatase, a β-glucuronidase, a β-galactosidase and a sulfatase.

[0047] In some embodiments, L 1 for in:

[0048] L a is a covalent bond or is selected from C 1-6 Alkylene,

[0049] wherein each a is independently selected from 1-6 (e.g. 1, 2, 3, 4, 5 or 6), each b is independently selected from 1-10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g. 8), and each c is independently selected from 0-6 (e.g. 0, 1, 2, 3, 4, 5 or 6).

[0050] In some embodiments, L a Selected from covalent bonds,

[0051] L bis selected from the group consisting of a covalent bond, an amino acid fragment, a peptide fragment formed by two or more amino acids, wherein the amino acids are selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn;

[0052] In some embodiments, L b is a covalent bond or is selected from the following structures:

[0053]

[0054]

[0055] Among them, each R 2 Independently selected from H or C 1-6 alkyl;

[0056] In some embodiments, L b Selected from

[0057] Each R 2 Independently selected from H or C 1-3 alkyl.

[0058] In some embodiments, L c For covalent bonds, -NH-CH 2 - or selected from the following structures:

[0059]

[0060] In some embodiments, L c Selected from -NH-CH 2 -.

[0061] In some embodiments, L 1 Select from the following structures:

[0062]

[0063] wherein each b is independently selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), L 1 The 6th position is connected to Aa, and the 7th position is connected to D 1 In some embodiments, b is 8.

[0064] In some embodiments, L 1 for:

[0065]

[0066] Among them, L 1 The 6th position is connected to Aa, and the 7th position is connected to D1 connected.

[0067] In some embodiments, L 2 The invention relates to a method for preparing a cleavable linker comprising: selecting a non-cleavable linker and a cleavable linker, wherein the cleavable linker can be cleaved by acid or by an enzyme present in a pathological environment, wherein the enzyme is selected from a protease, a phosphatase, a pyrophosphatase, a β-glucuronidase, a β-galactosidase and a sulfatase.

[0068] In some embodiments, L 2 for in:

[0069] L d is a covalent bond or is selected from C 1-6 Alkylene,

[0070] wherein each a is independently selected from 1-6 (e.g., 1, 2, 3, 4, 5, or 6), each b is independently selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and each c is independently selected from 0-6 (e.g., 0, 1, 2, 3, 4, 5, or 6); in some embodiments, c is 1; in some embodiments, c is 2.

[0071] In some embodiments, L d Selected from

[0072] In some embodiments, L e Selected from a covalent bond, an amino acid fragment, a peptide fragment formed by two or more amino acids, wherein the amino acids are selected from Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn.

[0073] In some embodiments, L e is a covalent bond or is selected from the following structures:

[0074]

[0075] Among them, each R 2 Independently selected from H and C 1-6 alkyl.

[0076] In some embodiments, L e It is a covalent bond.

[0077] In some embodiments, L f Selected from covalent bonds, -NH-CH 2 - or the following structure:

[0078]

[0079] In some embodiments, L f It is a covalent bond.

[0080] In some embodiments, L 2 Select from the following structures:

[0081]

[0082] wherein each b is independently selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); L 2 The 8th position is connected to Aa, and the 9th position is connected to D 2 In some embodiments, b is 7; in some embodiments, b is 8; in some embodiments, b is 10.

[0083] In some embodiments, L 2 Selected from: Among them, L 2 The 8th position is connected to Aa, and the 9th position is connected to D 2 connected.

[0084] In some embodiments, L 2 Selected from: Among them, L 2 The 8th position is connected to Aa, and the 9th position is connected to D 2 connected.

[0085] In some embodiments, D 1 A fragment selected from cytotoxic drugs, wherein the cytotoxic drugs include but are not limited to tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, wherein the tubulin inhibitors include but are not limited to dolastatin, auristatin, maytansine, tubulysins and cryptomycins, wherein the DNA damaging agents include but are not limited to pyrrolobenzodiazepines. The topoisomerase inhibitors include, but are not limited to, camptothecin and its derivatives, epipodophyllotoxins, anthracyclines and anthraquinones.

[0086] In some embodiments, the microtubule protein inhibitor is selected from dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3 and DM4; the topoisomerase inhibitor is selected from camptothecin, SN-38, exitecan, topotecan, belotecan, rubitecan, diflutecan, 10-hydroxycamptothecin, 9-aminocamptothecin, Dxd, T030, doxorubicin, epirubicin and PNU-159682; the specific structures of the microtubule protein inhibitor and topoisomerase inhibitor are shown in the table below.

[0087]

[0088]

[0089] In some embodiments, D 1 Select from the following structures:

[0090]

[0091] In some embodiments, D 1 Select from the following structures:

[0092]

[0093] In some embodiments, D 2 It is a TLR agonist fragment, wherein the TLR agonist is selected from a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist and a TLR9 agonist.

[0094] In some embodiments, D 2 It is a TLR agonist fragment, wherein the TLR agonist is selected from a TLR7 agonist, a TLR8 agonist and a TLR7 / 8 agonist.

[0095] In some embodiments, D 2 Select from the following structures:

[0096]

[0097] In some embodiments, D2 is selected from

[0098] In some embodiments, D2 is

[0099] In some embodiments, the Ab is a targeting group that targets a cell surface receptor or a tumor surface antigen.

[0100] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof; the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a murine antibody, a humanized antibody, a fully human antibody, and a fusion protein comprising the antigen-binding portion of an antibody; the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, a disulfide-linked Fv, and scFv.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-Her-2 antibody.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0103] (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0104] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 3 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 4 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

[0105] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0106] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 9 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 10 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

[0107] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0108] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; or

[0109] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0110] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises: VH shown in SEQ ID NO: 18 or a variant thereof, and / or, VL shown in SEQ ID NO: 19 or a variant thereof.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain of VH shown in SEQ ID NO: 18 or a variant thereof and a heavy chain constant region (CH) shown in SEQ ID NO: 20 or a variant thereof, and / or a light chain of VL shown in SEQ ID NO: 19 or a variant thereof and a light chain constant region (CL) shown in SEQ ID NO: 21 or a variant thereof.

[0113] In some embodiments, the anti-Her-2 antibody is trastuzumab.

[0114] In some embodiments, the amino acid sequence of the heavy chain of trastuzumab is shown in SEQ ID NO:1, and the amino acid sequence of the light chain of trastuzumab is shown in SEQ ID NO:2.

[0115] In some embodiments, the antibody drug conjugate has a structure selected from the group consisting of:

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Each m is selected from 1 to 5.

[0130] In some embodiments, the Ab is trastuzumab.

[0131] In a second aspect, the present invention provides a composition of an antibody drug conjugate, comprising the above-mentioned antibody drug conjugate, wherein the DAR value of the composition is 1.0-5.0, such as 1.0-2.0, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 2.0-3.0, 2.0-3.5, 2.0-4.0, 2.0-4.5, 2.0-5.0, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 4.0-4.5, 4.5-5.0 or 4.0-5.0.

[0132] In a third aspect, the present application provides a compound of formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0133]

[0134] in,

[0135] M 1 Select from the following structures:

[0136]

[0137] M a Pentafluorophenoxy

[0138] A is unsubstituted or substituted with one or more R 1 A substituted 3-20 membered heterocyclic subunit, wherein the heterocyclic subunit contains at least one nitrogen atom;

[0139] R 1 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo;

[0140] a are each independently selected from 1-6 (e.g., 1, 2, 3, 4, 5 or 6);

[0141] b are each independently selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0142] c are each independently selected from 0-6 (e.g., 0, 1, 2, 3, 4, 5 or 6);

[0143] Aa, L 1 , L 2 , D 1 and D 2 As defined above.

[0144] In some embodiments, A is optionally substituted with one or more R 1 substituted 5-6 membered heterocyclic subunit or optionally substituted by one or more R 1 Substituted 18-20 membered heterocyclic subunits.

[0145] In some embodiments, the heterocyclic ring contains 1-2 nitrogen atoms.

[0146] Optionally, the heterocyclic ring further contains 1, 2, 3 or 4 other kinds of heteroatoms (eg, oxygen atoms or sulfur atoms).

[0147] In some embodiments, the heterocycle is selected from piperidine, piperazine, 1,4,10,13-tetraoxo-7,16-diaza-cyclooctadecane.

[0148] In some embodiments, A is selected from:

[0149]

[0150] In some embodiments, the 2' position is near the M in Ma or the position of the 2,4-divinyl-1,3,5-triazine ring.

[0151] In some embodiments, a is 1. In some embodiments, a is 2.

[0152] In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 8.

[0153] In some embodiments, c is 1. In some embodiments, c is 2.

[0154] In some embodiments, M 1 Select from the following structures:

[0155] In some embodiments, M 1 Select from the following structures:

[0156]

[0157] In some embodiments, the compound has a structure selected from:

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] In a fourth aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate of the present invention, a composition of an antibody-drug conjugate of the present invention, a compound of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, and optionally one or more pharmaceutical excipients.

[0173] In a fifth aspect, the present application provides use of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof in the preparation of an antibody-drug conjugate (e.g., the antibody-drug conjugate of the present invention).

[0174] In a sixth aspect, the present application provides the use of an antibody-drug conjugate of the present invention, a composition of an antibody-drug conjugate of the present invention, a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing cancer.

[0175] In the seventh aspect, the present application provides a method for treating and / or preventing cancer, comprising administering to a subject in need thereof a therapeutically and / or preventively effective amount of an antibody-drug conjugate of the present invention, a composition of the antibody-drug conjugate of the present invention, a compound of the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention.

[0176] In the sixth and seventh aspects, the cancer includes, but is not limited to, esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

[0177] In some embodiments, the cancer is selected from a HER2-positive cancer, such as HER2-positive gastric cancer, breast cancer, or non-small cell lung cancer.

[0178] In an eighth aspect, the present application relates to a method for preparing the antibody-drug conjugate described in the first aspect of the present application, the method comprising: conducting a coupling reaction between a reduced antibody or an antigen-binding fragment thereof and the compound described in the second aspect of the present application or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof to obtain the antibody-drug conjugate. The antibody or antigen-binding fragment thereof can be reduced by a thiol reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)).

[0179] definition

[0180] Unless otherwise defined below, the meanings of all technical terms and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the technology used herein is intended to refer to the technology generally understood in the art, including those changes in technology or replacement of equivalent technology that are obvious to those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0181] The terms "comprises," "comprising," "having," "containing," or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0182] The terms "conjugate" or "conjugate" are interchangeable and refer to a substance obtained by linking one or more heterologous molecules (heterologous molecule(s)) to an antibody, wherein the heterologous molecules include but are not limited to cytotoxic agents (cytotoxic agent), and the English name is ADC (antibody-drug conjugate).

[0183] The one or more heterologous molecules can be connected to the antibody via a linker. The linker can be connected to the antibody via various chemical bonds. For example, in some embodiments, the linker is connected by forming a thioether bond with the thiol group of the antibody. In some specific ADC molecular structures, -S- only represents the thioether bond formed by the linker and the thiol group of the antibody, and does not mean that -S- is part of the linker.

[0184] The structure of the antibody-drug conjugate of the present application can be represented by the general formula I, wherein m refers to the number of bioactive molecular fragments connected to each antibody molecule. During the preparation of the antibody-drug conjugate, each antibody molecule may be connected to a different number of bioactive molecular fragments. Therefore, in general, the composition of the antibody-drug conjugate is a mixture of antibody-drug conjugates with different drug-antibody coupling ratios. In practice, DAR is usually used to represent the average value of the number of drugs connected to the antibody.

[0185] As used herein, the term "antibody" is used in the broadest sense, including various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen binding activity. For example, an immunoglobulin molecule can be composed of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 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 3 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into highly variable regions (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of 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, or related research by AbM, Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272).Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0186] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or, Martin's related work (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272), which integrates part of the definitions of Kabat and Chothia, and was first used in the Oxford Molecular antibody modeling software (Martin AC R. Protein sequence and structure analysis of antibody variable domains [M] / / Antibody engineering. Springer, Berlin, Heidelberg, 2010: 33-51.). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to a person skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0187] The CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably identified by the Chothia numbering system.

[0188] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody, which includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds. For example, a polypeptide of a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0189] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". Wherein, "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N to C; and, when the full-length antibody is an IgE isotype, optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C. A "full-length light chain" or "light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N to C. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, such as human; or may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0190] Herein, the technology for obtaining antibodies can use conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) to obtain an antigen-binding fragment of the antibody (e.g., the above-mentioned antibody fragment) from a given antibody (e.g., an antibody provided by the present invention), and screen the antigen-binding fragment of the antibody for specificity in the same manner as for intact antibodies.

