IMMUNOSTIMULATING ANTIBODY CONJUGATE, METHOD OF ITS PRODUCTION AND ITS APPLICATION

Abstract

The present invention relates to an immunostimulatory antibody conjugate, a method for producing the same, and its use. In particular, the present invention relates to an immunostimulatory antibody conjugate represented by formula (I), a method for producing the same, and its use for producing a medicament for the treatment and / or prevention of cancer. The present invention also relates to a linker-drug represented by formula (II) and a compound represented by formula III-1, formula III-2, formula IV-1, or formula IV-2, a method for producing them, and their use for producing an immunostimulatory antibody conjugate.

Description

A class of immunostimulatory antibody conjugates, preparation methods and uses thereof

[0001] This application is based on the application with CN application number 202311788893.6 and application date December 22, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention belongs to the field of medicine, and specifically relates to a class of immunostimulatory antibody conjugates, a preparation method thereof, a pharmaceutical composition and uses thereof. Background Art

[0003] STING (Stimulator of interferon gene) is a key signaling molecule in immune responses. When activated by ligands (e.g., bacterial-derived cyclic dinucleotides (CDNs)), it upregulates the IRF3 and NF-κB signaling pathways. Activated STING recruits cytoplasmic TBK1, mediating TBK1's phosphorylation of IRF3, leading to the production of interferons and other cytokines. STING is widely involved in various pathological and physiological processes, including tumor immunity, autoimmune inflammation, and autophagy.

[0004] Currently available STING agonists primarily include two structural types: cyclic dinucleotides and acyclic dinucleotides. Compared to traditional cyclic dinucleotides, acyclic dinucleotide STING agonists theoretically possess superior stability and PK properties, and hold promising application prospects in the pharmaceutical industry.

[0005] Antibody-drug conjugates (ADCs) couple antibodies and small molecule cytotoxic drugs through specific linkers, using antibodies as carriers to deliver cytotoxic drugs to target cells, thereby reducing systemic exposure of cytotoxic drugs and improving safety. ADCs are a research hotspot in targeted tumor therapy.

[0006] With the advancement of ADC technology, a new class of immunostimulating antibody conjugates (ISACs) has been developed. These conjugates link immunomodulators, such as STING agonists, to antibodies via cleavable or non-cleavable linkers. These immunostimulating antibody conjugates combine the precise targeting of antibodies with the long-lasting anti-tumor activity and immune memory of immunomodulators, such as STING agonists, enabling systemic delivery while safely eliciting anti-tumor immune responses. Summary of the Invention

[0007] On the one hand, the present invention provides a class of immunostimulatory antibody conjugates with non-cyclic dinucleotide STING agonists as warheads, which can effectively exert the long-lasting anti-tumor activity and immune memory efficacy of STING agonists through the precise positioning of antibodies, and are of great significance for the prevention and treatment of tumors.

[0008] The first aspect of the present invention provides an immunostimulatory antibody conjugate of formula (I):

[0009]

[0010] in,

[0011] Ab is an antibody or antigen-binding fragment thereof that targets the target antigen;

[0012] M is a linker site for connecting an antibody or an antigen-binding fragment thereof;

[0013] L is a linker connecting M and D;

[0014] D is selected from the structures of formula (DI-1) and formula (DI-2):

[0015] in Indicates the connection point with L;

[0016] X 1 and X 3 are the same or different and are each independently selected from a covalent bond, -O-, -S- and -NR a -;

[0017] X 2 Selected from C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, cyano, C 1-6 Alkyl, -OR a and -NR a R b ;

[0018] X 4 and X 5 Each independently selected from -O- and -NR a -;

[0019] L 1Selected from covalent bonds and -(C(R 6 )2) j -;

[0020] L 2 Selected from covalent bonds and -(C(R 7 )2) k -;

[0021] R 1 and R 2 are the same or different and are each independently selected from H, halogen, cyano, -OR a 、-NR a R b 、-C(O)-OR a 、-C(O)-NR a R b 、-NR a -C(O)-R a 、C 1-6 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a and -NR a R b ;

[0022] R 3 Selected from H and C 1-6 alkyl;

[0023] R 4 Selected from H, -OR a 、-NR a R b 、-C(O)-OR a 、-OC(O)-R a 、-C(O)-NR a R b 、-NR a -C(O)-R a 、-OC(O)-NR a R b 、-NR a -C(O)-OR a 、-NR a -C(O)-NR a R b 、C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the C1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;

[0024] R 5 Selected from H, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, -OR a and -C(O)-OR a , the C 1-6 Alkyl, C 3- 10 The cycloalkyl and 3-10 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;

[0025] R 6 Each independently selected from H, -OR a 、-NR a R b 、C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;or

[0026] Two R on different carbon atoms 6 Together with the carbon atoms between them, they form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; or

[0027] Two R on the same carbon atom 6Together with the carbon atoms they are connected to form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group;

[0028] R 7 Each independently selected from H, -OR a 、-NR a R b 、C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ; or two R on the same carbon atom 7 Together with the carbon atoms they are connected to form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; or any one of R 7 and R 3 Together with the atoms between them, they form a 3-10 membered heterocyclic group;

[0029] R a and R b Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 alkyl, hydroxy, amino, and carboxyl groups;

[0030] j and k are each independently selected from 1, 2 and 3;

[0031] m and q are each independently selected from 0, 1 and 2;

[0032] n and p are each independently selected from 0, 1, 2 and 3;

[0033] z is selected from 1-10.

[0034] A second aspect of the present invention provides an immunostimulatory antibody conjugate selected from the following structures:

[0035] Each z is selected from 1 to 10;

[0036] Ab is selected from anti-Her-2 antibody.

[0037] The third aspect of the present invention provides a drug linker of formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: M 1 -LD (II)

[0038] in,

[0039] M 1 Selected from

[0040] wherein each a is independently selected from an integer of 1-6, and b is selected from an integer of 1-10;

[0041] L and D are as defined above.

[0042] The fourth aspect of the present invention provides a compound represented by formula III-1 or III-2 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof,

[0043] Among them, R 1 、R 2 、R 3 、R 4 、R 5 , L 1 , L 2 、X 1 、X 2 、X 3 、X 4 、X 5 , m, n, p and q are as defined above.

[0044] In a fifth aspect, the present invention provides a compound of formula IV-1 or IV-2, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof:

[0045] Among them, R 1 、R 2 、R 3 、R 4 、R 5 , L 1 , L 2 、X 1 、X 2 、X 3、X 4 、X 5 , m, n, p and q are as defined above;

[0046] R 8 and R 9 Each independently selected from -C 1-6 Alkylene-NR 12 -C(=O)-C 1-6 Alkylene-NR 10 R 11 、-C(=O)OC 6-10 Aryl, -C(=O)NR 12 -C 1-6 Alkylene-NR 10 R 11 、-C 1-6 Alkylene-OC 1-6 Alkyl and -C 1-6 Alkylene-C(=O)-C 6-10 aryl, wherein the aryl group is optionally substituted with amino or nitro;

[0047] R 10 and R 11 Each independently selected from H, C 1-6 Alkyl, Fmoc, and Boc;

[0048] R 12 Selected from H and C 1-6 alkyl.

[0049] In a sixth aspect, the present invention provides a composition of immunostimulatory antibody conjugates, comprising the above-mentioned immunostimulatory antibody conjugates, wherein the DAR value of the composition is 1.0-10.0.

[0050] The seventh aspect of the present invention provides a pharmaceutical composition comprising a preventively and / or therapeutically effective amount of the immunostimulatory antibody conjugate of the first or second aspect of the present invention, the drug linker of the third aspect of the present invention, or the compound of the fourth or fifth aspect or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or the immunostimulatory antibody conjugate of the sixth aspect of the present invention, and one or more pharmaceutical excipients.

[0051] In an eighth aspect, the present invention provides an immunostimulatory antibody conjugate of formula (I) of the first or second aspect of the present invention, a drug linker of the third aspect of the present invention, or a compound of the fourth or fifth aspect or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, a composition of the immunostimulatory antibody conjugate of the sixth aspect of the present invention, or a pharmaceutical composition of the seventh aspect of the present invention, for use 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).

[0052] In a ninth aspect, the present invention provides an immunostimulatory antibody conjugate of formula (I) of the first or second aspect of the present invention, a drug linker of the third aspect of the present invention, or a compound of the fourth or fifth aspect or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, a composition of the immunostimulatory antibody conjugate of the sixth aspect of the present invention, or a pharmaceutical composition of the seventh aspect of the present invention, for use in treating and / or preventing cancer (e.g., HER2-positive cancer, such as HER2-positive gastric cancer, breast cancer or non-small cell lung cancer).

[0053] In a tenth aspect, the present invention provides 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 an individual in need thereof a therapeutically and / or prophylactically effective amount of the immunostimulatory antibody conjugate of formula (I) of the first or second aspect of the present invention, the drug linker of the third aspect of the present invention, or the compound of the fourth or fifth aspect, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, the immunostimulatory antibody conjugate of the sixth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention.

[0054] In an eleventh aspect, the present invention provides use of the drug linker of the third aspect of the present invention or the compound of the fourth or fifth aspect, 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., an immunostimulatory antibody conjugate of the present invention).

[0055] definition

[0056] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of technology or substitutions of equivalent technology that would be apparent to those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

[0057] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit 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 regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved 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 the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0058] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in 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 the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0059] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof can be identified according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT or AbM numbering systems.

[0060] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0061] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., 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 techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0062] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0063] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0064] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0065] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0066] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0067] 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.

[0068] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0069] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0070] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase 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 the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0071] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. 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.

[0072] As used herein, the terms "comprises," "comprising," "having," "containing," or "involving," and variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0073] As used herein, the term "alkyl" is defined as a straight-chain or branched 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.

[0074] As used herein, the term "alkoxy" refers to "alkyl-O-," wherein "alkyl" is as defined above. For example, the term "C 1-6 "Alkoxy" refers to "C 1-6 Alkyl-O-", the "C 1-6 "C alkyl" is as defined above. 1-3 "Alkoxy" refers to "C 1-3 Alkyl-O-", the "C 1-3 "Alkyl" is as defined above. Exemplary C 1-6 Alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy or n-hexoxy.

[0075] The "C 1-6 "Alkylene" refers to a divalent group formed by removing two hydrogen atoms from a straight or branched alkane containing 1 to 6 carbon atoms, including "C 1-5 Alkylene", "C 1-4 Alkylene", "C 1-3 Alkylene", "C 1-2 "Alkylene", specific examples include but are not limited to: -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, etc.

[0076] As used herein, the term "alkenyl" refers to a straight or branched aliphatic hydrocarbon group having one or more carbon-carbon double bonds. 2-6 The term "alkenyl" refers to an alkenyl group having 2 to 6 carbon atoms and one, two or three carbon-carbon double bonds (e.g., ethenyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, etc.), which is optionally substituted with one or more (e.g., 1 to 3) substituents described herein.

[0077] The "C 2-6 "Alkenylene" refers to a divalent group formed by removing two hydrogen atoms from a straight-chain or branched aliphatic hydrocarbon having one or more carbon-carbon double bonds.

[0078] As used herein, the term "alkynyl" refers to a straight or branched aliphatic hydrocarbon group having one or more carbon-carbon triple bonds. 2-6 The term "alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms and one, two or three carbon-carbon triple bonds (e.g., ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, etc.), which is optionally substituted with one or more (e.g., 1 to 3) substituents described herein.

[0079] The "C 2-6 "Alkyne" refers to a divalent group formed by removing two hydrogen atoms from a straight-chain or branched aliphatic hydrocarbon having one or more carbon-carbon triple bonds.

[0080] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., a monocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or a bicyclic ring, including spirocyclic, fused or bridged systems such as bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl or bicyclo[5.2.0]nonyl, decahydronaphthyl, etc.), which is optionally substituted with one or more (such as 1 to 3) suitable substituents. For example, the term "C 3-6 "Cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring having 3 to 6 ring carbon atoms (for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents, for example methyl substituted cyclopropyl.

[0081] As used herein, the term "halogen" group is defined to include fluorine, chlorine, bromine, or iodine.

[0082] As used herein, the term "halo" refers to substitution with one or more (such as 1 to 3) the same or different halogen atoms.

[0083] 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. For example, the term "C 1-6The term "haloalkyl" refers to a halogenated alkyl group having 1 to 6 carbon atoms, for example, -CF3, -C2F5, -CHF2, -CH2F, -CH2CF3, -CH2Cl or -CH2CH2CF3.

[0084] As used herein, the term "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic group, for example, having 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms and one or more (e.g., 1, 2, 3 or 4) independently selected from N, O or S(O) in the ring. t (wherein t is 0, 1 or 2) heteroatom, such as 3-12 membered heterocyclyl, 3-7 membered heterocyclyl, 3-6 membered heterocyclyl, 5-6 membered heterocyclyl, etc., such as 5-10 membered nitrogen-containing heterocyclyl, 6-10 membered oxygen-containing heterocyclyl, 6-8 membered sulfur-containing heterocyclyl, 5-8 membered oxygen-containing heterocyclyl, etc. Representative examples of heterocyclyl include, but are not limited to, oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, hexahydro-1H-pyrroline, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, tetrahydropyridinyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, etc.

[0085] 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. For example, the term "C 6-10 Aryl" or "C 6-10 "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 one or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, C 1-6 alkyl, etc.) substituted.

[0086] In this article, term " heteroaryl " refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl oxazolyl, pyridinyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0087] Optionally, hydrogen in the groups involved in the present invention may be replaced by deuterium.

