Site-selective conjugate of antibody and functional substance, or its salt, and antibody derivative and compound or their salt used in the production thereof

Abstract

The present invention provides: a conjugate of a functional substance and an antibody, in which the binding ratio between the antibody and the functional substance is controlled to within a specific range, said conjugate being exceptional with respect to a desired property; or a salt of said conjugate. More specifically, the present invention provides: a regioselective conjugate of a functional substance and antibody or a salt of said conjugate, the conjugate including a structural unit represented by formula (I) (wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and is regioselectively bound to L1 adjacent to the Ig via an amino group in the side chain of a lysine residue in the two heavy chains; HG represents a hydrophilic group, etc.; RA represents a side chain of a valine residue; RB represents a side chain of a citrulline residue, etc.; ring A represents a divalent aromatic ring group optionally having a substituent; R1 and R2 independently represent a hydrogen atom or a monovalent group; L1 and L2 independently represent a divalent group; D represents a functional substance; and r is 1.5 to 2.5); and a substance relating to the conjugate and the salt.

Description

[Technical field]

[0001] The present invention relates to a site-selective conjugate of an antibody and a functional substance, or a salt thereof, as well as an antibody derivative and a compound or a salt thereof used in the production thereof. [Background technology]

[0002] In recent years, research and development of antibody drug conjugates (ADCs) has been actively conducted. As the name suggests, ADCs are drugs in which a drug (e.g., an anticancer drug) is conjugated to an antibody, and have direct cytocidal activity against cancer cells, etc. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)), jointly developed by Immunogene and Roche.

[0003] ADCs are produced by binding functional groups in the side chains of specific amino acid residues present in antibodies to drugs. An example of such a functional group used in producing ADCs is the amino group in the side chain of a lysine residue present in an antibody. Several techniques have been reported for site-selectively modifying lysine groups in antibodies (e.g., lysine residues at positions 246 / 248, 288 / 290, or 317) (e.g., Patent Documents 1 to 4).

[0004] In ADC, the antibody and the drug are linked via a linker. There are various types of linkers in ADC. For example, in ADC used as an anticancer drug, there is a linker containing a dipeptide (Val-Cit:VC structure) consisting of valine-citrulline as a linker that is stable in human plasma and has a structure that can be cleaved by a specific enzyme to release the drug in cancer cells. Although such a linker containing a dipeptide is stable in human plasma as shown in (A) below, as shown in (B) below, the VC structure is recognized by cathepsin B in the lysosome in human cancer cells, and the amide bond present on the carboxy terminal side of citrulline is cleaved. Therefore, an ADC having a linker containing such a dipeptide can release a drug in human cancer cells to exert its medicinal effect.

[0005] [ka]

[0006] [ka]

[0007] However, ADCs having a linker containing a dipeptide as described above are unstable in mouse plasma (Non-Patent Documents 1 and 2). This is because mouse plasma contains Ces1c, a carboxylase that recognizes a VC structure and cleaves the amide bond present at the carboxy-terminal side of citrulline, and the linker containing the dipeptide as described above is cleaved in plasma by Ces1c. Therefore, the pharmacokinetics of ADCs having a linker containing a dipeptide as described above is significantly different between mice and humans. Therefore, there is a problem that it is difficult to evaluate the drug efficacy in humans in mice.

[0008] [ka]

[0009] As described above, in order to improve the instability of ADCs having the structure "antibody-spacer-VC structure-spacer-drug" in mouse plasma, attempts have been made to stabilize the ADC by modifying the linker (i.e., spacer-VC structure-spacer), and from this perspective, ADCs in which an antibody and a drug or a mimic thereof are linked via the above linker have been reported. For example, the following has been reported as an ADC that contains the above linker not in the main chain linking the antibody and the drug or a mimic thereof, but in a side chain of the main chain (Patent Document 5).

[0010] [ka] Ab: Antibody Cbz: benzyloxycarbonyl Val: valine residue Cit: Citrulline residue

[0011] Incidentally, Non-Patent Document 3 describes that the higher the hydrophobicity of an ADC, the faster its plasma clearance, and that the hydrophobicity of an ADC can be evaluated by HIC (Hydrophobic Interaction Chromatography)-HPLC. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2018 / 199337 [Patent Document 2] International Publication No. 2019 / 240288 [Patent Document 3] International Publication No. 2019 / 240287 [Patent Document 4] International Publication No. 2020 / 090979 [Patent Document 5] International Publication No. 2015 / 038426 [Non-patent literature]

[0013] [Non-Patent Document 1] Dorywalska et al.,Bioconjugate Chem.,2015,26(4),650-659 [Non-Patent Document 2] Dorywalska et al.,Mol Cancer Ther.,2016,15(5),958-70 [Non-Patent Document 3] Lyon et al.,Nat Biotechnol.,2015,33(7),733-5 Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a conjugate of an antibody and a functional substance, or a salt thereof, which has excellent desired properties while controlling the binding ratio between the antibody and the functional substance within a specific range. [Means for solving the problem]

[0015] As a result of intensive research, the present inventors have found that a regioselective conjugate that contains a linker of a specific structure in the side chain of the main chain linking the antibody and the drug (or drug mimic) and has an average ratio of the bonds between the immunoglobulin unit and the functional substance (functional substance / immunoglobulin unit) in a desired range (1.5 to 2.5) has excellent properties. For example, such a regioselective conjugate or a salt thereof has excellent clearance (long residence time in the body), low aggregation rate (high monomer ratio), high cleavability by cathepsin B (high release ability of the functional substance in human cells), and high stability in mouse plasma. Such a regioselective conjugate or a salt thereof has a hydrophilic group at or near the end of the side chain that is easily exposed on the surface of the conjugate molecule, and therefore can efficiently improve the hydrophilicity of the entire molecule and can also exhibit the above-mentioned excellent properties.

[0016] The present inventors have also succeeded in developing antibody derivatives and compounds useful for preparing such regioselective conjugates. The regioselective conjugates, antibody derivatives, and compounds of the present invention represented by structures such as formulas (I) to (VII) have the technical feature of sharing partial structural units excluding X and Y among the structural units represented by formula (V). The present inventors have succeeded in developing a series of inventions having such technical features, and have completed the present invention. The prior art has neither described nor suggested the chemical structure of the regioselective conjugate of the present invention, nor the relationship between such chemical structure and the excellent properties described above. The prior art has also neither described nor suggested the antibody derivatives and compounds of the present invention that can be used for preparing such regioselective conjugates.

[0017] That is, the present invention is as follows.

[0018] In a first embodiment, the present invention provides a compound represented by the following formula (I): [ka] [During the ceremony, Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and is regioselectively linked to L1 adjacent to Ig via amino groups in the side chains of lysine residues in the two heavy chains; HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; R1 and R2 each independently represent a hydrogen atom or a monovalent group. L1 and L2 each independently represent a divalent group. D represents a functional substance, The average ratio r of the bonds per two heavy chains is 1.5 to 2.5. The present invention provides a site-selective conjugate of an antibody and a functional substance, comprising a structural unit represented by the following formula:

[0019] In certain embodiments, the structural unit represented by formula (I) has the following formula (I'): [ka] [During the ceremony, Ig, R A , R B , ring A, R1, R2, L1, L2, D, and r are the same as those shown in formula (I), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and the structural unit is represented by the following formula (1):

[0020] In a second embodiment, the present invention provides a compound represented by the following formula (II): [ka] [During the ceremony, Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and is regioselectively linked to L1 adjacent to Ig via amino groups in the side chains of lysine residues in the two heavy chains; HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; R1 and R2 each independently represent a hydrogen atom or a monovalent group. L1 and L2 each independently represent a divalent group. B2 represents a bioorthogonal functional group; The average ratio r of the bonds per two heavy chains is 1.5 to 2.5. The present invention provides an antibody derivative or a salt thereof which regioselectively has a bioorthogonal functional group, the antibody derivative comprising a structural unit represented by the following formula:

[0021] In certain embodiments, the structural unit represented by formula (II) is represented by the following formula (II'): [ka] [During the ceremony, Ig, R A , R B , ring A, R1, R2, L1, L2, B2, and r are the same as those shown in formula (II), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and the structural unit is represented by the following formula (1):

[0022] In a third embodiment, the present invention provides a compound represented by the following formula (III): [ka] [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; R1 and R2 each independently represent a hydrogen atom or a monovalent group. L1 and L2 each independently represent a divalent group. B1 represents a bioorthogonal functional group; and D represents a functional substance.], or a salt thereof, is provided.

[0023] In certain embodiments, the compound represented by formula (III) has the following formula (III'): [ka] [During the ceremony, R A , R B , ring A, R1, R2, L1, L2, B1, and D are the same as those shown in formula (III), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and wherein the compound is contained in one or more sites selected from the group consisting of:

[0024] In a fourth embodiment, the present invention provides an antibody derivatization reagent comprising a compound according to the third embodiment above, or a salt thereof.

[0025] In a fifth embodiment, the present invention relates to a compound represented by the following formula (IV):

[0026] [ka] [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; R1 and R2 each independently represent a hydrogen atom or a monovalent group. L1 and L2 each independently represent a divalent group. B1 represents a first bioorthogonal functional group; and B2 represents a second bioorthogonal functional group.], or a salt thereof.

[0027] In certain embodiments, the compound represented by formula (IV) has the following formula (IV'): [ka] [During the ceremony, R A , R B , ring A, R1, R2, L1, L2, B1, and B2 are the same as those shown in formula (IV), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and wherein the compound is contained in one or more sites selected from the group consisting of:

[0028] In a sixth embodiment, the present invention provides a derivatization reagent for an antibody or a functional substance, comprising a compound according to the above fifth embodiment or a salt thereof.

[0029] In a seventh embodiment, the present invention relates to a compound of formula (V): [ka] [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; X and Y each independently represent a monovalent group; or a salt thereof.

[0030] In certain embodiments, the compound represented by formula (V) is represented by formula (V'): [ka] [During the ceremony, R A , R B , ring A, X, and Y are the same as those shown in formula (V), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and wherein the compound is contained in one or more sites selected from the group consisting of:

[0031] In an eighth embodiment, the present invention relates to a compound of formula (VI): [ka] [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, RA indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; X represents a monovalent group; R2 represents a hydrogen atom or a monovalent group; L2 represents a divalent group, B2 represents a bioorthogonal functional group.] or a salt thereof.

[0032] In certain embodiments, the compound of formula (VI) has the following formula (VI'): [ka] [During the ceremony, R A , R B , ring A, and X are the same as those shown in formula (VI), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and R2 represents a hydrogen atom or a monovalent group; L2 represents a divalent group, B2 represents a bioorthogonal functional group.

[0033] In a ninth embodiment, the present invention relates to a compound represented by the following formula (VII): [ka] [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent; Y represents a monovalent group; R1 represents a hydrogen atom or a monovalent group; L1 represents a divalent group, B1 represents a bioorthogonal functional group.] or a salt thereof.

[0034] In certain embodiments, the compound represented by formula (VII) has the following formula (VII'): [ka] [During the ceremony, R A , R B , ring A, and Y are the same as those represented by formula (VII), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L HG , R HG1 , and R HG2 and R1 represents a hydrogen atom or a monovalent group; L1 represents a divalent group, B1 represents a bioorthogonal functional group.

[0035] In a preferred embodiment, the immunoglobulin unit may be a human immunoglobulin unit.

[0036] In a more preferred embodiment, the human immunoglobulin unit may be a human IgG antibody.

[0037] In a preferred embodiment, the lysine residue may be at position 246 / 248, 288 / 290, or 317 according to Eu numbering.

[0038] In a preferred embodiment, regioselective conjugation may be achieved through an amide bond by coupling the amino group in the side chain of a lysine residue and the carbonyl group in L1.

[0039] In a preferred embodiment, the above r may be 1.9 to 2.1.

[0040] In a preferred embodiment, the hydrophilic group may be one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety.

[0041] In a preferred embodiment, ring A may be a phenylene group which may have a substituent.

[0042] In a preferred embodiment, the functional substance may be a drug, a labeling substance, or a stabilizing agent.

[0043] In a preferred embodiment, the regioselective conjugate or antibody derivative may exhibit an aggregation rate of 2.6% or less when analyzed by size exclusion chromatography.

[0044] In a preferred embodiment, a divalent group (-L HG -) is represented by the following formula (a): -(C(R HG )2) n1 -(C=O) n2 -(NR HG ) n3 -(C(R HG )2) n4 - (a) [During the ceremony, Multiple R HG each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, n1 is an integer from 0 to 3, n2 is an integer of 0 or 1, n3 is an integer of 0 or 1, and n4 is an integer of 0 to 3.].

[0045] In a more preferred embodiment, the divalent group represented by formula (a) is represented by the following formula (a1), (a2), or (a3): (a1) -(C(R HG )2)-; (a2) -(C(R HG )2)-(C=O)-(NR HG )-(C(R HG )2)-;or (a3) -(C=O)-(C(R HG )2)2-; [During the ceremony, Multiple R HG each independently represents a hydrogen atom, a hydrophilic group, or an alkyl group having 1 to 6 carbon atoms and containing a hydrophilic group.

[0046] In a preferred embodiment, the hydrophilic groups may each independently be a carboxylic acid group, a sulfonic acid group, or a hydroxyl group.

[0047] In a more preferred embodiment, the hydrophilic group may be a carboxylic acid group.

[0048] In preferred embodiments, the bioorthogonal functional group may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. Effect of the Invention

[0049] The site-selective conjugate or a salt thereof of the present invention may have excellent properties such as a long residence time in the body, a high monomer ratio (low aggregation rate), a high release ability of a functional substance in human cells, and high stability in mouse plasma. The antibody derivatives and compounds of the present invention or their salts, and reagents are useful, for example, as synthetic intermediates in the preparation of the above-described regioselective conjugates. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 is a diagram showing the interrelationships between the regioselective conjugate of the present invention represented by formula (I), the antibody derivative of the present invention represented by formula (II), and the compounds of the present invention represented by formulas (III) to (VII). These substances share partial structural units excluding X and Y among the structural units represented by formula (V). These substances can be synthesized according to the scheme shown in FIG. 1. Thus, the present invention provides a series of inventions in the relationship between synthetic intermediates and final synthetic products. [Diagram 2] FIG. 2 shows a synthesis outline of the regioselective conjugate of the present invention represented by formula (I), the antibody derivative of the present invention represented by formula (II), and the compounds of the present invention represented by formulas (III) and (IV). [Diagram 3] 3 is a diagram showing an example of the synthesis outline of the compounds of the present invention represented by formulas (IV) to (VII). DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] 1. Definitions of General Terms In the present invention, the term "antibody" is as follows. The term "immunoglobulin unit" corresponds to a bivalent monomer unit that is a basic component of such an antibody, and is a unit containing two heavy chains and two light chains. Therefore, the definitions, examples, and preferred examples of the origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of lysine residue, and position selectivity of the immunoglobulin unit are the same as those of the antibody described below.

[0052] The origin of the antibody is not particularly limited, and may be derived from animals such as mammals and birds (e.g., chickens). Preferably, the immunoglobulin unit is derived from a mammal. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pet animals (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, more preferably humans.

[0053] The type of antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a specific glycosylation has been added (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylation consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, full-length antibodies or antibody fragments containing the variable region and the CH1 domain and CH2 domain can be used as monoclonal antibodies, with full-length antibodies being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0054] Any antigen can be used as the antigen of the antibody. For example, such antigens include proteins (including oligopeptides and polypeptides, and may be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low molecular weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0055] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of the disease target protein include the following:

[0056] (1) Cancer area PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1(CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfelin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Luis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 inhibitory protein, TAG-72 (CA72-4), CD70,

[0057] (2) Autoimmune diseases and inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP(Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT

[0058] (3) Neurological disorders CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0059] (4) Infectious disease Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0060] (5) Genetic and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0061] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0062] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0063] (8) Hematological diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0064] (9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

[0065] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab), , ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (if no IgG subtype is mentioned, it refers to IgG1).

[0066] The positions of amino acid residues in an antibody and the positions of the constant region of the heavy chain (e.g., CH2 domain) are based on EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, in the case of human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the CH2 region of human IgG, the lysine residue at position 248 corresponds to the 18th amino acid residue in the CH2 region of human IgG, the lysine residue at position 288 corresponds to the 58th amino acid residue in the CH2 region of human IgG, the lysine residue at position 290 corresponds to the 60th amino acid residue in the CH2 region of human IgG, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the CH2 region of human IgG. The designation 246 / 248 indicates that the lysine residue at position 246 or 248 is the target. The designation 288 / 290 indicates that the lysine residue at position 288 or 290 is the target.

[0067] According to the present invention, specific lysine residues (e.g., lysine residues at positions 246 / 248, 288 / 290, or 317) in the heavy chain of the immunoglobulin unit constituting the antibody can be regioselectively modified (see, e.g., WO 2018 / 199337, WO 2019 / 240288, WO 2019 / 240287, and WO 2020 / 090979). As used herein, "regioselective" or "regioselectivity" refers to a situation in which a specific structural unit capable of binding to a specific amino acid residue in an antibody is concentrated in a specific region in the antibody, even though a specific amino acid residue is not concentrated in a specific region in the antibody. Thus, expressions related to position selectivity, such as "having a position-selective property," "position-selective binding," and "binding with position selectivity," mean that the retention or binding rate of a given structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues that are the same type as the specific amino acid residues in the target region. Such position selectivity may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%.

[0068] In the present invention, as long as a specific lysine residue in the heavy chain of an antibody is regioselectively modified, a specific amino acid residue at another position may be further regioselectively modified.For example, a method for regioselectively modifying a specific amino acid residue at a predetermined position in an antibody is described in WO 2018 / 199337, WO 2019 / 240288, WO 2019 / 240287, and WO 2020 / 090979. Such specific amino acid residues may be amino acid residues having a side chain that is easily modified (e.g., an amino group, a carboxy group, an amide group, a hydroxy group, a thiol group) (e.g., a lysine residue, an aspartic acid residue, a glutamic acid residue, an asparagine residue, a glutamine residue, a threonine residue, a serine residue, a tyrosine residue, a cysteine ​​residue) and preferably a lysine residue having a side chain containing an amino group, a tyrosine residue, a serine residue, or a threonine residue having a side chain containing a hydroxy group, or a cysteine ​​residue having a side chain containing a thiol group, and more preferably a lysine residue (i.e., of the lysine residues at positions 246 / 248, 288 / 290, and 317, two lysine residues may be site-selectively doubly modified, and three lysine residues may be site-selectively triple modified).

[0069] (Halogen atom) Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0070] (Monovalent group) Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

[0071] The monovalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) substituents described below.

