Antibody-drug conjugate having linking system

Abstract

An antibody-drug conjugate (ADC) having a linking system, a preparation method therefor, and the use thereof. The linking system has high linking stability and enhanced solubility, allows efficient coupling of hydrophobic drugs, and effectively achieves intracellular delivery of drugs.

Description

Antibody-drug conjugates with linker systems

[0001] This application claims priority to an earlier application filed on November 1, 2024, with patent application number 202411553898.5 and entitled "Antibody-Drug Conjugate with Linkage System"; the entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of medicinal chemistry, and specifically relates to antibody-drug conjugates with a linker system. Background Technology

[0003] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active drugs via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of both normal and tumor cells, as well as the high efficacy of drugs, while avoiding the drawbacks of lower efficacy with antibodies and excessive toxicity with drugs. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

[0004] Microtubules are powerful, filamentous cytoskeletal proteins involved in a wide range of cellular functions, including intracellular migration and transport, cell signaling, and maintaining cell shape. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, necessary for chromosomes to divide into two daughter cells. The biological function of microtubules in all cells is largely regulated by their polymerization kinetics, which occur through the reversible, non-covalent addition of α- and β-tubulin dimers to the ends of the microtubules. This kinetic behavior and the resulting control over microtubule length are essential for the proper functioning of the mitotic spindle. Even minor alterations in microtubule kinetics can involve axon checkpoints, inhibiting cell cycle progression during mitosis and subsequently leading to cell death. Because cancer cells divide rapidly, they are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. Tubulin inhibitors, such as MMAE and eribulin, hold promise as potential drugs for cancer treatment.

[0005] Eribulin is a spongin-like microtubule dynamics inhibitor with unique binding properties. Besides its mechanism of action of inhibiting microtubule dynamic growth, non-clinical studies have shown that eribulin has unique effects on the tumor microenvironment, such as increasing vascular perfusion and permeability of the tumor core, promoting epithelial status, and reducing the migration ability of breast cancer cells. Currently, eribulin has been approved in more than 70 countries and regions, including Europe, the Americas, and Asia, for the treatment of breast cancer. The most common grade 3-4 adverse reactions in eribulin mesylate treatment are, in descending order, neutropenia (64%). Antibody-drug conjugates (ADCs) are conjugates of antibodies and small molecule drugs, combining the tumor-targeting activity of antibodies with the activity of bioactive molecules, becoming a kind of biological missile with very promising efficacy and safety advantages. Antibodies guide ADCs to bind to target cells, which are then internalized. The small molecule drug is then released intracellularly through enzymatic decomposition under the action of specific enzymes, treating the disease. Therefore, it is expected that the ADC composed of eribulin and tumor-targeting antibodies can maintain the therapeutic effect of eribulin while eliminating or reducing the toxic side effects caused by eribulin acting on non-disease tissues, thereby improving the therapeutic effect. Summary of the Invention

[0006] This invention provides compounds of formula (I), their racemates, stereoisomers, tautomers, solvates, polymorphs, pharmaceutically acceptable salts, or prodrug compounds thereof: MZ-Tr-LD (I)

[0007] Where M is the linker site that binds to the antibody or its antigen-binding fragment, selected from... Lg is a leaving group; R 21 R 22 Whether the two are the same or different, they are selected independently from H, CN, and C. 1-6 Alkyl-OC 1-6 Alkylene-, C 1-10 Alkyl group, -(OCH2CH2)m1-OCH3; t is selected from 1, 2, 3, 4, 5 or 6; m1 is selected from an integer from 1 to 36;

[0008] Z is selected from single bond, -N(R) z )-(CH2)n1-C(=O)-, -N(R1)-(CH2CH2O)z1-(CH2)n2-C(=O)-, Among them, ring A is selected from cyclohexane ring or six-membered heterocycle;

[0009] R1, R z They are either the same or different, and are independently selected from H, -(CH2CH2O)z3-CH3,

[0010] z1, z2, z3, z4, z5, and z6 may be the same or different, and are independently selected from integers from 1 to 36;

[0011] n1, n2, n3, n4, n5, n6, n7, and n8 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5, and 6;

[0012] Tr is a divalent trigger group, selected from divalent dipeptide or polypeptide residues;

[0013] L is the linker between Tr and the bioactive molecular structural fragment D, selected from single bonds, -NH-C... 1-10 Alkyl-, -N(CH3)-C 1-10 Alkylene-

[0014] D represents a bioactive molecular structural fragment.

[0015] According to an embodiment of the present invention, D is selected from...

[0016] According to an embodiment of the present invention, the leaving group Lg is selected from halogen, sulfone, trifluoromethanesulfonyl, and methanesulfonyl.

[0017] According to an embodiment of the present invention, the leaving group Lg is selected from methanesulfonyl.

[0018] According to an embodiment of the present invention, R 22 Selected from H.

[0019] According to an embodiment of the present invention, R 21 Selected from CN, CH3OCH2-, C 1-3 Alkyl group or -(OCH2CH2)m1-OCH3.

[0020] According to an embodiment of the present invention, R 21 Selected from methoxy, -(OCH2CH2)6-OCH3 or -(OCH2CH2)8-OCH3.

[0021] According to an embodiment of the present invention, t is selected from 2, 3 or 4.

[0022] According to an embodiment of the present invention, m1 is selected from an integer from 1 to 12.

[0023] According to an embodiment of the present invention, m1 is selected from 6, 7, 8, 9 or 10.

[0024] According to an embodiment of the present invention, M is selected from...

[0025] According to an embodiment of the present invention, ring A is selected from...

[0026] According to an embodiment of the present invention, R1 is selected from H.

[0027] According to an embodiment of the present invention, R z Selected from the following groups: -(CH2CH2O)z3-CH3,

[0028] According to an embodiment of the present invention, R z Selected from the following groups: -(CH2CH2O)9-CH3, -(CH2CH2O) 13 -CH3、

[0029] According to an embodiment of the present invention, z1, z2, z3, z4, z5, and z6 may be the same or different, and are independently selected from integers from 6 to 15.

[0030] According to an embodiment of the present invention, z1, z2, z3, z4, z5, and z6 may be the same or different, and are independently selected from integers of 8-14.

[0031] According to an embodiment of the present invention, n1, n2, n3, n4, n5, n6, n7, and n8 may be the same or different, and are independently selected from 0, 1, 2, and 3.

[0032] According to an embodiment of the present invention, Z is selected from single bonds, -N(R) z )-(CH2)n1-C(=O)-, -NH-(CH2CH2O)z1-(CH2)n2-C(=O)-, Wherein, X1 and X2 may be the same or different, and are independently selected from CH or N; preferably, at least one of X1 and X2 is an N atom; R z z1, z2, n1, n2, n3, and n4 have the definitions described herein.

[0033] According to an embodiment of the present invention, when X2 is CH, n4 is selected from 0.

[0034] According to an embodiment of the present invention, Z is selected from single bonds, -NH-(CH2CH2O)8-CH2CH2-C(=O)-, and -NH-(CH2CH2O). 10 -CH2CH2-C(=O)-、-N(R z -CH2-C(=O)-、-N(R) z )-(CH2)2-C(=O)-、-N(R z )-(CH2)3-C(=O)-、-N(Rz )-(CH2)4-C(=O)-、 Among them, R z It has the definition described in this article.

[0035] According to an embodiment of the present invention, Z is selected from single bonds,

[0036] According to an embodiment of the present invention, Tr is a divalent dipeptide or polypeptide residue, wherein the dipeptide or polypeptide is selected from glycine-glycine-phenylalanine-glycine (GGFG, SEQ ID NO.1), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG, SEQ ID NO.2), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG, SEQ ID NO.3), and lysine-glycine-glycine-phenylalanine-glycine (KGGFG, SEQ ID NO.4).

[0037] According to an embodiment of the present invention, Tr is selected from glycine-glycine-phenylalanine-glycine (GGFG) residues, i.e.

[0038] According to an embodiment of the present invention, Tr is selected from valine-alanine (VA) residues, that is:

[0039] According to an embodiment of the present invention, L is selected from a single bond.

[0040] According to an embodiment of the present invention, L is selected from

[0041] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0042] Among them, Z and R 21 R z X1, X2, Tr, L, D, z1, z3, n1, n3, n4 have the definitions described herein.

[0043] According to an embodiment of the present invention, the compound represented by formula (I) is selected from:

[0044] This invention also provides antibody-drug conjugates of formula (II), their racemic, stereoisomer, tautomer, solvate, polymorphs, and pharmaceutically acceptable salts, Ab-[M'-Z-Tr-LD]. β (II)

[0045] Wherein, Ab is an antibody or its antigen-binding fragment, M' is the fragment formed by the coupling of M and Ab, β is selected from integers or decimals between 1 and 10; Z, Tr, L, and D have the definitions described herein independently.

[0046] According to some implementation schemes, Ab is an antibody or antigen-binding fragment selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody, and sdAb; and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody.

[0047] According to some implementation schemes, Ab is an antibody against HER2 or its antigen-binding fragment.

[0048] According to an embodiment of the present invention, Ab is pertuzumab or its antigen-binding fragment.