[0191] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0192] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0193] For example, monoclonal antibodies can be prepared as follows. First, mice or other suitable host animals are immunized with immunogens (adjuvants are added when necessary). The injection method of immunogens or adjuvants is usually subcutaneous multi-point injection or intraperitoneal injection. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances that can inhibit the growth of unfused, parent myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin and thymidine (HAT culture medium) to the culture medium will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have characteristics such as high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among them, myeloma cells are preferably mouse myeloma, such as MOP-21 or MC-11 mouse tumor derivatives (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md. USA). In addition, there are also studies reporting the use of human myeloma and human-mouse heteromyeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium of growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).For example, the affinity of monoclonal antibodies can be determined by the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107: 220 (1980). After determining the specificity, affinity and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned by the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture fluids can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also grow in animals in the form of ascites tumors. Using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be separated from cell culture fluid, ascites or serum.

[0194] Monoclonal antibodies can also be obtained through genetic engineering recombination technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. The obtained DNA molecules are inserted into expression vectors, and then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.

[0195] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. The purification of immunoglobulins can be referred to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0196] As used herein, the term "mouse antibody" refers to the fusion of B cells from immunized mice with myeloma cells, followed by screening of mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation and antibody purification. Or, after antigens invade the mouse body, B cells differentiate and proliferate to form plasma cells, which can produce secretory antibodies. Produced by specific antigen stimulation, the production of antibodies is due to the interaction of various immune cells caused by antigens invading the human body, causing B cells in lymphocytes to differentiate and proliferate to form plasma cells, which can produce secretory antibodies.

[0197] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0198] Humanized antibodies can retain the expected properties of non-human donor antibodies (e.g., mouse antibodies) and can effectively reduce the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects, and therefore are particularly advantageous. However, due to the matching problem between the CDR of the donor antibody and the FR of the acceptor antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response) are usually lower than those of non-human donor antibodies (e.g., mouse antibodies).

[0199] Therefore, although researchers in this field have conducted in-depth studies on antibody humanization and have made some progress (see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000)), the prior art does not provide detailed guidance on how to fully humanize a donor antibody so that the generated humanized antibody has the highest possible degree of humanization and can retain the expected properties of the donor antibody as much as possible. Technicians need to explore, investigate and modify specific donor antibodies and put in a lot of creative work to obtain humanized antibodies that have a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization) and retain the expected properties of the specific donor antibodies.

[0200] In the present invention, in order to make the humanized antibody retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to increase the activity of immune cells and / or the ability to enhance the immune response), the framework region (FR) in the humanized antibody of the present invention may contain both the amino acid residues of the human acceptor antibody and the amino acid residues of the corresponding non-human donor antibody.

[0201] The humanized antibodies of the present invention can be prepared according to the sequence of the mouse monoclonal antibody prepared above.DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques.

[0202] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of the above transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996, Nature Biotech 14: 309).

[0203] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction.

[0204] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, bioluminescence interferometry (e.g., ForteBio Octet method) can be used to measure the dissociation constant. In addition, surface plasmon resonance technology (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0205] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0206] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0207] As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, a tumor) in a subject.

[0208] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" may also refer to extending survival compared to the expected survival (if not receiving treatment).

[0209] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.

[0210] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0211] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes blood tumors, especially hematological malignancies.

[0212] As used herein, the term "alkyl" is defined as a straight or branched chain saturated aliphatic hydrocarbon group. For example, as used herein, the term "C 1-6 "Alkyl" refers to a straight or branched chain group having 1 to 6 carbon atoms (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl), which is optionally substituted with one or more (e.g. 1 to 3) suitable substituents such as halogen. The term "C 1-3 "Alkyl" refers to a straight or branched chain group having 1 to 3 carbon atoms (eg methyl, ethyl, n-propyl, isopropyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogen.

[0213] The term "heterocyclic group" refers to a divalent group obtained by losing two hydrogen atoms from a heterocycle.

[0214] As used herein, the term "aryl" or "aromatic ring" refers to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated π electron system. 6-10 Aryl" or "C 6-10 The term "aromatic ring" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl (ring) or naphthyl (ring). The aryl group is optionally substituted by 1 or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO 2 , C 1-6 alkyl, etc.) substituted.

[0215] The term "heterocycle" refers to a saturated or partially saturated non-aromatic cyclic structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 3-20 membered heterocycles, 5-6 membered heterocycles, 5-6 membered nitrogen-containing heterocycles, 5-6 membered oxygen-containing heterocycles, 18-20 membered nitrogen-containing heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, piperazine, 1,4,10,13-tetrahydro-7,16-diaza-cyclooctadecane, tetrahydropyran, and the like.

[0216] As used herein, the term "halogen" group is defined to include fluorine, chlorine, bromine, or iodine.

[0217] As used herein, the term "halo" refers to substitution with one or more (such as 1 to 3) the same or different halogen atoms.

[0218] As used herein, the term "haloalkyl" refers to an alkyl group substituted by one or more (such as 1 to 3) the same or different halogen atoms. 1-6 "Haloalkyl" refers to a haloalkyl group having 1 to 6 carbon atoms, such as -CF 3 , -C 2 F 5 , -CHF 2 、-CH 2 F, -CH 2 CF 3 、-CH 2 Cl or -CH 2 CH 2 CF 3 wait.

[0219] Optionally, hydrogen in the groups referred to in the present invention may be replaced by deuterium.

[0220] As used herein, the term "oxo" refers to substitution with =0.

[0221] The term "substituted" means that one or more (e.g., 1, 2, 3, or 4) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds.

[0222] If a substituent is described as being "optionally substituted with," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced or unsubstituted, individually and / or together, with independently selected substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced or unsubstituted, with independently selected substituents.

[0223] If a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0224] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, where reasonable.

[0225] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0226] When a bond to a substituent is shown to pass through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

[0227] The present invention also includes all pharmaceutically acceptable isotope-labeled compounds, which are identical to the compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g. 2 H. 3 H, deuterium D, tritium T); isotopes of carbon (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (e.g. 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and15 N); isotopes of oxygen (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (e.g. 32 P); and sulfur isotopes (e.g. 35 S). Certain isotopically-labeled compounds of the invention (e.g., those incorporating a radioactive isotope) are useful in drug and / or substrate tissue distribution studies (e.g., assays). 3 H) and carbon-14 (i.e. 14 C) are particularly useful for this purpose because of their ease of incorporation and ease of detection. 11 C. 18 F. 15 O and 13 N) substitution can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotope-labeled compounds of the present invention can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations by using appropriate isotope-labeled reagents instead of the non-labeled reagents previously used. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be isotopically substituted, for example, D 2 O, acetone-d 6 or DMSO-d 6 .

[0228] The term "stereoisomer" means an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., 1, 2, 3, or 4) asymmetric centers, it can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of the present application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0229] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

[0230] It should also be understood that certain compounds of the present invention may exist in free form for treatment, or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites or prodrugs, which, after being administered to a patient in need thereof, can directly or indirectly provide a compound of the present invention or a metabolite or residue thereof. Therefore, when referring to "compounds of the present invention" herein, the above-mentioned various derivative forms of the compounds are also intended to be covered.

[0231] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. A review of suitable salts is provided in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002) by Stahl and Wermuth. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0232] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound. The amount of polar solvent, especially water, may be present in a stoichiometric or non-stoichiometric ratio.

[0233] Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; AR Katritzky and AJ Boulton, Eds., Academic Press; and GWH Cheeseman and ES G Werstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0234] Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the present invention, including compounds prepared by contacting the compounds of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0235] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may have less pharmacological activity or no pharmacological activity themselves, and when administered to the body or thereon, can be converted into compounds of the present invention having the desired activity by, for example, hydrolytic cleavage. Usually such prodrugs will be functional group derivatives of the compounds, which are easily converted into the desired therapeutically active compounds in vivo. For additional information on the use of prodrugs, see "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (EB Roche ed., American Pharmaceutical Association). The prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain parts known to those skilled in the art as "pro-moiety" (e.g., "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).

[0236] The present invention also encompasses compounds of the present invention containing protecting groups. In any process of preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive groups or reactive groups on any related molecules, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P. ​​GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Using methods known in the art, the protecting groups can be removed at an appropriate subsequent stage.

[0237] In this document (specification and claims), the singular forms "a", "an" and "the" include their plural forms unless the context clearly indicates otherwise, for example, "a host cell" includes a plurality of such host cells. In addition, there is no corresponding grammatical rule for singular and plural in Chinese. The singular and plural of a noun should be determined according to the context or actual situation. Therefore, in the English translation, adding "one or more than one" before a noun is likely to be correct. DETAILED DESCRIPTION

[0238] In order to make the purpose and technical scheme of the present invention clearer, the embodiments of the present invention are described in detail below in conjunction with examples. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Those who do not specify specific conditions in the examples are all carried out according to the conditions recommended by normal conditions or manufacturers. Those who do not specify manufacturers for reagents or instruments used are all conventional products that can be obtained commercially.

[0239] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS). 1 The H NMR was measured using a JEOL Eclipse 400 nuclear magnetic spectrometer and the solvent was deuterated methanol (CD 3 OD), deuterated chloroform (CDCl 3 ) or hexadeuterated dimethyl sulfoxide (DMSO-d 6 ), the internal standard was tetramethylsilane (TMS), and the chemical shifts (δ) were given in parts per million (ppm).

[0240] The MS measuring instrument is Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0241] Preparation method for high performance liquid chromatography:

[0242] Instrument model: Agilent 1260, chromatographic column: Waters SunFire Prep C18 OBD (19mm×150mm×5.0μm); chromatographic column temperature: 25°C; flow rate: 20.0mL / min; detection wavelength: 214nm; elution gradient: (0min: 10% A, 90% B; 16.0min: 90% A, 10% B); mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid aqueous solution.

[0243] Aluminum plates (20×20 cm) produced by Merck were used for thin layer chromatography silica gel plates (TLC), and the specifications used for thin layer chromatography separation and purification were GF 254 (1 mm) produced in Yantai.

[0244] The reaction is monitored by thin layer chromatography (TLC) or LC-MS; the developing solvent systems used include: dichloromethane and methanol system, n-hexane and ethyl acetate system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound or adjusted by adding triethylamine.

[0245] Reverse phase column chromatography preparation method:

[0246] Preparation method A:

[0247] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reverse column (Spherical; 20-35μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0mL / min; detection wavelength: 220nm; mobile phase A: acetonitrile; mobile phase B: water;

[0248] Preparation method B:

[0249] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reverse column (Spherical; 20-35 μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid aqueous solution;

[0250] Preparation Method C:

[0251] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reversed phase column (Spherical; 20-35 μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% hydrochloric acid aqueous solution;

[0252] Preparation method D:

[0253] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reverse column (Spherical; 20-35 μm; 100A); chromatographic column temperature: 25 °C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.05% NH 4 HCO 3 Aqueous solution;

[0254] Preparation method E:

[0255] Instrument model: Biotage fast medium pressure preparative chromatography, chromatographic column: Agela C18 reverse column (Spherical; 20-35 μm; 100A); chromatographic column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: 0.02% formic acid aqueous solution;

[0256] The microwave reaction was carried out using a Biotage Initiator+ (400 W, RT-300° C.) microwave reactor.

[0257] Column chromatography generally uses 200-300 mesh silica gel as a carrier. The eluent system includes: dichloromethane and methanol system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

[0258] Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C to 35°C).

[0259] The reagents used in the present invention are purchased from Acros Organics, Aldrich Chemical Company, Teber Chemical and other companies.

[0260] In the general synthesis methods, examples, and intermediate synthesis examples, the meanings of the abbreviations are as follows.

[0261]

[0262]

[0263] The information of the sequences involved in the present invention is described in the following table:

[0264]

[0265]

[0266] 1. Compound Examples

[0267] Intermediate Preparation Example 1: Preparation of Compound Int1

[0268]

[0269] Step 1: Preparation of compound Int1-2

[0270] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (276.97 mg, 644.45 μmol), N,N-diisopropylethylamine (209.93 mg, 1610 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (296.84 mg, 773.34 μmol) and compound Int1-1 (604 mg, 644.45 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and stirred at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25%-35%) to obtain the title compound of this step (747.9 mg, yield: 95.6%).

[0271] MS m / z(ESI):1214.6[M+H] + .

[0272] Step 2: Preparation of Compound Int1-3

[0273] Compound Int1-2 (460 mg, 378.75 μmol) was dissolved in a mixed solvent of anhydrous dichloromethane (6 mL) and trifluoroacetic acid (3 mL) and stirred at 25° C. for 2 hours. The reaction solution was concentrated to obtain the title compound of this step (400 mg, yield: 91.2%).

[0274] MS m / z(ESI):1158.6[M+H] + .

[0275] Step 3: Preparation of Compound Int1-4

[0276] Compound Int1-3 (113.74 mg, 91.19 μmol), N,N-diisopropylethylamine (23.24 mg, 178.03 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (51.28 mg, 133.52 μmol) and VC-PABC-MMAE (95 mg, 84.56 μmol) were added to anhydrous N,N-dimethylformamide (4 mL) and stirred at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 55%-65%) to obtain the title compound of this step (170 mg, yield: 93.5%).

