Conjugate of antibody and functional substance or salt thereof, and compound or salt thereof used in production thereof

Abstract

To provide a conjugate or a salt thereof of an antibody and a functional substance having excellent desired properties while controlling the binding ratio between the antibody and the functional substance within a desired range.SOLUTION: Provided is s a conjugate or a salt thereof of an antibody and a functional substance, which comprises a structural unit represented by the following formula (I): [where, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and an amide bond is regioselectively formed with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue in the two heavy chains; X represents a predetermined divalent group; D represents a functional substance; RA represents a side chain of a valine residue; RB represents a side chain of a citrulline residue or an alanine residue; n represents 0 or 1], at least one hydrophilic group being present in the structural unit.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a conjugate of an antibody and a functional substance, or a salt thereof, as well as 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 with dipeptide-containing linkers as described above showed poor activity in mouse plasma. It is unstable (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 carboxyl terminal side of citrulline, and the linker containing the above-mentioned dipeptide 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).

[0010] For example, Patent Document 5 and Non-Patent Document 3 disclose stabilizing the linker by introducing a hydrophilic group (PEG) into the spacer between the VC structure and the drug (in the vicinity of the cleavage site by Ces1c).

[0011] On the other hand, a technique for stabilizing a linker by introducing a specific group into the spacer between the antibody and the VC structure has also been reported. One example of a linker stabilized by such a technique is the following linker 1) to 4), which is characterized by including a structure in which at least one α-amino acid residue (i.e., X or NH-C(R)-CO) is linked to the antibody side position (amino group of V) of the VC structure via an amide bond (i.e., a structure represented by X-Val-Cit or NH-C(R)-CO-Val-Cit): 1) A linker containing a tripeptide structure (Glu-Val-Cit) in which a glutamic acid residue is linked to the N-terminus of Val (Patent Document 6); 2) a linker containing a structure in which an aspartic acid residue is linked to the N-terminus of Val (Asp-Val-Cit) (Non-Patent Document 4); 3) A linker having a structure represented by NH-C(R)-CO-Val-Cit (R represents a side chain having a hydrophilic group such as PEG) (Patent Documents 7 to 9); 4) A linker having a structure represented by NH-C(R)-CO-Val-Cit (R represents a side chain having a hydrophilic group such as a sulfonic acid group or a sugar) (Patent Document 10); and 5) A linker containing a structure represented by NH—C(R)—CO-Val-Cit (R represents a side chain having cyclodextrin) (Patent Document 11).

[0012] Furthermore, as other examples of linkers in which a specific group has been introduced into the spacer present between the antibody and the VC structure, the following linkers 6) to 8) have been reported: 6) A linker having a structure represented by C(M)-CO-Val-Cit (wherein C in C(M) represents a carbon atom, M in C(M) represents a stability adjusting group containing a side chain such as an aromatic ring group, and CO represents a carbonyl group that is linked to the amino group of a valine residue to form an amide bond) (Patent Document 12); 7) A highly controlled and special PEG linker that contains PEG in both the main chain and the side chain of the spacer between the antibody and the VC structure (Non-Patent Document 5); and 8) C(R i )-NH is a VC analogue structure (CO-R ii -CO-Cit) to the antibody side position via an amide bond, C(R i )-NH-CO-R ii -CO-Cit (where R i represents a thiophenyl group, and R ii represents a cyclobutyl ring) (Patent Document 13).

[0013] Incidentally, Non-Patent Document 6 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]

[0014] [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] US Patent Application Publication No. 2019 / 0365915 [Patent Document 6] International Publication No. 2018 / 218004 [Patent Document 7] International Publication No. 2019 / 094395 [Patent Document 8] US Patent Application Publication No. 2016 / 0310612 [Patent Document 9] International Publication No. 2020 / 252043 [Patent Document 10] International Publication No. 2020 / 236841 [Patent Document 11] International Publication No. 2018 / 213077 [Patent Document 12] JP 2016-050204 A [Patent Document 13] International Publication No. 2016 / 090038 [Non-patent literature]

[0015] [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] Poudel et al.,ACS Med Chem Lett.,2020,11(11),2190-2194 [Non-Patent Document 4] Ratnayake et al.,Bioconjug Chem.,2019,30(1),200-209 [Non-Patent Document 5] Walker et al.,Bioconjug Chem.2019,30(11), 2982-2988 [Non-Patent Document 6] Lyon et al.,Nat Biotechnol.,2015,33(7),733-5 Summary of the Invention [Problem to be solved by the invention]

[0016] 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 desired range. [Means for solving the problem]

[0017] As a result of extensive investigations, the present inventors have selected a lysine residue in the heavy chain of an immunoglobulin unit as the modification site of an antibody, and have determined that a specific structural unit [i.e., CO-(N-R1)nX-CONH-CH2(-R A )-CONH-CH2(-R B It has been found that by using a linker containing the linker (R)-CO) to link an immunoglobulin unit with a functional substance, it is possible to easily produce a conjugate represented by formula (I) or a salt thereof having excellent desired properties while highly controlling the average ratio of bonds between the immunoglobulin unit and the functional substance (functional substance / immunoglobulin unit) within a desired range (1.5 to 2.5). For example, such a conjugate or a salt thereof can have one or more desired properties selected from the group consisting of excellent clearance (i.e., long residence time in the body), low aggregation rate, high cleavability by cathepsin B, and high stability in mouse plasma.

[0018] The present inventors have also found that, among the conjugates represented by formula (I), the conjugates represented by formulas (I-1) to (I-3) are excellent in the above-mentioned desired properties, and have thus completed the present invention.

[0019] For example, the conjugates represented by formulae (I-1) and (I-2) have a structural unit in which a tertiary amide structure having a group containing a hydrophilic group is linked to a VC structure in a linker for linking an antibody to a functional substance, but the above prior art does not teach or suggest the conjugates of the present invention that include a linker having such a structural unit.

[0020] In addition, the conjugate represented by formula (I-3) has a structural unit in which a γ-glutamic acid residue is linked to a VC structure in a linker for linking an antibody and a functional substance, but the above prior art does not teach or suggest a conjugate containing a linker having such a structural unit. In fact, Patent Documents 6 to 11 disclose linkers containing a structural unit in which at least one α-amino acid residue (i.e., α-type structure) is linked to a VC structure, but do not teach or suggest a linker containing a structural unit in which a γ-glutamic acid residue is linked to a VC structure. Patent Documents 12 and 13 and Non-Patent Document 5 also do not teach or suggest a linker containing a structural unit in which a γ-glutamic acid residue is linked to a VC structure.

[0021] That is, the present invention is as follows. [1] A conjugate of an antibody and a functional substance, or a salt thereof, which comprises a structural unit represented by formula (I) and at least one hydrophilic group is present in the structural unit: [2] The conjugate or salt thereof according to [1], wherein the immunoglobulin unit is a human immunoglobulin unit. [3] The conjugate or salt thereof according to [2], wherein the human immunoglobulin unit is a human IgG antibody. [4] The conjugate according to any one of [1] to [3] or a salt thereof, wherein the lysine residue is present at position 246 / 248, position 288 / 290, or position 317 according to Eu numbering. [5] The conjugate or a salt thereof according to any one of [1] to [4], wherein r is 1.9 to 2.1. [6] The conjugate according to any one of [1] to [5] or a salt thereof, 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. [7] A conjugate or a salt thereof according to any one of [1] to [6], wherein L1 represents a divalent group represented by formula (i). [8] L3 and L4 are each independently -(C(R)2). m The conjugate of [7] or a salt thereof. [9] Y is represented by the following structural formula:

[0022] [ka]

[0023] [wherein the white and black circles represent bonds, When the bond of the white circle is bonded to L3, the bond of the black circle is bonded to L4, When the bond represented by the open circle is bonded to L4, the bond represented by the filled circle is bonded to L3. A conjugate of [7] or [8], or a salt thereof, wherein the conjugate is a divalent group represented by any one of the structural formulas.

[10] The conjugate or a salt thereof according to any one of [1] to [9], wherein the structural unit represented by formula (I) is a structural unit represented by formula (I-1), (I-2), (I-3), or (I-4).

[11] The conjugate or a salt thereof according to

[10] , wherein the structural unit represented by formula (I) is a structural unit represented by formula (I-1), (I-2), or (I-3).

[12] The conjugate according to

[10] or a salt thereof, wherein the structural unit represented by the formula (I-1), (I-2), (I-3), or (I-4) is a structural unit represented by the formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), (I-3b'), (I-4'), (I-4b'), or (I-4c').

[13] The structural unit represented by the formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), (I-3b'), or (I-4') is a structural unit represented by the formula (I-1a'- A conjugate of

[12] or a salt thereof, wherein the structural unit is represented by the following formula:

[12] , (I-1), (I-1a'-2), (I-1b'-1), (I-1c'-1), (I-2'-1), (I-2'-2), (I-3a'-1), (I-3a'-2), (I-3b'-1), (I-4a'-1), (I-4b'-1), (I-4c'-1), or (I-4c'-2):

[14] L2 is represented by the following structural formula:

[0024] [ka]

[0025] (Here, the black and white circles represent bonds, The bond marked with a black circle is bonded to the carbonyl group adjacent to L2. The bond marked with a white circle is bonded to D. E represents an electron withdrawing group; and n2 is an integer of 1 to 4. A conjugate or a salt thereof according to any one of [1] to

[13] , wherein n is a divalent group represented by the following formula:

[15] A compound represented by the formula (II-1), (II-2), (II-3), (II-4b'), or (II-4c') or a salt thereof.

[16] The compound according to

[15] or a salt thereof, wherein 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.

[17] The compound represented by formula (II-1), (II-2) or (II-3) is a compound represented by formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a') or (II-3b'), or a salt thereof, represented by formula (15) or (16):

[18] A compound of any one of

[15] to

[17] or a salt thereof, wherein the compound represented by the formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), (II-3b'), (II-4b'), or (II-4c') is represented by the formula (II-1a'-1), (II-1a'-2), (II-1b'-1), (II-1c'-1), (II-2'-1), (II-2'-2), (II-3a'-1), (II-3a'-2), (II-3b'-1), (II-4b'-1), (II-4c'-1), or (II-4c'-2):

[19] L2 is represented by the following structural formula:

[0026] [ka]

[0027] (Here, the black and white circles represent bonds, The bond marked with a black circle is bonded to the carbonyl group adjacent to L2. The bond marked with a white circle is bonded to D. E represents an electron withdrawing group; n2 is an integer of 1 to 4. A compound according to any one of

[15] to

[18] , or a salt thereof, wherein

[20] A reagent for derivatizing an antibody, comprising any one of the compounds according to

[15] to

[19] or a salt thereof. Effect of the Invention

[0028] The conjugate of the present invention or a salt thereof can exhibit excellent desired properties while the average bond ratio between the immunoglobulin unit and the functional substance (functional substance / immunoglobulin unit) is highly controlled within a desired range (1.5 to 2.5). The compounds of the present invention or salts thereof, and reagents are useful as synthetic intermediates in the preparation of the above-mentioned conjugates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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:

[0034] (1) Cancer area PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, PS, EpCAM, Platelet-derived growth factor receptorン, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEAC AM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105(エンドグリン), 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, Thoms en-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, ルイス blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9インテグリン, TAG-72 (CA72-4), CD70

[0035] (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

[0036] (3) Diseases caused by diseases of the mind and body CGRP, CD20, βアミロイド, βアミロイドプロトフィブリン, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing1), αシヌクレイン, extracellular tau, CD52, インスリン receptor, tauタンパク, TDP-43, SOD1, TauC3, JCウイルス

[0037] (4) Infections 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

[0038] (5) Genetic and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0039] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0040] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0041] (8) Hematological diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0042] (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

[0043] 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).