[0088] The term "substituted" means that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on the designated atom are replaced with a group selected from the indicated group, provided that the designated atom's normal valence in the current context is not exceeded and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0089] 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 substituents listed, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or collectively, with independently selected substituents or unsubstituted. If a nitrogen of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected substituent or unsubstituted.

[0090] 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.

[0091] 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.

[0092] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0093] 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.

[0094] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom 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 suitable isotopes 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); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S). Certain isotopically labeled compounds of the invention (e.g., those incorporating radioactive isotopes) are useful in drug and / or substrate tissue distribution studies (e.g., assays). The radioactive isotope tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) are particularly useful for this purpose because they are easy to incorporate and easy to detect. 11 C. 18 F. 15 O and 13N) substitution can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotopically labeled compounds of the present invention can be prepared by methods analogous to those described in the accompanying schemes and / or examples and preparations by using appropriate isotopically labeled reagents instead of the non-labeled reagents previously employed. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent is isotopically substituted, for example, D2O, acetone-d6 or DMSO-d6.

[0095] The term "stereoisomer" refers to 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, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). 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 this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0096] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

[0097] It should also be understood that certain compounds of the present invention may be used therapeutically in free form 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 that, upon administration to a patient in need thereof, are capable of directly or indirectly providing a compound of the present invention or a metabolite or residue thereof. Therefore, when reference is made herein to a "compound of the present invention," such various derivative forms of the compound are also intended to be encompassed.

[0098] 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. For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0099] 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 the polar solvent, especially water, may be present in a stoichiometric or non-stoichiometric ratio.

[0100] 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 and include 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 ESGWerstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0101] Also included within the scope of the present invention are metabolites of the compounds of the invention, i.e., substances formed in vivo upon administration of the compounds of the 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 invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0102] 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 themselves have little or no pharmacological activity, and when administered to the body or thereon, can be converted into the compounds of the present invention having the desired activity by, for example, hydrolytic cleavage. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted into the desired therapeutically active compounds in vivo. Further information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems," Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (E.B. 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 moieties known to those skilled in the art as "pro-moieties" (e.g., as described in "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)).

[0103] The present invention also encompasses compounds of the present invention that contain protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved using conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P.GM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0104] The term "about" means within ±10%, preferably within ±5%, and more preferably within ±2% of the stated numerical value.

[0105] The term "conjugate" refers to a substance resulting from the linkage of a small molecule drug to a targeting moiety. In some embodiments of the present invention, the small molecule drug and the targeting moiety are connected via a linker. The linker can be cleaved in specific environments (e.g., a low intracellular pH environment, a slightly acidic tumor microenvironment) or under specific effects (e.g., the action of lysosomal proteases), thereby separating the small molecule drug from the targeting moiety.

[0106] The term "linker" refers to a moiety that connects a small molecule drug to a targeting moiety.

[0107] The term "targeting moiety" refers to a portion of the conjugate that is capable of specifically binding to a target (or portion of a target) on the cell surface. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.

[0108] The term "fragment" refers to the portion of a compound molecule that remains after the compound has lost one or more atoms or groups of atoms (e.g., hydrogen atoms or hydroxyl groups). For example, a "compound fragment" refers to the portion of the compound described herein that remains after the compound has been linked to the linker in the immunostimulatory antibody conjugate, resulting from the loss of a hydrogen atom or hydroxyl group.

[0109] The structure of the immunostimulatory antibody conjugates of the present application can be represented by the general formula I, where z refers to the number of small molecule drug fragments attached to each antibody molecule. During the preparation of the immunostimulatory antibody conjugates, each antibody molecule may be attached to a different number of small molecule drug fragments. Therefore, generally speaking, the composition of the immunostimulatory antibody conjugates is a mixture of immunostimulatory antibody conjugates having different drug-antibody conjugation ratios. In practice, DAR is generally used to represent the average number of drugs attached to the antibody.

[0110] The linker can be attached to the antibody via various chemical bonds. For example, in some embodiments, the linker is attached via a thioether bond to a sulfhydryl group of the antibody. The -S- in some specific immunostimulatory antibody conjugates simply indicates the thioether bond formed between the linker and the sulfhydryl group of the antibody and does not indicate that the -S- is part of the linker.

[0111] Immunostimulatory antibody conjugates

[0112] An object of the present invention is to provide an immunostimulatory antibody conjugate of formula (I):

[0113]

[0114] in,

[0115] Ab is an antibody or antigen-binding fragment thereof that targets the target antigen;

[0116] M is a linker site for connecting an antibody or an antigen-binding fragment thereof;

[0117] L is a linker connecting M and D;

[0118] D is selected from the structures of formula (DI-1) and formula (DI-2):

[0119] in Indicates the connection point with L;

[0120] X 1 and X 3 are the same or different and are each independently selected from a covalent bond, -O-, -S- and -NR a -;

[0121] X 2 Selected from C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, cyano, C 1-6 Alkyl, -OR a and -NR a R b ;

[0122] X 4 and X 5 Each independently selected from -O- and -NR a -;

[0123] L 1 Selected from covalent bonds and -(C(R 6 )2) j -;

[0124] L 2 Selected from covalent bonds and -(C(R 7 )2) k -;

[0125] R 1 and R 2 are the same or different and are each independently selected from H, halogen, cyano, -OR a 、-NR a R b 、-C(O)-ORa 、-C(O)-NR a R b 、-NR a -C(O)-R a 、C 1-6 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a and -NR a R b ;

[0126] R 3 Selected from H and C 1-6 alkyl;

[0127] R 4 Selected from H, -OR a 、-NR a R b 、-C(O)-OR a 、-OC(O)-R a 、-C(O)-NR a R b 、-NR a -C(O)-R a 、-OC(O)-NR a R b 、-NR a -C(O)-OR a 、-NR a -C(O)-NR a R b 、C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;

[0128] R 5 Selected from H, C 1-6 Alkyl, C 3-10Cycloalkyl, 3-10 membered heterocyclic group, -OR a and -C(O)-OR a , the C 1-6 Alkyl, C 3- 10 The cycloalkyl and 3-10 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;

[0129] R 6 Each independently selected from H, -OR a 、-NR a R b 、C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;or

[0130] Two R on different carbon atoms 6 Together with the carbon atoms between them, they form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; or

[0131] Two R on the same carbon atom 6 Together with the carbon atoms they are connected to form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group;

[0132] R 7 Each independently selected from H, -OR a 、-NR a R b 、C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 2-6 Alkenyl, C2-6 Alkynyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ;or

[0133] Two R on the same carbon atom 7 Together with the carbon atoms they are connected to form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; or any one of R 7 and R 3 Together with the atoms between them, they form a 3-10 membered heterocyclic group;

[0134] R a and R b Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, the C 1-6 Alkyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 alkyl, hydroxy, amino, and carboxyl groups;

[0135] j and k are each independently selected from 1, 2 and 3;

[0136] m and q are each independently selected from 0, 1 and 2;

[0137] n and p are each independently selected from 0, 1, 2 and 3;

[0138] z is selected from 1-10.

[0139] In some embodiments of the present invention, M is selected from the following structures:

[0140] wherein the 1 position of M is connected to Ab, and the 2 position is connected to L; each a is independently selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6), and b is selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0141] In some embodiments of the present invention, M is selected from the following structures:

[0142] Among them, the 1 position of M is connected to Ab, and the 2 position is connected to L.

[0143] In some embodiments of the present invention, M is selected from the following structures:

[0144] Among them, the 1 position of M is connected to Ab, and the 2 position is connected to L.

[0145] In some embodiments of the present invention, M is Among them, the 1 position of M is connected to Ab, and the 2 position is connected to L.

[0146] In some embodiments of the present invention, M is Among them, the 1 position of M is connected to Ab, and the 2 position is connected to L.

[0147] In some embodiments of the present invention, L is selected from a non-cleavable linker and a cleavable linker, wherein the cleavable linker is cleaved 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.

[0148] In some embodiments of the present invention, L is -L a -L b -L c -,in:

[0149] L a is a covalent bond or is selected from

[0150] wherein each c is independently selected from an integer of 1-6, and each d is independently selected from an integer of 1-10;

[0151] L b 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;

[0152] L c is a covalent bond, -NH-CH2- or selected from the following structures:

[0153] In some embodiments of the present invention, L a For covalent bonds or

[0154] In some embodiments of the present invention, L a It is a covalent bond.

[0155] In some embodiments of the present invention, L bIt is a covalent bond or a peptide fragment formed by two or more amino acids, wherein the amino acids are selected from Val, Cit, Phe, Gly and Ala.

[0156] In some embodiments of the present invention, L b is a covalent bond or is selected from the following structures:

[0157] In some embodiments of the present invention, L b Selected from the following structures:

[0158] In some embodiments of the present invention, L c is a covalent bond, -NH-CH2- or selected from the following structures:

[0159] In some embodiments of the present invention, L c Selected from -NH-CH2- and

[0160] In some embodiments of the present invention, L is selected from the following structures:

[0161] Among them, the 3 position of L is connected to M, and the 4 position is connected to D.

[0162] In some embodiments of the present invention, X in Formula (DI-1) and Formula (DI-2) 1 and X 3 are each independently selected from -O- and -S-.

[0163] In some embodiments of the present invention, X in Formula (DI-1) and Formula (DI-2) 1 and X 3 All are -O-.

[0164] In some embodiments of the present invention, X in Formula (DI-1) and Formula (DI-2) 2 Selected from C 1-6 Alkylene, such as C 2-4 Alkylene.

[0165] In some embodiments of the present invention, X in Formula (DI-1) and Formula (DI-2) 2 Selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4- and -(CH2)5-.

[0166] In some embodiments of the present invention, D is selected from the structures of formula (D-II-1) and formula (D-II-2):

[0167] in, Represents the connection point with L, each group R 1 、R 2 、X 1 、X 2 、X 3 、X 4 、X 5 , n and p are as defined above.

[0168] In some embodiments of the present invention, D is selected from the structures of formula (D-III-1), formula (D-III-2), formula (D-III-3) and formula (D-III-4):

[0169] in, Represents the connection point with L, each group R 2 、X 1 、X 2 and X 3 As defined above.

[0170] In some embodiments of the present invention, D is selected from the following structures:

[0171] in, Indicates the connection point with L.

[0172] In some embodiments of the present invention, D is the following structure:

[0173] In some embodiments of the present invention, the Ab is an antibody or an antigen-binding fragment thereof; the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, murine antibodies, humanized antibodies, fully human antibodies and fusion proteins 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, disulfide-linked Fv and scFv.

[0174] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is an anti-Her-2 antibody.

[0175] In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises:

[0176] (1) The following heavy chain variable region (VH) and / or light chain variable region (VL):

[0177] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 3 or a variant thereof, CDR-H2 of SEQ ID NO: 4 or a variant thereof, and CDR-H3 of SEQ ID NO: 5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 8 or a variant thereof;

[0178] (2) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0179] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 9 or a variant thereof, CDR-H2 of SEQ ID NO: 10 or a variant thereof, and CDR-H3 of SEQ ID NO: 5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 8 or a variant thereof;

[0180] (3) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0181] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof, CDR-L2 of SEQ ID NO: 15 or a variant thereof, and CDR-L3 of SEQ ID NO: 8 or a variant thereof; or

[0182] (4) the following heavy chain variable region (VH) and / or light chain variable region (VL):

[0183] 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, a CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and a 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, a CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and a CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof.

[0184] In some embodiments of the invention, the antibody or antigen-binding fragment thereof comprises:

[0185] (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:

[0186] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 3 or a variant thereof, CDR-H2 of SEQ ID NO: 4 or a variant thereof, and CDR-H3 of SEQ ID NO: 5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 8 or a variant thereof;

[0187] (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:

[0188] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 9 or a variant thereof, CDR-H2 of SEQ ID NO: 10 or a variant thereof, and CDR-H3 of SEQ ID NO: 5 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 6 or a variant thereof, CDR-L2 of SEQ ID NO: 7 or a variant thereof, and CDR-L3 of SEQ ID NO: 8 or a variant thereof;

[0189] (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:

[0190] A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof, CDR-L2 of SEQ ID NO: 15 or a variant thereof, and CDR-L3 of SEQ ID NO: 8 or a variant thereof; or

[0191] (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:

[0192] 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, a CDR-H2 with a sequence of SEQ ID NO: 17 or a variant thereof, and a 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, a CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof, and a CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof.

[0193] In some embodiments of the present invention, 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.

[0194] In some embodiments of the present invention, 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 weight 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.

[0195] In some embodiments of the present invention, the anti-Her-2 antibody is trastuzumab.

[0196] In some embodiments of the present invention, 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.

[0197] The immunostimulatory antibody conjugates of the present invention encompass the immunostimulatory antibody conjugates of formula (I) obtained by any combination of the above preferred groups.

[0198] According to some embodiments of the present invention, the immunostimulatory antibody conjugate of the present invention is selected from the group consisting of:

[0199] wherein z is selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0200] In some embodiments of the invention, the Ab is trastuzumab.

[0201] The present invention provides the following immunostimulatory antibody conjugates:

[0202] wherein each z is selected from 1 to 10; and Ab is selected from anti-Her-2 antibody.

[0203] The present invention provides the following immunostimulatory antibody conjugates:

[0204] wherein each z is selected from 1 to 10; and Ab is selected from anti-Her-2 antibody.

[0205] The present invention provides the following immunostimulatory antibody conjugates:

[0206] wherein each z is selected from 1 to 10; and Ab is selected from anti-Her-2 antibody.

[0207] The present invention provides the following immunostimulatory antibody conjugates:

[0208] wherein each z is selected from 1 to 10; and Ab is selected from anti-Her-2 antibody.