[0277] MS m / z(ESI):1132.7[M / 2+H] + .

[0278] Step 4: Preparation of compound Int1

[0279] Compound Int1-4 (170 mg, 75.09 μmol) and diethylamine (27.74 mg, 375.47 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25° C. for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=55%-65%) to obtain the title compound (110 mg, yield: 71.9%).

[0280] MS m / z(ESI):1021.6[M / 2+H] + .

[0281] Intermediate Preparation Example 2: Preparation of Compound Int2

[0282]

[0283] Step 1: Preparation of compound Int2-2

[0284] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (91.93 mg, 216.06 μmol), compound Int2-1 (148.2 mg, 180.05 μmol), N,N-diisopropylethylamine (46.54 mg, 360.11 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75.26 mg, 198.06 μmol) were added to N,N-dimethylformamide (2 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with N,N-dimethylformamide and purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (152 mg, yield: 68.6%).

[0285] MS m / z(ESI):1230.7[M+H] + .

[0286] Step 2: Preparation of compound Int2-3

[0287] Compound Int2-2 (133 mg, 108.08 μmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and stirred at 25° C. for 2 hours. The reaction solution was concentrated to obtain the crude product of the title compound, which was used directly in the next step.

[0288] MS m / z(ESI):1174.6[M+H] + .

[0289] Step 3: Preparation of compound Int2-4

[0290] Compound Int2-3 (47.5 mg, 40.44 μmol), VC-PABC-MMAE (54.52 mg, 48.53 μmol), N,N-diisopropylethylamine (10.45 mg, 80.89 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (16.91 mg, 44.49 μmol) were added to N,N-dimethylformamide (3 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with N,N-dimethylformamide and purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound of this step (57.8 mg, yield: 62.7%).

[0291] MS m / z(ESI):1140.7[M / 2+H] + .

[0292] Step 4: Preparation of compound Int2

[0293] Compound Int2-4 (57.8 mg, 25.35 μmol) was dissolved in N,N-dimethylformamide (2 mL), diethylamine (9.27 mg, 126.76 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 40%-50%) to obtain the title compound of this step (39.6 mg, yield: 75.9%).

[0294] MS m / z(ESI):1029.6[M / 2+H] + .

[0295] Intermediate Preparation Example 3: Preparation of Compound Int3

[0296]

[0297] Step 1: Preparation of compound Int3-2

[0298] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (170 mg, 398.75 μmol), compound Int3-1 (152 mg, 199.38 μmol), N,N-diisopropylethylamine (51.5 mg, 398.75 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (98.09 mg, 299.07 μmol) were added to N,N-dimethylformamide (2 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with N,N-dimethylformamide and purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (159 mg, yield: 70.2%).

[0299] MS m / z(ESI):1133.6[M+H] + .

[0300] Step 2: Preparation of compound Int3-3

[0301] Compound Int3-2 (140 mg, 117.3 μmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (2 mL) was added, and stirred at 25° C. for 2 hours. The reaction solution was concentrated to obtain the crude product of the title compound of this step, which was directly used in the next step.

[0302] MS m / z(ESI):1077.6[M+H] + .

[0303] Step 3: Preparation of compound Int3-4

[0304] Compound Int3-3 (126.4 mg, 117.34 μmol), VC-PABC-MMAE (158.1 mg, 140.8 μmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.77 mg, 140.8 μmol) and N,N-diisopropylethylamine (30.34 mg, 234.68 μmol) were added to tetrahydrofuran (4 mL) and dimethyl sulfoxide (1.6 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with N,N-dimethylformamide and purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (180 mg, yield: 70.3%).

[0305] MS m / z(ESI):1092.2[M / 2+H] + .

[0306] Step 4: Preparation of compound Int3

[0307] Compound Int3-4 (180 mg, 82.47 μmol) was dissolved in N,N-dimethylformamide (2 mL), diethylamine (21.78 mg, 297.80 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 40%-50%) to obtain the title compound of this step (150 mg, yield: 93.8%).

[0308] MS m / z(ESI):1961.2[M+H] + .

[0309] Intermediate Preparation Example 4: Preparation of Compound Int4

[0310]

[0311] Step 1: Preparation of compound Int4-1

[0312] Compound Int1-1 (190 mg, 235.42 μmol), (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (100.17 mg, 235.42 μmol), N,N-diisopropylethylamine (33.47 mg, 258.98 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (73.85 mg, 194.23 μmol) were added to anhydrous tetrahydrofuran (6 mL) and anhydrous dimethyl sulfoxide (2 mL) and stirred at 0°C for 1 hour. The reaction solution was concentrated to remove tetrahydrofuran and then directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (180 mg, yield: 62.9%).

[0313] MS m / z(ESI):1214.6[M+H] + .

[0314] Step 2: Preparation of compound Int4-2

[0315] Compound Int4-1 (133 mg, 109.51 mmol) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (4 mL, 4 M) and stirred at 25°C for 2 hours. After the reaction solution was concentrated to remove 1,4-dioxane, an appropriate amount of acetonitrile was added to dissolve it and purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 50%-55%) to obtain the title compound of this step (114 mg, yield: 89.9%).

[0316] MS m / z(ESI):1158.6[M+H]+ .

[0317] Step 3: Preparation of compound Int4-3

[0318] Compound Int4-2 (100 mg, 86.33 μmol), VC-PABC-MMMAE (96.98 mg, 86.33 μmol), N,N-diisopropylethylamine (33.47 mg, 258.98 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (73.85 mg, 194.23 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL) and stirred at 0°C for 1 hour. The reaction solution was concentrated to remove tetrahydrofuran and then directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (150 mg, yield: 76.8%).

[0319] MS m / z(ESI):1132.7[M / 2+H] + .

[0320] Step 4: Preparation of compound Int4

[0321] Compound Int4-3 (150 mg, 66.26 μmol) and diethylamine (48.3 mg, 660.3 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound of this step (130 mg, yield: 96.1%).

[0322] MS m / z(ESI):1021.6[M / 2+H] + .

[0323] Intermediate Preparation Example 5: Preparation of Compound Int5

[0324]

[0325] Step 1: Preparation of compound Int5-1

[0326] Compound Int1-1 (400 mg, 446.06 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (203.92 mg, 446.06 μmol), N,N-diisopropylethylamine (290.62 mg, 2.24 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (256.82 mg, 669.1 μmol) were added to N,N-dimethylformamide (6 mL) and stirred at 25°C for 2 hours. The reaction solution was concentrated to obtain the crude product of the title compound of this step, which was directly used in the next step.

[0327] MS m / z(ESI):1200.6[M+H] + .

[0328] Step 2: Preparation of compound Int5-2

[0329] Compound Int5-1 (360.00 mg, 179.9 μmol) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (5 mL, 4 M) and stirred at 25° C. for 2 hours. The reaction solution was concentrated to obtain the crude product of the title compound, which was used directly in the next step.

[0330] MS m / z(ESI):1144.6[M+H] + .

[0331] Step 3: Preparation of compound Int5-3

[0332] Compound Int5-2 (144 mg, 125.84 μmol), VC-PABC-MMAE (124.48 mg, 125.84 μmol), N,N-diisopropylethylamine (32.52 mg, 251.66 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (71.78 mg, 182.74 μmol) were added to N,N-dimethylformamide (6 mL) and stirred at 25°C for 2 hours. The reaction solution was diluted with N,N-dimethylformamide and purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound of this step (140 mg, yield: 54.4%).

[0333] MS m / z(ESI):1125.7[M / 2+H] + .

[0334] Step 4: Preparation of compound Int5

[0335] Compound Int5-3 (93.6 mg, 41.6 μmol) was dissolved in N,N-dimethylformamide (0.5 mL), and diethylamine (30.43 mg, 416.04 μmol) was added and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 40%-50%) to obtain the title compound (57 mg, yield: 67.5%)

[0336] MS m / z(ESI):1014.6[M / 2+H] + .

[0337] Intermediate Preparation Example 6: Preparation of Compound Int6

[0338]

[0339] Step 1: Preparation of compound Int6-1

[0340] (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (61.20 mg, 148.74 μmol), compound Int1-1 (100.04 mg, 123.95 μmol), N,N-diisopropylethylamine (32.04 mg, 247.91 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (70.66 mg, 185.93 μmol) were added to N,N-dimethylformamide (6 mL) and stirred at 25° C. for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=30%-40%) to obtain the title compound of this step (120 mg, yield: 80.6%).

[0341] MS m / z(ESI):1200.6[M+H] + .

[0342] Step 2: Preparation of compound Int6-2

[0343] Compound Int6-1 (120 mg, 99.96 μmol) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (4 mL, 4 M) and stirred at 25° C. for 2 hours. The reaction solution was concentrated to obtain the crude product of the title compound, which was used directly in the next step.

[0344] MS m / z(ESI):1144.6[M+H] + .

[0345] Step 3: Preparation of compound Int6-3

[0346] Compound Int6-2 (120.7 mg, 124.09 μmol), VC-PABC-MMAE (139.40 mg, 136.6 μmol), N,N-diisopropylethylamine (29.22 mg, 248.17 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (70.78 mg, 186.13 μmol) were added to N,N-dimethylformamide (3 mL) and stirred at 25° C. for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=30%-40%) to obtain the title compound of this step (206.8 mg, yield: 74.1%).

[0347] MS m / z(ESI):1125.7[M / 2+H] + .

[0348] Step 4: Preparation of compound Int6

[0349] Compound Int6-3 (117.5 mg, 45.96 μmol) was dissolved in N,N-dimethylformamide (2 mL), diethylamine (35.38 mg, 149.6 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 40%-45%) to obtain the title compound (80.5 mg, yield: 96.6%)

[0350] MS m / z(ESI):1014.6[M / 2+H] + .

[0351] Intermediate Preparation Example 7: Preparation of Compound Int7

[0352]

[0353] Step 1: Preparation of compound Int7-2

[0354] Compound Int1-3 (75.54 mg, 61.78 μmol), N,N-diisopropylethylamine (15.97 mg, 123.56 μmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30.54 mg, 80.32 μmol) and compound Int7-1 (50 mg, 67.96 μmol) were added to anhydrous N,N-dimethylformamide (3 mL) and stirred at 0°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25%-35%) to obtain the title compound of this step (85 mg, yield: 77.2%).

[0355] MS m / z(ESI):1876.9[M+H] + .

[0356] Step 2: Preparation of compound Int7

[0357] Compound Int7-2 (85 mg, 45.3 μmol) and diethylamine (15.9 mg, 215.2 μmol) were added to N,N-dimethylformamide (2 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 45%-55%) to obtain the title compound (70 mg, yield: 98.3%).

[0358] MS m / z(ESI):1653.8[M+H] + .

[0359] Intermediate Preparation Example 8: Preparation of Compound Int8

[0360]

[0361] Step 1: Preparation of compound Int8-1

[0362] Compound Int2-3 (76 mg, 64.71 μmol), compound Int7-1 (57.13 mg, 77.65 μmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (27.05 mg, 71.18 μmol) and N,N-diisopropylethylamine (16.73 mg, 129.42 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 30%-35%) to obtain the title compound of this step (95 mg, yield: 77.6%).

[0363] MS m / z(ESI):947.0[M / 2+H] + .

[0364] Step 2: Preparation of compound Int8

[0365] Compound Int8-1 (105 mg, 52.71 μmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (19.27 mg, 263.55 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 40%-50%) to obtain the title compound (71.58 mg, yield: 81.3%).

[0366] MS m / z(ESI):1669.8[M+H] + .

[0367] Intermediate Preparation Example 9: Preparation of Compound Int9

[0368]

[0369] Step 1: Preparation of compound Int9-1

[0370] Compound Int3-3 (50.35 mg, 46.74 μmol), compound Int7-1 (37.83 mg, 51.41 μmol), N,N-diisopropylethylamine (12.08 mg, 93.48 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.33 mg, 56.09 μmol) were added to anhydrous tetrahydrofuran (2 mL) and anhydrous dimethyl sulfoxide (1 mL) in sequence, and stirred at 0°C for 1 hour. After the reaction solution was concentrated to remove tetrahydrofuran, it was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-35%) to obtain the title compound of this step (80 mg, yield: 95.4%).

[0371] MS m / z(ESI):898.0[M / 2+H] + .

[0372] Step 2: Preparation of compound Int9

[0373] Compound Int9-1 (76 mg, 42.34 μmol) and diethylamine (15.64 mg, 211.7 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25° C. for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A%=25%-30%) to obtain the title compound (50 mg, yield: 75.1%).

[0374] MS m / z(ESI):1572.8[M+H] + .

[0375] Intermediate Preparation Example 10: Preparation of Compound Int10

[0376]

[0377] Step 1: Preparation of compound Int10-1

[0378] Under ice bath, tert-butyl 1-hydroxy-3,6,9,12,15,18,21,24,27,30-decaoxatriacontanoate (600 mg, 1.01 mmol), 2,6-lutidine (219.16 mg, 2.02 mmol) and trifluoromethanesulfonic anhydride (346.07 mg, 1.21 mmol) were added to dichloromethane (20 mL), stirred at 0°C for 2 hours, and the reaction solution was directly used for the next step.