[0044] 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.

[0045] 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%.

[0046] In the present invention, as long as a specific lysine residue in the heavy chain of the antibody is site-selectively modified, Alternatively, specific amino acid residues at other positions may be regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are 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).

[0047] (Halogen atom) Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0048] (Monovalent group) Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

[0049] 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.

[0050] (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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0061] 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.

[0062] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0063] Among these, the monovalent hydrocarbon group is preferably an alkyl group, a cycloalkyl group, or an aryl group.

[0064] (Monovalent heterocyclic groups and related terms) A monovalent heterocyclic group is a group formed by removing one hydrogen atom from a heterocyclic ring of a heterocyclic compound. A monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. Heterocycle The heteroatom constituting the group preferably contains one or more types selected from the group consisting of oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably contains one or more types selected from the group consisting of oxygen atoms, sulfur atoms, and nitrogen atoms.

[0065] 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.

[0066] 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.

[0067] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0068] (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.

[0069] 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 linear alkynylene include ethynylene, n- Examples thereof include propynylene, n-butynylene, n-pentynylene, and n-hexynylene. The divalent linear hydrocarbon group is preferably a linear alkylene group.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] (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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] (hydrophilic group) The hydrophilic group is a group that can make the structural unit represented by formula (I) or a subordinate formula 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).

[0082] The polyethylene glycol 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 20 or less, preferably an integer of 15 or less, more preferably an integer of 12 or less, and even more preferably an integer of 10 or less. More specifically, k1 may be an integer of 3 to 20, preferably an integer of 4 to 15, more preferably an integer of 5 to 12, and even more preferably an integer of 6 to 10.

[0083] 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 20 or less, preferably an integer of 15 or less, more preferably an integer of 12 or less, and even more preferably an integer of 10 or less. More specifically, k2 may be an integer of 3 to 20, preferably an integer of 4 to 15, more preferably an integer of 5 to 12, and even more preferably an integer of 6 to 10.

[0084] The sugar moiety may be a monosaccharide, an oligosaccharide (e.g., a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide), or a polysaccharide. The sugar moiety may comprise an aldose or ketose, or a combination thereof. The sugar moiety may 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 comprising such a monosaccharide.

[0085] 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.

[0086] (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)).

[0087] 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 (L). 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.

[0088] Examples of such bioorthogonal functional groups include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, it is sufficient if they have a vinylene (ethenylene) moiety, which is the minimum unit having a double bond between carbon atoms; the same applies below), alkyne residues (in other words, it is sufficient if they have an ethynylene moiety, which is the minimum 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., having a fluorine atom, chlorine atom, bromine atom or iodine atom at the α-position), Examples of the carbonyl residue that is a carbonyl group that is an isonitrile residue, a sydnone residue, and a selenium residue are as follows:

[0089] More specifically, the bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of:

[0090] [ka]

[0091] [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.

[0092] 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, a keto group (e.g., acyl), and an alkyloxy group, and preferably a halogen atom, a boronic acid residue, mesyl, tosyl, or triflate.

[0093] 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. al.,Journal of American Chemical Society.132,1500(2010).;Bessodes M. et al.,Jour nal 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)).

[0094] Protected bioorthogonal functional groups include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

[0095] More specifically, the protected bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of:

[0096] [ka]

[0097] [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.

[0098] Preferably, the optionally protected bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0099] (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 drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked together.

[0100] 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 disease / inflammatory disease (e.g., allergic disease, rheumatoid arthritis, systemic lupus erythematosus), neurological disease (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious disease (e.g., bacterial infection, Examples of the diseases include diseases such as glaucoma, viral infections, hereditary diseases and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), diseases in the bone and orthopedic fields (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, and digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0101] 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., 153 Sm), Reniu Radioisotopes of γ- 186 Re), radioisotopes of astatine (e.g., 211 At), radioisotopes of bismuth (e.g., 212More specifically, the drugs include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBDs (pyrrolobenzodiazepines), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] (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. Salts with inorganic acids include, for example, salts with inorganic acids, salts with organic bases, and salts with amino acids. Examples of the 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 the salts with inorganic bases include salts with alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium and magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of the salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of the salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, and ornithine) and acidic amino acids (e.g., aspartic acid and glutamic acid). The salt is preferably a salt with an inorganic acid (eg, hydrogen chloride) or a salt with an organic acid (eg, trifluoroacetic acid).

[0106] 2. Conjugate or salt thereof The present invention provides a conjugate of an antibody and a functional substance, or a salt thereof, which comprises a structural unit represented by the following formula (I) and has at least one hydrophilic group present in the structural unit:

[0107] [ka]

[0108] [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and selectively forms an amide bond between the carbonyl group adjacent to Ig and the amino group in the side chain of the lysine residue in the two heavy chains, L1 and L2 each represent a divalent group. R1 represents a monovalent group which may contain a hydrophilic group, X represents a divalent group which may have a substituent, in which the divalent group has 1 to 3 carbon atoms constituting a main chain portion linking the two atoms on both sides of X, and the substituent is a monovalent group which may contain a hydrophilic group; D represents a functional substance, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, n is 0 or 1, and when n is 1, the substituents on X may be joined with R1 to form a ring which may contain a hydrophilic group; The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5.

[0109] 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.

[0110] An antibody comprises the above immunoglobulin unit. Such an antibody may, for example, comprise two Examples of the antibody include an IgG antibody, an IgD antibody, and an IgE antibody, which contain an immunoglobulin unit having a heavy chain and two light chains and disulfide bonds between the heavy chains and between the heavy chains and light chains, an IgA antibody, which contains four heavy chains and four light chains and contains an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy chains and light chains, and an IgM antibody, which contains eight heavy chains and eight light chains and contains an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy chains and light chains, but 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.

[0111] The divalent groups represented by L1 and L2 are the same as those described above.

[0112] The hydrophilic group and the monovalent group in R1 are the same as those described above.

[0113] The divalent group in X is a divalent group having 1 to 3 carbon atoms constituting the main chain portion connecting the two atoms on both sides of X. Such a main chain portion is composed of a chain structure, a cyclic structure, or a structure including a combination thereof. When the main chain portion is a chain structure not including a cyclic structure, the number of carbon atoms in the main chain portion can be determined by counting the number of carbon atoms in the chain structure. On the other hand, when the main chain portion is a structure including a cyclic structure, the number of carbon atoms constituting the cyclic structure can be determined by counting the number of carbon atoms in the main chain portion. Specifically, the number of carbon atoms in the main chain portion in the cyclic structure can be determined by counting the number of carbon atoms in the shortest path connecting two bonds in the cyclic structure (for example, see the bold paths in (a) to (d) below). When the main chain is a structure including a combination of a chain structure and a cyclic structure, the number of atoms in the main chain can be determined by adding the number of atoms in the chain structure not including a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure.

[0114] [ka]

[0115] · is a bond. In the case of (a), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of carbon atoms in the main chain is 2. In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of carbon atoms in the main chain is 3. In the case of (c), since both paths are the shortest paths (equidistant), the number of carbon atoms in the divalent cyclic structure counted as the number of carbon atoms in the main chain portion is 4 (therefore, in the present invention, such a structure is excluded from the divalent group in X). In the case of (d), since the path of the condensation site is the shortest path, the number of carbon atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4 (therefore, in the present invention, such a structure is excluded from the divalent group in X).

[0116] The divalent group for X can be selected from the above-mentioned divalent groups so that the main chain portion satisfies the above conditions.

[0117] Preferably, the divalent group in X may be: (1) a divalent linear hydrocarbon group having 1 to 3 carbon atoms; (2) a divalent cyclic hydrocarbon group; and (3) A divalent group consisting of a divalent cyclic hydrocarbon group and a divalent linear hydrocarbon group (1 or 2) having 1 or 2 carbon atoms linked together.

[0118] The divalent linear hydrocarbon group having 1 to 3 carbon atoms is a linear alkylene group having 1 to 3 carbon atoms (e.g., methylene, ethylene, n-propylene), a linear alkenylene group having 2 or 3 carbon atoms (e.g., ethenylene, propenylene), or a linear alkynylene group having 2 or 3 carbon atoms (e.g., ethynylene, n-propynylene). As the divalent linear hydrocarbon group having 1 to 4 carbon atoms, a linear alkylene group having 1 to 3 carbon atoms is preferred.

[0119] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. By appropriately setting two bonds in such a divalent cyclic hydrocarbon group, the number of atoms constituting the main chain as described above can be set to 3 to 5. The arylene is preferably an arylene having 6 to 10 carbon atoms, and more preferably an arylene having 6 carbon atoms. Examples of the arylene include phenylene and naphthylene. 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.

[0120] The divalent linear hydrocarbon group having 1 or 2 carbon atoms is a linear alkylene having 1 or 2 carbon atoms (e.g., methylene, ethylene), a linear alkenylene having 2 carbon atoms (ethenylene), or a linear alkynylene having 2 carbon atoms (ethynylene). As the divalent linear hydrocarbon group having 1 to 4 carbon atoms, a linear alkylene having 1 to 4 carbon atoms is preferred.

[0121] The divalent group for X is preferably (1) or (2) among the above (1) to (3), and more preferably (1).

[0122] Regarding the substituent that the divalent group in X may have, the hydrophilic group and the monovalent group are the same as those described above.

[0123] The functional substance represented by D is the same as that described above.

[0124] R A indicates the side chain of a valine residue (i.e., -CH(CH3)2).

[0125] R B indicates the side chain of a citrulline residue (i.e., -CH2CH2CH2NHCONH2) or the side chain of an alanine residue (i.e., -CH3).

[0126] n is 0 or 1. When n is 0, there is no N-R1 unit, and X is directly bonded to two carbonyl groups. When n is 1, the substituents on X may be combined with R1 to form a ring that may contain a hydrophilic group. Such a ring contains a nitrogen atom connected to R1 as a ring-constituting atom. As such a ring, a 5-membered or 6-membered ring is preferred. Such a ring may also be a non-aromatic ring or an aromatic ring, and a non-aromatic ring is preferred. (eg, pyrrolidine, piperazine) are preferred.

[0127] r represents the average ratio of the amide 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.

[0128] At least one (e.g., 1, 2, or 3) hydrophilic group is present in the structural unit represented by formula (I). Thus, at least one of the "monovalent group which may contain a hydrophilic group" represented by R1, the "monovalent group which may contain a hydrophilic group" for the "substituent" in the "divalent group which may have a substituent" represented by X, and the "ring which may contain a hydrophilic group" when the substituent in X is combined with R1 contains a hydrophilic group.

[0129] In certain embodiments, L1 may represent a divalent group represented by formula (i): -L4-Y-L3- (i) [During the ceremony, L3 and L4 each independently represent -(C(R)2) m -, -(OC(R)2-C(R)2) m- and -(C(R)2-C(R)2-O) m -, and combinations thereof; 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 20; Y is a divalent group formed by the reaction of two bioorthogonal functional groups capable of reacting with each other.