[0209] In some embodiments of the invention, the Ab comprises the VH set forth in SEQ ID NO:18, and / or the VL set forth in SEQ ID NO:19.

[0210] In some embodiments of the invention, the Ab comprises a heavy chain of VH set forth in SEQ ID NO: 18 and a heavy chain of CH set forth in SEQ ID NO: 20, and / or a light chain of VL set forth in SEQ ID NO: 19 and a light chain of CL set forth in SEQ ID NO: 21.

[0211] In some embodiments of the invention, the Ab is trastuzumab.

[0212] The present invention provides a composition of an immunostimulatory antibody conjugate, comprising the above-mentioned immunostimulatory antibody conjugate, wherein the DAR value of the composition is 1.0-10.0, for example, 1.0-2.0, 1.0-3.0, 1.0-3.5, 1.0-4.0, 1.0-4.5, 1.0-5.0, 1.0-5.5, 1.0-6.0, 1.0-6.5, 1.0-7.0, 1.0-7.5, 1.0-8.0, 1.0-8.5, 1.0-9.0, 1.0-9.5, 2.0-3.0, 2.0-3.5, 2.0-3.0, 2.0-3.5, 2.0-3.5, 2.0-3.5, 2.0-3.5, 2.0-3.5, 2.0-3.5, 2.0-3.5 .5, 2.0-4.0, 2.0-4.5, 2.0-5.0, 2.0-5.5, 2.0-6.0, 2.0-6.5, 2.0-7.0, 2.0-7.5, 2.0-8.0, 2.0-8.5, 2.0-9.0, 2.0-9.5, 2.0-10.0, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.0-6.5, 3.0-7.0, 3.0-7.5, 3.0-8.0, 3.0-8 .5, 3.0-9.0, 3.0-9.5, 3.0-10.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 4.0-4.5, 4.5-5.0, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 4.0-8.5, 4.0-9.0, 4.0-9.5, 4.0-10.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0 -7.5, 5.0-8.0, 5.0-8.5, 5.0-9.0, 5.0-9.5, 5.0-10.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.0-9.0, 6.0-9.5, 6.0-10.0, 7.0-7.5, 7.0-8.0, 7.0-8.5, 7.0-9.0, 7.0-9.5, 7.0-10.0, 8.0-8.5, 8.0-9.0, 8.0-9.5, 8.0-10.0.

[0213] In some embodiments, the DAR value of the composition of the immunostimulatory antibody conjugate is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0.

[0214] Drug-linker

[0215] The present invention also provides a drug linker of formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: M 1 -LD (II)

[0216] in,

[0217] M 1 Selected from

[0218] wherein each a is independently selected from an integer of 1-6, and b is selected from an integer of 1-10;

[0219] L and D are as defined above.

[0220] In some embodiments of the present invention, M 1 Selected from the following structures:

[0221] The drug linker of the present invention is selected from the following structures:

[0222] Compound

[0223] The present invention also provides a compound represented by Formula III-1 or III-2, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof:

[0224] Among them, R 1 、R 2 、R 3 、R 4 、R 5 , L 1 , L 2 、X 1 、X 2 、X 3 、X 4 、X 5 , m, n, p and q are as defined above.

[0225] In some embodiments of the present invention, R 4 and R 5 Each independently selected from -C(O)-OH and -C(O)-OC 1-6 alkyl.

[0226] In some embodiments of the present invention, R 4 and R 5 Each is independently selected from -C(O)-OH, -C(O)-O-methyl, -C(O)-O-ethyl, -C(O)-O-n-propyl, -C(O)-O-isopropyl and -C(O)-O-tert-butyl.

[0227] In some embodiments of the present invention, the compound of formula III-1 has a structure as shown in formula III-1-1, and the compound of formula III-2 has a structure as shown in formula III-2-1.

[0228] In some embodiments of the present invention, the compound of formula III-1 has a structure as shown in formula III-1-2, and the compound of formula III-2 has a structure as shown in formula III-2-2.

[0229] In some embodiments of the present invention, the compound of formula III-1 has a structure as shown in formula III-1-3-a to III-1-3-d, and the compound of formula III-2 has a structure as shown in formula III-2-3-a to III-2-3-d:

[0230] In some embodiments of the present invention, the compound represented by formula III-1 or formula III-2 is selected from the following structures:

[0231] The present invention also provides a compound represented by Formula IV-1 or IV-2 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0232] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、L 1 、L 2 、X 1 、X 2 、X 3 、X 4 、X 5 , m, n, p and q are as defined above;

[0233] R 8 and R 9 Each independently selected from -C 1-6 Alkylene-NR 12 -C(=O)-C 1-6 Alkylene-NR 10 R 11 、-C(=O)OC 6-10 Aryl, -C(=O)NR 12 -C 1-6 Alkylene-NR 10 R 11 、-C 1-6 Alkylene-OC 1-6 Alkyl and -C 1-6 Alkylene-C(=O)-C 6-10 aryl, wherein the aryl group is optionally substituted with amino or nitro;

[0234] R 10 and R 11 Each independently selected from H, C 1-6 Alkyl, Fmoc, and Boc;

[0235] R 12 Selected from H and C 1-6 alkyl.

[0236] In some embodiments of the present invention, the compound represented by formula IV-1 or IV-2 is selected from the following structures:

[0237] Pharmaceutical compositions and kits

[0238] Another object of the present invention is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of the immunostimulatory antibody conjugate of the present invention, a drug linker of Formula II of the present invention, a compound of Formula III-1, Formula III-2, Formula IV-1 or Formula IV-2, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, a combination of the antibody drug conjugate of the present invention, and one or more pharmaceutically acceptable carriers.

[0239] Another object of the present invention is to provide a pharmaceutical composition comprising the immunostimulatory antibody conjugate described in any one of the preceding claims. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutical excipients. In some embodiments, the DAR value of the pharmaceutical composition is 1.0-10.0, such as 6.0-9.0, 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, 1.0-5.5, 1.0-6.0, 1.0-6.5, 1.0-7.0, 1.0-7.5, 1.0-8.0, 1.0-8.5, 1.0-9.0, 1.0-9.5, 2.0-3.0, 2.0-3.5, 2.0-4.0, 2.0-4.5, 2.0-5.0, 2.0-5.5, 2.0-6.0, 2.0-6.5, 2.0- 7.0, 2.0-7.5, 2.0-8.0, 2.0-8.5, 2.0-9.0, 2.0-9.5, 2.0-10.0, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.0-6.5, 3.0-7.0, 3.0-7.5, 3.0-8.0, 3.0-8.5, 3.0-9.0, 3.0-9.5, 3.0-10.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 4.0-4.5, 4.5-5.0, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4. 0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 4.0-8.5, 4.0-9.0, 4.0-9.5, 4.0-10.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.0-8.5, 5.0-9.0, 5.0-9.5, 5.0-10.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.0-9.0, 6.0-9.5, 6.0-10.0, 7.0-7.5, 7.0-8.0, 7.0-8.5 , 7.0-9.0, 7.0-9.5, 7.0-10.0, 8.0-8.5, 8.0-9.0, 8.0-9.5, 8.0-10.0, and for example 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0. .

[0240] Another object of the present invention is to provide a drug kit comprising the immunostimulatory antibody conjugate of the present invention, a drug linker of Formula II, a compound of Formula III-1, Formula III-2, Formula IV-1, or Formula IV-2, and pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, or prodrugs thereof, a composition of the antibody-drug conjugate of the present invention, or a pharmaceutical composition of the present invention. Optionally, the drug kit further comprises instructions for use.

[0241] In the present invention, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle that is administered together with the therapeutic agent and is suitable for contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic response or other problems or complications corresponding to a reasonable benefit / risk ratio within the scope of reasonable medical judgment.

[0242] Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, sterile liquids. The pharmaceutical composition can be in the form of, for example, a solid preparation, a semi-solid preparation, a liquid preparation, or a gaseous preparation.

[0243] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered by a suitable route, for example by injection or percutaneous administration; or by oral administration or by inhalation.

[0244] The content or dosage of the compound of the present invention in the pharmaceutical composition may be about 0.001 mg to about 1000 mg, suitably 0.01-800 mg, preferably 0.05-500 mg.

[0245] In some embodiments, the present invention provides a method for preparing a pharmaceutical composition of the present invention, comprising combining a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof with one or more pharmaceutically acceptable carriers.

[0246] Treatment methods and uses

[0247] The present invention provides use of any of the above-described immunostimulatory antibody conjugates in the preparation of a medicament for treating and / or preventing cancer (eg, HER2-positive cancer, such as HER2-positive gastric cancer, breast cancer, or non-small cell lung cancer).

[0248] In another aspect, the present invention provides use of any of the above-described drug linkers, compounds, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof in the preparation of immunostimulatory antibody conjugates. In some embodiments, the immunostimulatory antibody conjugate comprises a small molecule ligand capable of interacting with STING. In some embodiments, the immunostimulatory antibody conjugate is as described above.

[0249] In another aspect, the 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 the pharmaceutical composition of the present invention has a strong agonist effect on the target STING.

[0250] In another aspect, the present invention provides an immunostimulatory antibody conjugate, drug linker, compound or pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug as described above, a composition of immunostimulatory antibody conjugates or a pharmaceutical composition of the present invention for 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).

[0251] In another aspect, the present invention provides an immunostimulatory antibody conjugate, drug linker, compound or pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug of any of the foregoing, a composition of an immunostimulatory antibody conjugate or a pharmaceutical composition of the present invention 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).

[0252] In another aspect, the present invention provides 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 an individual in need thereof a therapeutically and / or prophylactically effective amount of any of the foregoing immunostimulatory antibody conjugates, drug linkers, compounds, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs, compositions of immunostimulatory antibody conjugates, or pharmaceutical compositions of the present invention.

[0253] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired prophylactic or therapeutic effect, for example, to achieve relief of one or more symptoms associated with the disease being treated.

[0254] The dosage regimen can be adjusted to provide the optimal desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It should be noted that the dosage value can vary with the type and severity of the condition to be alleviated, and can include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the immunostimulatory antibody conjugate, compound, pharmaceutical composition of the present invention or supervising the administration of the immunostimulatory antibody conjugate, compound, pharmaceutical composition of the present invention.

[0255] The amount of the immunostimulatory antibody conjugate, compound or pharmaceutical composition of the invention administered will depend on the individual being treated, the severity of the disorder or condition, the rate of administration, the handling of the compound and the judgment of the prescribing physician. In some cases, a dosage level not exceeding the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be employed without causing any adverse side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

[0256] As used herein, unless otherwise indicated, the terms "treat," ...

[0257] The term "prevention" refers to inhibiting and delaying the onset of a disease, and includes not only prevention before the development of a disease but also prevention of recurrence of a disease after treatment.

[0258] As used herein, "subject" includes humans and non-human animals. Exemplary human subjects include human subjects suffering from diseases (e.g., the diseases described herein) (referred to as patients) or normal individuals. "Non-human animals" herein include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0259] FIG1 is a curve showing the changes in tumor volume over time in each group in Experimental Example 4. DETAILED DESCRIPTION

[0260] 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 embodiment.But those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unindicated specific conditions in the examples are all carried out according to the conditions of normal conditions or manufacturer's advice.Reagents used or instruments not indicated by manufacturer are all conventional products that can be obtained commercially.

[0261] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS). 1 H NMR measurements were performed on a JEOL Eclipse 400 nuclear magnetometer. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS). Chemical shifts (δ) were given in parts per million (ppm).

[0262] The MS measuring instrument is an Agilent (ESI) mass spectrometer, manufactured by Agilent, model: Agilent 6120B.

[0263] Preparation method for preparative high performance liquid chromatography:

[0264] Instrument model: Agilent 1260, chromatographic column: Waters SunFire Prep C18OBD (19 mm × 150 mm × 5.0 μm); column temperature: 25°C; flow rate: 20.0 mL / min; detection wavelength: 214 nm; elution gradient: (0 min: 10% A, 90% B; 16.0 min: 90% A, 10% B); mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid in water.

[0265] 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.

[0266] 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 by adding triethylamine.

[0267] Microwave reaction was carried out using Biotage Initiator+ (400W, RT-300°C) microwave reactor.

[0268] Column chromatography generally uses 200-300 mesh silica gel as a carrier. Eluent systems include: dichloromethane and methanol systems, and petroleum ether and ethyl acetate systems. 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.

[0269] Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C to 35°C);

[0270] The reagents used in the present invention were purchased from Acros Organics, Aldrich Chemical Company, Teber Chemical and other companies.

[0271] In general synthesis methods, examples, and intermediate synthesis examples, the meanings of the abbreviations are as follows.

[0272] The information of the sequences involved in the present invention is described in the following table:

[0273] 1. Intermediate Preparation Example

[0274] Intermediate Preparation Example 1.1: Preparation of ethyl 3-(6-methoxy-N-methyl-5-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propanoate

[0275] Step 1: Preparation of ethyl 3-(5-(3-hydroxypropoxy)-6-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propionate

[0276] Ethyl 3-(5-hydroxy-6-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propanoate (7.1 g, 21.0 mmol), 3-bromo-1-propanol (3.5 g, 25.2 mmol), potassium iodide (0.17 g, 1.05 mmol), and potassium carbonate (4.64 g, 33.6 mmol) were added sequentially to N,N-dimethylformamide (75 mL). The temperature was slowly raised to 50°C and stirred for 12 hours. After cooling the reaction system to room temperature, the reaction solution was poured into water (300 mL) and extracted three times with ethyl acetate (300 mL). The organic phases were combined and washed three times with saturated brine (100 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (6.3 g, yield: 75.9%).