[0379] MS m / z(ESI):719.3[M+H] + .

[0380] Step 2: Preparation of compound Int10-2

[0381] Under ice bath, (tert-butyloxycarbonyl)-L-homoserine methyl ester (275.36 mg, 1.17 mmol) and N,N-diisopropylethylamine (203.04 mg, 1.56 mmol) were added to the reaction solution of compound Int10-1 (700 mg, 779.11 μmol), stirred at 0°C for 1 hour, and naturally warmed to room temperature and stirred for 4 hours. The reaction was quenched with ice water and concentrated, and the obtained crude product was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-45%) to obtain the title compound of this step (185 mg, yield: 22.5%).

[0382] MS m / z(ESI):802.5[M+H] + .

[0383] Step 3: Preparation of compound Int10-3

[0384] Compound Int10-2 (185 mg, 207.62 μmol) was added to a solution of hydrogen chloride in 1,4-dioxane (2 mL, 4 M) and dichloromethane (5 mL), stirred at room temperature for 5 hours, and the reaction solution was concentrated to obtain the title compound of this step (142 mg, yield: 95.3%).

[0385] MS m / z(ESI):646.4[M+H] + .

[0386] Step 4: Preparation of compound Int10-4

[0387] Compound Int10-3 (142 mg, 197.91 μmol), triethylamine (60.69 mg, 593.74 μmol) and di-tert-butyl dicarbonate (77.73 mg, 356.25 μmol) were added to 1,4-dioxane (2 mL) in sequence and stirred at room temperature for 2 hours. The reaction solution was concentrated and the crude product was purified by reverse phase column chromatography (Preparation method D, elution gradient: A% = 18%-25%) to obtain the title compound of this step (121 mg, yield: 73.8%).

[0388] MS m / z(ESI):746.4[M+H] + .

[0389] Step 5: Preparation of compound Int10-6

[0390] Compound Int10-4 (115 mg, 138.77 μmol), compound Int10-5 (79.12 mg, 166.52 μmol), N,N-diisopropylethylamine (45.20 mg, 346.92 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (79.90 mg, 208.15 μmol) were added to N,N dimethylformamide (3 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method C, elution gradient: A% = 20%-25%) to obtain the title compound of this step (126 mg, yield: 70.7%).

[0391] MS m / z(ESI):1155.6[M+H] + .

[0392] Step 6: Preparation of compound Int10-7

[0393] Compound Int10-6 (126 mg, 98.14 μmol) was dissolved in N,N-dimethylformamide (1 mL), and the pH was adjusted to 9-10 with an aqueous solution of lithium hydroxide (1 mol / L), and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method C, elution gradient: A% = 20%-25%) to obtain the title compound of this step (90 mg, yield: 72.3%).

[0394] MS m / z(ESI):1141.6[M+H] + .

[0395] Step 7: Preparation of compound Int10-8

[0396] Compound Int10-7 (65 mg, 51.25 μmol), VC-PABC-MMAE (63.98 mg, 51.25 μmol), N,N-diisopropylethylamine (16.70 mg, 128.13 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (29.51 mg, 76.88 μmol) were added to N,N dimethylformamide (1.5 mL) and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 45%-48%) to obtain the title compound of this step (48 mg, yield: 37.5%).

[0397] MS m / z(ESI):1124.2[M / 2+H] + .

[0398] Step 8: Preparation of compound Int10

[0399] Compound Int10-8 (45 mg, 18.03 μmol) and zinc bromide (32.80 mg, 144.20 μmol) were added to dichloromethane (3 mL) and stirred at room temperature for 7 hours. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the title compound (35 mg, yield: 81.4%).

[0400] MS m / z(ESI):1074.2[M+H] + .

[0401] Intermediate Preparation Example 11: Preparation of Compound Int11

[0402]

[0403] Step 1: Preparation of compound Int11-1

[0404] 1-Hydroxy-3,6,9,12,15,18,21,24,27,30-decaoxatriacontanoic acid (170 mg, 320.39 μmol), compound Int10-5 (137 mg, 320.39 μmol), N,N-diisopropylethylamine (62.11 mg, 480.59 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (146.19 mg, 384.47 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL), and stirred at 25°C for 2 hours. After the reaction solution was concentrated to remove tetrahydrofuran, it was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 25%-30%) to obtain the title compound of this step (240 mg, yield: 79.7%).

[0405] MS m / z(ESI):940.5[M+H] + .

[0406] Step 2: Preparation of compound Int11-2

[0407] Compound Int11-1 (240 mg, 257.71 μmol) and triethylamine (38.75 mg, 379.07 μmol) were dissolved in dichloromethane (8 mL) at 0°C, p-toluenesulfonyl chloride (72.02 mg, 1.01 mmol) was added in batches, and stirred at 25°C for 12 hours. The reaction solution was washed with 1N hydrochloric acid, saturated sodium bicarbonate solution and saturated brine in sequence, and the crude product obtained by concentration of the organic phase was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 96 / 4 (v / v)) to obtain the title compound of this step (266 mg, yield: 96.2%).

[0408] MS m / z(ESI):1094.5[M+H] + .

[0409] Step 3: Preparation of compound Int11-3

[0410] (tert-Butyloxycarbonyl)-L-cysteine ​​ethyl ester (550 mg, 502.57 μmol), compound Int11-2 (187.95 mg, 753.86 μmol), N,N-diisopropylethylamine (129.9 mg, 1005.14 μmol) and potassium iodide (8.34 mg, 50.26 μmol) were added to anhydrous N,N-dimethylformamide (5 mL) and stirred at 80°C for 2.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (400 mg, yield: 67.9%).

[0411] MS m / z(ESI):1171.6[M+H] + .

[0412] Step 4: Preparation of compound Int11-4

[0413] Compound Int11-3 (400 mg, 341.44 μmol) was dissolved in a mixed solvent of methanol (4 mL) and water (1.5 mL), and lithium hydroxide monohydrate (71.7 mg, 1707.2 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound of this step (290 mg, yield: 74.3%).

[0414] MS m / z(ESI):1143.6[M+H] + .

[0415] Step 5: Preparation of compound Int11-5

[0416] Compound Int11-4 (240 mg, 208.57 μmol), VC-PABC-MMMAE (235.8 mg, 208.57 μmol), N,N-diisopropylethylamine (40.68 mg, 299.1 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (122 mg, 299.1 μmol) were added to anhydrous tetrahydrofuran (6 mL) and anhydrous dimethyl sulfoxide (2 mL) and stirred at 0°C for 1 hour. After the reaction solution was concentrated to remove tetrahydrofuran, it was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (350 mg, yield: 78.1%).

[0417] MS m / z(ESI):1125.2[M / 2+H] + .

[0418] Step 6: Preparation of compound Int11

[0419] Compound Int11-5 (340 mg, 143.62 μmol) and zinc bromide (130 mg, 574.52 μmol) were added to anhydrous dichloromethane (4 mL) and stirred vigorously at 25° C. for 4 hours. The crude product obtained by concentration of the organic phase was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 3 / 1 (v / v)) to obtain the title compound (280 mg, yield: 90.7%).

[0420] MS m / z(ESI):1075.1[M / 2+H] + .

[0421] Intermediate Preparation Example 12: Preparation of 2-((((4-(methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (Int12)

[0422]

[0423] Step 1: Preparation of methyl 1-(N-((2-(benzyloxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate

[0424] Under nitrogen protection at 0°C, chlorosulfonyl isocyanate (427.5 mg, 3.02 mmol) and benzyl 2-hydroxyacetate (456.3 mg, 2.75 mmol) were dissolved in anhydrous dichloromethane (10 mL). After stirring at 0°C for 1.5 hours, 4-piperidinyl carboxylate (471.79 mg, 3.30 mmol) and N,N-diisopropylethylamine (532.32 mg, 4.12 mmol) were added, and the reaction was slowly heated to 25°C and stirred for 2 hours. Appropriate amount of water and dichloromethane were added to the reaction solution for extraction, and the organic phases were combined and washed with brine. The crude product obtained by concentrating the organic phase was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 18 / 1 (v / v)) to obtain the title compound of this step (1.08 g, yield: 75.1%).

[0425] MS m / z(ESI):415.1[M+H] + .

[0426] Step 2: Preparation of 2-((((4-(methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid

[0427] Dissolve methyl 1-(N-((2-(benzyloxy)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (850 mg, 1.95 mmol) in methanol (10 mL), add 10% palladium on carbon (236.6 mg, 0.20 mol), replace the hydrogen with a hydrogen balloon, and stir at 25°C for 2 hours. Filter the reaction solution through celite pad, and concentrate the organic phase to obtain the title compound of this step (580 mg, yield: 91.8%).

[0428] MS m / z(ESI):325.1[M+H] + .

[0429] Intermediate Preparation Example 13: Preparation of 1-((4-(methoxycarbonyl)piperidine)-1-sulfonyl)-1-oxo-2,5,8,11-tetraoxatetradecane-14-oic acid (Int13)

[0430]

[0431] Step 1: Preparation of methyl 1-(N-(16,16-dimethyl-14-oxo-2,5,8,11,15-pentaoxaheptadecanoyl)sulfamoyl)piperidine-4-carboxylate

[0432] Chlorosulfonyl isocyanate (203.40 mg, 1.42 mmol) and 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionic acid (400 mg, 1.42 mmol) were added to anhydrous dichloromethane (10 mL) under ice-water bath conditions, stirred at 0°C for 30 minutes, and the reaction solution was added dropwise to an anhydrous dichloromethane (10 mL) solution of methyl 4-piperidinate (205.77 mg, 1.42 mmol) and triethylamine (145.42 mg, 1.42 mmol). The reaction temperature was controlled below 5°C during the addition process. After the addition was completed, the temperature was naturally raised to room temperature and stirred for 2 hours. The reaction was quenched with ice water, and the reaction solution was concentrated to obtain a crude product which was purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 38%-42%) to obtain the title compound of this step (630 mg, yield: 75.7%).

[0433] MS m / z(ESI):527.2[M+H] + .

[0434] Step 2: Preparation of 1-((4-(methoxycarbonyl)piperidine)-1-sulfonamido)-1-oxo-2,5,8,11-tetraoxatetradecane-14-oic acid

[0435] 1-(N-(16,16-dimethyl-14-oxo-2,5,8,11,15-pentaoxaheptadecanoyl)sulfamoyl)piperidine-4-carboxylic acid methyl ester (200 mg, 341.82 μmol) was added to a solution of hydrogen chloride in 1,4-dioxane (2 mL) and dichloromethane (6 mL) and stirred at room temperature for 2 hours. The reaction solution was concentrated and the crude product was purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 15%-25%) to obtain the title compound ((127 mg, yield: 71.1%)).

[0436] MS m / z(ESI):471.2[M+H] + .

[0437] 1 H NMR(400MHz,CD3OD)δ:4.27-4.25(m,2H),3.76-3.67(m,9H),3.64-3.59(m,8H ),3.05-2.97(m,2H),2.60-2.44(m,3H),1.99-196(m,2H),1.75-1.64(m,2H).

[0438] Intermediate Preparation Example 14: Preparation of 1-((4-(methoxycarbonyl)piperidine)-1-sulfonamido)-1-oxo-2,5,8,11,14,17,20,23,26-nonaoxaconicoside-29-oic acid (Int14)

[0439]

[0440] Step 1: Preparation of 1-(N-(31,31-dimethyl-29-oxo-2,5,8,11,14,17,20,23,26,30-decaoxatriadodecanoyl)sulfamoyl)piperidine-4-carboxylic acid methyl ester

[0441] Under nitrogen protection at 0°C, chlorosulfonyl isocyanate (205.93 mg, 1.45 mmol) and 1-hydroxy-3,6,9,12,15,18,21,24-octaoxacosane-27-acid tert-butyl ester (760 mg, 1.52 mmol) were dissolved in anhydrous dichloromethane (6 mL). After reacting at 0°C for 1.5 hours, 4-piperidinyl carboxylate (198.41 mg, 3.30 mmol) and N,N-diisopropylethylamine (358.17 mg, 2.77 mmol) were added, and the reaction was slowly heated to 25°C and stirred for 2 hours. An appropriate amount of water was added to quench the reaction, and the crude product obtained by concentration of the reaction solution was dissolved in methanol and directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound of this step (142.5 mg, yield: 77.1%).

[0442] MS m / z(ESI):747.4[M+H] + .