[0130] The alkyl having 1 to 6 carbon atoms, the alkenyl having 2 to 6 carbon atoms, or the alkynyl having 2 to 6 carbon atoms represented by R is the same as defined above.

[0131] m is an integer from 0 to 20. m may be preferably an integer of 1 or more, more preferably an integer of 2 or more, an integer of 3 or more, an integer of 4 or more, or an integer of 5 or more. m may also be preferably an integer of 15 or less, more preferably an integer of 12 or less, an integer of 10 or less, or an integer of 9 or less. In addition, m can be set to a different integer for the divalent groups represented by L3 and L4.

[0132] In certain embodiments, L3 and L4 are each independently -(C(R)2). m It may contain a divalent group represented by -.

[0133] Y is a divalent group generated by the reaction of two bioorthogonal functional groups capable of reacting with each other. Combinations of two bioorthogonal functional groups capable of reacting with each other are well known, so a person skilled in the art can appropriately select such combinations and appropriately set the divalent group generated by the reaction of two bioorthogonal functional groups capable of reacting with each other. Combinations of bioorthogonal functional groups capable of reacting with each other include, for example, 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, and a combination of a tetrazine residue and an alkyne residue. and a combination of a thiol residue and another thiol residue (disulfide bond). Thus, Y may be a group formed by the reaction of a thiol residue and a maleimide residue, a group formed by the reaction of a furan residue and a maleimide residue, a group formed by the reaction of a thiol residue and a halocarbonyl residue, a group formed by the reaction of an alkyne residue and an azide residue, or a group formed by the reaction of a tetrazine residue and an alkene residue, or a disulfide group formed by the combination of a thiol residue and another thiol residue.

[0134] In certain embodiments, Y may be a divalent group represented by any one of the following structural formulas:

[0135] [ka]

[0136] [wherein the white and black circles represent bonds, When the bond of the white circle is bonded to L3, the bond of the black circle is bonded to L4, When the open circle bond is bonded to L4, the black circle bond is bonded to L3.

[0137] In a preferred embodiment, the structural unit represented by the above formula (I) may be a structural unit represented by the following formula (I-1), (I-2), (I-3), or (I-4): Such a structural unit may have superior performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

[0138] [ka]

[0139] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I), R1 represents a monovalent group containing a hydrophilic group.

[0140] [ka]

[0141] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); R2 represents a monovalent group containing a hydrophilic group.

[0142] [ka]

[0143] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); R3 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group, R4 represents a monovalent group containing a hydrophilic group.

[0144] [ka]

[0145] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); R1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R6 represents a monovalent group containing a hydrophilic group.

[0146] In the above formula (I-1), (I-2), (I-3), or (I-4), The monovalent group contained therein and the alkyl having 1 to 6 carbon atoms are the same as those described above.

[0147] In a preferred embodiment, the structural unit represented by the above formula (I) may be a structural unit represented by the above formula (I-1), (I-2), or (I-3). Among the structural units represented by the above formula (I), such structural units may have excellent performance as a conjugate (see Examples).

[0148] In one embodiment, in the above formula (I-3), R3 may represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R4 may represent a monovalent group containing a hydrophilic group.

[0149] In another embodiment, in the above formula (I-3), R3 and R4 may each independently represent a monovalent group containing a hydrophilic group.

[0150] In a more preferred embodiment, the structural unit represented by the above formula (I-1), (I-2), (I-3), or (I-4) may be a structural unit represented by the following formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), (I-3b'), (I-4a'), (I-4b'), or (I-4c'). Such a structural unit may have excellent performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

[0151] [ka]

[0152] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0153] [ka]

[0154] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); Two L5's each independently represent a bond or a divalent group; Each of the two HGs independently represents a hydrophilic group.

[0155] [ka]

[0156] [During the ceremony, Ig, L2, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group; L 1a , and L 1b each independently represents a bond or a divalent group, HG' represents a divalent hydrophilic group.

[0157] [ka]

[0158] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0159] [ka]

[0160] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0161] [ka]

[0162] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); HG represents a hydrophilic group.

[0163] [ka]

[0164] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0165] [ka]

[0166] [During the ceremony, Ig, L2, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group; L 1a , and L 1b each independently represents a bond or a divalent group, HG′ represents a divalent hydrophilic group.

[0167] [ka]

[0168] [During the ceremony, Ig, L1, D, R A , R B , r is the same as in formula (I); L5 represents a bond or a divalent group; HG represents a hydrophilic group; L2' represents a bond or a divalent group, E represents an electron withdrawing group; n2 is an integer from 1 to 4.

[0169] In formulae (I-1a') to (I-4c'), the definitions, examples and preferred examples of the divalent group and the hydrophilic group are the same as those described above. Examples of the divalent hydrophilic group include the polyethylene glycol group, polysarcosine group and sugar moiety as described above for the hydrophilic group, and the polyethylene glycol group and polysarcosine group are preferred, and the polyethylene glycol group is more preferred. The electron-withdrawing group is the same as that described above. The bonding position of the electron-withdrawing group to the phenyl ring is the ortho-position, meta-position or para-position to the adjacent amino group (NH), and the ortho-position or para-position is preferred, and the ortho-position is preferred. n2 may preferably be 1 or 2. The definitions, examples and preferred examples of the divalent hydrophilic group, the electron-withdrawing group and its bonding position, as well as n2, are the same for the other formulae.

[0170] In a preferred embodiment, the structural unit represented by "-L5-HG" may be selected from the group consisting of the following: (a)-OH (b) -NH(CH2CH2O)8CH3 (c) -NHCH2CH2COOH

[0171] In certain embodiments, the structural unit represented by formula (I-1a′) may be the following structural unit:

[0172] [ka]

[0173] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0174] [ka]

[0175] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0176] In another particular embodiment, the structural unit represented by formula (I-1b') may be the following structural unit:

[0177] [ka]

[0178] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0179] [ka]

[0180] [During the ceremony, Ig, L2, D, R A , R B , r is the same as in formula (I); L 1a , and L 1b each independently represents a bond or a divalent group, n1 is an integer from 3 to 20.

[0181] In formula (I-1c'-1), the definition, examples and preferred examples of the divalent group are the same as those described above. n1 may be 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. n1 may also be an integer of 20 or less, preferably an integer of 15 or less, more preferably an integer of 12 or less, and even more preferably an integer of 10 or less. More specifically, n1 may be an integer of 3 to 20, preferably an integer of 4 to 15, more preferably an integer of 5 to 12, and even more preferably an integer of 6 to 10. The definition, examples and preferred examples of n1 are the same for the other formulae.

[0182] In yet another particular embodiment, the structural unit represented by formula (I-2′) may be the following structural unit:

[0183] [ka]

[0184] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0185] [ka]

[0186] [During the ceremony, Ig, L1, L2, D, RA , R B , r is the same as in formula (I).

[0187] In yet another particular embodiment, the structural unit represented by formula (I-3a′) may be the following structural unit:

[0188] [ka]

[0189] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0190] [ka]

[0191] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0192] In yet another particular embodiment, the structural unit represented by formula (I-3b') may be the following structural unit:

[0193] [ka]

[0194] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0195] In yet another particular embodiment, the structural unit represented by formula (I-4a′) may be the following structural unit:

[0196] [ka]

[0197] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in formula (I).

[0198] In yet another particular embodiment, the structural unit represented by formula (I-4b') may be the following structural unit:

[0199] [ka]

[0200] [During the ceremony, Ig, L2, D, R A , R B , r is the same as in formula (I); L 1a , and L 1b each independently represents a bond or a divalent group, n1 represents an integer of 3 to 20.

[0201] In yet another particular embodiment, the structural unit represented by formula (I-4c′) may be the following structural unit:

[0202] [ka]

[0203] [During the ceremony, Ig, L1, D, R A , R B , r is the same as in formula (I); L2' represents a bond or a divalent group; or

[0204] [ka]

[0205] [During the ceremony, Ig, L1, D, R A , R B , r is the same as in formula (I); L2' represents a bond or a divalent group.

[0206] In another preferred embodiment, in the above formula, L2 may be a divalent group represented by the following structural formula:

[0207] [ka]

[0208] (Here, the black and white circles represent bonds, The bond marked with a black circle is bonded to the carbonyl group adjacent to L2. The bond marked with a white circle is bonded to D. E represents an electron withdrawing group; n2 is an integer from 1 to 4. The definitions, examples, and preferred examples of the electron-withdrawing group and its bonding position, as well as n2, are the same as those described above.

[0209] In a more preferred embodiment, the structural unit represented by the above formula (I) may be a structural unit represented by the above formula (I-1a'), (I-1b'), (I-2'), (I-3a'), (I-3b'), (I-4b'), or (I-4c') or a formula corresponding to a subordinate concept of these formulas. Such a structural unit may have excellent performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

[0210] In certain embodiments, the conjugate or 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 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, 2.6% or less, 2.4% or less, 2.2% or less, or 2.0% 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).

[0211] The conjugate of the present invention or a salt thereof has the following formula (II):

[0212] [ka]

[0213] [During the ceremony, L2 and L3 each represent a divalent group. R1 represents a monovalent group which may contain a hydrophilic group, X represents a divalent group which may have a substituent, in which the divalent group has 1 to 3 carbon atoms constituting a main chain portion linking the two atoms on both sides of X, and the substituent is a monovalent group which may contain a hydrophilic group; B represents a bioorthogonal functional group; D represents a functional substance, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, n is 0 or 1, and when n is 1, the substituent in X may be combined with R1 to form a ring which may contain a hydrophilic group, or a salt thereof, The antibody can be produced by reacting a starting antibody containing an immunoglobulin unit including two heavy chains and two light chains (wherein the two heavy chains contain lysine residues regioselectively modified with a bioorthogonal functional group) with the L2, L3, R1, X, D, and R1 in the above formula (II). A , R B The definitions, examples and preferred examples of, and n are the same as those described above for formula (I).

[0214] The definition, examples and preferred examples of the bioorthogonal functional group represented by B are the same as those described above.

[0215] In certain embodiments, the bioorthogonal functional group represented by B 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. The bioorthogonal functional group represented by B may be selected so as to be mutually reactive with the bioorthogonal functional group (e.g., the bioorthogonal functional group represented by B') in the starting antibody described below.

[0216] In a preferred embodiment, the compound represented by formula (II) or a salt thereof may be a compound represented by formula (II-1), (II-2), (II-3), or (II-4) below or a salt thereof. Such a compound or a salt thereof is useful as a synthetic intermediate in the production of a conjugate that may have excellent performance among the compounds represented by formula (II) or salts thereof (see Examples).

[0217] [ka]

[0218] [During the ceremony, L2 and L3 each represent a divalent group. R1 represents a monovalent group which may contain a hydrophilic group, D represents a functional substance, R A indicates the side chain of a valine residue, R B represents the side chain of a citrulline or alanine residue, B represents a bioorthogonal functional group.

[0219] [ka]

[0220] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); R2 represents a monovalent group which may contain a hydrophilic group.

[0221] [ka]

[0222] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); R3 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group, R4 represents a monovalent group containing a hydrophilic group.

[0223] [ka]

[0224] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); R1 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R6 represents a monovalent group containing a hydrophilic group.

[0225] In the above formula (II-1), (II-2), (II-3), or (II-4), the monovalent group containing a hydrophilic group and the alkyl having 1 to 6 carbon atoms are the same as those described above.

[0226] In one embodiment, in the above formula (II-3), R3 may represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R4 may represent a monovalent group containing a hydrophilic group.

