Class of degrader-antibody conjugates, preparation method therefor, and use thereof

Abstract

Provided are a class of degrader-antibody conjugates, a preparation method therefor, and use thereof. Specifically, provided are a degrader-antibody conjugate represented by formula II, a preparation method therefor, and use thereof in the preparation of a drug for treating and / or preventing cancer.

Description

A class of degradation agent antibody conjugates, preparation methods and applications

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to the following applications: CN application No. 202510123117.7, filed January 24, 2025; CN application No. 202510202502.0, filed February 24, 2025; CN application No. 202510376723.X, filed March 27, 2025; CN application No. 202510560621.3, filed April 29, 2025; and CN application No. 202511096745.7, filed August 5, 2025. The contents of the above applications are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the pharmaceutical field, specifically relating to a class of degradation agent antibody conjugates, their preparation methods, and their applications. Background Technology

[0004] Common protein degraders include proteolysis-targeting chimeras (PROTACs) and molecular glutens. PROTAC is a bifunctional molecule with a structure consisting of three parts: (1) a part that binds to the target protein substrate; (2) a part that binds to E3 ubiquitin ligase; and (3) a chain connecting the first two parts. PROTAC can recognize both the target protein and the E3 ubiquitin ligase, bringing them closer together. By inducing the recruitment of the E3 ubiquitin ligase to the target protein surface, it triggers ubiquitination and induces target protein degradation.

[0005] Degrader-Antibody Conjugates (DACs) are a novel type of targeted therapy drug, consisting of an antibody and a protein degrader linked by a linker unit. Utilizing the high specificity of antibodies, they recognize and bind to specific antigens on the cell surface, thereby precisely delivering the protein degrader into the target cell to degrade the target protein. This design not only improves drug targeting but also addresses the issues of low bioavailability and poor pharmacokinetic properties of protein degraders themselves through antibody internalization.

[0006] RAS is a guanine nucleotide-binding protein with GTPase activity and an intracellular anchoring function. RAS proteins can switch between an inactive state bound to GDP and an active state bound to GTP, influencing multiple downstream signaling pathways such as Raf, PI3K, and RalGDS, and regulating protein synthesis, gene transcription, cell growth, differentiation, apoptosis, and migration.

[0007] RAS mutations lead to persistent activation of downstream signaling pathways, promoting tumor development and progression. In all tumor types, RAS mutations primarily occur in the KRAS (85%). In KRAS-mutant tumor cells, KRAS GTPase activity is decreased, thus maintaining a persistently activated state. KRAS mutations are closely related to the development of various cancers, including lung cancer, pancreatic cancer, and colorectal cancer. Summary of the Invention

[0008] This invention provides a class of degradation agent antibody conjugates with degradation agents targeting KRAS proteins as loads, their preparation methods, and their applications in the prevention and / or treatment of cancer.

[0009] The first aspect of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:

[0010] M 1 -LD formula (I)

[0011] Among them, M 1 It is a precursor for the linker site to which an antibody or its antigen-binding fragment is attached;

[0012] L is connected to M 1 Connector to D;

[0013] D is a fragment of a degrader targeting the KRAS protein. Preferably, the degrader targeting the KRAS protein is selected from compounds represented by formula DI, D-II, or D-III.

[0014] Where ring A is C 3-6 Cycloalkyl or 3-6 membered heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally surrounded by one or more elements selected from halogens, C 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0015] Ring B is a 3-12 member nitrogen-containing heterocyclic group;

[0016] Ring D is a 4-8 membered heterocyclic group, wherein the heterocyclic group is optionally surrounded by one or more elements selected from halogens, C 1-6 Alkyl and C1-6 Substituents of haloalkyl groups;

[0017] Ring E is C 6-10 Aryl, 5-12-membered heteroaryl, or 5-12-membered heterocyclic group, wherein the aryl, heteroaryl, or heterocyclic group is optionally composed of one or more elements selected from halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, oxo groups, -OC 1-6 Alkyl, -OC 1-6 Halogenated alkyl groups and -OC 3-6 Substituents of cycloalkyl groups;

[0018] X 1 and X 2 Each is independently selected from O and N;

[0019] X 3 X 4 and X 5 Each independently selected from CR 7 and N;

[0020] X 6 Selected from CH and N;

[0021] L 10 It is a covalent bond, or selected from O, S and NR. 8 ;

[0022] L 11 and L 21 Each was independently selected from O, S, and NR. 9 ;

[0023] L 12 It is a covalent bond, or selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0024] L 13 It is a covalent bond, or selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0025] L 14 It is a covalent bond, or selected from O, NR 10 C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-NR 10 -、-C 1-6 Alkylene-OC 1-6 Alkylene-, C 3-6Cycloalkyl and 3-10 membered heterocyclic groups, wherein the cycloalkyl and heterocyclic groups are optionally selected from one or more halogens, hydroxyl groups, cyano groups, C6 groups, and C7 groups. 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0026] L 15 Selected from -C(=O)-NR 11 -、-C 1-6 Alkylene-C(=O)-NR 11 - and 5-10 heteroaryl groups, wherein the heteroaryl group is optionally surrounded by one or more groups selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0027] L 22 Selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0028] L 23 Selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0029] L 24 It is a covalent bond, or selected from O, NR 10 C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1- 6-alkylene-, -C(=O)-, -C(=O)-C 1-6 Alkylene and 3-12 membered heterocyclic groups, wherein the heterocyclic group is optionally surrounded by one or more groups selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0030] L 25 It is a covalent bond, or selected from O, NR 10 C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1- 6-alkylene-, -C(=O)-, -C(=O)-C 1-6 Alkylene and 3-12 membered heterocyclic groups, wherein the heterocyclic group is optionally surrounded by one or more groups selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0031] L 26 and L 36 Each is independently a covalent bond, or selected from O, NR 10 and -C(=O)-NR 10 -;

[0032] L 31 It is a covalent bond, or selected from C 1-6 Alkylene, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1-6 alkylene- and -C 1-6 Alkylene -C(=O)-; the alkylene group is optionally surrounded by one or more halogens, cyano groups, or C. 1-6 Alkyl substitution;

[0033] L 32 It is a covalent bond, or selected from C 3-6 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the cycloalkyl and heterocyclic groups are optionally selected from one or more halogens, C 1-6 Alkoxy, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0034] L 33 It is a covalent bond, or selected from C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1-6 Alkylene-, -C(=O)-, -C 1-6 Alkylene-C(=O)- and -C(=O)-C 1-6 Alkylene; the alkylene group is optionally oxidized by one or more halogens, cyano groups, or C. 1- 6-alkoxy substitution;

[0035] L 34 Selected from C 2-6 Ethyne group, C 3-6 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the cycloalkyl and heterocyclic groups are optionally composed of one or more groups selected from halogen, alkoxy, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0036] L 35 It is a covalent bond, or selected from C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylenes -O-, -C(=O)- and -C(=O)-C 1-6Alkylene; the alkylene group is optionally oxidized by one or more halogens, cyano groups, or C. 1-6 Alkyl substitution;

[0037] Each R 1 Selected independently from hydrogen, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OC 1-6 Alkyl, -OC 1-6 Halogenated alkyl groups, -OC 3-6 cycloalkyl groups and 3-6 membered heterocyclic groups;

[0038] R 2 Selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 3-6 cycloalkyl, C 6-10 Aryl, 3-12 membered heterocyclic, 5-10 membered heteroaryl, -C 1-6 Alkylene-C 3-6 cycloalkyl, -C 1-6 alkylene-3-10-membered heterocyclic groups and -C 1-6 Alkylene-5-10-membered heteroaryl, wherein the alkyl, alkylene, cycloalkyl, aryl, heterocyclic, and heteroaryl groups are optionally selected from one or more halogens, hydroxyl groups, amino groups, cyano groups, C6 groups, and C7 groups. 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OC 1-6 Alkyl, -C 1-6 Alkylene -OH, -OC 1-6 Haloalkyl, C 3-6 cycloalkyl, C 6-10 Aryl, 3-10 heterocyclic, 5-10 heteroaryl, -NR 12a R 12b -C 1-6 Alkylene-OC 1-6 Alkyl groups and -C(=O)-NR 12a R 12b Substituents of the substituents;

[0039] R 3 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl and C 3-6 cycloalkyl;

[0040] R 4a and R 4b Each is independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Haloalkyl, -C 1-6 alkylene-OH and C 3-6 cycloalkyl; or,

[0041] R4a and R 4b C forms with the carbon atoms that are bonded together. 3-6 Cycloalkyl or 3-6 membered heterocyclic groups;

[0042] R 5 Selected from halogens, phenyl groups, and 5-6-membered heteroaryl groups, wherein the phenyl and heteroaryl groups are optionally separated by one or more groups selected from halogens, hydroxyl groups, cyano groups, C6 groups, and C7 groups. 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0043] Each R 6 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups; or,

[0044] R 5 and adjacent R 6 The carbon atoms attached to them together form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring and heteroaromatic ring are optionally bonded by one or more elements selected from halogens, C, and other elements. 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0045] Each R 7 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 cycloalkyl, C 1-6 Alkoxy and C 1-6 Halogenated alkyl-O-;

[0046] R 8 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl and C 3-6 cycloalkyl;

[0047] R 9 Selected from hydrogen and C 1-6 alkyl;

[0048] R 10 Selected from hydrogen and C 1-6 alkyl;

[0049] R 11 Selected from hydrogen and C 1-6 alkyl;

[0050] R 12a and R 12b Each is independently selected from hydrogen and C. 1-6 Alkyl, C 3-6 cycloalkyl, -C 1-6 alkylene -OH and -C 1-6 Alkylene-OC1-6 Alkyl, or R 12a and R 12b The N atom attached to it forms a 3-6 member nitrogen-containing heterocyclic group;

[0051] p is selected from 0, 1, 2, 3, 4, 5, and 6;

[0052] q is selected from 0, 1, 2, 3 and 4.

[0053] A second aspect of the invention provides a degradation agent antibody conjugate of Formula II.

[0054] Among them, Ab 1 For targeting groups;

[0055] M is the junction site that connects to the target group;

[0056] L is the connector that connects M and D;

[0057] D is as defined above;

[0058] m is selected from 1-20.

[0059] A third aspect of the invention provides a composition comprising the degradation agent antibody conjugate described in the second aspect, wherein the composition has a DAR value of 1.0-20.0, preferably 1.0-10.0.

[0060] A fourth aspect of the invention provides a pharmaceutical composition comprising the compound of the first aspect or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, the degradation agent antibody conjugate of the second aspect, or the antibody or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

[0061] The fifth aspect of the invention provides the use of the compound described in the first aspect or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug or the antibody described herein or an antigen-binding fragment thereof in the preparation of the degrading agent antibody conjugate described in the second aspect.

[0062] The sixth aspect of the invention provides the use of the compounds described in the first aspect or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, the degradation agents antibody conjugates described in the second aspect, the compositions described in the third aspect, and the pharmaceutical compositions described in the fourth aspect in the preparation of medicaments, particularly in medicaments for the treatment and / or prevention of cancer (e.g., cancers associated with KRAS mutations).

[0063] The seventh aspect of the invention provides the compounds described in the first aspect or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, the degradation agent antibody conjugates described in the second aspect, the compositions described in the third aspect, and the pharmaceutical compositions described in the fourth aspect for the treatment and / or prevention of cancer (e.g., cancers associated with KRAS mutations).

[0064] The eighth aspect of the invention provides a method for treating and / or preventing cancer (e.g., cancer associated with KRAS mutations), comprising administering to a subject in need a therapeutically and / or preventively effective amount of the compound of the first aspect or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, a degrading agent antibody conjugate of the second aspect, a composition of the third aspect, or a pharmaceutical composition of the fourth aspect. Detailed Implementation

[0065] definition

[0066] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better interpret this disclosure.

[0067] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0068] The terms "conjugate" and "combination" are used interchangeably, referring to a substance obtained by linking one or more heterologous molecules(s) to an antibody. These heterologous molecules can be, for example, bioactive molecules or drugs, including but not limited to cytotoxic agents and protein degraders. Conjugates targeting antibodies are also called antibody-drug conjugates (ADCs). The term "degrader-antibody conjugates (DACs)" specifically refers to conjugates obtained by linking protein degraders to antibodies.

[0069] The one or more heterologous molecules can be linked to a target moiety (e.g., an antibody) via a linker. The linker can be linked to the target moiety via various chemical bonds. For example, in some embodiments, when the target moiety is an antibody, the linker is linked by forming a thioether bond with the thiol group of the antibody. In some specific ADC molecular structures, -S- only represents the thioether bond formed between the linker and the thiol group of the antibody, and does not mean that -S- is part of the linker.

[0070] The structure of the conjugates in this application can be represented by general formula II, where m refers to the number of bioactive molecular fragments linked to each target molecule. During the preparation of antibody-drug conjugates, each antibody molecule may link to different numbers of bioactive molecular fragments; therefore, generally speaking, antibody-drug conjugates are mixtures of antibody-drug conjugates with different drug-antibody conjugation ratios. In practice, DAR is usually used to represent the average number of drugs linked to the antibody.

[0071] As used herein, the term "antibody" is used in the broadest sense to encompass a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can consist of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, 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 variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain 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 in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the 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, or related studies by AbM and Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272).In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0072] As used in this article, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region responsible for antigen binding. The precise boundaries of these amino acid residues 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), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA). 86:9268–9272), this definition method integrates parts of the definitions from Kabat and Chothia, and was first applied in the Oxford Molecular antibody modeling software (Martin AC R. Protein sequence and structure analysis of antibody variable domains[M] / / Antibody engineering. Springer, Berlin, Heidelberg, 2010:33-51.). For a given antibody, those skilled in the art will easily 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).

[0073] The CDR contained in the antibodies or antigen-binding fragments thereof disclosed herein can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibodies or antigen-binding fragments thereof disclosed herein is preferably determined using the Chothia numbering system.

[0074] As used herein, the term “antigen-binding fragment” of an antibody refers to a molecule other than the full-length antibody, which includes a portion of the full-length antibody that binds to the antigen bound to the full-length antibody. For example, a polypeptide fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound to the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, is also referred to as an “antigen-binding moiety.” See also 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 by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody, linear antibody, nanobody (technology from Domantis), domain antibody (technology from Ablynx), 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.

[0075] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; 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, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0076] 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 (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0077] The term "Fc" refers to an antibody fragment formed by 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 various functions but does not participate in antigen binding.

[0078] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). 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 (SEQ ID NO:64) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 65) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0079] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". A "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that, in the N-terminal to C-terminal direction, comprises a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" or "light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody disclosed herein can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody disclosed herein comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0080] In this document, the techniques for obtaining antibodies may use conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods) to obtain antigen-binding fragments of the antibody (e.g., the antibody fragments described above) from a given antibody (e.g., the antibody provided in this disclosure), and the antigen-binding fragments of the antibody may be specifically screened in the same manner as those used for intact antibodies.

[0081] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0082] The monoclonal antibodies disclosed herein can be prepared using a variety of techniques, such as hybridoma techniques (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA techniques (see, for example, U.S. Patent Application 4,816,567), or phage antibody library techniques (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0083] For example, monoclonal antibodies can be prepared as follows: First, immunize mice or other suitable host animals with an immunogen (with adjuvants added if necessary). The immunogen or adjuvant is usually injected subcutaneously at multiple sites or intraperitoneally. The immunogen can be pre-conjugated to certain known proteins, such as serum albumin or soybean trypsin inhibitors, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After immunization, the animal will produce lymphocytes that secrete antibodies specifically binding to the immunogen. Alternatively, lymphocytes can be obtained through in vitro immunization. Collect the target lymphocytes and fuse them with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared above can be seeded into a suitable culture medium for growth, preferably containing one or more substances that can inhibit the growth of unfused maternal myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine-guanine phosphotransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium can inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should possess characteristics such as high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture medium. Among these, murine myeloma cells are preferred, such as MOP-21 or MC-11 mouse tumor-derived lines (THE Salk Institute Cell Distribution Center, San Diego, Calif.USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md.USA). Other studies have reported the preparation of human monoclonal antibodies using human myeloma and human-mouse heterologous myeloma cell lines (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987). The culture medium for growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al. in Anal. Biochem. 107:220 (1980). Once the specificity, affinity, and reactivity of the antibodies produced by the hybridoma have been determined, the target cell line can be subcloned using the standard limiting dilution method described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. Suitable culture media may include DMEM or RPMI-1640. Additionally, hybridoma cells can also grow in animals in the form of ascites tumors. Monoclonal antibodies secreted by subclonal cells can be separated from cell culture medium, ascites, or serum using conventional immunoglobulin purification methods such as protein A agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0084] Monoclonal antibodies can also be obtained through recombinant genetic engineering. PCR amplification using nucleic acid primers that specifically bind to the heavy and light chain genes of monoclonal antibodies allows the isolation of DNA molecules encoding these genes from hybridoma cells. The resulting DNA molecules are then inserted into an expression vector, transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other non-immunoglobulin-producing myeloma cells), and cultured under suitable conditions to obtain recombinantly expressed target antibodies.

[0085] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to purify the immunospecific antibody. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0086] As used in this article, the term "mouse antibody" refers to a mouse hybrid fusion cell derived from the fusion of B cells and myeloma cells from immunized mice. These cells are then screened to identify those capable of unlimited proliferation and antibody secretion, followed by screening, antibody preparation, and purification. Alternatively, antibodies may be produced by the differentiation and proliferation of B cells after antigen invasion of the mouse, forming plasma cells that can secrete antibodies. Antibodies are also produced by stimulation with specific antigens. Antibody production occurs because antigen invasion of the human body triggers interactions among various immune cells, causing B cells in lymphocytes to differentiate and proliferate, forming plasma cells that can secrete antibodies.

[0087] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with human antibodies. Typically, 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 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, ability to enhance immune cell activity, and ability to enhance immune responses. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance immune responses).

[0088] Humanized antibodies are particularly advantageous because they retain the intended properties of non-human donor antibodies (e.g., murine antibodies) while effectively reducing their immunogenicity in human subjects. However, due to the matching problem between the CDR of the donor antibody and the FR of the receptor antibody, the intended properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response) are generally lower than those of non-human donor antibodies (e.g., murine antibodies).

[0089] Therefore, although researchers in this field have conducted in-depth research on antibody humanization and made some progress (see, for example, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000)), existing technologies do not provide detailed guidance on how to fully humanize a donor antibody so that the resulting humanized antibody has the highest possible degree of humanization while retaining the expected properties of the donor antibody as much as possible. Technicians need to explore, investigate, and modify specific donor antibodies, and it takes a lot of creative work to obtain humanized antibodies that have a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) while retaining the expected properties of the specific donor antibody.

[0090] In this disclosure, in order to preserve as much of the properties of the donor antibody as possible (including, for example, antigen specificity, affinity, reactivity, ability to enhance immune cell activity and / or ability to enhance immune response), the framework region (FR) of the humanized antibody of this disclosure may contain both amino acid residues of the human receptor antibody and the corresponding non-human donor antibody.

[0091] The humanized antibodies disclosed herein can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. The DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0092] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter's U.S. Patent No. 5,225,539; Queen et al.'s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used. For example, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice can completely suppress the production of endogenous antibodies, and then transfer of human germline immunoglobulin gene arrays into said germline mutant mice will cause the mice to produce human antibodies upon encountering antigen stimulation (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2551; Jakobovits et al., 1993, Nature 362: 255-258; Bruggermann et al., 1993, Year in Immunology 7: 33; and Duchosal et al., 1992, Nature 355: 258). Non-limiting examples of the aforementioned transgenic animals include the HuMAb mouse (Medarex, Inc.) containing a miniloci of the human immunoglobulin gene encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, coupled with targeted mutations that inactivate the endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or the “KM mouse™” carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0093] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effective concentration (EC50) of the interaction.

[0094] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured using bioluminescent interferometry (e.g., the ForteBio Octet method). Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0095] The twenty common amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala.

[0096] As used herein, the term “pharmaceuticalally acceptable carrier” means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0097] As used herein, the term “prevention” refers to methods implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a tumor) in a subject.

[0098] As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of disease, stabilization (i.e., cessation of disease progression) of the disease state, delay or slowing of disease progression, improvement or relief of the disease state, and relief of symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also mean prolongation of survival compared to expected survival (if no treatment was received).

[0099] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor, or is at risk of having the aforementioned disease.

[0100] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0101] The terms "cancer" and "tumor" are used interchangeably to refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant sites of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies, especially hematologic malignancies.

[0102] As used herein, the term "alkyl" is defined as a straight-chain or branched saturated aliphatic hydrocarbon group. For example, as used herein, the term "C" is... 1-6 "Alkyl" refers to a straight-chain or branched group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl), optionally substituted with one or more (such as 1 to 3) suitable substituents such as halogens. The term "C" 1-3 "Alkyl" refers to a straight-chain or branched group having 1 to 3 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogens.

[0103] As used herein, the term "alkoxy" refers to "alkyl-O-", where "alkyl" is as defined above. For example, the term "C" 1-6 "Alkoxy" refers to "C 1-6 alkyl-O-", the "C" 1-6 "alkyl" is as defined above. "C" as used in this disclosure 1-3 "Alkoxy" refers to "C 1-3 alkyl-O-", the "C" 1-3 "Alkyl" is as defined above. An exemplary C 1-6 Alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-hexoxy.

[0104] The term "alkylene" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched alkane, including, for example, "C". 1-20 Alkylene, C 1- 10 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, C 1-3 "alkylene", etc., specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene, etc.

[0105] The term "alkenyl" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C". 2-6 "Ideinyl", "C" 2-4 Examples of these include, but are not limited to: vinylidene, 1-propenyne, 2-propenyne, 1-butenyne, 2-butenyne, 1,3-butadiene, 1-pentenyne, 2-pentenyne, 3-pentenyne, 1,3-pentadiene, 1,4-pentadiene, 1-hexenyne, 2-hexenyne, 3-hexenyne, 1,4-hexadiene, etc.

[0106] The term "acetylenic" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-6 "Isynyne", "C" 4-6 Examples of "ethynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentynyl, 1,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexynyl, etc.

[0107] As used herein, the term "aryl" or "aromatic ring" refers to a fully carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated π-electron system. For example, the term "C 6-10 "Aryl" or "C" 6-10 "Aromatic ring" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl (cyclic) or naphthyl (cyclic). The aryl group is optionally substituented by one or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, C). 1-6 Alkyl groups, etc., are substituted.

[0108] In this document, the term "heteroaryl" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atom (e.g., a carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may be oxidized. Specific examples include, but are not limited to, 5-10-membered heteroaryl, 5-10-membered nitrogen-containing heteroaryl, 6-10-membered oxygen-containing heteroaryl, 6-8-membered nitrogen-containing heteroaryl, 5-8-membered oxygen-containing heteroaryl, etc., such as furanyl, thiophene, pyrrole, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazole, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl Azolyl, pyridyl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxazinyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, aziridine-heptanetrienyl, 1,3-diazacycloheptanetrienyl, aziridine-octatetraenyl, etc.

[0109] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl groups (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C" 3-10 cycloalkyl (e.g., C10) 3-8 "Cycloalkyl" refers to cycloalkyl groups having 3 to 10 (e.g., 3-8) cyclic carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0110] The term "heterocyclic group" refers to a group formed by a saturated or partially saturated non-aromatic cyclic structure containing at least one ring member selected from N, O, and S. The term "3-12 membered heterocyclic group (e.g., 3-10 membered heterocyclic group, 3-8 membered heterocyclic group)" refers to a heterocyclic group having 3-12 (e.g., 3-10, 3-8) ring atoms. Specific examples include, but are not limited to, 5-6 membered heterocyclic groups, 5-6 membered nitrogen-containing heterocyclic groups, and 5-6 membered oxygen-containing heterocyclic groups, such as ethylene oxide, aziridinyl, azetidinyl, oxetanyl, thiohexacyclic, pyrrolyl, hexahydro-1H-pyrrololine, pyrrolidone, imidazoalkyl, pyrazolyl, dihydropyrroleyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyridinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydropyridinyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, or piperazine, etc.