[0443] Step 2: Preparation of 1-((4-(methoxycarbonyl)piperidine)-1-sulfonamido)-1-oxo-2,5,8,11,14,17,20,23,26-nonaoxaconicoside-29-oic acid

[0444] 1-(N-(31,31-dimethyl-29-oxo-2,5,8,11,14,17,20,23,26,30-decaoxatriacontriadecanoyl)sulfamoyl)piperidine-4-carboxylic acid methyl ester (200 mg, 267.79 mmol) was dissolved in dichloromethane (4 mL), and a solution of hydrogen chloride in 1,4-dioxane (3 mL, 4 M) was added, and the mixture was stirred at 25°C for 12 hours. Saturated sodium bicarbonate solution was added to the reaction solution to adjust the pH to about 6, and the dichloromethane was removed by concentration. The crude product obtained by dissolving in an appropriate amount of acetonitrile was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound (142.5 mg, yield: 77.1%).

[0445] MS m / z(ESI):691.3[M+H] + .

[0446] Intermediate Preparation Example 15: Preparation of 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (Int15)

[0447]

[0448] Step 1: Preparation of tert-butyl 4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazine-1-carboxylate

[0449] Under ice bath conditions, piperazine-1-carboxylic acid tert-butyl ester (4.50 g, 24.1 mmol) in acetonitrile (50 mL) was slowly added dropwise to a solution of 2,4,6-trichloro-1,3,5-triazine (4.95 g, 26.84 mmol) and N,N-diisopropylethylamine (3.46 g, 26.8 mmol) in acetonitrile (50 mL), and reacted at 0°C for 30 minutes. The reaction solution was concentrated to obtain the crude product of the title compound of this step, which was directly used in the next step.

[0450] MS m / z(ESI):334.1[M+H] + .

[0451] Step 2: Preparation of tert-butyl 4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-carboxylate

[0452] tert-Butyl 4-(4,6-dichloro-1,3,5-triazine-2-yl)piperazine-1-carboxylate (8.07 g, 24.2 mmol), potassium vinyl trifluoroborate (11.3 g, 84.6 mmol), potassium carbonate (10.0 g, 72.5 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium(II) (1.77 g, 2.42 mmol) were added to 1,4-dioxane (100 mL) and water (10 mL), replaced with nitrogen three times, and reacted at 80 °C for 6 hours. After the reaction solution was cooled to room temperature, it was diluted with water (100 mL), extracted three times with ethyl acetate (100 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue which was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 88 / 12 (v / v)) to give the title compound of this step (4.75 g, yield: 61.9%).

[0453] MS m / z(ESI):318.2[M+H] + .

[0454] 1 H NMR (400 MHz, CDCl 3)δ6.73(dd,J=16.0,1.6Hz,2H),6.61(dd,J=16.0,8.0Hz,2H),5.78(dd,J=12.0,2.0Hz,2H),3.99-3.81(m,4H),3.56-3.43(m,4H),1.49(s,9H).

[0455] Step 3: Preparation of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine

[0456] Dissolve tert-butyl 4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-carboxylate (475 mg, 1.50 mmol) in anhydrous dichloromethane (5 mL), add trifluoroacetic acid (1.54 g, 13.5 mmol), and react at 25° C. for 4 hours. The reaction solution is concentrated to obtain the crude product of the title compound of this step.

[0457] MS m / z(ESI):218.1[M+H] + .

[0458] Step 4: Preparation of tert-butyl 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetate

[0459] At 0°C, tert-butyl 2-hydroxyacetate (47.9 mg, 0.36 mmol) and chlorosulfonyl isocyanate (51.3 mg, 0.36 mmol) were added to anhydrous dichloromethane (2 mL), replaced with nitrogen three times, reacted at 0°C for 1 hour, and then a solution of 2-(piperazine-1-yl)-4,6-divinyl-1,3,5-triazine (100 mg, 0.30 mmol) and N,N-diisopropylethylamine (214 mg, 6.03 mmol) in dichloromethane (1 mL) was added and reacted at 0°C for 1 hour. The residue obtained by concentrating the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (85.4 mg, yield: 62.1%).

[0460] MS m / z(ESI):455.2[M+H] + .

[0461] 1 H NMR (400MHz, CD 3OD)δ6.73(d,J=2.0Hz,1H),6.69(d,J=2.0Hz,1H),6.60(d,J=10.4Hz,1H),6.56(d,J=10.4Hz,1H),5.80(d,J =2.0Hz, 1H), 5.78 (d, J = 2.0Hz, 1H), 4.31 (s, 2H), 4.01 (d, J = 5.2Hz, 4H), 3.28 (d, J = 5.2Hz, 4H), 1.37 (s, 9H).

[0462] Step 5: Preparation of 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazine-1-yl)sulfonyl)carbamoyl)oxy)acetic acid

[0463] Tert-butyl 2-((((4-(4,6-divinyl-1,3,5-triazine-2-yl)piperazin-1-yl)sulfonyl)carbamoyl)oxy)acetate (85.4 mg, 0.19 mmol) was dissolved in anhydrous dichloromethane (1 mL), trifluoroacetic acid (69.2 mg, 0.61 mmol) was added, and the mixture was reacted at 25° C. for 4 hours. The reaction solution was concentrated to obtain a crude product of the title compound.

[0464] MS m / z(ESI):399.1[M+H] + .

[0465] Intermediate Preparation Example 16: Preparation of Compound Int16

[0466]

[0467] Compound Int16-1 (114 mg, 71.35 μmol) was dissolved in dichloromethane (4 mL) at 0°C, and trifluoroacetic acid (78.26 mg, 679.5 μmol) was added and stirred at 0°C for 1 hour. After the reaction solution was concentrated, it was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 50%-55%) to obtain the title compound (85.5 mg, yield: 94.9%).

[0468] MS m / z(ESI):1325.6[M+H] + .

[0469] Example 1-1: Preparation of Compound C-1

[0470]

[0471] Step 1: Preparation of compound C-1-1

[0472] Compound Int1 (114 mg, 55.84 μmol), compound Int12 (36.21 mg, 111.68 μmol), N,N-diisopropylethylamine (10.82 mg, 83.76 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25.48 mg, 67.01 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL), and stirred at 0°C for 1 hour. The reaction solution was concentrated to obtain the title compound of this step (110 mg, yield: 83.9%).

[0473] MS m / z(ESI):1174.7[M / 2+H] + .

[0474] Step 2: Preparation of compound C-1-2

[0475] Compound C-1-1 (110 mg, 46.85 μmol) was dissolved in a mixed solvent of methanol (3 mL) and water (1 mL), lithium hydroxide monohydrate (5.61 mg, 234.25 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (100 mg, yield: 91.5%).

[0476] MS m / z(ESI):1167.7[M / 2+H] + .

[0477] Step 3: Preparation of Compound C-1

[0478] Compound C-1-2 (100 mg, 42.58 μmol), pentafluorophenol (11.83 mg, 64.27 μmol) and dicyclohexylcarbodiimide (9.72 mg, 47.12 μmol) were dissolved in anhydrous dichloromethane (6 mL) and stirred at 25°C for 2 hours. The reaction solution was concentrated, acetonitrile was added to the obtained crude product, filtered, and the filtrate was purified by preparative high performance liquid chromatography to obtain the title compound (26.13 mg, yield: 23.2%).

[0479] MS m / z(ESI):1250.6[M / 2+H] + .

[0480] Example 1-2: Preparation of Compound C-3

[0481]

[0482] Step 1: Preparation of compound C-3-1

[0483] Compound Int1 (80 mg, 39.19 μmol), compound Int13 (18.44 mg, 39.19 μmol), N,N-diisopropylethylamine (12.76 mg, 97.96 μmol), 1-hydroxybenzotriazole (5.88 mg, 43.10 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.34 mg, 58.78 μmol) were added to N,N-dimethylformamide (1.5 mL), stirred at room temperature for 2 hours, and the reaction solution was directly used for the next step.

[0484] MS m / z(ESI):1247.7[M / 2+H] + .

[0485] Step 2: Preparation of compound C-3-2

[0486] The pH of the reaction solution of the first step compound C-3-1 was adjusted to 9-10 with an aqueous solution of lithium hydroxide (1 mol / L), and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-45%) to obtain the title compound of this step (65 mg, yield: 87.2%).

[0487] MS m / z(ESI):1240.7[M / 2+H] + .

[0488] Step 3: Preparation of compound C-3

[0489] Compound C-3-2 (30 mg, 12.10 μmol), pentafluorophenol (2.45 mg, 13.31 μmol) and N,N'-diisopropylcarbodiimide (2.31 mg, 18.15 μmol) were added to dichloromethane (1 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method E, elution gradient: A% = 45%-50%) to obtain the title compound (22.10 mg, yield: 61.1%).

[0490] MS m / z(ESI):1323.7[M / 2+H] + .

[0491] Example 1-3: Preparation of Compound C-4

[0492]

[0493] Step 1: Preparation of compound C-4-1

[0494] Compound Int2 (50 mg, 21.87 μmol), compound Int13 (17.15 mg, 32.81 μmol), N,N-diisopropylethylamine (8.55 mg, 65.61 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.59 mg, 32.81 μmol) were added to N,N-dimethylformamide (1.5 mL), stirred at room temperature for 2 hours, and the reaction solution was directly used for the next step.

[0495] MS m / z(ESI):1255.7[M / 2+H] + .

[0496] Step 2: Preparation of compound C-4-2

[0497] The pH of the reaction solution of the first step compound C-4-1 was adjusted to 9-10 with an aqueous solution of lithium hydroxide (1 mol / L) and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-45%) to obtain the title compound of this step (31 mg, yield: 76.0%).

[0498] MS m / z(ESI):1248.7[M / 2+H] + .

[0499] Step 3: Preparation of compound C-4

[0500] Compound C-4-2 (31 mg, 11.18 μmol), pentafluorophenol (2.45 mg, 13.31 μmol) and N,N'-diisopropylcarbodiimide (2.31 mg, 18.15 μmol) were added to dichloromethane (1 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method E, elution gradient: A% = 35%-40%) to obtain the title compound (13.5 mg, yield: 42.4%).

[0501] MS m / z(ESI):1331.7[M / 2+H] + .

[0502] Example 1-4: Synthesis of Compound C-5

[0503]

[0504] Step 1: Preparation of compound C-5-1

[0505] Compound Int3 (53.2 mg, 27.14 μmol) and compound Int13 (25.5 mg, 54.27 μmol) were dissolved in tetrahydrofuran (3 mL) and dimethyl sulfoxide (1 mL), and N,N-diisopropylethylamine (7.01 mg, 54.27 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15.5 mg, 40.71 μmol) were added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (58.5 mg, yield: 89.3%).

[0506] MS m / z(ESI):1207.2[M / 2+H] + .

[0507] Step 2: Preparation of compound C-5-2

[0508] Compound C-5-1 (58.5 mg, 24.24 μmol) was dissolved in anhydrous methanol (3 mL), and an aqueous solution of lithium hydroxide monohydrate (7.13 mg, 169.71 μmol) was added under ice bath, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (36 mg, yield: 61.9%).

[0509] MS m / z(ESI):1200.2[M / 2+H] + .

[0510] Step 5: Preparation of C-5

[0511] Compound C-5-2 (9.5 mg, 3.96 μmol) and pentafluorophenol (1.45 mg, 7.92 μmol) were added to dichloromethane (0.4 mL), and N,N'-diisopropylcarbodiimide (999.55 μg, 7.92 μmol) was added, and stirred at 25°C for 2 hours. Water was added to quench the reaction, and the dichloromethane was extracted. The organic phase was dried and concentrated to obtain the title compound C-5 (9.8 mg, yield: 87.0%).

[0512] MS m / z(ESI):1283.2[M / 2+H] + .

[0513] Example 1-5: Preparation of Compound C-6

[0514]

[0515] Step 1: Preparation of compound C-6-1

[0516] Compound Int1 (38 mg, 18.61 μmol), compound Int14 (25.72 mg, 37.23 μmol), N,N-diisopropylethylamine (3.61 mg, 27.92 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.61 mg, 27.92 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL) in sequence and stirred at 0°C for 1 hour. The reaction solution was concentrated to obtain the title compound of this step (45 mg, yield: 89.1%).

[0517] MS m / z(ESI):1357.8[M / 2+H] + .

[0518] Step 2: Preparation of compound C-6-2

[0519] Compound C-6-1 (45 mg, 16.58 μmol) was dissolved in a mixed solvent of methanol (2 mL) and water (1 mL), and lithium hydroxide monohydrate (53.48 mg, 82.89 μmol) was added and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (35 mg, yield: 78.3%).

[0520] MS m / z(ESI):1350.8[M / 2+H] + .

[0521] Step 3: Preparation of compound C-6

[0522] Compound C-6-2 (27.55 mg, 10.20 μmol), pentafluorophenol (2.06 mg, 11.22 μmol) and dicyclohexylcarbodiimide (2.34 mg, 11.32 μmol) were dissolved in anhydrous dichloromethane (4 mL) and stirred at 25°C for 2 hours. The reaction solution was concentrated, acetonitrile was added to the obtained crude product, filtered, and the filtrate was purified by preparative high performance liquid chromatography to obtain the title compound (4.78 mg, yield: 16.3%).