[0227] In another embodiment, in the above formula (II-3), R3 and R4 may each independently represent a monovalent group containing a hydrophilic group.

[0228] In a preferred embodiment, the compound represented by formula (II) or its salt may be a compound represented by formula (II-1), (II-2), or (II-3) or its salt. Such a compound or its salt is useful as a synthetic intermediate in the production of a conjugate that may have excellent performance among the compounds represented by formula (II) or their salts (see Examples).

[0229] In a more preferred embodiment, the compound or salt thereof represented by the above formula (II-1), (II-2), (II-3), or (II-4) may be a compound or salt thereof represented by the following formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), (II-3b'), (II-4a'), (II-4b'), or (II-4c'). Such a compound or salt thereof is useful as a synthetic intermediate in the production of a conjugate having excellent performance among the compounds or salts thereof represented by the above formula (II) (see Examples).

[0230] [ka]

[0231] [During the ceremony, L2, L3, D, RA , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0232] [ka]

[0233] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); Two L5's each independently represent a bond or a divalent group; Each of the two HGs independently represents a hydrophilic group.

[0234] [ka]

[0235] [During the ceremony, L2, D, R A , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group; L 3a , and L 3b each independently represents a bond or a divalent group, HG′ represents a divalent hydrophilic group.

[0236] [ka]

[0237] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0238] [ka]

[0239] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0240] [ka]

[0241] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); HG represents a hydrophilic group.

[0242] [ka]

[0243] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group.

[0244] [ka]

[0245] [During the ceremony, L2, D, R A , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group; L 3a , and L 3b each independently represents a bond or a divalent group, HG′ represents a divalent hydrophilic group.

[0246] [ka]

[0247] [During the ceremony, L3, D, R A , R B and B are the same as in formula (II-1); L5 represents a bond or a divalent group; HG represents a hydrophilic group; L2' represents a bond or a divalent group, E represents an electron withdrawing group; n2 is an integer from 1 to 4.

[0248] In formulae (II-1a') to (II-4c'), the definitions, examples, and preferred examples of the structural units represented by the divalent group and the hydrophilic group are the same as those described above. The definitions, examples, and preferred examples of the divalent hydrophilic group, the electron-withdrawing group and its bonding position, and n2 are the same as those described above.

[0249] In a preferred embodiment, the structural unit represented by "-L5-HG" may be selected from the group consisting of the following: (a)-OH (b) -NH(CH2CH2O)8CH3 (c) -NHCH2CH2COOH

[0250] In certain embodiments, the compound represented by formula (II-1a′) may be:

[0251] [ka]

[0252] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0253] [ka]

[0254] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0255] In another particular embodiment, the compound of formula (II-1b') may be:

[0256] [ka]

[0257] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0258] In another particular embodiment, the compound of formula (II-1c') may be:

[0259] [ka]

[0260] [During the ceremony, L2, D, R A , R B and B are the same as in formula (II-1); L 3a , and L 3b each independently represents a bond or a divalent group, n1 is an integer from 3 to 20.

[0261] L 3a , and L 3b The definitions, examples, and preferred examples of the divalent group represented by the formula: and n1 are the same as those described above (the same applies hereinafter).

[0262] In another particular embodiment, the compound of formula (II-2') may be:

[0263] [ka]

[0264] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0265] [ka]

[0266] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0267] In yet another particular embodiment, the compound of formula (II-3a') may be:

[0268] [ka]

[0269] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0270] [ka]

[0271] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0272] In yet another particular embodiment, the compound of formula (II-3b') may be:

[0273] [ka]

[0274] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0275] In yet another particular embodiment, the compound of formula (II-4a′) may be:

[0276] [ka]

[0277] [During the ceremony, L2, L3, D, R A , R B and B are the same as in formula (II-1).

[0278] In yet another particular embodiment, the compound of formula (II-4b') may be:

[0279] [ka]

[0280] [During the ceremony, L2, D, R A , R Band B are the same as in formula (II-1); L 3a , and L 3b each independently represents a bond or a divalent group, n1 is an integer from 3 to 20.

[0281] In yet another particular embodiment, the compound of formula (II-4c') may be:

[0282] [ka]

[0283] [During the ceremony, L3, D, R A , R B and B are the same as in formula (II-1); L2' represents a bond or a divalent group; or

[0284] [ka]

[0285] [During the ceremony, L3, D, R A , R B and B are the same as in formula (II-1); L2' represents a bond or a divalent group.

[0286] In a preferred embodiment, in the above formula, L2 may be a divalent group represented by the following structural formula:

[0287] [ka]

[0288] (Here, the black and white circles represent bonds, The bond marked with a black circle is bonded to the carbonyl group adjacent to L2. The bond marked with a white circle is bonded to D. E represents an electron withdrawing group; n2 is an integer from 1 to 4. The definitions, examples, and preferred examples of the electron-withdrawing group and its bonding position, as well as n2, are the same as those described above.

[0289] In a more preferred embodiment, the compound represented by formula (II) or its salt may be a structural unit represented by formula (II-1a'), (II-1b'), (II-2'), (II-3a'), (II-3b'), (II-4b'), or (II-4c'), or a formula corresponding to a subordinate concept of these formulas. Such a compound or its salt is useful as a synthetic intermediate in the production of a conjugate that may have excellent performance among the compounds represented by formula (II) or its salts (see Examples).

[0290] The starting antibody contains a lysine residue regioselectively modified with a bioorthogonal functional group. The bioorthogonal functional group in the starting antibody 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. The bioorthogonal functional group in the starting antibody may be selected so as to be mutually reactive with the bioorthogonal functional group in the above-mentioned compound or its salt (e.g., the bioorthogonal functional group represented by B).

[0291] In certain embodiments, the starting antibody has the following formula (III):

[0292] [ka]

[0293] [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and selectively forms an amide bond between the carbonyl group adjacent to Ig and the amino group in the side chain of the lysine residue in the two heavy chains, L4 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 B; The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5. The definitions, examples and preferred examples of Ig, L4, R, m and r in the above formula (III) are the same as those described above.

[0294] The bioorthogonal functional group denoted by B' is the same as the bioorthogonal functional group described above for the starting antibody.

[0295] 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).

[0296] The production of the conjugate or its salt can be confirmed by, for example, reverse phase HPLC under reducing conditions or mass spectrometry, depending on the specific raw material and the molecular weight of the product. The conjugate or its salt can be appropriately purified by any method such as chromatography (e.g., affinity chromatography).

[0297] The conjugate of the present invention or a salt thereof can be used, for example, as a medicine or a reagent (eg, diagnostic agent, research reagent).

[0298] 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).

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 3. Compounds or their salts, and reagents containing them The present invention also provides a compound represented by the above formula (II-1), (II-2), or (II-3) or a salt thereof. The details of these compounds or salts thereof are the same as those described above.

[0303] In a preferred embodiment, the compound or salt thereof represented by the above formula (II-1), (II-2), or (II-3) may be a compound or salt thereof represented by the above formula (II-1a'), (II-1b'), (II-2'), (II-3a'), or (II-3b'), or a formula corresponding to a subordinate concept of these formulas. The details of these compounds or salts are the same as those described above.

[0304] The compounds of the present invention, or salts thereof, are useful as synthetic intermediates in the preparation of conjugates that may have superior performance (see Examples).

[0305] The present invention also relates to a derivatization reagent for an antibody, comprising a compound represented by the above formula (II-1), (II-2), (II-3), (II-4b'), or (II-4c') or a salt thereof. Provided.

[0306] The starting antibody or its salt to be derivatized with the reagent of the present invention, and the conjugate or its salt obtained by the derivatization reaction of the starting antibody or its salt are as described above in detail.

[0307] The reagent of the present invention may be provided in the form of a composition further containing other components. Such other components include, for example, a solution and a stabilizer (e.g., an antioxidant, a preservative). The solution is preferably an aqueous solution. Examples of the aqueous solution include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), and buffer solutions (e.g., aqueous phosphate solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), with buffer solutions being preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder). EXAMPLES

[0308] 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.

[0309] Example 1: Synthesis of Linker-payload mimic (1-1) Synthesis of Linker-payload mimic (1) The linker-payload mimic(1) was synthesized as follows:

[0310] [ka]

[0311] (1-1-1) Synthesis of pyrene (2)

[0312] [ka]

[0313] Fmoc-Val-Cit-PAB-PNP(CAS No:863971-53-2 , 121.2 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (5 mL), and known (described in WO2018218004A1) sarcosine-pyrene (59.2 mg, 0.196 mmol), N,N-diisopropylethylamine (39 μL, 0.227 mmol), and 4-dimethylaminopyridine (3.7 mg, 0.03 mmol) were added and stirred at room temperature for 2 hours, after which diethylamine (2 mL, 18.95 mmol) was added and stirred at room temperature for 1.5 hours. After concentrating under reduced pressure, it 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 (2) (73.7 mg, 0.104 mmol).

[0314] 1 H NMR(400MHz,DMSO-d6)δ10.21(s,1H),8.68(s,1H),8.43-7.93(m,12H),7.60(t, J=7.1Hz,2H),7.31(m,2H),6.04(s,1H),5.48(s,2H),5.02(d,J=16.1Hz,4H),4.5 4(s,1H),3.96(s,2H),3.66(s,2H),2.92(d,J=6.1Hz,3H),2.08(q,J=6.6Hz,1H) ,1.80-1.56(m,2H),1.46(s,2H),1.21-1.13(m,1H),0.94(dt,J=6.8,3.0Hz,6H).

[0315] MS(ESI)m / z:708.80[M+H] +

[0316] (1-1-2) Synthesis of pyrene (3)

[0317] [ka]

[0318] Fmoc-Glu(OtBu)-OH·HO (11.1 mg, 0.025 mmol) was dissolved in dimethylformamide (1 mL), pyrene (2) (17.3 mg, 0.024 mmol), 1-hydroxy-7-azabenzotriazole (5.1 mg, 0.037 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.3 mg, 0.038 mmol), and triethylamine (7.1 μL, 0.51 mmol) were added, and the mixture was stirred at room temperature for 2.5 hours. Diethylamine (0.2 mL, 1.91 mmol) was then added and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, 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 pyrene (3) (15.6 mg, 0.017 mmol).

[0319] 1 H NMR(400MHz,Chloroform-d)δ10.87(s,1H),8.68(m,1H),8.41-7.97(m,13H),7.60-7.58(m,2 H),7.30(m,2H),6.00(t,J=6.2Hz,1H),5.45(s,2H),5.05-4.99(m,4H),4.46(m,1H),4.26(m,1 H),3.96(s,2H),3.88(m,1H),3.07(m,1H),2.96(m,1H),2.93(d,J=5.6Hz,3H),2.68(t,J=1.8Hz,1H ),2.34-2.30(m,3H),2.03(m,1H),1.93-1.90(m,2H),1.30(s,9H),1.15(s,1H),0.92-0.86(m,6H).