[0111] As used herein, the term "halogen" is defined as including fluorine, chlorine, bromine, or iodine.

[0112] As used in this article, the term "halogenation" refers to the substitution of one or more (such as 1 to 3) identical or different halogen atoms.

[0113] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (such as 1 to 3) identical or different halogen atoms. For example, the term "C" 1-6 "Halogenated alkyl" refers to alkyl halogroups having 1 to 6 carbon atoms, such as -CF3, -C2F5, -CHF2, -CH2F, -CH2CF3, -CH2Cl, or -CH2CH2CF3.

[0114] Optionally, the hydrogen in the groups involved in this disclosure may be replaced by deuterium.

[0115] The term "substitution" refers to the selective replacement of one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound.

[0116] Unless otherwise defined, the terms "substituent" or "suitable substituent" as used herein refer to modifications of a compound that can be made by those skilled in the art to suit the needs of the compound's substituents, including oxo (=O), halogen, cyano, amino, C... 1-6 Alkylamino, carboxyl, mercapto, hydroxyl, ester (e.g., -C) 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 Halogenated alkoxy groups, etc.

[0117] If a substituent is described as “optionally substituted with…”, then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together with independently selected substituents or not substituted. If the nitrogen of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted with independently selected substituents or not substituted.

[0118] If a substituent is described as being "independently selected" from a group of groups, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0119] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, six, seven, eight, nine, or ten.

[0120] Unless otherwise specified, as used herein, the connection point of a substituent may be located at any suitable position of the substituent.

[0121] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0122] This disclosure also includes all pharmaceutically acceptable isotopically labeled compounds identical to those of this disclosure, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of this disclosure include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 37Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Certain isotope-labeled compounds of this disclosure (e.g., those doped with radioisotopes) can be used in drug and / or substrate tissue distribution studies (e.g., analysis). Radioisotope tritium (i.e. 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and detection. Using positron-emitting isotopes (e.g.) 11 C 18 F, 15 O and 13 Substitution of N) can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds of this disclosure can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations, by replacing previously used unlabeled reagents with appropriate isotopically labeled reagents. Pharmaceutically acceptable solvates of this disclosure include those in which the crystallization solvent can be isotopically substituted, for example, D2O, acetone-d6, or DMSO-d6.

[0123] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of this disclosure can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0124] This disclosure covers all possible crystalline forms or polymorphs of the compounds disclosed herein, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0125] It should also be understood that certain compounds of this disclosure may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In this disclosure, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites, or prodrugs that, upon administration to a patient in need, can directly or indirectly provide the compounds of this disclosure or their metabolites or residues. Therefore, when referring herein to “compounds of this disclosure,” it is also intended to encompass the various derivative forms of the compounds described above.

[0126] Pharmaceutically acceptable salts of the compounds disclosed herein include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. A review of suitable salts can be found in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds disclosed herein are known to those skilled in the art.

[0127] The compounds disclosed herein may exist as solvates (preferably hydrates), wherein the compounds of this disclosure contain a polar solvent as a structural element of the compound's crystal lattice. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0128] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form N-oxides because nitrogen requires available lone pairs of electrons to be oxidized into oxides; those skilled in the art will identify nitrogen-containing heterocycles that can form N-oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750; A.R. Katritzky and A.J. Boulton, Eds., Academic Press; and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R. Katritzky and A.J. Boulton, Eds., Academic Press.

[0129] The scope of this disclosure also includes metabolites of the compounds of this disclosure, i.e., substances formed in the body upon administration of the compounds of this disclosure. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this disclosure includes metabolites of the compounds of this disclosure, including compounds prepared by methods that expose the compounds of this disclosure to mammals for a time sufficient to produce their metabolites.

[0130] This disclosure further includes, within its scope, prodrugs of the compounds of this disclosure, which are certain derivatives of the compounds of this disclosure that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of this disclosure having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (EB Roche, editor, American Pharmaceutical Association). Prodrugs of this disclosure can be prepared, for example, by replacing suitable functional groups present in the compounds of this disclosure with certain portions known to those skilled in the art as “pro-moiety” (e.g., as described in “Design of Prodrugs,” H. Bundgaard (Elsevier, 1985)).

[0131] This disclosure also covers compounds of this disclosure containing protecting groups. In any process of preparing the compounds of this disclosure, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of this disclosure. This can be achieved by conventional protecting groups, for example, those described in *Protective Groups in Organic Chemistry*, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P. ​​G.W. M. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0132] In this document (specification and claims), the singular forms "a," "an," and "the" are used unless the context clearly indicates otherwise, including their plural forms, such as "a host cell" which includes multiple such host cells. Furthermore, there are no direct equivalent singular / plural grammatical rules in Chinese; the singular or plural form of a noun must be determined based on the context or actual situation. Therefore, in the English translation, adding "one or more than one" before a noun is likely correct.

[0133] Whether explicitly stated or not, all numerical values ​​in this application are modified by the term “about”. The term “about” means within ±20%, ±10%, ±5%, or ±2% of the stated numerical value.

[0134] Drug linker

[0135] This invention provides compounds of formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof:

[0136] M 1 -LD formula (I)

[0137] Among them, M 1 It is a precursor for the linker site to which an antibody or its antigen-binding fragment is attached;

[0138] L is connected to M 1 Connector to D;

[0139] D is a fragment of a degrader targeting the KRAS protein. Preferably, the degrader targeting the KRAS protein is selected from compounds represented by formula DI, D-II, or D-III.

[0140] Where ring A is C 3-6 Cycloalkyl or 3-6 membered heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally surrounded by one or more elements selected from halogens, C 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0141] Ring B is a 3-12 member nitrogen-containing heterocyclic group;

[0142] Ring D is a 4-8 membered heterocyclic group, wherein the heterocyclic group is optionally surrounded by one or more elements selected from halogens, C 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0143] Ring E is C 6-10Aryl, 5-12-membered heteroaryl, or 5-12-membered heterocyclic group, wherein the aryl, heteroaryl, or heterocyclic group is optionally composed of one or more elements selected from halogens, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, oxo groups, -OC 1-6 Alkyl, -OC 1-6 Halogenated alkyl groups and -OC 3-6 Substituents of cycloalkyl groups;

[0144] X 1 and X 2 Each is independently selected from O and N;

[0145] X 3 X 4 and X 5 Each independently selected from CR 7 and N;

[0146] X 6 Selected from CH and N;

[0147] L 10 It is a covalent bond, or selected from O, S and NR. 8 ;

[0148] L 11 and L 21 Each was independently selected from O, S, and NR. 9 ;

[0149] L 12 It is a covalent bond, or selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0150] L 13 It is a covalent bond, or selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0151] L 14 It is a covalent bond, or selected from O, NR 10 C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-NR 10 -、-C 1-6 Alkylene-OC 1-6 Alkylene-, C 3-6 Cycloalkyl and 3-10 membered heterocyclic groups, wherein the cycloalkyl and heterocyclic groups are optionally selected from one or more halogens, hydroxyl groups, cyano groups, C6 groups, and C7 groups. 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0152] L 15 Selected from -C(=O)-NR 11 -、-C 1-6 Alkylene-C(=O)-NR 11 - and 5-10 heteroaryl groups, wherein the heteroaryl group is optionally surrounded by one or more groups selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0153] L 22 Selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0154] L 23 Selected from C 1-6 Alkylene and Halogenated C 1-6 Alkylene;

[0155] L 24 It is a covalent bond, or selected from O, NR 10 C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1- 6-alkylene-, -C(=O)-, -C(=O)-C 1-6 Alkylene and 3-12 membered heterocyclic groups, wherein the heterocyclic group is optionally surrounded by one or more groups selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0156] L 25 It is a covalent bond, or selected from O, NR 10 C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1- 6-alkylene-, -C(=O)-, -C(=O)-C 1-6 Alkylene and 3-12 membered heterocyclic groups, wherein the heterocyclic group is optionally surrounded by one or more groups selected from halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0157] L 26 and L 36 Each is independently a covalent bond, or selected from O, NR 10 and -C(=O)-NR 10 -;

[0158] L 31 It is a covalent bond, or selected from C 1-6 Alkylene, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1-6 alkylene- and -C 1-6 Alkylene -C(=O)-; the alkylene group is optionally surrounded by one or more halogens, cyano groups, or C. 1-6 Alkyl substitution;

[0159] L 32 It is a covalent bond, or selected from C 3-6 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the cycloalkyl and heterocyclic groups are optionally selected from one or more halogens, C 1-6 Alkoxy, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0160] L 33 It is a covalent bond, or selected from C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylene -O-, -C 1-6 Alkylene-OC 1-6 Alkylene-, -C(=O)-, -C 1-6 Alkylene-C(=O)- and -C(=O)-C 1-6 Alkylene; the alkylene group is optionally oxidized by one or more halogens, cyano groups, or C. 1- 6-alkoxy substitution;

[0161] L 34 Selected from C 2-6 Ethyne group, C 3-6 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the cycloalkyl and heterocyclic groups are optionally composed of one or more groups selected from halogen, alkoxy, cyano, C 1-6 Alkyl, C 1-6 Substituents of haloalkyl groups;

[0162] L 35 It is a covalent bond, or selected from C 1-6 Alkylene, -OC 1-6 alkylene-, -C 1-6 Alkylenes -O-, -C(=O)- and -C(=O)-C 1-6 Alkylene; the alkylene group is optionally oxidized by one or more halogens, cyano groups, or C. 1-6 Alkyl substitution;

[0163] Each R 1 Selected independently from hydrogen, halogen, C1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OC 1-6 Alkyl, -OC 1-6 Halogenated alkyl groups, -OC 3-6 cycloalkyl groups and 3-6 membered heterocyclic groups;

[0164] R 2 Selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 3-6 cycloalkyl, C 6-10 Aryl, 3-12 membered heterocyclic, 5-10 membered heteroaryl, -C 1-6 Alkylene-C 3-6 cycloalkyl, -C 1-6 alkylene-3-10-membered heterocyclic groups and -C 1-6 Alkylene-5-10-membered heteroaryl, wherein the alkyl, alkylene, cycloalkyl, aryl, heterocyclic, and heteroaryl groups are optionally selected from one or more halogens, hydroxyl groups, amino groups, cyano groups, C6 groups, and C7 groups. 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OC 1-6 Alkyl, -C 1-6 Alkylene -OH, -OC 1-6 Haloalkyl, C 3-6 cycloalkyl, C 6-10 Aryl, 3-10 heterocyclic, 5-10 heteroaryl, -NR 12a R 12b -C 1-6 Alkylene-OC 1-6 Alkyl groups and -C(=O)-NR 12a R 12b Substituents of the substituents;

[0165] R 3 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl and C 3-6 cycloalkyl;

[0166] R 4a and R 4b Each is independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Haloalkyl, -C 1-6 alkylene-OH and C 3-6 cycloalkyl; or,

[0167] R 4a and R 4b C forms with the carbon atoms that are bonded together. 3-6 Cycloalkyl or 3-6 membered heterocyclic groups;

[0168] R 5Selected from halogens, phenyl groups, and 5-6-membered heteroaryl groups, wherein the phenyl and heteroaryl groups are optionally separated by one or more groups selected from halogens, hydroxyl groups, cyano groups, C6 groups, and C7 groups. 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0169] Each R 6 Independently selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl and C 1-6 Halogenated alkyl groups; or,

[0170] R 5 and adjacent R 6 The carbon atoms attached to them together form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring and heteroaromatic ring are optionally bonded by one or more elements selected from halogens, C, and other elements. 1-6 Alkyl and C 1-6 Substituents of haloalkyl groups;

[0171] Each R 7 Independently selected from hydrogen and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 cycloalkyl, C 1-6 Alkoxy and C 1-6 Halogenated alkyl-O-;

[0172] R 8 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl and C 3-6 cycloalkyl;

[0173] R 9 Selected from hydrogen and C 1-6 alkyl;

[0174] R 10 Selected from hydrogen and C 1-6 alkyl;

[0175] R 11 Selected from hydrogen and C 1-6 alkyl;

[0176] R 12a and R 12b Each is independently selected from hydrogen and C. 1-6 Alkyl, C 3-6 cycloalkyl, -C 1-6 alkylene -OH and -C 1-6 Alkylene-OC 1-6 Alkyl, or R 12a and R 12b The N atom attached to it forms a 3-6 member nitrogen-containing heterocyclic group;

[0177] p is selected from 0, 1, 2, 3, 4, 5, and 6;

[0178] q is selected from 0, 1, 2, 3 and 4.

[0179] When multiple R 10 When they exist, each R 10 They can be the same or different.

[0180] When multiple R 12a When they exist, each R 12a They can be the same or different.

[0181] When multiple R 12b When they exist, each R 12b They can be the same or different. In some implementations, M 1 Selected from the following structures

[0182] In this case, each 'a' is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and each 'b' is selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0183] In some implementation schemes, M 1 Selected from the following structures

[0184] In this case, each 'a' is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and each 'b' is selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0185] In some implementation schemes, M 1 Selected from

[0186] In some implementation schemes, M 1 Selected from

[0187] In some embodiments, L is selected from non-breakable linkers and breakable linkers, wherein the breakable linker is cleaved by an enzyme present in the pathological environment, wherein the enzyme is selected from proteases, phosphatases, pyrophosphatases, β-glucuronidase, β-galactosidase, and sulfatases.

[0188] In some implementations, L is -(L 2 ) r -L 3 -L 4 -,in:

[0189] L 2 The bonds are covalent or each can be independently selected from the following structures:

[0190] R 12 Selected from H and -L 2a -L 2b -(Q) n ,in;

[0191] L 2a It is a covalent bond or selected from the following structures:

[0192] For example, L 2a It is a covalent bond or selected from the following structures:

[0193] L 2b It is a covalent bond or selected from the following structures:

[0194] Q is a hydroxyl group or selected from the following structures:

[0195] For example, Q is a hydroxyl group or selected from the following structures:

[0196] Where r is selected from 1 to 3 (e.g., 1, 2, or 3); n is independently selected from 1 to 9 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9); each c is independently selected from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6); and each d is independently selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0197] L 3 Fragments selected from amino acids or their derivatives and peptide fragments formed from two or more amino acids or their derivatives, wherein the amino acids are selected from Val, Cit, Glu, Lys, Arg, Phe, Leu, Gly, Ala, and Asn; and the amino acid derivatives are selected from... R' is independently selected from hydrogen, C 1-6 Alkyl, -C 1-6 Alkylene-OC 1-6 Alkyl, -C 1-6 Alkylene-NHC 1-6 Alkyl, -C 1-6 Alkylene-N(C) 1-6 Alkyl)2;

[0198] L 4 It is a covalent bond or selected from the following structures:

[0199] In some implementations, L 2 The bonds are covalent or each can be independently selected from the following structures:

[0200] R 12 Selected from H and -L 2a -L 2b -(Q) n ,in;

[0201] L 2a It is a covalent bond or selected from the following structures:

[0202] L 2b It is a covalent bond or selected from the following structures:

[0203] Q is a hydroxyl group or selected from the following structures:

[0204] For example, Q is a hydroxyl group or selected from the following structures:

[0205] Wherein, n is independently selected from integers 1 to 9 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9), each c is independently selected from integers 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from integers 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0206] In some implementations, L 2 The bonds are covalent or each can be independently selected from the following structures:

[0207] R 12 Selected from H and -L 2a -L 2b -(Q) n ,in;

[0208] L 2a It is a covalent bond or selected from the following structures: Preferably, L 2a It is a covalent bond or selected from the following structures:

[0209] L 2b Covalent bond or selected from:

[0210] Q is a hydroxyl group or selected from the following structures:

[0211] For example, Q is a hydroxyl group or selected from the following structures:

[0212] Wherein, n is independently selected from integers 1 to 9 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9), each c is independently selected from integers 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from integers 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0213] In some implementations, L 2 The bonds are covalent or each can be independently selected from the following structures:

[0214] R 12 Selected from H and -L 2a -L 2b -(Q) n ,in;

[0215] L 2a It is a covalent bond or selected from the following structures: Preferably, L 2a It is a covalent bond or selected from the following structures:

[0216] L 2b Covalent bond or selected from:

[0217] Q is a hydroxyl group or selected from the following structures:

[0218] For example, Q is a hydroxyl group or selected from the following structures:

[0219] Wherein, n is independently selected from integers 1 to 9 (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9), each c is independently selected from integers 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from integers 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0220] In some implementations, L 2a Covalent bond or selected from

[0221] In some implementations, L 2a Covalent bond or selected from

[0222] In some implementations, L 2b Covalent bond or selected from: Position 6 is related to L 2a Connected, position 7 is connected to Q.

[0223] In some implementations, L 2b Covalent bond or selected from: Position 6 is related to L 2a Connected, position 7 is connected to Q.

[0224] In some implementation schemes, R 12 Selected from the following structures:

[0225] In some implementation schemes, R 12 Selected from the following structures:

[0226] In some implementation schemes, R 12 Selected from the following structures:

[0227] In some implementations, L 2 It is a covalent bond or selected from the following structures: Each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0228] In some implementations, L 2 It is a covalent bond or selected from the following structures: Each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0229] In some implementations, L 2 It is a covalent bond or selected from the following structures: Each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0230] In some implementations, -(L 2 ) r - It is a covalent bond or selected from the following structures:

[0231] Among them, position 4 and M 1 Connected, position 8 and L 3 Connected.

[0232] In some implementations, -(L 2 ) r - It is a covalent bond or selected from the following structures:

[0233] Among them, position 4 and M 1 Connected, position 8 and L 3 Connected.

[0234] In some implementations, -(L 2 ) r - It is a covalent bond or selected from the following structures:

[0235] Among them, position 4 and M 1 Connected, position 8 and L 3 Connected.

[0236] In some implementations, L 3 Selected from the following structures:

[0237] In some implementations, L 3 Selected from the following structures:

[0238] In some implementations, L 3 for

[0239] In some implementations, L 3 for

[0240] In some implementations, L 4 For covalent bonds or

[0241] In some implementations, L is selected from the following structures:

[0242] Among them, the 4th position of L is related to M 1 The 5th position is connected to the D; each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0243] In some implementations, L is selected from the following structures:

[0244] Among them, the 4th position of L is related to M 1 The 5th position is connected to the D; each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0245] In some implementations, L is selected from the following structures:

[0246] Among them, the 4th position of L is related to M 1 The 5th position is connected to the D; each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0247] In some implementations, L is selected from the following structures:

[0248] Among them, the 4th position of L is related to M 1 The 5th position is connected to the D; each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0249] In some implementations, L is selected from the following structures:

[0250] Among them, the 4th position of L is related to M 1 Connected, position 5 is connected to D.

[0251] In some implementations, L is selected from the following structures:

[0252] Among them, the 4th position of L is related to M 1 Connected, position 5 is connected to D.

[0253] In some implementations, L is selected from the following structures:

[0254] Among them, the 4th position of L is related to M 1 Connected, position 5 is connected to D.

[0255] In some implementations, L is selected from the following structures: Among them, the 4th position of L is related to M 1 The 5th position is connected to the D; each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0256] In some implementations, c is 2. In some implementations, d is 8.

[0257] In some implementations, L is selected from the following structures:

[0258] Among them, the 4th position of L is related to M 1The 5th position is connected to the D; each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0259] In some implementations, c is 2. In some implementations, d is 8.

[0260] In some embodiments, the KRAS protein-targeting degrader is selected from:

[0261] In some embodiments, D is the remainder obtained after the degrading agent targeting the KRAS protein loses one or more atoms or groups of atoms (e.g., hydrogen or hydroxyl groups).

[0262] In some embodiments, D is a monovalent structure obtained by losing an H from a -OH, -NH2, or secondary amino group on a degrader targeting the KRAS protein.

[0263] In some implementations, D is selected from the following structures:

[0264] In some implementations, D is selected from the following structures:

[0265] In some implementations, D is selected from the following structures:

[0266] In some implementations, D is selected from the following structures:

[0267] In some embodiments, the compounds of formula (I) are selected from:

[0268] Coupled

[0269] The present invention provides a degradation agent antibody conjugate of Formula II,

[0270] Among them, Ab 1 For targeting groups;

[0271] M is the junction site that connects to the target group;

[0272] L and D are as defined above;

[0273] m is selected from 1-20.

[0274] In some implementations, M is selected from the following structures:

[0275] Where, the 2nd position of M is related to Ab 1 Connected, position 3 is connected to L; each a is independently selected from integers from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and b is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0276] In some implementations, M is selected from the following structures:

[0277] Where, the 2nd position of M is related to Ab 1 Connected, position 3 is connected to L; each a is independently selected from integers from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and b is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0278] In some implementations, M is selected from

[0279] In some implementations, M is selected from

[0280] In some implementations, M is selected from

[0281] In some implementation schemes, Ab 1 The antibody is an antibody or its antigen-binding fragment; the antibody is selected from monoclonal antibodies, polyclonal antibodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, murine antibodies, humanized antibodies, fully human antibodies, and fusion proteins containing the antigen-binding portion of the antibody; the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, disulfide-linked Fv and scFv.

[0282] In Ab 1 When the substance is an antibody or its antigen-binding fragment, the compound represented by Formula I is a degradation agent antibody conjugate.

[0283] In some implementation schemes, Further for Where Ab-(W- is Ab) 1 , represents an antibody or its antigen-binding fragment, and -(W- represents the linking portion of the amino acid residues in the antibody or its antigen-binding fragment to M.

[0284] In some embodiments, the amino acid residue is a cysteine, lysine, serine, or threonine residue.

[0285] In some implementations, W is S, NH, or O.

[0286] In some implementation schemes, Ab 1 It is an anti-EGFR antibody or its antigen-binding fragment.

[0287] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0288] a1) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 2; and / or having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 1; or

[0289] a2) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 23; and / or having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 22; or

[0290] a3) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 42; and / or having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 41; or

[0291] a4) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 65; and / or, having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 64; or

[0292] a5) having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in any one of a1) to a4); and / or having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in any one of a1) to a4).