[0049] According to embodiments of the present invention, β is selected from an integer or decimal between 1 and 10, preferably from an integer or decimal between 2 and 8, and more preferably from an integer or decimal between 2 and 5; for example, β is selected from 2, 3, 4, 5, 6, 7, 8; or β is selected from 2.93, 2.94, 3.22, 3.33, 3.38, 3.48, 3.52, 3.60, 3.64, 3.66, 3.67, 3.68, 3.93, 3.92, 3.96, 4.06, 4.07, 4.21, such as 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2.

[0050] According to an embodiment of the present invention, M'-Z-Tr-LD in antibody-drug conjugate formula (II) has the following structure:

[0051] Among them, Z and R 21 R z X1, X2, Tr, L, D, z1, z3, n1, n3, and n4 have the definitions described herein, and the wavy line is the connection point with Ab.

[0052] According to an embodiment of the present invention, M'-Z-Tr-L in antibody-drug conjugate formula (II) has the following structure:

[0053] According to an embodiment of the present invention, the antibody-drug conjugate represented by formula (II) has the following structure:

[0054] Among them, Ab and R 21 R Z z1, z3, X1, X2, n1, n3, n4, Tr, L, and β each have the definitions described above independently.

[0055] According to an embodiment of the present invention, the antibody-drug conjugate represented by formula (II) has the following structure:

[0056] Among them, Ab and β independently have the definitions described above.

[0057] According to an embodiment of the present invention, the antibody-drug conjugate represented by formula (II) has the following structure:

[0058] Wherein, β has the definition described above.

[0059] According to an embodiment of the present invention, the antibody-drug conjugate is selected from the antibody-drug conjugates in the examples: ADC-001, ADC-002, ADC-003, ADC-004, ADC-005, ADC-007, ADC-009, ADC-010, ADC-014, ADC-019, ADC-020, ADC-021, ADC-022, ADC-023, and ADC-024.

[0060] In some embodiments, the antibody-drug conjugate of formula (II) is covalently linked to an amino or thiol residue on an antibody or its antigen-binding fragment by a compound of formula (I); preferably, the compound of formula (I) is covalently linked to a thiol residue on an antibody or its antigen-binding fragment; more preferably, the compound of formula (I) is covalently linked to a thiol residue formed after the interchain disulfide bond on the antibody or its antigen-binding fragment is opened.

[0061] In some embodiments, when the compound of formula (I) is covalently coupled to an antibody Ab, the alkylsulfonyl (e.g., methanesulfonyl) pyrimidine linker loses the alkylsulfonyl (e.g., methanesulfonyl) group, and the pyrimidine group is covalently linked to the thiol residue formed after the interchain disulfide bond on the antibody is opened. For example, when compound 001 is covalently coupled to Pertuzumab, the resulting ADC structure...

[0062] and They can represent the same structure.

[0063] In the ADC formed above, the -S- is not a thiol group introduced into the Ab, nor is it the -S- contained in the compound structure shown in formula (I), but rather a thiol residue formed after the interchain disulfide bond on the antibody or its antigen-binding fragment is opened.

[0064] This invention also provides the connecting sub-formula shown in formula (III): MZ-Tr-L' (III)

[0065] Wherein, M, Z, and Tr have the definitions described in this paper, and L' is the reaction form of L;

[0066] According to an embodiment of the present invention, when L is a single bond, L' is the reaction form of Tr, for example, Tr-L' is a peptide with a carboxyl group or active ester at the carbon terminus.

[0067] According to an embodiment of the present invention, when L is L' is the carbonate active ester form of p-aminobenzyl alcohol, for example The wavy lines represent the connection sites with peptide residues.

[0068] The present invention also provides a connector as shown below, which is used to obtain an antibody-drug conjugate formed by linking an antibody and a drug via the connector.

[0069] Among them, Z and R 21 R z X1, X2, Tr, L, z1, n1, n3, and n4 have the definitions described in this paper, with bit 1 connected to Ab and bit 2 connected to D.

[0070] According to some implementation schemes, the connector is as follows:

[0071] The present invention also provides the following intermediates:

[0072] Among them, Lg and R 21 R z X1, X2, z3, n3, n4 have the definitions described herein, and Y1 is selected from hydroxyl, methoxy, ethoxy, isopropoxy, tert-butoxy, or OSu.

[0073] According to some implementation schemes, the intermediate is selected from the following groups:

[0074] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate of formula (II).

[0075] According to embodiments of the present invention, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0076] According to embodiments of the present invention, the pharmaceutical composition may further contain one or more additional therapeutic agents.

[0077] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of at least one of the antibody-drug conjugate of formula (II), its racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound thereof.

[0078] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of the above-described pharmaceutical composition.

[0079] The tumor diseases mentioned are selected from breast cancer, stomach cancer, lung cancer, colorectal cancer, colon cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, or leukemia.

[0080] In some implementations, the patient includes mammals, preferably humans.

[0081] The present invention also provides at least one of the following: an antibody-drug conjugate of formula (II) for treating tumor diseases, a racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or a prodrug compound thereof, or a pharmaceutical composition thereof.

[0082] The present invention also provides the use of at least one of the antibody-drug conjugate of formula (II), its racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound, or the above-described pharmaceutical compositions in the preparation of a topoisomerase I inhibitor and / or in the preparation of a medicament for the prevention or treatment of diseases or conditions related to topoisomerase I.

[0083] In some implementations, the disease or condition is a tumor, including breast cancer, stomach cancer, lung cancer, colorectal cancer, colon cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, or leukemia. Beneficial effects

[0084] This invention provides antibody-drug conjugates (ADCs) with linker systems, their preparation methods, and applications. These linker systems exhibit high linker stability and enhanced solubility, allowing for efficient conjugation of hydrophobic drugs and effective intracellular drug delivery. Attached Figure Description

[0085] Figure 1 shows the in vivo tumor suppression effect of ADC-006 on the NCI-N87 transplantation model.

[0086] Figure 2 shows the in vivo tumor suppression effect of ADC-006 on the JIMT-1 transplantation model.

[0087] Figure 3 shows the in vivo tumor inhibition effect of ADC-006 and ADC-027 on the human gastric cancer LD1-0017-411335PDX model.

[0088] Terminology Definitions and Explanations

[0089] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0090] The "antibody-drug conjugate" (ADC) described in this invention refers to a target portion that is linked to a biologically active drug via a stable linker unit.

[0091] Unless otherwise stated, the numerical ranges described in this specification and claims are equivalent to describing at least each specific integer value therein. For example, the numerical range "1-15" is equivalent to describing each integer value in the numerical range "1-36", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. Furthermore, when certain numerical ranges are defined as "numbers", it should be understood that they describe the two endpoints of the range, each integer within the range, and each decimal within the range.

[0092] The term "integers from 0 to 10" refers to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0093] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0094] “C 1-10 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. 1-8 "Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. 1-6 "Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers.

[0095] Wavy lines intersecting chemical bonds Used to indicate the connection position of a group with other atoms or groups in a general formula.

[0096] The term "alkoxy" refers to -O-(alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkyloxy, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, or heterocycloalkyloxy.

[0097] The term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen via at least one antigen-binding site located in its variable region. When the term "antibody" is used, unless the context explicitly indicates otherwise, it includes not only the complete antibody but also the antigen-binding fragment capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). The antibody light chain can be classified as κ (kappa) and λ (lambda) light chains. The heavy chain can be classified as μ, δ, γ, α, or ε, and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various 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 be further subdivided into highly degenerated regions (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 sites. The allocation of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3.The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those 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). In this invention, the CDR contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the Kabat numbering system. As used herein, the term “frame region” or “FR” residue refers to those amino acid residues in the variable region of the antibody other than the CDR residues as defined above. The term “antibody” is not limited to any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; this fragment is also referred to as the “antigen-binding moiety.” See 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 generated through recombinant DNA technology or through enzymatic or chemical fragmentation of the intact antibody.Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabody, single-domain antibody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136. As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; and the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of the VH and CH1 domains). As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site. As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has a variety of functions but does not participate in antigen binding. "Effective functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. The Fc domain can include both native and variant Fc regions. Native Fc regions contain amino acid sequences consistent with those found in naturally occurring Fc regions, such as the native human IgG1 Fc region; the native human IgG2 Fc region; the native human IgG3 Fc region; and the native human IgG4 Fc region, as well as their naturally occurring variants. Variant Fc regions contain amino acid sequences that differ from the amino acid sequence of native Fc regions due to at least one amino acid modification. In some implementations, the variant Fc region may have altered effector functions compared to the native Fc region (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function).As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence (SEQ ID NO. 5) or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also exist between the VH and VL of the scFv. In some embodiments of the invention, scFv may form di-scFv, which refers to two or more individual scFvs linked in tandem to form an antibody. In some embodiments of the invention, scFv may form (scFv)2, which refers to two or more individual scFvs linked in parallel to form an antibody. As used herein, the term "biantibody" means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)). As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind the same antigen bound by the full-length antibody. As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity to at least two (e.g., three or four) different antigens (or epitopes). Bispecific or multispecific antibodies contain multiple antigen-binding domains with binding specificity to different antigens (or epitopes), thereby enabling them to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain in a bispecific or multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or its antigen-binding fragments (e.g., Fv fragments, Fab fragments, F(ab')2 fragments, or scFv). In some cases, the individual antigen-binding domains are linked by peptide linkers. Each of these antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific antigen binding.Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody fragments provided in this invention) can be obtained from a given antibody (e.g., the antibody fragments described above) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as for intact antibodies. As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Generally, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or the constant region) is derived from a human immunoglobulin (receptor antibody). In some embodiments, the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or the constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. In this application, the donor antibody may be a murine antibody with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human frame sequence using methods known in the art. In some cases, the human frame sequence may contain amino acid mutations substituted by corresponding non-human residues. Furthermore, the humanized antibody may also contain residues not found in either the variable region (e.g., the light chain variable region or the heavy chain variable region) of the initial donor antibody or in the human frame sequence to further improve or optimize the performance of the humanized antibody. As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains binding activity to the target antigen in any case. In some embodiments, the term "chimeric antibody" may include an antibody in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, while the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0098] The term "peptide" refers to a peptide compound with a specific amino acid sequence, formed by three or more amino acids linked linearly or in a branched manner through peptide bonds (-CO-NH-). Preferably, it is a peptide compound with a specific amino acid sequence formed by 3-50 amino acids linked linearly or in a branched manner through peptide bonds (-CO-NH-).