[0320] MS(ESI)m / z:893.45[M+H] +

[0321] (1-1-3) Synthesis of pyrene (4)

[0322] [ka]

[0323] Pyrene (3) (15.6 mg, 0.017 mmol) was dissolved in dimethylformamide (1.5 mL), 6-maleimidohexanoic acid (3.7 mg, 0.018 mmol), 1-hydroxy-7-azabenzotriazole (3.5 mg, 0.025 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.8 mg, 0.025 mmol), and triethylamine (4.8 μL, 0.34 mmol) were added, and the mixture was stirred at room temperature for 3 hours. Then, 6-maleimidohexanoic acid (1.8 mg, 0.009 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 mg, 0.012 mmol), and triethylamine (2.4 μL, 0.17 mmol) were added, and the mixture was stirred at room temperature for 1.5 hours. Then, 4-dimethylaminopyridine (0.5 mg, 0.004 mmol) was added and the mixture was stirred for another 2.5 hours. After concentration under reduced pressure, the mixture was purified by column chromatography (dichloromethane:methanol=9:1). The fraction was collected and concentrated under reduced pressure to obtain the above pyrene (4) (8.0 mg, 0.007 mmol).

[0324] MS(ESI)m / z:1086.60[M+H] +

[0325] (1-1-4) Synthesis of Linker-payload mimic (1)

[0326] [ka]

[0327] Pyrene (4) (9.7 mg, 0.0089 mmol) was dissolved in 1,4-dioxane (1 mL) and hydrogen chloride-1,4-dioxane solution (1 mL) and stirred in an ice-water bath for 2 hours. Dimethylformamide (1 mL) was added, and the mixture was warmed to room temperature and then concentrated under reduced pressure. It was then 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 lyophilized to obtain the linker-payload mimic (1) (2.0 mg, 0.002 mmol).

[0328] MS(ESI)m / z:1030.50[M+H] +

[0329] (1-2) Synthesis of Linker-payload mimic (5) The linker-payload mimic (5) was synthesized as follows:

[0330] [ka]

[0331] (1-2-1) Synthesis of Maleimide (6)

[0332] [ka]

[0333] Fmoc-Glu-OtBu (213.0 mg, 0.50 mmol) was dissolved in dichloromethane (2 mL), N-(5-aminopentyl)maleimide hydrochloride (108.7 mg, 0.48 mmol), 1-hydroxybenzotriazole (101.7 mg, 0.753 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (143.6 mg, 0.749 mmol), triethylamine (155 μL, 1.12 mmol), and 4-dimethylaminopyridine (7.2 mg, 0.0589 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the mixture was purified by column chromatography (hexane: ethyl acetate = 4:1). The fraction containing the product was collected and concentrated under reduced pressure to obtain the above maleimide (6) (254.1 mg, 0.431 mmol).

[0334] 1 H NMR(400 MHz,Methanol-d4)δ7.82(d,J=7.6 Hz,3H),7.70(t,J=6.7 Hz,2H),7.41(t,J=7.5 Hz,2H),7.37-7.29(m,2H),6.79(s,2H),4.43(dd,J=10.4,6.8 Hz,1H),4.34(dd,J=10.4,7.0 Hz,1H),4.24(t,J=6.9 Hz,1H),4.18-4.00(m,1H),3.50(t,J=7.1 Hz,2H),3.16(td,J=6.9,2.0 Hz,2H),2.28(t,J=7.5 Hz,2H),2.24-2.08(m,1H),1.99-1.83(m,1H),1.48(s,13H),1.28(dt, J=17.1,7.1 Hz,2H).

[0335] MS(ESI)m / z:590.25[M+H] +

[0336] (1-2-2) Synthesis of maleimide (7)

[0337] [ka]

[0338] Maleimide (6) (253.1 mg, 0.41 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, 1 M hydrochloric acid (2 mL) was added, the mixture was stirred at room temperature for 3 minutes, concentrated under reduced pressure, and then freeze-dried to obtain the above maleimide (7) (244.6 mg, 0.458 mmol).

[0339] 1 H NMR(400 MHz,Methanol-d4)δ7.82(d,J=7.5 Hz,2H),7.70(dt,J=9.3,4.7 Hz,2H),7.41(t,J=7.5 Hz,2H),7.33(t,J=7.4 Hz,2H),6.79(s,2H),4.49-4.30(m,2H),4.30-4.11(m,2H),3.50(t,J=7.0 Hz,2H),3.16(t,J=7.2 Hz,2H),2.36-2.09(m,3H),2.09-1.86(m,1H),1.56(dt,J=25.5,7.5 Hz,5H),1.39-1.23(m,2H).

[0340] MS(ESI)m / z:534.20[M+H] +

[0341] (1-2-3) Synthesis of pyrene (8)

[0342] [ka]

[0343] Maleimide (7) (31.5 mg, 0.052 mmol) was dissolved in N,N-dimethylformamide (2 mL), pyrene (2) (35.5 mg, 0.50 mmol) synthesized in Example 1-1-1, 1-hydroxybenzotriazole (10.1 mg, 0.0747 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (15.0 mg, 0.0782 mmol), triethylamine (14 μL, 0.10 mmol), and 4-dimethylaminopyridine (0.7 mg, 0.00573 mmol) were added, and the mixture was stirred at room temperature for 2 hours, and then purified by column chromatography (hexane: ethyl acetate = 15:85). The fraction containing the product was collected and concentrated under reduced pressure to obtain the pyrene (8) (7.4 mg, 6.05 nmol).

[0344] MS(ESI)m / z:1223.65[M+H] + ,612.65[M+2H] 2+

[0345] (1-2-4) Synthesis of Linker-payload mimic (5)

[0346] [ka]

[0347] Pyrene (8) (6.3 mg, 0.0051 mmol) was dissolved in N,N-dimethylformamide (0.6 mL), dicyclohexylamine (0.12 mL, 0.6 mmol) was added, and the mixture was stirred at room temperature for 3 hours. Succinic anhydride (1.0 mg, 0.0095 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. The mixture was 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 linker-payload mimic (5) (1.10 mg, 0.991 nmol).

[0348] 11H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.59 (d, J = 6.2 Hz, 1H), 8.36 - 7.87 (m, 10H), 7.67 (t, J = 8.7 Hz, 2H), 7.22 (dd, J = 26.1, 8.1 Hz, 3H), 6.91 (s, 2H), 5.92 (s, 1H), 4.94 (d, J = 18.4 Hz, 4H), 4.33 (d, J = 7.5 Hz, 1H), 3.88 (s, 2H), 3.10 (s, 3H), 3.05 - 2.78 (m, 7H), 2.69 (d, J = 22.3 Hz, 1H), 2.38 - 2.22 (m, 3H), 2.15 - 1.89 (m, 4H), 1.81 (d, J = 14.3 Hz, 1H), 1.72 - 1.50 (m, 3H), 1.48 - 1.22 (m, 6H), 1.14 (dd, J = 35.5, 8.5 Hz, 4H), 0.92 (s, 1H), 0.85 - 0.68 (m, 6H).

[0349] MS (ESI) m / z: 1101.55 [M + H] +

[0350] (1 - 3) Synthesis of Linker - payload mimic (9) Linker - payload mimic (9) was synthesized as follows.

[0351]

Chemical Structure

[0352] (1 - 3 - 1) Synthesis of maleimide (10)

[0353]

Chemical Structure

[0354] 6-Maleimidohexanoic acid (300mg, 1.420mmol) was dissolved in dimethylformamide (2mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (647.8mg, 1.704mmol), iminodiacetic acid (189.0mg, 1.420mmol), N,N-diisopropylethylamine (0.29mL, 1.704mmol), 4-dimethylaminopyridine (34.7mg, 0.284mmol) were added, and the mixture was stirred for 1 hour. After concentrating under reduced pressure, 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 maleimide (10) (65.9mg, 0.202mmol).

[0355] 1 H NMR(400 MHz,DMSO-d6)δ6.79(s,2H),4.25( s,2H),4.11(s,2H),3.49(t,J=7.0Hz,2H),2.35(t,J=7.4Hz,2H),1.16(m,4H),1.33(m,2H)

[0356] MS(ESI)m / z:327.00[M+H] +

[0357] (1-3-2) Synthesis of Linker-payload mimic (9)

[0358] [ka]

[0359] Maleimide (10) (46.2 mg, 0.142 mmol) was dissolved in dimethylformamide (0.3 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (54.0 mg, 0.142 mmol) was added, and the mixture was stirred for 1 hour. After that, pyrene (2) (50.1 mg, 0.71 mmol) synthesized in Example 1-1-1, N,N-diisopropylethylamine (0.025 mL, 0.142 mmol) was added in dimethylformamide solution (0.3 mL), and the mixture was stirred for another 3 hours. After purification by reversed-phase preparative chromatography, the fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain Linker-payload mimic (9) (24.4 mg, 0.024 mmol).

[0360] MS(ESI)m / z:1016.50[M+H] +

[0361] Example 2: Synthesis of Linker-Payload (2-1) Synthesis of Linker-payload (11) Linker-payload (11) was synthesized according to Example 1-1 using the known compound Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518).

[0362] [ka]

[0363] MS(ESI)m / z:1445.8[M+H] +

[0364] (2-2) Synthesis of Linker-payload (12) Linker-payload (12) was synthesized as follows.

[0365] [ka]

[0366] [ka]

[0367] Fmoc-Glu(OMe)-H (56 mg, 0.15 mmol) was dissolved in dimethylformamide (1 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (56 mg, 0.15 mmol) and N,N-diisopropylethylamine (0.046 mL, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 minute. The reaction mixture was mixed with the known compound Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) (150 mg, 0.15 mmol). A DMF solution (1 mL) of 1.13 mmol of 1,2-dichlorophenyl ether (1.0 mmol) was added and stirred for an additional 30 minutes. After completion of the reaction, the reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic acid) and purified by reverse phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 14 (180 mg, 0.12 mmol).

[0368] MS(ESI)m / z:1488.2[M+H] +

[0369] [ka]

[0370] Lithium hydroxide (8.7 mg, 0.36 mmol) and compound 14 (180 mg, 0.12 mmol) were dissolved in 1:1 water:THF (8 mL) and stirred at room temperature for 15 minutes. After completion of the reaction, 1M aqueous hydrochloric acid was carefully added to the reaction solution to adjust the pH to 5-6. This 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 compound 15 (100 mg, 0.068 mmol).

[0371] MS(ESI)m / z:1474.4[M+H] +

[0372] [ka]

[0373] Compound 15 (100 mg, 0.068 mmol) was dissolved in dimethylformamide (2 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (0.035 mL, 0.20 mmol) were added, and the mixture was stirred at room temperature for 1 minute. N-(5-aminopentyl)maleimide hydrochloride (30 mg, 0.10 mmol) was added to the reaction mixture, and the mixture was stirred for another 5 minutes. After the reaction was completed, the reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic acid) and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain Compound 16 (83 mg, 0.051 mmol).

[0374] MS(ESI)m / z:1638.6[M+H] +

[0375] [ka]

[0376] Compound 16 (21 mg, 0.013 mmol) was dissolved in dimethylformamide (1 mL), diazabicycloundecene (0.0042 mL, 0.028 mmol) was added, and the mixture was stirred at room temperature for 2 minutes. After the reaction was completed, 1M aqueous hydrochloric acid was carefully added to the reaction solution to adjust the pH to 5-6. The reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic 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 compound 17 (10 mg, 0.0071 mmol).

[0377] MS(ESI)m / z:1416.5[M+H] +

[0378] [ka]

[0379] Under an argon atmosphere, compound (17) (21 mg, 0.015 mmol) was dissolved in N,N-dimethylformamide (2 mL), 4-methylmorpholine (0.082 mL, 0.074 mmol) and succinic anhydride (7.4 mg, 0.074 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was then diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic 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 Linker-payload (12) (17.5 mg, 0.012 nmol).