[0293] In some implementations, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0294] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0295] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0296] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:9 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:13 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:3 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0297] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:30 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:24 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:26 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:28 or a variant thereof; or,

[0298] (1c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:49 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:53 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:56 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:43 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:45 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:47 or a variant thereof; or,

[0299] (1d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:71 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:75 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:78 or a variant thereof; and / or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:66 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:68 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0300] or,

[0301] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0302] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:15 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:3 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0303] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:32 or a variant thereof, CDR-H2 with sequence SEQ ID NO:36 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:24 or a variant thereof, CDR-L2 with sequence SEQ ID NO:26 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:28 or a variant thereof; or,

[0304] (2c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:51 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:55 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:56 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:43 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:45 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:47 or a variant thereof; or,

[0305] (2d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:73 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:77 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:78 or a variant thereof; and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:66 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:68 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0306] or,

[0307] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0308] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:3 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0309] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:29 or a variant thereof, CDR-H2 with sequence SEQ ID NO:33 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:24 or a variant thereof, CDR-L2 with sequence SEQ ID NO:26 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:28 or a variant thereof; or,

[0310] (3c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:48 or a variant thereof, CDR-H2 with sequence SEQ ID NO:52 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:56 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:43 or a variant thereof, CDR-L2 with sequence SEQ ID NO:45 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:47 or a variant thereof; or,

[0311] (3d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:70 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:74 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:78 or a variant thereof; and / or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:66 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:68 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0312] or,

[0313] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0314] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:10 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:17 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0315] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:31 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:35 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:38 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:25 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:27 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:28 or a variant thereof; or,

[0316] (4c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:50 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:54 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:57 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:44 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:46 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:47 or a variant thereof; or,

[0317] (4d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:72 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:76 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:79 or a variant thereof; and / or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:67 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0318] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0319] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0320] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0321] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:9 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:13 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:3 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0322] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:30 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:24 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:26 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:28 or a variant thereof; or,

[0323] (1c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:49 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:53 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:56 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:43 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:45 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:47 or a variant thereof; or,

[0324] (1d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:71 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:75 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:78 or a variant thereof; and / or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:66 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:68 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0325] or,

[0326] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0327] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:15 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:3 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0328] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:32 or a variant thereof, CDR-H2 with sequence SEQ ID NO:36 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:24 or a variant thereof, CDR-L2 with sequence SEQ ID NO:26 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:28 or a variant thereof; or,

[0329] (2c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:51 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:55 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:56 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:43 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:45 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:47 or a variant thereof; or,

[0330] (2d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:73 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:77 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:78 or a variant thereof; and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:66 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:68 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0331] or,

[0332] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0333] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:3 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0334] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:29 or a variant thereof, CDR-H2 with sequence SEQ ID NO:33 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:24 or a variant thereof, CDR-L2 with sequence SEQ ID NO:26 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:28 or a variant thereof; or,

[0335] (3c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:48 or a variant thereof, CDR-H2 with sequence SEQ ID NO:52 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:56 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:43 or a variant thereof, CDR-L2 with sequence SEQ ID NO:45 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:47 or a variant thereof; or,

[0336] (3d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:70 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:74 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:78 or a variant thereof; and / or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:66 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:68 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0337] or,

[0338] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0339] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:10 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:17 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:7 or a variant thereof; or,

[0340] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:31 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:35 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:38 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:25 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:27 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:28 or a variant thereof; or,

[0341] (4c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:50 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:54 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:57 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:44 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:46 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:47 or a variant thereof; or,

[0342] (4d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:72 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:76 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:79 or a variant thereof; and / or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:67 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:69 or a variant thereof;

[0343] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0344] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0345] (a) VH or a variant thereof shown in SEQ ID NO: 2, and / or VL or a variant thereof shown in SEQ ID NO: 1; or

[0346] (b) VH or a variant thereof shown in SEQ ID NO: 23, and / or VL or a variant thereof shown in SEQ ID NO: 22; or

[0347] (c) VH or a variant thereof shown in SEQ ID NO: 42, and / or VL or a variant thereof shown in SEQ ID NO: 41; or,

[0348] (d) VH or a variant thereof shown in SEQ ID NO: 65, and / or VL or a variant thereof shown in SEQ ID NO: 64;

[0349] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0350] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0351] The light chain of the light chain constant region (CL) shown in SEQ ID NO: 18, and the heavy chain of the heavy chain constant region (CH) shown in SEQ ID NO: 19, SEQ ID NO: 60, SEQ ID NO: 61 or SEQ ID NO: 80.

[0352] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0353] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 2 and the heavy chain constant region (CH) shown in SEQ ID NO: 19, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 1 and the light chain constant region (CL) shown in SEQ ID NO: 18; or

[0354] (2) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 23 and the heavy chain constant region (CH) shown in SEQ ID NO: 19, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 22 and the light chain constant region (CL) shown in SEQ ID NO: 18; or

[0355] (3) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 42 and the heavy chain constant region (CH) shown in SEQ ID NO: 60, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 41 and the light chain constant region (CL) shown in SEQ ID NO: 18; or

[0356] (4) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 2 and the heavy chain constant region (CH) shown in SEQ ID NO: 61, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 1 and the light chain constant region (CL) shown in SEQ ID NO: 18; or

[0357] (5) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 23 and the heavy chain constant region (CH) shown in SEQ ID NO: 61, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 22 and the light chain constant region (CL) shown in SEQ ID NO: 18; or

[0358] (6) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 65 and the heavy chain constant region (CH) shown in SEQ ID NO: 80, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 64 and the light chain constant region (CL) shown in SEQ ID NO: 18.

[0359] In some implementations, the anti-EGFR antibody or its antigen-binding fragment comprises:

[0360] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 21, and the light chain comprising the sequence shown in SEQ ID NO: 20; or

[0361] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 40, and the light chain comprising the sequence shown in SEQ ID NO: 39; or

[0362] (3) The heavy chain comprising the sequence shown in SEQ ID NO: 59, and the light chain comprising the sequence shown in SEQ ID NO: 58; or

[0363] (4) The heavy chain comprising the sequence shown in SEQ ID NO: 62, and the light chain comprising the sequence shown in SEQ ID NO: 20; or

[0364] (5) The heavy chain comprising the sequence shown in SEQ ID NO: 63, and the light chain comprising the sequence shown in SEQ ID NO: 39; or

[0365] (6) The heavy chain comprising the sequence shown in SEQ ID NO: 82, and the light chain comprising the sequence shown in SEQ ID NO: 81.

[0366] In some embodiments, the N-terminal glutamine of the VH or variant thereof of the sequence shown in SEQ ID NO:2, 23, 42 or 65, or the heavy chain or variant thereof of the sequence shown in SEQ ID NO:21, 40, 59 or 82, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0367] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 19 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 21, 40, 59 or 82 or a variant thereof, lacks a C-terminal lysine residue.

[0368] In some implementations, the anti-EGFR antibody is selected from cetuximab, zalutumumab, necitumumab, and panitumumab or variants thereof;

[0369] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0370] In some implementations, the anti-EGFR antibody is selected from cetuximab, zalutumumab, necitumumab, or variants thereof;

[0371] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0372] In some embodiments, the zalutumumab variant has one or more amino acid substitutions compared to the constant region of zalutumumab, for example, substitutions of 1-10 amino acids; preferably, the substitutions are conservative substitutions.

[0373] In some embodiments, the zalutumumab variant has one or more amino acid substitutions compared to the heavy chain constant region of zalutumumab, for example, substitutions of 1-10 amino acids; preferably, the substitutions are conservative substitutions.

[0374] In some implementations, the zalutumumab variant has the same heavy chain variable region and light chain variable region as zalutumumab.

[0375] In some embodiments, the necitumumab variant has one or more amino acid substitutions compared to the constant region of necitumumab, for example, substitutions of 1-10 amino acids; preferably, the substitutions are conservative substitutions.

[0376] In some embodiments, the necitumumab variant has one or more amino acid substitutions compared to the heavy chain constant region of necitumumab, for example, substitutions of 1-10 amino acids; preferably, the substitutions are conservative substitutions.

[0377] In some implementations, the necitumumab variant has the same heavy chain variable region and light chain variable region as necitumumab.

[0378] In some implementations, the anti-EGFR antibody is selected from cetuximab, zalutumumab, nexituzumab, and panitumumab.

[0379] In some implementations, the anti-EGFR antibody is selected from cetuximab, zalutumumab, and nexituzumab.

[0380] In some implementations, the anti-EGFR antibody is selected from cetuximab, zalutumumab, or variants thereof.

[0381] In some implementations, the anti-EGFR antibody is selected from cetuximab and zalutumumab.

[0382] In some embodiments, the compound represented by Formula II is selected from:

[0383] Each m is selected from 1 to 10, and Ab-(S-) is an antibody or its antigen-binding fragment as defined in any of the preceding items.

[0384] In some implementations, the S in Ab-(S-) originates from the thiol group in the antibody or its antigen-binding fragment.

[0385] In some embodiments, the degrading agent antibody conjugate is selected from DAC-5, DAC-6, DAC-7, DAC-8, DAC-9, and DAC-10, wherein Ab-(S-) is cetuximab or a variant thereof. In some embodiments, Ab comprises a heavy chain including the sequence shown in SEQ ID NO: 59, and a light chain including the sequence shown in SEQ ID NO: 58.

[0386] In some embodiments, the degrading agent antibody conjugate is selected from: DAC-8, DAC-9, DAC-10, DAC-11, DAC-12, DAC-13, DAC-14, DAC-15, DAC-16, DAC-17, DAC-18, DAC-20, DAC-23, DAC-25, DAC-27, DAC-28, DAC-29, DAC-30, DAC-36, DAC-39, DAC-40, DAC-41, DAC-42, DAC-43, DAC-44, DAC-45, DAC-46, DAC-47, DAC-48, DAC-49, DAC-52, DAC... C-53, DAC-55, DAC-58, DAC-59, DAC-60, DAC-61, DAC-62, DAC-63, DAC-64, DAC-68, DAC-69, DAC-70, DAC-71, DAC-72, DAC-73, DAC-74, DAC-75, DAC-76, DAC-77, DAC-78, DAC-79, DAC-80, DAC-81, DAC-82, DAC-83, DAC-84, and DAC-85; wherein, Ab-(S-) is zalutumumab or a variant thereof. In some embodiments, Ab comprises a heavy chain including the sequence shown in SEQ ID NO: 21, and a light chain including the sequence shown in SEQ ID NO: 20.

[0387] In some embodiments, the degrading agent antibody conjugate is selected from DAC-8, DAC-10, and DAC-11; wherein Ab-(S-) is necitumumab or a variant thereof. In some embodiments, Ab comprises a heavy chain including the sequence shown in SEQ ID NO: 40, and a light chain including the sequence shown in SEQ ID NO: 39.

[0388] In some embodiments, the degrading agent antibody conjugate is selected from DAC-10 and DAC-43; wherein Ab-(S-) is panitumumab or a variant thereof. In some embodiments, Ab comprises a heavy chain including the sequence shown in SEQ ID NO: 82, and a light chain including the sequence shown in SEQ ID NO: 81.

[0389] In some embodiments, the degrading agent antibody conjugate is selected from: DAC-8, DAC-9, DAC-10, DAC-12, DAC-14, DAC-23, DAC-43, DAC-44, DAC-45, DAC-73, DAC-74, DAC-75, DAC-77, and DAC-81, wherein Ab-(S-) is zalutumumab or a variant thereof. In some embodiments, Ab comprises a heavy chain including the sequence shown in SEQ ID NO: 21, and a light chain including the sequence shown in SEQ ID NO: 20.

[0390] In some embodiments, the degrading agent antibody conjugate is selected from D-8 and D-9, wherein Ab-(S-) is cetuximab or a variant thereof. In some embodiments, Ab comprises a heavy chain including the sequence shown in SEQ ID NO: 59, and a light chain including the sequence shown in SEQ ID NO: 58.

[0391] Composition of degradation agent antibody conjugate

[0392] In some embodiments, this disclosure provides compositions of degrading agent antibody conjugates comprising the degrading agent antibody conjugate as defined in any of the preceding embodiments, wherein the composition has a DAR value of 1.0-20.0, preferably 1.0-10.0, for example 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.5-4.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.0, 5.5-6.0. 0.5, 5.5–7.0, 5.5–7.5, 5.5–8.0, 6.0–6.5, 6.0–7.0, 6.0–7.5, 6.0–8.0, 6.5–7.0, 6.5–7.5, 6.5–8.0, 7.0–7.5, 7.0–8.0, 7.5–8.0, for example, about 1.0, about 1.01, about 1.02 Approximately 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23. Approximately 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44. Approximately 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, and so on. 1.66, approximately 1.67, approximately 1.68, approximately 1.69, approximately 1.7, approximately 1.71, approximately 1.72, approximately 1.73, approximately 1.74, approximately 1.75, approximately 1.76, approximately 1.77, approximately 1.78, approximately 1.79, approximately 1.8, approximately 1.81, approximately 1.82, approximately 1.83, approximately 1.84, approximately 1.85, approximately 1.86, approximately 1 .87, about 1.88, about 1.89, about 1.9, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, about 2.0, about 2.01, about 2.02, about 2.03, about 2.04, about 2.05, about 2.06, about 2.07, about 2.08, approximately 2.09, approximately 2.1, approximately 2.11, approximately 2.12, approximately 2.13, approximately 2.14, approximately 2.15, approximately 2.16, approximately 2.17, approximately 2.18, approximately 2.19, approximately 2.2, approximately 2.21, approximately 2.22, approximately 2.23, approximately 2.24, approximately 2.25, approximately 2.26, approximately 2.27, approximately 2.28, approximately 2. 29, approximately 2.3, approximately 2.31, approximately 2.32, approximately 2.33, approximately 2.34, approximately 2.35, approximately 2.36, approximately 2.37, approximately 2.38, approximately 2.39, approximately 2.4, approximately 2.41, approximately 2.42, approximately 2.43, approximately 2.44, approximately 2.45, approximately 2.46, approximately 2.47, approximately 2.48, approximately 2.49, approximately 2.5 Approximately 2.51, 2.52, 2.53, 2.54, 2.55, 2.56, 2.57, 2.58, 2.59, 2.6, 2.61, 2.62, 2.63, 2.64, 2.65, 2.66, 2.67, 2.68, 2.69, 2.7, 2.71 Approximately 2.72, 2.73, 2.74, 2.75, 2.76, 2.77, 2.78, 2.79, 2.8, 2.81, 2.82, 2.83, 2.84, 2.85, 2.86, 2.87, 2.88, 2.89, 2.9, 2.91, 2.92. Approximately 2.93, 2.94, 2.95, 2.96, 2.97, 2.98, 2.99, 3.0, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.1, 3.11, 3.12, 3.13. Approximately 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.2, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.3, 3.31, 3.32, 3.33, 3.34, 3. 3.35, approximately 3.36, approximately 3.37, approximately 3.38, approximately 3.39, approximately 3.4, approximately 3.41, approximately 3.42, approximately 3.43, approximately 3.44, approximately 3.45, approximately 3.46, approximately 3.47, approximately 3.48, approximately 3.49, approximately 3.5, approximately 3.51, approximately 3.52, approximately 3.53, approximately 3.54, approximately 3.55, approximately 3 .56, approximately 3.57, approximately 3.58, approximately 3.59, approximately 3.6, approximately 3.61, approximately 3.62, approximately 3.63, approximately 3.64, approximately 3.65, approximately 3.66, approximately 3.67, approximately 3.68, approximately 3.69, approximately 3.7, approximately 3.71, approximately 3.72, approximately 3.73, approximately 3.74, approximately 3.75, approximately 3.76, approximately 3.77, approximately 3.78, approximately 3.79, approximately 3.8, approximately 3.81, approximately 3.82, approximately 3.83, approximately 3.84, approximately 3.85, approximately 3.86, approximately 3.87, approximately 3.88, approximately 3.89, approximately 3.9, approximately 3.91, approximately 3.92, approximately 3.93, approximately 3.94, approximately 3.95, approximately 3.96, approximately 3.97, approximately 3. 98, approximately 3.99, approximately 4.0, approximately 4.01, approximately 4.02, approximately 4.03, approximately 4.04, approximately 4.05, approximately 4.06, approximately 4.07, approximately 4.08, approximately 4.09, approximately 4.1, approximately 4.11, approximately 4.12, approximately 4.13, approximately 4.14, approximately 4.15, approximately 4.16, approximately 4.17, approximately 4.18, approximately 4.1 9. Approximately 4.2, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 4.3, 4.31, 4.32, 4.33, 4.34, 4.35, 4.36, 4.37, 4.38, 4.39, 4.4 Approximately 4.41, 4.42, 4.43, 4.44, 4.45, 4.46, 4.47, 4.48, 4.49, 4.5, 4.51, 4.52, 4.53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, 4.6, 4.61. Approximately 4.62, 4.63, 4.64, 4.65, 4.66, 4.67, 4.68, 4.69, 4.7, 4.71, 4.72, 4.73, 4.74, 4.75, 4.76, 4.77, 4.78, 4.79, 4.8, 4.81, 4.82. Approximately 4.83, 4.84, 4.85, 4.86, 4.87, 4.88, 4.89, 4.9, 4.91, 4.92, 4.93, 4.94, 4.95, 4.96, 4.97, 4.98, 4.99, 5.0, 5.01, 5.02, 5.03, approximately 5.04, approximately 5.05, approximately 5.06, approximately 5.07, approximately 5.08, approximately 5.09, approximately 5.1, approximately 5.11, approximately 5.12, approximately 5.13, approximately 5.14, approximately 5.15, approximately 5.16, approximately 5.17, approximately 5.18, approximately 5.19, approximately 5.2, approximately 5.21, approximately 5.22, approximately 5.23, approximately 5.24, approximately 5 .25, about 5.26, about 5.27, about 5.28, about 5.29, about 5.3, about 5.31, about 5.32, about 5.33, about 5.34, about 5.35, about 5.36, about 5.37, about 5.38, about 5.39, about 5.4, about 5.41, about 5.42, about 5.43, about 5.44, about 5.45, about 5.46, approximately 5.47, approximately 5.48, approximately 5.49, approximately 5.5, approximately 5.51, approximately 5.52, approximately 5.53, approximately 5.54, approximately 5.55, approximately 5.56, approximately 5.57, approximately 5.58, approximately 5.59, approximately 5.6, approximately 5.61, approximately 5.62, approximately 5.63, approximately 5.64, approximately 5.65, approximately 5.66, approximately 5. 67, approximately 5.68, approximately 5.69, approximately 5.7, approximately 5.71, approximately 5.72, approximately 5.73, approximately 5.74, approximately 5.75, approximately 5.76, approximately 5.77, approximately 5.78, approximately 5.79, approximately 5.8, approximately 5.81, approximately 5.82, approximately 5.83, approximately 5.84, approximately 5.85, approximately 5.86, approximately 5.87, approximately 5.8 8, approximately 5.89, approximately 5.9, approximately 5.91, approximately 5.92, approximately 5.93, approximately 5.94, approximately 5.95, approximately 5.96, approximately 5.97, approximately 5.98, approximately 5.99, approximately 6.0, approximately 6.01, approximately 6.02, approximately 6.03, approximately 6.04, approximately 6.05, approximately 6.06, approximately 6.07, approximately 6.08, approximately 6.0 9. Approximately 6.1, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.2, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29, 6.3 Approximately 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 6.4, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48, 6.49, 6.5, 6.51. Approximately 6.52, approximately 6.53, approximately 6.54, approximately 6.55, approximately 6.56, approximately 6.57, approximately 6.58, approximately 6.59, approximately 6.6, approximately 6.61, approximately 6.62, approximately 6.63, approximately 6.64, approximately 6.65, approximately 6.66, approximately 6.67, approximately 6.68, approximately 6.69, approximately 6.7, approximately 6.71, approximately 6.72, approximately 6.73, approximately 6.74, approximately 6.75, approximately 6.76, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6 .94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7.0, approximately 7.01, approximately 7.02, approximately 7.03, approximately 7.04, approximately 7.05, approximately 7.06, approximately 7.07, approximately 7.08, approximately 7.09, approximately 7.1, approximately 7.11, approximately 7.12, approximately 7.13, approximately 7.14, approximately 7.15, approximately 7.16, approximately 7.17, approximately 7.18, approximately 7.19, approximately 7.2, approximately 7.21, approximately 7.22, approximately 7.23, approximately 7.24, approximately 7.25, approximately 7.26, approximately 7.27, approximately 7.28, approximately 7.29, approximately 7.3, approximately 7.31, approximately 7.32, approximately 7.33, approximately 7.34, approximately 7.35, approximately 7.36 Approximately 7.37, approximately 7.38, approximately 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54, approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7. 79, approximately 7.8, approximately 7.81, approximately 7.82, approximately 7.83, approximately 7.84, approximately 7.85, approximately 7.86, approximately 7.87, approximately 7.88, approximately 7.89, approximately 7.9, approximately 7.91, approximately 7.92, approximately 7.93, approximately 7.94, approximately 7.95, approximately 7.96, approximately 7.97, approximately 7.98, approximately 7.99, approximately 8.0.

[0393] In some embodiments, the compounds shown in Formula I or II of this disclosure are optionally substituted with one or more suitable substituents.

[0394] Connection unit

[0395] The present invention provides a connection unit represented by formula -ML-, wherein M and L are as described in any of the preceding descriptions.

[0396] In some embodiments, the connecting unit -ML - is used to connect the target group and the load to obtain a coupling compound.

[0397] In some embodiments, in the connecting unit -ML-, M is the connector site that connects to the target group, and L is the connecting portion that connects to the load.

[0398] In some embodiments, in the connecting unit -ML-, the M is used to connect with the targeting group described in any one of the present invention, and the L is used to connect with the D described in any one of the present application.

[0399] In some embodiments, this disclosure provides a formula M 1 The connection unit shown in -L-, where M 1 And L as described in any of the above.

[0400] In some implementations, the connection unit M 1 -L- is used to connect the target group and the load to obtain a coupling compound.

[0401] In some implementations, the connection unit M 1 In -L-, the M 1 The structure is designed to react with and connect to the target group, and L is the connection portion that is connected to the load.

[0402] In some implementations, the connection unit M 1 In -L-, the M 1 The L is used to react with and connect to the target group described in any one of the present invention, wherein the L is used to connect to the D described in any one of the present applications.

[0403] In some embodiments, this disclosure provides a connecting unit of formula -L-, wherein L is as described in any of the preceding embodiments. In some embodiments, the connecting unit -L- is used to connect a target group and a load to obtain a coupling. In some embodiments, one end of the connecting unit -L- is connected to the target group via a connector that connects to the target group, and the other end is connected to the load. In some embodiments, one end of the connecting unit -L- is connected to the target group described in any of the preceding embodiments via a connector that connects to the target group as described in any of the preceding embodiments, and the other end is connected to D as described in any of the preceding embodiments.

[0404] In some embodiments, this disclosure provides a connecting unit of formula -LD, wherein L and D are as described in any of the preceding embodiments. In some embodiments, the connecting unit is used to connect a target group via a connector site to obtain a coupling.

[0405] In some embodiments, this disclosure provides a linker unit of formula -MLD, wherein M, L, and D are as described in any of the preceding embodiments. In some embodiments, the linker unit is used to link a target group to obtain a conjugate.

[0406] In some implementations, the connection unit shown by equation -L- is -(L 2 ) r -L 3 -L 4 -, where L 2 L 3 L 4 And r as defined above.

[0407] In some implementations, the connection unit shown in Equation-L- includes the following structure:

[0408] In some implementations, the connection unit shown in Equation-L- has the following structure:

[0409] Among them, the connector at position 4 is connected to the target group (e.g., M in this application). 1 Or M) connection, position 5 is connected to the load (e.g., D in this application); each c is independently selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6), and each d is independently selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0410] In some implementations, the connection unit shown in Equation-L- has the following structure:

[0411] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising -L-, -ML-, M-, ... 1 Connection units with structures shown as -L-, -LD, or -MLD.

[0412] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof is provided, comprising -L-, -ML-, M-, ... 1 -L- The connection unit of the structure shown in Ab and Ab 1 And / or D, the connecting unit and Ab 1And / or D connection; where Ab 1 Or D as defined in any of the preceding items.

[0413] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug is provided, comprising a linker unit of the structure shown in -LD or -MLD and Ab. 1 The connection unit and Ab 1 Connect; where Ab 1 As defined in any of the preceding items.

[0414] intermediate

[0415] In some embodiments, this disclosure provides intermediate compounds with the structures shown below, or their salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, or isotopically labeled compounds:

[0416] PG 1 -L 2b -(Q) n PG 1 -L 3 -L 4 -D, PG 1 -(L 2 ) r -L 3 -L 4 -D,

[0417] Among them, PG 1 Each group is independently an H or amino protecting group, such as alkoxycarbonyl amino protecting groups, for example benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl); acyl amino protecting groups, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-)nitrobenzenesulfonyl (Ns), tert-pentanoyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as triphenylmethyl (Trt), C 1-6 Alkyl-substituted triphenylmethyl, p-methoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

[0418] PG 2 and PG 3 Each group is independently protected by an H, OH, or carboxyl group, such as a C group. 1-6 Alkyl-O-, allyl-O-, benzyl-O-, 2,4-dimethoxybenzyl-O-, p-methoxybenzyl-O-, methoxyethoxymethyl-O-, pentafluorophenyl-O-, 4-p-methylbenzyloxybenzyl-O-;

[0419] The remaining groups are as defined above.

[0420] In some embodiments, this disclosure provides intermediate compounds with the structures shown below, or their salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, or isotopically labeled compounds:

[0421] In some embodiments, this disclosure provides the use of the intermediate compound as described above, or its salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, in the preparation of the compound of Formula I or its pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, or the degrading agent antibody conjugate of Formula II.