[0277] MS m / z(ESI):396.2[M+H] + .

[0278] Step 2: Preparation of ethyl 3-(6-methoxy-N-methyl-5-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propanoate

[0279] Ethyl 3-(5-(3-hydroxypropoxy)-6-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propanoate (6.0 g, 15.2 mmol) and triethylamine (7.7 g, 76.0 mmol) were added sequentially to dichloromethane (100 mL). The mixture was cooled in an ice-water bath. p-Toluenesulfonyl chloride (5.8 g, 30.4 mmol) was slowly added portionwise to the reaction mixture at 0°C. The temperature was slowly raised to room temperature and stirred for 12 hours. The reaction system was concentrated to remove the solvent, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:1, volume ratio) to obtain the title compound (6.97 g, yield: 83.4%).

[0280] MS m / z(ESI):550.1[M+H] + .

[0281] Intermediate Preparation Example 1.2: Preparation of ethyl 3-(6-methoxy-N-methyl-5-(2-(toluenesulfonyloxy)ethoxy)benzo[b]thiophene-2-carboxamido)propanoate

[0282] The synthetic route in Intermediate Preparation Example 1.1 was adopted, and the reaction raw material 3-bromo-1-propanol was replaced with 2-bromoethanol to obtain the title compound (136 mg, yield: 81.9%).

[0283] MS m / z(ESI):536.1[M+H] + .

[0284] Intermediate Preparation Example 1.3: Preparation of ethyl 3-(6-methoxy-N-methyl-5-(4-(toluenesulfonyloxy)butoxy)benzo[b]thiophene-2-carboxamido)propanoate

[0285] The synthetic route in Intermediate Preparation Example 1.1 was adopted, and the reaction raw material 3-bromo-1-propanol was replaced with 4-bromo-1-butanol to obtain the title compound (25 mg, yield: 35.9%).

[0286] MS m / z(ESI):564.2[M+H] + .

[0287] Intermediate Preparation Example 1.4: Preparation of ethyl 3-(5-methoxy-N-methyl-6-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propanoate

[0288] Using the synthetic route in Intermediate Preparation Example 1.1, the reaction raw material 3-(5-hydroxy-6-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propionic acid ethyl ester was replaced with 3-(6-hydroxy-5-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propionic acid ethyl ester to obtain the title compound (25 mg, yield: 35.9%).

[0289] MS m / z(ESI):550.1[M+H] + .

[0290] Intermediate Preparation Example 1.5: Preparation of ethyl 4-(5-hydroxy-6-(methoxymethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0291] Step 1: Preparation of 5-bromo-2-fluoro-4-methoxybenzaldehyde

[0292] Completely dissolve 2-fluoro-4-methoxybenzaldehyde (20.0 g, 0.13 mol) in methanol (150 mL). Cool in an ice-water bath. Slowly add bromine (41.5 g, 0.26 mol) dropwise to the reaction mixture at 0°C. After addition, slowly warm to room temperature and stir for 12 hours. Saturated aqueous sodium bisulfite solution (300 mL) is slowly added to the reaction mixture, followed by water (150 mL) until a white solid precipitates. Filter with suction, wash the filter cake with water (200 mL), and dry in a vacuum at 50°C to obtain the title compound (29.3 g, yield: 96.7%).

[0293] MS m / z(ESI):234.9[M+H] + .

[0294] Step 2: Preparation of ethyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate

[0295] 5-Bromo-2-fluoro-4-methoxybenzaldehyde (28.0 g, 0.12 mol), ethyl mercaptoacetate (15.9 g, 0.13 mol), and potassium carbonate (33.2 g, 0.24 mol) were added sequentially to N,N-dimethylacetamide (250 mL). The temperature was raised to 100°C and stirred for 12 hours. After cooling the reaction system to room temperature, the reaction solution was slowly poured into water (1000 mL), and a white solid precipitated. The filter cake was filtered, washed with water (200 mL), and dried under vacuum at 50°C to obtain the title compound (28.0 g, yield: 74.0%).

[0296] MS m / z(ESI):315.0[M+H] + .

[0297] Step 3: Preparation of 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid

[0298] Ethyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (28.0 g, 88.8 mmol) was completely dissolved in a mixture of methanol (150 mL) and tetrahydrofuran (150 mL). Sodium hydroxide (17.8 g, 0.44 mol) was also completely dissolved in water (50 mL). The aqueous sodium hydroxide solution was slowly added dropwise to the reaction system at 0°C, and the temperature was slowly raised to room temperature with stirring for 2 hours. Water (200 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. The residue was adjusted to pH 4 with dilute hydrochloric acid (1N, 300 mL), filtered, and the filter cake was washed with water (300 mL). The residue was then dried under vacuum at 50°C. The resulting residue was purified by slurrying with methyl tert-butyl ether (100 mL) to afford the title compound (24.2 g, yield: 94.8%).

[0299] MS m / z(ESI):286.9[M+H] + .

[0300] Step 4: Preparation of 5-bromo-6-methoxybenzo[b]thiophene

[0301] 5-Bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (24.0 g, 83.6 mmol) and cuprous oxide (23.9 g, 167.2 mmol) were added sequentially to N,N-dimethylacetamide (250 mL), heated to 180°C, and stirred for 4 hours. The reaction system was cooled to room temperature and filtered. Water (1000 mL) was added to the filtrate, and the mixture was extracted three times with ethyl acetate (500 mL). The organic phases were combined and washed three times with saturated brine (200 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:20, volume ratio) to obtain the title compound (17.1 g, yield: 84.2%).

[0302] MS m / z(ESI):242.9[M+H] + .

[0303] Step 5: Preparation of 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0304] Succinic anhydride (4.2 g, 42.0 mmol) was added to dichloromethane (200 mL). The mixture was cooled in an ice-water bath. Aluminum trichloride powder (16.8 g, 126.0 mmol) was slowly added portionwise to the reaction mixture at 0°C. Separately, 5-bromo-6-methoxybenzo[b]thiophene (10.2 g, 42.0 mmol) was completely dissolved in dichloromethane (100 mL) and slowly added dropwise to the reaction mixture at 0°C. After completion of the dropwise addition, the mixture was slowly warmed to room temperature and stirred for 1 hour. Dilute hydrochloric acid (1N, 300 mL) was slowly added to the reaction mixture. The mixture was extracted three times with dichloromethane (200 mL). The organic phases were combined and washed three times with saturated brine (150 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (12.4 g, yield: 86.2%).

[0305] MS m / z(ESI):343.0[M+H] + .

[0306] Step 6: Preparation of ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0307] 4-(5-Bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (12.4 g, 36.1 mmol) was completely dissolved in anhydrous ethanol (150 mL). The mixture was cooled in an ice-water bath, and thionyl chloride (21.5 g, 180.7 mmol) was slowly added dropwise to the reaction mixture at 0°C. The temperature was slowly raised to room temperature and stirred for 4 hours. The solvent was then concentrated to remove the solvent. The residue was dissolved in ethyl acetate (300 mL) and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane:petroleum ether = 2:1, volume ratio) to obtain the title compound (13.0 g, yield: 97.0%).

[0308] MS m / z(ESI):371.0[M+H] + .

[0309] Step 7: Preparation of ethyl 4-(5-bromo-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0310] Ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (10.0 g, 26.9 mmol) and aluminum chloride (35.9 g, 269.4 mmol) were added sequentially to dichloromethane (300 mL), heated to 40°C, and stirred for 12 hours. After cooling the reaction system to room temperature, dilute hydrochloric acid (1N, 100 mL) was added to the reaction solution. The mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. Ethanol (300 mL) and a solution of hydrogen chloride in 1,4-dioxane (4N, 20 mL) were added to the residue, and the temperature was raised to 60°C and stirred for 2 hours. After cooling the reaction system to room temperature, the solvent was concentrated, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:4, volume ratio) to obtain the title compound (7.2 g, yield: 75.0%).

[0311] MS m / z(ESI):357.1[M+H] + .

[0312] Step 8: Preparation of ethyl 4-(5-bromo-6-(methoxymethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0313] Ethyl 4-(5-bromo-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (7.0 g, 19.6 mmol) and N,N-diisopropylethylamine (7.6 g, 58.8 mmol) were added sequentially to tetrahydrofuran (150 mL). The mixture was cooled in an ice-water bath. Bromomethyl methyl ether (4.9 g, 39.2 mmol) was slowly added dropwise at 0°C. The temperature was slowly raised to room temperature and stirred for 4 hours. The solvent was removed by concentration, and the resulting residue was dissolved in ethyl acetate (200 mL) and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the title compound (7.5 g, yield: 95.4%).

[0314] MS m / z(ESI):401.0[M+H] + .

[0315] Step 9: Preparation of ethyl 4-(6-(methoxymethoxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0316] Ethyl 4-(5-bromo-6-(methoxymethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (7.0 g, 17.4 mmol), pinacol diboron (8.9 g, 34.9 mmol), potassium acetate (3.4 g, 34.9 mmol), and 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (0.63 g, 0.87 mmol) were added sequentially to 1,4-dioxane (100 mL). The reaction system was sparged with nitrogen for 5 minutes, then heated to 100°C and stirred for 4 hours. The reaction system was cooled to room temperature and concentrated to remove the solvent. The resulting residue was dissolved in ethyl acetate (200 mL) and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the title compound (6.8 g, yield: 87.3%).

[0317] MS m / z(ESI):449.1[M+H] + .

[0318] Step 10: Preparation of ethyl 4-(5-hydroxy-6-(methoxymethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0319] Ethyl 4-(6-(methoxymethoxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-2-yl)-4-oxobutanoate (6.5 g, 14.5 mmol) was completely dissolved in a mixture of acetone (280 mL) and water (70 mL). Potassium peroxosulfate (25.1 g, 72.5 mmol) was slowly added portionwise at room temperature, and the reaction system was stirred at room temperature for 12 hours. The reaction system was concentrated to remove as much acetone as possible, and the resulting residue was dissolved in ethyl acetate (150 mL) and washed three times with saturated brine (60 mL). The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:2, volume ratio) to obtain the title compound (2.1 g, yield: 43.8%).

[0320] MS m / z(ESI):339.1[M+H] + .

[0321] Intermediate Preparation Example 1.6: Preparation of ethyl 4-(6-hydroxy-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0322] Step 1: Preparation of ethyl 4-(6-methoxy-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0323] Dissolve ethyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (25 g, 80.27 mmol) in N,N-dimethylformamide (100 mL), add 2-bromo-1-phenylethane-1-one (19.56 g, 96.32 mmol) and potassium carbonate (22.41 g, 160.53 mmol), and stir at room temperature for 6 hours. The reaction solution is slowly poured into water (2500 mL), and a yellow solid precipitates. Filter the mixture, wash the filter cake three times with water (600 mL), and dry it in vacuo at 50°C to obtain the title compound (33.0 g, yield: 95.4%).

[0324] MS m / z(ESI):427.1[M+H] + .

[0325] Step 2: Preparation of ethyl 4-(6-hydroxy-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0326] Ethyl 4-(6-methoxy-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (10 g, 23.21 mmol) and anhydrous aluminum chloride (12.51 g, 92.85 mmol) were added to 1,2-dichloroethane (80 mL) and stirred at 80°C for 2 hours. Hydrochloric acid (2N, 80 mL) was added to the reaction solution, stirred for 10 minutes, and then extracted three times with dichloromethane (80 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product, which was slurried with methyl tert-butyl ether to obtain the title compound (8.0 g, yield: 83.7%).

[0327] MS m / z(ESI):413.1[M+H] + .

[0328] Intermediate Preparation Example 1.7: Preparation of ethyl 3-(6-bromo-5-hydroxy-N-methylbenzo[b]thiophene-2-carboxamido)propionate

[0329] Step 1: Preparation of ethyl 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylate

[0330] 4-Bromo-2-fluoro-5-methoxybenzaldehyde (5.0 g, 21.46 mmol), ethyl mercaptoacetate (3.1 g, 25.79 mmol), and potassium carbonate (8.9 g, 64.45 mmol) were added sequentially to N,N-dimethylacetamide (50 mL). The temperature was raised to 60°C and stirred for 15 hours. After cooling the reaction system to room temperature, the reaction solution was slowly poured into water (250 mL), resulting in the precipitation of a white solid. The filter cake was filtered, washed with water (200 mL), and dried under vacuum at 50°C to obtain the title compound (6.0 g, yield: 88.7%).

[0331] MS m / z(ESI):315.0[M+H] + .

[0332] Step 2: Preparation of 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid

[0333] Ethyl 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylate (6.0 g, 19.04 mmol) was completely dissolved in a mixture of methanol (25 mL) and tetrahydrofuran (25 mL). Lithium hydroxide monohydrate (4.0 g, 95.24 mmol) was also completely dissolved in water (10 mL). The aqueous lithium hydroxide solution was slowly added dropwise to the reaction system at 0°C, and the temperature was slowly raised to room temperature with stirring for 2 hours. Water (50 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. The residue was adjusted to pH 4 with dilute hydrochloric acid (1N, 300 mL), filtered, and the filter cake was washed with water (100 mL). The resulting residue was then dried under vacuum at 50°C. The resulting residue was purified by slurrying with methyl tert-butyl ether (20 mL) to obtain the title compound (2.7 g, yield: 49.4%).

[0334] MS m / z(ESI):286.9[M+H] + .