[0072] (Monovalent Hydrocarbon Radicals and Related Terms) Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0073] The monovalent chain-like hydrocarbon group means a hydrocarbon group that is composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain-like hydrocarbon group include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0074] The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms. When the alkyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0075] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. When the alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0076] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. When the alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0077] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0078] The monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it is not necessary to be composed only of alicyclic hydrocarbons, and it may contain a chain structure as a part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0079] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. When the cycloalkyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0080] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 to 6 carbon atoms. When the cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0081] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 to 6 carbon atoms. When the cycloalkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0082] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0083] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it is not necessary to be composed of only an aromatic ring, and it may contain a chain structure or an alicyclic hydrocarbon as a part thereof, and the aromatic ring may be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is even more preferable. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. As the aryl having 6 to 12 carbon atoms, for example, phenyl and naphthyl can be mentioned.

[0084] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0085] Among these, the monovalent hydrocarbon group is preferably an alkyl group, a cycloalkyl group, or an aryl group.

[0086] (Monovalent heterocyclic groups and related terms) The monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocyclic group, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom.

[0087] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0088] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0089] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0090] (Divalent group) The divalent group is a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR7-, -C(=O)-NR7-, -NR7-C(=O)-, -C(=S)-NR7-, -NR7-C(=S)-, -O-, -S-, -(O-R8) m - and - (S-R8) m1 - is one group selected from the group consisting of, or a group having a main chain structure containing two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2 or 3). R7 represents a hydrogen atom or a substituent described later. R8 represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. m1 represents an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, and particularly preferably an integer of 1 to 3.

[0091] The divalent straight chain hydrocarbon group is a straight chain alkylene, a straight chain alkenylene, or a straight chain alkynylene. The straight-chain alkylene is a straight-chain alkylene having 1 to 6 carbon atoms, and preferably a straight-chain alkylene having 1 to 4 carbon atoms. Examples of the straight-chain alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, and preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethylenylene, n-propenylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, and preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of the linear alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. The divalent linear hydrocarbon group is preferably a linear alkylene group.

[0092] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of the arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene group.

[0093] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent aromatic heterocyclic group include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferable. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. The divalent heterocyclic group is preferably a divalent aromatic heterocyclic group.

[0094] Preferably, the divalent group is alkylene, arylene, -C(=O)-, -NR7-, -C(=O)-NR7-, -NR7-C(=O)-, -O-, and -(O-R8). m -, or a divalent group having a main chain structure containing one group selected from the group consisting of Alkylene, arylene, -C(=O)-, -NR7-, -C(=O)-NR7-, -NR7-C(=O)-, -O-, and -(O-R8) m1 - is a divalent group having a main chain structure containing two or more groups selected from the group consisting of R7 is a hydrogen atom or an alkyl group; R8 is alkylene or arylene; m1 may be an integer from 1 to 5 (ie, 1, 2, 3, 4, or 5). The alkylene, arylene and alkyl are the same as those defined above.

[0095] The main chain structure in the divalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) substituents described below.

[0096] (substituent) Substituents include: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) aralkyl; (v)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or a monovalent hydrocarbon group; or (vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or a monovalent hydrocarbon group; (vii) Nitro, sulfate, sulfonate, cyano, and carboxyl groups.

[0097] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, and monovalent heterocyclic group in the above-mentioned substituents are the same as those described above.

[0098] The aralkyl refers to an arylalkyl. The definitions, examples and preferred examples of aryl and alkyl in the arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such an aralkyl include benzoyl, phenethyl, naphthylmethyl and naphthylethyl.

[0099] Preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; or (vii) The same groups as those enumerated in (vii) above.

[0100] More preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0101] Even more preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 6 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R care the same or different and each represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0102] Particularly preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 4 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0103] (hydrophilic group) The hydrophilic group is a group that can make the structural unit represented by formulae (I) to (VII) or their subordinate formulae more hydrophilic. By having a hydrophilic group at a predetermined site in the structural unit, the conjugate can be more stabilized in mouse plasma. Examples of such hydrophilic groups include a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety. The conjugate may contain one or more hydrophilic groups (e.g., 1, 2, 3, 4, or 5).

[0104] The polyethylene glycol (PEG) group is -(CH2-CH2-O-) k1- is a divalent group represented by the formula: When the conjugate has a polyethylene glycol group, the conjugate may have a monovalent group in which one bond of the polyethylene glycol group is bonded to a hydrogen atom or a monovalent group (e.g., a monovalent hydrocarbon group). k1 may be, for example, an integer of 3 or more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more. k1 may also be an integer of 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k1 may be an integer of 3 to 15, preferably an integer of 4 to 12, more preferably an integer of 5 to 10, and even more preferably an integer of 4 to 9.

[0105] The polysarcosine group is -(NCH3-CH2-CO-) k2 - is a divalent group represented by the formula: -. The polysarcosine group can be used as an alternative to PEG. k2 may be, for example, an integer of 3 or more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more. k2 may also be an integer of 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k2 may be an integer of 3 to 15, preferably an integer of 4 to 12, more preferably an integer of 5 to 10, and even more preferably an integer of 4 to 9.

[0106] The sugar moiety is a monosaccharide, an oligosaccharide (e.g., a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide), or a polysaccharide. The sugar moiety can include an aldose or a ketose, or a combination thereof. The sugar moiety can be a monosaccharide, such as ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, or fructose, or an amino sugar (e.g., glucosamine), or an oligosaccharide or polysaccharide containing such a monosaccharide.

[0107] In certain embodiments, the sugar moiety may be a low molecular weight hydrophilic group. A low molecular weight hydrophilic group refers to a hydrophilic group having a molecular weight of 1500 or less. The molecular weight of the low molecular weight hydrophilic group may be preferably 1200 or less, 1000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less. Examples of low molecular weight hydrophilic groups include carboxylic acid groups, sulfonic acid groups, and hydroxyl groups, as well as polyethylene glycol groups, polysarcosine groups, and sugar moieties (e.g., monosaccharides and oligosaccharides) that satisfy the above molecular weight.

[0108] (Bio-orthogonal functional groups) A bioorthogonal functional group refers to a group that does not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates) or reacts slowly with biological components, but selectively reacts with components other than biological components. Bioorthogonal functional groups are well known in the art (e.g., see Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

[0109] In the present invention, a bioorthogonal functional group for a protein is used as the bioorthogonal functional group. This is because the thiol group-introduced antibody to be derivatized by the reagent of the present invention is a protein. The bioorthogonal functional group for a protein is a group that does not react with the side chain of the 20 natural amino acid residues that constitute the protein, or reacts slowly with the side chain, but reacts with the functional group of interest. The 20 natural amino acids that constitute the protein are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Among these 20 naturally occurring amino acids, glycine, which has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, which have a hydrocarbon side chain (i.e., do not contain a heteroatom selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inactive to normal reactions. Therefore, the bioorthogonal functional group for proteins is a group that does not react or reacts slowly with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, in addition to the side chains of these amino acids that have side chains that are inactive to normal reactions, but reacts with the functional group of interest.

[0110] Examples of such bioorthogonal functional groups include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, any residue that has a vinylene (ethenylene) moiety, which is the smallest unit having a double bond between carbon atoms; the same applies below), alkyne residues (in other words, any residue that has an ethynylene moiety, which is the smallest unit having a triple bond between carbon atoms; the same applies below), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α position; the same applies below), isonitrile residues, sydnone residues, and selenium residues.

[0111] More specifically, the bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [ka] [Where: R 1a , single or multiple R 1b , and one or more R 1c are the same or different and are the above-mentioned substituents or electron-withdrawing groups, · is a bond.

[0112] Examples of the electron-withdrawing group include a halogen atom, an alkyl substituted with a halogen atom (e.g., trifluoromethyl), a boronic acid residue, mesyl, tosyl, triflate, nitro, cyano, a phenyl group, and a keto group (e.g., acyl), and preferred are a halogen atom, a boronic acid residue, mesyl, tosyl, and triflate.

[0113] In certain embodiments, the bioorthogonal functional group may be protected. The bioorthogonal functional group that may be protected refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. The unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above. The protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of the protecting group. The cleavage of the protecting group can be performed by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of an amide bond). Examples of such a specific treatment include (a) treatment with one or more substances selected from the group consisting of an acidic substance, a basic substance, a reducing agent, an oxidizing agent, and an enzyme, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) leaving the product when a cleavable linker containing a self-degrading cleavable portion is used. Such protecting groups and their cleavage conditions are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Jounal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society. 132, 17928 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)).

[0114] Protected bioorthogonal functional groups include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

[0115] More specifically, the protected bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Single or multiple R 2a are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents, · is a bond.

[0116] Preferably, the optionally protected bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0117] (Functional substance) The functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and examples thereof include a drug, a labeling substance, an affinity substance, a transport substance, and a stabilizer, but it may be preferably a drug, a labeling substance, an affinity substance, or a transport substance, or it may be a drug or a labeling substance. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked.

[0118] The drug may be a drug for any disease. Examples of such diseases include cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), genetic and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0119] More specifically, the drug may be an anticancer drug. Examples of the anticancer drug include chemotherapeutic agents, toxins, radioisotopes, or substances containing the same. Examples of the chemotherapeutic agents include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of the toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of the radioisotopes include radioisotopes of hydrogen atoms (e.g., 3 H), radioisotopes of carbon atoms (e.g., 14 C), radioisotopes of phosphorus atoms (e.g., 32 P), radioisotopes of sulfur atoms (e.g., 35 S ), radioisotopes of yttrium (e.g., 90 Y), radioisotopes of technetium (e.g., 99m Tc), radioisotopes of indium (e.g., 111 In), radioisotopes of iodine atoms (e.g., 123 I, 125 I, 129 I, 131 I), radioisotopes of samarium (e.g., 153Sm), radioisotopes of rhenium (e.g., 186 Re), radioisotopes of astatine (e.g., 211 At), radioisotopes of bismuth (e.g., 212 More specifically, the drugs include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBDs (pyrrolobenzodiazepines), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0120] A labeling substance is a substance that allows detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2′-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), or substances containing the same.

[0121] The affinity substance is a substance that has affinity for a target. Examples of the affinity substance include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary chains to target nucleic acids. The affinity substance is preferably an affinity protein or affinity peptide, and more preferably an antibody. The animal species from which the antibody used as the functional substance is derived are the same as those described above.

[0122] The type of antibody used as the functional substance may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a specific sugar chain has been added (e.g., antibodies modified to have a sugar chain consensus sequence such as an N-type sugar chain consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Examples of antibodies used as the functional substance include full-length antibodies and fragments thereof (fragment antibodies). The antibody fragment may be any fragment that maintains binding ability to a desired antigen, and examples of such fragments include Fab, Fab', F(ab')2, and scFv.

[0123] The antigenicity of the antibody used as the functional substance may be the same as or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, and is preferably different. The origin of the antibody used as the functional substance may be the same as or different from the origin of the immunoglobulin unit, and is preferably different. Therefore, the antibody used as the functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody mentioned in the above-mentioned specific example of the monoclonal antibody, or an antibody derived therefrom. The antibody used as the functional substance may also be an IgG1, IgG2, IgG3, or IgG4 mentioned in the above-mentioned specific example of the monoclonal antibody, or an antibody derived therefrom.

[0124] The transport substance is a substance capable of transporting a compound. The transport substance is preferably a substance capable of encapsulating a compound in a protein shell (e.g., a multimer) (e.g., ferritin such as human ferritin, virus particles, virus-like particles).

[0125] Stabilizers are substances that allow for the stabilization of antibodies, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0126] The functional substance may also be a peptide, a protein, a nucleic acid, a low molecular weight organic compound, a sugar chain, a lipid, a high molecular weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Examples of nucleic acids also include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of low molecular weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.

[0127] In certain embodiments, the functional substance may be a substance having an aromatic ring, such as monomethylauristatin (e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF)) or exatecan.

[0128] (salt) In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0129] 2. Regioselective conjugate or salt thereof The present invention provides a regioselective conjugate of an antibody and a functional substance, which comprises a structural unit represented by the above formula (I), or a salt thereof. The regioselectivity of the conjugate of the present invention is as described above.

[0130] In formula (I) and other formulae presented in connection with the present invention, a hyphen (-) indicates that the two units (eg, atoms, groups) on either side of it are covalently bonded.

[0131] The antibody comprises an immunoglobulin unit as described above. Examples of such antibodies include an IgG antibody, an IgD antibody, and an IgE antibody, which comprise an immunoglobulin unit comprising two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy chains and the light chains; an IgA antibody, which comprises four heavy chains and four light chains and having an immunoglobulin unit comprising disulfide bonds between the heavy chains and between the heavy chains and the light chains; and an IgM antibody, which comprises eight heavy chains and eight light chains and having an immunoglobulin unit comprising disulfide bonds between the heavy chains and between the heavy chains and the light chains; and an IgG antibody (e.g., IgG1, IgG2, IgG3, IgG4) is preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0132] The site-selective bond is preferably achieved by a bond between an amino group in the side chain of the lysine residue and an atom or group capable of bonding therewith (e.g., a carbonyl group, a thiocarbonyl group), and more preferably by an amide bond between an amino group and a carbonyl group in the side chain of the lysine residue.

[0133] In formula (I), HG represents a hydrophilic group or a monovalent group containing a hydrophilic group. The hydrophilic group and the monovalent group are as described above. Preferably, HG may represent a monovalent group containing a hydrophilic group.

[0134] R A represents the side chain of a valine residue (i.e., -CH(CH3)2). Alternatively, R A R may be the side chain of a phenylalanine, threonine, leucine, or alanine residue. A The configuration of the amino acid residues in may be either L- or D-form, with the L-form being preferred.

[0135] R B represents the side chain of a citrulline residue (i.e., -CH2CH2CH2NHCONH2) or the side chain of an alanine residue (i.e., -CH3). Alternatively, R BR may be the side chain of a glutamic acid, glutamine, lysine, arginine, threonine, or methionine residue. B The configuration of the amino acid residues in may be either L- or D-form, with the L-form being preferred.

[0136] R A and R B The combination of R A is the side chain of a valine residue, and R B is preferably the side chain of a citrulline or alanine residue. A and R B Other preferred examples of combinations are: (a)R A is the side chain of a valine residue, and R B is the side chain of a glutamic acid residue, a lysine residue, an arginine residue, or a threonine residue; (b)R A is the side chain of a phenylalanine residue, and R B is the side chain of a lysine, arginine, or glutamine residue; (c)R A is the side chain of a threonine residue, and R B is the side chain of a threonine or methionine residue; (d)R A is the side chain of a leucine residue, and R B is the side chain of a glutamic acid residue; and (e)R A is the side chain of an alanine residue, and R B is the side chain of an alanine residue.

[0137] Ring A represents a divalent aromatic ring group which may have a substituent. The divalent aromatic ring group is the above-mentioned arylene or divalent aromatic heterocycle. The position of the divalent aromatic ring group to which two adjacent atoms (carbon atom and nitrogen atom) are bonded is not particularly limited, as long as cleavage occurs between the oxygen atom and the carbonyl group in -O-(C=O)- due to the conjugation of π electrons when the amide bond present on the carboxyl terminal side of citrulline is cleaved by cathepsin B (for example, see the cleavage reaction shown in "(B) Description in lysosomes in human cancer cells" in the Background Art). Such a position is technical common knowledge in the field, and can be easily determined by a person skilled in the art depending on factors such as the type of the divalent aromatic ring group.

[0138] Preferably, ring A is a divalent monocyclic aromatic ring group which may have a substituent. The divalent aromatic ring group is a phenylene group or a divalent monocyclic aromatic heterocyclic group.

[0139] More preferably, ring A may be a divalent 6-membered aromatic ring group. Examples of the 6-membered aromatic ring group include the various groups described above. In this case, the position of the divalent 6-membered aromatic ring group in which two adjacent atoms are bonded is the ortho position or the para position, and preferably the para position.

[0140] More preferably, ring A may be a phenylene group which may have a substituent. In this case, the position of the phenylene group to which two adjacent atoms are bonded is the ortho position or the para position, and preferably the para position.

[0141] The substituent in the optionally substituted divalent aromatic ring group is as described above. Such a substituent may be an electron-withdrawing group as described above.

[0142] R1 and R2 each independently represent a hydrogen atom or a monovalent group. The monovalent group is as described above. The monovalent group in R1 and R2 is preferably a monovalent hydrocarbon group that may have a substituent, more preferably an alkyl that may have a substituent, and even more preferably an alkyl. The alkyl is preferably one that has been described above.

[0143] In certain embodiments, the monovalent groups represented by R1 and R2 may be a protecting group for an amino group. Examples of such protecting groups include an alkylcarbonyl group (acyl group) (e.g., an acetyl group, a propoxy group, a butoxycarbonyl group such as a tert-butoxycarbonyl group), an alkyloxycarbonyl group (e.g., a fluorenylmethoxycarbonyl group), an aryloxycarbonyl group, and an arylalkyl(aralkyl)oxycarbonyl group (e.g., a benzyloxycarbonyl group).

[0144] In a preferred embodiment, R1 and R2 each independently represent a hydrogen atom or a protecting group for an amino group. Preferably, R1 and R2 each may be a hydrogen atom.

[0145] The divalent groups represented by L1 and L2 are as described above.

[0146] In certain embodiments, the divalent groups represented by L1 and L2 may each contain a moiety generated by the reaction of two bioorthogonal functional groups capable of reacting with each other. Since combinations of two bioorthogonal functional groups capable of reacting with each other are well known, those skilled in the art can appropriately select such combinations and appropriately set divalent groups containing a moiety generated by the reaction of two bioorthogonal functional groups capable of reacting with each other. Examples of combinations of bioorthogonal functional groups capable of reacting with each other include a combination of a thiol residue and a maleimide residue, a combination of a furan residue and a maleimide residue, a combination of a thiol residue and a halocarbonyl residue (a halogen is replaced by a thiol by a substitution reaction), a combination of an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted by a substituent as described above) and an azide residue, a combination of a tetrazine residue and an alkene residue, a combination of a tetrazine residue and an alkyne residue, and a combination of a thiol residue and another thiol residue (disulfide bond). Thus, the moiety may be a group formed by the reaction of a thiol residue with a maleimide residue, a group formed by the reaction of a furan residue with a maleimide residue, a group formed by the reaction of a thiol residue with a halocarbonyl residue, a group formed by the reaction of an alkyne residue with an azide residue, or a group formed by the reaction of a tetrazine residue with an alkene residue, or a disulfide group formed by the combination of a thiol residue with another thiol residue.

[0147] In certain embodiments, the moiety can be a divalent group represented by any one of the following structural formulas: [ka] (Here, the white and black circles represent bonds.)