[0422] The present invention also provides HL as shown in the example. 2a -L 2b -(Q) n or HL 2b -(Q) n The compound shown, or its salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, or isotopically labeled compounds, wherein L 2a L 2b Q and n are as defined above.

[0423] In some implementation schemes, this application is HL 2a -L 2b -(Q) n In the compound shown, the L 2a Selected from covalent bonds or c is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and d is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0424] In some implementation schemes, this application is HL 2a -L 2b -(Q) n In the compound shown, the L 2a Selected from covalent bonds or

[0425] In some implementation schemes, this application is HL 2a -L 2b -(Q) n In the compound shown, the L 2a Selected from covalent bonds or c is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and d is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0426] In some implementation schemes, this application is HL 2a -L 2b -(Q) n In the compound shown, the L 2a Selected from covalent bonds or

[0427] In some implementation schemes, this application is HL 2a -L 2b -(Q) n or HL 2b -(Q) n The compounds shown are selected from:

[0428] In some implementations, this disclosure provides HL 2a -L 2b -(Q) n or HL 2b -(Q) n Use of the compound shown or its salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, or isotopically labeled compounds in the preparation of couplings, for example, in the preparation of couplings as described above.

[0429] In some embodiments, this disclosure provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, the compound being selected from the following structures:

[0430] In this context, each 'a' is independently selected from integers from 1 to 6, and each 'b' is selected from integers from 1 to 10.

[0431] In some implementations, the compound is selected from...

[0432] In some embodiments, this disclosure provides the use of the above-described compounds or their salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, and isotopically labeled compounds in the preparation of couplings, for example, in the preparation of couplings as described above.

[0433] Pharmaceutical Composition

[0434] The present invention provides a pharmaceutical composition comprising the compound described above or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, the degradation agent antibody conjugate described in the second aspect, or the antibody or antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.

[0435] The above-described pharmaceutical compositions can act systemically and / or locally, which can be achieved through suitable dosage forms. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0436] The pharmaceutical composition described above may contain 0.01 mg to 1000 mg of at least one of the compounds disclosed herein or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, or the described degradation agent antibody conjugate.

[0437] This disclosure also provides a method for preparing the above-described pharmaceutical composition or its corresponding formulation, comprising combining at least one of the compounds of this disclosure or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug or said degrading agent antibody conjugate with one or more pharmaceutically acceptable carriers.

[0438] Treatment methods and uses

[0439] The present invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug or the antibody or an antigen-binding fragment thereof in the preparation of a degrading agent antibody-drug conjugate of Formula II.

[0440] The present invention provides the use of compounds of Formula I or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, degrading agents of Formula II, antibody conjugates, and pharmaceutical compositions thereof in the preparation of medicaments, particularly in medicaments for the treatment and / or prevention of cancer (e.g., cancers associated with KRAS mutations).

[0441] The present invention provides compounds of Formula I or thereof, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, degrading agent antibody conjugates of Formula II, or pharmaceutical compositions thereof, for the treatment and / or prevention of cancer (e.g., cancers associated with KRAS mutations).

[0442] The present invention provides a method for treating and / or preventing cancer (e.g., cancer associated with KRAS mutations), comprising administering to a subject in need a therapeutically and / or preventively effective amount of a compound of Formula I or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, degrader antibody conjugate of Formula II, or pharmaceutical composition thereof.

[0443] In some implementations, the KRAS mutation is a G12D mutation.

[0444] In some implementations, the cancer is pancreatic cancer. Beneficial effects

[0445] The compounds disclosed herein have one or more of the following beneficial effects, but are not limited to:

[0446] (1) It has a good degradation effect on KRAS protein;

[0447] (2) It has a strong inhibitory activity against tumor cell proliferation;

[0448] (3) It has a good tumor-suppressing effect;

[0449] (4) Excellent physicochemical properties (e.g., solubility, physical and / or chemical stability);

[0450] (5) Excellent pharmacokinetic properties (e.g., good bioavailability, appropriate half-life and duration of action);

[0451] (6) Excellent safety profile (lower toxicity and / or fewer side effects, wider therapeutic window), etc.

[0452] Example

[0453] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention.

[0454] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0455] The structure of the compound was determined by nuclear magnetic resonance (NMR). 1 Determined by 1H NMR and / or mass spectrometry (MS).

[0456] 1 The 1H NMR was performed using a JEOL Eclipse 400 NMR spectrometer. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS). Chemical shifts (δ) were given in parts per million (ppm).

[0457] The instrument used for MS measurements was an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0458] Reversed-phase column chromatography was performed using an Agela C18 reversed-phase Flash column (20-35 μm). ).

[0459] The preparative high-performance liquid chromatography (HPLC) conditions are as follows:

[0460] (1) Instrument model: Agilent 1260; Column: Waters SunFire Prep C 18OBD (19mm×150mm×5.0μm); Column temperature: 25℃; Flow rate: 20.0mL / min; Detection wavelength: 214nm; Elution gradient: (0min: 10% A, 90% B; 16.0min: 90% A, 10% B); Mobile phase A: acetonitrile; Mobile phase B: 0.05% formic acid aqueous solution;

[0461] (2) Instrument model: Agilent 1260, chromatographic column: Waters XBridge Prep C 18 OBD (19mm×150mm×5.0μm); Column temperature: 25℃; Flow rate: 20.0mL / min; Detection wavelength: 214nm; Elution gradient: (0min: 10%A, 90%B; 16.0min: 90%A, 10%B); Mobile phase A: Acetonitrile; Mobile phase B: 0.05% ammonium bicarbonate aqueous solution.

[0462] The thin-layer chromatography silica gel plates (TLC) used were Merck aluminum plates (20×20cm), and the TLC separation and purification used Yantai-made GF 254 (1mm) plates.

[0463] The reaction was monitored using thin-layer chromatography (TLC) or LC-MS. The developing solvent systems used included dichloromethane and methanol, n-hexane and ethyl acetate, and petroleum ether and ethyl acetate. The volume ratio of the solvent was adjusted according to the polarity of the compound or by adding triethylamine, etc.

[0464] Column chromatography typically uses 200-300 mesh silica gel as the support. Eluent systems include dichloromethane and methanol systems, and petroleum ether and ethyl acetate systems. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and a small amount of triethylamine can also be added for adjustment.

[0465] Unless otherwise specified, the reaction temperature is room temperature (20℃-35℃).

[0466] The reagents used in the examples were purchased from Acros Organics, Aldrich Chemical Company, and TEB Chemicals.

[0467] The meanings of the abbreviations used in this article are shown in the table below.

[0468] Information about the sequences involved in this invention is described in the table below:

[0469] Preparation of intermediates

[0470] Example 1: Preparation of intermediate 1

[0471] Step 1: Preparation of compound Int1-2

[0472] Int1-1 (5.0 g, 10.6 mmol) and tris(hydroxymethyl)aminomethane (4.49 g, 37.1 mmol) were dissolved in DMF (50 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (14.1 g, 37.1 mmol) and N,N-diisopropylethylamine (6.85 g, 53 mmol) were added. The mixture was reacted at 40 °C for 12 hours. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (2.0 g, yield: 24.1%).

[0473] MS m / z (ESI): 781.3 [M+H] + .

[0474] Step 2: Preparation of compound Int1

[0475] Compound Int1-2 (2.0 g, 2.56 mmol) was dissolved in anhydrous methanol (20 mL), and 10% palladium on carbon (500 mg) was added. The reaction was carried out at 25 °C for 12 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (1.4 g, yield: 84.3%).

[0476] MS m / z (ESI): 647.3 [M+H] + .

[0477] Example 2: Preparation of intermediate 2: Preparation of compound Int2

[0478] Step 1: Preparation of compound Int2-2

[0479] Int2-1 (5.0 g, 9.0 mmol) was dissolved in acetonitrile (60 mL), and potassium carbonate (4.98 g, 36.0 mmol), potassium iodide (1.49 g, 9.0 mmol), and 4-ethynylpiperidine hydrochloride (1.97 g, 13.5 mmol) were added. The mixture was stirred at 65 °C for 8 hours. The reaction mixture was cooled to room temperature, and water (100 mL) was added to the reaction solution. The mixture was extracted with ethyl acetate (100 mL), and the organic phase was washed with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to give the title compound (4.55 g, yield: 88.7%).

[0480] MS m / z (ESI): 569.3 [M+H] + .

[0481] Step 2: Preparation of compound Int2

[0482] Compound Int2-2 (4.5 g, 7.9 mmol), ammonium acetate (3.65 g, 47.4 mmol), and sulfur (1.52 g, 47.4 mmol) were added to ethanol (50 mL), and the mixture was stirred at 60 °C for 15 minutes. Then, a solution of malononitrile (4.17 g, 63.2 mmol) in ethanol (10 mL) was added dropwise, and the mixture was stirred at 80 °C for 16 hours. The reaction mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1, v / v) to give the title compound (3.5 g, yield: 68.2%).

[0483] MS m / z (ESI): 649.3 [M+H] + .

[0484] Example 3: Preparation of intermediate Int3

[0485] Using the synthetic route in Intermediate Preparation Example 2, the first-step reaction starting material Int2-1 was replaced with Int3-1 to obtain the title compound (1.5 g, yield: 65.3%).

[0486] MS m / z (ESI): 555.3 [M+H] + .

[0487] Example 4: Preparation of intermediate Int4

[0488] Step 1: Preparation of compound Int4-1

[0489] Compound Int2 (900 mg, 1.32 mmol) and potassium carbonate (909.33 mg, 6.59 mmol) were mixed in tetrahydrofuran (5 mL), and (9H-fluorene-9-yl)methyl(S)-(1-chloro-1-oxopropane-2-yl)carbamate (2.0 g, 5.76 mmol) was added. The reaction mixture was reacted at 25 °C for 12 hours. The reaction mixture was added dropwise to water (50 mL), extracted with ethyl acetate (30 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 1 / 3, v / v) to give the title compound (1.1 g, yield: 84.2%).

[0490] MS m / z (ESI): 942.4 [M+H] + .

[0491] Step 2: Preparation of compound Int4-2

[0492] Compound Int4-1 (1.1 g, 1.11 mmol) was dissolved in DMF (10 mL), and diethylamine (162.26 mg, 2.22 mmol) was added. The reaction mixture was reacted at 25 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (800 mg, yield: 95.18%).

[0493] MS m / z(ESI): 720.3 [M+H] + .

[0494] Step 3: Preparation of compound Int4-3

[0495] Compound Int4-2 (800 mg, 1.06 mmol), 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine ester (727.58 mg, 1.58 mmol), and N,N-diisopropylethylamine (413.48 mg, 3.17 mmol) were dissolved in DMF (10 mL) and stirred at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (700 mg, yield: 60.49%).

[0496] MS m / z (ESI): 1041.4 [M+H] + .

[0497] Step 4: Preparation of compound Int4-4

[0498] Compound Int4-3 (700 mg, 638.67 μmol) and (2S,4R)-1-((S)-2-azido-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl))ethyl)pyrrolidine-2-carboxamide (300 mg, 605.63 μmol) were dissolved in dimethyl sulfoxide (10 mL) and water (1 mL). Sodium ascorbate (183.64 mg, 908.44 μmol) and copper sulfate pentahydrate (308.60 mg, 1.21 mmol) were added, and the mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (700 mg, yield: 88.19%).

[0499] MS m / z (ESI): 1511.7 [M+H] + .

[0500] Step 5: Preparation of compound Int4

[0501] Compound Int4-4 (200 mg, 125.68 μmol) was dissolved in DMF (3 mL), and diethylamine (18.38 mg, 251.35 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (160 mg, yield: 93.79%).

[0502] MS m / z (ESI): 1289.6 [M+H] + .

[0503] Example 5: Preparation of intermediate Int5

[0504] Step 1: Preparation of compound Int5-2

[0505] Compound Int5-1 (2.0 g, 4.89 mmol), N,N-diisopropylethylamine (1.26 g, 9.28 mmol), and amino-octavalent polyethylene glycol tert-butyl ester (2.44 g, 4.89 mmol) were dissolved in dimethyl sulfoxide (20 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.64 g, 6.96 mmol) was added. The reaction mixture was reacted at 25 °C for 3 h. The reaction solution was purified by high performance liquid chromatography (condition 1) to give the title compound (3.5 g, yield: 80.5%).

[0506] MS m / z (ESI): 889.5 [M+H]+ .

[0507] Step 2: Preparation of compound Int5-3

[0508] Compound Int5-2 (3.5 g, 3.74 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (10 mL) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (2.7 g, yield 72.42%).

[0509] MS m / z (ESI): 833.4 [M+H] + .

[0510] Step 3: Preparation of compound Int5-4

[0511] Compound Int5-3 (1.1 g, 1.25 mmol) and N,N-diisopropylethylamine (597.38 mg, 4.39 mmol) were dissolved in dimethyl sulfoxide (10 mL). Compound Int1 (1 g, 1.47 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (714.4 mg, 1.88 mmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (1.2 g, yield: 62.17%).

[0512] MS m / z (ESI): 1461.7 [M+H] + .

[0513] Step 4: Preparation of compound Int5

[0514] Compound Int5-4 (1.2 g, 779.97 μmol) was dissolved in anhydrous DMF (10 mL), and 1,3-dimethylbarbitine (365.03 mg, 2.34 mmol) and tetrakis(triphenylphosphine)palladium (180.26 mg, 155.99 μmol) were added. The system was purged with nitrogen, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (1.1 g, yield: 94.25%).

[0515] MS m / z (ESI): 1421.7 [M+H] + .

[0516] Example 6: Preparation of intermediate 6

[0517] Step 1: Preparation of compound Int6-2

[0518] (2S,4R)-1-((S)-2-azido-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (400 mg, 807.50 μmol) and (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (1.30 g, 3.23 mmol) were dissolved in dichloromethane (10 mL), and then trifluoroacetic acid (1 mL) was added. The mixture was stirred at 25 °C for 3 hours. The reaction solution was filtered, the filtrate was diluted with dichloromethane (30 mL), the organic phase was washed with water (30 mL), the aqueous phase was extracted with dichloromethane (20 mL), the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1, v / v) to give the title compound (1 g, yield: 78.09%).

[0519] MS m / z(ESI): 793.3 [M+H] + .

[0520] Step 2: Preparation of compound Int6-3

[0521] Compound Int6-2 (1 g, 630.56 μmol) was dissolved in DMF (10 mL), and diethylamine (92.24 mg, 1.26 mmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 4 / 6, v / v) to give the title compound (350 mg, yield: 92.40%).

[0522] MS m / z (ESI): 571.3 [M+H] + .

[0523] Step 3: Preparation of compound Int6-4

[0524] Compound Int6-3 (200 mg, 245.31 μmol), N,N-diisopropylethylamine (66.75 mg, 490.62 μmol), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (148.12 mg, 367.97 μmol) were dissolved in DMF (3 mL). 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (139.84 mg, 367.97 μmol) was added, and the reaction was carried out at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (60 mg, yield: 26.16%).

[0525] MS m / z (ESI): 935.4 [M+H] + .

[0526] Step 4: Preparation of compound Int6-5

[0527] Compounds Int6-4 (450 mg, 457.17 μmol) and Int2 (374.65 mg, 548.61 μmol) were dissolved in dimethyl sulfoxide (3 mL) and water (0.3 mL). Sodium ascorbate (110.90 mg, 548.61 μmol) and copper sulfate pentahydrate (139.77 mg, 548.61 μmol) were added to the system, and the mixture was stirred at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (450 mg, yield: 78.7%).

[0528] MS m / z (ESI): 1583.7 [M+H] + .

[0529] Step 5: Preparation of compound Int6

[0530] Compound Int6-5 (250 mg, 149.95 μmol) was dissolved in DMF (3 mL), and diethylamine (21.93 mg, 299.90 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 4 / 6, v / v) to give the title compound (200 mg, yield: 93.0%).

[0531] MS m / z (ESI): 1361.6 [M+H] + .

[0532] Example 7: Preparation of intermediate Int7

[0533] Step 1: Preparation of compound Int7-1

[0534] Compound Int5 (180 mg, 120.29 μmol), N,N-diisopropylethylamine (32.73 mg, 240.59 μmol), and compound Int6 (200 mg, 139.54 μmol) were dissolved in dimethyl sulfoxide (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (72.18 mg, 180.44 μmol) were added. The mixture was reacted at 25 °C for 3 hours. The reaction solution was purified by high performance liquid chromatography (condition 1) to give the title compound (180 mg, yield: 51.4%).

[0535] MS m / z(ESI): 2765.3 [M+H] + .

[0536] Step 2: Preparation of compound Int7

[0537] Compound Int7-1 (180 mg, 61.84 μmol) was dissolved in DMF (2 mL), and diethylamine (9.05 mg, 123.68 μmol) was added. The mixture was reacted at 25 °C for 1 hour. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (150 mg, yield: 90.6%).

[0538] MS m / z(ESI): 2543.3 [M+H] + .

[0539] Example 8: Preparation of compound Int8

[0540] Dissolve (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-lysine (1 g, 2.14 mmol), 3-methoxypropionaldehyde (753.8 mg, 8.56 mmol), and acetic acid (385.3 mg, 6.42 mmol) in a mixed solvent of dichloromethane (10 mL) and methanol (2 mL). Stir the mixture at 25 °C for 1 hour. Add sodium cyanoborohydride (672 mg, 10.69 mmol) and stir the mixture at 25 °C for 12 hours. Concentrate the reaction mixture under reduced pressure. Purify the residue by silica gel column chromatography (eluent: dichloromethane / methanol = 5 / 1, v / v) to give the title compound (807 mg, yield: 61.7%).

[0541] MS m / z (ESI): 612.1 [M+H] + .

[0542] Example 9: Preparation of intermediate: Int9

[0543] Using the synthetic route in Intermediate Preparation Example 6, the first-step reaction starting material (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester was replaced with (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate, and the third-step reaction starting material (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine was replaced with compound Int8, yielding the title compound (70 mg, yield: 61.1%).

[0544] MS m / z (ESI): 1576.8 [M+H] + .

[0545] Intermediate Preparation Example 10: Preparation of Compound Int10

[0546] Using the synthetic route in Intermediate Preparation Example 7, the starting material Int6 in the first step was replaced with compound Int9 to obtain the title compound (50 mg, yield: 98.2%).

[0547] MS m / z(ESI): 2758.4 [M+H] + .

[0548] Example 11: Preparation of intermediate: Int11

[0549] Step 1: Preparation of compound Int11-1

[0550] Compounds Int5 (176.37 mg, 117.87 μmol), N,N-diisopropylethylamine (56.12 mg, 412.54 μmol), and Int4 (160 mg, 117.87 μmol) were dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (67.2 mg, 176.80 μmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (280 mg, yield: 83.8%).

[0551] MS m / z(ESI): 2693.3 [M+H] + .

[0552] Step 2: Preparation of compound Int11-2

[0553] Compound Int11-1 (280 mg, 98.77 μmol) was dissolved in DMF (2 mL), and diethylamine (14.45 mg, 197.54 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (150 mg, yield: 58.4%).

[0554] MS m / z(ESI): 2470.3 [M+H] + .

[0555] Step 3: Preparation of compound Int11-3

[0556] Compound Int5 (27.62 mg, 18.46 μmol), N,N-diisopropylethylamine (7.32 mg, 53.83 μmol), and compound Int11-2 (40 mg, 15.38 μmol) were dissolved in DMF (3 mL). 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.77 mg, 23.07 μmol) was added, and the reaction was carried out at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (50 mg, yield: 79.72%).

[0557] MS m / z (ESI): 1936.9 [M / 2+H] + .

[0558] Step 4: Preparation of compound Int11

[0559] Compound Int11-3 (50 mg, 12.26 μmol) was dissolved in DMF (2 mL), and diethylamine (1.79 mg, 24.52 μmol) was added. The mixture was reacted at 20 °C for 1 hour. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (45 mg, yield: 95.5%).

[0560] MS m / z(ESI): 3650.8 [M+H] + .

[0561] Example 12: Preparation of intermediate: Int12

[0562] Step 1: Preparation of compound Int12-1

[0563] Compound Int4 (320 mg, 248.3 μmol), N,N-diisopropylethylamine (96 mg, 744.9 μmol), and (3S)-7-(tert-butoxycarbonylamino)-3-(9H-fluorene-9-ylmethoxycarbonylamino)heptanoic acid (179.5 mg, 372.45 μmol) were added to dimethyl sulfoxide (2 mL), followed by 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (141.6 mg, 372.45 μmol). The reaction mixture was reacted at 25 °C for 3 hours. The reaction solution was purified by high-performance liquid chromatography (condition 2) to give the title compound (300 mg, yield: 68.8%).

[0564] MS m / z (ESI): 1754.8 [M+H] + .

[0565] Step 2: Preparation of compound Int12-2

[0566] Compound Int12-1 (300 mg, 171.0 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound (275 mg, yield: 97.2%).

[0567] MS m / z(ESI): 1652.8 [M+H] + .

[0568] Step 3: Preparation of compound Int12-3

[0569] Compound Int12-2 (275 mg, 166.4 μmol) was dissolved in DMF (3 mL), and N,N-diisopropylethylamine (107.5 mg, 831.9 μmol) and 2-[4,10-bis(carboxymethyl)-7-[2-(2,5-dioxopyrrolidone-1-yl)oxy-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1-yl]acetic acid (159.18 mg, 301.54 μmol) were added. The mixture was stirred at 25 °C for 6 hours. The reaction solution was concentrated under reduced pressure. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (100 mg, yield: 29.4%).

[0570] MS m / z(ESI): 2041.5 [M+H] + .

[0571] Step 4: Preparation of compound Int12

[0572] Compound Int12-3 (100 mg, 46.56 μmol) was dissolved in DMF (2 mL), and diethylamine (6.81 mg, 93.12 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (85 mg, yield: 95.39%).

[0573] MS m / z (ESI): 1817.9 [M+H] + .

[0574] Preparation of intermediates Example 13: Preparation of (S)-2-amino-3'-(4-((S)-2-methyl-1,4-diazacycloheptane-1-yl)-6-(pyrimidin-5-yloxy)pyrimidin-2-yl)-5',6,6',7-tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-nitrile

[0575] Step 1: Preparation of compound Int13-2

[0576] Compound Int13-1 (1.1 g, 2.35 mmol) and N,N-diisopropylethylamine (606.9 mg, 4.7 mmol) were dissolved in dimethyl sulfoxide (10 mL). Compound β-alanine-2-propen-1-yl ester (607.1 mg, 4.7 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.34 g, 3.53 mmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 7 / 3, v / v) to give the title compound (1.0 g, yield: 73.5%).

[0577] MS m / z (ESI): 580.3 [M+H] + .

[0578] Step 2: Preparation of compound Int13

[0579] Compound Int13-2 (1.0 g, 1.7 mmol) was dissolved in anhydrous DMF (10 mL), and 1,3-dimethylbarbitine (806.5 mg, 5.2 mmol) and tetrakis(triphenylphosphine)palladium (196.4 mg, 170 μmol) were added. The system was purged with nitrogen three times, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (0.7 g, yield: 75.3%).

[0580] MS m / z (ESI): 540.3 [M+H] + .

[0581] Intermediate Preparation Example 14: Preparation of Compound Int14

[0582] Using the synthetic route in Intermediate Preparation Example 12, the first-step reaction starting material (3S)-7-(tert-butoxycarbonylamino)-3-(9H-fluorene-9-ylmethoxycarbonylamino)heptanoic acid was replaced with compound Int13 to obtain the title compound (15 mg, yield: 30.2%).

[0583] MS m / z (ESI): 1810.8 [M+H] + .

[0584] Intermediate Preparation Example 15: Preparation of Compound Int15

[0585] Step 1: Preparation of compound Int15-1

[0586] Compounds Int6-4 (300 mg, 321.2 μmol) and Int3 (177.9 mg, 321.2 μmol) were dissolved in dimethyl sulfoxide (3 mL) and water (0.3 mL). Sodium ascorbate (95.4 mg, 481.8 μmol) and copper sulfate pentahydrate (160.6 mg, 624.4 μmol) were added to the system, and the mixture was stirred at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (333 mg, yield: 69.7%).