[0099] In this invention, the compounds involved also include isotopically labeled compounds, which are the same as those shown in Formula I, but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds of this invention include isotopes of H, C, N, O, S, F, and Cl, respectively such as 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 32 P, 35 S, 18 F and 36 Cl. Compounds of the present invention, their prodrugs, or pharmaceutically acceptable salts of said compounds or prodrugs containing the aforementioned isotopes and / or other isotopes are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those doped with radioactive isotopes (e.g.,... 3 H and 14 Compounds in (C) can be used for drug and / or substrate tissue distribution assays. Tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Furthermore, heavier isotopes (such as deuterium, i.e., 2 Hydrogen (H or D) substitution can provide certain therapeutic advantages derived from greater metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain circumstances. The compounds of the invention claimed in the claims are particularly defined as being substituted with deuterium or tritium. Furthermore, the presence of hydrogen in the substituents without a separate mention of the terms deuterium or tritium does not exclude deuterium or tritium, but may also include deuterium or tritium.

[0100] Those skilled in the art will understand that the compounds shown in formula (I) can exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they can form acid addition salts; if these compounds have an acidic center, they can form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they can also form internal salts.

[0101] The compounds of the present invention may exist as solvates (such as hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0102] Depending on their molecular structure, the compounds of the present invention can be chiral, and therefore may exist in various enantiomeric forms. Thus, these compounds can exist in racemic or optically active forms. The compounds of the present invention encompass isomers of each chiral carbon in the R or S configuration, or mixtures thereof, and racemates. The compounds of the present invention or their intermediates can be isolated as enantiomeric compounds by chemical or physical methods known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are obtained from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as tartaric acid in both R and S forms, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g., N-benzoylproline or N-benzenesulfonylproline), or various optically active camphorsulfonic acids. Chromatographic enantiomeric separation can also be advantageously performed using optically active resolving agents (e.g., dinitrobenzoylphenylglycine immobilized on silica gel, cellulose triacetate or other carbohydrate derivatives, or chiral derivatized isobutylene ester polymers). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, such as hexane / isopropanol / acetonitrile.

[0103] The corresponding stable isomers can be separated using known methods, such as extraction, filtration, or column chromatography.

[0104] The term "patient" refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, with humans being the most preferred.

[0105] The term “therapeutic effective amount” refers to the amount of an active compound or drug that researchers, veterinarians, physicians, or other clinicians are searching for in tissues, systems, animals, individuals, or humans to elicit a biological or medical response. It includes one or more of the following: (1) prevention of disease: e.g., prevention of disease, disorder, or condition in individuals susceptible to disease, disorder, or symptom but not yet experiencing or exhibiting the pathology or symptoms of the disease; (2) suppression of disease: e.g., suppression of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., prevention of further development of the pathology and / or symptoms); (3) relief of disease: e.g., relief of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., reversal of the pathology and / or symptoms). Detailed Implementation

[0106] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0107] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0108] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0109] MS determination was performed using an Agilent 1260 / 1290 Infinity II liquid chromatography system (manufacturer: Agilent, MS model: 1260 / 1290 Infinity II) and a Shimadzu Prominence UFLC+LCMS-2020 system (manufacturer: Shimadzu, MS model: Prominence UFLC+LCMS-2020).

[0110] High performance liquid chromatography (HPLC) analysis was performed using a Thermo UltiMate 3000 (manufacturer: Thermo, MS model: UltiMate 3000).

[0111] Chiral HPLC analysis was performed using a YMC K-PrepLAB100G high-performance liquid chromatograph.

[0112] High performance liquid chromatography (HPLC) preparation was performed using a Shimadzu LH-40 Liquid Handler, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, GX-281 Liquid Handler without pump, 4020 Syringe pump, 333-H3 Pump, 334-H3 Pump, and 1741 UV Detector preparative chromatograph.

[0113] The rapid chromatograph used is the Biotage Isolera One rapid chromatograph.

[0114] The silica gel plates used for thin-layer chromatography are Yantai Xinnuo GF254 silica gel plates. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15mm to 0.2mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4mm to 0.5mm.

[0115] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0116] Mean inhibition rate of kinases and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0117] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0118] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0119] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0120] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0121] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0122] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0123] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0124] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0125] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0126] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0127] Synthesis of intermediate Int 1

[0128] 2,5-Dioxopyrrolidone-1-yl-6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate Int-1

[0129] Step 1: 5-Bromo-2-chloro-4-methoxypyrimidine Int-1b

[0130] 5-Bromo-2,4-dichloropyrimidine Int-1a (5.0 g, 22 mmol) was dissolved in methanol (60 mL), and a 30% sodium methoxide methanol solution (4.0 g, 22.0 mol) was added to the solution. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was concentrated, extraction was inhibited by acetic acid, and the organic phase was dried and concentrated to give the title compound Int-1b (5.0 g, yield: 100%).

[0131] MS m / z(ESI): 223.1(M+1) + .

[0132] Step 2: 5-Bromo-4-methoxy-2-(methylthio)pyrimidine Int-1c

[0133] Sodium methanethiol (1.52 g, 22 mmol) was added to N,N-dimethylformamide (25 mL) to react with compound Int-1b (5.0 g, 22.0 mmol). The reaction mixture was stirred at 40 °C for 1 hour. After the reaction was completed, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried and concentrated. The crude product was purified by silica gel column chromatography system B to give the title compound Int-1c (3.0 g, yield: 60%).

[0134] MS m / z(ESI): 235.0(M+1) + .

[0135] Step 3: 6-(4-methoxy-2-(methylthio)pyrimidin-5-yl)hexyl-5-alkynic acid Int-1d

[0136] Compound Int-1c (3.0 g, 12.8 mmol) was dissolved in isopropanol (20 mL), and hexano-5-acetylic acid (1.43 g, 12.8 mmol), cuprous iodide (243 mg, 1.29 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) chloride (300 mg, 0.41 mmol), and sodium carbonate solution (5 M, 8 mL) were added. The reaction mixture was stirred at 80 °C for 16 hours under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was filtered, and the filtrate was extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried and concentrated. The crude product was purified by silica gel column chromatography system B to give the title compound Int-1d (3.0 g, yield: 87%).

[0137] MS m / z(ESI): 267.1(M+1) +.

[0138] Step 4: 5-Bromo-2,4-dichloropyrimidine 6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-alkynic acid Int-1e

[0139] Compound Int-2d (3.0 g, 11.2 mmol) was dissolved in a mixed solvent of methanol and water (40 mL, V / V = 1:1), and potassium peroxymonosulfate (11.6 g, 33.6 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was poured into water and extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried over sodium sulfate in wastewater to obtain the crude product. The crude product was purified by silica gel column chromatography system B to obtain the title compound Int-1e (2.5 g, yield: 75%).

[0140] MS m / z(ESI): 299.1(M+H) + .

[0141] Step 5: 2,5-Dioxopyrrolidone-1-yl-6-(4-methoxy-2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate Int-1

[0142] N,N-diisopropylcarbodiimide (434 mg, 3.45 mmol) was added to a tetrahydrofuran (10 mL) solution of compound Int-1e (700 mg, 2.3 mmol) and N-hydroxysuccinimide (396 mg, 3.45 mmol), and the reaction was stirred at room temperature for 3 hours. After the reaction was completed, the crude product obtained by direct concentration of the reaction solution was purified by silica gel column chromatography system B to give the title compound Int-1 (600 mg, yield: 60%).

[0143] MS m / z(ESI): 395.5(M+1) + .