[0380] 11H NMR (500 MHz, DMSO-d6) δ 12.06 (br.s, 1H), 9.94 - 10.05 (m, 1H), 8.26 - 8.33 (m, 0.5H), 8.13 - 8.20 (m, 1H), 8.10 (d, J = 7.8 Hz, 2H), 8.03 - 8.07 (m, 0.5H), 7.86 - 7.90 (m, 0.5H), 7.69 - 7.77 (m, 2H), 7.60 - 7.64 (m, 0.5H), 7.55 - 7.60 (m, 2H), 7.29 - 7.35 (m, 3H), 7.23 - 7.29 (m, 3H), 7.17 (s, 1H), 6.99 (s, 2H), 5.94 - 6.02 (m, 1H), 5.39 - 5.44 (m, 2H), 5.34 (d, J = 4.9 Hz, 1H), 4.94 - 5.12 (m, 2H), 4.70 - 4.77 (m, 1H), 4.58 - 4.68 (m, 1H), 4.49 - 4.52 (m, 1H), 4.40 - 4.45 (m, 1H), 4.33 - 4.40 (m, 1H), 4.21 - 4.29 (m, 2H), 4.18 - 4.21 (m, 1H), 3.92 - 4.02 (m, 2H), 3.76 - 3.80 (m, 1H), 3.53 - 3.61 (m, 1H), 3.41 - 3.50 (m, 1H), 3.36 (t, J = 7.1 Hz, 2H), 3.29 - 3.31 (m, 1H), 3.21 - 3.26 (m, 4H), 3.20 (s, 2H), 3.17 (s, 2H), 3.11 (s, 2H), 2.90 - 3.07 (m, 5H), 2.82 - 2.8 9 (m, 3H), 2.31 - 2.45 (m, 5H), 2.22 - 2.30 (m, 1H), 2.02 - 2.17 (m, 3H), 1.97 - 2.01 (m, 2H), 1.83 - 1.91 (m, 1H), 1.75 - 1.82 (m, 1H), 1.65 - 1.75 (m, 2H), 1.50 - 1.62 (m, 2H), 1.42 - 1.50 (m, 4H), 1.26 - 1.39 (m, 3H), 1.13 - 1.22 (m, 2H), 0.96 - 1.06 (m, 6H), 0.72 - 0.89 (m, 24H)

[0381] MS (ESI) m / z: 1516.4 [M + H] +

[0382] (2 - 3) Linker - payload (18) synthesis Linker-Payload (18) was synthesized as follows.

[0383] [ka]

[0384] [ka]

[0385] Linker-payload (12) (10 mg, 0.0066 mmol) was dissolved in dimethylformamide (1 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.8 mg, 0.0073 mmol) and N,N-diisopropylethylamine (0.0023 mL, 0.013 mmol) were added, and the mixture was stirred at room temperature for 1 minute. Methoxy-PEG8-amine (3.8 mg, 0.0099 mmol) was added to the reaction mixture, and the mixture was stirred for another 2 minutes. After the reaction was completed, the reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic acid) and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the linker payload (18) (12.1 mg, 0.064 mmol).

[0386] 1 H NMR(500 MHz,DMSO-d6)δ9.89-10.00(m,1H),8.26-8.35(m,1H),8.10-8.15(m,1H),8.03-8.10(m,1H),7.92-7.96(m,1H),7.86-7.92(m,0.5 H), 7.71 - 7.78 (m, 2H), 7.60 - 7.65 (m, 0.5H), 7.53 - 7.60 (m, 2H), 7.23 - 7.36 (m, 6H), 7.13 - 7.20 (m, 1H), 6.99 (s, 2H), 5.93 - 6.00 (m, 1H), 5.40 - 5.43 (m, 2H), 5.32 - 5.36 (m, 1H), 4.92 - 5.14 (m, 2H), 4.69 - 4.78 (m, 1H), 4.58 - 4.68 (m, 1H), 4.45 - 4.51 (m, 1H), 4.39 - 4.45 (m, 1H), 4.31 - 4.39 (m, 1H), 4.24 - 4.30 (m, 1H), 4.19 - 4.24 (m, 1H), 4.13 - 4.18 (m, 1H), 3.91 - 4.05 (m, 2H), 3.53 - 3.61 (m, 1H), 3.46 - 3.53 (m, 36H), 3.40 - 3.44 (m, 3H), 3.33 - 3.40 (m, 3H), 3.21 - 3.26 (m, 6H), 3.14 - 3.21 (m, 4H), 3.11 (s, 1H), 2.91 - 3.06 (m, 4H), 2.82 - 2.90 (m, 2H), 2.22 - 2.44 (m, 6H), 2.05 - 2.16 (m, 3H), 1.94 - 2.04 (m, 2H), 1.84 - 1.93 (m, 1H), 1.75 - 1.83 (m, 1H), 1.63 - 1.74 (m, 2H), 1.50 - 1.62 (m, 2H), 1.40 - 1.50 (m, 4H), 1.31 - 1.40 (m, 4H), 1.13 - 1.21 (m, 2H), 0.95 - 1.07 (m, 6H), 0.71 - 0.90 (m, 24H)

[0387] MS(ESI) m / z: 1882.6 [M + H] +

[0388] (2 - 4) Linker - payload(19) synthesis Linker - Payload(19) was synthesized as follows.

[0389]

Chemical Structure

[0390]

Chemical Structure

[0391] Maleimide (10) (8 mg, 0.0245 mmol) was dissolved in dimethylformamide (0.2 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (4.7 mg, 0.0123 mmol) and N,N-diisopropylethylamine (0.0085 mL, 0.0491 mmol) were added, and the mixture was stirred at room temperature for 5 minutes. The reaction mixture was treated with the known compound Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) (13.8 mg, 0.0123 mmol) and N,N-diisopropylethylamine (0.00 After addition of 86 mL, 0.0491 mmol), the mixture was stirred for an additional 2 hours. After completion of the reaction, 1 M aqueous hydrochloric acid was carefully added to the reaction solution to adjust the pH to 5-6. The reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic 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 linker-payload (19) (12.1 mg, 0.064 mmol).

[0392] 1H NMR (500 MHz, DMSO-d6) δ 9.98 (brs, 1H), 9.77 (brs, 1H), 8.40 - 8.27 (m, 1H), 8.21 (d, J = 7.3 Hz, 1H), 8.10 - 8.02 (m, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.63 - 7.57 (m, 3H), 7.34 - 7.24 (m, 6H), 7.21 - 7.13 (m, 1H), 6.99 (d, J = 2.4 Hz, 2H), 6.04 - 5.96 (m, 1H), 5.46 - 5.40 (m, 2H), 5.13 - 4.93 (m, 4H), 4.79 - 4.60 (m, 2H), 4.53 - 4.35 (m, 3H), 4.34 - 4.23 (m, 3H), 4.21 - 3.88 (m, 4H), 3.78 (dd, J = 9.5, 2.2 Hz, 1H), 3.65 - 3.53 (m, 1H), 3.51 - 3.43 (m, 2H), 3.40 - 3.34 (m, 2H), 3.28 - 3.16 (m, 8H), 3.14 - 3.10 (m, 2H), 3.08 - 2.81 (m, 5H), 2.41 - 1.90 (m, 8H), 1.87 - 1.64 (m, 3H), 1.64 - 1.41 (m, 6H), 1.40 - 1.13 (m, 5H), 1.11 - 0.95 (m, 6H), 0.95 - 0.65 (m, 24H).

[0393] MS (ESI) m / z: 1431.2 [M + H] +

[0394] (2 - 5) Synthesis of Linker - payload(20) Linker - Payload(20) was synthesized as follows.

[0395]

Chemical Structure

[0396]

Chemical Structure

[0397] Linker-payload (19) (14.6 mg, 0.0102 mmol) was dissolved in dimethylformamide (0.12 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (4.7 mg, 0.0122 mmol) and N,N-diisopropylethylamine (0.0035 mL, 0.0204 mmol) were added, and the mixture was stirred at room temperature for 5 minutes. Methoxy-PEG8-amine (4.7 mg, 0.0122 mmol) and N,N-diisopropylethylamine (0.0035 mL, 0.0204 mmol) were added to the reaction mixture, and the mixture was stirred for another 10 minutes. After the reaction was completed, 1M aqueous hydrochloric acid was carefully added to the reaction solution to adjust the pH to 5-6. The reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic 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 Linker-payload (20) (12.9 mg, 0.072 mmol).

[0398] MS(ESI)m / z:1796.9[M+H] +

[0399] (2-6) Synthesis of Linker-payload (21) Linker-Payload (21) was synthesized as follows.

[0400] [ka]

[0401] Linker-Payload (21) was synthesized according to the following scheme.

[0402] [ka]

[0403] The MS analysis results of the Linker-Payload (21) were as follows.

[0404] MS(ESI)m / z:1544.8[M+H] +

[0405] (2-7) Synthesis of Linker-payload (32) Linker-Payload (32) was synthesized as follows.

[0406] [ka]

[0407] [ka]

[0408] Compound (17) (15 mg, 0.011 mmol) was dissolved in acrylic acid (1.5 mL) and stirred at 40° C. for 24 hours, after which the reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic 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 Linker-payload (32) (2.5 mg, 0.0016 mmol).

[0409] 1 H NMR(500 MHz,DMSO-d6)δ10.58(brs,2H),9.59(brs,1H),8.99-9.07(m,1H),7.98-8.04(m,1H),7.85-7.97(m,2H),7.72-7 .78(m,1H),7.59-7.68(m,2H),7.22-7.36(m,8H),7.11-7.20(m,1H),6.99(s,2H),6.80-6.93(m,1H),5.49-5.89( m, 4H), 4.91 - 5.14 (m, 2H), 4.69 - 4.78 (m, 1H), 4.57 - 4.67 (m, 1H), 4.35 - 4.53 (m, 3H), 4.18 - 4.31 (m, 2H), 3.89 - 4.08 (m, 2H), 3.74 - 3.81 (m, 1H), 3.51 - 3.66 (m, 1H), 3.34 - 3.39 (m, 3H), 3.25 - 3.11 (m, 10H), 2.91 - 3.07 (m, 5H), 2.84 - 2.90 (m, 3H), 2.65 - 2.83 (m, 4H), 2.37 - 2.43 (m, 5H), 2.22 - 2.33 (m, 1H), 2.09 - 2.20 (m, 4H), 1.96 - 2.09 (m, 2H), 1.83 - 1.96 (m, 2H), 1.69 - 1.83 (m, 2H), 1.59 - 1.68 (m, 1H), 1.53 - 1.58 (m, 1H), 1.42 - 1.52 (m, 4H), 1.26 - 1.40 (m, 3H), 1.14 - 1.25 (m, 2H), 0.94 - 1.06 (m, 6H), 0.71 - 0.89 (m, 24H)

[0410] MS(ESI) m / z: 1560.3 [M + H] +

[0411] (2 - 8) Linker - payload(33) synthesis Linker - Payload(33) was synthesized as follows.