[0335] Step 3: Preparation of ethyl 3-(6-bromo-5-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propionate

[0336] Dissolve 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid (1.5 g, 5.22 mmol) and ethyl 3-(methylamino)propionate (1.0 g, 7.62 mmol) in N,N-dimethylacetamide (20 mL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.53 g, 7.98 mol) and 4-dimethylaminopyridine (1.0 g, 8.18 mmol) were added sequentially. The mixture was stirred at 25°C for 15 hours. The reaction mixture was poured into water (100 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed three times with saturated brine (10 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:1, volume ratio) to obtain the title compound (1.5 g, yield: 71.8%).

[0337] MS m / z(ESI):400.0[M+H] + .

[0338] Step 4: Preparation of ethyl 3-(6-bromo-5-hydroxy-N-methylbenzo[b]thiophene-2-carboxamido)propionate

[0339] Ethyl 3-(6-bromo-5-methoxy-N-methylbenzo[b]thiophene-2-carboxamido)propanoate (860.0 mg, 2.15 mmol) was added to dichloromethane (30 mL). A 1 M dichloromethane solution of boron tribromide (11 mL) was slowly added dropwise to the reaction mixture, and the mixture was stirred at 25°C for 12 hours. Dilute hydrochloric acid (1N, 10 mL) was added to the reaction mixture, which was then poured into water (100 mL) and extracted three times with dichloromethane (30 mL). The organic phases were combined and washed three times with saturated brine (10 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to afford the title compound (800.0 mg, yield: 96.4%).

[0340] MS m / z(ESI):386.0[M+H] + .

[0341] Intermediate Preparation Example 1.8: Preparation of 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl) glycyl glycyl-L-phenylalaninate

[0342] Step 1: Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine methyl ester

[0343] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycine (1.0 g, 2.82 mmol), L-phenylalanine methyl ester (531.0 mg, 2.96 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (1.18 g, 3.10 mmol) were added to N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (728.9 mg, 5.64 mmol) was added at room temperature, and the reaction system was stirred at room temperature for 2 hours. The reaction solution was poured into water (150 mL) and extracted three times with ethyl acetate (100 mL). The organic phases were combined and washed three times with saturated brine (60 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: 100% ethyl acetate) to give the title compound (1.28 g, yield: 88.0%).

[0344] MS m / z(ESI):516.2[M+H] + .

[0345] Step 2: Preparation of Glycylglycyl-L-phenylalanine

[0346] (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine methyl ester (1.2 g, 2.33 mmol) was completely dissolved in a mixture of tetrahydrofuran (15 mL) and methanol (15 mL). Separately, lithium hydroxide monohydrate (0.49 g, 11.64 mmol) was completely dissolved in water (2 mL). The aqueous lithium hydroxide solution was slowly added dropwise to the reaction system at 0°C, and the reaction solution was slowly warmed to room temperature and stirred for 2 hours. Water (25 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. The residue was adjusted to pH 4-5 with dilute hydrochloric acid (1N, 100 mL) and filtered. The resulting filtrate was concentrated under reduced pressure to obtain the title compound (0.44 g, yield: 68.2%).

[0347] MS m / z(ESI):280.1[M+H] + .

[0348] Step 3: Preparation of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine

[0349] Glycylglycyl-L-phenylalanine (1.5 g, 5.37 mmol) and N,N-diisopropylethylamine (1.39 g, 10.74 mmol) were dissolved in N,N-dimethylformamide (15 mL). 2,5-Dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (1.66 g, 5.37 mmol) was added portionwise to the reaction mixture at room temperature. The mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated to remove the solvent, and the resulting residue was purified by preparative HPLC to obtain the title compound (1.2 g, 47.2% yield).

[0350] MS m / z(ESI):473.2[M+H] + .

[0351] Step 4: Preparation of 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl) glycyl glycyl-L-phenylalanine ester

[0352] (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine (1.0 g, 2.12 mmol) and N-hydroxysuccinimide (0.29 g, 2.54 mmol) were dissolved in N,N-dimethylformamide (10 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.61 g, 3.18 mmol) was added portionwise to the reaction mixture at room temperature and stirred at room temperature for 12 hours. The reaction mixture was concentrated to remove the solvent, and the resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (0.95 g, 78.7% yield).

[0353] MS m / z(ESI):570.1[M+H] + .

[0354] Intermediate Preparation Example 1.9: Preparation of benzyl 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)methyl(2-(methylamino)ethyl)carbamate

[0355] Step 1: Preparation of 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl(4-nitrophenyl)carbonate

[0356] (S)-2-((S)-3,5-Azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (1.2 g, 1.31 mmol) and N,N-diisopropylethylamine (0.34 g, 2.62 mmol) were dissolved in N,N-dimethylformamide (5 mL). The mixture was cooled in an ice-water bath. Di(p-nitrobenzene) carbonate (0.6 g, 1.97 mmol) was added to the reaction solution at 0°C, and the temperature was slowly raised to room temperature and stirred for 3 hours. The reaction solution was poured into water (50 mL) and extracted three times with ethyl acetate (60 mL). The organic phases were combined and washed three times with saturated brine (30 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:1, volume ratio) to give the title compound (0.9 g, yield: 63.8%).

[0357] MS m / z(ESI):1081.5[M+H] + .

[0358] Step 2: Preparation of benzyl 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)methyl(2-(methylamino)ethyl)carbamate

[0359] 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl(4-nitrophenyl)carbonate (350.0 mg, 0.32 mmol) and N,N-diisopropylethylamine (125.5 mg, 0.97 mmol) were dissolved in N,N-dimethylformamide (6 mL). The mixture was cooled in an ice-water bath. A solution of N,N'-dimethylethylenediamine (225.7 mg, 2.56 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise to the reaction solution at 0°C. The temperature was slowly raised to room temperature and stirred for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (160.5 mg, yield: 48.7%).

[0360] MS m / z(ESI):1030.6[M+H] + .

[0361] Intermediate Preparation Example 1.10: Preparation of 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0362] Step 1: Preparation of ethyl 4-(6-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0363] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (464 mg, 0.69 mmol) was dissolved in trifluoroacetic acid (12 mL). Methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (508 mg, 1.38 mmol) was slowly added to the reaction system in four portions at room temperature, with intervals of 0.5 hours. The reaction system was stirred at room temperature for 1 hour. The solvent was concentrated, and the resulting residue was purified by silica gel column chromatography (eluent: dichloromethane:ethyl acetate = 1:1, volume ratio) to give the title compound (254.3 mg, 37.6% yield).

[0364] MS m / z(ESI):980.2[M+H] + .

[0365] Step 2: Preparation of 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0366] Ethyl 4-(6-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (245 mg, 0.25 mmol) was dissolved in a mixed solvent of methanol (2 mL), tetrahydrofuran (2 mL) and water (0.5 mL). Lithium hydroxide monohydrate (31.5 mg, 0.75 mmol) was added to the reaction solution at room temperature, and stirring was maintained at room temperature for 4 hours. The solvent was removed by concentration, and water (5 mL) was added to the residue. The pH was adjusted to 5 with dilute hydrochloric acid (1N, 2 mL). The residue was filtered, and the filter cake was washed with water (5 mL). The filter cake was dried under vacuum at 50°C to give the title compound (115 mg, yield: 65.5%).

[0367] MS m / z(ESI):702.0[M+H] + .

[0368] Intermediate Preparation Example 1.11: Preparation of ethyl 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0369] Ethyl 4-(6-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (140 mg, 142.84 μmol) and diethylamine (31.34 mg, 428.49 μmol) were dissolved in N,N-dimethylformamide (2 mL) and stirred at 20°C for 3 hours. The reaction solution was purified by preparative HPLC to give the title compound (50 mg, yield: 46.2%).

[0370] MS m / z(ESI):758.2[M+H] + .

[0371] Intermediate Preparation Example 1.12: Preparation of (S)-4-(6-((2-aminopropionamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester

[0372] Step 1: Preparation of ethyl (S)-4-(6-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0373] The synthetic method of the first step in Intermediate Preparation Example 1.10 was used to replace the starting material of the first step, 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester with 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester. The title compound (300 mg, yield: 40.5%) was obtained by replacing methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate with methyl (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)acetate.

[0374] MS m / z(ESI):994.3[M+H] + .

[0375] Step 2: Preparation of ethyl (S)-4-(6-((2-aminopropionamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0376] The synthetic method in Intermediate Preparation Example 1.11 was used to replace the reaction raw material 4-(6-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester with ( S)-4-(6-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid, ethyl ester, to give the title compound (100 mg, yield: 42.9%).

[0377] MS m / z(ESI):772.2[M+H] + .

[0378] Intermediate Preparation Example 1.13: Preparation of (S)-4-(6-((2-aminopropionamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester

[0379] Using the synthetic route in Intermediate Preparation Example 1.12, the first step reaction raw material 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester to obtain the title compound (224 mg, yield: 55.9%).

[0380] MS m / z(ESI):772.2[M+H] + .

[0381] Intermediate Preparation Example 1.14: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0382] Step 1: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((4-nitrophenoxy)carbonyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0383] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (300 mg, 446.57 μmol) and N,N-diisopropylethylamine (116.6 mg, 902.20 μmol) were dissolved in N,N-dimethylformamide (3 mL). The mixture was cooled in an ice-water bath. Bis(4-nitrophenyl) carbonate (286 mg, 940.14 μmol) was added to the reaction solution at 0°C, and the temperature was slowly raised to room temperature and stirred for 3 hours. The reaction solution was poured into water (10 mL) and extracted three times with ethyl acetate (6 mL). The organic phases were combined and washed three times with saturated brine (5 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:1, volume ratio) to give the title compound (230 mg, yield: 61.5%).

[0384] MS m / z(ESI):837.2[M+H] + .

[0385] Step 2: Preparation of ethyl 4-(6-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0386] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((4-nitrophenoxy)carbonyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (200.0 mg, 238.98 μmol) and N,N-diisopropylethylamine (98.0 mg, 758.28 μmol) were dissolved in N,N-dimethylformamide (4 mL). The mixture was cooled in an ice-water bath. A solution of tert-butyl methyl(2-(methylamino)ethyl)carbamate (58.5 mg, 310.72 μmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise at 0°C. The temperature was slowly raised to room temperature and stirred for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (182.0 mg, yield: 85.9%).

[0387] MS m / z(ESI):886.3[M+H] + .

[0388] Step 3: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0389] Ethyl 4-(6-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (90 mg, 101.58 μmol) was dissolved in dichloromethane (7 mL). Trifluoroacetic acid (0.6 mL) was added to the reaction mixture at 0°C, and the temperature was slowly raised to room temperature with stirring for 1 hour. Dichloromethane (20 mL) was added to the reaction mixture for dilution, and the solvent was concentrated to obtain the title compound (80.0 mg, yield: 87.5%).

[0390] MS m / z(ESI):786.3[M+H] + .

[0391] 2. Compound Examples

[0392] Example 2.1: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-2)

[0393] Step 1: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(methoxymethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0394] Ethyl 4-(5-hydroxy-6-(methoxymethoxy)benzo[b]thiophene-2-yl)-4-oxobutanoate (2.0 g, 5.91 mmol), ethyl 3-(6-methoxy-N-methyl-5-(3-(tosyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propanoate (3.25 g, 5.91 mmol) and potassium carbonate (1.63 g, 11.83 mmol) were added sequentially to N,N-dimethylformamide (50 mL), and the temperature was raised to 50°C and stirred for 12 hours. After the reaction system was cooled to room temperature, the reaction solution was poured into water (300 mL) and extracted three times with ethyl acetate (300 mL). The organic phases were combined and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:1, volume ratio) to give the title compound (3.2 g, yield: 75.6%).

[0395] MS m / z(ESI):716.2[M+H] + .

[0396] Step 2: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0397] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(methoxymethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (3.0 g, 4.19 mmol) was completely dissolved in dichloromethane (50 mL). Trifluoroacetic acid (5 mL) was added at room temperature, and the reaction system was stirred at room temperature for 2 hours. The reaction system was concentrated to remove the solvent to obtain the title compound (2.68 g, yield: 95.3%).

[0398] MS m / z(ESI):672.2[M+H] + .

[0399] Example 2.2: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-3)

[0400] Completely dissolve ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.0 g, 2.98 mmol) in a mixture of tetrahydrofuran (40 mL) and water (10 mL). Separately, completely dissolve lithium hydroxide monohydrate (0.29 g, 11.9 mmol) in water (2 mL). Slowly add the lithium hydroxide aqueous solution dropwise to the reaction system at 0°C, slowly warm to room temperature, and stir for 12 hours. Water (50 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. The residue was adjusted to pH 4 with dilute hydrochloric acid (1N, 100 mL) and filtered. The filter cake was washed with water (100 mL) and dried in vacuo at 50°C to give the title compound (1.5 g, yield: 81.3%).

[0401] MS m / z(ESI):616.1[M+H] + .

[0402] 1 H NMR (400MHz, DMSO-d6): δ12.21(br,2H),9.72(s,1H),8.07(s,1H),7.56(s,1H),7.49(s,1H),7.44(s,1H),7.40(s,1H),7 .27(s,1H),4.21-4.14(m,4H),3.77(s,3H),3.65-3.58(m,2H),3.20-3.15(m,5H),2.56-2.48(m,4H),2.25-2.21(m,2H).

[0403] Example 2.3: Preparation of tert-butyl 4-(5-(3-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-1)

[0404] Dissolve 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (0.5 g, 0.81 mmol) and O-tert-butyl-N,N'-diisopropylisourea (0.65 g, 3.24 mmol) in tetrahydrofuran (5 mL), warm to 50°C, and stir for 12 hours. The solvent was removed by concentration, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:1, volume ratio) to obtain the title compound (267 mg, yield: 45.3%).

[0405] MS m / z(ESI):728.2[M+H] + .