[0144] Example 1

[0145] Synthesis of compound (001)

[0146] Synthesis of compound (001b) in step one

[0147] Eribulin 001a methanesulfonic acid (700 mg, 0.85 mmol) and N-[fluorenylmethoxycarbonyl]-L-valine-L-alanine (369 mg, 0.90 mmol) were dissolved in N,N-dimethylformamide (5 mL), and HATU (486 mg, 1.28 mmol) and DIPEA (348 mg, 2.7 mmol) were added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, water was added, and the system was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography to obtain compound 001b (900 mg, yield: 94%).

[0148] MS m / z(ESI): 1122.5(M+1) + .

[0149] The second step involves the synthesis of compound (001).

[0150] Compound 001b (70 mg, 0.062 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (0.2 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was evaporated, and the residue was redissolved in DMF (1 mL). 2,5-Dioxopyrrolidone-1-yl 6-(4-methoxy-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylacetate Int 1 (29 mg, 0.074 mmol) and DIPEA (17 mg, 0.128 mmol) were added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x 19mm, 5μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 50%-60%, flow rate: 20mL / min) to obtain compound 001 (28mg, yield: 38%).

[0151] MS m / z(ESI): 1180.4(M+1) + .

[0152] 1H NMR(400MHz,DMSO-d6)δ8.82(s,1H),7.98(d,1H),7.90(d,1H),7.74(t,1H),5.0 2(d,2H),4.79(d,2H),4.67–4.58(m,2H),4.55(t,1H),4.31–4.22(m,2H),4.21–4 .14(m,2H),4.12–4.09(m,2H),4.08(s,3H),4.06–3.97(m,1H),3.84–3.77(m,1H) ,3.77–3.72(m,1H),3.72–3.65(m,1H),3.55–3.46(m,3H),3.39(s,3H),3.25(s,4 H),3.15–3.06(m,1H),3.02–2.95(m,1H),2.84(d,1H),2.78–2.65(m,2H),2.58– 2.53(m,2H),2.42–2.35(m,1H),2.36–2.17(m,7H),2.17–2.08(m,1H),2.05–1.95 (m,3H),1.94–1.85(m,4H),1.82–1.74(m,2H),1.73–1.62(m,5H),1.59–1.43(m,3 H),1.36–1.28(m,2H),1.19(d,3H),1.03(d,3H),1.01–0.92(m,1H),0.84(m,6H).

[0153] Example 2

[0154] Synthesis of compound (002)

[0155] Synthesis of compound (002b) in step one

[0156] Compound 002a (144 mg, 0.49 mmol) was dissolved in N,N-dimethylformamide (2 mL), and then Int 1 (214 mg, 0.54 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was directly evaporated to dryness and purified by column chromatography separation system B to obtain compound 002b (100 mg, yield: 36%).

[0157] MS m / z (ESI): 596.2 (M+Na) + .

[0158] The synthesis of the second compound (002c)

[0159] Compound 002b (100 mg, 0.17 mmol) was dissolved in DMF (2 mL), and bis(4-nitrophenyl) carbonate (61 mg, 0.21 mmol) and triethylamine (53 mg, 0.5 mmol) were added. The reaction was stirred at room temperature for 16 hours. After the reaction was completed, the mixture was purified by column chromatography using system B to obtain compound 002c (120 mg, yield: 93%).

[0160] MS m / z(ESI): 761.2(M+1) + .

[0161] The synthesis of compound (002) in the third step.

[0162] Compound 002c (50 mg, 0.07 mmol) was dissolved in N,N-dimethylformamide (3 mL), and 001a (54 mg, 0.07 mmol) and N,N-diisopropylethylamine (26 mg, 0.2 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated to dryness to obtain the crude product. The crude product was prepared by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x 19 mm; mobile phase 1: water (containing 0.1% formic acid); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 55%-75%, flow rate: 20 mL / min) to obtain compound 002 (41 mg, yield: 45%).

[0163] MS m / z (ESI): 1352.4 (M+Na) + .

[0164] 1H NMR(400MHz, CDCl3)δ8.57(d,2H),7.50(s,2H),7.31–7.27(m,1H),7.21–6.99(m,1H),6.64–6.36(m,1H),5.37(s,1H),5.09–4.98(m, 3H),4.88–4.84(m,1H),4.81–4.76(m,1H),4.70–4.66(m,2H),4.63–4.56(m,1H),4.43–4.30(m,3H),4.21–4.09(m,5H),4.05–3.79(m, 7H),3.67–3.59(m,2H),3.41(s,3H),3.36–3.25(m,5H),3.20–3.14(m,1H),2.87(d,2H),2.73–2.67(m,1H),2.61–2.42(m,6H),2.29–2 .13(m,8H),2.10–2.06(m,2H),1.98–1.89(m,6H),1.77–1.65(m,7H),1.61–1.56(m,2H),1.51–1.34(m,6H),1.09(d,4H),0.93(s,4H).

[0165] Example 3

[0166] Synthesis of compound (003)

[0167] Synthesis of compound (003b) in step one

[0168] 2,5,8,11,14,17-hexadecan-19-ol (1 g, 3.4 mmol) was dissolved in THF (10 mL), and NaH (272 mg, 6.8 mmol) was slowly added at 0 °C with stirring for 1 hour. 003a (809 mg, 3.4 mmol) was added while maintaining 0 °C, and the mixture was then slowly raised to room temperature for 3 hours. The reaction was allowed to proceed to completion, quenched with water, and extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography system B to give compound 003b (1 g, yield: 59%).

[0169] MS m / z(ESI): 499.1(M+1) + .

[0170] The second step involves the synthesis of compound (003c).

[0171] Compound 003b (500 mg, 1.0 mmol) was dissolved in tetrahydrofuran (10 mL), and hexano-5-acetylic acid (224 mg, 2.0 mmol), cuprous iodide (19 mg, 0.1 mmol), bis(triphenylphosphine) palladium dichloride (14 mg, 0.2 mmol), and triethylamine (303 mg, 3 mmol) were added. The reaction mixture was stirred at 60 °C for 3 hours under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was filtered, and the filtrate was separated into layers with ethyl acetate and water. The aqueous phase was extracted with ethyl acetate (20 mL x 3), and the combined organic phases were dried and concentrated. The crude product was purified by silica gel column chromatography system A to give compound 003c (200 mg, yield: 38%).

[0172] MS m / z(ESI): 531.2(M+1) + .

[0173] The synthesis of the third compound (003d)

[0174] Compound 003c (200 mg, 0.38 mmol) was dissolved in a mixed solvent of acetone (10 mL) and water (10 mL), and potassium peroxide monosulfonate (7.6 g, 1.9 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was poured into water and extracted three times with ethyl acetate, washed once with saturated brine, and concentrated under reduced pressure. The crude product obtained by concentration was purified by silica gel column chromatography system A to give compound 003d (100 mg, yield: 47%).

[0175] MS m / z(ESI): 563.2(M+1) + .

[0176] Synthesis of compound (003) in step four

[0177] Compound 001b (70 mg, 0.062 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (0.2 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was evaporated, and the residue was redissolved in DMF (1 mL). Compound 003d (50 mg, 0.09 mmol), HATU (51 mg, 0.14 mmol), and DIPEA (23 mg, 0.18 mmol) were added. The reaction was stirred at room temperature for 30 minutes. After the reaction was complete, the reaction solution was purified by high-performance liquid chromatography (HPLC) using Waters MS-triggered Prep-LC with SQD2 detector (column: Xbridge 5u C18 150 x 19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 45%-55%, flow rate: 20 mL / min) to obtain compound 003 (10 mg, yield: 8%).

[0178] MS m / z(ESI): 1444.6(M+1) + .

[0179] 1 H NMR(400MHz, CDCl3)δ8.56(s,1H),6.66–6.58(m,1H),5.08(s,1H),4.94(s,1H),4.90–4.76(m,3H),4.75–4.67 (m,3H),4.60(t,1H),4.43(s,1H),4.38–4.26(m,3H),4.24–4.16(m,2H),4.11(s,1H),4.07–3.98(m,2H),3.99– 3.84(m,6H),3.74–3.53(m,22H),3.40(d,8H),3.31(s,3H),2.87(d,2H),2.71(s,2H),2.65–2.39(m,8H),2.17 (s,6H),2.12–2.06(m,2H),2.04–1.90(m,8H),1.65–1.51(m,5H),1.38–1.23(m,6H),1.09(d,3H),0.95(s,6H).

[0180] Example 4

[0181] Synthesis of compound (004)

[0182] Using a synthetic route similar to that in Example 3, the final product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: Xbridge 5u C18 150x 19mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 45%-65%, flow rate: 20 mL / min) to obtain compound 004 (36 mg, yield: 40%).

[0183] MS m / z(ESI): 1532.8(M+1) + .

[0184] 1 H NMR(400MHz, CDCl3)δ8.56(s,1H),6.66–6.57(m,3H),6.24(d,1H),5.97(d,1H),5.08–5.05(m,1H),4.94–4.86(m,2H),4.81–4.7 9(m,2H),4.72–4.68(m,2H),4.43(s,1H),4.25–4.17(m,1H),4.06–4.03(m,2H),3.96–3.85(m,3H),3.74–3.72(m,2H),3.66–3.6 4(m,27H),3.57–3.53(m,2H),3.42(s,3H),3.38(s,3H),3.31(s,3H),3.26(s,1H),3.18–3.15(m,1H),2.93–2.69(m,4H),2.64–2 .47(m,8H),2.38–2.03(m,12H),2.01–1.85(m,8H),1.80–1.61(m,8H),1.40–1.39(m,4H),1.11–1.04(m,4H),0.99–0.93(m,7H).