[0148] In L1, when the bond of the white circle is bonded to an atom on the Ig bond side, the bond of the black circle may be bonded to an atom on the nitrogen atom (N) bond side in "N-R1", When the bond indicated by the white circle is bonded to an atom present on the nitrogen atom (N) bonding side in "N-R1," the bond indicated by the black circle may be bonded to an atom present on the Ig bonding side.

[0149] In L2, when the bond of the white circle is bonded to an atom present on the nitrogen atom (N) bond side in "N-R2", the bond of the black circle may be bonded to an atom present on the functional substance (D) bond side, When the bond indicated by the white circle is bonded to an atom present on the bonding side of the functional substance (D), the bond indicated by the black circle may be bonded to an atom present on the bonding side of the nitrogen atom (N) in "N-R2."

[0150] The functional substance represented by D is as described above.

[0151] r represents the average ratio of the bonds per two heavy chains, and is 1.5 to 2.5. Such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1.

[0152] In certain embodiments, the regioselective conjugate or its salt of the present invention has the desired property of being less likely to aggregate, and therefore can be identified by the aggregation rate. More specifically, the aggregation rate of the conjugate or its salt of the present invention may be 5% or less. This is because, according to the present invention, antibody aggregation is easily avoided. The aggregation rate is preferably 4.8% or less, more preferably 4.6% or less, even more preferably 4.4% or less, particularly preferably 4.2% or less, 4.0% or less, 3.8% or less, 3.6% or less, 3.4% or less, 3.2% or less, 3.0% or less, 2.8% or less, or 2.6% or less. The aggregation rate of the antibody can be measured by size exclusion chromatography (SEC)-HPLC (see Examples and ChemistrySelect, 2020, 5, 8435-8439).

[0153] In a preferred embodiment, the regioselective conjugate or salt thereof of the present invention may have an aggregation rate of 2.6% or less. The aggregation rate may also be 2.4% or less, 2.2% or less, 2.0% or less, 1.8% or less, or 1.6% or less.

[0154] Preferably, the structural unit represented by formula (I) may be represented by formula (I'). Ig, R represented by formula (I') A , R B , ring A, R1, R2, L1, L2, D, and r are each the same as those represented in formula (I).

[0155] In formula (I'), L HG represents a bond or a divalent group which may contain a hydrophilic group. The hydrophilic group and the divalent group are as described above. The divalent group which may contain a hydrophilic group is L HG It may be contained in the main chain linking the nitrogen atom and carbon atom adjacent to the main chain or in a side chain of the main chain, and is preferably contained in a side chain of the main chain.

[0156] Preferably, the divalent group (-L HG -) may be a divalent group represented by formula (a): -(C(R HG )2) n1 -(C=O) n2 -(NR HG ) n3 -(C(R HG )2) n4 - (a) (The hyphens (-) at both ends indicate bonds.)

[0157] In formula (a), a plurality of R HG each independently represents a hydrogen atom or a monovalent group which may contain a hydrophilic group. The hydrophilic group and the monovalent group are as described above.

[0158] n1 is an integer of 0 to 3, preferably an integer of 0 to 2, and more preferably an integer of 0 or 1.

[0159] n2 is an integer of 0 or 1.

[0160] n3 is an integer of 0 or 1.

[0161] n4 is an integer of 0 to 3, preferably an integer of 0 to 2, and more preferably an integer of 0 or 1.

[0162] Even more preferably, the divalent group (-L HG -) may be a divalent group represented by the following formula (a1), (a2), or (a3): (a1) -(C(R HG )2)-; (a2) -(C(R HG )2)-(C=O)-(NR HG )-(C(R HG )2)-;or (a3) -(C=O)-(C(R HG )2)2-.

[0163] In formula (a1), (a2), or (a3), a plurality of R HGeach independently represents a hydrogen atom, a hydrophilic group, or an alkyl group containing a hydrophilic group and having 1 to 6 carbon atoms. The hydrophilic group and the alkyl group having 1 to 6 carbon atoms are as described above.

[0164] In formula (I'), R HG1 , and R HG2 each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group that may contain a hydrophilic group. The hydrophilic group and the monovalent group are as described above.

[0165] In certain embodiments, R HG1 , and R HG2 The monovalent group represented by the formula (I) which may contain a hydrophilic group may be a protecting group for an amino group. Examples of the protecting group for an amino group include those described above for R1 and R2. For example, R HG1 , and R HG2 One of these may be a hydrogen atom and the other a protecting group for an amino group.

[0166] Or, R HG1 , and R HG2 One of the groups may be a hydrogen atom, and the other may be a monovalent group containing a hydrophilic group. Examples of the monovalent group containing a hydrophilic group include an alkyl group containing a hydrophilic group, a carboxyl group containing a hydrophilic group, an alkylcarbonyl group containing a hydrophilic group (e.g., the groups described above), an alkyloxycarbonyl group containing a hydrophilic group, and an oxycarbonyl group containing a hydrophilic group.

[0167] In formula (I'), at least one hydrophilic group is L HG , R HG1 , and R HG2 The hydrophilic group is contained in one or more sites selected from the group consisting of: Examples of the site containing at least one hydrophilic group and combinations thereof include the following: (i)L HG Alone; (ii)R HG1 Alone; (iii)R HG2 Alone; (iv) LHG and L HG1 A combination of; (v)L HG and L HG2 A combination of; (vi) L HG1 and L HG2 combination of; (vii) L HG , L HG1 and L HG2 A combination of. These L HG Part, R HG1 Part, R HG2 Each of the moieties may contain one hydrophilic group, or may contain two or more hydrophilic groups.

[0168] The definitions, examples, and preferred examples of the symbols and elements in the symbols (eg, specific elements such as cleavable sites, or specific formulas) described in the above series of formulas apply similarly to the other formulas.

[0169] The regioselective conjugate of the present invention or a salt thereof is useful, for example, as a medicine or a reagent (eg, a diagnostic agent, a research reagent).

[0170] The conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate of the present invention or a salt thereof, a pharma- ceutically acceptable carrier. Examples of medicamentously acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cacao butter, polyethylene glycol, and white kerosene. However, these are not limited to these. The conjugate of the present invention or a salt thereof may also have an optional modification that provides stability (eg, PEGylation).

[0171] Formulations suitable for oral administration include liquid preparations in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granules; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0172] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0173] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to which the composition is administered, the drug tolerance, body weight, age, etc. of the subject, but can be determined appropriately.

[0174] In one embodiment, the regioselective conjugate or its salt of the present invention can be prepared by reacting an antibody derivative or its salt having a regioselective bioorthogonal functional group with a functional substance (FIG. 2). Such a reaction can proceed by a reaction between the bioorthogonal functional group in the antibody derivative and the functional substance.

[0175] When the functional substance has a functional group that is easily reactive with the bioorthogonal functional group, the functional group of the functional substance can be appropriately reacted with the bioorthogonal functional group in the antibody derivative. The functional group that is easily reactive with the bioorthogonal functional group can also vary depending on the specific type of the bioorthogonal functional group. Those skilled in the art can appropriately select an appropriate functional group as a functional group that is easily reactive with the bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Examples of functional groups that easily react with bioorthogonal functional groups include, but are not limited to, alkyne residues when the bioorthogonal functional group is an azide residue, maleimide residues and disulfide residues when the bioorthogonal functional group is a thiol residue, hydrazine residues when the bioorthogonal functional group is an aldehyde residue or a ketone residue, azide residues when the bioorthogonal functional group is a norbornene residue, and alkyne residues when the bioorthogonal functional group is a tetrazine residue. Of course, in the above combinations of bioorthogonal functional groups and functional groups that easily react with them, it is also possible to replace the combination. Therefore, when the first example in the above combination is replaced, a combination of an alkyne residue as the bioorthogonal functional group and an azide residue as the functional group that easily reacts with the bioorthogonal functional group can be used.

[0176] If the functional substance does not have a functional group that is easily reactive with the bioorthogonal functional group in the antibody derivative, the drug may be derivatized to have such a functional group. Derivatization is common knowledge in the art (e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be performed using any crosslinking agent. Alternatively, derivatization may be performed using a specific linker that has a desired functional group. In the present invention, the derivatized functional substance is simply referred to as a "functional substance" because it is just one type of functional substance.

[0177] The above reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in an appropriate reaction system, for example, a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0178] In another embodiment, the regioselective conjugate or salt thereof of the present invention can be prepared by reacting a compound or salt thereof having a bioorthogonal functional group and a functional substance with a starting antibody having an Ig (immunoglobulin unit) (FIG. 2).

[0179] The starting antibody contains a lysine residue regioselectively modified with a bioorthogonal functional group. The bioorthogonal functional group in the starting antibody can be selected so as to be capable of reacting with the bioorthogonal functional group and the bioorthogonal functional group in the compound having a functional substance. As the bioorthogonal functional group in the starting antibody, the various bioorthogonal functional groups described above can be used. From the viewpoint of versatility, the bioorthogonal functional group in the starting antibody may be a leimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.

[0180] In certain embodiments, the starting antibody has the following formula (VIII): [ka] [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and that regioselectively forms amide bonds between amino groups in the side chains of lysine residues in the two heavy chains and carbonyl groups adjacent to Ig; L is -(C(R)2) m -, -(OC(R)2-C(R)2) m - and -(C(R)2-C(R)2-O) m - is a divalent group selected from the group consisting of R's are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; m is an integer from 0 to 10; B is a bioorthogonal functional group capable of reacting with the bioorthogonal functional group represented by B1; The average ratio r of the bonds per two heavy chains is 1.5 to 2.5. The definitions, examples and preferred examples of Ig and r in the above formula (VIII) are the same as those described above.

[0181] m may preferably be an integer of 1 or greater, more preferably an integer of 2 or greater, an integer of 3 or greater, an integer of 4 or greater, or an integer of 5 or greater. m may also preferably be an integer of 9 or less, more preferably an integer of 8 or less, an integer of 7 or less, or an integer of 6 or less. In certain cases, m may be an integer of 1 to 8 (preferably an integer of 2 to 6).

[0182] The bioorthogonal functional group represented by B is the same as the bioorthogonal functional group described above.

[0183] The reaction between the compound having a bioorthogonal functional group and a functional substance or its salt and the above-mentioned starting antibody can be appropriately carried out under the above-mentioned conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds).

[0184] The confirmation of the formation of the regioselective conjugate or its salt can be carried out, for example, by reversed-phase HPLC under reducing conditions or mass spectrometry, depending on the specific raw material and the molecular weight of the product. The confirmation of the regioselectivity can be carried out, for example, by peptide mapping. Peptide mapping can be carried out, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. The protease is preferably an endoprotease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The conjugate or its salt can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, high-performance liquid chromatography, affinity chromatography).

[0185] 3. Antibody derivatives or salts thereof The present invention also provides an antibody derivative or a salt thereof having a bioorthogonal functional group regioselectively, the bioorthogonal functional group comprising a structural unit represented by the above formula (II). The regioselectivity of the antibody derivative of the present invention is as described above.

[0186] In formula (II), Ig, HG, R A , R B , ring A, R1, R2, L1, L2, and r are as described above in formula (I). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I).

[0187] The bioorthogonal functional group represented by B2 is as described above.

[0188] In certain embodiments, the bioorthogonal functional group may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. These bioorthogonal functional groups are preferred due to their excellent reaction efficiency and versatility.

[0189] Preferably, the structural unit represented by formula (II) may be represented by formula (II'). Ig, R represented by formula (II') A , R B , ring A, R1, R2, L1, L2, B2, and r are each the same as those represented in formula (II).

[0190] In formula (II'), L HG , R HG1 , and R HG2 are as described above in formula (I'). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I').

[0191] The antibody derivative of the present invention or a salt thereof is useful, for example, as an intermediate for producing the regioselective conjugate of the present invention or a salt thereof.

[0192] The antibody derivative or its salt of the present invention can be produced, for example, by reacting a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or its salt with a starting antibody having an Ig (immunoglobulin unit) (Figure 2). The starting antibody is the same as that described above.

[0193] The reaction between the compound having the first bioorthogonal functional group and the second bioorthogonal functional group or its salt and the starting antibody can be appropriately carried out under the above-mentioned conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds).

[0194] The production of the antibody derivative or its salt can be confirmed in the same manner as described for the regioselective conjugate of the present invention (the regioselectivity can also be confirmed). The antibody derivative or its salt can be appropriately purified by any purification method as described for the regioselective conjugate of the present invention.

[0195] 4. Compounds having bioorthogonal functional groups and functional substances or their salts The present invention also provides a compound having a bioorthogonal functional group and a functional substance, represented by the above formula (III), or a salt thereof.

[0196] In formula (III), H G, R A , R B , ring A, R1, R2, L1, L2, and D are as described above in formula (I). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I).

[0197] B1 represents a bioorthogonal functional group. The bioorthogonal functional group is as described above.

[0198] In certain embodiments, the bioorthogonal functional group may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. These bioorthogonal functional groups are preferred due to their excellent reaction efficiency and versatility.

[0199] Preferably, the compound represented by formula (III) may be represented by formula (III'). R represented by formula (III') A , R B , ring A, R1, R2, L1, L2, B1, and D are each the same as those represented in formula (III).

[0200] In formula (III'), L HG , R HG1 , and R HG2 are as described above in formula (I'). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I').

[0201] The compound of the present invention represented by formula (III) or a salt thereof is useful, for example, as an intermediate for preparing the regioselective conjugate of the present invention. The compound of the present invention represented by formula (III) or a salt thereof is also useful, for example, for derivatizing any substance such as a biomolecule (e.g., a protein such as an antibody, a sugar, a nucleic acid, or a lipid).

[0202] The compound or its salt having a bioorthogonal functional group and a functional substance can be produced, for example, by reacting a compound or its salt having a first bioorthogonal functional group and a second bioorthogonal functional group with a functional substance (FIG. 2). Details of the functional substance are as described above.

[0203] The reaction of the compound having the first and second bioorthogonal functional groups or a salt thereof with the functional substance can be carried out in an appropriate reaction system, such as an organic solvent system or an aqueous solution (e.g., a buffer solution) system, at an appropriate temperature (e.g., about 15 to 200°C). The reaction system may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. Of course, such a reaction can also be carried out under the above-mentioned mild conditions.

[0204] The confirmation of the production of a compound or its salt having a bio-orthogonal functional group and a functional substance can be carried out, for example, by NMR, HPLC, or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Such a compound or its salt can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, high performance liquid chromatography, affinity chromatography).

[0205] 5. Compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof The present invention also provides a compound having a first bioorthogonal functional group and a second bioorthogonal functional group, represented by the above formula (IV), or a salt thereof.

[0206] In formula (IV), H G, R A , R B , ring A, R1, R2, L1, L2, and D are as described above in formula (I). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I).

[0207] B1 represents the first bioorthogonal functional group. The first bioorthogonal functional group is the same as that described above for the bioorthogonal functional group.

[0208] B2 represents a second bioorthogonal functional group. The second bioorthogonal functional group is the same as that described above for the bioorthogonal functional group.

[0209] Preferably, the second bioorthogonal functional group may be a bioorthogonal functional group that does not react with the first bioorthogonal functional group or has low reactivity with the first bioorthogonal functional group. In this case, the intermolecular reaction of the compound represented by formula (IV) or a salt thereof can be suppressed. Therefore, the first and second bioorthogonal functional groups can be used in a combination that does not react with each other or has low reactivity with each other. Such combinations of bioorthogonal functional groups are well known in the art. For example, examples of such combinations for the preferred bioorthogonal functional groups, maleimide residue, thiol residue, furan residue, halocarbonyl residue, alkene residue, alkyne residue, azide residue, and tetrazine residue, are as follows:

[0210] [Table A]

[0211] Preferably, the compound represented by formula (IV) may be represented by formula (IV'). R represented by formula (IV') A , R B , ring A, R1, R2, L1, L2, B1, and B2 are each the same as those represented in formula (IV).

[0212] In formula (IV'), L HG , R HG1 , and R HG2 are as described above in formula (I'). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I').

[0213] The compound of the present invention represented by formula (IV) or a salt thereof is useful, for example, as an intermediate for producing the antibody derivative of the present invention and the compound of the present invention represented by formula (III). The compound of the present invention represented by formula (IV) or a salt thereof is also useful, for example, for derivatizing any substance such as a biomolecule (e.g., a protein such as an antibody, a sugar, a nucleic acid, a lipid), and a functional substance.

[0214] In one embodiment, a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof can be prepared by reacting a compound of formula (VI) having a bioorthogonal functional group or a salt thereof with a compound represented by B1-L1-NH-R1 (FIG. 3). The definitions, examples, and preferred examples of B1, L1, and R1 are as described above.

[0215] In another embodiment, a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof can be prepared by reacting a compound of formula (VII) having a bioorthogonal functional group or a salt thereof with bis(4-nitrophenyl)carbonate and N,N-diisopropylethylamine (DIPEA), and then reacting with a compound represented by B2-L2-NH-R2 (FIG. 3). The definitions, examples, and preferred examples of B2, L2, and R2 are as described above.

[0216] The above reaction can be carried out in an appropriate reaction system, such as an organic solvent system or an aqueous solution (e.g., a buffer solution) system, at an appropriate temperature (e.g., about 15 to 200°C). The reaction system may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. Of course, such a reaction can also be carried out under the above-mentioned mild conditions.

[0217] The confirmation of the production of the compound or its salt having the first and second bioorthogonal functional groups can be carried out, for example, by NMR, HPLC, or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Such a compound or its salt can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, high performance liquid chromatography, affinity chromatography).

[0218] 6. A series of compounds or their salts (1) A compound or a salt thereof The present invention also provides a compound represented by the above formula (V) or a salt thereof.

[0219] In formula (V), H G, R A , R B and ring A are as described above in formula (I). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I).

[0220] X and Y each independently represent a monovalent group. The monovalent group is as described above.

[0221] Preferably, the compound represented by formula (V) may be represented by formula (V'). R represented by formula (V') A , R B , ring A, X, and Y are the same as those shown in formula (IV).

[0222] In formula (V'), L HG , R HG1 , and R HG2 are as described above in formula (I'). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I').

[0223] The compound represented by formula (V) or a salt thereof is useful, for example, as a synthetic intermediate for the regioselective conjugates, antibody derivatives, and other compounds of the present invention.

[0224] The compound represented by formula (V) or a salt thereof can be, for example, a compound represented by the following formula (V-1): [ka] [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A indicates the side chain of a valine residue, R B represents a side chain of a citrulline residue or an alanine residue, or a salt thereof, The following formula (V-2): [ka] [During the ceremony, Ring A represents a divalent aromatic ring group which may have a substituent; X and Y each independently represent a monovalent group. ] or a salt thereof (Figure 3). A , and R B The definitions, examples, and preferred examples of rings A, X, and Y in formula (V-2) are as described above. Such a reaction can be carried out under the same reaction conditions as those described above for the preparation of a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof.