[0523] MS m / z(ESI):1434.7[M / 2+H] + .

[0524] Example 1-6: Preparation of Compound C-9

[0525]

[0526] Step 1: Preparation of compound C-9-1

[0527] Compound Int4 (114 mg, 55.84 μmol), compound Int12 (36.21 mg, 111.68 μmol), N,N-diisopropylethylamine (10.82 mg, 83.76 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25.48 mg, 67.01 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL), and stirred at 0°C for 1 hour. The reaction solution was concentrated to obtain the title compound of this step (120 mg, yield: 91.5%).

[0528] MS m / z(ESI):1174.7[M / 2+H] + .

[0529] Step 2: Preparation of compound C-9-2

[0530] Compound C-9-1 (120 mg, 51.11 μmol) was dissolved in a mixed solvent of methanol (3 mL) and water (1 mL), lithium hydroxide monohydrate (6.18 mg, 255.55 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (105.6 mg, yield: 88.5%).

[0531] MS m / z(ESI):1167.7[M / 2+H] + .

[0532] Step 3: Preparation of compound C-9

[0533] Compound C-9-2 (100 mg, 42.58 μmol), pentafluorophenol (11.83 mg, 64.27 μmol) and dicyclohexylcarbodiimide (9.72 mg, 47.12 μmol) were dissolved in anhydrous dichloromethane (4 mL) and stirred at 25°C for 2 hours. The reaction solution was concentrated, acetonitrile was added to the obtained crude product, filtered, and the filtrate was purified by preparative high performance liquid chromatography to obtain the title compound (8.18 mg, yield: 7.6%).

[0534] MS m / z(ESI):1250.6[M / 2+H] + .

[0535] Example 1-7: Preparation of Compound C-11

[0536]

[0537] Step 1: Preparation of compound C-11-1

[0538] Compound Int5 (54 mg, 26.63 μmol) and compound Int12 (17.27 mg, 53.27 μmol) were dissolved in dimethyl sulfoxide (0.5 mL) and tetrahydrofuran (1.5 mL), and N,N-diisopropylethylamine (6.887 mg, 53.27 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15.2 mg, 39.95 μmol) were added, and stirred at 25°C for 2 hours. The reaction solution was concentrated and the residue was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 50%-60%) to obtain the title compound of this step (56.7 mg, yield: 91.2%).

[0539] MS m / z(ESI):1167.6[M / 2+H] + .

[0540] Step 2: Preparation of compound C-11-2

[0541] Compound C-11-1 (63 mg, 24.29 μmol) was dissolved in anhydrous methanol (3 mL), and an aqueous solution of lithium hydroxide monohydrate (10.31 mg, 242.95 μmol) was added under ice bath, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (60 mg, yield: 90.5%).

[0542] MS m / z(ESI):1160.7[M / 2+H] + .

[0543] Step 3: Preparation of Compound C-11

[0544] Compound C-11-2 (60 mg, 25.61 μmol) and pentafluorophenol (7.14 mg, 38.41 μmol) were dissolved in dichloromethane (2 mL), N, N-dicyclohexylcarbodiimide (5.87 mg, 28.17 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound (8.89 mg, yield: 13.0%).

[0545] MS m / z(ESI):1243.6[M / 2+H] + .

[0546] Example 1-8: Preparation of Compound C-13

[0547]

[0548] Step 1: Preparation of compound C-13-1

[0549] Compound Int5 (40 mg, 17.76 μmol), compound Int13 (13.92 mg, 26.63 μmol), N,N-diisopropylethylamine (6.94 mg, 53.27 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.22 mg, 26.63 μmol) were added to N,N-dimethylformamide (1.5 mL), stirred at room temperature for 2 hours, and the reaction solution was directly used for the next step.

[0550] MS m / z(ESI):1240.7[M / 2+H] + .

[0551] Step 2: Preparation of compound C-13-2

[0552] The pH of the reaction solution of the first step compound C-13-1 was adjusted to 9-10 with an aqueous solution of lithium hydroxide (1 mol / L) and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-45%) to obtain the title compound of this step (37 mg, yield: 90.8%).

[0553] MS m / z(ESI):1233.7[M / 2+H] + .

[0554] Step 3: Preparation of Compound C-13

[0555] Compound C-13-2 (30 mg, 10.95 μmol), pentafluorophenol (3.40 mg, 16.42 μmol) and N,N'-diisopropylcarbodiimide (2.51 mg, 19.71 μmol) were added to dichloromethane (1 mL) and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method E, elution gradient: A% = 40%-50%) to obtain the title compound (19.05 mg, yield: 54.6%).

[0556] MS m / z(ESI):1316.7[M / 2+H] + .

[0557] Example 1-9: Preparation of Compound C-15

[0558]

[0559] Step 1: Preparation of compound C-15-1

[0560] Compound Int5 (40 mg, 17.76 μmol), compound Int14 (13.92 mg, 26.63 μmol), N,N-diisopropylethylamine (5.78 mg, 44.39 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (10.22 mg, 26.63 μmol) were added to N,N-dimethylformamide (1.5 mL), stirred at room temperature for 2 hours, and the reaction solution was directly used for the next step.

[0561] MS m / z(ESI):1350.8[M / 2+H] + .

[0562] Step 2: Preparation of compound C-15-2

[0563] The pH of the reaction solution of the first step compound C-15-1 was adjusted to 9-10 with an aqueous solution of lithium hydroxide (1 mol / L) and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 38%-42%) to obtain the title compound of this step (23 mg, yield: 69.4%).

[0564] MS m / z(ESI):1343.7[M / 2+H] + .

[0565] Step 3: Preparation of Compound C-15

[0566] Compound C-15-2 (23 mg, 7.71 μmol), pentafluorophenol (2.15 mg, 11.56 μmol) and N,N'-diisopropylcarbodiimide (1.96 mg, 15.41 μmol) were added to dichloromethane (1 mL) in sequence and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method E, elution gradient: A% = 35%-45%) to obtain the title compound (13.1 mg, yield: 55.7%).

[0567] MS m / z(ESI):1426.7[M / 2+H] + .

[0568] Example 1-10: Preparation of Compound C-18

[0569]

[0570] Step 1: Preparation of compound C-18-1

[0571] Compound Int6 (76 mg, 37.48 μmol) and compound Int12 (36.47 mg, 112.45 μmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (10.01 mg, 74.97 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.28 mg, 56.23 μmol) were added, and stirred at 25°C for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (63 mg, yield: 72.0%).

[0572] MS m / z(ESI):1167.7[M / 2+H] + .

[0573] Step 2: Preparation of compound C-18-2

[0574] Compound C-18-1 (63 mg, 26.99 μmol) was dissolved in anhydrous methanol (3 mL), and an aqueous solution of lithium hydroxide monohydrate (11.34 mg, 269.94 μmol) was added under ice bath, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 30%-40%) to obtain the title compound of this step (60 mg, yield: 95.8%).

[0575] MS m / z(ESI):1160.7[M / 2+H] + .

[0576] Step 3: Preparation of Compound C-18

[0577] Compound C-18-2 (60 mg, 25.86 μmol) and pentafluorophenol (7.14 mg, 38.80 μmol) were dissolved in dichloromethane (2 mL), N,N-dicyclohexylcarbodiimide (5.87 mg, 28.45 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-50%) to obtain the title compound (14.71 mg, yield: 22.5%).

[0578] MS m / z(ESI):1243.6[M / 2+H] + .

[0579] Example 1-11: Preparation of Compound C-20

[0580]

[0581] Step 1: Preparation of compound C-20-1

[0582] Compound Int10 (30 mg, 12.58 μmol), compound Int13 (9.86 mg, 18.87 μmol), N,N-diisopropylethylamine (4.10 mg, 31.44 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.24 mg, 18.87 μmol) were added to N,N dimethylformamide (1.5 mL) and stirred at room temperature for 1.5 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-48%) to obtain the title compound of this step (21 mg, yield: 57.8%).

[0583] MS m / z(ESI):1300.2[M / 2+H] + .

[0584] Step 2: Preparation of compound C-20-2

[0585] Compound C-20-1 (21 mg, 7.27 μmol) was dissolved in tetrahydrofuran (2 mL), and the pH was adjusted to 9-10 with an aqueous solution of lithium hydroxide (1 mol / L), and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40%-48%) to obtain the title compound of this step (13 mg, yield: 62.2%).

[0586] MS m / z(ESI):1293.2[M / 2+H] + .

[0587] Step 3: Preparation of Compound C-20

[0588] Compound C-20-2 (13 mg, 4.53 μmol), pentafluorophenol (1.01 mg, 5.43 μmol) and N,N'-diisopropylcarbodiimide (864.03 μg, 6.79 μmol) were added to N,N-dimethylformamide (1 mL) in sequence and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method E, elution gradient: A% = 35%-45%) to obtain the title compound (2.1 mg, yield: 14.9%).

[0589] MS m / z(ESI):1376.2[M / 2+H] + .

[0590] Example 1-12: Preparation of Compound C-21

[0591]

[0592] Step 1: Preparation of compound C-21-1

[0593] Compound Int11 (200 mg, 93.07 μmol), compound Int13 (65.69 mg, 139.62 μmol), N,N-diisopropylethylamine (13.53 mg, 99.48 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (39.81 mg, 99.48 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL) in sequence and stirred at 0°C for 1 hour. The reaction solution was concentrated to obtain the title compound of this step (190 mg, yield: 99.9%).

[0594] MS m / z(ESI):1301.3[M / 2+H] + .

[0595] Step 2: Preparation of compound C-21-2

[0596] Compound C-21-1 (190 mg, 73.04 μmol) was dissolved in a mixed solvent of methanol (4 mL) and water (2 mL), and lithium hydroxide monohydrate (14.61 mg, 365.21 μmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound of this step (95 mg, yield: 50.3%).

[0597] MS m / z(ESI):1294.3[M / 2+H] + .

[0598] Step 3: Preparation of Compound C-21

[0599] Compound C-21-2 (95 mg, 36.72 μmol), pentafluorophenol (7.44 mg, 40.39 μmol) and dicyclohexylcarbodiimide (9.72 mg, 47.12 μmol) were dissolved in anhydrous dichloromethane (4 mL) in sequence and stirred at 25°C for 2 hours. The reaction solution was concentrated, acetonitrile was added to the obtained crude product, filtered, and the filtrate was purified by preparative high performance liquid chromatography to obtain the title compound (14 mg, yield: 13.8%).

[0600] MS m / z(ESI):1377.3[M / 2+H] + .

[0601] Example 1-13: Preparation of Compound C-23

[0602]

[0603] Compound Int7 (47.5 mg, 28.72 μmol) and compound Int15 (22.08 mg, 43.08 μmol) were dissolved in N,N-dimethylformamide (4 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.00 mg, 31.59 μmol) and N,N-diisopropylethylamine (7.42 mg, 57.44 μmol) were added, and stirred at 25°C for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 35%-40%) to obtain the title compound (11.4 mg, yield: 19.1%).

[0604] MS m / z(ESI):1017.8[M / 2+H] + .

[0605] Example 1-14: Preparation of Compound C-24

[0606]

[0607] Compound Int8 (68 mg, 40.72 μmol) and compound Int15 (19.46 mg, 48.86 μmol) were dissolved in N,N-dimethylformamide (4 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.57 mg, 48.86 μmol) and N,N-diisopropylethylamine (10.53 mg, 81.44 μmol) were added, and stirred at 25°C for 2 hours. The residue obtained by concentration of the reaction solution was purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-40%) to obtain the title compound (19.95 mg, yield: 23.4%).

[0608] MS m / z(ESI):684.3[M / 3+H] + .

[0609] Example 1-15: Preparation of Compound C-25

[0610]

[0611] Compound Int9 (38 mg, 24.16 μmol), compound Int15 (10.6 mg, 26.58 μmol), N,N-diisopropylethylamine (6.57 mg, 48.32 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.6 mg, 28.99 μmol) were added to anhydrous tetrahydrofuran (2 mL) and anhydrous dimethyl sulfoxide (1 mL) and stirred at 0°C for 2 hours. The reaction solution was concentrated to remove tetrahydrofuran and purified by preparative high performance liquid chromatography to obtain the title compound (14.14 mg, yield: 28.3%).

[0612] MS m / z(ESI):977.0[M / 2+H] + .

[0613] Example 1-16: Preparation of Compound C-33

[0614]

[0615] Step 1: Preparation of compound C-33-1

[0616] Compound Int1-3 (120 mg, 102.56 μmol), propargylamine (8.56 mg, 153.83 μmol), N,N-diisopropylethylamine (26.78 mg, 205.11 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (47.27 mg, 123.07 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL), stirred at 0°C for 2 hours, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (94.54 mg, 246.14 μmol) was added to quench the excess propargylamine. The reaction solution was concentrated to remove tetrahydrofuran and then directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A%=30%-35%) to obtain the title compound of this step (95 mg, yield: 77.5%).

[0617] MS m / z(ESI):1195.6[M+H] + .