[0185] Example 5

[0186] Synthesis of compound (006)

[0187] Synthesis of compound (006b) in step one

[0188] Compound 006a (100 mg, 0.201 mmol) and compound Int-1 (87 mg, 0.221 mmol) were dissolved in N,N-dimethylformamide (1 mL). N,N-diisopropylethylamine (78 mg, 0.603 mol) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was heated to 25 °C and stirred for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation. The mixture was dissolved in water (20 mL) and extracted three times with dichloromethane (30 mL). The organic phase was washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate. The product was then evaporated to dryness to give compound 006b (142 mg, yield: 91%), which was used directly in the next reaction without purification.

[0189] MS m / z(ESI): 722.3(M+1-56) + .

[0190] The second step involves the synthesis of compound (006c).

[0191] Compound 006b (60 mg, 0.077 mmol) was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (0.2 mL, 298 mg, 2.612 mmol) was added at 0 °C. The mixture was then heated to 25 °C and stirred for 1 hour. After the reaction was complete, the reaction solution was evaporated to dryness to obtain compound 006c (50 mg). The product was used directly in the next reaction without purification.

[0192] MS m / z(ESI): 722.2(M+1) + .

[0193] Synthesis of the third compound (006)

[0194] Compound 001b (70 mg, 0.062 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (0.2 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was evaporated, and the residue was redissolved in DMF (1 mL). Compound 006c (50 mg, 0.062 mmol), HATU (35 mg, 0.093 mmol), and DIPEA (17 mg, 0.128 mmol) were added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x 19mm, 5μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 42%-57%, flow rate: 20mL / min) to obtain compound 006 (21.4mg, yield: 21%).

[0195] MS m / z(ESI): 1604.6(M+1) + .

[0196] 1 H NMR(400MHz,DMSO-d6)δ8.81(s,1H),7.97(d,1H),7.94–7.90(m,1H),7.87(d,1H) ,7.75–7.69(m,1H),5.03–5.00(m,2H),4.79–4.72(m,2H),4.63(s,1H),4.58–4.5 3(m,1H),4.30–4.22(m,2H),4.21–4.14(m,2H),4.11–4.06(m,5H),4.05–3.98(m, 1H),3.84–3.65(m,3H),3.58(t,2H),3.54–3.44(m,32H),3.42–3.37(m,6H),3.25( s,4H),3.23–3.16(m,3H),3.14–3.06(m,1H),3.04–2.95(m,1H),2.84–2.80(m,1H ),2.81–2.62(m,2H),2.42–2.29(m,4H),2.29–2.18(m,5H),2.17–2.08(m,1H),2. 01–1.95(m,4H),1.94–1.85(m,4H),1.81–1.75(m,2H),1.74–1.60(m,4H),1.58–1 .41(m,4H),1.24(s,4H),1.19–1.12(m,3H),1.03-1.00(m,3H),0.82–0.80(m,6H).

[0197] Example 6

[0198] Synthesis of compound (005)

[0199] Using a synthetic route similar to that in Example 5, the reaction solution was purified by high performance liquid chromatography (HPLC) using Waters MS-triggered Prep-LC with SQD2 detector. The chromatographic column was an Xbridge 5u C18 150x 19mm. The mobile phase 1 was water (containing 0.1% FA), and the mobile phase 2 was acetonitrile. The chromatographic gradient was 15 minutes, with a gradient ratio of 45%-60% acetonitrile phase and a flow rate of 20 mL / min, to obtain compound 005 (30 mg).

[0200] MS m / z(ESI): 846.6(M / 2+1) + .

[0201] 1 H NMR(400MHz,DMSO-d6)δ8.82(s,1H),7.97(d,3H),7.92(t,1H),7.87(d,1H ),7.73(t,1H),5.03–5.00(m,2H),4.79–4.75(m,2H),4.66–4.61(m,1H),4 .55–4.50(m,1H),4.29–4.22(m,2H),4.20–4.14(m,2H),4.11–4.09(m,2H) ,4.08(s,3H),3.84–3.64(m,5H),3.62–3.56(m,3H),3.52–3.47(m,40H),3. 40(s,6H),3.25(s,4H),3.22–3.18(m,2H),3.14–2.95(m,3H),2.84–2.80( m,1H),2.81–2.70(m,2H),2.69–2.65(m,1H),2.62–2.52(m,4H),2.48–2.41 (m,2H),2.41–2.29(m,4H),2.24(s,6H),1.92(s,7H),1.82–1.72(m,3H),1 .40–1.24(m,4H),1.19–1.12(m,3H),1.03–1.00(m,3H),0.83–0.80(m,5H).

[0202] Example 7

[0203] Synthesis of compound (007)

[0204] Synthesis of compound (007b) in step one

[0205] Compound 007a (200 mg, 1.04 mmol), 28-bromo-2,5,8,11,14,17,20,23,26-nonoxyoctadecane (561 mg, 1.14 mmol), and N,N-diisopropylethylamine (270.4 mg, 2.08 mmol) were dissolved in DMF (5 mL) and stirred at 60 °C for 16 hours. After the reaction was complete, the solution was concentrated under reduced pressure. The crude product 007b (400 mg) obtained from the concentration was used directly in the next reaction without purification.

[0206] MS m / z(ESI): 604.4(M+1) + .

[0207] The second step involves the synthesis of compound (007c).

[0208] The 007b (400 mg) obtained in the previous step was dissolved in methanol (20 mL), and Pd / C (40 mg) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The crude 007c (300 mg) obtained from the concentration was directly used in the next step of the reaction without purification.

[0209] MS m / z(ESI): 514.3(M+1) + .

[0210] Synthesis of the third compound (007d)

[0211] The crude product obtained in the previous step was dissolved in DMF (5 mL), and Int 1 (410 mg, 1.04 mmol) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction solution was purified by high-performance liquid chromatography (HPLC) using Waters MS-triggered Prep-LC with QDA detector. The chromatographic column was a Welch Ultimate C18 21.2*250 mm 10 μm column; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 30%-50%, flow rate: 20 mL / min, to obtain compound 007d (20 mg).

[0212] MS m / z(ESI): 794.3(M+1) + .

[0213] Synthesis of compound (007) in step four

[0214] Compound 001b (30 mg, 0.026 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (0.2 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was evaporated, and the residue was redissolved in DMF (1 mL). Compound 007d (20 mg, 0.03 mmol), HATU (19 mg, 0.05 mmol), and DIPEA (8 mg, 0.06 mmol) were added. The reaction was stirred at room temperature for 30 minutes. After the reaction was complete, the reaction solution was purified by high-performance liquid chromatography (HPLC) using Waters MS-triggered Prep-LC with SQD2 detector. The chromatographic column was an Xbridge 5u C18 150 x 19 mm column; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 50%-60%, flow rate: 20 mL / min) to obtain compound 007 (10 mg, yield: 22%).

[0215] MS m / z(ESI): 838.5(M / 2+1)+ .

[0216] 1 H NMR(400MHz,DMSO-d6)δ8.81(d,1H),7.98(s,1H),7.88(s,1H),7.72(s,1H),5.03–4.95(m,2H),4.79–4.70(m,3H),4.63(s,1H), 4.55(s,1H),4.29–4.22(m,2H),4.21–4.13(m,2H),4.11–4.07(m,4H),4.06–3.96(m,2H),3.86–3.64(m,6H),3.54–3.47(m,32H), 3.23(s,7H),3.10(s,2H),2.99(s,2H),2.87–2.71(m,4H),2.55(s,3H),2.46–2.42(m,2H),2.29–2.05(m,8H),2.03–1.87(m,8H), 1.83–1.74(m,3H),1.66(s,5H),1.45(s,4H),1.27(s,4H),1.24(s,4H),1.19–1.12(m,3H),1.03–0.95(m,3H),0.88–0.78(m,6H).

[0217] Example 8

[0218] Synthesis of compound (009)

[0219] Using a synthetic route similar to that in Example 7, the final product was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x 19mm, 5μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 57%-100%, flow rate: 20mL / min) to obtain compound 009 (15mg, yield: 28%).

[0220] MS m / z(ESI):824.4(M / 2+1)+.

[0221] 1H NMR(400MHz,DMSO-d6)δ8.80(d,1H),8.10–8.01(m,1H),8.01–7.78(m,1H),7. 76–7.58(m,1H),5.02(d,2H),4.79(d,2H),4.69–4.54(m,3H),4.32–4.23(m,2H ),4.22–4.14(m,2H),4.12–4.05(m,6H),4.04–3.96(m,2H),3.86–3.76(m,2H) ,3.75–3.64(m,3H),3.57–3.52(m,4H),3.52–3.47(m,30H),3.44–3.40(m,3H), 3.39(s,4H),3.24(d,7H),3.18–3.05(m,2H),3.03–2.92(m,2H),2.84(d,1H), 2.78–2.62(m,3H),2.60–2.55(m,3H),2.42–2.18(m,7H),2.05–1.94(m,4H),1. 92(s,4H),1.83–1.74(m,2H),1.75–1.63(m,5H),1.56–1.39(m,4H),1.39–1.28 (m,4H),1.27–1.15(m,8H),1.03(d,3H),1.00–0.93(m,1H),0.89–0.76(m,6H).