[0225] (2) A compound having a bioorthogonal functional group represented by formula (VI) or a salt thereof The present invention also provides a compound represented by the above formula (VI) or a salt thereof.

[0226] In formula (VI), H G, R A , R B , ring A, R2, and L2 are as described above in formula (I). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I).

[0227] The monovalent group represented by X is as described above.

[0228] The bioorthogonal functional group represented by B2 is as described above.

[0229] Preferably, the compound represented by formula (VI) may be represented by formula (VI'). A , R B , ring A, X, R2, L2, and B2 are the same as those shown in formula (IV).

[0230] In formula (VI'), L HG , R HG1 , and R HG2 are as described above in formula (I'). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I').

[0231] The compound represented by formula (VI) or a salt thereof is useful, for example, as a synthetic intermediate for the regioselective conjugate of the present invention, the antibody derivative, and the specific compound of the present invention. Such a compound or a salt thereof is also useful, for example, for derivatization of a functional substance.

[0232] The compound represented by formula (VI) or a salt thereof can be prepared, for example, by reacting the compound represented by formula (V) or a salt thereof with bis(4-nitrophenyl)carbonate and N,N-diisopropylethylamine (DIPEA), and then reacting with the compound represented by B2-L2-NH-R2 (FIG. 3). The definitions, examples, and preferred examples of B2, L2, and R2 are as described above. Such a reaction can be carried out under the same reaction conditions as those described above for the preparation of the compound having the first bioorthogonal functional group and the second bioorthogonal functional group or a salt thereof.

[0233] (3) A compound having a bioorthogonal functional group represented by formula (VII) or a salt thereof The present invention also provides a compound represented by the above formula (VII) or a salt thereof.

[0234] In formula (VII), H G, R A , R B , ring A, R1, and L1 are as described above in formula (I). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I).

[0235] The monovalent group represented by Y is as described above.

[0236] The bioorthogonal functional group represented by B1 is as described above.

[0237] Preferably, the compound represented by formula (VII) may be represented by formula (VII'). A , R B , ring A, Y, R1, L1, and B1 are each the same as those represented in formula (IIV).

[0238] In formula (VII'), L HG , R HG1 , and R HG2are as described above in formula (I'). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I').

[0239] The compound represented by formula (VII) or a salt thereof is useful, for example, as a regioselective conjugate of the present invention, an antibody derivative, and a synthetic intermediate of a specific compound of the present invention. Such a compound or a salt thereof is also useful, for example, for derivatizing any substance such as a biomolecule (e.g., a protein such as an antibody, a sugar, a nucleic acid, a lipid).

[0240] The compound represented by formula (VII) or a salt thereof can be prepared, for example, by reacting the compound represented by formula (V) or a salt thereof with the compound represented by B1-L1-NH-R1 (FIG. 3). The definitions, examples, and preferred examples of B1, L1, and R1 are as described above. Such a reaction can be carried out under the same reaction conditions as those described above for the preparation of the compound having the first bioorthogonal functional group and the second bioorthogonal functional group or a salt thereof.

[0241] The production of the compound represented by formula (V), (VI), or (VII) or a salt thereof can be confirmed by, for example, NMR, HPLC, or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Such a compound or a salt thereof can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, high performance liquid chromatography, affinity chromatography). EXAMPLES

[0242] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0243] The following peptides in Example 1, Ac-Glu(OtBu)-Val-Cit-OH, Z-Glu(OtBu)-Val-Cit-OH, Ac-Glu(OtBu)-Glu(OtBu)-Val-Cit-OH, and SCA(OtBu)-Glu(OtBu)-Val-Cit-OH, were all prepared in the same manner. Note that SCA(OtBu) is an abbreviation for mono-tert-butyl succinate, and SCA is an abbreviation for succinic acid. By solid-phase peptide synthesis using Cl-TCP(Cl) ProTide Resin (CEM) by the Fmoc method, N-terminal capped with acetyl group and N-terminal capped with succinic acid were prepared, and the amino acid side chain was cleaved from the resin while being protected by stirring overnight in a 20% HFIP / dichloromethane solution. The resin was removed by filtration, and the solution was concentrated, and then purified by preparative HPLC to obtain the product peptide.

[0244] Ac-Glu(OtBu)-Val-Cit-OH

[0245] 1 H NMR(400 MHz,DMSO-d6)δ12.50(brs,1H),8.22(d,J=7.2Hz,1H),8.05(d,J=8.0Hz,1H),7.69(d,J=8.8H z,1H),5.95-5.93(m,1H),5.38(brs,2H),4.33-4.27(m,1H),4.22-4.18(m,1H),4.15-4.10(m ,1H),2.96-2.95(m,2H),2.25-2.19(m,2H),2.00-1.93(m,1H),1.89-1.80(m,4H),1.73-1.66 (m,2H),1.61-1.51(m,1H),1.46-1.34(m,11H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).

[0246] MS(ESI)m / z:502.30[M+H] +

[0247] Z-Glu(OtBu)-Val-Cit-OH

[0248] 1 H NMR(400 MHz,DMSO-d6)δ12.50(brs,1H),7.93-7.37(m,8H),6.05-6.00(m,1H),5.44(brs,2H),5.08(s,2H),4.17-3.81(m,3H),3.00-2.90(m,2H),2.31-2.27(m,2H),2.10-1.34(m,16H),0.89-0.83(m,6H).

[0249] MS(ESI)m / z:594.30[M+H] +

[0250] Ac-Glu(OtBu)-Glu(OtBu)-Val-Cit-OH

[0251] 1 H NMR(400 MHz,DMSO-d6)δ12.50(brs,1H),8.21(d,J=7.2Hz,1H),8.08(d,J=8.0Hz,1H),8.04(d,J=8.0Hz,1H),7.68(d,J=8.0Hz,1H),5.95(brs,1H),5.37(brs,2H),4.32-4.19(m,3H),4.16-4.11(m,1H),2.98-2.94(m,2H),2.27-2.13(m,4H),2.00-1.79(m,5H),1.76-1.52(m,5H),1.42-1.36(m,20H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.8Hz,3H).

[0252] MS(ESI)m / z:687.35[M+H] +

[0253] SCA(OtBu)-Glu(OtBu)-Val-Cit-OH

[0254] 11H NMR (400 MHz, DMSO-d6) δ 12.70 (brs, 1H), 8.21 (d, J = 7.2 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 5.96 (brs, 1H), 5.25 (brs, 2H), 4.35 - 4.30 (m, 1H), 4.21 - 4.18 (m, 1H), 4.15 - 4.10 (m, 1H), 2.98 - 2.94 (m, 2H), 2.40 - 2.28 (m, 4H), 2.24 - 2.18 (m, 2H), 2.01 - 1.93 (m, 1H), 1.90 - 1.81 (m, 1H), 1.73 - 1.63 (m, 2H), 1.61 - 1.51 (m, 1H), 1.40 - 1.31 (m, 20H), 0.86 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H).

[0255] MS (ESI) m / z: 616.30 [M + H] +

[0256] Ac-Glu(OtBu)-Val-Ala-OH

[0257] 1 1H NMR (400 MHz, DMSO-d6) δ 12.50 (brs, 1H), 8.25 (d, J = 6.8 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H), 4.33 - 4.27 (m, 1H), 4.22 - 4.16 (m, 2H), 2.23 - 2.18 (m, 2H), 1.99 - 1.94 (m, 1H), 1.89 - 1.80 (m, 4H), 1.73 - 1.65 (m, 1H), 1.39 (s, 9H), 1.27 (d, J = 7.2 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H).

[0258] MS (ESI) m / z: 416.20 [M + H] +

[0259] Example 1: Synthesis of Linker-payload mimic (1-1) Synthesis of Linker-payload mimic (1) The linker-payload mimic(1) was synthesized as follows: [ka]

[0260] (1-1-1) Synthesis of alcohol (2) [ka]

[0261] Ac-Glu(OtBu)-Val-Cit-OH (19.9 mg, 39.7 μmol) was dissolved in N,N-dimethylformamide (400 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (18.1 mg, 47.6 μmol) and 2,4,6-trimethylpyridine (6.27 μL, 47.6 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 4-aminomandelate methyl (8.63 mg, 47.6 μmol) was added, and the mixture was stirred at room temperature for 21.5 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (2) (28.5 mg, quant).

[0262] 1H NMR (400 MHz, DMSO-d6) δ9.95(s,1H),8.07(d,J=7.4 Hz,1H),7.99(d,J=8.0 Hz,1H),7.66(d,J=8.4 Hz,1H),7.50(d,J=8.4 Hz,2H),7.25(d,J=8.4 Hz,2H),5.92(brs,1H),5.36(brs,2H),5.01(s,1H),4.34-4.29(m,1H),4.26-4.20(m,1H),4.14-4.10(m,1H),3.53(s,3H), 3.00-2.83(m,2H),2.18-2.13(m,2H),1.94-1.89(m,2H),1.84-1.23(m,17H),0.79(d,J=6.8Hz,3H),0.75(d,J=6.8Hz,3H).

[0263] MS (ESI) m / z: 665.30 [M+H] +

[0264] (1-1-2)ピレン(3)のSynthesis

change

[0265] The alcohol (2) (28.5 mg) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (430 μL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (26.6 mg, 85.7 μmol) and N,N-diisopropylethylamine (11.1 μL, 64.4 μmol) were added and stirred at room temperature for 1.5 hours. The reaction was tracked by LCMS and found to have residual raw materials, so bis(4-nitrophenyl)carbonate (13.3 mg, 42.9 μmol) and N,N-diisopropylethylamine (5.54 μL, 32.2 μmol) were added and stirred at room temperature for 5 hours. After that, the mixture was cooled on ice, and sarcosin-pyrene (64.9 mg, 215 μmol), 1-hydroxybenzotriazole (8.7 mg, 64 μmol), and N,N-diisopropylethylamine (57.2 μL, 333 μmol) were added, followed by stirring at room temperature for 16 hours. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (3) (26.1 mg, 26.3 μmol).

[0266] 1 H NMR(400 MHz,DMSO-d6)δ10.09-10.07(m,1H),8.63-8.60(m,1H),8.33-7.65(m,13H),7.6 0-7.57(m,2H),7.37-7.32(m,2H),5.94-5.91(m,1H),5.72-5.70(m,1H),5.37(br s,2H),4.98-4.96(m,1H),4.93-4.00(m,4H),3.85-3.75(m,1H),3.56-3.55(m,3 H),2.97-2.87(m,5H),2.17-2.13(m,2H),1.93-1.17(m,19H),0.80-0.73(m,6H).

[0267] MS(ESI)m / z:993.40[M+H] +

[0268] (1-1-3) Synthesis of pyrene (4) [ka]

[0269] Pyrene (3) (10.8 mg, 10.9 μmol) was dissolved in tetrahydrofuran (700 μL) and water (400 μL), and the mixture was stirred for 5 minutes under ice cooling, after which 1 M lithium hydroxide aqueous solution (109 μL, 109 μmol) was added and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was adjusted to about pH 6 using 0.1 M hydrochloric acid, and purified by reversed phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above pyrene (4) (4.5 mg, 4.6 μmol).

[0270] 1 H NMR(400 MHz,DMSO-d6)δ13.05(brs,1H),10.08-10.05(m,1H),8.62-8.59(m,1H),8.33-7.56(m,15H),7.37-7.35(m,2H),5.92(brs,1H),5.61-5.60 (m,1H),5.36(brs,2H),4.98-4.03(m,5H),3.88-3.74(m,1H),2.99-2.83(m,5H),2.15-2.13(m,2H),1.93-1.14(m,19H),0.84-0.73(m,6H).

[0271] MS(ESI)m / z:979.40[M+H] +

[0272] (1-1-4) Synthesis of pyrene (5) [ka]

[0273] Pyrene (4) (3.7 mg, 3.8 μmol) was dissolved in N,N-dimethylformamide (400 μL) and then cooled on ice, and N,N-diisopropylethylamine (1.9 μL, 11 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.9 mg, 5.6 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (1.3 mg, 5.7 μmol) was added, and the mixture was returned to room temperature and stirred for 2 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (5) (1.3 mg, 1.1 μmol).

[0274] 1 H NMR(400 MHz,DMSO-d6)δ10.02-9.99(m,1H),8.85-7.88(m,14H),7.67-7.65(m,1H),7.60-7.51(m,2H),7.33 -7.27(m,2H),6.91-6.87(m,2H),5.92-5.91(m,1H),5.63-5.62(m,1H),5.36(brs,2H),5.07-4.92( m,2H),4.35-3.76(m,5H),3.18-3.14(m,1H),2.99-2.83(m,7H),2.17-2.13(m,2H),1.95-1.89(m,1 H),1.85-1.72(m,4H),1.66-1.45(m,3H),1.40-1.17(m,15H),1.05-1.01(m,2H),0.83-0.73(m,6H).

[0275] MS(ESI)m / z:1143.45[M+H] +

[0276] (1-1-5) Synthesis of Linker-payload mimic (1) [ka]

[0277] 1,4-dioxane (380 μL) and 4M hydrogen chloride / dioxane solution (95 μL, 380 μmol) were added to pyrene (5) (2.2 mg, 1.9 μmol) in that order, and the mixture was stirred at room temperature for 4 hours. After cooling on ice, N,N-diisopropylethylamine (71.8 μL, 418 μmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain Linker-payload mimic (1) (2.1 mg, 1.9 μmol).

[0278] 1 H NMR(400 MHz,DMSO-d6)δ12.06(brs,1H),10.03-10.00(m,1H),8.84-7.88(m,14H),7.67-7.64(m,1H),7.56-7.5 1(m,2H),7.33-7.27(m,2H),6.90-6.87(m,2H),5.92-5.90(m,1H),5.63-5.61(m,1H),5.36(brs,2H),5. 08-4.96(m,2H),4.35-3.76(m,5H),3.18-3.14(m,1H),2.97-2.83(m,7H),2.20-2.16(m,2H),1.93-1.88 (m,1H),1.81-1.78(m,4H),1.69-1.53(m,3H),1.35-1.17(m,6H),1.08-1.01(m,2H),0.81-0.73(m,6H).

[0279] MS(ESI)m / z:1087.45[M+H] +

[0280] (1-2) Synthesis of Linker-payload mimic (6) The linker-payload mimic (6) was synthesized as follows: [ka]

[0281] (1-2-1) Synthesis of alcohol (7) [ka]

[0282] Z-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 84.3 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (38.4 mg, 101 μmol) and 2,4,6-trimethylpyridine (13.3 μL, 101 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 4-aminomandelate methyl (18.3 mg, 101 μmol) was added, and the mixture was stirred at room temperature for 16 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (7) (49.1 mg, 64.9 μmol).

[0283] 1 H NMR(400 MHz,DMSO-d6)δ10.04-9.95(m,1H),8.40-7.28(m,12H),6.00-5.97(m,1H),5.43(brs,2H),5.08-4.97(m,3H),4.43-4.37(m,1H),4. 24-4.20(m,1H),4.16-4.05(m,1H),3.60-3.59(m,3H),3.04-2.91(m,2H),2.26-2.20(m,2H),2.03-1.22(m,16H),0.88-0.78(m,6H).

[0284] MS(ESI)m / z:757.30[M+H] +

[0285] (1-2-2) Synthesis of pyrene (8) [ka]

[0286] Alcohol (7) (44.6 mg, 58.9 μmol) was dissolved in N,N-dimethylformamide (650 μL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (53.8 mg, 177 μmol) and N,N-diisopropylethylamine (22.5 μL, 133 μmol) were added and stirred at room temperature for 4 hours. Then, the mixture was ice-cooled, and Sarcosin-Pyrene (89.1 mg, 295 μmol), 1-hydroxybenzotriazole (11.9 mg, 88.4 μmol), and N,N-diisopropylethylamine (77.7 μL, 457 μmol) were added and stirred at room temperature for 18 hours. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (8) (49.2 mg, 45.3 μmol).

[0287] 1 H NMR(400MHz,DMSO-d6)δ10.11-9.97(m,1H),8.64-8.60(m,1H),8.36-7.95(m,11H),7.8 2-7.74(m,1H),7.65-7.57(m,2H),7.49-7.19(m,8H),5.91-5.90(m,1H),5.72-5.70(m, 1H),5.36(brs,2H),4.98-4.86(m,4H),4.41-4.00(m,3H),3.85-3.75(m,1H),3.56-3.5 4(m,3H),3.00-2.82(m,5H),2.19-2.12(m,2H),1.92-1.17(m,16H),0.80-0.73(m,6H).

[0288] MS(ESI)m / z:1085.45[M+H] +

[0289] (1-2-3) Synthesis of pyrene (9) [ka]

[0290] Pyrene (8) (44.8 mg, 41.3 μmol) was dissolved in tetrahydrofuran (3.75 mL) and water (1.25 mL) and stirred for 5 minutes under ice cooling, after which lithium hydroxide monohydrate (8.7 mg, 210 μmol) was added and stirred at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to about 6 using 0.1 M hydrochloric acid and purified by reversed phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above pyrene (9) (18.4 mg, 17.2 μmol).

[0291] 1 H NMR(400 MHz,DMSO-d6)δ13.02(brs,1H),10.09-9.97(m,1H),8.62-8.59(m,1H),8.32-7.78(m,12H),7 .63-7.44(m,3H),7.37-7.35(m,2H),7.29-7.20(m,5H),5.91(brs,1H),5.61-5.60(m,1H),5. 36(brs,2H),4.98-4.86(m,4H),4.45-4.30(m,1H),4.24-4.15(m,1H),4.07-4.02(m,1H),3.8 4-3.74(m,1H),2.97-2.82(m,5H),2.19-2.12(m,2H),1.93-1.11(m,16H),0.80-0.72(m,6H).

[0292] MS(ESI)m / z:1071.45[M+H] +

[0293] (1-2-4) Synthesis of pyrene (10) [ka]

[0294] Pyrene (9) (15.6 mg, 14.6 μmol) was dissolved in N,N-dimethylformamide (1.0 mL) and then cooled on ice, and N,N-diisopropylethylamine (5.0 μL, 29 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (11.4 mg, 21.9 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.8 mg, 22 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (10) (15.4 mg, 12.5 μmol).

[0295] 1 H NMR(400 MHz,DMSO-d6)δ10.11-10.01(m,1H),8.91-8.63(m,1H),8.40-7.96(m,12H),7.72-7.27(m,11H),6.97-6 .94(m,2H),5.99(brs,1H),5.71-5.69(m,1H),5.43(brs,2H),5.14-4.96(m,4H),4.49-4.40(m,1H),4.3 0-4.23(m,1H),4.16-3.83(m,3H),3.25-3.22(m,1H),3.02-2.90(m,7H),2.26-2.22(m,2H),2.01-1.98( m,1H),1.92-1.84(m,1H),1.77-1.66(m,2H),1.65-1.04(m,16H),1.12-1.04(m,2H),0.88-0.80(m,6H).