[0587] MS m / z (ESI): 1489.6 [M+H] + .

[0588] Step 2: Preparation of compound Int15

[0589] Compound Int15-1 (333 mg, 223.5 μmol) was dissolved in DMF (3 mL), and diethylamine (32.8 mg, 447.1 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 4 / 6, v / v) to give the title compound (183 mg, yield: 64.7%).

[0590] MS m / z (ESI): 1267.6 [M+H] + .

[0591] Example 16: Preparation of compound Int16

[0592] Step 1: Preparation of compound Int16-2

[0593] 1-(3-(2,6-dioxopiperidin-3-yl)-1-methyl-1H-indazol-6-yl)piperidin-4-carboxylic acid tert-butyl ester (280 mg, 625 μmol) was dissolved in THF (5 mL). Sodium hydride (200 mg, 5.0 mmol) was added in an ice bath and stirred for 10 minutes. Then (9H-fluorene-9-yl)methyl(S)-(1-((chloromethyl)amino)-1-oxopropane-2-yl)carbamate (470 mg, 1.25 mmol) was added and reacted at 25 °C for 1 hour. The reaction was quenched dropwise with 0.5 M hydrochloric acid (10 mL) in an ice bath, extracted with ethyl acetate (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 1 / 3, v / v) to give the title compound (220 mg, yield: 88.0%).

[0594] MS m / z (ESI): 749.3 [M+H] + .

[0595] Step 2: Preparation of compound Int16-3

[0596] Compound Int16-2 (220 mg, 293.8 μmol) was dissolved in DMF (5 mL), and diethylamine (180 mg, 2.9 mmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium formate) = 7 / 3, v / v) to give the title compound (120 mg, yield: 78%).

[0597] MS m / z (ESI): 527.3 [M+H]+ .

[0598] Step 3: Preparation of compound Int16-4

[0599] Compound Int16-3 (110 mg, 200 μmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (120 mg, 300 μmol) were dissolved in DMF (5 mL). N,N-diisopropylethylamine (53 mg, 400 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (115 mg, 300 μmol) were added. The mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium formate) = 7 / 3, v / v) to give the title compound (150 mg, yield: 80%).

[0600] MS m / z (ESI): 891.4 [M+H] + .

[0601] Step 4: Preparation of compound Int16-5

[0602] Compound Int16-4 (150 mg, 162 μmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (2 mL). The reaction was carried out at 5 °C for 12 hours. The reaction solution was concentrated under reduced pressure to give the title compound (135 mg, yield: 96%).

[0603] MS m / z (ESI): 835.3 [M+H] + .

[0604] Step 5: Preparation of compound Int16-6

[0605] Compound Int16-5 (80 mg, 95.8 μmol) and (S)-2-amino-3'-(4-((S)-2-methyl-4-(3-azaspiro[5.5]undecane-9-yl)-1,4-diazacycloheptane-1-yl)-6-(pyrimidin-5-yloxy)pyrimidin-2-yl)-5',6,6',7-tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-nitrile (76 mg, 105 μmol) were dissolved in DMF (3 mL), and N,N-diisopropylethylamine (24 mg, 182 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (53 mg, 136.5 μmol) were added. The mixture was stirred at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (50 mg, yield: 34%).

[0606] MS m / z (ESI): 1537.7 [M+H] + .

[0607] Step 6: Preparation of compound Int16-7

[0608] Compound Int16-6 (50 mg, 32.5 μmol) was dissolved in DMF (2 mL), and diethylamine (23.8 mg, 325 μmmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (36 mg, yield: 84%).

[0609] MS m / z (ESI): 1315.6 [M+H] + .

[0610] Step 7: Preparation of compound Int16-8

[0611] Compounds Int16-7 (32 mg, 24.3 μmol) and Int5 (34.6 mg, 24.3 μmol) were dissolved in DMF (3 mL), and N,N-diisopropylethylamine (6.3 mg, 48.6 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (13.8 mg, 36.5 μmol) were added. The mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (43 mg, yield: 65%).

[0612] MS m / z(ESI): 2719.3 [M+H] + .

[0613] Step 8: Preparation of compound Int16

[0614] Compound Int16-8 (43 mg, 15.8 μmol) was dissolved in DMF (2 mL), and diethylamine (11.6 mg, 158 μmmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (30 mg, yield: 76%).

[0615] MS m / z(ESI): 2497.2 [M+H] + .

[0616] Intermediate Preparation Example 17: Preparation of Compound Int17

[0617] Step 1: Preparation of compound Int17-1

[0618] Compound Int6-4 (100 mg, 102 μmol) was dissolved in DMF (3 mL), and diethylamine (74.3 mg, 1.02 mmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium formate) = 4 / 6, v / v) to give the title compound (70 mg, yield: 92%).

[0619] MS m / z (ESI): 713.3 [M+H] + .

[0620] Step 2: Preparation of compound Int17-2

[0621] Compound Int17-1 (70 mg, 94 μmol) and N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (67 mg, 140 μmol) were dissolved in DMF (3 mL). N,N-diisopropylethylamine (25 mg, 188 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (55 mg, 140 μmol) were added. The mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (72 mg, yield: 63%).

[0622] MS m / z (ESI): 1163.5 [M+H] + .

[0623] Step 3: Preparation of compound Int17-3

[0624] Compound Int17-2 (72 mg, 61.9 μmol) was dissolved in DMF (3 mL), and diethylamine (45 mg, 619 μmol) was added. The reaction was carried out at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium formate) = 4 / 6, v / v) to give the title compound (57 mg, yield: 97%).

[0625] MS m / z (ESI): 927.5 [M+H] + .

[0626] Step 4: Preparation of compound Int17

[0627] Compound Int17-3 (57 mg, 60.5 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (22 mg, 60.5 μmol) were dissolved in DMF (2 mL), and N,N-diisopropylethylamine (15.6 mg, 121 μmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (62 mg, yield: 85%).

[0628] MS m / z (ESI): 1191.5 [M+H] + .

[0629] Intermediate Preparation Example 18: Preparation of Compound Int18

[0630] Step 1: Preparation of compound Int18-1

[0631] Compounds Int7 (150 mg, 58.40 μmol) and Int5 (84 mg, 58.40 μmol) were dissolved in dimethyl sulfoxide (2 mL), and N,N-diisopropylethylamine (16 mg, 116.80 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (34 mg, 87.60 μmol) were added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (170 mg, yield: 73.0%).

[0632] MS m / z (ESI): 3946.5 [M+H] + .

[0633] Step 2: Preparation of compound Int18

[0634] Compound Int18-1 (170 mg, 42.65 μmol) was dissolved in DMF (2 mL), and diethylamine (9.5 mg, 127.94 μmol) was added. The mixture was reacted at 25 °C for 3 hours. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (28 mg, yield: 17.4%).

[0635] MS m / z(ESI): 3724.5 [M+H] + .

[0636] Intermediate Preparation Example 19: Preparation of Compound Int19

[0637] Step 1: Preparation of compound Int19-1

[0638] Compound Int5-3 (200 mg, 0.24 mmol) and N,N-diisopropylethylamine (93 mg, 0.72 mmol) were dissolved in dimethyl sulfoxide (3 mL). Compound D-glucosamine (65 mg, 0.36 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (136.8 mg, 0.36 mmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (150 mg, yield: 62.8%).

[0639] MS m / z (ESI): 996.5 [M+H] + .

[0640] Step 2: Preparation of compound Int19-2

[0641] Compound Int19-1 (150 mg, 150.6 μmol) was dissolved in anhydrous DMF (2 mL), and 1,3-dimethylbarbitine (70.5 mg, 451.8 μmol) and tetrakis(triphenylphosphine)palladium (17.4 mg, 15.1 μmol) were added. The system was purged with nitrogen three times, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (100 mg, yield: 69.4%).

[0642] MS m / z (ESI): 956.5 [M+H] + .

[0643] Step 3: Preparation of compound Int19-3

[0644] Compound Int19-2 (100 mg, 0.1 mmol) and N,N-diisopropylethylamine (38.8 mg, 0.3 mmol) were dissolved in dimethyl sulfoxide (3 mL). Compound Int15 (126.7 mg, 0.1 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (57.1 mg, 0.15 mmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (101 mg, yield: 43.7%).

[0645] MS m / z(ESI): 2206.5 [M+H] + .

[0646] Step 4: Preparation of compound Int19

[0647] Compound Int19-3 (101 mg, 45.8 μmol) was dissolved in DMF (2 mL), and diethylamine (8.3 mg, 114 μmol) was added. The mixture was reacted at 25 °C for 3 hours. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (50 mg, yield: 55.1%).

[0648] MS m / z(ESI):1984.4 [M+H] + .

[0649] Intermediate Preparation Example 20: Preparation of Compound Int20

[0650] Int20-1 (300 mg, 870 μmol) and 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolyl) ester (349 mg, 957 μmol) were dissolved in DMF (2 mL), and N,N-diisopropylethylamine (337 mg, 2.61 mmol) was added. The reaction mixture was reacted at 25 °C for 2 h. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (296 mg, yield: 57.1%).

[0651] MS m / z (ESI): 596.3 [M+H]+ .

[0652] Intermediate Preparation Example 21: Preparation of Compound Int21

[0653] Step 1: Preparation of compound Int21-2

[0654] Compound Int21-1 (2.0 g, 3.86 mmol) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (2.45 g, 9.65 mmol) were dissolved in anhydrous toluene (30 mL). Tris(dibenzylacetone)dipalladium (353 mg, 386 μmol), 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl (361 mg, 772 μmol), and sodium tert-butoxide (1.85 g, 19.3 mmol) were added sequentially. The mixture was purged three times with nitrogen and reacted at 90 °C for 2 hours. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1, v / v) to give the title compound (1.84 g, yield: 68.9%).

[0655] MS m / z (ESI): 692.4 [M+H] + .

[0656] Step 2: Preparation of compound Int21-3

[0657] Compound Int21-2 (1.84 g, 2.66 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and 10% palladium on carbon (500 mg) was added. The mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (1.16 g, yield: 84.7%).

[0658] MS m / z (ESI): 514.3 [M+H] + .

[0659] Step 3: Preparation of compound Int21

[0660] Compound Int21-3 (1.16 g, 2.25 mmol) was dissolved in dichloromethane (25 mL), and a solution of 1,4-dioxane in 4 M hydrogen chloride (12 mL) was added. The reaction was carried out at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound (910 mg, yield: 93.4%).

[0661] MS m / z (ESI): 414.3 [M+H] + .

[0662] Intermediate Preparation Example 22: Preparation of Compound Int22

[0663] Step 1: Preparation of compound Int22-2

[0664] (S)-3-(4,6-dichloropyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-2'-one (8 g, 22.8 mmol) was dissolved in DMF (80 mL), and 1,1-cyclopropanediethanol (3.49 g, 34.2 mmol) and cesium carbonate (18.6 g, 57 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with water (300 mL), extracted three times with ethyl acetate (100 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 3 / 1, v / v) to give the title compound (6.7 g, yield: 70.5%).

[0665] MS m / z (ESI): 418.1 [M+H] + .

[0666] Step 2: Preparation of compound Int22-3

[0667] Compound Int22-2 (5.0 g, 11.99 mmol), 5-hydroxypyrimidine (1.50 g, 15.59 mmol), triethylenediamine (201.74 mg, 1.80 mmol), and potassium carbonate (1.99 g, 14.39 mmol) were suspended in acetonitrile (75 mL) and stirred at 60 °C for 8 hours. The reaction mixture was cooled to room temperature and poured into water (750 mL), precipitating a solid. The solid was filtered, collected, and dried to give the title compound (5.4 g, yield: 94.4%).

[0668] MS m / z (ESI): 478.2 [M+H] + .

[0669] Step 3: Preparation of compound Int22-4

[0670] Compound Int22-3 (5.4 g, 11.32 mmol), ammonium acetate (3.49 g, 45.28 mmol), and sulfur (1.45 g, 45.28 mmol) were dissolved in ethanol (35 mL) and stirred at 60 °C for 15 min. Then, a solution of malononitrile (3.74 g, 56.6 mmol) in ethanol (20 mL) was added dropwise, and the mixture was stirred at 80 °C for 8 h. The reaction mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give the title compound (4.3 g, yield: 68.2%).

[0671] MS m / z (ESI): 558.2 [M+H] + .

[0672] Step 4: Preparation of compound Int22-5

[0673] Compound Int22-4 (4.3 g, 7.7 mmol) was dissolved in tetrahydrofuran (50 mL), and N,N-dimethylformamide dimethyl acetal (1.1 g, 9.24 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated to give the title compound (4.6 g, yield: 97.9%).

[0674] MS m / z (ESI): 613.2 [M+H] + .

[0675] Step 5: Preparation of compound Int22

[0676] Compound Int22-5 (3 g, 4.9 mmol) was dissolved in dichloromethane (30 mL), and Dys-Martin oxidant (3.12 g, 7.35 mmol) was added. The mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with water (50 mL), extracted three times with dichloromethane (50 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 3 / 2, v / v) to give the title compound (2.3 g, yield: 76.9%).

[0677] MS m / z (ESI): 611.3 [M+H] + .

[0678] Example 23: Preparation of intermediate 23: Preparation of compound Int23

[0679] Step 1: Preparation of compound Int23-1

[0680] Compounds Int22 (400 mg, 656 μmol) and Int21 (272 mg, 656 μmol) were dissolved in dimethyl sulfoxide (2 mL), and tetraethyl titanate (298.4 mg, 1.31 mmol) was added. The reaction mixture was reacted at 25 °C for 30 min, and sodium triacetoxyborohydride (277.6 mg, 1.31 mmol) was added. The reaction mixture was then reacted at 25 °C for 5 h. The reaction mixture was filtered to remove the solids, and the filtrate was purified by reversed-phase column chromatography (eluent: acetonitrile / water (0.05% TFA) = 4 / 6, v / v) to give the title compound (360 mg, yield: 54.5%).

[0681] MS m / z (ESI): 1008.4 [M+H] + .

[0682] Step 2: Preparation of compound Int23

[0683] Compound Int23-1 (360 mg, 357 μmol) was dissolved in tetrahydrofuran (10 mL), and 2 M dilute hydrochloric acid (5 mL) was added. The mixture was reacted at 65 °C for 3 hours. The reaction solution was concentrated, and the residue was purified by high performance liquid chromatography (condition 1) to give the title compound (180 mg, yield: 52.9%).

[0684] MS m / z (ESI): 953.4 [M+H] + .

[0685] Intermediate Preparation Example 24: Preparation of Compound Int24

[0686] Step 1: Preparation of compound Int24-1

[0687] (9H-fluorene-9-yl)methyl(S)-(1-chloro-1-oxopropane-2-yl)carbamate (143.7 mg, 435.76 μmol) and potassium carbonate (68.78 mg, 498.38 μmol) were mixed in tetrahydrofuran (2 mL), and a tetrahydrofuran (1 mL) solution of compound Int23 (95 mg, 99.68 μmol) was added. The reaction was carried out at 25 °C for 12 hours. The reaction solution was filtered to remove the solids, and the filtrate was purified by reversed-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 9 / 1, v / v) to give the title compound (114 mg, yield: 91.8%).

[0688] MS m / z (ESI): 1246.6 [M+H] + .

[0689] Step 2: Preparation of compound Int24-2

[0690] Compound Int24-1 (114 mg, 91.46 μmol) was dissolved in DMF (2 mL), and diethylamine (13.38 mg, 182.93 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reversed-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (85.5 mg, yield: 91.3%).

[0691] MS m / z (ESI): 1024.4 [M+H] + .

[0692] Step 3: Preparation of compound Int24-3

[0693] Compound Int24-2 (85.5 mg, 87.88 μmol) and (2,5-dioxopyrrolidone-1-yl)-(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methylbutyrate (43.7 mg, 105.45 μmol) were dissolved in dimethyl sulfoxide (1 mL), and N,N-diisopropylethylamine (22.7 mg, 175.75 μmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction solution was purified by high performance liquid chromatography (condition 1) to give the title compound (55 mg, yield: 46.52%).

[0694] MS m / z (ESI): 1345.6 [M+H] + .

[0695] Step 4: Preparation of compound Int24

[0696] Compound Int24-3 (55 mg, 38.83 μmol) was dissolved in DMF (2 mL), and diethylamine (13.38 mg, 182.93 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reversed-phase column chromatography (eluent: water (0.05% trifluoroacetic acid) / acetonitrile = 4 / 6, v / v) to give the title compound (42.8 mg, yield: 98.1%).

[0697] MS m / z (ESI): 1123.5 [M+H] + .

[0698] Intermediate Preparation Example 25: Preparation of Compound Int25

[0699] Using the synthetic route in Intermediate Preparation Example 7, the starting material Int6 in the first step was replaced with compound Int24 to obtain the title compound (25 mg, yield: 91.5%).

[0700] MS m / z (ESI): 1152.6 [M / 2+H] + .

[0701] Intermediate Preparation Example 26: Preparation of Compound Int26

[0702] Using the synthetic route in Example 4 of intermediate preparation, the starting material Int2 in the first step was replaced with compound Int3 to obtain the title compound (200 mg, yield: 63.8%).

[0703] MS m / z (ESI): 1195.6 [M+H] + .

[0704] Intermediate Preparation Example 27: Preparation of Compound Int27

[0705] Step 1: Preparation of compound Int27-1

[0706] Compounds Int26 (200 mg, 167.4 μmol), N,N-diisopropylethylamine (86.4 mg, 669.6 μmol), and Int4 (237.8 mg, 167.4 μmol) were dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (95.4 mg, 251.1 μmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (325 mg, yield: 74.7%).

[0707] MS m / z(ESI): 2599.3 [M+H] + .

[0708] Step 2: Preparation of Compound 27

[0709] Compound Int27-1 (325 mg, 125 μmol) was dissolved in DMF (2 mL), and diethylamine (18.3 mg, 250 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (266 mg, yield: 89.6%).

[0710] MS m / z(ESI): 2377.4 [M+H] + .

[0711] Intermediate Preparation Example 28: Preparation of Compound Int28

[0712] Compound Int28-1 (2 g, 4.4 mmol) was dissolved in methanol (20 mL), and 10% palladium on carbon (500 mg) was added. The mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (1.25 g, yield: 78.1%).

[0713] MS m / z(ESI): 362.2 [M+H] + .

[0714] Intermediate Preparation Example 29: Preparation of Compound Int29

[0715] Step 1: Preparation of compound Int29-1

[0716] Compound Int5-3 (581 mg, 0.69 mmol), N,N-diisopropylethylamine (226 mg, 1.73 mmol), and compound Int28 (210 mg, 0.58 mmol) were dissolved in dimethyl sulfoxide (5 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (332 mg, 0.86 mmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 3 / 1, v / v) to give the title compound (560 mg, yield: 82.0%).

[0717] MS m / z (ESI): 1176.6 [M+H] + .

[0718] Step 2: Preparation of compound Int29-2

[0719] Compound Int29-1 (560 mg, 0.48 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at 25 °C for 5 hours. The reaction solution was concentrated to give the title compound (500 mg, yield: 98.7%).

[0720] MS m / z (ESI): 1064.5 [M+H] + .

[0721] Step 3: Preparation of compound Int29-3

[0722] Compound Int29-2 (300 mg, 279 μmol) and N,N-diisopropylethylamine (292 mg, 2.23 mmol) were dissolved in dimethyl sulfoxide (10 mL). Compound Int1 (547 mg, 837 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (321.6 mg, 837 μmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (460 mg, yield: 70.3%).

[0723] MS m / z(ESI): 2322.2 [M+H] + .

[0724] Step 4: Preparation of compound Int29

[0725] Compound Int29-3 (460 mg, 198 μmol) was dissolved in anhydrous DMF (10 mL), and 1,3-dimethylbarbitine (93.2 mg, 594 μmol) and tetrakis(triphenylphosphine)palladium (45.7 mg, 39.6 μmol) were added. The system was purged with nitrogen three times, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (450 mg, yield: 99.6%).

[0726] MS m / z(ESI): 2282.2 [M+H] + .

[0727] Preparation of intermediates Example 30: Preparation of compound Int30

[0728] Step 1: Preparation of compound Int30-1

[0729] Compounds Int29 (177 mg, 77.5 μmol), N,N-diisopropylethylamine (29.8 mg, 231.3 μmol), and Int4 (100 mg, 77.5 μmol) were dissolved in DMF (3 mL). 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (43.9 mg, 115.4 μmol) was added, and the reaction was carried out at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (40 mg, yield: 14.5%).

[0730] MS m / z (ESI): 3552.7 [M+H] + .

[0731] Step 2: Preparation of compound Int30

[0732] Compound Int30-1 (40 mg, 11.3 μmol) was dissolved in DMF (2 mL), and diethylamine (2.5 mg, 33.9 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (35 mg, yield: 93.3%).

[0733] MS m / z(ESI): 3330.6 [M+H] + .

[0734] Intermediate Preparation Example 31: Preparation of Compound Int31

[0735] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int4 in the first step was replaced with compound Int15 to obtain the title compound (38 mg, yield: 92.5%).

[0736] MS m / z(ESI): 3402.6 [M+H] + .

[0737] Intermediate Preparation Example 32: Preparation of Compound Int32

[0738] Step 1: Preparation of compound Int32-2

[0739] Compound Int32-1 (3.0 g, 15.4 mmol) and potassium bicarbonate (6.15 g, 61.5 mmol) were dissolved in DMF (30 mL). The system was cooled to 0 °C, and then tert-butyl bromoacetate (6.76 g, 34.65 mmol) was added dropwise. After the addition was complete, the reaction was carried out at 25 °C for 12 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed once with saturated sodium bicarbonate solution (50 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to give the title compound (4.6 g, yield: 70.7%).

[0740] MS m / z(ESI): 423.3 [M+H] + .

[0741] Step 2: Preparation of compound Int32

[0742] Compound Int32-2 (4.6 g, 10.9 mmol) was dissolved in ethanol (60 mL), and 10% palladium on carbon catalyst (500 mg) was added. The reaction was carried out at 25 °C for 16 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (2.77 g, yield: 88.2%).

[0743] MS m / z(ESI): 289.2 [M+H] + .

[0744] Preparation of intermediates Example 33: Preparation of compound Int33

[0745] Step 1: Preparation of compound Int33-1

[0746] Compounds Int5-1 (100 mg, 244 μmol), N,N-diisopropylethylamine (94.6 mg, 733.4 μmol), and Int32 (98.4 mg, 341.6 μmol) were dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (148.4 mg, 390.4 μmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 8 / 2, v / v) to give the title compound (150 mg, yield: 90.4%).

[0747] MS m / z (ESI): 680.4 [M+H] + .

[0748] Step 2: Preparation of compound Int33-2

[0749] Compound Int33-1 (150 mg, 0.22 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at 25 °C for 5 hours. The reaction solution was concentrated to give the title compound (120 mg, yield: 96%).

[0750] MS m / z (ESI): 568.2 [M+H] + .

[0751] Step 3: Preparation of compound Int33-3

[0752] Compounds Int33-2 (125 mg, 0.22 mmol), N,N-diisopropylethylamine (341.3 mg, 2.64 mmol), and Int32 (254.3 mg, 0.88 mmol) were dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (334.5 mg, 0.88 mmol) were added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 8 / 2, v / v) to give the title compound (170 mg, yield: 69.7%).

[0753] MS m / z (ESI): 1108.6 [M+H] + .

[0754] Step 4: Preparation of compound Int33-4

[0755] Compound Int33-3 (170 mg, 153.4 μmol) was dissolved in anhydrous DMF (2 mL), and 1,3-dimethylbarbitine (71.8 mg, 460 mmol) and tetrakis(triphenylphosphine)palladium (35.5 mg, 30.7 μmol) were added. The system was purged with nitrogen three times, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 6 / 4, v / v) to give the title compound (132 mg, yield: 80.5%).

[0756] MS m / z (ESI): 1068.6 [M+H] + .