[0618] Step 2: Preparation of compound C-33-2

[0619] Compound C-33-1 (95 mg, 79.47 μmol) and diethylamine (29.35 mg, 397.34 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 25%-30%) to obtain the title compound of this step (63 mg, yield: 81.5%).

[0620] MS m / z(ESI):973.5[M+H] + .

[0621] Step 3: Preparation of compound C-33-3

[0622] Compound C-33-2 (50 mg, 50.86 μmol), compound Int15 (20.47 mg, 50.86 μmol), N,N-diisopropylethylamine (13.28 mg, 101.72 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.44 mg, 61.03 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL) and stirred at 0°C for 2 hours. The reaction solution was concentrated to remove tetrahydrofuran and then purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 25%-30%) to obtain the title compound of this step (60 mg, yield: 87.1%).

[0623] MS m / z(ESI):1353.7[M+H] + .

[0624] Step 4: Preparation of compound C-33

[0625] Compound C-33-3 (33.25 mg, 24.56 μmol) and compound Int16 (32.56 mg, 24.56 μmol) were dissolved in dimethyl sulfoxide (2 mL) at 0°C, and sodium L-ascorbate (9.73 mg, 49.13 μmol), copper sulfate pentahydrate (12.27 mg, 49.13 μmol) and water (0.5 mL) were added, and stirred at 0°C for 2 hours. The reaction solution was filtered and directly purified by preparative high performance liquid chromatography to obtain the title compound (10 mg, yield: 15.2%).

[0626] MS m / z(ESI):1340.1[M / 2+H] + .

[0627] Example 1-17: Preparation of Compound C-34

[0628]

[0629] Step 1: Preparation of compound C-34-1

[0630] Compound Int2-3 (150 mg, 126.44 μmol), propargylamine (10.55 mg, 189.66 μmol), N,N-diisopropylethylamine (33.01 mg, 252.89 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (63.13 mg, 164.38 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL), stirred at 0°C for 2 hours, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (126.26 mg, 328.76 μmol) was added to quench the excess propargylamine. The reaction solution was concentrated to remove tetrahydrofuran and then directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 40% - 50%) to obtain the title compound of this step (95 mg, yield: 77.5%).

[0631] MS m / z(ESI):1211.6[M+H] + .

[0632] Step 2: Preparation of compound C-34-2

[0633] Compound C-34-1 (114 mg, 94.1 μmol) and diethylamine (34.76 mg, 470.49 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 25%-30%) to obtain the title compound of this step (55 mg, yield: 59.1%).

[0634] MS m / z(ESI):989.7[M+H] + .

[0635] Step 3: Preparation of compound C-34-3

[0636] Compound C-34-2 (49.4 mg, 49.94 μmol), compound Int15 (19.89 mg, 49.94 μmol), N,N-diisopropylethylamine (13.04 mg, 99.87 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.4 mg, 64.92 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL) and stirred at 0°C for 2 hours. The reaction solution was concentrated to remove tetrahydrofuran and then purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 30%-35%) to obtain the title compound of this step (35 mg, yield: 51.2%).

[0637] MS m / z(ESI):1369.6[M+H] + .

[0638] Step 4: Preparation of compound C-34

[0639] Compound C-34-3 (33.25 mg, 24.28 μmol) and compound Int16 (32.56 mg, 24.28 μmol) were dissolved in dimethyl sulfoxide (2 mL) at 0°C, and sodium L-ascorbate (9.62 mg, 48.55 μmol), copper sulfate pentahydrate (12.12 mg, 48.55 μmol) and water (0.5 mL) were added, and stirred at 0°C for 2 hours. The reaction solution was filtered and directly purified by preparative high performance liquid chromatography to obtain the title compound (13 mg, yield: 19.9%).

[0640] MS m / z(ESI):1348.2[M / 2+H] + .

[0641] Example 1-18: Preparation of Compound C-38

[0642]

[0643] Step 1: Preparation of compound C-38-1

[0644] 26-Amino-3,6,9,12,15,18,21,24-octaoxahexacosacan-1-ol (427 mg, 948.97 μmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pent-4-ynoic acid (322 mg, 960.19 μmol), N,N-diisopropylethylamine (496 mg, 3.84 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (547 mg, 1.44 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 35%-45%) to obtain the title compound of this step (510 mg, yield: 72.7%).

[0645] MS m / z(ESI):731.5[M+H] + .

[0646] Step 2: Preparation of compound C-38-2

[0647] Compound C-38-1 (510 mg, 697.83 μmol) and Dess-Martin periodinane (384 mg, 905.36 μmol) were added to anhydrous dichloromethane (20 mL) and stirred at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 92 / 8 (v / v)) to obtain the title compound of this step (520 mg, yield: 98.1%).

[0648] MS m / z(ESI):729.5[M+H] + .

[0649] Step 3: Preparation of compound C-38-3

[0650] Compound C-38-2 (520 mg, 713.48 μmol) and compound Int10-5 (305 mg, 713.28 μmol) were added to anhydrous methanol (10 mL), and sodium cyanoborohydride (212 mg, 3.37 mmol) was added, and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 20%-40%) to obtain the title compound of this step (250 mg, yield: 30.7%).

[0651] MS m / z(ESI):1140.6[M+H] + .

[0652] Step 4: Preparation of compound C-38-4

[0653] Compound C-38-3 (250 mg, 219.22 μmol) and diethylamine (80 mg, 1.09 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method A, elution gradient: A% = 20%-40%) to obtain the title compound of this step (145 mg, yield: 72.0%).

[0654] MS m / z(ESI):918.6[M+H] + .

[0655] Step 5: Preparation of compound C-38-5

[0656] Compound C-38-4 (100 mg, 108.91 μmol), compound Int15 (62 mg, 115.63 μmol), N,N-diisopropylethylamine (43 mg, 332.71 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (62 mg, 163.06 μmol) were added to dimethyl sulfoxide (3 mL) and stirred at 25°C for 2 hours. The reaction solution was directly purified by reverse phase column chromatography (Preparation method B, elution gradient: A% = 20%-35%) to obtain the title compound of this step (90 mg, yield: 63.6%).

[0657] MS m / z(ESI):649.8[M / 2+H] + .

[0658] Step 6: Preparation of compound C-38

[0659] Compound C-38-5 (80 mg, 61.61 μmol), compound Int16 (74 mg, 55.83 μmol), sodium L-ascorbate (22 mg, 111.05 μmol), copper sulfate pentahydrate (28 mg, 112.14 μmol) were added to dimethyl sulfoxide (2 mL) and water (0.2 mL) and stirred at 25°C for 2 hours. The reaction solution was purified twice by preparative high performance liquid chromatography to obtain the title compound (7 mg, yield: 4.33%).

[0660] MS m / z(ESI):875.4[M / 3+H] + .

[0661] Example 1-19: Preparation of Compound C-44

[0662]

[0663] Compound Int1 (25.7 mg, 12.56 μmol), compound Int15 (7.5 mg, 18.85 μmol), N,N-diisopropylethylamine (3.25 mg, 25.13 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.26 mg, 13.82 μmol) were added to anhydrous tetrahydrofuran (2 mL) and anhydrous dimethyl sulfoxide (1 mL) and stirred at 25° C. for 2 hours. The reaction solution was concentrated to remove tetrahydrofuran and purified by preparative high performance liquid chromatography to obtain the title compound (11.4 mg, yield: 33.1%).

[0664] MS m / z(ESI):1211.6[M / 2+H] + .

[0665] Example 1-20: Preparation of Compound C-45

[0666]

[0667] Compound Int2 (28.0 mg, 13.61 μmol), compound Int15 (6.0 mg, 14.97 μmol), N,N-diisopropylethylamine (3.52 mg, 27.22 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.21 mg, 16.33 μmol) were added to anhydrous tetrahydrofuran (1.5 mL) and anhydrous dimethyl sulfoxide (0.5 mL), and stirred at 25° C. for 2 hours. The reaction solution was concentrated to remove tetrahydrofuran and purified by preparative high performance liquid chromatography to obtain the title compound (13.3 mg, yield: 39.4%).

[0668] MS m / z(ESI):1219.7[M / 2+H] + .

[0669] Example 1-21: Preparation of Compound C-46

[0670]

[0671] Compound Int3 (95 mg, 48.46 μmol), compound Int15 (23.2 mg, 58.15 μmol), N,N-diisopropylethylamine (12.53 mg, 96.92 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.95 mg, 63.00 μmol) were added to anhydrous tetrahydrofuran (3 mL) and anhydrous dimethyl sulfoxide (1 mL) and stirred at 25°C for 2 hours. The reaction solution was concentrated to remove tetrahydrofuran and purified by preparative high performance liquid chromatography to obtain the title compound (22.1 mg, yield: 17.6%).

[0672] MS m / z(ESI):1171.2[M / 2+H] + .

[0673] 2. Preparation of Antibody Drug Conjugates

[0674] The antibody-drug conjugate was prepared using Trastuzumab as the antibody. The amino acid sequences of the heavy chain and light chain of Trastuzumab are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0675] Example 2-1 Trastuzumab-ADC-23

[0676]

[0677] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-23 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.3mg / mL, 1.5mL) of Trastuzumab-ADC-23, which was stored at 4°C.

[0678] Example 2-2 Trastuzumab-ADC-24

[0679]

[0680] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-24 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.3mg / mL, 1.5mL) of Trastuzumab-ADC-24, which was stored at 4°C.

[0681] Example 2-3 Trastuzumab-ADC-25

[0682]

[0683] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-25 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (2.8mg / mL, 1.5mL) of Trastuzumab-ADC-25, which was stored at 4°C.

[0684] Example 2-4 Trastuzumab-ADC-34

[0685]

[0686] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-34 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (6.4mg / mL, 1.5mL) of Trastuzumab-ADC-34, which was stored at 4°C.

[0687] Example 2-5 Trastuzumab-ADC-44

[0688]

[0689] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-44 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (7.8mg / mL, 1.5mL) of Trastuzumab-ADC-44, which was stored at 4°C.

[0690] Example 2-6 Trastuzumab-ADC-45

[0691]

[0692] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-45 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (5.0mg / mL, 1.5mL) of Trastuzumab-ADC-45, which was stored at 4°C.

[0693] Example 2-7 Trastuzumab-ADC-46

[0694]

[0695] The Trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.58 with 1M disodium hydrogen phosphate solution, and then the volume was fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a Trastuzumab monoclonal antibody buffer solution. 0.1M EDTA was added to the obtained Trastuzumab monoclonal antibody buffer solution, and TCEP (6eq, 10mM) was added at the same time, and the mixture was shaken and reacted at 25°C for 2 hours. A DMSO solution (10mM) of compound C-46 with a molar equivalent of 10 times of Trastuzumab monoclonal antibody was added thereto, and the mixture was shaken and reacted at 37°C for 16 hours. The mixture was purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution (7.9mg / mL, 1.5mL) of Trastuzumab-ADC-46, which was stored at 4°C.

[0696] Example 2-8 Trastuzumab-ADC-3

[0697]

[0698] The trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.40 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a trastuzumab monoclonal antibody buffer solution. A DMSO solution (10 mM) of compound C-3 with a molar equivalent of 10 times of trastuzumab monoclonal antibody was added thereto, shaken well, reacted at 25°C for 18 hours, purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution of trastuzumab-ADC-3 (6.4 mg / mL, 1.5 mL), which was stored at -20°C.

[0699] Example 2-9 Trastuzumab-ADC-4

[0700]

[0701] The trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.40 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a trastuzumab monoclonal antibody buffer solution. A DMSO solution (10 mM) of compound C-4 with a molar equivalent of 10 times of trastuzumab monoclonal antibody was added thereto, shaken well, reacted at 25°C for 18 hours, purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution of trastuzumab-ADC-4 (5.4 mg / mL, 1.5 mL), which was stored at -20°C.

[0702] Example 2-10 Trastuzumab-ADC-13

[0703]

[0704] The trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.40 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a trastuzumab monoclonal antibody buffer solution. A DMSO solution (10 mM) of compound C-13 with a molar equivalent of 10 times of trastuzumab monoclonal antibody was added thereto, shaken well, reacted at 25°C for 18 hours, purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution of trastuzumab-ADC-13 (6.3 mg / mL, 1.5 mL), which was stored at -20°C.

[0705] Example 2-11 Trastuzumab-ADC-21

[0706]

[0707] The trastuzumab monoclonal antibody stock solution was adjusted to pH = 7.40 with 1M disodium hydrogen phosphate solution, and then fixed to 10 mg / mL with phosphate buffered saline (PBS) to obtain a trastuzumab monoclonal antibody buffer solution. A DMSO solution (10 mM) of compound C-21 with a molar equivalent of 10 times of trastuzumab monoclonal antibody was added thereto, shaken well, reacted at 25°C for 18 hours, purified with a NAP-10 gel column, and washed with a pH = 6.0-6.1 histidine hydrochloride buffer solution, and the filtrate was collected to obtain a histidine hydrochloride buffer solution of trastuzumab-ADC-21 (7.1 mg / mL, 1.5 mL), which was stored at -20°C.