[0296] MS(ESI)m / z:1235.50[M+H] +

[0297] (1-2-5) Synthesis of Linker-payload mimic (6) [ka]

[0298] 1,4-dioxane (2.0 mL) and 4M hydrogen chloride / dioxane solution (490 μL, 1.96 mmol) were added to pyrene (10) (12.1 mg, 9.79 μmol) in that order, and the mixture was stirred at room temperature for 4 hours. After cooling on ice, N,N-diisopropylethylamine (366 μL, 2.15 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain Linker-payload mimic (6) (6.0 mg, 5.1 μmol).

[0299] 1 H NMR(400 MHz,DMSO-d6)δ12.05(brs,1H),10.04-9.94(m,1H),8.84-8.57(m,1H),8.32-7.89(m,12 H),7.72-7.20(m,11H),6.90-6.86(m,2H),5.90(brs,1H),5.64-5.62(m,1H),5.36(brs, 2H),5.07-4.88(m,4H),4.42-4.29(m,1H),4.21-4.15(m,1H),4.08-3.76(m,3H),3.18-3 .14(m,1H),2.95-2.82(m,7H),2.21-2.16(m,2H),1.92-0.97(m,13H),0.82-0.74(m,6H).

[0300] MS(ESI)m / z:1179.50[M+H] +

[0301] (1-3) Synthesis of Linker-payload mimic (11) Linker-payload mimic (11) was synthesized as follows. [ka]

[0302] (1-3-1) Synthesis of alcohol (12) [ka]

[0303] Ac-Glu(t-Bu)-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 72.8 μmol) was dissolved in N,N-dimethylformamide (800 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (33.2 mg, 87.4 μmol) and 2,4,6-trimethylpyridine (11.5 μL, 87.4 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 4-aminomandelate methyl (15.8 mg, 87.4 μmol) was added, and the mixture was stirred at room temperature for 16 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (12) (54.0 mg, 63.5 μmol).

[0304] 1 H NMR(400 MHz,DMSO-d6)δ10.00(s,1H),8.26-7.88(m,3H),7.68-7.60(m,1H),7.57(d,J=8.4 Hz,2H),7.32(d,J=8.4 Hz,2H),6.00-5.97(m,1H),5.43(brs,2H),5.08(s,1H),4.40-4.37(m,1H),4.32-4.19(m,3H),3.60(s,3H),3.09-2. 90(m,2H),2.25-2.18(m,4H),2.03-1.53(m,10H),1.46-1.36(m,20H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.8Hz,3H).

[0305] MS(ESI)m / z:850.40[M+H] +

[0306] (1-3-2) Synthesis of pyrene (13) [ka]

[0307] Alcohol (12) (50.3 mg, 59.2 μmol) was dissolved in N,N-dimethylformamide (650 μL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (54.0 mg, 178 μmol) and N,N-diisopropylethylamine (22.7 μL, 133 μmol) were added and stirred at room temperature for 5 hours. Then, the mixture was ice-cooled, and Sarcosin-Pyrene (89.5 mg, 296 μmol), 1-hydroxybenzotriazole (12.0 mg, 88.8 μmol), and N,N-diisopropylethylamine (78.1 μL, 459 μmol) were added and stirred at room temperature for 18 hours. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (13) (34.0 mg, 28.9 μmol).

[0308] 1 H NMR(400 MHz,DMSO-d6)δ10.14-10.12(m,1H),8.69-8.67(m,1H),8.40-7.82(m,13H),7.76-7. 72(m,1H),7.68-7.65(m,2H),7.44-7.39(m,2H),5.99(brs,1H),5.79(d,J=6.4Hz,1H) ,5.44(brs,2H),5.05-4.97(m,2H),4.44-4.08(m,4H),3.93-3.80(m,1H),3.63-3.62 (m,3H),3.05-2.92(m,5H),2.25-2.14(m,4H),2.00-1.28(m,30H),0.87-0.80(m,6H).

[0309] MS(ESI)m / z:1178.50[M+H] +

[0310] (1-3-3) Synthesis of pyrene (14) [ka]

[0311] Pyrene (13) (30.7 mg, 26.1 μmol) was dissolved in tetrahydrofuran (2.25 mL) and water (0.75 mL) and stirred for 5 minutes under ice cooling, after which lithium hydroxide monohydrate (5.5 mg, 0.13 mmol) was added and stirred at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to about 6 using 0.1 M hydrochloric acid and purified by reversed phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and the product was freeze-dried to obtain the above pyrene (14) (17.2 mg, 14.8 μmol).

[0312] 1 H NMR(400 MHz,DMSO-d6)δ13.06(brs,1H),10.13-10.10(m,1H),8.69-8.66(m,1H),8.40-7.85 (m,13H),7.77-7.73(m,1H),7.67-7.64(m,2H),7.44-7.42(m,2H),5.99(brs,1H),5 .68-5.67(m,1H),5.44(brs,2H),5.05-4.96(m,2H),4.46-4.10(m,4H),3.92-3.81( m,1H),3.05-2.90(m,5H),2.25-2.14(m,4H),2.03-1.29(m,30H),0.88-0.80(m,6H).

[0313] MS(ESI)m / z:1164.55[M+H] +

[0314] (1-3-4) Synthesis of pyrene (15) [ka]

[0315] Pyrene (14) (14.7 mg, 12.6 μmol) was dissolved in N,N-dimethylformamide (1.0 mL), cooled on ice, and N,N-diisopropylethylamine (4.29 μL, 25.2 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (9.8 mg, 19 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.1 mg, 19 μmol) was added, and the mixture was returned to room temperature and stirred for 3.5 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (15) (7.0 mg, 9.2 μmol).

[0316] 1 H NMR(400 MHz,DMSO-d6)δ10.08-10.05(m,1H),8.92-7.95(m,15H),7.75-7.73(m,1H),7.63-7.59(m ,2H),7.40-7.34(m,2H),6.97-6.94(m,2H),6.00-5.98(m,1H),5.70-5.69(m,1H),5.43(br s,2H),5.14-5.01(m,2H),4.45-4.38(m,1H),4.32-3.83(m,5H),3.25-3.20(m,1H),3.10- 2.90(m,7H),2.25-2.18(m,4H),2.08-1.24(m,34H),1.12-1.04(m,2H),0.88-0.81(m,6H).

[0317] MS(ESI)m / z:1328.60[M+H] +

[0318] (1-3-5) Synthesis of Linker-payload mimic (11) [ka]

[0319] 1,4-dioxane (920 μL) and 4M hydrogen chloride / dioxane solution (230 μL, 918 μmol) were added to pyrene (15) (6.1 mg, 4.6 μmol) in that order, and the mixture was stirred at room temperature for 4 hours. After cooling on ice, N,N-diisopropylethylamine (172 μL, 1.10 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain Linker-payload mimic (11) (3.7 mg, 3.0 μmol).

[0320] 1 H NMR(400 MHz,DMSO-d6)δ12.04(brs,2H),10.01-9.98(m,1H),8.83-7.89(m,15H),7.71-7.69(m,1H),7 .56-7.51(m,2H),7.33-7.26(m,2H),6.90-6.87(m,2H),5.91-5.90(m,1H),5.64-5.62(m,1H), 5.36(brs,2H),5.08-4.92(m,2H),4.35-4.32(m,1H),4.25-3.76(m,5H),3.18-3.14(m,1H),2. 99-2.82(m,7H),2.21-2.15(m,4H),1.93-1.17(m,16H),1.05-1.01(m,2H),0.82-0.74(m,6H).

[0321] MS(ESI)m / z:1216.45[M+H] +

[0322] (1-4) Synthesis of Linker-payload mimic (16) Linker-payload mimic (16) was synthesized as follows. [ka]

[0323] (1-4-1) Synthesis of alcohol (17) [ka]

[0324] SCA(t-Bu)-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 81.2 μmol) was dissolved in N,N-dimethylformamide (890 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (37.0 mg, 97.4 μmol) and 2,4,6-trimethylpyridine (12.8 μL, 97.4 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 4-aminomandelate methyl (17.6 mg, 97.4 μmol) was added, and the mixture was stirred at room temperature for 16 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (17) (60.4 mg, 77.5 μmol).

[0325] 1 H NMR(400 MHz,DMSO-d6)δ10.01(s,1H),8.13(d,J=7.2 Hz,1H),8.07(d,J=8.4 Hz,1H),7.73(d,J=8.8 Hz,1H),7.57(d,J=8.4 Hz,2H),7.32(d,J=8.4 Hz,2H),5.99(brs,1H),5.43(brs,2H),5.08(s,1H),4.41-4.30(m,2H),4.21-4.17(m,1H),3.60(s,3H),3.04-2.95(m,2H),2.44-2.1 5(m,6H),2.03-1.95(m,1H),1.92-1.83(m,1H),1.74-1.58(m,3H),1.46-1.34(m,20H),0.86(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).

[0326] MS(ESI)m / z:779.40[M+H] +

[0327] (1-4-2) Synthesis of pyrene (18) [ka]

[0328] Alcohol (17) (58.0 mg, 74.5 μmol) was dissolved in N,N-dimethylformamide (820 μL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (68.0 mg, 223 μmol) and N,N-diisopropylethylamine (28.5 μL, 168 μmol) were added and stirred at room temperature for 6 hours. Then, the mixture was ice-cooled, and sarcosin-pyrene (113 mg, 373 μmol), 1-hydroxybenzotriazole (15.1 mg, 112 μmol), and N,N-diisopropylethylamine (98.3 μL, 578 μmol) were added and stirred at room temperature for 17 hours. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (18) (37.1 mg, 33.5 μmol).

[0329] 1 H NMR(400 MHz,DMSO-d6)δ10.15-10.13(m,1H),8.70-8.67(m,1H),8.40-8.03(m,11H),7.90-7.86(m,1H),7.75-7. 72(m,1H),7.68-7.64(m,2H),7.44-7.39(m,2H),5.99(brs,1H),5.79(d,J=6.8Hz,1H),5.44(brs,2H),5. 05-4.98(m,2H),4.40-4.08(m,3H),3.93-3.80(m,1H),3.63-3.62(m,3H),3.05-2.90(m,5H),2.44-2.15 (m,6H),2.02-1.98(m,1H),1.91-1.85(m,1H),1.75-1.55(m,3H),1.50-1.30(m,20H),0.88-0.80(m,6H).

[0330] MS(ESI)m / z:1107.55[M+H] +

[0331] (1-4-3) Synthesis of pyrene (19) [ka]

[0332] Pyrene (18) (17.4 mg, 15.7 μmol) was dissolved in tetrahydrofuran (675 μL) and water (225 μL) and stirred for 5 minutes under ice cooling, then 1 M lithium hydroxide aqueous solution (86.4 μL, 86.4 μmol) was added and stirred for 5 hours under ice cooling. After the reaction was completed, the pH was adjusted to about 6 using 0.1 M hydrochloric acid and purified by reversed phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (19) (17.2 mg, 15.7 μmol).

[0333] 1 H NMR(400 MHz,DMSO-d6)δ13.06(brs,1H),10.14-10.11(m,1H),8.68-8.66(m,1H),8.40-8.01(m,11H),7.89-7.8 5(m,1H),7.75-7.72(m,1H),7.67-7.64(m,2H),7.44-7.41(m,2H),5.99(brs,1H),5.68-5.67(m,1H),5 .44(brs,2H),5.05-4.93(m,2H),4.44-4.10(m,3H),3.92-3.81(m,1H),3.05-2.94(m,5H),2.43-2.15( m,6H),2.02-1.97(m,1H),1.88-1.85(m,1H),1.77-1.66(m,3H),1.61-1.30(m,20H),0.88-0.80(m,6H).

[0334] MS(ESI)m / z:1093.50[M+H] +

[0335] Synthesis of (1-4-4)pyrene (20) [ka]

[0336] Pyrene (19) (16.3 mg, 14.9 μmol) was dissolved in N,N-dimethylformamide (1.0 mL) and then cooled on ice. N,N-diisopropylethylamine (5.1 μL, 30 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (11.7 mg, 22.4 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.9 mg, 22 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (20) (12.1 mg, 9.62 μmol).

[0337] 1 H NMR(400 MHz,DMSO-d6)δ10.01-9.98(m,1H),8.85-7.88(m,14H),7.68-7.65(m,1H),7.56-7.51(m,2H),7.33 -7.27(m,2H),6.90-6.87(m,2H),5.92-5.90(m,1H),5.63-5.62(m,1H),5.36(brs,2H),5.08-4.92( m,2H),4.36-3.76(m,5H),3.18-3.14(m,1H),3.00-2.83(m,7H),2.36-2.08(m,6H),1.95-1.89(m,1 H),1.84-1.78(m,1H),1.68-1.50(m,3H),1.42-1.17(m,24H),1.07-0.97(m,2H),0.81-0.74(m,6H).

[0338] MS(ESI)m / z:1257.55[M+H] +

[0339] (1-4-5) Synthesis of Linker-payload mimic (16) [ka]

[0340] Ethyl acetate (1.7 mL) and 4M hydrogen chloride / ethyl acetate (2.09 mL, 8.36 mmol) were added to pyrene (20) (10.5 mg, 8.35 μmol) in that order, and the mixture was stirred at room temperature for 4.5 hours. After cooling on ice, N,N-diisopropylethylamine (781 μL, 4.59 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain Linker-payload mimic (16) (7.5 mg, 6.6 μmol).

[0341] 1 H NMR(400 MHz,DMSO-d6)δ12.02(brs,2H),10.04-9.98(m,1H),8.85-7.89(m,14H),7.65-7.62(m,1H), 7.56-7.52(m,2H),7.33-7.27(m,2H),6.90-6.87(m,2H),5.92(brs,1H),5.63-5.62(m,1H),5 .37(brs,2H),5.08-4.93(m,2H),4.36-3.76(m,5H),3.18-3.14(m,1H),3.02-2.80(m,7H),2. 38-2.16(m,6H),1.96-1.77(m,2H),1.70-1.17(m,9H),1.06-0.98(m,2H),0.81-0.74(m,6H).

[0342] MS(ESI)m / z:1145.45[M+H] +

[0343] (1-5) Synthesis of Linker-payload mimic (21) Linker-payload mimic (21) was synthesized as follows. [ka]

[0344] (1-5-1) Synthesis of alcohol (22) [ka]

[0345] Ac-Glu(OtBu)-Val-Ala-OH (20.9 mg, 50.3 μmol) was dissolved in N,N-dimethylformamide (700 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (29.0 mg, 76.3 μmol) and 2,4,6-trimethylpyridine (10.1 μL, 76.7 μmol) were added, and the mixture was stirred at room temperature for 12 minutes. Subsequently, 4-aminomandelate methyl (11.0 mg, 60.7 μmol) was added, and the mixture was stirred at room temperature for 18 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (22) (20.3 mg, 35.1 μmol).

[0346] 1 H NMR(400 MHz,DMSO-d6)δ9.98-9.89(m,1H),8.32-8.18(m,1H),8.07-7.85(m,1H),7.73-7.59(m,1H),7.55(d,J=8.4 Hz,2H),7.32(d,J=8.4 Hz,2H),5.08(s,1H),4.45-4.34(m,1H),4.32-4.27(m,1H),4.20-4.09(m,1H),3.59(s,3H),2.24-2.20(m,2H),2.01-1.96(m,1H) ),1.90-1.80(m,4H),1.72-1.67(m,1H),1.39-1.36(m,9H),1.30(d,J=7.2Hz,3H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.8Hz,3H).

[0347] MS(ESI)m / z:579.30[M+H] +

[0348] (1-5-2) Synthesis of pyrene (23) [ka]

[0349] Alcohol (22) (17.5 mg, 30.2 μmol) was dissolved in N,N-dimethylformamide (174 μL) and stirred for 5 minutes under ice cooling, then bis(4-nitrophenyl)carbonate (18.9 mg, 60.8 μmol) and N,N-diisopropylethylamine (7.80 μL, 45.4 μmol) were added and stirred at room temperature for 30 minutes. LCMS showed that the raw material remained, so bis(4-nitrophenyl)carbonate (9.5 mg, 31 μmol) and N,N-diisopropylethylamine (3.90 μL, 22.7 μmol) were added and stirred at room temperature for 1 hour. After that, the mixture was cooled on ice, and sarcosin-pyrene (36.2 mg, 120 μmol), 1-hydroxybenzotriazole (6.3 mg, 47 μmol), and N,N-diisopropylethylamine (40.3 μL, 235 μmol) were added, followed by stirring at room temperature for 1 hour. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (23) (12.9 mg, 14.2 μmol).

[0350] 1 H NMR(400 MHz,DMSO-d6)δ10.05-10.02(m,1H),8.61-8.60(m,1H),8.30-7.78(m,12H),7.67-7.64(m,1 H),7.57(d,J=8.8Hz,2H),7.37-7.33(m,2H),5.72-5.70(m,1H),4.98-4.89(m,2H),4.34-4. 00(m,3H),3.85-3.75(m,1H),3.56-3.55(m,3H),2.93-2.87(m,3H),2.17-2.13(m,2H),1.94 -1.91(m,1H),1.85-1.75(m,4H),1.65-1.61(m,1H),1.31-1.23(m,12H),0.80-0.73(m,6H).

[0351] MS(ESI)m / z:907.35[M+H] +

[0352] (1-5-3) Synthesis of pyrene (24) [ka]

[0353] Pyrene (23) (11.8 mg, 13.0 μmol) was dissolved in tetrahydrofuran (800 μL) and water (400 μL), and then cooled on ice. 1 M lithium hydroxide aqueous solution (65 μL, 65 μmol) was added and stirred for 30 minutes. After the reaction was completed, the pH was adjusted to about 6 using 0.1 M hydrochloric acid, and purified by reversed phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (24) (8.9 mg, 10.0 μmol).

[0354] 1 H NMR(400 MHz,DMSO-d6)δ13.05(brs,1H),10.04-9.91(m,1H),8.63-8.60(m,1H),8.33-7.77(m ,12H),7.69-7.55(m,3H),7.37-7.35(m,2H),5.61-5.60(m,1H),4.98-4.86(m,2H),4. 26-4.03(m,3H),3.84-3.74(m,1H),2.93-2.87(m,3H),2.18-2.13(m,2H),1.95-1.92 (m,1H),1.85-1.77(m,4H),1.67-1.62(m,1H),1.31-1.24(m,12H),0.81-0.75(m,6H).