[0412]

Chemical Structure

[0413]

Chemical Structure

[0414] Iminodiacetic acid (212 mg, 1.59 mmol) and sodium bicarbonate (534 mg, 6.36 mmol) were dissolved in water (4 mL). After the foaming had subsided, THF (2 mL) and Boc2O (416 mg, 1.91 mmol) were added and stirred at room temperature for 72 hours. After removing THF by concentration under reduced pressure, the reaction solution was adjusted to about pH 1 with 1N hydrochloric acid. The aqueous layer was extracted with ethyl acetate (15 mL x 3), the organic layer was dried over magnesium sulfate, and the filtered organic layer was concentrated to obtain compound (34) (205 mg, 0.877 mmol).

[0415] MS(ESI)m / z:256.0[M+Na] +

[0416] [ka]

[0417] Compound (34) (14.6 mg, 0.0625 mmol), HATU (26.2 mg, 0.0689 mmol), and DIPEA (21.8 μL, 0.125 mmol) were dissolved in DMF (1.5 ml) and stirred at room temperature for 5 minutes. A solution of H2N-PEG8-OMe (52.7 mg, 0.138 mmol) and DIPEA (21.8 μL, 0.125 mmol) dissolved in DMF (1 mL) was added to the above reaction solution and stirred at room temperature for 1 hour. HATU (26.2 mg, 0.0689 mmol) and DIPEA (21.8 μL, 0.125 mmol) were further added to the reaction solution and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with a 1:1 water:acetonitrile solution, and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound (35) (33.2 mg, 0.0344 mmol).

[0418] MS(ESI)m / z:964.4[M+H] +

[0419] [ka]

[0420] Compound (35) (33.2 mg, 0.0344 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain compound (36) (33.6 mg, quant).

[0421] MS(ESI)m / z:864.2[M+H] +

[0422] [ka]

[0423] Linker-payload (19) (6.6 mg, 0.0046 mmol), HATU (4.4 mg, 0.0046 mmol), and DIPEA (1.6 μL, 0.0092 mmol) were dissolved in DMF (0.15 ml) and stirred at room temperature for 5 minutes. A solution of compound (36) (5.0 mg, 0.0058 mmol) and DIPEA (1.6 μL, 0.0058 mmol) dissolved in DMF (0.15 mL) was added to the above reaction solution and stirred at room temperature for 3 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain Linker-payload (33) (2.0 mg, 0.00088 mmol).

[0424] MS(ESI)m / z:2277.8[M+H] +

[0425] (2-9) Synthesis of Linker-payload (37) Linker-Payload (37) was synthesized as follows.

[0426] [ka]

[0427] [ka]

[0428] NHS-PEG9-NHS (164 mg, 0.231 mmol) was dissolved in DMF (1 mL). In a separate flask, compound (38) (35.5 mg, 0.112 mmol) was dissolved in DMF (1.5 mL), and this solution was added to the above-mentioned NHS-PEG9-NHS solution. The reaction solution was stirred at room temperature for 30 minutes, diluted with water (8 mL), and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound (39) (56.8 mg, 0.0623 mmol).

[0429] MS(ESI)m / z:912.2[M+H] +

[0430] [ka]

[0431] Fmoc-iminodiacetic acid (13.6 mg, 0.0382 mmol), HATU (14.5 mg, 0.0382 mmol), and DIPEA (10.0 μL, 0.057 mmol) were dissolved in DMF (0.75 ml) and stirred at room temperature for 5 minutes. In another flask, a solution of H2N-VC-PAB-MMAE (42.9 mg, 0.0382 mmol) and DIPEA (10.0 μL, 0.057 mmol) dissolved in DMF (0.75 mL) was added to the above-mentioned Fmoc-iminodiacetic acid solution and stirred at room temperature for 18 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound (40) (21.2 mg, 0.0143 mmol).

[0432] MS(ESI)m / z:1460.4[M+H]+

[0433] [ka]

[0434] Compound (40) (100 mg, 0.0695 mmol) was dissolved in DMF (2.5 mL), DBU (31 μL, 0.205 mmol) was added, and the mixture was stirred at room temperature for 5 minutes. The reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05% formic 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 compound (41) (86.6 mg, 0.0674 mmol).

[0435] MS(ESI)m / z:1238.4[M+H] +

[0436] [ka]

[0437] Compound (39) (22.9 mg, 0.0185 mmol) and compound (41) (33.7 mg, 0.0178 mmol) were dissolved in a 1:1 water:acetonitrile solution (2 mL), sodium bicarbonate (1.9 mg, 0.0222 mmol) was added, and the mixture was stirred at room temperature for 23 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution 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 Linker-payload (37) (7.3 mg, 0.0036 mmol).

[0438] MS(ESI)m / z:2035.5[M+H] +

[0439] (2-10) Synthesis of Linker-payload (42) Linker-Payload (42) was synthesized as follows.

[0440] [ka]

[0441] [ka]

[0442] Compound (39) (23.4 mg, 0.0257 mmol) and EVC-PAB-MMAE (21.4 mg, 0.0171 mmol) were dissolved in a 1:1 water:acetonitrile solution (3 mL), sodium bicarbonate (10 mg, 0.119 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was adjusted to about pH 4 with 1N hydrochloric acid, diluted with a 1:1 water:acetonitrile solution, and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain Linker-payload (42) (16.4 mg, 0.008 mmol).

[0443] MS(ESI)m / z:2049.8[M+H] +

[0444] (2-11) Synthesis of Linker-payload (43) Linker-Payload (43) was synthesized as follows.

[0445] [ka]

[0446] [ka]

[0447] Linker-payload (21) (15.6 mg, 0.074 mmol), HATU (5.4 mg, 0.014 mmol), and DIPEA (4.5 μL, 0.026 mmol) were dissolved in DMF (0.5 mL) and stirred at room temperature for 1 minute. m-PEG8-amine (BroadPharm #BP-21111) (7.5 mg, 0.0194 mmol) was added to the reaction solution and stirred at room temperature for 20 minutes. The reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05% formic 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 Linker-payload (43) (5.2 mg, 0.0027 mmol).

[0448] MS(ESI)m / z:955.2[M+2H] 2+

[0449] (1-4) Synthesis of Linker-payload mimic (44) Linker-Payload mimic (44) was synthesized as follows.

[0450] [ka]

[0451] [ka]

[0452] Compound (45) (20.2 mg, 0.0293 mmol), HATU (13.4 mg, 0.0352 mmol), and DIPEA (6.0 μL, 0.035 mmol) were dissolved in DMF (0.3 mL) and stirred at room temperature for 1 minute. Compound (46) (5.4 mg, 0.035 mmol) was added and stirred at room temperature for 17 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound (47) (23.9 mg, quant).

[0453] MS(ESI)m / z:817.4[M+H] +

[0454] [ka]

[0455] Compound (47) (23.9 mg, 0.0293 mmol) was dissolved in DMF (0.45 mL), and then bis(4-nitrophenyl)carbonate (27.3 mg, 0.0879 mmol) and DIPEA (11.4 μL, 0.0657 mmol) were added and stirred at room temperature for 3 hours. Sarucosine-pyrene (44.3 mg, 0.147 mmol), HOBt (5.9 mg, 0.044 mmol), and DIPEA (39.4 μL, 0.227 mmol) were added to the reaction solution and stirred at room temperature for 20 hours. Diethylamine (93.0 μL, 0.882 mmol) was added to the reaction solution, stirred at room temperature for 3 hours, and concentrated under reduced pressure. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile. The solvent was removed and the mixture was lyophilized to obtain compound (48) (1.9 mg, 0.0021 mmol).

[0456] MS(ESI)m / z:923.5[M+H] +

[0457] [ka]

[0458] Compound (48) (1.9 mg, 0.0021 mmol) was dissolved in DMF (0.4 mL), and then 4-maleimidohexanoic acid (0.7 mg, 0.003 mmol), HOAt (0.4 mg, 0.003 mmol), WSC·HCl (0.9 mg, 0.005 mmol), triethylamine (0.89 μL, 0.0063 mmol), and DMAP (0.1 mg, 0.001 mmol) were added and stirred at room temperature for 5 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution 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 compound (49) (0.3 mg, 0.0003 mmol).

[0459] MS(ESI)m / z:1116.5[M+H] +

[0460] [ka]

[0461] Compound (49) (0.3 mg, 0.0003 mmol) was dissolved in 1,4-dioxane (0.3 mL), and then 4N hydrogen chloride dioxane solution (315 μL, 1.26 mmol) was added and stirred at room temperature for 5 hours. The reaction solution was cooled to 0° C., DIPEA (238 μL, 1.39 mmol) was added, and then stirred at room temperature for 1 minute. The reaction solution was concentrated, then diluted with a 1:1 water:acetonitrile solution, 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 Linker-payload (44) (0.3 mg, 0.0003 mmol).

[0462] MS(ESI)m / z:1060.5[M+H] +

[0463] (1-5) Synthesis of Linker-payload mimic (50) Linker-Payload mimic (50) was synthesized as follows.

[0464] [ka]

[0465] [ka]

[0466] Compound (45) (24.8 mg, 0.0367 mmol), HATU (16.7 mg, 0.0440 mmol), and collidine (5.8 μL, 0.044 mmol) were suspended in acetonitrile (0.5 mL) and stirred at room temperature for 1 minute. Compound (51) (6.2 mg, 0.044 mmol) was added and stirred at room temperature for 24 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution 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 compound (52) (11.4 mg, 0.0142 mmol).

[0467] MS(ESI)m / z:805.4[M+H] +

[0468] [ka]

[0469] Compound (52) (11.4 mg, 0.0142 mmol) was dissolved in DMF (0.3 mL), and then bis(4-nitrophenyl)carbonate (12.6 mg, 0.0426 mmol) and DIPEA (5.4 μL, 0.0318 mmol) were added thereto, followed by stirring at room temperature for 4 hours. Further, bis(4-nitrophenyl) carbonate (6.5 mg, 0.0213 mmol) and DIPEA (2.7 μL, 0.016 mmol) were added and stirred at room temperature for 3 hours. Sarucosine-pyrene (42.9 mg, 0.142 mmol), HOBt (2.9 mg, 0.021 mmol), and DIPEA (38.4 μL, 0.226 mmol) were added to the reaction solution and stirred at room temperature for 16 hours. Diethylamine (29.7 μL, 0.284 mmol) was added to the reaction solution and stirred at room temperature for 3 hours and concentrated under reduced pressure. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized. By carrying out the above procedure, compound (53) (10.0 mg, 0.0110 mmol) was obtained.

[0470] MS(ESI)m / z:911.4[M+H] +

[0471] [ka]

[0472] Compound (54) (10.0 mg, 0.0110 mmol) was dissolved in DMF (1 mL), and then 4-maleimidohexanoic acid (3.5 mg, 0.017 mmol), HOAt (2.2 mg, 0.017 mmol), WSC·HCl (4.8 mg, 0.025 mmol), triethylamine (4.6 μL, 0.033 mmol), and DMAP (0.3 mg, 0.002 mmol) were added and stirred at room temperature for 4 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution 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 compound (55) (1.6 mg, 0.0014 mmol).

[0473] MS(ESI)m / z:1104.5[M+H] +

[0474] [ka]

[0475] Compound (55) (1.6 mg, 0.0014 mmol) was suspended in 1,4-dioxane (0.2 mL), and then 4N hydrogen chloride dioxane solution (181 μL, 724 mmol) was added and stirred at room temperature for 5 hours. The reaction solution was cooled to 0° C., DIPEA (136 μL, 798 mmol) was added, and then stirred at room temperature for 1 minute. The reaction solution was concentrated, then diluted with a 1:1 water:acetonitrile solution, 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 Linker-payload (50) (0.81 mg, 0.00077 mmol).