[0406] 1 H NMR (400MHz, DMSO-d6): δ9.77(s,1H),8.13(s,1H),7.62(s,1H),7.56(s,1H),7.50(s,1H),7.47(s,1H),7.33(s,1H),4.27-4.22( m,4H),3.84(s,3H),3.70-3.62(m,2H),3.34(s,3H),3.23-3.20(m,2H),2.58-2.50(m,4H),2.30-2.27(m,2H),1.39-1.37(m,18H).

[0407] Example 2.4: Preparation of ethyl 4-(5-(2-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-4)

[0408] Using the synthetic route in Example 2.1, the first step reaction raw material 3-(6-methoxy-N-methyl-5-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester was replaced with 3-(6-methoxy-N-methyl-5-(2-(toluenesulfonyloxy)ethoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester to obtain the title compound (20.0 mg, yield: 84.5%).

[0409] MS m / z(ESI):658.2[M+H] + .

[0410] Example 2.5: Preparation of 4-(5-(2-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-5)

[0411] Using the synthetic route in Example 2.2, the reaction raw material 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with 4-(5-(2-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester to give the title compound (10.0 mg, yield: 54.7%).

[0412] MS m / z(ESI):602.1[M+H] + .

[0413] 1 H NMR (400MHz, DMSO-d6): δ8.15(s,1H),7.65(s,1H),7.59-7.57(m,3H),7.36(s,1 H),4.43(s,4H),3.85(s,3H),3.71(s,2H),3.27-3.12(m,5H),2.60-2.57(m,4H).

[0414] Example 2.6: Preparation of ethyl 4-(5-(4-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)butoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-6)

[0415] Using the synthetic route in Example 2.1, the first step reaction raw material 3-(6-methoxy-N-methyl-5-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester was replaced with 3-(6-methoxy-N-methyl-5-(4-(toluenesulfonyloxy)butoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester to give the title compound (32 mg, yield: 94.6%).

[0416] MS m / z(ESI):686.2[M+H] + .

[0417] Example 2.7: Preparation of 4-(5-(4-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)butoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-7)

[0418] Using the synthetic route in Example 2.2, the reaction raw material 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with 4-(5-(4-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)butoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester to give the title compound (16 mg, yield: 52.8%).

[0419] MS m / z(ESI):630.1[M+H] + .

[0420] 1 H NMR (400MHz, DMSO-d6): δ12.25(s,2H),9.85(s,1H),8.13(s,1H),7.64(s,1H),7.56(s,1H),7.47(s,1H),7.45(s, 1H),7.33(s,1H),4.20-4.35(m,4H),3.84(s,3H),3.71(s,2H),3.09-3.28(m,5H),2.56-2.63(m,4H),1.98(s,4H).

[0421] Example 2.8: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-8)

[0422] Using the synthetic route in Example 2.1, the first step reaction raw material 3-(6-methoxy-N-methyl-5-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester was replaced with 3-(5-methoxy-N-methyl-6-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester to give the title compound (40 mg, yield: 96.8%).

[0423] MS m / z(ESI):672.2[M+H] + .

[0424] Example 2.9: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-9)

[0425] Using the synthetic route in Example 2.2, the reaction raw material 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester to give the title compound (15 mg, yield: 38.8%).

[0426] MS m / z(ESI):616.1[M+H] + .

[0427] 1 H NMR (400MHz, DMSO-d6): δ9.85(s,1H),8.12(s,1H),7.65(s,1H),7.61(s,1H),7.50(s,1H),7.43(s,1H),7.34( s,1H),4.21-4.31(m,4H),3.83(s,3H),3.70(s,2H),3.21-3.24(m,5H),2.56-2.60(m,4H),2.27-2.33(m,2H).

[0428] Example 2.10: Preparation of ethyl 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-10)

[0429] Step 1: Preparation of ethyl 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0430] Ethyl 4-(6-hydroxy-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (97.0 mg, 235.17 μmol), ethyl 3-(6-methoxy-N-methyl-5-(3-(tosyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propanoate (129.2 mg, 235.06 μmol), potassium carbonate (65.0 mg, 471.01 μmol) and potassium iodide (39.4 mg, 237.35 μmol) were added sequentially to N,N-dimethylformamide (1 mL), and the temperature was raised to 50°C and stirred for 12 hours. After the reaction system was cooled to room temperature, the reaction solution was poured into water (10 mL) and extracted three times with ethyl acetate (10 mL). The organic phases were combined and washed three times with saturated brine (10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 3:1, volume ratio) to give the title compound (36 mg, yield: 19.4%).

[0431] MS m / z(ESI):790.2[M+H] + .

[0432] Step 2: Preparation of ethyl 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0433] Ethyl 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-(2-oxo-2-phenylethoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (36 mg, 45.57 μmol) was completely dissolved in tetrahydrofuran (3 mL). Acetic acid (1 mL) was added at room temperature, followed by zinc powder (59 mg, 907.69 μmol). The reaction mixture was stirred at 60°C for 6 hours. The reaction mixture was filtered, the filtrate was collected, and the solvent was concentrated to obtain the title compound (21.0 mg, yield: 68.6%).

[0434] MS m / z(ESI):672.2[M+H] + .

[0435] Example 2.11: Preparation of 4-(6-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-11)

[0436] Completely dissolve ethyl 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (21.0 mg, 31.26 μmol) in a mixture of tetrahydrofuran (2 mL) and water (0.3 mL). Separately, completely dissolve lithium hydroxide monohydrate (5.3 gm, 126.31 μmol) in water (0.2 mL). Slowly add the lithium hydroxide aqueous solution dropwise to the reaction system at 0°C, slowly warm to room temperature, and stir for 12 hours. Water (10 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. The residue was adjusted to pH 4 with dilute hydrochloric acid (1N, 10 mL) and filtered. The filter cake was washed with water (10 mL) and dried in vacuo at 50°C to give the title compound (10.0 mg, yield: 52.0%).

[0437] MS m / z(ESI):616.1[M+H] + .

[0438] 1 H NMR (400MHz, DMSO-d6): δ12.22(s,2H),9.32(s,1H),8.16(s,1H),7.63(s,1H),7.59(s,1H),7.56(s,1H),7.47(s,1H), 7.34(s,1H),4.25(t,J=5.2Hz,4H),3.84(s,3H),3.70(s,2H),3.16-3.27(m,5H),2.56-2.62(m,4H),2.23-2.33(m,2H).

[0439] Example 2.12: Preparation of ethyl 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-12)

[0440] Using the synthetic route in Example 2.10, the first step reaction raw material 3-(6-methoxy-N-methyl-5-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester was replaced with 3-(5-methoxy-N-methyl-6-(3-(toluenesulfonyloxy)propoxy)benzo[b]thiophene-2-carboxamido)propionic acid ethyl ester to obtain the title compound (18 mg, yield: 47.5%).

[0441] MS m / z(ESI):672.2[M+H] + .

[0442] Example 2.13: Preparation of 4-(6-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-13)

[0443] Using the synthetic route in Example 2.11, the reaction raw material 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with 4-(6-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-5-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester to give the title compound (16 mg, yield: 52.9%).

[0444] MS m / z(ESI):616.1[M+H] + .

[0445] 1 H NMR (400MHz, DMSO-d6): δ12.20(s,2H),9.31(s,1H),8.16(s,1H),7.65(s,1H),7.61(s,1H),7.59(s,1H),7.42(s,1H),7.34 (s,1H),4.22-4.31(m,4H),3.82(s,3H),3.70(s,2H),3.16-3.28(m,5H),2.56-2.61(m,4H),2.29(dd,J=16.0,10.4Hz,2H).

[0446] Example 2.14: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-hydroxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (C-14)

[0447] Step 1: Preparation of ethyl 4-(5-(3-((6-bromo-2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)benzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0448] Ethyl 3-(6-bromo-5-hydroxy-N-methylbenzo[b]thiophene-2-carboxamido)propanoate (750.0 mg, 1.94 mmol), ethyl 4-(6-methoxy-5-(3-(tosyloxy)propoxy)benzo[b]thiophene-2-yl)-4-oxobutanoate (1.0 g, 1.92 mmol) and potassium carbonate (536.0 mg, 3.88 mmol) were added sequentially to N,N-dimethylformamide (10 mL), and the temperature was raised to 50°C and stirred for 12 hours. After the reaction system was cooled to room temperature, the reaction solution was poured into water (50 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed three times with saturated brine (10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 2:1, volume ratio) to give the title compound (1.1 g, yield: 77.1%).

[0449] MS m / z(ESI):734.1[M+H] + .

[0450] Step 2: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)benzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0451] Ethyl 4-(5-(3-((6-bromo-2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)benzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (590 mg, 803.08 μmol), pinacol diboron (306 mg, 1.20 mmol), potassium acetate (160 mg, 1.63 mmol) and 1,1'-bisdiphenylphosphinoferrocenepalladium dichloride (120 mg, 164 μmol) were added to 1,4-dioxane (6 mL) in sequence. The reaction system was bubbling with nitrogen for 5 minutes, then heated to 100 °C and stirred for 4 hours. After the reaction system was cooled to room temperature, the solvent was removed by concentration. The residue was dissolved in ethyl acetate (20 mL) and washed three times with saturated brine (10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:1, volume ratio) to give the title compound (60 mg, yield: 9.6%).

[0452] MS m / z(ESI):782.3[M+H] + .

[0453] Step 3: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-hydroxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

[0454] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (35 mg, 44.77 μmol) was completely dissolved in a mixed solution of acetone (2 mL) and water (0.5 mL). Potassium peroxosulfate (40 mg, 115.51 μmol) was slowly added in portions at room temperature, and the reaction system was stirred at room temperature for 4 hours. The reaction system was concentrated to remove acetone as much as possible. The residue was dissolved in ethyl acetate (5 mL) and washed three times with saturated brine (6 mL). The residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 2:1, volume ratio) to give the title compound (12 mg, yield: 40.0%).

[0455] MS m / z(ESI):672.2[M+H] + .

[0456] Example 2.15: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-hydroxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (C-15)

[0457] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-hydroxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (12.0 mg, 17.86 μmol) was completely dissolved in a mixed solvent of tetrahydrofuran (2 mL) and water (0.3 mL). Separately, lithium hydroxide monohydrate (3.3 mg, 78.64 μmol) was completely dissolved in water (0.2 mL). The lithium hydroxide aqueous solution was slowly added dropwise to the reaction system at 0°C, and the temperature was slowly raised to room temperature and stirred for 12 hours. Water (5 mL) was added to the reaction solution, and the mixture was concentrated under reduced pressure at 40°C to remove the organic solvent. The residue was adjusted to pH 4 with dilute hydrochloric acid (1N, 10 mL) and filtered. The filter cake was washed with water (10 mL) and dried in vacuo at 50°C to give the title compound (2.0 mg, yield: 18.2%).

[0458] MS m / z(ESI):616.1[M+H] + .

[0459] 3. Preparation of drug-linker

[0460] Example 3.1: 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((2-((((4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3- Preparation of (4-(2-(triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-1)

[0461] Step 1: Preparation of tert-butyl 4-(5-(3-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((4-nitrophenoxy)carbonyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0462] Tert-butyl 4-(5-(3-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-hydroxybenzo[b]thiophen-2-yl)-4-oxobutanoate (86 mg, 0.12 mmol) and N,N-diisopropylethylamine (31 mg, 0.24 mmol) were dissolved in N,N-dimethylformamide (1 mL). Di(p-nitrobenzene) carbonate (73 mg, 0.24 mmol) was slowly added to the reaction solution at 0°C, and the temperature was slowly raised to room temperature and stirred for 3 hours. The reaction solution was poured into water (20 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed three times with saturated brine (15 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:1, volume ratio) to give the title compound (81 mg, yield: 75.8%).

[0463] MS m / z(ESI):893.3[M+H] + .

[0464] Step 2: Preparation of tert-butyl 4-(6-(((2-((((4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl))-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0465] Tert-butyl 4-(5-(3-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((4-nitrophenoxy)carbonyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (80 mg, 0.09 mmol) and N,N-diisopropylethylamine (35 mg, 0.27 mmol) were dissolved in N,N-dimethylformamide ( To the reaction mixture (1 mL), benzyl 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)methyl(2-(methylamino)ethyl)carbamate (93 mg, 0.09 mmol) was slowly added at 0°C. The reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was purified by preparative high-performance liquid chromatography to obtain the title compound (109 mg, yield: 67.7%).

[0466] MS m / z(ESI):1783.8[M+H] + .

[0467] Step 3: 4-(5-(3-((2-((3-(tert-Butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((2-(((4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl))hex-5-ynamide)methyl)-1H-1 Preparation of tert-butyl (2,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0468] 4-(6-(((2-((((4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl))-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((3-(tert-butyloxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl) Tert-butyl ((2-( ...

[0469] MS m / z(ESI):2089.9[M+H] + .

[0470] Step 4: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((2-((((4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatriacontamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0471] 4-(5-(3-((2-((3-(tert-butoxy)-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((2-(((4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl))hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl) Tert-butyl (51 mg, 0.024 mmol) of (1,2-(4-((2-( ...

[0472] MS m / z(ESI):1976.7[M+H] + .

[0473] Example 3.2: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-((methyl(2-(methyl(((4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)propionamido)benzyl)oxy)carbonyl)amino)ethyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-2)

[0474] Step 1: Preparation of ethyl 4-(6-(((2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0475] (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (27.5 mg, 40.40 μmol) and N,N-diisopropylethylamine (11 mg, 85.11 μmol) were dissolved in N,N-dimethylformamide (1 mL). To the reaction solution was slowly added ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-((methyl(2-(methylamino)ethyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (31.75 mg, 40.40 μmol) at 0°C. The reaction solution was stirred at 20°C for 2 hours. The reaction solution was purified by preparative HPLC to obtain the title compound (12 mg, yield: 22.4%).