[0355] MS(ESI)m / z:893.35[M+H] +

[0356] (1-5-4) Synthesis of pyrene (25) [ka]

[0357] Pyrene (24) (6.7 mg, 7.5 μmol) was dissolved in N,N-dimethylformamide (400 μL) and then cooled on ice, and N,N-diisopropylethylamine (3.9 μL, 22 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (5.9 mg, 11 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (2.4 mg, 11 μmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (25) (4.1 mg, 3.9 μmol).

[0358] 1 H NMR(400 MHz,DMSO-d6)δ9.98-9.95(m,1H),8.83-7.77(m,14H),7.67-7.65(m,1H),7.56-7.50(m,2 H),7.34-7.27(m,2H),6.89-6.86(m,2H),5.63-5.61(m,1H),5.06-4.93(m,2H),4.37-3.76 (m,5H),3.17-3.14(m,1H),2.95-2.83(m,5H),2.17-2.10(m,2H),1.95-1.89(m,1H),1.85- 1.75(m,4H),1.65-1.62(m,1H),1.31-1.17(m,16H),1.02-1.00(m,2H),0.81-0.74(m,6H).

[0359] MS(ESI)m / z:1057.45[M+H] +

[0360] (1-5-5) Synthesis of Linker-payload mimic (21) [ka]

[0361] Pyrene (25) (2.4 mg, 2.3 μmol) was added with 1,4-dioxane (454 μL) and 4M hydrogen chloride / dioxane solution (568 μL, 2.27 mmol) in that order, and stirred at room temperature for 4 hours. N,N-dimethylformamide (300 μL) was added, and then N,N-diisopropylethylamine (214 μL, 1.25 mmol) was added under ice cooling, and stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload mimic (21) (1.1 mg, 1.1 μmol).

[0362] 1 H NMR(400 MHz,DMSO-d6)δ11.99(brs,1H),9.98-9.96(m,1H),8.84-7.88(m,14H),7.66-7.63(m,1H),7.5 4-7.50(m,2H),7.34-7.27(m,2H),6.90-6.87(m,2H),5.63-5.61(m,1H),5.07-4.93(m,2H),4. 36-3.76(m,5H),3.17-3.14(m,1H),2.95-2.83(m,5H),2.20-2.16(m,2H),1.95-1.90(m,1H),1 .88-1.76(m,4H),1.67-1.62(m,1H),1.32-1.17(m,7H),1.02-1.01(m,2H),0.81-0.74(m,6H).

[0363] MS(ESI)m / z:999.35[MH] ―

[0364] Comparative Example 1: Synthesis of Linker-payload (1-1) Synthesis of Linker-Payload (26) Linker-Payload (26) was synthesized as follows. [ka]

[0365] Linker-Payload (26) was synthesized according to the following scheme. [ka]

[0366] The MS analysis results of the Linker-Payload (26) were as follows.

[0367] MS(ESI)m / z:1050.55[M+H] +

[0368] Example 2: Synthesis of ADC mimics (2-1) Synthesis of ADC mimics In the following Comparative Examples and Examples, the antibody derivative (thiol group-introduced trastuzumab) described in Examples 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used as the thiol group-introduced antibody. This antibody derivative has the following structure in which a thiol group is regioselectively introduced into trastuzumab (humanized IgG1 antibody) via the amino group of the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue is according to EU numbering). [ka] (In the above structure, NH-CH2-CH2-CH2-CH2- extending from the antibody heavy chain corresponds to the side chain of a lysine residue, and a thiol-containing group, HS-CH2-CH2-C(=O), is added to the amino group in the side chain of this lysine residue. Since no modification with other lysine residues was detected by peptide mapping method, this antibody is considered to have 100% position selectivity at positions 246 and 248 of the antibody heavy chain.)

[0369] To a solution (20 μM) of the thiol group-introduced antibody in buffer (pH 7.4 PBS buffer), 10 equivalents of a DMF solution (10 mM) of the linker-payload mimic synthesized in Example 1 was added, and the mixture was left to stand at room temperature for 2 hours. After that, the mixture was purified using NAP-5 Columns (GE Healthcare) to obtain an ADC mimic.

[0370] ADC mimic 1 having the following structure was synthesized from the linker-payload mimic (1) synthesized in Example 1-1 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 150-350, where two linker-payload mimics (1) were introduced. [ka]

[0371] Similarly, ADC mimic 2 having the following structure was synthesized from the linker-payload mimic (6) of Example 1-2 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and peaks were confirmed at 150535 for the reaction product, where two linker-payload mimics (6) were introduced. [ka]

[0372] Similarly, ADC mimic 3 having the following structure was synthesized from the linker-payload mimic (11) of Example 1-3 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and a peak was confirmed at 150609, where two linker-payload mimics (11) were introduced, for the reaction product. [ka]

[0373] Similarly, ADC mimic 4 having the following structure was synthesized from the linker-payload mimic (16) of Example 1-4 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 150466, where two linker-payload mimics (16) were introduced. [ka]

[0374] Similarly, ADC mimic 5 having the following structure was synthesized from the linker-payload mimic (26) of Comparative Example 1 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and peaks were confirmed at 150276 for the reaction product, in which two linker-payload mimics (26) were introduced. [ka]

[0375] (2-2)DAR analysis of ADC mimics ESI-TOFMS analysis of the ADC mimic synthesized in Example 2-1 was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2.

[0376] [Table 1]

[0377] Example 3: Evaluation of hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC) HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). Measurements were performed under the following conditions. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram.

[0378] Measurement system: Chromaster (registered trademark) (Hitachi) Column: Tosoh Biobuthyl NPR 2.5μm 4.6×35mm column manufactured by Tosoh Biosciences Gradient: Linear gradient of eluent A / B Flow rate: 0.8mL / min Eluent A: 1.1M (NH4)2SO4, 25mM Na2HPO4 / NaH2PO4 (pH6.0) Eluent B: 25 mM Na2HPO4 / NaH2PO4 (pH 6.0, 25 v / v% isopropanol added) Detector: UV (280 nm)

[0379] [Table 2]

[0380] As a result, it was confirmed that the ADC mimics synthesized in Examples 1-1, 1-2, 1-3, and 1-4 tended to have a short retention time, indicating that they have high hydrophilicity. Therefore, it was confirmed that the ADC mimics synthesized in Examples 1-1, 1-2, 1-3, and 1-4 are preferable ADCs because they are considered to have a slow plasma clearance and a long retention time in the body.

[0381] Example 4: Assessment of aggregation rates of ADCs and ADC mimics by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to a previous report (Chemistry Select, 2020, 5, 8435-8439). The measurement was performed under the following conditions.

[0382] Measurement system: 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300Å 2.7μm, 4.6mm×150mm Flow rate: 0.25mL / min Eluent: 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride aqueous solution (pH 6.8), 10% v / v isopropanol Detector: UV (280 nm)

[0383] [Table 3]

[0384] Example 5: Evaluation of ADC mimics using the enzyme cathepsin B The cleavage ability of various ADC mimics by cathepsin B was evaluated by analyzing the amount of fluorescent molecules shed from the ADC mimics as described below.

[0385] (5-1) Cathepsin B cleavage assay The experiment was carried out as follows according to a previous report (Nature Communications 2018,9,2512). ADC mimic was added to 180 μL of MES buffer (10 mM MES, 40 μM DTT, pH 5.0) to a concentration of 1 mg / mL, and then 30 μL was dispensed into six Eppendorf tubes. Three of the six samples were immediately added with 100 μL of acetonitrile at 0 °C, stirred with a vortex, and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and analyzed by HPLC. The remaining three were incubated at 37 °C for 6 hours. Each sample was added with 100 μL of acetonitrile, stirred with a vortex, and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and analyzed by HPLC.

[0386] (5-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis The measurement was performed by measuring the molecular weight of the fluorescence dropped from the ADC mimic using liquid chromatography / fluorescence detection. Three samples to which acetonitrile was immediately added at 0°C in Example 7-1 were set as 0 hour samples, and three samples incubated at 37°C for 6 hours as described in Example 7-1 were set as 6 hour samples, and the difference in fluorescence intensity between the 6 hour samples and the 0 hour samples was analyzed.

[0387] Separately, the correlation between the area of ​​fluorescence intensity by HPLC and the concentration was calculated using pyrene. The difference in fluorescence intensity of each ADC mimic was converted to concentration using this calculation formula. The dropout rate was calculated as the ratio of the difference in fluorescence intensity to the concentration at 0 time, which was set to 100%.

[0388] [Table 4]

[0389] As shown in Table 4, the synthesized ADC mimics were found to have sufficient cathepsin B cleavage.

[0390] Example 6: Evaluation of ADC mimics using mouse plasma (6-1) Plasma stability test of ADC mimics ADC mimic was added to 500 μL of mouse plasma (Charles River) to a concentration of 0.1 mg / mL, and then sterilized and filtered. This solution was dispensed into six Eppendorf tubes at 50 μL each. Three of the six samples were stored in an incubator set at 37°C for four days. The remaining three were similarly stored in a freezer at -80°C for four days. Each sample was added with 100 μL of acetonitrile, stirred with a vortex mixer, and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0391] (6-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis The measurement was performed by using liquid chromatography / fluorescence detection to measure the molecular weight of the fluorescence dropped from the ADC mimic. The three samples stored in the freezer in Example 9-1 were taken as Day = 0, and the three samples stored at 37°C in Example 9-1 were taken as Day = 4, and the difference in fluorescence intensity between Day = 4 and Day = 0 was analyzed.

[0392] The loss rate of fluorescent molecules was calculated according to Example 5-2. The loss rate of fluorescent molecules was evaluated as shown in the table below.

[0393] [Table 5]

[0394] As a result, compared to the ADC mimic synthesized in Comparative Example 1, the ADC mimics synthesized in Examples 1-1 and 1-2 exhibited three times or more greater stability, and the ADC mimics synthesized in Examples 1-3 and 1-4 exhibited ten times or more greater stability.

[0395] Example 7: Synthesis of Linker-payload (7-1) Synthesis of Linker-payload (35) Linker-payload (35) was synthesized as follows. [ka]

[0396] (7-1-1) Synthesis of carbonate (36) [ka]

[0397] The alcohol (2) (105 mg, 0.158 mmol) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (2 mL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (100 mg, 0.329 mmol) and N,N-diisopropylethylamine (83 μL, 0.48 mmol) were added and stirred for 19.5 hours at room temperature under a nitrogen atmosphere. After removing the N,N-dimethylformamide with an evaporator, the obtained crude product was dissolved in ethyl acetate (3 mL) and then diethyl ether (3 mL) was added. The obtained solution was filtered to remove the residue, and the organic solvent was removed by a vacuum pump to obtain carbonate (36) (102 mg, 0.123 mmol).

[0398] MS(ESI) m / z:830.1[M+H] + ,852.1[M+Na] +

[0399] (7-1-2) Synthesis of compound (37) [ka]

[0400] Compound (36) (69 mg, 0.083 mmol) obtained in (7-1-1) was dissolved in N,N-dimethylformamide (3.5 mL), and 1-hydroxybenzotriazole (16 mg, 0.10 mmol) and commercially available monomethylauristatin E (MMAE, 61 mg, 0.085 mmol) were added at room temperature. Diisopropylethylamine (29 μL, 0.17 mmol) was then added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 22.5 hours. After removing the organic solvent with an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reversed phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 37 (79 mg, 0.056 mmol).

[0401] MS(ESI) m / z:1408.9[M+H] +

[0402] (7-1-3) Synthesis of compound (38) [ka]

[0403] Compound (37) (97 mg, 0.069 mmol) obtained in (7-1-2) was dissolved in tetrahydrofuran (7 mL) and water (2 mL), lithium hydroxide (1.0 M, 1.4 mL, 1.4 mmol) was added under ice cooling, and the mixture was stirred for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 6, and then acetonitrile:water = 1:1 was added and the mixture was purified by reversed phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and the mixture was lyophilized to obtain compound 38 (70 mg, 0.050 mmol).

[0404] MS(ESI) m / z:1394.7[M+H] +

[0405] (7-1-4) Synthesis of compound (39) [ka]

[0406] Compound (38) (34 mg, 0.024 mmol) obtained in (7-1-3) was dissolved in N,N-dimethylformamide (3 mL), cooled on ice, and N,N-diisopropylethylamine (25 μL, 0.14 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (20 mg, 0.038 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (8.7 mg, 0.040 mmol) was added, and the mixture was returned to room temperature and stirred for 20 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain compound (39) (29 mg, 0.019 mmol).

[0407] MS(ESI) m / z:1558.9[M+H] +

[0408] (7-1-5) Synthesis of Linker-payload (35) [ka]

[0409] Acetonitrile (500 μL) and 85 wt% aqueous phosphoric acid solution (0.50 mL, 7.3 mmol) were added to the compound (39) (29 mg, 0.019 mmol) obtained in (7-1-4) and stirred at room temperature for 3 hours. After the reaction was completed, water (2 mL) was added, the reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload (35) (18 mg, 0.012 mmol).

[0410] MS(ESI) m / z:1502.9[M+H] +

[0411] (7-2) Synthesis of Linker-payload (40) Linker-payload (40) was synthesized as follows. [ka]

[0412] (7-2-1) Synthesis of compound (41) [ka]

[0413] Compound (38) (10 mg, 0.0072 mmol) obtained in (7-1-3) was dissolved in N,N-dimethylformamide (1 mL) and then cooled on ice, and N,N-diisopropylethylamine (10 μL, 0.057 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (7.0 mg, 0.013 mmol) were added. Next, a solution of DBCO-hexylamine (4.7 mg, 0.015 mmol) in N,N-dimethylformamide (0.5 mL) was added, and the mixture was returned to room temperature and stirred for 20 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain compound (41) (5.8 mg, 0.0034 mmol).

[0414] MS(ESI) m / z:1696.0[M+H] +

[0415] (7-2-2) Synthesis of Linker-payload (40) [ka]

[0416] Acetonitrile (500 μL) and 85 wt% phosphoric acid aqueous solution (0.50 mL, 7.3 mmol) were added to the compound (41) (13 mg, 0.0077 mmol) obtained in (7-2-1) and stirred at room temperature for 4 hours. After the reaction was completed, water (2 mL) was added, the reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload (40) (7.4 mg, 0.0045 mmol).

[0417] MS(ESI) m / z:1638.9[M+H] +

[0418] (7-3) Synthesis of Linker-payload (42) Linker-payload (42) was synthesized as follows. [ka]

[0419] (7-3-1) Synthesis of carbonate (43) [ka]

[0420] The alcohol (2) (140 mg, 0.165 mmol) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (4 mL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (108 mg, 0.355 mmol) and N,N-diisopropylethylamine (100 μL, 0.574 mmol) were added and stirred for 18 hours at room temperature under a nitrogen atmosphere. After removing the organic solvent with an evaporator, a solution of acetonitrile:water = 1:1 was added and purified by reverse phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 43 (130 mg, 0.128 mmol).

[0421] MS(ESI) m / z:1015.6[M+H] +

[0422] (7-3-2) Synthesis of compound (44) [ka]

[0423] Compound (43) (85 mg, 0.084 mmol) obtained in (7-3-1) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (20 mg, 0.13 mmol) and commercially available monomethylauristatin E (MMAE, 63 mg, 0.088 mmol) were added at room temperature. Diisopropylethylamine (75 μL, 0.43 mmol) was then added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 23 hours. After removing the organic solvent with an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reversed phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 44 (94 mg, 0.059 mmol).

[0424] MS(ESI) m / z:1593.6[M+H] +

[0425] (7-3-3) Synthesis of compound (45) [ka]

[0426] Compound (44) (94 mg, 0.059 mmol) obtained in (7-3-2) was dissolved in tetrahydrofuran (5 mL) and water (2 mL), lithium hydroxide (1.0 M, 0.6 mL, 0.6 mmol) was added under ice cooling, and the mixture was stirred for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 5, and then acetonitrile:water = 1:1 was added and the mixture was purified by reversed phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 45 (77 mg, 0.049 mmol).

[0427] MS(ESI) m / z:1579.7[M+H] +

[0428] (7-3-4) Synthesis of compound (46) [ka]

[0429] Compound (45) (77 mg, 0.049 mmol) obtained in (7-3-3) was dissolved in N,N-dimethylformamide (3 mL), cooled on ice, and N,N-diisopropylethylamine (50 μL, 0.29 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (87 mg, 0.17 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (35 mg, 0.16 mmol) was added, and the mixture was returned to room temperature and stirred for 20 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain compound (46) (65 mg, 0.037 mmol).

[0430] MS(ESI) m / z:1744.7[M+H] +

[0431] (7-3-5) Synthesis of Linker-payload (42) [ka]

[0432] Acetonitrile (2 mL) and 85 wt% phosphoric acid aqueous solution (1.00 mL, 14.6 mmol) were added to the compound (46) (65 mg, 0.037 mmol) obtained in (7-3-4) and stirred at room temperature for 6 hours. After the reaction was completed, water (2 mL) was added, the reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload (42) (49 mg, 0.030 mmol).

[0433] MS(ESI) m / z:1631.6[M+H] +

[0434] (7-4) Synthesis of Linker-payload (47) Linker-payload (47) was synthesized as follows. [ka]

[0435] (7-4-1) Synthesis of compound (48) [ka]

[0436] Compound (43) (67 mg, 0.066 mmol) obtained in (7-3-1) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (17 mg, 0.11 mmol) and commercially available Exatecan mesylate (CAS: 169869-90-3, 35 mg, 0.066 mmol) were added at room temperature. Diisopropylethylamine (50 μL, 0.29 mmol) was then added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. After removing the organic solvent with an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reverse phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 48 (57 mg, 0.043 mmol).

[0437] MS(ESI) m / z:1311.7[M+H] +

[0438] (7-4-2) Synthesis of compound (49) [ka]

[0439] Compound (48) (57 mg, 0.043 mmol) obtained in (7-4-1) was dissolved in tetrahydrofuran (3 mL) and water (1.5 mL), and lithium hydroxide (1.0 M, 0.5 mL, 0.5 mmol) was added under ice cooling, followed by stirring for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 5, and then acetonitrile:water = 1:1 was added, followed by purification by reversed phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 49 (45 mg, 0.035 mmol).

[0440] MS(ESI) m / z:1297.6[M+H] +

[0441] (7-4-3) Synthesis of compound (50) [ka]

[0442] Compound (49) (45 mg, 0.035 mmol) obtained in (7-4-2) was dissolved in N,N-dimethylformamide (3 mL), cooled on ice, and N,N-diisopropylethylamine (30 μL, 0.17 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (55 mg, 0.11 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (22 mg, 0.10 mmol) was added, and the mixture was returned to room temperature and stirred for 18 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain compound (50) (22 mg, 0.015 mmol).