[0222] Example 9

[0223] Synthesis of compound (010)

[0224] Using a synthetic route similar to that in Example 7, the reaction solution was prepared by high-performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x 19mm, 5μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 10-minute gradient, gradient ratio: acetonitrile phase 45%-55%, flow rate: 20mL / min) to obtain 23R,24aS,25S,26aR,27S,28R,29aS,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctahydro-25H-2,27:5,8:11,15-triepoxy-23,25-ethanol-2,28-methylfuran[3,2] -i]furan[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentadioct-18-yl)propyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-oxopropyl)-6-(4-methoxy-2-(methylsulfonyl))pyrimidin-5-yl)-N-(2,5,8,11,14,17,20,23,26-nonaoxooctadecane-28-yl)hex-5-acetamide 010 (11.7 mg, yield: 11%).

[0225] MS m / z(ESI): 831.7(M / 2+1) + .

[0226] 1H NMR(400MHz, CDCl3)δ8.61(d,1H),7.16–6.80(m,2H),6.82–6.67(m,1H),5.08(s,1H),4 .95(s,1H),4.87(s,1H),4.80(s,1H),4.72–4.65(m,1H),4.62–4.57(m,1H),4.54–4.41 (m,1H),4.39–4.25(m,3H),4.21–4.15(m,1H),4.14(s,4H),4.11(s,1H),4.06–4.00(m, 1H),3.98–3.91(m,1H),3.92–3.83(m,2H),3.82–3.73(m,2H),3.69–3.58(m,32H),3.56– 3.52(m,3H),3.41(s,4H),3.37(s,3H),3.33(s,3H),3.25(s,2H),2.86–2.80(m,2H),2. 75–2.65(m,2H),2.59–2.53(m,6H),2.50–2.42(m,2H),2.41–2.31(m,2H),2.30–2.23(m, 3H),2.22–2.12(m,5H),2.11–2.04(m,2H),2.00–1.85(m,5H),1.83–1.64(m,5H),1.62– 1.51(m,3H),1.49–1.36(m,6H),1.34–1.20(m,3H),1.13–1.06(m,1H),0.99–0.80(m,6H)

[0227] Example 10

[0228] Synthesis of compound (014)

[0229] Using a synthetic route similar to that in Example 7, the crude product was prepared by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x 19mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 55%-65%, flow rate: 20mL / min) to obtain compound 014 (10.4mg, yield: 9%).

[0230] MS m / z(ESI): 873.4(M / 2+1) + .

[0231] 1H NMR(400MHz, CDCl3)δ8.57(s,1H),7.35–7.27(m,1H),7.19–7.05(m,1H),6.90–6.78(m,1H), 5.09(s,1H),4.95(s,1H),4.87(s,1H),4.80(s,1H),4.71–4.66(m,1H),4.63–4.59(m,1H),4 .53–4.44(m,1H),4.38–4.27(m,3H),4.23–4.17(m,2H),4.17–4.13(m,3H),4.13–4.07(m,1H ),4.05–4.01(m,1H),3.99–3.92(m,2H),3.91–3.85(m,2H),3.83–3.75(m,4H),3.69–3.58(m ,35H),3.56–3.53(m,2H),3.41(s,3H),3.37(s,3H),3.33(s,3H),3.26(s,1H),3.22–3.12(m ,1H),3.04–2.93(m,1H),2.90–2.82(m,2H),2.75–2.66(m,2H),2.61–2.54(m,4H),2.50–2.4 6(m,1H),2.37–2.33(m,1H),2.32–2.25(m,2H),2.23–2.06(m,10H),2.01–1.87(m,6H),1.81 –1.63(m,13H),1.60–1.54(m,2H),1.46–1.31(m,6H),1.29–1.21(m,1H),1.12–1.07(m,5H).

[0232] Example 11

[0233] Synthesis of compound (019)

[0234] Synthesis of compound (019a) in step one

[0235] Compound 009d (120 mg, 0.16 mmol) was dissolved in N,N-dimethylformamide (2 mL), followed by the addition of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (46 mg, 0.16 mmol), oxo-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (120 mg, 0.31 mmol), and N,N-diisopropylethylamine (41 mg, 0.31 mmol). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was directly filtered and evaporated to dryness, and purified by reverse column chromatography to obtain compound 019a (150 mg, yield: 92%).

[0236] MS m / z(ESI): 1063.4(M+1) + .

[0237] The second step involves the synthesis of compound (019b).

[0238] Compound 019a (90 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (5 mL), and bis(4-nitrophenyl) carbonate (79 mg, 0.26 mmol) and triethylamine (53 mg, 0.52 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the solvent was directly evaporated to dryness, and the crude product was purified by silica gel column chromatography system B to give compound 019b (40 mg, yield: 38%).

[0239] MS m / z (ESI): 1228.4 (M+Na) + .

[0240] Synthesis of the third compound (019)

[0241] Compound 019b (35 mg, 0.03 mmol) was dissolved in N,N-dimethylformamide (2 mL), and eribulin methanesulfonate 001a (24 mg, 0.03 mmol) and triethylamine (6 mg, 0.06 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated to dryness to obtain the crude product. The crude product was prepared by high performance liquid chromatography (Waters MS-triggered Prep-LC with SQD2 detector, column: Xbridge 5u C18 150x19 mm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; gradient ratio: acetonitrile phase 52%-62%, flow rate: 20 mL / min) to obtain compound 019 (10.5 mg, yield: 20%).

[0242] MS m / z (ESI): 921.2 (M / 2 + Na)+ .

[0243] 1 H NMR (400MHz, CDCl3) δ8.57–8.48(m,2H),7.77(d,2H),7.50–7.44(m,1H),7.25–7.23(m ,1H),6.77–6.71(m,1H),5.32(s,1H),5.08(s,1H),5.01(s,2H),4.94(s,1H),4.88(s,1 H),4.81(s,1H),4.72–4.67(m,2H),4.61(s,1H),4.38–4.32(m,1H),4.31–4.26(m,2H), 4.23–4.17(m,1H),4.16–4.09(m,2H),4.06–4.01(m,2H),4.00–3.88(m,4H),3.86–3.78 (m,2H),3.72–3.59(m,30H),3.56–3.53(m,2H),3.42(s,3H),3.38(s,3H),3.33(s,3H), 3.27(s,1H),3.22–3.12(m,3H),2.91–2.85(m,2H),2.77–2.66(m,2H),2.51–2.40(m,6H ),2.31–2.14(m,8H),2.12–2.05(m,2H),2.03–1.90(m,5H),1.75–1.68(m,6H),1.55–1. 49(m,5H),1.49–1.39(m,4H),1.39–1.25(m,4H),1.11–1.08(m,3H),1.06–0.95(m,6H).

[0244] Example 12

[0245] Synthesis of compound (024)

[0246] Synthesis of compound (024a) in step one

[0247] Compound 006c (210 mg, 0.29 mmol), (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (85 mg, 0.29 mmol), and HATU (166 mg, 0.436 mmol) were dissolved in DMF (8 mL), and DIEA (75 mg, 0.582 mmol) was added. The reaction was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was evaporated to dryness to obtain the crude product. The crude product was purified by reversed-phase column chromatography to give product 024a (90 mg, yield: 27%).

[0248] MS m / z(ESI): 997.3(M+H) + .

[0249] The second step involves the synthesis of compound (024b).

[0250] Compound 024a (50 mg, 0.04 mmol) and bis(4-nitrophenyl) carbonate (50 mg, 0.04 mmol) were dissolved in DMF, and DIEA (50 mg, 0.04 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography system A to give compound 024b (60 mg, yield: 54%).

[0251] MS m / z(ESI): 1162.2(M+H) + .

[0252] The synthesis of compound (024) in the third step.

[0253] 024b (60 mg, 0.05 mmol) and eribulin mesylate 001a (38 mg, 0.05 mmol) were dissolved in DMF (4 mL), and DIEA (10 mg, 0.08 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was purified by high performance liquid chromatography (Waters MS-triggered Prep-LC with QDA detector, column: WELCH Xtimate C18 21.2*250 mm 10 μm; mobile phase 1: water (containing 0.1% FA); mobile phase 2: acetonitrile; 15-minute gradient, gradient ratio: acetonitrile phase 22%-80%, flow rate: 15 mL / min) to obtain compound 024 (16.7 mg, yield: 18%).

[0254] MS m / z (ESI): 898.9 (M / 2 + Na) + .