[0476] MS m / z(ESI):1327.5[M+H] + .

[0477] Step 2: Preparation of 4-(6-(((2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0478] Ethyl 4-(6-(((2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (12 mg, 9.04 μmol) was dissolved in a mixed solvent of methanol (1 mL), tetrahydrofuran (1 mL) and water (0.05 mL). Lithium hydroxide monohydrate (3.0 mg, 71.42 μmol) was added to the reaction solution at room temperature, and the mixture was stirred at room temperature for 4 hours. The solvent was removed by concentration, and water (5 mL) was added to the residue. The pH was adjusted to 5 with dilute hydrochloric acid (1N, 2 mL). The mixture was filtered, and the filter cake was washed with water (5 mL) and dried to give the title compound (9 mg, yield: 94.9%).

[0479] MS m / z(ESI):1049.4[M+H] + .

[0480] Step 3: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-((methyl(2-(methyl(((4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butyramido)propionamido)benzyl)oxy)carbonyl)amino)ethyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0481] 4-(6-(((2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl )-4-oxobutanoic acid (9 mg, 8.58 μmol) and N,N-diisopropylethylamine (5.8 mg, 44.88 μmol) were dissolved in N,N-dimethylformamide (1 mL). 2,5-Dioxopyrrolidin-1-yl 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoate (5.3 mg, 14.51 μmol) was added to the reaction mixture at room temperature and stirred at room temperature for 4 hours. The solvent was removed by concentration, and the resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (3 mg, 26.9% yield).

[0482] MS m / z(ESI):1299.4[M+H] + .

[0483] Example 3.3: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-((methyl(2-(methyl(((4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)carbamoyl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-3)

[0484] Using the synthetic route in Example 3.2, the first step reaction raw material (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobutan-2-yl)carbamate was replaced with (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate to give the title compound (2 mg, yield: 12.2%).

[0485] MS m / z(ESI):1385.5[M+H] + .

[0486] Example 3.4: Preparation of (S)-4-(6-((7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaeicosyl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-4)

[0487] 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (100 mg, 0.14 mmol) and N,N-diisopropylethylamine (37 mg, 0.28 mmol) were dissolved in N,N-dimethylformamide (2 mL). 2,5-dioxopyrrolidin-1-yl (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalaninate (187 mg, 0.34 mmol) was added to the reaction mixture at room temperature, and stirring was maintained at room temperature for 4 hours. The solvent was removed by concentration, and the resulting residue was purified by preparative high performance liquid chromatography to give the title compound (36 mg, yield: 11.3%).

[0488] MS m / z(ESI):1156.4[M+H] + .

[0489] Example 3.5: Preparation of (S)-4-(6-((7-benzyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicos-19-yn-1-yl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-5)

[0490] Step 1: Preparation of ethyl (S)-4-(6-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl)oxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

[0491] Ethyl 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (50.0 mg, 65.97 μmol), (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-[[(9H-fluoren-9-yl)methoxy]carbonyl] ... 4-Phenylalanine (45.0 mg, 89.73 μmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (53.0 mg, 139.39 μmol) were added to N,N-dimethylformamide (3 mL). N,N-diisopropylethylamine (42.6 mg, 329.62 μmol) was added at room temperature, and the reaction system was stirred at 25°C for 2 hours. The reaction solution was poured into water (15 mL) and extracted three times with ethyl acetate (10 mL). The organic phases were combined and washed three times with saturated brine (6 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: 100% ethyl acetate) to obtain the title compound (50.0 mg, yield: 68.4%).

[0492] MS m / z(ESI):1241.4[M+H] + .

[0493] Step 2: Preparation of (S)-4-(6-((13-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0494] (S)-ethyl 4-(6-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl)oxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (50 mg, 40.28 μmol) was dissolved in a mixed solvent of methanol (1 mL), tetrahydrofuran (1 mL) and water (0.1 mL). Lithium hydroxide monohydrate (13.5 mg, 321.43 μmol) was added to the reaction solution at room temperature, and the mixture was stirred at room temperature for 4 hours. The solvent was removed by concentration, and the resulting residue was purified by preparative HPLC (30 mg, yield: 77.3%).

[0495] MS m / z(ESI):963.3[M+H] + .

[0496] Step 3: Preparation of (S)-4-(6-((7-benzyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicos-19-yn-1-yl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

[0497] (S)-4-(6-((13-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)oxy)-5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (10 mg, 10.38 μmol) and N,N-diisopropylethylamine (2.7 mg, 20.89 μmol) were dissolved in N,N-dimethylformamide (1 mL). 2,5-dioxopyrrolidin-1-yl 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoate (5.3 mg, 14.51 μmol) was added to the reaction mixture at room temperature and stirred at room temperature for 4 hours. The solvent was removed by concentration, and the resulting residue was purified by preparative high performance liquid chromatography to give the title compound (4 mg, yield: 31.7%).

[0498] MS m / z(ESI):1213.3[M+H] + .

[0499] Example 3.6: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-6-(((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecan-16-yn-1-yl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-6)

[0500] The synthetic route in Example 3.5 was used, and the first step reaction raw material 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced by (S)-4-(6-((2-aminopropionamido)methoxy)-5-(3-((2- Ethyl ((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propyloxy)benzo[b]thiophen-2-yl)-4-oxobutanoate, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine was replaced with (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine to give the title compound (8 mg, yield: 29.4%).

[0501] MS m / z(ESI):1108.3[M+H]+ .

[0502] Example 3.7: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-(((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecan-16-yn-1-yl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-9)

[0503] Using the synthetic route in Example 3.5, the first step reaction raw material 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with (S)-4-(6-((2-aminopropionamido)methoxy)-5-(3-((2-( (3-Ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propyloxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine was replaced with (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine to give the title compound (5.8 mg, yield: 64.2%).

[0504] MS m / z(ESI):1108.3[M+H] + .

[0505] Example 3.8: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-(((4S,7R,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecan-16-yn-1-yl)oxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid (DL-10)

[0506] Using the synthetic route in Example 3.5, the first step reaction raw material 4-(6-((2-aminoacetamido)methoxy)-5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester was replaced with (S)-4-(6-((2-aminopropionamido)methoxy)-5-(3-((2-( (3-Ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propyloxy)benzo[b]thiophen-2-yl)-4-oxobutanoic acid ethyl ester, (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine was replaced with (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-D-alanine to give the title compound (14.0 mg, yield: 29.2%).

[0507] MS m / z(ESI):1108.3[M+H] + .

[0508] 4. Preparation of Immunostimulatory Antibody Conjugates (ISACs)

[0509] Preparation method A:

[0510] The trastuzumab monoclonal antibody stock solution was adjusted to a pH of 7.6-7.7 with 1M sodium hydrogen phosphate solution and then diluted to 10 mg / mL with phosphate buffered saline (PBS) at pH 7.6-7.7 to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA and TCEP (6 eq, 10 mM) were added to the trastuzumab monoclonal antibody buffer solution, shaken well, and reacted at room temperature for 2 hours. A DMSO solution (10 eq, 10 mM) of the drug linker was added to the reaction system at room temperature, shaken well, and reacted at room temperature for 2 hours. The solution was then purified using a NAP-10 gel column and washed with histidine hydrochloride buffer at pH 6.0-6.1. The filtrate was collected to obtain the histidine hydrochloride buffer solution for trastuzumab-ISAC and stored at 4°C.

[0511] Preparation method B:

[0512] Adjust the IgG1 mAb stock solution to a pH of 7.6-7.7 with 1M sodium hydrogen phosphate solution, then dilute to 10 mg / mL with phosphate-buffered saline (PBS) at pH 7.6-7.7 to obtain an IgG1 mAb buffer solution. Add 0.1M EDTA and TCEP (6 eq, 10 mM) to the IgG1 mAb buffer solution, shake well, and react at room temperature for 2 hours. Add a DMSO solution (10 eq, 10 mM) of the drug linker to the reaction system at room temperature, shake well, and react at room temperature for 2 hours. Purify the reaction using a NAP-10 gel column and wash with histidine hydrochloride buffer at pH 6.0-6.1. Collect the filtrate to obtain the histidine hydrochloride buffer for the IgG1-ISAC and store at 4°C.

[0513] 5. Drug / Antibody Ratio of Immunostimulatory Antibody Conjugates (ISACs): Determination of DAR Value

[0514] The molecular weight of ISAC was determined by LC-MS, and the drug / antibody ratio (DAR) was calculated.

[0515] Chromatographic determination conditions:

[0516] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0517] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0518] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0519] Mass spectrometry conditions:

[0520] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0521] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0522] m / z 600-4000; Time bins to sum 40.

[0523] The average DAR values ​​of immunostimulatory antibody conjugates (ISACs) were calculated by CE-SDS. The results are shown in Table 1:

[0524] Table 1. Average DAR value test results of immunostimulatory antibody conjugates (ISACs)

[0525] Biological tests

[0526] Experimental Example 1. Effect of Compounds on THP1-Blue TM STING-mediated agonism of interferon (IFN) signaling reporter genes in ISG cells

[0527] In this experiment, THP1-Blue TM The activity of the SEAP (secreted embryonic alkaline phosphatase) reporter gene regulated by IFN regulatory factor was detected in ISG cells (InvivoGen) to evaluate the stimulatory effect of the test compound on the STING-mediated IFN signaling pathway at the cellular level.

[0528] THP1-Blue in the logarithmic growth phase TM ISG cells (InvivoGen) were centrifuged and resuspended in cell culture medium to a density of 2×10 6 / mL, and inoculated into 96-well cell culture plates (Corning) at 50 μL / well. The compound stock solution was serially diluted with cell culture medium to 2× working concentration dilutions of 60, 7.5, 0.94, 0.12, 0.015, 0.0018, 0.00023, 0.00003 and 0 μM, and 50 μL / well of the compound 2× working concentration dilution was added to the 96-well plate (the final DMSO concentration was 1%). The culture plate was placed in a cell culture incubator and incubated for 16 hours. After the cell incubation was completed, 10 μL of cell culture supernatant was transferred to a 96-well plate, 90 μL / well of QUANTI-Blue (InvivoGen) solution was added, and the plate was incubated at 37°C for 3 hours. The absorbance value (OD) at 620 nm was read using a microplate reader (BMG LABTECH). 620nm ).

[0529] EC 50 The log(agonist) vs. response--Variable slope fitting calculation was performed using Graphpad Prism software. Emax is the OD at which the drug activation effect reaches the maximum in this test. 620nm value.

[0530] Experimental results:

[0531] The effects of the compounds on THP1-Blue were determined according to the above method. TM The activation activity of ISG cells was measured by EC 50 and Emax, the results are shown in Table 2:

[0532] Table 2 Agonistic effects of test compounds on STING-mediated Interferon (IFN) signaling pathway

[0533] The results showed that compounds C-3, C-7, C-9 and C-11 had a significant effect on THP1-Blue TM The STING-mediated interferon (IFN) signaling pathway in ISG cells has a strong stimulatory effect.

[0534] Experimental Example 2. Activation of the Compound on the Expression of STING Pathway Cytokine mIFN-β in Murine Raw264.7 Cells

[0535] In this experiment, the expression of the STING signaling pathway cytokine mIFN-β was detected in Raw264.7 cells (Nanjing Kebai) by ELISA, thereby evaluating the stimulatory effect of the test compound on the expression of the STING pathway cytokine mIFN-β at the cellular level.

[0536] Raw264.7 cells (Nanjing Kebai) in the logarithmic growth phase were centrifuged and resuspended in cell culture medium to a density of 8×10 6 / mL, 250μL / well cell suspension was inoculated into a 24-well cell culture plate (Corning). The test compound was diluted with cell culture medium to a 2× working concentration of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09 and 0μM. 250μL of the test compound dilution was added to the 24-well plate (the final DMSO concentration was 1%), mixed thoroughly with the cells, and placed in a cell culture incubator for 6 hours. After the cell incubation was completed, the supernatant was collected by centrifugation at 300g x 5min, 100μL of cell culture medium was aspirated, and the primary antibody and HRP-labeled secondary antibody were incubated according to the steps of the Mouse IFN-β ELISA kit (PBL-42400-2), color was developed, and the absorbance at a wavelength of 450nm (OD 450nm ).

[0537] EC was calculated by log(agonist) vs. response--Variable slope fitting using Graphpad Prism software. 50 , Emax is the secretion amount of mIFN-β that reaches the maximum activation effect of the drug in this trial.

[0538] Experimental results:

[0539] The activation activity of the compound on the STING pathway cytokine mIFN-β in mouse Raw264.7 cells was determined according to the above method. The activity of the compound was measured by EC 50 and Emax, the results are shown in Table 3:

[0540] Table 3 Activation activity of test compounds on the expression of STING pathway cytokine mIFN-β in mouse Raw264.7 cells

[0541] The results showed that compounds C-3, C-9 and C-11 had a strong stimulatory effect on the expression of STING pathway cytokine mIFN-β in mouse Raw264.7 cells.