[0443] MS(ESI) m / z:1462.7[M+H] +

[0444] (7-3-5) Synthesis of Linker-payload (47) [ka]

[0445] Acetonitrile (1 mL) and 85 wt% phosphoric acid aqueous solution (1.0 mL, 14.6 mmol) were added to the compound (50) (22 mg, 0.015 mmol) obtained in (7-4-3) in order, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, water (1 mL) was added, and the reaction solution was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload (47) (18.8 mg, 0.0139 mmol).

[0446] 1 H NMR(300MHz;DMSO-d6) δ 10.04(s,1H),8.10-8.05(m,4H),7.80-7.77(m,2H),7.59-7.55(m,2H),7.35-7.30(m,3H),6 .99(d,J=8.8Hz,2H),6.54-6.53(m,1H),6.01(brs,1H),5.74(d,J=9.0Hz,1H),5.43(brs,4H) ,5.33-5.28(m,2H),4.29-4.15(m,4H),3.19-2.98(m5H),2.43-2.38(m,5H),2.27-2.20(m,7 H),1.95-1.83(m,8H),1.73-1.62(m,4H),1.42-1.30(m,7H),1.23-1.13(m,3H),0.84(m,9H).

[0447] MS(ESI) m / z:1349.2[M+H] +

[0448] (7-5) Synthesis of Linker-payload(125) The linker-payload (125) shown below was synthesized in a similar manner to the synthesis of the linker-payload mimic (120), using MMAE instead of sarcosine-pyrene. [ka]

[0449] MS(ESI)m / z:1968.14[M+H] +

[0450] (7-6) Synthesis of Linker-payload(126) The linker-payload (126) shown below was synthesized in the same manner as in the synthesis of the linker-payload mimic (35), except that Exatecan mesylate was used instead of MMAE. [ka]

[0451] MS(ESI)m / z:1220.50[M+H] +

[0452] Example 8: Synthesis of ADCs (8-1) Synthesis of ADC4 In the following Comparative Examples and Examples, the antibody derivative (thiol group-introduced trastuzumab) described in Examples 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used as the thiol group-introduced antibody. This antibody derivative has the following structure in which a thiol group is regioselectively introduced into trastuzumab (humanized IgG1 antibody) via the amino group of the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue is according to EU numbering). [ka] (In the above structure, NH-CH2-CH2-CH2-CH2- extending from the antibody heavy chain corresponds to the side chain of a lysine residue, and a thiol-containing group, HS-CH2-CH2-C(=O), is added to the amino group in the side chain of this lysine residue. Since no modification with other lysine residues was detected by peptide mapping method, this antibody is considered to have 100% position selectivity at positions 246 and 248 of the antibody heavy chain.)

[0453] 10 equivalents of a DMF solution (10 mM) of the linker payload (35) synthesized in Example 12-1 was added to a buffer (pH 7.4 PBS buffer) solution (20 μM) of the thiol group-introduced antibody, and the mixture was left to stand at room temperature for 2 hours, and then purified using NAP-5 Columns (GE Healthcare) to obtain ADC 4. ESI-TOFMS analysis was performed, and peaks were confirmed at 151414, where two linker payloads (35) were introduced, for the reaction product. [ka]

[0454] (8-2) Synthesis of ADC5 According to (8-1), ADC5 was obtained from Linker-payload (42). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151673, where two Linker-payloads (42) were introduced. [ka]

[0455] (8-3) Synthesis of ADC6 According to (13-1), ADC6 was obtained from Linker-payload (47). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 151109, where two Linker-payloads (47) were introduced. [ka]

[0456] (8-4) Synthesis of ADC8 According to (13-1), ADC8 was obtained from Linker-payload (125). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 150524, where two Linker-payloads (125) were introduced. [ka]

[0457] (8-5) Synthesis of ADC11 According to (13-1), ADC11 was obtained from Linker-payload (126). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 150615, where two Linker-payloads (126) were introduced. [ka]

[0458] Example 9: HIC-HPLC analysis of ADCs HIC-HPLC analysis was carried out using the conditions of Example 3. [Table 6]

[0459] Next, the hydrophobicity of the ADC was evaluated using HIC-HPLC. The measurement was performed according to Example 3. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram. Trastuzumab, the raw material antibody, was used for comparison. [Table 7]

[0460] The exo-ADCs ADC4, 5, and 6 have retention times in the HIC chromatogram that are comparable to those of the raw antibody, indicating that they are more hydrophilic ADCs.

[0461] Example 10: Assessment of aggregation rate of ADCs by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was carried out according to Example 4. [Table 8]

[0462] As a result, it was confirmed that the ADCs synthesized in (8-1), (8-2) and (8-3) tended to have a low aggregation rate and were more stable. Therefore, it was confirmed that the ADCs synthesized in Example 8 were preferable ADCs.

[0463] Example 11: Synthesis of Linker-payload mimic (11-1) Synthesis of Linker-payload mimic (56) Linker-payload mimic (56) was synthesized as follows. [ka]

[0464] (11-1-1) Synthesis of alcohol (57) [ka]

[0465] Ac-Asp(OtBu)-Val-Cit-OH (51.7 mg, 103 μmol) was dissolved in N,N-dimethylformamide (520 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (46.9 mg, 123 μmol) and 2,4,6-trimethylpyridine (15.9 μL, 123 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 4-aminomandelate methyl (22.3 mg, 123 μmol) was added, and the mixture was stirred at room temperature for 19 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (57) (56.3 mg, 86.5 μmol).

[0466] 1H NMR(400MHz,DMSO-d6)δ9.99(s,1H),8.27(d,J=8.4Hz,1H),8.17(d,J=8.4Hz,1H),7.59(d,J=8.8Hz,1H),7.56(d,J =8.8Hz,2H),7.31(d,J=8.8Hz,2H),5.98(brs,1H),5.41(brs,2H),5.08(s,1H),4.64-4.59(m,1H),4.39-4.34(m,1 H),4.22-4.18(m,1H),3.59(s,3H),3.01-2.94(m,2H),2.69-2.63(m,1H),2.44-2.38(m,1H),2.00-1.95(m,1H),1. 83(s,3H),1.69-1.66(m,1H),1.62-1.53(m,1H),1.43-1.34(m,11H),0.84(d,J=6.8Hz,3H),0.79(d,J=6.8Hz,3H).

[0467] MS (ESI) m / z: 651.35 [M+H] +

[0468] (11-1-2) Synthesis of ピレン(58)

change

[0469] Alcohol (57) (55.0 mg, 84.5 μmol) was dissolved in N,N-dimethylformamide (423 μL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (77.1 mg, 254 μmol) and N,N-diisopropylethylamine (32.3 μL, 190 μmol) were added and stirred at room temperature for 2 hours. Then, the mixture was ice-cooled, and sarcosin-pyrene (76.7 mg, 254 μmol), 1-hydroxybenzotriazole (17.1 mg, 127 μmol), and N,N-diisopropylethylamine (53.9 μL, 317 μmol) were added and stirred at room temperature for 1.5 hours. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (58) (60.9 mg, 62.2 μmol).

[0470] 1 H NMR(400MHz,DMSO-d6)δ10.14-10.11(m,1H),8.68-8.66(m,1H),8.39-8.01(m,10H),7.89-7.85(m,1H),7.67-7.5 8(m,3H),7.44-7.39(m,2H),5.98(brs,1H),5.79-5.77(m,1H),5.42(brs,2H),5.04-4.97(m,2H),4.65-4.59(m,1 H),4.39-4.36(m,1H),4.28-4.07(m,2H),3.92-3.82(m,1H),3.62-3.61(m,3H),2.99-2.91(m,5H),2.69-2.63(m, 1H),2.44-2.38(m,1H),2.01-1.97(m,1H),1.83(s,3H),1.70-1.60(m,2H),1.44-1.30(m,11H),0.86-0.77(m,6H).

[0471] MS(ESI)m / z:979.45[M+H] +

[0472] (11-1-3) Synthesis of pyrene (59) [ka]

[0473] Pyrene (58) (24.9 mg, 25.4 μmol) was dissolved in tetrahydrofuran (1.88 mL) and water (625 μL), and the mixture was stirred for 5 minutes under ice cooling, after which 1 M lithium hydroxide aqueous solution (30.5 μL, 30.5 μmol) was added and the mixture was stirred at room temperature for 50 minutes. After the reaction was completed, the mixture was adjusted to about pH 6 using 0.1 M hydrochloric acid, and purified by reversed phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above pyrene (59) (8.3 mg, 8.6 μmol).

[0474] 1 H NMR(400MHz,DMSO-d6)δ13.06(brs,1H),10.13-10.09(m,1H),8.68-8.65(m,1H),8.39-8.00(m,10H),7.88-7.84 (m,1H),7.67-7.59(m,3H),7.44-7.41(m,2H),5.98(brs,1H),5.68-5.67(m,1H),5.42(brs,2H),5.04-4.93(m,2H) ),4.64-4.61(m,1H),4.41-4.35(m,1H),4.32-4.09(m,2H),3.91-3.80(m,1H),2.99-2.91(m,5H),2.70-2.64(m,1 H),2.44-2.38(m,1H),2.01-1.98(m,1H),1.83(s,3H),1.70-1.57(m,2H),1.45-1.30(m,11H),0.86-0.77(m,6H).

[0475] MS(ESI)m / z:965.45[M+H] +

[0476] (11-1-4) Synthesis of pyrene (60) [ka]

[0477] Pyrene (59) (7.2 mg, 7.5 μmol) was dissolved in N,N-dimethylformamide (150 μL) and then cooled on ice, and N,N-diisopropylethylamine (2.5 μL, 14.9 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (5.8 mg, 11.2 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (2.4 mg, 11.2 μmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (60) (5.7 mg, 5.1 μmol).

[0478] 1 H NMR(400MHz,DMSO-d6)δ10.07-10.04(m,1H),8.91-8.88(m,1H),8.65-7.95(m,12H),7.62-7.58(m,3H),7.40-7.36(m, 2H),6.96-6.94(m,2H),5.97(brs,1H),5.70-5.68(m,1H),5.41(brs,2H),5.14-5.00(m,2H),4.65-4.61(m,1H),4.42- 4.37(m,1H),4.25-4.19(m,1H),4.15-3.82(m,2H),3.30-3.21(m,2H),3.04-2.89(m,7H),2.70-2.64(m,1H),2.44-2.3 8(m,1H),2.01-1.97(m,1H),1.83(s,3H),1.75-1.60(m,2H),1.48-1.24(m,15H),1.12-1.03(m,2H),0.86-0.78(m,6H).

[0479] MS(ESI)m / z:1129.50[M+H] +

[0480] (11-1-5) Synthesis of Linker-payload mimic (56) [ka]

[0481] Acetonitrile (208 μL) was added to pyrene (60) (4.7 mg, 4.2 μmol), and 85% phosphoric acid solution (72.4 μL, 1.25 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain Linker-payload mimic (56) (3.1 mg, 2.9 μmol).

[0482] 1 H NMR(400MHz,DMSO-d6)δ12.38(brs,1H),10.05-10.01(m,1H),8.91-8.88(m,1H),8.65-7.94(m,12H),7.63-7.58(m,3H) ),7.40-7.34(m,2H),6.96-6.93(m,2H),6.00(brs,1H),5.70-5.68(m,1H),5.44(brs,2H),5.15-4.99(m,2H),4.64-4.6 0(m,1H),4.43-4.37(m,1H),4.25-4.22(m,1H),4.15-3.82(m,2H),3.30-3.21(m,2H),3.04-2.89(m,7H),2.73-2.69(m, 1H),2.46-2.44(m,1H),2.04-1.96(m,1H),1.84-1.83(m,3H),1.77-1.59(m,2H),1.44-1.04(m,8H),0.86-0.77(m,6H).

[0483] MS(ESI)m / z:1073.50[M+H] +

[0484] (11-2) Synthesis of Linker-payload mimic (66) The linker-payload mimic(1) was synthesized as follows: [ka]

[0485] (11-2-1) Synthesis of alcohol (67) [ka]

[0486] Ac-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Val-Cit-OH (50.2 mg, 47.3 μmol) was dissolved in N,N-dimethylformamide (237 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (21.6 mg, 56.8 μmol) and 2,4,6-trimethylpyridine (7.48 μL, 56.8 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 4-aminomandelate methyl (10.3 mg, 56.8 μmol) was added, and the mixture was stirred at room temperature for 20 hours, and then purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the above alcohol (67) (47.7 mg, 39.1 μmol).

[0487] MS(ESI)m / z:1220.65[M+H] +

[0488] (11-2-2) Synthesis of pyrene (68) [ka]

[0489] The alcohol (67) (46.3 mg, 37.9 μmol) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (380 μL) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (34.6 mg, 114 μmol) and N,N-diisopropylethylamine (14.5 μL, 85.3 μmol) were added and stirred at room temperature for 2 hours. Then, the mixture was ice-cooled, and Sarcosin-Pyrene (34.5 mg, 114 μmol), 1-hydroxybenzotriazole (7.7 mg, 56.9 μmol), and N,N-diisopropylethylamine (24.2 μL, 142 μmol) were added and stirred at room temperature for 2 hours. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above pyrene (68) (37.1 mg, 24.0 μmol).

[0490] MS(ESI)m / z:775.30[M+H] +

[0491] (11-2-3) Synthesis of pyrene (69) [ka]

[0492] Pyrene (68) (20.3 mg, 13.1 μmol) was dissolved in tetrahydrofuran (983 μL) and water (327 μL), and the mixture was stirred for 5 minutes under ice cooling, after which 1M lithium hydroxide aqueous solution (31.4 μL, 31.4 μmol) was added and the mixture was stirred at room temperature for 2.5 hours. After the reaction was completed, the mixture was adjusted to about pH 6 using 1M hydrochloric acid, and purified by reversed phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above pyrene (69) (16.7 mg, 10.9 μmol).

[0493] MS(ESI)m / z:1534.85[M+H] +

[0494] (11-2-4) Synthesis of pyrene (70) [ka]

[0495] Pyrene (69) (14.9 mg, 9.71 μmol) was dissolved in N,N-dimethylformamide (486 μL) and then cooled on ice, and N,N-diisopropylethylamine (3.3 μL, 19.4 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (7.6 mg, 14.6 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (3.2 mg, 14.6 μmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (70) (13.1 mg, 7.71 μmol).

[0496] MS(ESI)m / z:1698.90[M+H] +

[0497] (11-2-5) Synthesis of Linker-payload mimic (66) [ka]

[0498] Acetonitrile (309 μL) was added to pyrene (70) (5.25 mg, 3.09 μmol), and 85% phosphoric acid solution (53.8 μL, μ927 mol) was added under ice cooling, and the mixture was stirred at room temperature for 25 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload mimic (66) (3.7 mg, 2.51 μmol).

[0499] MS(ESI)m / z:1474.00[M+H] +

[0500] (11-3) Synthesis of Linker-payload mimic (11) Linker-payload mimic (11) was also synthesized via a route different from that of (1-3) as follows. [ka]

[0501] (11-3-1) Synthesis of alcohol (96) [ka]

[0502] The alcohol (12) (80.0 mg, 94.1 μmol) obtained in Example (1-3-1) was dissolved in tetrahydrofuran (7.0 mL) and water (2.35 mL), and stirred for 5 minutes under ice cooling, after which 1M lithium hydroxide aqueous solution (226 μL, 226 μmol) was added and stirred at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to about 6 using 1M hydrochloric acid, and purified by reversed phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (96) (61.5 mg, 73.6 μmol).

[0503] MS(ESI)m / z:836.40[M+H] +

[0504] (11-3-2) Synthesis of alcohol (97) [ka]

[0505] Alcohol (96) (60.5 mg, 72.4 μmol) was dissolved in N,N-dimethylformamide (3.6 mL), cooled on ice, and N,N-diisopropylethylamine (25 μL, 145 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (56.5 mg, 109 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (23.7 mg, 109 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain alcohol (97) (68.0 mg, 72.4 μmol).

[0506] MS(ESI)m / z:1000.50[M+H] +

[0507] (11-3-3) Synthesis of compound (98) [ka]

[0508] Alcohol (97) (10.0 mg, 10.0 μmol) was dissolved in N,N-dimethylformamide (0.1 mL) and then cooled on ice. Bis(4-nitrophenyl) carbonate (30.4 mg, 100 μmol) and N,N-diisopropylethylamine (3.8 μL, 22.5 μmol) were added and stirred at room temperature for 3 hours. After the reaction, the mixture was purified by normal phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and the mixture was freeze-dried to obtain the above compound (98) (5.6 mg, 4.8 μmol).

[0509] MS(ESI)m / z:1163.50[M+H] +

[0510] (11-3-4) Synthesis of pyrene (15) [ka]

[0511] Compound (98) (10.0 mg, 8.6 μmol) was dissolved in N,N-dimethylformamide (86 μL) and then cooled on ice. Sarcosin-Pyrene (2.2 mg, 7.2 μmol), 1-hydroxybenzotriazole (1.5 mg, 11 μmol), and N,N-diisopropylethylamine (1.8 μL, 11 μmol) were added and stirred at room temperature for 4 hours. After the reaction, the mixture was purified by normal phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above pyrene (15) (3.0 mg, 2.3 μmol).

[0512] MS(ESI)m / z:1328.60[M+H] +

[0513] (11-3-5) Synthesis of Linker-payload mimic (15) Linker-payload mimic (15) was synthesized in a manner similar to that in Example (1-3-5).

[0514] (11-4) Synthesis of Linker-payload mimic (120) Linker-payload mimic(120) was synthesized as follows. [ka]

[0515] (11-4-1) Synthesis of compound (122) [ka]

[0516] 21-[(tert-butoxycarbonyl)amino]-4,7,10,13,16,19-hexaoxaheneicosanoic acid (121) (70.0 mg, 154 μmol) was dissolved in N,N-dimethylformamide (7.72 mL) and then cooled on ice, and N,N-diisopropylethylamine (52.0 μL, 309 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (120 mg, 232 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (50.6 mg, 232 μmol) was added, and the mixture was returned to room temperature and stirred for 4 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain compound (122) (83.4 mg, 135 μmol).

[0517] MS(ESI)m / z:618.50[M+H] +

[0518] (11-4-2) Synthesis of compound (123) [ka]

[0519] Compound (122) (82.0 mg, 133 μmol) was dissolved in dichloromethane (13.3 mL) and trifluoroacetic acid (6.64 mL) and stirred at room temperature for 30 minutes. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by vacuum concentration to obtain the above compound (123) (71.8 mg, quant).