[0255] 1H NMR(400MHz, CDCl3)δ8.71(s,1H),8.56(s,1H),7.71(d,2H),7.29(s,2H),7.23–7.09(m, 1H),6.64(s,1H),5.29(s,1H),5.08(s,1H),5.03(s,2H),4.94(s,1H),4.88(s,1H),4.81 (s,1H),4.69(t,1H),4.61(t,1H),4.34(t,3H),4.22–4.17(m,2H),4.16(s,3H),4.12(s, 1H),4.06–4.01(m,1H),3.98–3.89(m,3H),3.86–3.78(m,2H),3.63–3.55(m,33H),3.45( s,2H),3.42(s,3H),3.33(s,3H),3.27(s,1H),3.19(s,1H),2.89–2.83(m,2H),2.75–2.6 8(m,2H),2.59–2.56(m,2H),2.51–2.44(m,3H),2.43–2.33(m,3H),2.32–2.24(m,3H),2. 23–2.13(m,5H),2.11–2.08(m,1H),2.03–1.90(m,8H),1.57–1.55(m,1H),1.51–1.40(m, 6H),1.39–1.29(m,4H),1.25(s,3H),1.14–1.06(m,4H),1.05–0.97(m,6H),0.88(s,1H).

[0256] Following a similar method disclosed above, the following compounds were synthesized, and their structural characterization data are shown in the table below:

[0257] Preparation of antibody-drug conjugates

[0258] Example A-1 Preparation of ADC-006

[0259] Pertuzumab (20 mg) was placed in a 50 mL centrifuge tube, and 50 mM PBS was added to adjust the pH to 8.0 until the antibody concentration was 5 mg / mL. 0.2 M EDTA was added to bring the final EDTA concentration to 2 mM. TCEP (4 times the molar amount of the monoclonal antibody) was added, and the mixture was incubated at 37°C for 3 h, continuously mixing. Compound 006 (prepared with DMA) solution was added under ice bath conditions at a drug-to-antibody molar ratio of 13:1. DMA was added to the mixture at 10% of the total reaction volume, and the mixture was shaken and incubated at 22°C for 16 h. The reaction solution was purified using a Zeba desalting column, and the sample was concentrated using Amicon buffer and the buffer was changed to a 30 mM His / HAc, pH 5.5 buffer system to obtain ADC-006 (13 mg). The average DAR value β of the ADC was calculated to be 3.7 using RP-HPLC.

[0260] Referring to the method of Example A-1, compound 006 was replaced with compounds from other examples of this application to prepare the following antibody-drug conjugates, the structures of which and their DAR values ​​(β) are shown in the table below.

[0261] The results showed that the cytotoxic drug-linker compounds of the present invention can be successfully conjugated with antibodies to obtain antibody-drug conjugates.

[0262] The preparation process of ADC-027 was obtained by referring to the patent WO2023001300A1.

[0263] Biological evaluation

[0264] Test Example 1: ADC Bioactivity Detection

[0265] The purpose of this experiment was to detect the inhibitory activity of ADC compounds on the in vitro proliferation of HER2-expressing NCI-N87 cells, JIMT-1 cells, and HER2-negative MDA-MB-468 cells. Cells were treated with different concentrations of the compounds in vitro, and after 5 days of culture, CTG was used for cell proliferation analysis. The Luminescent Cell Viability Assay detects cell proliferation based on IC50. 50 The value was used to evaluate the in vitro activity of the compound.

[0266] 2. Testing Methods

[0267] (1) On the first day, tumor cells were seeded on 96-well plates, with 5000 cells / 100μL of culture medium in each well, and 100μL of DPBS in each empty well at the edge. The plates were incubated overnight at 37°C.

[0268] (2) On the second day, first aspirate 50 μL of the old culture medium per well; add ADC at different concentration gradients, with the initial concentration of ADC being 200 nM, diluted 5 times, resulting in 9 concentrations. The volume of drug added is 50 μL per well.

[0269] (3) On the sixth day, thaw CellTiter-Glo Buffer and CellTiter-Glo Substrate reagent at 4°C. Before use, aspirate 10 ml of Buffer and add it to Substrate, mix well, and equilibrate to room temperature.

[0270] (4) On the seventh day, equilibrate the 96-well plate at room temperature for 30 minutes, and add 100 μL of Cell-Titer-Glo to each well. Shake at room temperature in the dark for 5 minutes, incubate for 10 minutes, transfer 100 μL of the liquid in the well to the white plate, and then use a microplate reader to detect chemiluminescence.

[0271] 3. Data Analysis

[0272] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5 to test the inhibitory activity of the ADC compound on the in vitro proliferation of NCI-N87 cells, JIMT-1 cells, and MDA-MB-468 cells. The results are shown in Table 1 below.

[0273] Table 1: Inhibitory activity of ADCs on in vitro proliferation

[0274] Conclusion: The antibody-drug conjugates targeting HER2 in this invention have significant inhibitory activity against the proliferation of HER2-positive cells JIMT-1 and NCI-N87; at the same time, they have weak inhibitory activity against the proliferation of HER2-negative cells MDA-MB-468; and they have good selectivity.

[0275] Test Example 2: ADC Plasma Stability Experiment

[0276] ADC DAR value test and results

[0277] ADC-006 was added to the above-mentioned fresh sterile human plasma at a final concentration of 200 μg / mL and incubated in a cell culture incubator at 37°C. The day of incubation was marked as day 0. Samples were then collected on days 1, 4, 7, 14, and 21, and the changes in ADC DAR values ​​were measured using mass spectrometry. DAR LC = Total L1 / (Total L0 + Total L1), DAR HC = (3*Total H3 + 2*Total H2 + Total H1) / (Total H3 + Total H2 + Total H1 + Total H0), DAR = 2*(DAR LC + DAR HC).

[0278] Table 2 shows the experimental results of ADC DAR value changes: the conjugate formed by the small molecule linker of the present invention exhibits excellent plasma stability in human plasma DAR value, which confirms the stability of the small molecule linker of the present invention.

[0279] Table 2: Plasma stability of ADC (DAR value changes)

[0280] Test Example 3: Efficacy Evaluation of NCI-N87 Tumor-Bearing Mice

[0281] Experimental Objective

[0282] Balb / c nude mice were used as test animals to evaluate the efficacy of the ADC in this application.

[0283] 1. Test drug

[0284] ADC-006: 3 mg / kg

[0285] 2. Preparation method: All preparations are made by diluting with PBS.

[0286] 3. Test methods

[0287] NCI-N87 cells were subcutaneously injected into the right rib area of ​​mice. After 7 days of tumor growth, the animals were randomly divided into 3 groups of 6 mice each (2 experimental groups + 1 blank control group).

[0288] The drug was administered via tail vein injection once. Tumor volume and body weight were measured twice weekly for five weeks, and data were recorded. Data were statistically analyzed using Excel 2023 software: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT; and p-values ​​for inter-group differences were calculated using TTEST. The experimental results are shown in Table 3 and Figure 1. The ADC molecules of this application significantly reduced tumor volume.

[0289] Table 3: In vivo tumor-suppressive effect of ADC on NCI-N87 transplantation model

[0290] Test Example 4: Evaluation of JIMT-1 Efficacy in Tumor-Bearing Mice

[0291] Experimental Objective

[0292] The efficacy of the ADC in this application was evaluated using SCID Beige mice as test animals.

[0293] 1. Test drug

[0294] ADC-006: 1.1 mg / kg

[0295] Blank control: PBS

[0296] 2. Preparation method: All preparations are made by diluting with PBS.

[0297] 3. Test methods

[0298] JIMT-1 cells were subcutaneously injected into the right rib area of ​​mice. After 8 days of tumor growth, the animals were randomly divided into two groups of 6 mice each (1 experimental group + 1 blank control group).

[0299] The drug was administered via tail vein injection once. Tumor volume and body weight were measured twice weekly for five weeks, and data were recorded. Data were statistically analyzed using Excel 2023 software: mean value was calculated as average (avg); SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT; and p-value for inter-group differences was calculated as TTEST. The experimental results are shown in Table 4 and Figure 2. The ADC molecules of this application significantly reduced tumor volume.

[0300] Table 4: In vivo tumor-suppressive effect of ADC on JIMT-1 transplantation model

[0301] Test Example 5: Efficacy Evaluation of Human Gastric Cancer LD1-0017-411335PDX in Tumor-Bearing Mice

[0302] Experimental Objective

[0303] The efficacy of the ADC in this application was evaluated using NU / NU mice as test animals.

[0304] 1. Test drug

[0305] ADC-006: 8.7 mg / kg

[0306] ADC-027: 10 mg / kg

[0307] Blank control: PBS

[0308] 2. Preparation method: All preparations are made by diluting with PBS.

[0309] 3. Test methods

[0310] Human gastric cancer tumor tissue derived from LD1-0017-411335 was uniformly cut into tumor blocks approximately 3mm × 3mm × 3mm (approximately 30–60 mg) and subcutaneously injected into the right side of NU / NU mice. The mice's condition after injection was then observed, and their weight changes and tumor growth were monitored. When the average tumor volume of the grouped mice reached 140.81 mm², the tumors were successfully treated. 3 Animals were randomly stratified and divided into two groups of six each, based on tumor size. Grouping was considered day 0. Drug administration was performed via tail vein injection twice (days 0 and 21). Tumor volume and body weight were measured twice weekly for four weeks, and data were recorded. Data were analyzed using Excel 2023 statistical software: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT; and p-values ​​for inter-group differences were calculated using TTEST. The experimental results are shown in Table 5 and Figures 3a and 3b. The ADC molecules described in this application significantly reduced tumor volume.