[0708] III. Determination of the Drug / Antibody Ratio (DAR) of Antibody Drug Conjugates

[0709] 1. The molecular weight of the product of the coupling reaction was determined by LC-MS, and the drug / antibody ratio DAR value was calculated.

[0710] Chromatographic conditions:

[0711] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0712] Mobile phase A: 0.1% FA / H 2 O; mobile phase B: 0.1% FA / ACN;

[0713] Flow rate: 0.25 ml / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 2 μl;

[0714] Time (minutes) 2 20 22 25 26 30 Mobile phase A (volume %) 75 60 5 5 75 75 Mobile phase B (volume %) 25 40 95 95 25 25

[0715] Mass spectrometry conditions:

[0716] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0717] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0718] m / z 600-4000; Time bins to sum 40.

[0719] Table 1 Drug / Antibody Ratio (DAR) Results of Antibody Drug Conjugates

[0720]

[0721]

[0722] Biological tests

[0723] Experimental Example 1: Antibody-drug conjugates inhibit proliferation of HER2-positive and HER2-negative tumor cells

[0724] Experimental steps:

[0725] 1. Collect HER2-positive tumor cells SK-OV-3 (Nanjing Kebai) and NCI-N87 (ATCC) in the logarithmic growth phase and count them.

[0726] 2. Tumor cells were inoculated into 96-well plates (Corning), with 1000 cells (SK-OV-3) or 3000 cells (NCI-N87) per well, in a volume of 80 μL, and placed at 37°C, 5% CO 2 Culture in an incubator overnight to allow cells to adhere (18 to 24 hours).

[0727] 3. Prepare 5× drug working solution with gradient concentration according to the experimental design. Take out the 96-well cell plate, transfer 20 μL working solution from the compound plate and add it to the cell plate. Place the cell plate at 37°C and 5% CO 2 Culture in an incubator for 96 h.

[0728] 4. Test plate: Take out the cell plate and observe the cell status under a microscope. 2.0 kit (Novozyme) instructions, add detection solution to the cell plate, and obtain raw data through PHERA Star FS.

[0729] 5. Calculate the inhibition rate (IR%) = 1-(average luminescence value of experimental wells / average luminescence value of DMSO control wells)*100%. Use Graphpad Prism 8.0 software for data analysis and plotting, and calculate the IC by fitting the logarithm of cell survival rate and drug concentration through a four-parameter curve. 50 value.

[0730] Experimental Example 2: Determination of the ability of immunomodulatory antibody drug conjugate (iADC) to stimulate peripheral blood mononuclear cells (PBMC) to secrete TNF-α in the presence of tumor cells

[0731] Experimental steps:

[0732] 1. Collect HCC1954 tumor cells (Connotai) expressing HER2 in the logarithmic growth phase, wash twice with PBS, and resuspend the cells in RPMI1640 + 10% heat-inactivated FBS complete medium to adjust the cell density to 2×10 5 HCC1954 tumor cells were inoculated in a 96-well plate (Corning), with 1×10 per well. 4 cells.

[0733] 2. Resuscitate PBMC (Sai ​​Li Biotechnology), resuspend the cells in RPMI1640 + 10% heat-inactivated FBS complete medium, and adjust the density to 6×10 5 3×10 / mL were added to a 96-well plate inoculated with tumor cells. 4 PBMC cells.

[0734] 3. Add 100 μL of the drug to be tested at different concentrations into the well plate to make the final concentrations 500 nM, 166.7 nM, 55.6 nM, 18.52 nM, 6.17 nM, 2.06 nM, and 0.69 nM, respectively. Add 100 μL of RPMI1640 + 10% heat-inactivated FBS complete culture medium into the blank well.

[0735] 4. Place in 5% CO 2 , cultured in a 37°C incubator. After 24 hours of culture, 50 μL of supernatant was taken from each well for detection. The primary antibody and HRP-labeled secondary antibody were incubated according to the steps of the human TNF-α ELISA detection kit (Invitrogen), color was developed, and the absorbance at a wavelength of 450 nm was read. The concentration of TNF-α was calculated based on the standard curve using Graphpad Prism software.

[0736] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. An antibody-drug conjugate as shown in formula (I): in, Ab is the targeting group; Aa is an amino acid fragment; L 1 To connect Aa and D 1 The linker of L 2 To connect Aa and D 2 The linker of D 1 It is a cytotoxic drug fragment; D 2 is a TLR agonist fragment; m is selected from 1 to 5; M is selected from the following structures: The 1 position of M is connected to Ab, and the 2 position is connected to Aa; A is unsubstituted or substituted with one or more R 1 A substituted 3-20 membered heterocyclic subunit, wherein the heterocyclic subunit contains at least one nitrogen atom; R 1 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo; a are each independently selected from 1-6 (eg, 1, 2, 3, 4, 5 or 6, such as 1, such as 2); b are each independently selected from 1-10; c are each independently selected from 0-6.

2. The antibody-drug conjugate according to claim 1, wherein: M is selected from the following structures: wherein each b is independently selected from 1-10, and the 1 position of M is connected to Ab, and the 2 position is connected to Aa; Preferably, M is selected from the following structures: Among them, the 1 position of M is connected to Ab, and the 2 position is connected to Aa.

3. The antibody-drug conjugate according to any one of claims 1 to 2, wherein: Aa is selected from the following structures: Among them, position 3 is connected to M, and position 4 is connected to L 1 and L 2 One of them is connected, and position 5 is connected to L 1 and L 2 Preferably, the 3 position is connected to M and the 4 position is connected to L 1 Connected, 5 position and L 2 connected.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein: L 1 The invention relates to a method for preparing a cleavable linker comprising: selecting a non-cleavable linker and a cleavable linker, wherein the cleavable linker can be cleaved by acid or by an enzyme present in a pathological environment, wherein the enzyme is selected from a protease, a phosphatase, a pyrophosphatase, a β-glucuronidase, a β-galactosidase and a sulfatase.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein: L 1 for in: L a is a covalent bond or is selected from C 1-6 Alkylene, wherein each a is independently selected from 1-6, each b is independently selected from 1-10, and each c is independently selected from 0-6; L b is selected from the group consisting of a covalent bond, an amino acid fragment, a peptide fragment formed by two or more amino acids, wherein the amino acids are selected from the group consisting of Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; Preferably, L b is a covalent bond or is selected from the following structures: Among them, each R 2 Independently selected from H or C 1-6 alkyl; Preferably, L b Selected from L c is a covalent bond, -NH-CH2- or selected from the following structures:

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein: L 1 Select from the following structures: wherein each b is independently selected from 1-10, L 1 The 6th position is connected to Aa, and the 7th position is connected to D 1 connected; Preferably, L 1 for: Among them, L 1 The 6th position is connected to Aa, and the 7th position is connected to D 1 connected.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein: L 2 The invention relates to a method for preparing a cleavable linker comprising: selecting a non-cleavable linker and a cleavable linker, wherein the cleavable linker can be cleaved by acid or by an enzyme present in a pathological environment, wherein the enzyme is selected from a protease, a phosphatase, a pyrophosphatase, a β-glucuronidase, a β-galactosidase and a sulfatase.

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein: L 2 for in: L d is a covalent bond or is selected from C 1-6 Alkylene, wherein each a is independently selected from 1-6, each b is independently selected from 1-10, and each c is independently selected from 0-6; L e is a covalent bond or is selected from an amino acid fragment and a peptide fragment formed by two or more amino acids, wherein the amino acid is selected from Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala and Asn; Preferably, L e is a covalent bond or is selected from the following structures: Among them, each R 2 Independently selected from H and C 1-6 alkyl; L f Selected from covalent bonds, -NH-CH2- or the following structures:

9. The antibody-drug conjugate according to any one of claims 1 to 8, wherein: L 2 Select from the following structures: wherein each b is independently selected from 1-10, L 2 The 8th position is connected to Aa, and the 9th position is connected to D 2 connected; Preferably, L 2 for: Among them, L 2 The 8th position is connected to Aa, and the 9th position is connected to D 2 connected.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein: D 1 A fragment selected from cytotoxic drugs, wherein the cytotoxic drugs include but are not limited to tubulin inhibitors, DNA damaging agents and topoisomerase inhibitors, wherein the tubulin inhibitors include but are not limited to dolastatin and auristatin, maytansine, tubulysins and cryptomycins, wherein the DNA damaging agents include but are not limited to pyrrolobenzodiazepines Pyrrolobenzodiazepine, duocarmycin and calicheamicin, the topoisomerase inhibitors include but are not limited to camptothecin and its derivatives, epipodophyllotoxin, anthracyclines and anthraquinones; Preferably, the tubulin inhibitor is selected from the group consisting of dolastatin 10, MMAE, MMAF, maytansine, DM1, DM3 and DM4, and the topoisomerase inhibitor is selected from the group consisting of camptothecin, SN-38, exitecan, topotecan, belotecan, rubitecan, diflutecan, 10-hydroxycamptothecin, 9-aminocamptothecin, Dxd, T030, doxorubicin, epirubicin and PNU-159682.

11. The antibody-drug conjugate according to any one of claims 1 to 10, wherein: D 1 Select from the following structures:

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein: D 2 is a TLR agonist fragment, wherein the TLR agonist is selected from a TLR2 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist and a TLR9 agonist; Preferably, the TLR agonist is selected from a TLR7 agonist, a TLR8 agonist and a TLR7 / 8 agonist.

13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein: D 2 Select from the following structures:

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein: Ab is a targeting group that targets cell surface receptors or tumor surface antigens; Preferably, Ab is an antibody or an antigen-binding fragment thereof; the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a murine antibody, a humanized antibody, a fully human antibody, and a fusion protein comprising an antigen-binding portion of an antibody; the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, a disulfide-bonded Fv, and scFv; Preferably, the antibody or antigen-binding fragment thereof is an anti-Her-2 antibody; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 3 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 4 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 9 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 10 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; or (4) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 16 or a variant thereof, CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; Preferably, the antibody or antigen-binding fragment thereof comprises: VH or a variant thereof as shown in SEQ ID NO: 18, and / or VL or a variant thereof as shown in SEQ ID NO: 19; Preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain of VH or a variant thereof shown in SEQ ID NO: 18 and a weight constant region (CH) or a variant thereof shown in SEQ ID NO: 20, and / or a light chain of VL or a variant thereof shown in SEQ ID NO: 19 and a light chain of (light chain constant region) CL or a variant thereof shown in SEQ ID NO: 21; Preferably, the anti-Her-2 antibody is trastuzumab; Preferably, the amino acid sequence of the heavy chain of trastuzumab is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain of trastuzumab is as shown in SEQ ID NO:

2.

15. The antibody drug conjugate of any one of claims 1 to 14, having a structure selected from the group consisting of: Each m is selected from 1 to 5; Preferably, the Ab is trastuzumab.

16. A composition of an antibody drug conjugate, comprising the antibody drug conjugate of any one of claims 1-15, wherein the DAR value of the composition is 1.0-5.0, such as 1.0-2.0, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 2.0-3.0, 2.0-3.5, 2.0-4.0, 2.0-4.5, 2.0-5.0, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 4.0-4.5, 4.5-5.0 or 4.0-5.

0.

17. A compound of formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: in, M 1 Select from the following structures: M a Pentafluorophenoxy A is unsubstituted or substituted with one or more R 1 A substituted 3-20 membered heterocyclic subunit, wherein the heterocyclic subunit contains at least one nitrogen atom; R 1 Each independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and oxo; a are each independently selected from 1-6; b are each independently selected from 1-10; c are each independently selected from 0-6; Aa, L 1 , L 2 , D 1 and D 2 As defined in any one of claims 1 to 15.

18. The compound of claim 17 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein: M 1 Select from the following structures:

19. The compound of claim 17 or 18, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, wherein the compound has a structure selected from the following:

20. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1 to 15, the composition of claim 16, or the compound of any one of claims 17 to 19, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, and optionally one or more pharmaceutical excipients.

21. Use of the compound of any one of claims 17 to 19 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof in the preparation of an antibody drug conjugate (e.g., the antibody drug conjugate of any one of claims 1 to 15).

22. Use of the antibody drug conjugate of any one of claims 1 to 15, the composition of claim 16, the compound of any one of claims 17 to 19 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or the pharmaceutical composition of claim 20 in the preparation of a medicament for treating and / or preventing cancer (e.g., HER2-positive cancer, such as HER2-positive gastric cancer, breast cancer or non-small cell lung cancer).

23. A method for treating and / or preventing cancer (e.g., HER2-positive cancer, such as HER2-positive gastric cancer, breast cancer, or non-small cell lung cancer), comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of an antibody drug conjugate according to any one of claims 1-15, a composition according to claim 16, a compound according to any one of claims 17-19, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition according to claim 20.

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