[0542] Experimental Example 3: Determination of the ability of immunostimulatory antibody conjugates (ISACs) to stimulate TNF-α secretion from human peripheral blood mononuclear cells (PBMCs) in the presence of tumor cells

[0543] Experimental steps:

[0544] NCI-N87 tumor cells (ATCC) expressing HER2 in the logarithmic growth phase were collected, washed twice with PBS, and resuspended in RPMI1640+10% heat-inactivated FBS complete medium to adjust the cell density to 2×10 6 NCI-N87 tumor cells were seeded in 96-well plates (Corning) at 1×10 cells / mL. 5 PBMC (Aoneng Biotech) were resuspended in RPMI1640+10% heat-inactivated FBS complete medium and the density was adjusted to 1×10 7 5 × 10 cells / mL were added to a 96-well plate inoculated with tumor cells. 5 PBMC cells were added to the wells. Samples of trastuzumab monoclonal antibody and immunostimulatory antibody conjugate (ISAC) diluted in 100 μL of RPMI1640 + 10% heat-inactivated FBS complete medium were added to the wells to final concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, 0.39 nM, and 0.20 nM, respectively. 100 μL of RPMI1640 + 10% heat-inactivated FBS complete medium was added to the blank wells. The cells were incubated in a 5% CO2, 37°C incubator for 24 hours. 100 μL of supernatant was collected from each well and assayed. Primary antibody and HRP-labeled secondary antibody were incubated according to the human TNF-α ELISA kit (R&D system). Color was developed and absorbance was read at 450 nm. TNF-α concentrations were calculated using Graphpad Prism software based on the standard curve.

[0545] EC 50 The log(agonist) vs. response--Variable slope fitting calculation was performed using Graphpad Prism software, and Emax was the maximum secretion amount of TNF-α when the drug activated the test drug.

[0546] Experimental results:

[0547] The ability of trastuzumab and various immunostimulatory antibody conjugates (ISACs) to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete TNF-α in the presence of NCI-N87 tumor cells was determined according to the above method. The results are shown in Table 4:

[0548] Table 4. Results of TNF-α secretion stimulated by each test substance in the in vitro NCI-N87 tumor cell and PBMC co-incubation system

[0549] The results showed that the immunostimulatory antibody conjugates Trastuzumab-ISAC-2, Trastuzumab-ISAC-3 and Trastuzumab-ISAC-4 can effectively stimulate PBMC to secrete TNF-α in the in vitro NCI-N87 tumor cell and PBMC co-incubation system.

[0550] Experimental Example 4. In vivo efficacy study of immunostimulatory antibody conjugate (ISAC) in NCI-N87 cell subcutaneous tumor-bearing mouse model

[0551] Experimental steps:

[0552] Each BALB / c Nude mouse (Jicui Yaokang) was subcutaneously inoculated with 5×10 6 NCI-N87 cells (suspended in 0.1 mL PBS). 3 Mice with irregular, small, or large tumors were removed. The remaining mice were randomly divided into groups based on tumor volume and body weight. They were intraperitoneally injected (ip) with 0.9% sodium chloride injection (vehicle control), isotype control IgG1-ISAC-4 (10 mg / kg), and immunostimulatory antibody conjugate Trastuzumab-ISAC-4 (10 mg / kg) once weekly for two doses. The efficacy of the test drugs in this tumor-bearing mouse model and the animals' tolerance to the test drugs were observed. Mice were weighed and tumor volumes were measured twice weekly, and the data were recorded.

[0553] Data statistics, tumor volume (V) calculation formula: V = 1 / 2 × a × b 2, where a and b represent the length and width respectively. The anti-tumor drug efficacy was evaluated by the tumor growth inhibition rate TGI (%), and the calculation formula: TGI (%) (瘤体积) =[1-(T Vt -T V0 ) / (C Vt -C V0 )]×100%, T V0 is the average tumor volume of the test compound group during grouped administration, T Vt is the average tumor volume of the test compound group at t days after administration; C V0 is the average tumor volume of the vehicle group during grouped administration; C Vt is the average tumor volume of the vehicle group at t days after administration. When the tumor regresses, TGI (%) (瘤体积) =100%-(T Vt -T V0 ) / T V0 ×100%. If the tumor shrinks compared to the initial volume, i.e., V t <V0, it is defined as partial regression (PR) of the tumor; if the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0554] Experimental results:

[0555] Figure 1 is a curve showing the change of tumor volume over time in each group in Experimental Example 4, which shows the in vivo efficacy of each test drug on the subcutaneous inoculation of NCI-N87 human gastric cancer xenograft tumor model in BALB / c Nude mice. After 28 days of administration, the immunostimulatory antibody conjugate Trastuzumab-ISAC-4 could significantly inhibit tumor growth. Compared with the vehicle control group, the TGI of tumor volume was 99.6%, P<0.001, and the effect of the isotype control IgG1-ISAC-4 was significantly weaker (Figure 1, Table 5). The results showed that in the subcutaneous inoculation of NCI-N87 human gastric cancer xenograft tumor model in BALB / c Nude mice, intraperitoneal injection (i.p.) of the immunostimulatory antibody conjugate Trastuzumab-ISAC-4, once a week for a total of two times (QW×2), showed a very significant tumor inhibitory effect, significantly better than the vehicle control group and the isotype control group.

[0556] Table 5. Efficacy of immunostimulatory antibody conjugates on the subcutaneous tumor-bearing mouse model of NCI-N87 cells PR: indicates partial regression; CR: indicates complete regression.

[0557] The above-described embodiments do not limit the solutions of the present application in any way. In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including all patents, patent applications, journal articles, books and any other disclosures) is incorporated herein by reference in its entirety.

Claims

1. Immunostimulatory antibody conjugate of formula (I): in, Ab is an antibody or antigen-binding fragment thereof that targets the target antigen; M is a linker site for connecting an antibody or an antigen-binding fragment thereof; L is a linker connecting M and D; D is selected from the structure of formula (DI-1) and formula (DI-2): in represents the connection point with L; X 1 and X 3 are the same or different and are each independently selected from a covalent bond, -O-, -S- and -NR a -; X 2 Selected from C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group, the C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted by one or more substituents independently selected from the group consisting of H, halogen, cyano, C 1-6 Alkyl, -OR a and-NR a R b ; X 4 and X 5 are each independently selected from -O- and -NR a -; L 1 Selected from covalent bonds and -(C(R 6 )2) j -; L 2 Selected from covalent bonds and -(C(R 7 )2) k -; R 1 and R 2 are the same or different and are each independently selected from H, halogen, cyano, -OR a 、-NR a R b 、-C(O)-OR a 、-C(O)-NR a R b 、-NR a -C(O)-R a , C 1-6 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group, the C 1-6 Alkyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted by one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a and-NR a R b ; R 3 Selected from H and C 1-6 alkyl; R 4 Selected from H, -OR a 、-NR a R b 、-C(O)-OR a 、-OC(O)-R a 、-C(O)-NR a R b 、-NR a -C(O)-R a 、-OC(O)-NR a R b 、-NR a -C(O)-OR a 、-NR a -C(O)-NR a R b , C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the C 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ; R 5 Selected from H, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, -OR a and -C(O)-OR a , the C 1-6 Alkyl, C 3- 10 The cycloalkyl and 3-10 membered heterocyclyl groups are each optionally substituted with one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ; R 6 Each independently selected from H, -OR a 、-NR a R b , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group, the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted by one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ; or Two R on different carbon atoms 6 Together with the carbon atoms between them, they form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; or Two R on the same carbon atom 6 Together with the carbon atoms they are connected to, they form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; R 7 Each independently selected from H, -OR a 、-NR a R b , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group, the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted by one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, -OR a 、-NR a R b and -C(O)-OR a ; or Two R on the same carbon atom 7 Together with the carbon atoms they are connected to, they form C 3-6 Cycloalkyl or 3-6 membered heterocyclic group; or any one of R 7 and R 3 Together with the atoms between them, they form a 3-10 membered heterocyclic group; R a and R b Each independently selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic group, the C 1-6 Alkyl, C 3-6 The cycloalkyl and 3-6 membered heterocyclyl groups are each optionally substituted by one or more substituents independently selected from the group consisting of H, halogen, C 1-6 Alkyl, hydroxy, amino and carboxyl groups; j and k are each independently selected from 1, 2 and 3; m and q are each independently selected from 0, 1 and 2; n and p are each independently selected from 0, 1, 2 and 3; z is selected from 1-10.

2. The immunostimulatory antibody conjugate according to claim 1, wherein M is selected from the following structures: Wherein, the 1 position of M is connected to Ab, and the 2 position is connected to L; each a is independently selected from an integer of 1-6, and b is selected from an integer of 1-10; 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 L.

3. The immunostimulatory antibody conjugate according to any one of claims 1 to 2, wherein L is selected from a non-cleavable linker and a cleavable linker, wherein the cleavable linker is cleaved 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; Preferably, L is -L a -L b -L c -,in: L a is a covalent bond or is selected from wherein each c is independently selected from an integer of 1-6, and each d is independently selected from an integer of 1-10; L b 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; L c is a covalent bond, -NH-CH2- or selected from the following structures: Preferably, L a is a covalent bond or Preferably, L a It is a covalent bond; Preferably, L b is a covalent bond or is selected from the following structures: Preferably, L b Selected from 4. The immunostimulatory antibody conjugate according to any one of claims 1 to 3, wherein L is selected from the following structures: Among them, the 3 position of L is connected to M, and the 4 position is connected to D.

5. The immunostimulatory antibody conjugate according to any one of claims 1 to 4, wherein D is selected from the structure of formula (D-II-1) and formula (D-II-2): in, Represents the connection point with L, each group R 1 , R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , n and p are as defined in any one of claims 1 to 4; Preferably, D is selected from the structures of formula (D-III-1), formula (D-III-2), formula (D-III-3) and formula (D-III-4): in, Represents the connection point with L, each group R 2 , X 1 , X 2 and X 3 As defined in any one of claims 1 to 4.

6. The immunostimulatory antibody conjugate according to any one of claims 1 to 5, wherein D is selected from the following structures: in, represents the connection point with L; Preferably, D is 7. The immunostimulatory antibody conjugate according to any one of claims 1 to 6, wherein 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): 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): 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): 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): 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.

8. The immunostimulatory antibody conjugate according to any one of claims 1 to 7, selected from the following structures: Each z is selected from 1 to 10; Preferably, the Ab is trastuzumab.

9. An immunostimulatory antibody conjugate selected from the following structures: Each z is selected from 1 to 10; Ab is selected from anti-Her-2 antibody; Preferably, the Ab contains the VH shown in SEQ ID NO: 18, and / or the VL shown in SEQ ID NO: 19; Preferably, the Ab comprises the heavy chain of VH shown in SEQ ID NO: 18 and CH shown in SEQ ID NO: 20, and / or the light chain of VL shown in SEQ ID NO: 19 and CL shown in SEQ ID NO: 21; Preferably, the Ab is trastuzumab.

10. The drug linker of formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: M 1 -LD (II) in, M 1 Selected from wherein each a is independently selected from an integer of 1-6, and b is selected from an integer of 1-10; L and D are as defined in any one of claims 1-9.

11. The drug linker according to claim 10, selected from the following structures:

12. A compound represented by formula III-1 or III-2 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: in, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , X 1 , X 2 , X 3 , X 4 , X 5 , m, n, p and q are as defined in any one of claims 1 to 9; Preferably, the compound is selected from the following structures:

13. A compound of formula IV-1 or IV-2 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: in, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , X 1 , X 2 , X 3 , X 4 , X 5 , m, n, p and q are as defined in any one of claims 1 to 9; R 8 and R 9 Each independently selected from -C 1-6 Alkylene-NR 12 -C(=O)-C 1-6 Alkylene-NR 10 R 11 、-C(=O)OC 6-10 Aryl, -C(=O)NR 12 -C 1-6 Alkylene-NR 10 R 11 , -C 1-6 Alkylene-OC 1-6 Alkyl and -C 1-6 Alkylene-C(=O)-C 6-10 Aryl, wherein the aryl is optionally substituted with amino or nitro; R 10 and R 11 Each independently selected from H, C 1-6 Alkyl, Fmoc and Boc; R 12 Selected from H and C 1-6 alkyl; Preferably, the compound is selected from the following structures:

14. Use of the drug linker of claim 10 or 11 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or the compound of claim 12 or 13 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof in the preparation of an immunostimulatory antibody conjugate; Preferably, the immunostimulatory antibody conjugate is as described in any one of claims 1-9.

15. A pharmaceutical composition comprising the immunostimulatory antibody conjugate according to any one of claims 1 to 9; Preferably, the DAR value of the pharmaceutical composition is 1.0-10.0, such as 6.0-9.0; Preferably, the pharmaceutical composition further contains one or more pharmaceutical excipients.

16. A pharmaceutical composition comprising the immunostimulatory antibody conjugate of any one of claims 1 to 9, the drug linker of claim 10 or 11 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the compound of claim 12 or 13 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, and one or more pharmaceutical excipients.

17. Use of an immunostimulatory antibody conjugate as described in any one of claims 1 to 9, a drug linker as described in claim 10 or 11, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, a compound as described in claim 11 or 12, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition as described in claim 15 or 16 in the preparation of a medicament for treating and / or preventing cancer (e.g., HER2-positive cancer, e.g., HER2-positive gastric cancer, breast cancer or non-small cell lung cancer).

18. An immunostimulatory antibody conjugate according to any one of claims 1 to 9, a drug linker according to claim 10 or 11, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, a compound according to claim 12 or 13, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition according to claim 15 or 16, for use in treating and / or preventing cancer (e.g., HER2-positive cancer, such as HER2-positive gastric cancer, breast cancer or non-small cell lung cancer).

19. 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 an individual in need thereof a therapeutically and / or prophylactically effective amount of an immunostimulatory antibody conjugate as described in any one of claims 1 to 9, a drug linker as described in claim 10 or 11, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, a compound as described in claim 12 or 13, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or a pharmaceutical composition as described in claim 15 or 16.

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