[0757] Step 5: Preparation of compound Int33-5

[0758] Compounds Int33-4 (42 mg, 39.3 μmol), N,N-diisopropylethylamine (17.8 mg, 137.6 μmol), and Int4 (50.6 mg, 39.3 μmol) were dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (22.4 mg, 58.9 μmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 6 / 4, v / v) to give the title compound (50 mg, yield: 54.6%).

[0759] MS m / z(ESI): 2326.2 [M+H] + .

[0760] Step 6: Preparation of compound Int33-6

[0761] Compound Int33-5 (50 mg, 21.5 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.8 mL) was added. The mixture was stirred at 25 °C for 5 hours. The reaction solution was concentrated to give the title compound (39 mg, yield: 85.9%).

[0762] MS m / z(ESI): 2115.9 [M+H] + .

[0763] Step 7: Preparation of compound Int33

[0764] Compound Int33-6 (39 mg, 18.4 μmol) was dissolved in DMF (2 mL), and diethylamine (2.7 mg, 36.8 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (29 mg, yield: 83.1%).

[0765] MS m / z (ESI): 1892.8 [M+H] + .

[0766] Intermediate Preparation Example 34: Preparation of Compound Int34

[0767] Using the synthetic route in Intermediate Preparation Example 33, the starting material Int5-1 in the first step was replaced with compound Int5-3 to obtain the title compound (31 mg, yield: 52.3%).

[0768] MS m / z(ESI): 2317.1 [M+H] + .

[0769] Preparation of intermediates Example 35: Preparation of compound Int35

[0770] Step 1: Preparation of compound Int35-2

[0771] Compound Int35-1 (1.0 g, 3.98 mmol), N,N-diisopropylethylamine (1.54 g, 11.9 mmol), and tris(hydroxymethyl)aminomethane (1.2 g, 9.95 mmol) were dissolved in DMF (15 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.78 g, 9.95 mmol) was added. The reaction mixture was reacted at 25 °C for 3 h. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 4 / 6, v / v) to give the title compound (700 mg, yield: 38.5%).

[0772] MS m / z (ESI): 458.2 [M+H] + .

[0773] Step 2: Preparation of compound Int35

[0774] Compound Int35-2 (700 mg, 1.53 mmol) was dissolved in methanol (10 mL), and 10% palladium on carbon (500 mg) was added. The mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (500 mg, yield: 88.9%).

[0775] MS m / z (ESI): 368.2 [M+H] + .

[0776] Intermediate Preparation Example 36: Preparation of Compound Int36

[0777] Step 1: Preparation of compound Int36-1

[0778] Compound Int28-1 (590 mg, 130 μmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at 25 °C for 5 hours. The reaction solution was concentrated to give the title compound (419 mg, yield: 94.4%).

[0779] MS m / z (ESI): 340.2 [M+H] + .

[0780] Step 2: Preparation of compound Int36-2

[0781] Compound Int36-1 (419 mg, 1.23 mmol), N,N-diisopropylethylamine (637 mg, 4.93 mmol), and tris(hydroxymethyl)aminomethane (372 mg, 3.08 mmol) were dissolved in DMF (10 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.17 g, 3.08 mmol) was added. The reaction mixture was reacted at 25 °C for 3 h. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 4 / 6, v / v) to give the title compound (270 mg, yield: 40.1%).

[0782] MS m / z (ESI): 546.3 [M+H] + .

[0783] Step 3: Preparation of compound Int36

[0784] Compound Int36-2 (270 mg, 0.49 mmol) was dissolved in methanol (5 mL), and 10% palladium on carbon (50 mg) was added. The mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (203 mg, yield: 90.2%).

[0785] MS m / z (ESI): 456.2 [M+H] + .

[0786] Intermediate Preparation Example 37: Preparation of Compound Int37

[0787] Step 1: Preparation of compound Int37-1

[0788] Compound Int29-2 (200 mg, 188 μmol) and N,N-diisopropylethylamine (72.9 mg, 564 μmol) were dissolved in dimethyl sulfoxide (5 mL). Compound Int35 (172.9 mg, 470 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (178.6 mg, 470 μmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (268 mg, yield: 80.9%).

[0789] MS m / z (ESI): 1762.9 [M+H] + .

[0790] Step 2: Preparation of compound Int37

[0791] Compound Int29-3 (268 mg, 152 μmol) was dissolved in anhydrous DMF (5 mL), and 1,3-dimethylbarbitine (47.4 mg, 304 μmol) and tetrakis(triphenylphosphine)palladium (35.1 mg, 30.4 μmol) were added. The system was purged with nitrogen three times, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (163 mg, yield: 62.2%).

[0792] MS m / z(ESI): 1722.9 [M+H] + .

[0793] Intermediate Preparation Example 38: Preparation of Compound Int38

[0794] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 in the first step was replaced with compound Int36 to obtain the title compound (88 mg, yield: 69.3%).

[0795] MS m / z(ESI): 1898.9 [M+H] + .

[0796] Intermediate Preparation Example 39: Preparation of Compound Int39

[0797] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int37 to obtain the title compound (38 mg, yield: 59.2%).

[0798] MS m / z(ESI): 2772.4 [M+H] + .

[0799] Intermediate Preparation Example 40: Preparation of Compound Int40

[0800] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int38 to obtain the title compound (15 mg, yield: 68.5%).

[0801] MS m / z(ESI): 2948.4 [M+H] + .

[0802] Intermediate Preparation Example 41: Preparation of Compound Int41

[0803] Using the synthetic route in Intermediate Preparation Example 36, the reactant in the second step, tris(hydroxymethyl)aminomethane, was replaced with compound Int1 to obtain the title compound (360 mg, yield: 81.2%).

[0804] MS m / z (ESI): 1506.7 [M+H] + .

[0805] Intermediate Preparation Example 42: Preparation of Compound Int42

[0806] Step 1: Preparation of compound Int42-1

[0807] Compounds Int5-1 (50 mg, 122 μmol), N,N-diisopropylethylamine (47.3 mg, 366 μmol), and Int41 (275.5 mg, 183 μmol) were dissolved in DMF (3 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (69.5 mg, 183 μmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 8 / 2, v / v) to give the title compound (60 mg, yield: 25.9%).

[0808] MS m / z(ESI): 1897.9 [M+H] + .

[0809] Step 2: Preparation of compound Int42-2

[0810] Compound Int42-1 (60 mg, 31.3 μmol) was dissolved in anhydrous DMF (2 mL), and 1,3-dimethylbarbitine (14.8 mg, 93.9 μmol) and tetrakis(triphenylphosphine)palladium (7.31 mg, 6.26 μmol) were added. The system was purged with nitrogen three times, and the reaction was carried out at 20 °C for 4 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6, v / v) to give the title compound (55 mg, yield: 93.6%).

[0811] MS m / z (ESI): 1857.9 [M+H] + .

[0812] Step 3: Preparation of compound Int42-3

[0813] Compounds Int42-2 (55 mg, 29.3 μmol), N,N-diisopropylethylamine (11.5 mg, 87.9 μmol), and Int4 (40 mg, 30.7 μmol) were dissolved in DMF (2 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (16.9 mg, 43.9 μmol) were added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 6 / 4, v / v) to give the title compound (65 mg, yield: 70.2%).

[0814] MS m / z(ESI): 3129.4 [M+H] + .

[0815] Step 4: Preparation of compound Int42

[0816] Compound Int42-3 (65 mg, 20.6 μmol) was dissolved in DMF (2 mL), and diethylamine (4.6 mg, 61.8 μmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (42 mg, yield: 70.1%).

[0817] MS m / z(ESI): 2907.4 [M+H] + .

[0818] Intermediate Preparation Example 43: Preparation of Compound Int43

[0819] Using the synthetic route in Intermediate Preparation Example 33, the starting material Int5-1 of the first step reaction was replaced with compound Int5-3, and the starting material Int32 of the first step reaction was replaced with di-tert-butyl 3,3-[[2-amino-2-[[3-(tert-butoxy)-3-oxopropoxy]methyl]propane-1,3-diyl]bis(oxy)]dipropionate, to obtain the title compound (15 mg, yield: 53.8%).

[0820] MS m / z(ESI): 2636.2 [M+H] + .

[0821] Intermediate Preparation Example 44: Preparation of Compound Int44

[0822] Using the synthetic route in Intermediate Preparation Example 33, the starting material in the third step, compound Int33-2, was replaced with compound Int29-2 to obtain the title compound (30 mg, yield: 60.5%).

[0823] MS m / z(ESI): 2390.2 [M+H] + .

[0824] Intermediate Preparation Example 45: Preparation of Compound Int45

[0825] Step 1: Preparation of compound Int45-1

[0826] Compounds Int35-1 (400 mg, 1.6 mmol), N,N-diisopropylethylamine (1.65 g, 12.8 mmol), and Int32 (1.15 g, 4.0 mmol) were dissolved in DMF (10 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.52 g, 4.0 mmol) was added. The reaction mixture was reacted at 25 °C for 12 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 6 / 4, v / v) to give the title compound (600 mg, yield: 47.6%).

[0827] MS m / z (ESI): 792.5 [M+H] + .

[0828] Step 2: Preparation of compound Int45-2

[0829] Compound Int45-1 (600 mg, 130 μmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at 25 °C for 5 hours. The reaction solution was concentrated to give the title compound (396 mg, yield: 91.9%).

[0830] MS m / z (ESI): 568.3 [M+H] + .

[0831] Step 3: Preparation of compound Int45-3

[0832] Compound Int45-2 (396 mg, 0.7 mmol), N,N-diisopropylethylamine (720.8 mg, 5.6 mmol), and tris(hydroxymethyl)aminomethane were used.

[0833] (508.2 mg, 4.2 mmol) was dissolved in DMF (10 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.6 g, 4.2 mmol) was added. The reaction mixture was reacted at 25 °C for 6 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 3 / 7, v / v) to give the title compound (195 mg, yield: 28.5%).

[0834] MS m / z (ESI): 980.5 [M+H] + .

[0835] Step 4: Preparation of compound Int45

[0836] Compound Int45-3 (195 mg, 0.2 mmol) was dissolved in methanol (5 mL), and 10% palladium on carbon (60 mg) was added. The mixture was reacted at 25 °C for 12 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (160 mg, yield: 90.4%).

[0837] MS m / z (ESI): 890.5 [M+H] + .

[0838] Intermediate Preparation Example 46: Preparation of Compound Int46

[0839] Using the synthetic route in Intermediate Preparation Example 42, the starting material Int5-1 of the first step reaction was replaced with compound Int5-3, and the starting material Int41 of the first step reaction was replaced with compound Int45, to obtain the title compound (8.5 mg, yield: 43.2%).

[0840] MS m / z(ESI): 2714.3 [M+H] + .

[0841] Intermediate Preparation Example 47: Preparation of Compound Int47

[0842] Step 1: Preparation of compound Int47-1

[0843] Compound Int22-1 (2 g, 5.70 mmol) was dissolved in 1,4-dioxane (20 mL), and (S)-3-methyl-1,4-diazacycloheptan-1-carboxylic acid tert-butyl ester (1.58 g, 7.4 mmol) and N,N-diisopropylethylamine (2.21 g, 17.1 mmol) were added. The mixture was stirred at 80 °C for 6 hours. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (30 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 7 / 3, v / v) to give the title compound (2.6 g, yield: 86.3%).

[0844] MS m / z (ESI): 530.2 [M+H] + .

[0845] Step 2: Preparation of compound Int47-2

[0846] Compound Int47-1 (2.6 g, 4.9 mmol), 5-hydroxypyrimidine (0.57 g, 5.88 mmol), triethylenediamine (54.9 mg, 0.49 mmol), and cesium carbonate (3.19 g, 9.8 mmol) were added to DMF (25 mL) and stirred at 100 °C for 16 hours. The reaction mixture was cooled to room temperature, diluted with water (100 mL), extracted three times with ethyl acetate (30 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give the title compound (2.3 g, yield: 79.6%).

[0847] MS m / z (ESI): 590.3 [M+H] + .

[0848] Step 3: Preparation of compound Int47-3

[0849] Compound Int47-2 (2.3 g, 3.9 mmol), ammonium acetate (1.5 g, 19.5 mmol), and sulfur (0.62 g, 19.5 mmol) were added to ethanol (15 mL), and the mixture was stirred at 60 °C for 15 min. Then, a solution of malononitrile (1.54 g, 23.4 mmol) in ethanol (10 mL) was added dropwise, and the mixture was stirred at 80 °C for 16 h. The reaction mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1, v / v) to give the title compound (2.2 g, yield: 84.3%).

[0850] MS m / z (ESI): 670.3 [M+H] + .

[0851] Step 4: Preparation of compound Int47

[0852] Compound Int47-3 (2.2 g, 3.28 mmol) was dissolved in dichloromethane (10 mL) and methanol (10 mL), and a 1,4-dioxane solution of 4 M hydrogen chloride (20 mL) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in water (30 mL), extracted once with ethyl acetate (20 mL), and then the pH of the aqueous phase was adjusted to greater than 12 with solid potassium carbonate, extracted with ethyl acetate (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (1.6 g, yield: 85.6%).

[0853] MS m / z (ESI): 570.3 [M+H] + .

[0854] Intermediate Preparation Example 48: Preparation of Compound Int48

[0855] Step 1: Preparation of compound Int48-1

[0856] Compound Int47 (1.15 g, 2.00 mmol) was dissolved in dimethyl sulfoxide (15 mL), 2 drops of acetic acid were added, and the reaction was carried out at 25 °C for 1 hour. Sodium triacetoxyborohydride (856 mg, 4.00 mmol) was then added, and the reaction was carried out at 25 °C for 5 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (1.1 g, yield: 66.3%).

[0857] MS m / z (ESI): 821.5 [M+H] + .

[0858] Step 2: Preparation of compound Int48-2

[0859] Compound Int48-1 (300 mg, 361 μmol) and (9H-fluorene-9-yl)methyl(S)-(1-chloro-1-oxopropane-2-yl)carbamate (145 mg, 434 μmol) were dissolved in tetrahydrofuran (5 mL), and anhydrous potassium carbonate (101 mg, 723 μmol) was added. The reaction mixture was reacted at 25 °C for 12 hours. The reaction solution was filtered to remove the solids, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to give the title compound (230 mg, yield: 56.5%).

[0860] MS m / z (ESI): 1114.5 [M+H] + .

[0861] Step 3: Preparation of compound Int48-3

[0862] Compound Int48-2 (230 mg, 204 μmol) was dissolved in DMF (5 mL), and diethylamine (45 mg, 613 μmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (140 mg, yield: 76.0%).

[0863] MS m / z (ESI): 892.5 [M+H] + .

[0864] Step 4: Preparation of compound Int48-4

[0865] Compound Int48-3 (140 mg, 155 μmol) and (2,5-dioxopyrrolidone-1-yl)-(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methylbutyrate (82 mg, 186 μmol) were dissolved in DMF (4 mL), and N,N-diisopropylethylamine (60 mg, 465 μmol) was added. The reaction mixture was reacted at 25 °C for 2 h. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (160 mg, yield: 84.0%).

[0866] MS m / z(ESI):1213.6[M+H]+.

[0867] Step 5: Preparation of compound Int48-5

[0868] Compound Int48-4 (160 mg, 130 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (2 mL) was added. The reaction was carried out at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound (130 mg, yield: 88.5%).

[0869] MS m / z(ESI): 1114.5[M+H]+.

[0870] Step 6: Preparation of compound Int48-6

[0871] Compound Int48-5 (130 mg, 115 μmol) and 1-(3-(2,6-dioxopiperidin-3-yl)-1-methyl-1H-indazol-6-yl)piperidin-4-carboxylic acid (90 mg, 150 μmol) were dissolved in DMF (4 mL). N,N-diisopropylethylamine (45 mg, 346 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (66 mg, 173 μmol) were added sequentially. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (110 mg, yield: 64.2%).

[0872] MS m / z(ESI):1466.6[M+H]+.

[0873] Step 7: Preparation of compound Int48

[0874] Compound Int48-6 (110 mg, 74 μmol) was dissolved in DMF (2 mL), and diethylamine (16 mg, 222 μmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 6 / 4, v / v) to give the title compound (80 mg, yield: 85.7%).

[0875] MS m / z(ESI):1243.6[M+H]+.

[0876] Intermediate Preparation Example 49: Preparation of Compound Int49

[0877] Using the synthetic route in Intermediate Preparation Example 7, the starting material Int6 in the first step was replaced with compound Int48 to obtain the title compound (70 mg, yield: 88.5%).

[0878] MS m / z(ESI): 2425.4 [M+H] + .

[0879] Intermediate Preparation Example 50: Preparation of Compound Int50

[0880] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int4 in the first step was replaced with compound Int48 to obtain the title compound (31 mg, yield: 81.3%).

[0881] MS m / z(ESI): 3284.6 [M+H] + .

[0882] Intermediate Preparation Example 51: Preparation of Compound Int51

[0883] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int4 in the first step was replaced with compound Int26 to obtain the title compound (55 mg, yield: 76.3%).

[0884] MS m / z(ESI): 3236.6 [M+H] + .

[0885] Intermediate Preparation Example 52: Preparation of Compound Int52

[0886] Step 1: Preparation of compound Int52-2

[0887] Compound Int52-1 (2 g, 8.04 mmol), N,N-disuccinimidyl carbonate (6.17 g, 24.11 mmol), and triethylamine (2.44 g, 24.11 mmol) were dissolved in dimethyl sulfoxide (30 mL) under ice bath conditions and reacted at 25 °C for 2 h. 2-Amino-2-(hydroxymethyl)propane-1,3-diol (2.95 g, 24.11 mmol) was added to the above solution, and the reaction was carried out at 25 °C for 3 h. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (350 mg, yield: 8.1%).

[0888] MS m / z (ESI): 541.3 [M+H] + .

[0889] Step 2: Preparation of compound Int52

[0890] Compound Int52-2 (350 mg, 647.42 μmol) was dissolved in dichloromethane (1 mL), and 1,4-dioxane hydrochloride (1 mL) was added. The reaction was carried out at 25 °C for 1 hour. The reaction solution was concentrated to give the title compound (300 mg, yield: 97.2%).

[0891] MS m / z (ESI): 441.3 [M+H] + .

[0892] Example 53: Preparation of compound Int53

[0893] Step 1: Preparation of compound Int53-2

[0894] Compound Int53-1 (700 mg, 1.58 mmol) was dissolved in dichloromethane (5 mL) and dioxane hydrochloride solution (5 mL), and stirred at 25 °C for 2 hours. The solution was concentrated under reduced pressure to give the title compound (420 mg, yield: 96.8%).

[0895] MS m / z(ESI): 238.1 [M+H] + .

[0896] Step 2: Preparation of compound Int53-3

[0897] Triphosgene (424.4 mg, 1.4 mmol) was dissolved in tetrahydrofuran (10 mL) at 0 °C, N,N-diisopropylethylamine (276.8 mg, 2.1 mmol) was added, followed by the slow addition of compound Int53-2 (193 mg, 700 μmol), and the reaction was carried out at 25 °C for 3 hours. 2-Amino-2-(hydroxymethyl)propane-1,3-diol (180 mg, 1.47 mmol) was added to the above reaction solution at 25 °C, and the reaction was carried out at 25 °C for 2 hours. The reaction solution was purified by high-performance liquid chromatography (condition 2) to give the title compound (150 mg, yield: 40.3%).

[0898] MS m / z (ESI): 532.2 [M+H] + .

[0899] Step 3: Preparation of compound Int53

[0900] Compound Int53-3 (150 mg, 279.4 μmol) was dissolved in methanol (5 mL), and 10% palladium on carbon (15 mg) was added. The mixture was reacted at 25 °C for 16 hours under a hydrogen atmosphere. The solid was removed by filtration, and the filtrate was concentrated to give the title compound (105 mg, yield: 94.6%).

[0901] MS m / z(ESI): 398.2 [M+H] + .

[0902] Intermediate Preparation Example 54: Preparation of Compound Int54

[0903] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 in the first step was replaced with compound Int52 to obtain the title compound (30 mg, yield: 61.29%).

[0904] MS m / z(ESI): 1869.1 [M+H] + .

[0905] Intermediate Preparation Example 55: Preparation of Compound Int55

[0906] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 of the first step reaction was replaced with compound Int52, and the starting material Int29-2 of the first step reaction was replaced with compound Int43-2, to obtain the title compound (10 mg, yield: 17.1%).

[0907] MS m / z(ESI): 2379.6 [M+H] + .

[0908] Intermediate Preparation Example 56: Preparation of Compound Int56

[0909] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int4 in the first step was replaced with compound Int24 to obtain the title compound (55 mg, yield: 97.9%).

[0910] MS m / z (ESI): 3164.5 [M+H] + .

[0911] Intermediate Preparation Example 57: Preparation of Compound Int57

[0912] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int55 to obtain the title compound (6 mg, yield: 91.3%).

[0913] MS m / z(ESI): 3428.9 [M+H] + .

[0914] Intermediate Preparation Example 58: Preparation of Compound Int58

[0915] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int54 to obtain the title compound (25 mg, yield: 59.8%).

[0916] MS m / z(ESI): 2918.4 [M+H] + .

[0917] Intermediate Preparation Example 59: Preparation of Compound Int59

[0918] Using the synthetic route in Intermediate Preparation Example 33, the starting material Int32 of the first step reaction was replaced with di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate, and the starting material Int32 of the third step reaction was replaced with compound Int28, to obtain the title compound (45 mg, yield: 76.2%).

[0919] MS m / z(ESI): 2431.7 [M+H] + .

[0920] Intermediate Preparation Example 60: Preparation of Compound Int60

[0921] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 in the first step was replaced with compound Int53 to obtain the title compound (320 mg, yield: 75.3%).

[0922] MS m / z (ESI): 1782.9 [M+H] + .

[0923] Example 61: Preparation of intermediate 61

[0924] Using the synthetic route in Intermediate Preparation Example 30, the starting material compound Int29 in the first step was replaced with compound Int60 to obtain the title compound (140 mg, yield: 88.8%).

[0925] MS m / z(ESI): 2832.4 [M+H] + .

[0926] Preparation of intermediates Example 62: Preparation of compound Int62

[0927] Using the synthetic route in Intermediate Preparation Example 33, the starting material Int5-1 of the first step reaction was replaced with compound Int5-3, the starting material Int32 of the first step reaction was replaced with di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate, and the starting material Int32 of the third step reaction was replaced with compound Int28, to obtain the title compound (30 mg, yield: 64.6%).

[0928] MS m / z(ESI): 2855.3 [M+H]+ .

[0929] Preparation of intermediates Example 63: Preparation of compound Int63

[0930] Step 1: Preparation of compound Int63-2

[0931] Compound Int63-1 (350 mg, 1.31 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (482 mg, 1.31 mmol) and N,N-diisopropylethylamine (341 mg, 2.62 mmol) were added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was purified by high performance liquid chromatography (condition 1) to give the title compound (580 mg, yield: 85.2%).

[0932] MS m / z (ESI): 516.2 [M+H] + .

[0933] Step 2: Preparation of compound Int63

[0934] Compound Int63-2 (50 mg, 96.01 μmol) was dissolved in tetrahydrofuran (2 mL), and 1-hydroxypyrrolidine-2,5-dione (13 mg, 112.95 μmol) and N,N'-diisopropylcarbodiimide (18 mg, 142.63 μmol) were added sequentially. The mixture was reacted at 25 °C for 2 hours to give the title compound (58 mg, yield: 97.6%).

[0935] MS m / z (ESI): 613.4 [M+H] + .

[0936] Preparation of intermediates Example 64: Preparation of compound Int64

[0937] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 of the first step reaction was replaced with compound Int53, and the starting material Int29-2 of the first step reaction was replaced with compound Int43-2, to obtain the title compound (150 mg, yield: 63.6%).

[0938] MS m / z(ESI): 2250.4 [M+H] + .

[0939] Preparation of intermediates Example 65: Preparation of compound Int65

[0940] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int64 to obtain the title compound (75 mg, yield: 72.7%).