[0311] Table 5: In vivo tumor-suppressive effect of ADC on human gastric cancer LD1-0017-411335PDX model

[0312] The above description provides an exemplary account of the implementation methods of the technical solution disclosed herein. It should be understood that the scope of protection of this disclosure is not limited to the above-described embodiments. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this application.

Claims

1. The compound represented by formula (I), its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound: MZ-Tr-LD (I) in, M is the linker site for binding to antibodies or their antigen fragments, selected from... Lg is a leaving group; R 21 R 22 Whether the two are the same or different, they are selected independently from H, CN, and C. 1-6 Alkyl-OC 1-6 Alkylene-, C 1-10 Alkyl group, -(OCH2CH2)m1-OCH3; t is selected from 1, 2, 3, 4, 5 or 6; m1 is selected from an integer from 1 to 36; Z is selected from single bond, -N(R) z )-(CH2)n1-C(=O)-, -N(R1)-(CH2CH2O)z1-(CH2)n2-C(=O)-, Among them, ring A is selected from cyclohexane ring or six-membered heterocycle; R1, R z They are either the same or different, and are independently selected from H, -(CH2CH2O)z3-CH3, z1, z2, z3, z4, z5, and z6 may be the same or different, and are independently selected from integers from 1 to 36; n1, n2, n3, n4, n5, n6, n7, and n8 may be the same or different, and are independently selected from 0, 1, 2, 3, 4, 5, and 6; Tr is a divalent trigger group, selected from divalent dipeptide or polypeptide residues; L is the linker between Tr and the bioactive molecular structural fragment D, selected from single bonds, -NH-C... 1-10 Alkyl-, -N(CH3)-C 1-10 Alkylene- D is a bioactive molecular structural fragment; preferably, D is selected from...

2. The compound of formula (I) as claimed in claim 1, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound, characterized in that, The leaving group Lg is selected from halogen, sulfone, trifluoromethanesulfonyl, and methanesulfonyl groups; Preferably, the leaving group Lg is selected from methanesulfonyl; Preferably, R 22 Selected from H; Preferably, R 21 Selected from CN, CH3OCH2-, C 1-3 alkoxy or -(OCH2CH2)m1-OCH3; Preferably, R 21 Selected from methoxy, -(OCH2CH2)6-OCH3 or -(OCH2CH2)8-OCH3; Preferably, t is selected from 2, 3, or 4; Preferably, m1 is selected from integers from 1 to 12; Preferably, m1 is selected from 6, 7, 8, 9 or 10; Preferably, M is selected from Preferably, ring A is selected from Preferably, R1 is selected from H; Preferably, R z Selected from the following groups: -(CH2CH2O)z3-CH3, Preferably, R z Selected from the following groups: -(CH2CH2O)9-CH3, -(CH2CH2O) 13 -CH3、 Preferably, z1, z2, z3, z4, z5, and z6 are the same or different, and are independently selected from integers between 6 and 15; Preferably, z1, z2, z3, z4, z5, and z6 are the same or different, and are independently selected from integers of 8-14; Preferably, n1, n2, n3, n4, n5, n6, n7, and n8 are the same or different, and are independently selected from 0, 1, 2, and 3; Preferably, Z is selected from single bonds, -N(R) z )-(CH2)n1-C(=O)-, -NH-(CH2CH2O)z1-(CH2)n2-C(=O)-, Wherein, X1 and X2 may be the same or different, and are independently selected from CH or N; preferably, at least one of X1 and X2 is an N atom; R z z1, z2, n1, n2, n3, and n4 have the definitions described above; Preferably, when X2 is CH, n4 is selected from 0; Preferably, Z is selected from single bonds, -NH-(CH2CH2O)8-CH2CH2-C(=O)-, and -NH-(CH2CH2O). 10 -CH2CH2-C(=O)-、-N(R z -CH2-C(=O)-、-N(R) z )-(CH2)2-C(=O)-、-N(R z )-(CH2)3-C(=O)-、-N(R z )-(CH2)4-C(=O)-、 Among them, R z It has the definition described above; Preferably, Z is selected from single bonds, Preferably, the Tr is selected from dipeptide or polypeptide residues, wherein the dipeptide or polypeptide is selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), valine-alanine (VA), aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG), and lysine-glycine-glycine-phenylalanine-glycine (KGGFG). Preferably, Tr is selected from glycine-glycine-phenylalanine-glycine (GGFG) residues, i.e. Preferably, Tr is selected from valine-alanine (VA) residues, that is: Preferably, L is selected from single bonds; Preferably, L is selected from 3. The compound of formula (I) as described in claim 1 or 2, its racemate, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound, characterized in that, The compound shown in formula (I) is selected from the following structures: Among them, Z and R 21 R z X1, X2, Tr, L, D, z1, z3, n1, n3, n4 have the definitions as described in claim 1 or 2; Preferably, the compound represented by formula (I) is selected from:

4. The antibody-drug conjugate shown in formula (II), its racemic, stereoisomer, tautomer, solvate, polymorph, and pharmaceutically acceptable salt. Ab-[M'-Z-Tr-LD] β (II) in, Ab is the antibody or its antigen-binding fragment, M' is the fragment formed after M is coupled with Ab, β is selected from integers or decimals between 1 and 10; Z, Tr, L 、 D independently has the definition as described in any one of claims 1-3; Preferably, Ab is an antibody or antigen-binding fragment, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody, and sdAb; and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, or a multispecific antibody; Preferably, Ab is an antibody against HER2 or its antigen-binding fragment; Preferably, Ab is pertuzumab or its antigen-binding fragment; Preferably, β is selected from an integer or decimal between 1 and 10; more preferably, it is selected from an integer or decimal between 2 and 8; more preferably, it is selected from an integer or decimal between 2 and 5; for example, β is selected from 2, 3, 4, 5, 6, 7, 8, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2; Preferably, M'-Z-Tr-LD has the following structure: Among them, Z and R 21 R z X1, X2, Tr, L, D, z1, z3, n1, n3, n4 have the definitions as described in any one of claims 1-3, and the wavy line is the connection point with Ab; Preferably, the M'-Z-Tr-L in the antibody-drug conjugate has the following structure:

5. The antibody-drug conjugate of formula (II) according to claim 4, its racemic, stereoisomer, tautomer, solvate, polymorph, or pharmaceutically acceptable salt, wherein, The antibody-drug conjugate shown in formula (II) is selected from the following structures: Among them, Ab and R 21 R Z z1, z3, X1, X2, n1, n3, n4, Tr, L, and β each have the definition as described in any one of claims 1-4 independently; Preferably, the antibody-drug conjugate shown in formula (II) has the following structure: Wherein, Ab and β independently have the definitions described in any one of claims 1-4; Preferably, the antibody-drug conjugate shown in formula (II) has the following structure: Wherein, β has the definition as described in any one of claims 1-4; More preferably, the antibody-drug conjugate is selected from the antibody-drug conjugates in the examples: ADC-001, ADC-002, ADC-003, ADC-004, ADC-005, ADC-007, ADC-009, ADC-010, ADC-014, ADC-019, ADC-020, ADC-021, ADC-022, ADC-023, and ADC-024.

6. Connector shown in equation (III): MZ-Tr-L' (III) in, M, Z, and Tr have the definitions described in any one of claims 1-5, and L' is the reaction form of L; Preferably, when L is a single bond, L' is the reaction form of Tr, for example, Tr-L' is a peptide with a carboxyl group or active ester at the carbon terminus; Preferably, when L is L' is the carbonate active ester form of p-aminobenzyl alcohol, for example The wavy lines represent the connection sites with peptide residues.

7. The connector shown below, in, Z, R 21 R z X1, X2, Tr, L, z1, n1, n3, n4 have the definitions as described in any one of claims 1-7, with 1 bit connected to Ab and 2 bits connected to D; Preferably, the connector is selected from the following structures:

8. The intermediate shown below: in, Lg, R 21 R z X1, X2, Z3, n3, and n4 have the definitions described in any one of claims 1-7, and Y1 is selected from hydroxyl, methoxy, ethoxy, isopropoxy, tert-butoxy, or Osu; Preferably, the intermediate is selected from the following structures:

9. A pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate of formula (II) as claimed in claim 4 or 5, a racemic, stereoisomer, tautomer, solvate, polymorph, or pharmaceutically acceptable salt thereof.

10. The use of at least one of the antibody-drug conjugate of formula (II) as claimed in claim 4 or 5, its racemic, stereoisomer, tautomer, solvate, polymorph, pharmaceutically acceptable salt, or the pharmaceutical composition of claim 9 in the preparation of a topoisomerase I inhibitor and / or in the preparation of a medicament for the prevention or treatment of diseases or conditions related to topoisomerase I. Preferably, the disease or symptom is a tumor, including breast cancer, stomach cancer, lung cancer, colorectal cancer, colon cancer, ovarian cancer, liver cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, pancreatic cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, or leukemia.

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