[0520] MS(ESI)m / z:518.40[M+H] +

[0521] (11-4-3) Synthesis of pyrene (124) [ka]

[0522] Pyrene (14) (16.0 mg, 14.0 μmol) was dissolved in N,N-dimethylformamide (690 μL) and then cooled on ice, and N,N-diisopropylethylamine (9.3 μL, 55.0 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (11 mg, 21.0 μmol) were added. Next, PEG6 (11.0 mg, 21.0 μmol) was added, and the mixture was returned to room temperature and stirred for 2.5 hours. After the reaction was completed, the product was purified by reversed-phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain pyrene (124) (11.2 mg, 6.73 μmol).

[0523] MS(ESI)m / z:1664.80[M+H] +

[0524] (11-4-4) Synthesis of Linker-payload mimic (120) [ka]

[0525] Acetonitrile (200 μL) was added to pyrene (124) (5.0 mg, 3.0 μmol), and 85% phosphoric acid solution (60.0 μL, 880 μmol) was added under ice cooling, and the mixture was stirred at room temperature for 25 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain Linker-payload mimic (120) (2.4 mg, 1.5 μmol).

[0526] MS(ESI)m / z:1552.65[M+H] +

[0527] Example 12: Synthesis of ADC mimics (12-1)Synthesis of ADC mimics In the following Examples, ADC mimics were prepared in the same manner as in Example 2.

[0528] The ADC mimic 22 with the following structure was synthesized from the linker-payload mimic (56) of (11-1) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 150322, where two linker-payload mimics (56) were introduced. [ka]

[0529] Similarly, the ADC mimic 24 with the following structure was synthesized from the linker-payload mimic (66) of (11-2) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 151125, where two linker-payload mimics (66) were introduced. [ka]

[0530] Similarly, the ADC mimic 33 with the following structure was synthesized from the linker-payload mimic (120) of (11-4) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had a peak at 151279, where two linker-payload mimics (120) were introduced. [ka]

[0531] (12-2)DAR analysis of ADC mimics ESI-TOFMS analysis of the ADC mimic synthesized in Example 12-1 was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2. [Table 9]

[0532] Example 13: Evaluation of hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC) HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). Measurements were performed under the following conditions. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram.

[0533] Measurement system: Chromaster (registered trademark) (Hitachi) Column: Tosoh Biobuthyl NPR 2.5μm 4.6×35mm column manufactured by Tosoh Biosciences Gradient: Linear gradient of eluent A / B Flow rate: 0.8mL / min Eluent A: 1.1M (NH4)2SO4, 25mM Na2HPO4 / NaH2PO4 (pH6.0) Eluent B: 25 mM Na2HPO4 / NaH2PO4 (pH 6.0, 25 v / v% isopropanol added) Detector: UV (280 nm) [Table 10]

[0534] As a result, it was confirmed that the ADC mimics synthesized in Examples 11-1, 11-2, and 11-4 tended to have a short retention time, indicating that they had high hydrophilicity. Therefore, it was confirmed that the ADC mimics synthesized in Examples 11-1, 11-2, and 11-4 were preferable ADCs because they are considered to have a slow plasma clearance and a long retention time in the body.

[0535] Example 14: Assessment of aggregation rates of ADCs and ADC mimics by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to a previous report (Chemistry Select, 2020, 5, 8435-8439). The measurement was performed under the following conditions.

[0536] Measurement system: 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300Å 2.7μm, 4.6mm×150mm Flow rate: 0.25mL / min Eluent: 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride in water (pH 6.8), 10% v / v isopropanol Detector: UV (280 nm) [Table 11]

[0537] Example 15: Evaluation of ADC mimics using the enzyme cathepsin B The cleavage ability of various ADC mimics by cathepsin B was evaluated by analyzing the amount of fluorescent molecules shed from the ADC mimics as described below.

[0538] (15-1) Cathepsin B cleavage assay The test was carried out in the same manner as in Example 5.

[0539] (15-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis The analysis was carried out in the same manner as in Example 5. [Table 12]

[0540] As shown in Table 12, the synthesized ADC mimics were found to have sufficient cathepsin B cleavage.

[0541] Example 16: Evaluation of ADC mimics using mouse plasma (16-1) Plasma stability test of ADC mimics The test was carried out in the same manner as in Example 6.

[0542] (16-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis The analysis was carried out in the same manner as in Example 6. [Table 13]

[0543] As a result, compared with the ADC mimic synthesized in Comparative Example 1, the ADC mimic synthesized in Example 12-4 exhibited more than twice the stability, and the ADC mimics synthesized in Examples 12-1 and 12-2 exhibited more than 10 times the stability.

[0544] Example 17: Synthesis of Linker-payload The NMR spectrum data of the linker-payload (42) synthesized in Example (7-3-5) is as follows.

[0545] 1 H NMR (300MHz;DMSO-d6) δ12.06(brs,2H),10.05-10.07(m,1H),8.47-8.28(m,1H),8.29-8.19(m,1H),8.13-8.04(m,3H),7.91-7.56(m,5H), 7.39-7.17(m,7H),6.99(s,2H),5.99(s,1H),5.86-5.67(m,1H),5.43-5.35(m,3H),4.77-4.16(m,6H),4.00-3.98(m ,2H),3.80-3.76(m,1H),3.52-3.18(m,12H),3.01-2.73(m,9H),2.26-2.14(m,6H),2.12-2.09(m,2H),1.97-1.90(m ,2H),1.84(s,6H),1.76-1.69(m,5H),1.43-1.35(m,10H),1.23-1.14(m,3H),1.01-0.96(m,7H),0.83-0.68(m,24H).

[0546] Example 18: Synthesis of ADC (18-1) Synthesis of Linker Intermediate (18-1-1) Synthesis of linker intermediate (115) [ka]

[0547] 5-Azidopentanoic acid (800 mg, 5.59 mmol) was dissolved in THF (14 mL), isobutyl chloroformate (808 μL, 6.15 mmol), N-methylmorpholine (873 μL, 8.39 mmol) were added, and the mixture was stirred at 0° C. for 30 minutes. Hydrazine hydrate (1.36 g, 6.71 mmol) dissolved in 1M NaOH aqueous solution (4 mL) was then added and stirred at room temperature for 3 hours. After concentration under reduced pressure, 1M NaOH aqueous solution was added, the pH of the system was adjusted to pH 10, and the mixture was washed with ethyl acetate. 1M HCl aqueous solution was added to the aqueous layer, the pH of the system was adjusted to 3.0, ethyl acetate was added for washing, and sodium sulfate was added to the obtained ethyl acetate solution. Sodium sulfate was removed by filtration, and the mixture was purified by vacuum concentration column chromatography (dichloromethane:methanol = 10:1). The fraction containing the product was collected and concentrated under reduced pressure to obtain the linker intermediate (115).

[0548] 1 H NMR(400MHz,Chloroform-d)δ6.29(d,J=7.7Hz,1H),4.56(td,J=8.0,4.9Hz,1H),3.32(t,J=6.6Hz,2H),2.53 -2.38(m,3H),2.36-2.16(m,3H),2.12(s,2H),1.96(dq,J=14.7,7.6Hz,1H),1.84-1.59(m,4H),1.50(s,9H).

[0549] MS(ESI)m / z:329[M+H] +

[0550] (18-1-2) Synthesis of linker intermediate (117) [ka]

[0551] Linker intermediate (116) (2.41 g, 5.59 mmol) was dissolved in dichloromethane (28 mL), thiophenol (627 μL, 6.15 mmol), benzotriazol-1-yloxy (3.49 g, 6.71 mmol), and DIPEA (1.42 mL, 8.39 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate=4:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (117) (2.20 g, 5.23 mmol).

[0552] 1 H NMR(400MHz,Chloroform-d)δ7.43(s,5H),6.10(d,J=7.8Hz,1H),4.55(td,J=7.7,4.9Hz,1H),3.31(t,J=6.7Hz,2H),2.87-2.63( m,2H),2.28(dd,J=8.7,5.9Hz,2H),2.16-1.98(m,1H),1.83-1.58(m,4H),1.50(s,9H),1.37-1.22(m,2H),0.91(t,J=6.7Hz,1H).

[0553] MS(ESI)m / z:421[M+H] +

[0554] (18-1-3) Synthesis of linker intermediate (118) [ka]

[0555] Linker intermediate (117) (2.20 g, 5.23 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to remove dichloromethane, and water was added and the mixture was freeze-dried to obtain linker intermediate (118) (1.98 g, 5.43 mmol).

[0556] 1H NMR(400MHz,Chloroform-d)δ7.44(s,J=6.3,4.6,2.4Hz,5H),6.76(s,1H),4.62(td,J=7.5,4.9Hz,1H),3.31(t,J=6.6Hz,2H),2.88(qt,J=16.8 ,6.8Hz,2H),2.33(dt,J=12.4,6.8Hz,3H),2.18(dq,J=14.4,7.4Hz,1H),1.74(dq,J=11.8,7.5,6.9Hz,2H),1.63(ddd,J=17.7,10.5,4.8Hz,2H).

[0557] MS(ESI)m / z:365[M+H] +

[0558] (18-1-4) Synthesis of linker intermediate (119) [ka]

[0559] The linker intermediate (118) (100 mg, 0.274 mmol) was dissolved in dichloromethane (3 mL), and (40.6 μL, 0.280 mmol), benzotriazol-1-yloxy (150 mg, 0.288 mmol), and DIPEA (70.1 μL, 0.412 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The pH of the system was adjusted to 3 by adding 1M HCl aqueous solution, and dichloromethane was added for dilution. After washing with water and saline, sodium sulfate was added. After removing sodium sulfate by filtration, the mixture was concentrated under reduced pressure and purified by column chromatography (hexane: ethyl acetate = 4:1). The fraction containing the product was collected and concentrated under reduced pressure to obtain the linker intermediate (119) (84.7 mg, 0.171 mmol).

[0560] 1H NMR(400MHz,Chloroform-d)δ7.50-7.38(m,5H),6.33(d,J=8.4Hz,1H),4.78(tdd,J=7.8,4.6,3.0Hz,1H),3.70-3.54(m,2 H),3.32(dt,J=9.1,6.7Hz,2H),2.96-2.67(m,2H),2.30(pd,J=7.1,4.5Hz,2H),1.85-1.60(m,6H),1.49(d,J=2.8Hz,9H).

[0561] MS(ESI)m / z:495[M+H] +

[0562] (18-1-5) Synthesis of linker intermediate (120) [ka]

[0563] Linker intermediate (119) (84.7 mg, 0.171 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added, and the mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure to remove dichloromethane, water was added, and the mixture was lyophilized, followed by purification by column chromatography (dichloromethane:methanol = 10:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (120) (46.8 mg, 0.107 mmol).

[0564] 1 H NMR(400MHz,Methanol-d4)δ7.44(dq,J=2.3,1.5Hz,5H),4.69-4.57(m,1H),3.79-3.67(m,2H), 3.40-3.30(m,2H),2.89-2.71(m,2H),2.44-2.23(m,4H),2.08-1.95(m,1H),1.82-1.61(m,4H).

[0565] MS(ESI)m / z:439[M+H] +

[0566] (18-2) Preparation of affinity reagent (18) having an azide group [ka] (The above amino acid sequence is the amino acid sequence of SEQ ID NO: 1.)

[0567] Ac-RGNCAYHKGQIIWCTYH-NH2 (SEQ ID NO: 1, 30.9 mg, 14.9 μmol, where the 4th and 14th cysteines are each intramolecularly disulfide bonded) described in the previous report (WO2019 / 240287A1) was dissolved in dimethylformamide (468 μL), and the linker intermediate (120) (46.8 mg, 0.107 mmol) synthesized in Example 18-1-5) and WSC·HCl (29.7 mg, 0.155 mmol) were added, stirred at room temperature for 5 hours, and then eluted by reversed phase preparative chromatography. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above modification reagent (121) (15.1 mg, 6.02 μmol).

[0568] (18-3) Introduction of two peptide molecules into Trastuzumab [ka] (The above amino acid sequence is the amino acid sequence of SEQ ID NO: 1.)

[0569] Next, conjugation was performed on trastuzumab using the peptide reagent (121) prepared in Example 18-2 according to the method previously reported (WO2019 / 240287A1). As a result, an antibody into which the modification reagent (121) was introduced was obtained. DAR analysis of the antibody into which the peptide reagent (121) was introduced was performed according to a previously reported method (Anal.Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two peptide reagents were introduced.

[0570] (18-4) Synthesis of azido-trastuzumab (T-1) [ka] (The above amino acid sequence is the amino acid sequence of SEQ ID NO: 1.)

[0571] A cleavage reaction was carried out by adding a methoxyamine solution to the antibody to which the modification reagent (121) obtained in Example 18-3 was introduced, and shaking at room temperature for 3 hours, in accordance with the method previously reported (WO2019 / 240287A1). As a result, an antibody to which an azide group was introduced was obtained. Analysis was performed according to a previously reported method (Anal.Chem.,2019,91,20,12724-12732), and HIC-HPLC analysis was performed to confirm that an azide group had been introduced.

[0572] (18-5) Synthesis of ADC7 Linker-payload (40) was added to the above azide-introduced antibody to obtain ADC (7). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151276, where two Linker-payloads (40) were introduced. In addition, ESI-TOFMS analysis was performed according to a previous report (WO2019 / 240287A1), and it was confirmed that the DAR was 2. [ka]

[0573] Example 19: Evaluation of ADC using mouse plasma (19-1) Plasma stability test of ADC ADC mimic was added to 500 μL of mouse plasma (Charles River) to a concentration of 0.1 mg / mL, and then sterilized and filtered. This solution was dispensed into six Eppendorf tubes at 50 μL each. Three of the six samples were stored in an incubator set at 37°C for four days. The remaining three were similarly stored in a freezer at -80°C for four days. Each sample was added with 100 μL of acetonitrile, stirred with a vortex mixer, and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0574] (19-2) Analysis of the amount of shed payload using HPLC analysis The amount of payload dropped from the ADC was measured using liquid chromatography mass spectrometry (including tandem mass spectrometry). The sample stored in a freezer at -80°C for 4 days in the same manner as in Example 19-1 was taken as the 0th day, and the three samples incubated at 37°C for 4 days in Example 19-1 were taken as the 4th day. The MS intensities of the payload detected from the 4th day sample and the 0th day sample were calculated by extracted ion chromatograms, and the difference between them was analyzed.

[0575] Separately, using MMAE, the correlation between the area of ​​the TIC by HPLC and the concentration was calculated. Using this calculation formula, the TIC of the fluorescence intensity of each ADC was converted to concentration. The dropout rate was calculated as the ratio of the difference in the above ion chromatogram, with the concentration on Day 0 taken as 100%. [Table 14]

[0576] As a result, it was found that the ADCs synthesized in Examples 7-1, 7-2, and 7-3 had high stability.

Claims

1. The following formula (I'): 【Chemistry 1】 [During the ceremony, Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and is regioselectively linked to L1 adjacent to Ig via amino groups in the side chains of lysine residues at positions 246 / 248, 288 / 290, or 317 according to Eu numbering in the two heavy chains; R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, Ring A is a phenylene group which may have a substituent, R １ , and R ２ each independently represents a hydrogen atom or an alkyl group; L １ has a carbonyl group, and the site-selective bond with Ig is via the amino group in the side chain of the lysine residue, and L １ represents a divalent group achieved by an amide bond through the bonding of a carbonyl group in L ２ represents a divalent group, D represents a drug, a labeling substance, or a stabilizer; the average ratio r of said bonds per two heavy chains is between 1.5 and 2.5; (-L ＨＧ -) is represented by the following formula (a): -(C(R ＨＧ ) ２ ) ｎ１ -(C=O) ｎ２ -(NR ＨＧ ) ｎ３ -(C(R ＨＧ ) ２ ) ｎ４ - (a) [During the ceremony, Multiple R ＨＧ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, n1 is an integer from 0 to 3, n2 is an integer of 0 or 1; n3 is an integer of 0 or 1; n4 is an integer of 0 to 3. R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L ＨＧ , R ＨＧ１ , and R ＨＧ２ and wherein the hydrophilic group is one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety.

2. The divalent group represented by formula (a) is represented by the following formula (a1), (a2), or (a3): (a1) -(C(R ＨＧ ) ２ )-; (a2) -(C(R ＨＧ ) ２ )-(C=O)-(NR ＨＧ )-(C(R ＨＧ ) ２ )-;or (a3) -(C=O)-(C(R ＨＧ ) ２ ) ２ -; [During the ceremony, Multiple R ＨＧ each independently represents a hydrogen atom, a hydrophilic group, or an alkyl group having 1 to 6 carbon atoms and containing a hydrophilic group.

3. The regioselective conjugate or salt thereof according to claim 1 or 2, wherein the hydrophilic groups are each independently a carboxylic acid group, a sulfonic acid group, or a hydroxyl group.

4. The regioselective conjugate or salt thereof according to claim 3 , wherein the hydrophilic group is a carboxylic acid group.

5. The following formula (III'): 【Chemistry 2】 [During the ceremony, R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, Ring A is a phenylene group which may have a substituent, R １ , and R ２ each independently represents a hydrogen atom or an alkyl group; L １ , and L ２ each independently represents a divalent group, B １ represents a bioorthogonal functional group, D represents a drug, a labeling substance, or a stabilizer; (-L ＨＧ -) is represented by the following formula (a): -(C(R ＨＧ ) ２ ) ｎ１ -(C=O) ｎ２ -(NR ＨＧ ) ｎ３ -(C(R ＨＧ ) ２ ) ｎ４ - (a) [During the ceremony, Multiple R ＨＧ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, n1 is an integer from 0 to 3, n2 is an integer of 0 or 1; n3 is an integer of 0 or 1; n4 is an integer of 0 to 3. R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is L ＨＧ , R ＨＧ１ , and R ＨＧ２ and the hydrophilic group is one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety; The bioorthogonal functional group is a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue, or a salt thereof.

6. A reagent for derivatizing an antibody, comprising the compound according to claim 5 or a salt thereof.

7. The following compound (3) or a salt thereof: (3) The following formula (VII): 【Chemistry 3】 [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, the hydrophilic group is one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polysarcosine group, and a sugar moiety; R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent, Y represents a monovalent group; R １ represents a hydrogen atom or a monovalent group, L １ represents a divalent group, B １ represents a bioorthogonal functional group, The bioorthogonal functional group is a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.

8. The compound according to claim 7, wherein the compound (3) is the following compound (3') or a salt thereof: (3') The following formula (VII'): 【Chemistry 4】 [During the ceremony, R Ａ , R Ｂ , ring A, and Y are the same as those represented by formula (VII), L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, R １ , L １ , and B １ and R 1 and R 2 are the same as those represented by formula (VII).

9. A compound selected from the group consisting of the following formulas (1) to (6): 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】

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