[0941] MS m / z(ESI): 3299.7 [M+H] + .

[0942] Preparation of intermediates Example 66: Preparation of compound Int66

[0943] Using the synthetic route in Intermediate Preparation Example 36, the starting material In28-1 in the first step was replaced with compound Int32-2 to obtain the title compound (173 mg, yield: 38.6%).

[0944] MS m / z (ESI): 383.2 [M+H] + .

[0945] Preparation of intermediates Example 67: Preparation of compound Int67

[0946] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 of the first step reaction was replaced with compound Int66, and the starting material Int29-2 of the first step reaction was replaced with compound Int43-2, to obtain the title compound (84 mg, yield: 53.5%).

[0947] MS m / z(ESI): 2205.4 [M+H] + .

[0948] Intermediate Preparation Example 68: Preparation of Compound Int68

[0949] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 of the first step reaction was replaced with compound Int36, and the starting material Int29-2 of the first step reaction was replaced with compound Int43-2, to obtain the title compound (56 mg, yield: 68.8%).

[0950] MS m / z(ESI): 2424.6 [M+H] + .

[0951] Preparation of intermediates Example 69: Preparation of compound Int69

[0952] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int67 to obtain the title compound (35 mg, yield: 58.5%).

[0953] MS m / z(ESI): 3254.7 [M+H] + .

[0954] Intermediate Preparation Example 70: Preparation of Compound Int70

[0955] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int68 to obtain the title compound (55 mg, yield: 72.3%).

[0956] MS m / z(ESI): 3474.1 [M+H] + .

[0957] Intermediate Preparation Example 71: Preparation of Compound Int71

[0958] Using the synthetic route in Example 33, the starting compound Int32 in the third step was replaced with tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine to give the title compound (100 mg, yield: 73.4%).

[0959] MS m / z (ESI): 2215.4 [M+H] + .

[0960] Intermediate Preparation Example 72: Preparation of Compound Int72

[0961] Using the synthetic route in Example 33 of intermediate preparation, the starting material in the first step, Int5-1, was replaced with compound Int5-3, and the starting material in the third step, Int32, was replaced with tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine, to obtain the title compound (20 mg, yield: 43.4%).

[0962] MS m / z(ESI): 2317.1 [M+H] + .

[0963] Intermediate Preparation Example 73: Preparation of Compound Int73

[0964] Step 1: Preparation of compound Int73-2

[0965] Compound Int73-1 (1.41 g, 7.16 mmol) was dissolved in tetrahydrofuran (50 mL), sodium hydride (391 mg, 9.76 mmol) was added, and the reaction was carried out at 25 °C for 0.5 h. Ethyl glycol monomethyl ether (500 mg, 6.51 mmol) was added, and the reaction was carried out at 25 °C for 3 h. The reaction solution was quenched with saturated ammonium chloride aqueous solution (50 mL), ethyl acetate (50 mL) was added, the mixture was separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 1, v / v) to give the title compound (1.52 g, yield: 97.2%).

[0966] MS m / z(ESI): 235.1 [M+H] + .

[0967] Step 2: Preparation of compound Int73-3

[0968] Compound Int73-2 (1.52 g, 6.39 mmol) was dissolved in DMF (15 mL) and tetrahydrofuran (15 mL), followed by the addition of [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (937 mg, 1.28 mmol) and triethylamine (3.27 g, 31.96 mmol). Carbon monoxide gas was introduced, and the reaction was carried out at 85 °C for 12 hours. The reaction solution was filtered to remove the solids, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 2 / 1, v / v) to give the title compound (1.22 g, yield: 73.1%).

[0969] MS m / z (ESI): 259.1 [M+H] + .

[0970] Step 3: Preparation of compound Int73-4

[0971] Compound Int73-3 (1.22 g, 4.68 mmol) was dissolved in tetrahydrofuran (30 mL) and water (10 mL), and lithium hydroxide monohydrate (595 mg, 14.03 mmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The tetrahydrofuran was removed by concentration under reduced pressure, and the pH was adjusted to 3 with 2 M dilute hydrochloric acid. A solid precipitated, and the solid was collected by filtration to give the title compound (1.01 g, yield: 87.5%).

[0972] MS m / z (ESI): 245.1 [M+H] + .

[0973] Step 4: Preparation of compound Int73-5

[0974] Compound Int73-4 (300 mg, 1.22 mmol) and amino-polyethylene glycol-tert-butyl propionate (279 mg, 1.46 mmol) were dissolved in DMF (5 mL). N,N-diisopropylethylamine (317 mg, 2.43 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (700 mg, 1.82 mmol) were added sequentially, and the reaction was carried out at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (500 mg, yield: 97.9%).

[0975] MS m / z (ESI): 416.3 [M+H] + .

[0976] Step 5: Preparation of compound Int73-6

[0977] Compound Int73-5 (500 mg, 1.19 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (430 mg, yield: 99.5%).

[0978] MS m / z(ESI): 360.2 [M+H] + .

[0979] Step 6: Preparation of compound Int73-7

[0980] Compound Int73-6 (430 mg, 1.19 mmol) was dissolved in dichloromethane (10 mL), and m-chloroperoxybenzoic acid (527 mg, 2.61 mmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (400 mg, yield: 86.3%).

[0981] MS m / z(ESI): 392.4 [M+H] + .

[0982] Step 7: Preparation of compound Int73

[0983] Compound Int73-7 (30 mg, 76.1 μmol) was dissolved in tetrahydrofuran (2 mL), and 1-hydroxypyrrolidine-2,5-dione (9.7 mg, 83.6 μmol) and N,N'-diisopropylcarbodiimide (14.5 mg, 114.1 μmol) were added sequentially. The mixture was reacted at 25 °C for 2 hours to give the title compound (37 mg, yield: 98.7%).

[0984] MS m / z (ESI): 489.2 [M+H] + .

[0985] Intermediate Preparation Example 74: Preparation of Compound Int74

[0986] Step 1: Preparation of compound Int74-2

[0987] Compound Int74-1 (500 mg, 2.29 mmol) was dissolved in acetonitrile (10 mL), and N,N-diisopropylethylamine (887.6 mg, 6.87 mmol) and 2-methoxyethylamine (258 mg, 3.44 mmol) were added. The mixture was reacted at 80 °C for 12 hours. The reaction solution was quenched with saturated ammonium chloride aqueous solution (60 mL), and ethyl acetate (100 mL) was added. The mixture was separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solution was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% ammonium bicarbonate) = 1 / 1, v / v) to give the title compound (89 mg, yield: 15.1%).

[0988] MS m / z(ESI): 258.3 [M+H] + .

[0989] Step 2: Preparation of compound Int74-3

[0990] Compound Int74-2 (89 mg, 0.34 mmol) was dissolved in methanol (3 mL) and water (1 mL), and lithium hydroxide monohydrate (43.5 mg, 1.03 mmol) was added. The reaction mixture was reacted at 25 °C for 3 hours. The reaction mixture was filtered and purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 1 / 3, v / v) to give the title compound (84 mg, yield: 99.8%).

[0991] MS m / z(ESI): 244.3 [M+H] + .

[0992] Step 3: Preparation of compound Int74-4

[0993] Compound Int74-3 (84 mg, 0.34 mmol) and amino-polyethylene glycol-tert-butyl propionate (78.2 mg, 0.41 mmol) were dissolved in DMF (5 mL). N,N-diisopropylethylamine (132 mg, 1.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (194 mg, 0.51 mmol) were added sequentially, and the reaction was carried out at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 7 / 3, v / v) to give the title compound (115 mg, yield: 80.5%).

[0994] MS m / z (ESI): 415.3 [M+H] + .

[0995] Step 4: Preparation of compound Int74-5

[0996] Compound Int74-4 (115 mg, 0.28 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1.5 mL) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (99 mg, yield: 99.5%).

[0997] MS m / z (ESI): 359.4 [M+H] + .

[0998] Step 5: Preparation of compound Int74-6

[0999] Compound Int74-5 (99 mg, 0.28 mmol) was dissolved in dichloromethane (10 mL), and m-chloroperoxybenzoic acid (123 mg, 0.61 mmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 4 / 6, v / v) to give the title compound (50 mg, yield: 46.4%).

[1000] MS m / z (ESI): 391.4 [M+H] + .

[1001] Step 6: Preparation of compound Int74

[1002] Compound Int74-6 (30 mg, 76.1 μmol) was dissolved in tetrahydrofuran (2 mL), and 1-hydroxypyrrolidine-2,5-dione (9.7 mg, 83.6 μmol) and N,N'-diisopropylcarbodiimide (14.5 mg, 114.1 μmol) were added sequentially. The mixture was reacted at 25 °C for 2 hours to give the title compound (37 mg, yield: 98.6%).

[1003] MS m / z (ESI): 488.4 [M+H] + .

[1004] Intermediate Preparation Example 75: Preparation of Compound Int75

[1005] Using the synthetic route in Example 33, the starting material Int32 of the first step reaction was replaced with tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine to obtain the title compound (45 mg, yield: 76.2%).

[1006] MS m / z(ESI): 2416.1 [M+H] + .

[1007] Intermediate Preparation Example 76: Preparation of Compound Int76

[1008] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 of the first step reaction was replaced with compound Int66, and the starting material Int29-2 of the first step reaction was replaced with compound Int34-2, to obtain the title compound (80 mg, yield: 52.3%).

[1009] MS m / z (ESI): 1680.8 [M+H] + .

[1010] Intermediate Preparation Example 77: Preparation of Compound Int77

[1011] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int76 to obtain the title compound (30 mg, yield: 72.1%).

[1012] MS m / z(ESI): 2729.3 [M+H] + .

[1013] Intermediate Preparation Example 78: Preparation of Compound Int78

[1014] Step 1: Preparation of compound Int78-1

[1015] Compound Int5-1 (2.0 g, 4.89 mmol), N,N-diisopropylethylamine (1.26 g, 9.28 mmol), and propargylamine (0.35 g, 6.36 mmol) were dissolved in dimethyl sulfoxide (20 mL), and 2-(7-azo-4-triazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.64 g, 6.96 mmol) was added. The reaction mixture was reacted at 25 °C for 3 h. The reaction solution was added dropwise to water, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 4, v / v) to give the title compound (2.6 g, yield: 97.7%).

[1016] MS m / z (ESI): 447.2 [M+H] + .

[1017] Step 2: Preparation of compound Int78-2

[1018] Compound Int78-1 (2.6 g, 5.82 mmol) and O-(2-azidoethyl)-O-[2-(dihydroxyacetyl-amino)ethyl]heptaethylene glycol (3.23 g, 5.82 mmol) were dissolved in dimethyl sulfoxide (20 mL) and water (2 mL). Copper sulfate pentahydrate (2.91 g, 11.64 mmol) and sodium ascorbate (2.31 g, 11.64 mmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (3.1 g, yield: 53.8%).

[1019] MS m / z (ESI): 1001.5 [M+H] + .

[1020] Step 3: Preparation of compound Int78-3

[1021] Compound Int78-2 (1.5 g, 1.5 mmol) and N,N-diisopropylethylamine (581.6 mg, 4.5 mmol) were dissolved in dimethyl sulfoxide (10 mL). Compound Int28 (541.5 mg, 1.5 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (855.5 mg, 2.25 mmol) were added, and the reaction was carried out at 25 °C for 3 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 7 / 3, v / v) to give the title compound (1.91 g, yield: 95.0%).

[1022] MS m / z (ESI): 1344.5 [M+H] + .

[1023] Step 4: Preparation of compound Int78

[1024] Compound Int78-3 (1.91 g, 1.42 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (10 mL) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 1 / 1, v / v) to give the title compound (1.5 g, yield: 85.7%).

[1025] MS m / z(ESI): 1232.6 [M+H] + .

[1026] Intermediate Preparation Example 79: Preparation of Compound Int79

[1027] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 was replaced with compound Int52, and the starting material Int29-2 was replaced with compound Int78, to obtain the title compound (192 mg, yield: 53.1%).

[1028] MS m / z(ESI): 2037.2 [M+H] + .

[1029] Example 80: Preparation of compound Int80

[1030] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 of the first step was replaced with compound Int1, and the starting material Int29-2 was replaced with compound Int78, to obtain the title compound (150 mg, yield: 44.6%).

[1031] MS m / z(ESI): 2449.6 [M+H] + .

[1032] Example 81: Preparation of intermediate 81

[1033] Step 1: Preparation of compound Int81-1

[1034] Compound Int5-1 (1.0 g, 2.44 mmol), N,N-diisopropylethylamine (0.63 g, 4.88 mmol), and tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate (0.78 g, 2.44 mmol) were dissolved in dimethyl sulfoxide (10 mL), and 2-(7-azo-4-triazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.39 g, 3.66 mmol) were added. The mixture was reacted at 25 °C for 3 hours. The reaction solution was added dropwise to water (60 mL), extracted twice with ethyl acetate (40 mL), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 7 / 3, v / v) to give the title compound (1.3 g, yield: 74.7%).

[1035] MS m / z (ESI): 713.4 [M+H] + .

[1036] Step 2: Preparation of compound Int81

[1037] Compound Int81-1 (1.3 g, 1.81 mmol) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (8 mL) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 1 / 1, v / v) to give the title compound (1.1 g, yield: 91.7%).

[1038] MS m / z (ESI): 657.3 [M+H] + .

[1039] Example 82: Preparation of intermediate: Int82

[1040] Using the synthetic route in Intermediate Preparation Example 29, the starting material In5-3 of the first step reaction was replaced with compound Int81 to obtain the title compound (150 mg, yield: 44.2%).

[1041] MS m / z(ESI): 2105.3 [M+H] + .

[1042] Example 83: Preparation of intermediate 83

[1043] Using the synthetic route in Intermediate Preparation Example 37, the starting material Int35 in the first step was replaced with compound Int52, and the starting material Int29-2 was replaced with compound Int82-2, to obtain the title compound (130 mg, yield: 35.7%).

[1044] MS m / z (ESI): 1692.8 [M+H] + .

[1045] Preparation of intermediates Example 84: Preparation of compound Int84

[1046] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int82, and compound Int4 was replaced with compound Int26, to obtain the title compound (40 mg, yield: 56.2%).

[1047] MS m / z (ESI): 3060.5 [M+H] + .

[1048] Preparation of intermediates Example 85: Preparation of compound Int85

[1049] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int83, and compound Int4 was replaced with compound Int26, to obtain the title compound (55 mg, yield: 60.2%).

[1050] MS m / z(ESI): 2648.2 [M+H] + .

[1051] Preparation of intermediates Example 86: Preparation of compound Int86

[1052] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int82, and compound Int4 was replaced with compound Int24, to obtain the title compound (80 mg, yield: 44.9%).

[1053] MS m / z(ESI): 2988.3 [M+H] + .

[1054] Preparation of intermediates Example 87: Preparation of compound Int87

[1055] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int83, and compound Int4 was replaced with compound Int24, to obtain the title compound (66 mg, yield: 58.7%).

[1056] MS m / z (ESI): 2575.9 [M+H] + .

[1057] Example 88: Preparation of compound Int88

[1058] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int79, and compound Int4 was replaced with compound Int24, to obtain the title compound (50 mg, yield: 56.0%).

[1059] MS m / z(ESI): 2920.3 [M+H] + .

[1060] Example 89: Preparation of compound Int89

[1061] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int79, and compound Int4 was replaced with compound Int26, to obtain the title compound (45 mg, yield: 44.4%).

[1062] MS m / z(ESI): 2992.5 [M+H] + .

[1063] Preparation of intermediates Example 90: Preparation of compound Int90

[1064] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int80, and compound Int4 was replaced with compound Int26, to obtain the title compound (60 mg, yield: 58.1%).

[1065] MS m / z(ESI): 3404.9 [M+H] + .

[1066] Example 91: Preparation of intermediate 91

[1067] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int80, and compound Int4 was replaced with compound Int24, to obtain the title compound (55 mg, yield: 56.0%).

[1068] MS m / z(ESI): 3332.6 [M+H] + .

[1069] Example 92: Preparation of intermediate: Int92

[1070] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int54, and compound Int4 was replaced with compound Int26, to obtain the title compound (60 mg, yield: 58.3%).

[1071] MS m / z(ESI): 2824.4 [M+H] + .

[1072] Example 93: Preparation of intermediate 93: Preparation of compound Int93

[1073] Using the synthetic route in Intermediate Preparation Example 30, the starting material Int29 in the first step was replaced with compound Int54, and compound Int4 was replaced with compound Int24, to obtain the title compound (60 mg, yield: 58.3%).

[1074] MS m / z(ESI): 2752.2 [M+H] + .

[1075] Preparation of intermediates Example 94: Preparation of compound Int94

[1076] Using the synthetic route in Example 1 of intermediate preparation, the first-step reaction starting material tris(hydroxymethyl)aminomethane was replaced with meglumine to obtain the title compound (263 mg, yield: 83.2%).

[1077] MS m / z (ESI): 869.4 [M+H] + .

[1078] Preparation of intermediates Example 95: Preparation of compound Int95

[1079] Step 1: Preparation of compound Int95-1

[1080] Compounds Int5-3 (70 mg, 79.84 μmol), Int94 (73 mg, 79.84 μmol), and N,N-diisopropylethylamine (16 mg, 119.76 μmol) were dissolved in N,N-dimethylformamide (2 mL), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (42 mg, 103.79 μmol) was added. The mixture was stirred at room temperature for 3 hours. The reaction solution was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (120 mg, yield: 87.5%).

[1081] MS m / z (ESI): 1685.3 [M+H] + .

[1082] Step 2: Preparation of compound Int95

[1083] Compound Int95-1 (120 mg, 69.84 μmol) and 1,3-dimethylbarbituric acid (33 mg, 209.52 μmol) were dissolved in N,N-dimethylformamide (2 mL). The mixture was purged three times with nitrogen at room temperature, and tetrakis(triphenylphosphine)palladium (17 mg, 13.97 μmol) was added. The mixture was stirred at room temperature for 8 hours. The reaction solution was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 1 / 1, v / v) to give the title compound (112 mg, yield: 92.68%).

[1084] MS m / z (ESI): 1644.3 [M+H] + .

[1085] Preparation of intermediates Example 96: Preparation of compound Int96

[1086] Step 1: Preparation of compound Int96-1

[1087] Compounds Int95 (56 mg, 32.36 μmol), Int26 (45 mg, 32.36 μmol), and N,N-diisopropylethylamine (8 mg, 61.28 μmol) were dissolved in N,N-dimethylformamide (1 mL). Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (17 mg, 42.47 μmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature. The reaction solution was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% trifluoroacetic acid) = 53 / 47, v / v) to give the title compound (49 mg, yield: 53.1%).

[1088] MS m / z(ESI): 2821.3 [M+H] + .

[1089] Step 2: Preparation of compound Int96

[1090] Compound Int96-1 (49 mg, 16.50 μmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (2 mg, 27.05 μmol) was added at room temperature. The mixture was stirred for 1 hour at room temperature. The solution was concentrated under reduced pressure to give the title compound (45 mg, yield: 99.7%).

[1091] MS m / z(ESI): 2599.3 [M+H] + .

[1092] Preparation of intermediates Example 97: Preparation of compound Int97

[1093] Using the synthetic route in Intermediate Preparation Example 96, the starting material Int26 in the first step was replaced with compound Int24 to obtain the title compound (42 mg, yield: 99.4%).

[1094] MS m / z (ESI): 2527.5 [M+H] + .

[1095] Synthesis Examples

[1096] Example 1-1: Preparation of compound D-1

[1097] Compound Int15 (14 mg, 11.1 μmol) and 2,5-dioxopyrrolidone-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionate (14 mg, 11.1 μmol) were dissolved in DMF (2 mL), and N,N-diisopropylethylamine (2.9 mg, 22.2 μmol) was added. The reaction was carried out at 25 °C for 2 hours. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (3 mg, yield: 15.7%).

[1098] MS m / z (ESI): 1418.6 [M+H] + .

[1099] Examples 1-2: Preparation of compound D-6

[1100] Compound Int14 (25 mg, 13.3 μmol) and 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolyl) ester (4.86 mg, 13.3 μmol) were dissolved in DMF (2 mL), and N,N-diisopropylethylamine (3.4 mg, 26.6 μmol) was added. The reaction was carried out at 25 °C for 2 hours. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (5 mg, yield: 17.7%).

[1101] MS m / z(ESI): 2126.6 [M+H] + .

[1102] Examples 1-3: Preparation of compound D-3

[1103] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int19 to obtain the title compound (2.3 mg, yield: 12.5%).

[1104] MS m / z(ESI): 2234.1 [M+H] + .

[1105] Examples 1-4: Preparation of compound D-4

[1106] Step 1: Preparation of compound D-4-1

[1107] Compounds Int9-2 (80 mg, 143.7 μmol) and Int20 (85.6 mg, 147.7 μmol) were dissolved in DMF (3 mL), and N,N-diisopropylethylamine (37.1 mg, 287.4 μmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (82 mg, 215.6 μmol) were added. The mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6) to give the title compound (80 mg, yield: 49.0%).

[1108] MS m / z (ESI): 1134.5 [M+H] + .

[1109] Step 2: Preparation of compound D-4-2

[1110] Compound D-4-1 (50 mg, 41.9 μmol) and compound Int3 (27 mg, 41.9 μmol) were dissolved in DMSO (3 mL) and water (0.3 mL), followed by the addition of sodium ascorbate (12.7 mg, 62.8 μmol) and anhydrous copper sulfate (21.3 mg, 83.7 μmol). The mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 7 / 3) to give the title compound (50 mg, yield: 67%).

[1111] MS m / z (ESI): 1688.7 [M+H] + .

[1112] Step 3: Preparation of compound D-4

[1113] Compound D-4-2 (50 mg, 29 μmol) was dissolved in dichloromethane (3 mL), and then zinc bromide (95 mg, 422 μmol) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was purified by high performance liquid chromatography (condition 2) to give the title compound (15 mg, yield: 32%).

[1114] MS m / z (ESI): 1588.7 [M+H] + .

[1115] Examples 1-5: Preparation of compound D-7

[1116] Step 1: Preparation of compound D-7-1

[1117] Compounds Int17 (30 mg, 25 μmol) and Int2 (20 mg, 25 μmol) were dissolved in DMSO (3 mL) and water (0.3 mL). Sodium ascorbate (7.2 mg, 36 μmol) and anhydrous copper sulfate (12.2 mg, 48 μmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 7 / 3) to give the title compound (38 mg, yield: 82.1%).

[1118] MS m / z (ESI): 1839.8 [M+H] + .

[1119] Step 2: Preparation of compound D-7-2

[1120] Compound 7-1 (38 mg, 20.6 μmol) was dissolved in dichloromethane (2 mL), and then zinc bromide (66 mg, 295 μmol) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified by reverse-phase column chromatography (eluent: acetonitrile / water (0.05% formic acid) = 4 / 6) to give the title compound (16 mg, yield: 44.6%).

[1121] MS m / z (ESI): 1739.7 [M+H] + .

[1122] Step 3: Preparation of compound D-7

[1123] Compound 7-2 (14 mg, 7.9 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (8 mg, 15.8 μmol) were dissolved in DMF (2 mL), and N,N-diisopropylethylamine (3 mg, 23.7 μmol) was added. The reaction was carried out at 25 °C for 2 h. The reaction solution was purified by high performance liquid chromatography (condition 1) to give the title compound (5 mg, yield: 29.2%).

[1124] MS m / z(ESI): 2126.9 [M+H] + .

[1125] Examples 1-6: Preparation of compound D-5

[1126] Using the synthetic route in Examples 1-5, the reactant compound Int2 was replaced with compound Int3 to obtain the title compound (4 mg, yield: 32.4%).

[1127] MS m / z(ESI): 2032.9 [M+H] + .

[1128] Examples 1-7: Preparation of compound D-13

[1129] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int16 to obtain the title compound (7 mg, yield: 20.6%).

[1130] MS m / z(ESI): 2747.3 [M+H] + .

[1131] Examples 1-8: Preparation of compound D-14

[1132] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int18 to obtain the title compound (8.0 mg, yield: 24.8%).

[1133] MS m / z(ESI): 3973.8 [M+H] + .

[1134] Examples 1-9: Preparation of compound D-2

[1135] Using the synthetic route in Example 1-1, the reactant compound Int15 was replaced with compound Int6 to obtain the title compound (10 mg, yield: 36.2%).

[1136] MS m / z (ESI): 1512.6 [M+H] + .

[1137] Examples 1-10: Preparation of compound D-8

[1138] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int7 to obtain the title compound (45 mg, yield: 27.3%).

[1139] MS m / z(ESI): 2793.3 [M+H] + .

[1140] Examples 1-11: Preparation of compound D-9

[1141] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int12 to obtain the title compound (80 mg, yield: 82.7%).

[1142] MS m / z(ESI): 2068.9 [M+H] + .

[1143] Examples 1-12: Preparation of compound D-11

[1144] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int10 to obtain the title compound (11 mg, yield: 20.2%).

[1145] MS m / z (ESI): 3008.5 [M+H] + .

[1146] Examples 1-13: Preparation of compound D-10

[1147] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int11-2 to obtain the title compound (35 mg, yield: 55.8%).

[1148] MS m / z(ESI): 2721.3 [M / 2+H] + .

[1149] Examples 1-14: Preparation of compound D-12

[1150] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int11 to obtain the title compound (6 mg, yield: 12.5%).

[1151] MS m / z(ESI): 3901.8 [M+H] + .

[1152] Examples 1-15: Preparation of compound D-17

[1153] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int25 to obtain the title compound (13 mg, yield: 46.9%).

[1154] MS m / z (ESI): 2555.3 [M+H] + .

[1155] Examples 1-16: Preparation of compound D-23

[1156] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int30 to obtain the title compound (10.86 mg, yield: 28.3%).

[1157] MS m / z (ESI): 3580.6 [M+H] + .

[1158] Examples 1-17: Preparation of compound D-15

[1159] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int27 to obtain the title compound (50 mg, yield: 60.5%).

[1160] MS m / z(ESI): 2627.2 [M+H] + .

[1161] Examples 1-18: Preparation of compound D-18

[1162] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int31 to obtain the title compound (4.8 mg, yield: 14.6%).

[1163] MS m / z(ESI): 3652.7 [M+H] + .

[1164] Examples 1-19: Preparation of compound D-16

[1165] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int49 to obtain the title compound (23 mg, yield: 29.5%).

[1166] MS m / z(ESI): 2675.3 [M+H] + .

[1167] Examples 1-20: Preparation of compound D-20

[1168] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int40 to obtain the title compound (9.9 mg, yield: 75.2%).

[1169] MS m / z(ESI): 3198.5 [M+H] + .

[1170] Examples 1-21: Preparation of compound D-25

[1171] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int34 to obtain the title compound (20 mg, yield: 55.3%).

[1172] MS m / z(ESI): 2567.2 [M+H] + .

[1173] Examples 1-22: Preparation of compound D-27

[1174] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int43 to obtain the title compound (3.5 mg, yield: 16.9%).

[1175] MS m / z(ESI): 2886.3 [M+H] + .

[1176] Examples 1-23: Preparation of compound D-29

[1177] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int46 to obtain the title compound (3.1 mg, yield: 33.8%).

[1178] MS m / z(ESI): 2964.4 [M+H] + .

[1179] Examples 1-24: Preparation of compound D-35

[1180] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int39 to obtain the title compound (9 mg, yield: 32.7%).

[1181] MS m / z(ESI): 3022.4 [M+H] + .

[1182] Examples 1-25: Preparation of compound D-36

[1183] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int33 to obtain the title compound (16 mg, yield: 46.3%).

[1184] MS m / z(ESI): 2143.4 [M+H] + .

[1185] Examples 1-26: Preparation of compound D-39

[1186] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int42 to obtain the title compound (25 mg, yield: 38.9%).

[1187] MS m / z (ESI): 3157.6 [M+H] + .

[1188] Examples 1-27: Preparation of compound D-40

[1189] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int44 to obtain the title compound (15 mg, yield: 29.7%).

[1190] MS m / z(ESI): 2640.4 [M+H] + .

[1191] Examples 1-28: Preparation of compound D-44

[1192] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int51 to obtain the title compound (11 mg, yield: 43.8%).

[1193] MS m / z (ESI): 3487.2 [M+H] + .

[1194] Examples 1-29: Preparation of compound D-45

[1195] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int50 to obtain the title compound (19 mg, yield: 31.5%).

[1196] MS m / z (ESI): 3535.1 [M+H] + .

[1197] Examples 1-30: Preparation of compound D-43

[1198] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int56 to obtain the title compound (40 mg, yield: 64.1%).

[1199] MS m / z (ESI): 3414.8 [M+H] + .

[1200] Examples 1-31: Preparation of compound D-46

[1201] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int57 to obtain the title compound (3 mg, yield: 44.3%).

[1202] MS m / z(ESI): 3679.2 [M+H] + .

[1203] Examples 1-32: Preparation of compound D-42

[1204] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int58 to obtain the title compound (8 mg, yield: 28.0%).

[1205] MS m / z(ESI): 3168.7 [M+H] + .

[1206] Examples 1-33: Preparation of compound D-49

[1207] Using the synthetic route in Example 1-1, the reactant compound Int15 was replaced with compound Int11-2 to obtain the title compound (3 mg, yield: 26.8%).

[1208] MS m / z(ESI): 2622.1 [M+H] + .

[1209] Examples 1-34: Preparation of compound D-48

[1210] Using the synthetic route in Example 1-1, the reactant compound Int15 was replaced with compound Int11-2, and the reactant 2,5-dioxopyrrolidone-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionate was replaced with 6-(maleimide)hexanoic acid succinimide ester, to obtain the title compound (5 mg, yield: 44.1%).

[1211] MS m / z(ESI): 2664.1 [M+H] + .

[1212] Examples 1-35: Preparation of compound D-41

[1213] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int61 to obtain the title compound (13 mg, yield: 23.9%).

[1214] MS m / z (ESI): 3082.4 [M+H] + .

[1215] Examples 1-36: Preparation of compound D-62

[1216] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int62 to obtain the title compound (14 mg, yield: 41.4%).

[1217] MS m / z (ESI): 3105.4 [M+H] + .

[1218] Examples 1-37: Preparation of compound D-60

[1219] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int59 to obtain the title compound (28 mg, yield: 55.5%).

[1220] MS m / z(ESI): 2682.1 [M+H] + .

[1221] Examples 1-38: Preparation of compound D-52

[1222] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int11-2, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (5 mg, yield: 19.9%).

[1223] MS m / z(ESI): 2968.4 [M+H] + .

[1224] Examples 1-39: Preparation of compound D-47

[1225] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int65 to obtain the title compound (25 mg, yield: 53.2%).

[1226] MS m / z (ESI): 3550.1 [M+H] + .

[1227] Examples 1-40: Preparation of compound D-53

[1228] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int25, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (28 mg, yield: 48.4%).

[1229] MS m / z(ESI): 2802.2 [M+H] + .

[1230] Examples 1-41: Preparation of compound D-30

[1231] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int69 to obtain the title compound (26 mg, yield: 78.6%).

[1232] MS m / z (ESI): 3504.9 [M+H] + .

[1233] Examples 1-42: Preparation of compound D-64

[1234] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int70 to obtain the title compound (5.6 mg, yield: 19.3%).

[1235] MS m / z(ESI): 3724.3 [M+H] + .

[1236] Examples 1-43: Preparation of compound D-61

[1237] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int71 to obtain the title compound (77 mg, yield: 69.2%).

[1238] MS m / z(ESI): 2466.1 [M+H] + .

[1239] Examples 1-44: Preparation of compound D-63

[1240] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int72 to obtain the title compound (15 mg, yield: 68.5%).

[1241] MS m / z(ESI): 2889.4 [M+H] + .

[1242] Examples 1-45: Preparation of compound D-55

[1243] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int25, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int73, to obtain the title compound (5.1 mg, yield: 17.1%).

[1244] MS m / z(ESI): 2678.4 [M+H] + .

[1245] Examples 1-46: Preparation of compound D-58

[1246] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int25, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int74, to obtain the title compound (5 mg, yield: 21.1%).

[1247] MS m / z(ESI): 2678.2 [M+H] + .

[1248] Examples 1-47: Preparation of compound D-59

[1249] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int75 to obtain the title compound (19 mg, yield: 41.9%).

[1250] MS m / z(ESI): 2463.0 [M+H] + .

[1251] Examples 1-48: Preparation of compound D-28

[1252] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int77 to obtain the title compound (4 mg, yield: 12.2%).

[1253] MS m / z(ESI): 2979.4 [M+H] + .

[1254] Examples 1-49: Preparation of compound D-73

[1255] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int84, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (38 mg, yield: 69.0%).

[1256] MS m / z(ESI): 3558.2 [M+H] + .

[1257] Examples 1-50: Preparation of compound D-74

[1258] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int51, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (28 mg, yield: 41.9%).

[1259] MS m / z(ESI): 3734.3 [M+H] + .

[1260] Examples 1-51: Preparation of compound D-71

[1261] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int85, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (15 mg, yield: 27.1%).

[1262] MS m / z (ESI): 3145.7 [M+H] + .

[1263] Examples 1-52: Preparation of compound D-79

[1264] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int92, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (15 mg, yield: 27.1%).

[1265] MS m / z(ESI): 3321.9 [M+H] + .

[1266] Examples 1-53: Preparation of compound D-77

[1267] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int86, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (18 mg, yield: 18.5%).

[1268] MS m / z (ESI): 3485.9 [M+H] + .

[1269] Examples 1-54: Preparation of compound D-75

[1270] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int56, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (28 mg, yield: 41.9%).

[1271] MS m / z(ESI): 3662.1 [M+H] + .

[1272] Examples 1-55: Preparation of compound D-76

[1273] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int91, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (30 mg, yield: 40.1%).

[1274] MS m / z(ESI): 3830.3 [M+H] + .

[1275] Examples 1-56: Preparation of compound D-72

[1276] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int90, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (27 mg, yield: 39.2%).

[1277] MS m / z(ESI): 3902.5 [M+H] + .

[1278] Examples 1-57: Preparation of compound D-78

[1279] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int93, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (8 mg, yield: 33.4%).

[1280] MS m / z(ESI): 3249.7 [M+H] + .

[1281] Examples 1-58: Preparation of compound D-70

[1282] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int89, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (6 mg, yield: 23.1%).

[1283] MS m / z(ESI): 3490.1 [M+H] + .

[1284] Examples 1-59: Preparation of compound D-68

[1285] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int88, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (9 mg, yield: 25.4%).

[1286] MS m / z (ESI): 3417.9 [M+H] + .

[1287] Examples 1-60: Preparation of compound D-69

[1288] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int87, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int63, to obtain the title compound (22 mg, yield: 37.1%).

[1289] MS m / z (ESI): 3073.5 [M+H] + .

[1290] Examples 1-61: Preparation of compound D-80

[1291] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int51, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int74, to obtain the title compound (13 mg, yield: 46.7%).

[1292] MS m / z(ESI): 3069.2 [M+H] + .

[1293] Examples 1-62: Preparation of compound D-81

[1294] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int56, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int74, to obtain the title compound (24 mg, yield: 33.2%).

[1295] MS m / z(ESI): 3536.9 [M+H] + .

[1296] Examples 1-63: Preparation of compound D-82

[1297] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int56, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int73, to obtain the title compound (20 mg, yield: 27.7%).

[1298] MS m / z (ESI): 3537.9 [M+H] + .

[1299] Examples 1-64: Preparation of compound D-83

[1300] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int51, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int73, to obtain the title compound (15.4 mg, yield: 47.8%).

[1301] MS m / z(ESI): 3610.2 [M+H] + .

[1302] Examples 1-65: Preparation of compound D-84

[1303] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int96 to obtain the title compound (16.8 mg, yield: 35.1%).

[1304] MS m / z(ESI): 2849.3 [M+H] + .

[1305] Examples 1-66: Preparation of compound D-85

[1306] Using the synthetic route in Examples 1-2, the reactant compound Int14 was replaced with compound Int97, and the reactant 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolidinyl) ester was replaced with compound Int74, to obtain the title compound (16.8 mg, yield: 34.4%).

[1307] MS m / z(ESI): 2899.3 [M+H] + .

[1308] II. Preparation of Degrading Agent Antibody Conjugate (DAC)

[1309] Construction and expression of recombinant anti-EGFR monoclonal antibodies

[1310] The variable region sequences of the anti-EGFR monoclonal antibodies Zalutumumab, Necitumumab, and Panitumumab were obtained from the IMGT website. The INN number of Zalutumumab is 8605, the INN number of Necitumumab is 9083, and the INN number of Panitumumab is 8499. The variable regions of the zaltumumab light chain (SEQ ID NO: 1), necitumumab light chain (SEQ ID NO: 22), and panitumumab light chain (SEQ ID NO: 64) were fused with the constant region of the human kappa light chain (SEQ ID NO: 18) to obtain the zaltumumab-Fc mutant light chain, necitumumab-Fc mutant light chain, and panitumumab-Fc mutant light chain, respectively. The variable regions of the zaltumumab heavy chain (SEQ ID NO: 2) and necitumumab heavy chain (SEQ ID NO: 23) were fused with the constant region of the mutant human IgG1 heavy chain (SEQ ID NO: 19) to obtain the zaltumumab-Fc mutant heavy chain and necitumumab-Fc mutant heavy chain, respectively. The variable region of the panitumumab heavy chain (SEQ ID NO: 65) was fused with the constant region 2 of the mutant human IgG1 heavy chain (SEQ ID NO: 80) to obtain the panitumumab-Fc mutant heavy chain. The above sequences were codon-optimized and DNA synthesized. The light and heavy chains of the antibodies were cloned into the pKLGS expression vector (containing two expression cassettes. The promoter of the first expression cassette is mouse CMV, and the polyA is sv40 polyA; the promoter of the second expression cassette is mouse CMV, and the polyA is TK polyA). Plasmids expressing the light and heavy chains of Zaltumumab-Fc mutant, Necitumumab-Fc mutant, and Panitumumab-Fc mutant were transfected into CHO-K1 cells. After expression for a period of time, the supernatant was harvested and purified using Protein A (MabSelect SuRe, GE) to obtain the antibody proteins of Zaltumumab-Fc mutant, Necitumumab-Fc mutant, and Panitumumab-Fc mutant. Cetuximab is cetuximab manufactured by Kelun Biotech Co., Ltd., under the trade name Datailai.

[1311] Example 2-1: Preparation of Cetuximab-D-5

[1312] Adjust the pH of the Cetuximab stock solution to 7.69 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Cetuximab stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-6 (equivalent to 12 times the molar amount of Cetuximab) to the reduced Cetuximab buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-5 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Cetuximab-D-5 histidine hydrochloride buffer (5 mg / mL, 1 mL), and store at -20°C.

[1313] Example 2-2: Preparation of Cetuximab-D-6

[1314] Adjust the pH of the Cetuximab stock solution to 7.63 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Cetuximab stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-2 (equivalent to 12 times the molar amount of Cetuximab) to the reduced Cetuximab buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-25 / NAP-5 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Cetuximab-D-6 in histidine hydrochloride buffer (1.3 mg / mL, 3.5 mL), and store at -20°C.

[1315] Examples 2-3: Preparation of Cetuximab-D-7

[1316] Adjust the pH of the Cetuximab stock solution to 7.63 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Cetuximab stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-5 (equivalent to 12 times the amount of Cetuximab) to the reduced Cetuximab buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-5 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Cetuximab-D-7 in histidine hydrochloride buffer (0.3 mg / mL, 1 mL), and store at -20°C.

[1317] Examples 2-4: Preparation of Cetuximab-D-8

[1318] Adjust the pH of the Cetuximab stock solution to 7.63 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Cetuximab stock solution. After shaking and reacting at room temperature for 2 hours, add 15 mM of the DMSO solution of the target compound from Examples 1-10 (equivalent to 13.5 times the molar amount of Cetuximab) to the reduced Cetuximab buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-25 / NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Cetuximab-D-8 in histidine hydrochloride buffer (2.2 mg / mL, 8.5 mL), and store at -20°C.

[1319] Examples 2-5: Preparation of Zalutumumab-D-8

[1320] Adjust the pH of the Zalutumumab-Fc mutant stock solution (including the heavy chain of the sequence shown in SEQ ID NO: 21 and the light chain of the sequence shown in SEQ ID NO: 20) to 7.56 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add 10 mM of DMSO solution of the target compound from Examples 1-10 (equivalent to 11 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-8 (3.0 mg / mL, 1.25 mL), and store at -20°C.

[1321] Examples 2-6: Preparation of Necitumumab-D-8

[1322] Adjust the pH of the Necitumumab-Fc mutant stock solution (including the heavy chain of the sequence shown in SEQ ID NO: 40 and the light chain of the sequence shown in SEQ ID NO: 39) to 6.77 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Necitumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add 10 mM of DMSO solution of the target compound from Examples 1-10 (equivalent to 10 times the amount of Necitumumab-Fc mutant) to the reduced Necitumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Necitumumab-D-8 in histidine hydrochloride buffer (3.1 mg / mL, 4.5 mL), and store at -20°C.

[1323] Examples 2-7: Preparation of Cetuximab-D-9

[1324] Adjust the pH of the Cetuximab stock solution to 7.56 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Cetuximab stock solution. After shaking and reacting at room temperature for 2 hours, add 15 mM of the DMSO solution of the target compound from Examples 1-11 (equivalent to 13.5 times the molar amount of Cetuximab) to the reduced Cetuximab buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Cetuximab-D-9 in histidine hydrochloride buffer (1.3 mg / mL, 12 mL), and store at -20°C.

[1325] Examples 2-8: Preparation of Zalutumumab-D-9

[1326] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.56 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-11 to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-9 (2.8 mg / mL, 1.25 mL), and store at -20°C.

[1327] Examples 2-9: Preparation of Cetuximab-D-10

[1328] Adjust the pH of the Cetuximab stock solution to 7.56 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Cetuximab stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-13 (equivalent to 10 times the amount of Cetuximab) to the reduced Cetuximab buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Cetuximab-D-10 in histidine hydrochloride buffer (1.3 mg / mL, 1.25 mL), and store at -20°C.

[1329] Examples 2-10: Preparation of Zalutumumab-D-10

[1330] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 6.76 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-13 (equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-10 (3.0 mg / mL, 4.5 mL), and store at -20°C.

[1331] Example 2-11: Preparation of Necitumumab-D-10

[1332] Adjust the pH of the Necitumumab-Fc mutant stock solution to 6.87 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Necitumumab-Fc mutant stock solution. Shake well and react at room temperature for 2 hours. Add 10 mM of DMSO solution (equivalent to 10 times the molar amount of Necitumumab-Fc mutant) of the target compound from Examples 1-13 to the reduced Necitumumab-Fc mutant buffer solution. Shake well and react at room temperature for 2 hours. Purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Necitumumab-D-10 in histidine hydrochloride buffer (3.0 mg / mL, 4.5 mL), and store at -20°C.

[1333] Example 2-12: Preparation of Zalutumumab-D-11

[1334] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 6.83 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-12 to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-11 (3.3 mg / mL, 4.5 mL), and store at -20°C.

[1335] Examples 2-13: Preparation of Necitumumab-D-11

[1336] Adjust the pH of the Necitumumab-Fc mutant stock solution to 6.87 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Necitumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-12 (equivalent to 10 times the amount of Necitumumab-Fc mutant) to the reduced Necitumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain Necitumumab-D-11 in histidine hydrochloride buffer (3.2 mg / mL, 4.5 mL), and store at -20°C.

[1337] Examples 2-14: Preparation of Zalutumumab-D-12

[1338] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 6.83 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-14 to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-12 (3.1 mg / mL, 4.5 mL), and store at -20°C.

[1339] Examples 2-15: Preparation of Zalutumumab-D-13

[1340] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 6.83 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-7 to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-13 (3.1 mg / mL, 4.5 mL), and store at -20°C.

[1341] Examples 2-16: Preparation of Zalutumumab-D-14

[1342] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 6.89 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of the DMSO solution of the target compound from Examples 1-8 to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, purify using a NAP-10 gel column and wash with histidine hydrochloride buffer solution at pH 6.0-6.1. Collect the filtrate to obtain histidine hydrochloride buffer solution of Zalutumumab-D-14 (3.4 mg / mL, 4.5 mL), and store at -20°C.

[1343] Example 2-17: Preparation of Zalutumumab-D-17

[1344] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.67 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-15 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The solution was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-23 (3.0 mg / mL, 1.5 mL), which was stored at -20 °C.

[1345] Example 2-18: Preparation of Zalutumumab-D-23

[1346] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.67 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-16 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The solution was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-23 (3.0 mg / mL, 1.5 mL), which was stored at -20 °C.

[1347] Example 2-19: Preparation of Zalutumumab-D-15

[1348] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.67 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-17 (10 mM, equivalent to 10 times the molar amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The sample was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-15 (3.0 mg / mL, 1.5 mL), which was stored at -20°C.

[1349] Examples 2-20: Preparation of Zalutumumab-D-18

[1350] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.67 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-18 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The solution was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-18 (3.0 mg / mL, 1.5 mL), which was stored at -20 °C.

[1351] Example 2-21: Preparation of Zalutumumab-D-29

[1352] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.67 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-23 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The sample was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-29 (3.0 mg / mL, 1.5 mL), which was stored at -20 °C.

[1353] Example 2-22: Preparation of Zalutumumab-D-39

[1354] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.51 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-26 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The sample was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-39 (2.8 mg / mL, 1.5 mL), which was stored at -20 °C.

[1355] Examples 2-23: Preparation of Zalutumumab-D-20

[1356] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.51 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-20 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The solution was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-20 (2.8 mg / mL, 1.5 mL), which was stored at -20 °C.

[1357] Examples 2-24: Preparation of Zalutumumab-D-27

[1358] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.51 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA and TCEP (5.5 eq, 10 mM) to the Zalutumumab-Fc mutant stock solution. After shaking and reacting at room temperature for 2 hours, add 10 mM of DMSO solution of the target compound from Examples 1-22 (equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP TM The sample was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-27 (2.9 mg / mL, 1.5 mL), which was stored at -20 °C.

[1359] Examples 2-25: Preparation of Zalutumumab-D-45

[1360] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.51 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-29 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The sample was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-45 (3.1 mg / mL, 1.5 mL), which was stored at -20 °C.

[1361] Examples 2-26: Preparation of Zalutumumab-D-16

[1362] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.57 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-19 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The solution was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-16 (2.3 mg / mL, 1.5 mL), which was stored at -20 °C.

[1363] Example 2-27: Preparation of Zalutumumab-D-36

[1364] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.57 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-25 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The solution was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-36 (2.1 mg / mL, 1.5 mL), which was stored at -20 °C.

[1365] Examples 2-28: Preparation of Zalutumumab-D-25

[1366] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.57 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumumab-Fc mutant stock solution, along with TCEP (5.5 eq, 10 mM). After shaking and reacting at room temperature for 2 hours, add a DMSO solution of the target compound from Examples 1-21 (10 mM, equivalent to 10 times the amount of Zalutumumab-Fc mutant) to the reduced Zalutumumab-Fc mutant buffer solution. After shaking and reacting at room temperature for 2 hours, use NAP... TM The sample was purified by gel column chromatography (Cytiva) and washed with histidine hydrochloride buffer solution at pH 6.0-6.1. The filtrate was collected to obtain histidine hydrochloride buffer solution of Zalutumumab-D-25 (2.1 mg / mL, 1.5 mL), which was stored at -20 °C.

[1367] Example 2-29: Preparation of Zalutumumab-D-44

[1368] Adjust the pH of the Zalutumumab-Fc mutant stock solution to 7.57 using 1M disodium hydrogen phosphate solution. Add 0.1M EDTA to the Zalutumum...

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