[0476] MS(ESI)m / z:1048.5[M+H] +

[0477] Comparative Example 1: Synthesis of Linker-payload mimic (28) Maleimide-VC-pyrene (28) was synthesized in one step from commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) and known sarcosine-pyrene (WO2018218004A1).

[0478] [ka]

[0479] [ka]

[0480] Commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) (15.5 mg, 0.021 mmol) was dissolved in dichloromethane (1 mL), N,N-diisopropylethylamine (0.025 mL, 0.142 mmol), known Sarcosine-pyrene (WO2018218004A1) (7.6 mg, 0.025 mmol), and a dimethylformamide solution (0.5 mL) were added and stirred for 17 hours. After purification by reversed-phase preparative chromatography, the fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain Linker-payload mimic (28) (7.3 mg, 0.008 mmol).

[0481] 1 H NMR(400 MHz,DMSO-d6)δ9.98(s,1H),8.34(d,J=9.2Hz,2H),8.32-8.23(m,4H),8.16(s,2H),8.10-8.00(m,4H),7.80(d ,J=8.8Hz,1H),7.59(d,J=8.4Hz,2H),7.31(d,J=8.0Hz,2H),6.99(s,2H),5.96(m,1H),5.40(s,2H),5.01(s,2H) ),4.95(d,J=6.0Hz,2H),4.38(m,1H),4.19(m,1H),3.03-2.92(m,3H),2.67(m,1H),2.33(m,1H),2.20-2.07(m, 2H),1.97(m,1H),1.67(m,1H),1.59(m,1H),1.51-1.45(m,6H),1.26-1.15(m,3H),0.83(dd,J=12.8,6.8Hz,6H)

[0482] MS(ESI)m / z:901.45[M+H] +

[0483] Comparative Example 2: Synthesis of Linker-payload (2-1) Synthesis of Linker-Payload (29) Linker-Payload (29) was synthesized as follows.

[0484] [ka]

[0485] Linker-Payload (29) was synthesized according to the following scheme.

[0486] [ka]

[0487] The MS analysis results of Linker-Payload (29) were as follows.

[0488] MS(ESI)m / z:1447.8[M+H] +

[0489] Example 3: Synthesis of ADC mimics (3-1) 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).

[0490] [ka]

[0491] (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.)

[0492] Ten equivalents of a DMF solution (1.25 mM) of the linker-payload mimic synthesized in Comparative Example 1 and Example 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. After that, the mixture was purified using NAP-5 Columns (GE Healthcare) to obtain an ADC mimic.

[0493] 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 a peak was confirmed at 150641 for the reaction product, in which two linker-payload mimics (1) were introduced.

[0494] [ka]

[0495] Similarly, ADC mimic 2 having the following structure was synthesized from the linker-payload mimic (5) of Example 1-2 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and a peak was confirmed at 150803, where two linker-payload mimics (5) were introduced, for the reaction product.

[0496] [ka]

[0497] Similarly, ADC mimic 3 having the following structure was synthesized from the linker-payload mimic (9) of Example 1-3 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and peaks were confirmed at 150620 for the reaction product, where two linker-payload mimics (9) were introduced.

[0498] [ka]

[0499] Similarly, ADC mimic 4 having the following structure was synthesized from the linker-payload mimic (28) of Comparative Example 1 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and peaks were confirmed at 150244 for the reaction product, where two linker-payload mimics (28) were introduced.

[0500] [ka]

[0501] (3-2)DAR analysis of ADC mimics ESI-TOFMS analysis of the ADC mimic synthesized in Example 3-1 was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2.

[0502] [Table 1]

[0503] Example 4: Synthesis of ADCs (4-1) Synthesis of ADC To a buffer (pH 7.4 PBS buffer) solution (20 μM) of the thiol group-introduced antibody, 10 equivalents of a DMF solution (1.25 mM) of the linker-payload mimic synthesized in Comparative Example 2 and 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.

[0504] ADC1 with the following structure was synthesized from the Linker-payload (11) synthesized in Example 2-1 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 151230, where two Linker-payloads (11) were introduced.

[0505] [ka]

[0506] ADC2 having the following structure was synthesized from the Linker-payload (12) synthesized in Example 2-2 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151443, where two Linker-payloads (12) were introduced.

[0507] [ka]

[0508] ADC3 with the following structure was synthesized from the Linker-payload (18) synthesized in Example 2-3 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 152173, where two Linker-payloads (18) were introduced.

[0509] [ka]

[0510] ADC4 with the following structure was synthesized from the Linker-payload (19) synthesized in Example 2-4 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151270, where two Linker-payloads (19) were introduced.

[0511] [ka]

[0512] ADC5 having the following structure was synthesized from the Linker-payload (20) synthesized in Example 2-5 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 152005, where two Linker-payloads (20) were introduced.

[0513] [ka]

[0514] ADC6 with the following structure was synthesized from the Linker-payload (21) synthesized in Example 2-6 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151492, where two Linker-payloads (21) were introduced.

[0515] [ka]

[0516] ADC7 having the following structure was synthesized from the Linker-payload (29) synthesized in Comparative Example 2 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 151295, where two Linker-payloads (29) were introduced.

[0517] [ka]

[0518] ADC 8 with the following structure was synthesized from commercially available MC-VC-MMAE (CAS no: 646502-53-6) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had a peak at 151091, where two MC-VC-MMAE units were introduced.

[0519] [ka]

[0520] Similarly, ADC mimic 9 having the following structure was synthesized from the Linker-payload (32) of Example 2-7 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and peaks were confirmed at 152261 for the reaction product, where two Linker-payloads (32) were introduced.

[0521] [ka]

[0522] (4-2)DAR analysis of ADC ESI-TOFMS analysis of the ADC synthesized in Example 4-1 was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2.

[0523] [Table 2]

[0524] Example 5: 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.

[0525] 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)

[0526] [Table 3]

[0527] Example 6: 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.

[0528] 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)

[0529] [Table 4]

[0530] Example 7: 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.

[0531] (7-1) 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.

[0532] (7-2) 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.

[0533] 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%.

[0534] [Table 5]

[0535] As a result, the ADC mimic synthesized in Example 1 was highly reactive to cathepsin and immediately released fluorescent molecules, whereas the ADC mimics synthesized in Comparative Examples 1-1 and 1-2 were low in reactivity to cathepsin.

[0536] Example 8: Evaluation of ADCs using the enzyme cathepsin B The cleavage ability of various ADCs by cathepsin B was evaluated by analyzing the amount of fluorescent molecules shed from the ADCs as described below.

[0537] (8-1) Cathepsin B cleavage assay This was carried out as previously reported (Nature Communications 2018, 9, 2512).

[0538] (8-2) 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). Three samples to which acetonitrile was immediately added at 0°C in Example 8-1 were taken as 0 hour samples, and three samples incubated at 37°C for 6 hours in Example 8-1 were taken as 6 hour samples. The MS intensities of the payload detected from the 6 hour samples and the 0 hour samples were calculated by extracted ion chromatograms, and the difference between them was analyzed.

[0539] 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%.

[0540] [Table 6]

[0541] As a result, it was found that the ADC mimic synthesized in Example 2 was highly reactive to cathepsin and quickly released the payload.

[0542] Example 9: Evaluation of ADC mimics using mouse plasma (9-1) 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.

[0543] (9-2) 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.

[0544] The loss rate of fluorescent molecules was calculated according to Example 7-2. The loss rate of fluorescent molecules was evaluated as shown in the table below.

[0545] [Table 7]

[0546] Example 10: Evaluation of ADC using mouse plasma (10-1) 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.

[0547] (10-2) 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). Three samples to which acetonitrile was immediately added at 0°C in Example 10-1 were taken as 0-hour samples, and three samples incubated at 37°C for 6 hours in Example 8-1 were taken as 6-hour samples. The MS intensities of the payload detected from the 6-hour sample and the 0-hour sample were calculated by extracted ion chromatograms, and the difference between them was analyzed. The calculation of the dropout rate of the payload was performed according to Example 8-2.

[0548] [Table 8]

Claims

1. The following formula (I): 【Chemistry 1】 [During the ceremony, Ig represents an immunoglobulin unit including two heavy chains and two light chains, and selectively forms an amide bond between the amino groups in the side chains of lysine residues in the two heavy chains and the carbonyl groups adjacent to Ig; the immunoglobulin unit is a human immunoglobulin unit, L １ is represented by the following formula (i): -L ４ -Y-L ３ - (i) [During the ceremony, L ３ , and L ４ Each independently represents -(C(R) ２ ) ｍ -,-(OC(R) ２ -C(R) ２ ) ｍ - and - (C(R) ２ -C(R) ２ -O) ｍ -, -, and combinations thereof; R 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 20; Y is represented by the following structural formula: 【Chemistry 2】 [wherein the white and black circles represent bonds, The bond in the white circle is L ３ If the bond is bonded to L, the bond of the black circle is L. ４ is bound to The bond in the white circle is L ４ If the bond is bonded to L, the bond of the black circle is L. ３ is bonded to a divalent group represented by any one of the structural formulae: L ２ represents a divalent group, R １ represents a monovalent group which may contain a hydrophilic group, X represents a divalent group which may have a substituent, in which the divalent group has 1 to 3 carbon atoms constituting a main chain portion linking the two atoms on both sides of X, and the substituent is a monovalent group which may contain a hydrophilic group; D represents a functional substance; R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, n is 0 or 1, where when n is 1, the substituents on X are R １ may together form a ring which may contain a hydrophilic group, the average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5; and at least one hydrophilic group is present in the structural unit; a lysine residue is present at position 246 / 248, 288 / 290, or 317 according to Eu numbering; A conjugate of an antibody and a functional substance or a salt thereof, The structural unit represented by formula (I) is a structural unit represented by the following formula (I-1), (I-2), or (I-3): 【Chemistry 3】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); R １ represents a monovalent group containing a hydrophilic group. 【Chemistry 4】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); R ２ represents a monovalent group containing a hydrophilic group; or 【Chemistry 5】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); R ３ represents a monovalent group containing a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydrophilic group, R ４ represents a monovalent group containing a hydrophilic group. The structural unit represented by formula (I-1), (I-2), or (I-3) is a structural unit represented by the following formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), or (I-3b'): 【Chemistry 6】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); L ５ represents a bond or a divalent group, HG represents a hydrophilic group. 【Chemistry 7】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); Two L's ５ each independently represents a bond or a divalent group, Each of the two HGs independently represents a hydrophilic group. 【Chemistry 8】 [During the ceremony, Ig, L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); L ５ represents a bond or a divalent group, HG represents a hydrophilic group, L １ａ , and L １ｂ each independently represents a bond or a divalent group, HG′ represents a divalent hydrophilic group. 【Chemistry 9】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); L ５ represents a bond or a divalent group, HG represents a hydrophilic group. 【Chemistry 10】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); L ５ represents a bond or a divalent group, HG represents a hydrophilic group; or 【Chemistry 11】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); HG represents a hydrophilic group. The structural unit represented by the formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), or (I-3b') is represented by the following formula (I-1a'-1), (I-1a'-2), (I-1b'-1), (I-1c'-1), (I-2'-1), (I-2'-2), (I-3a'-1), (I-3a'-2), or (I-3b'-1): A conjugate of an antibody and a functional substance, which is a structural unit, or a salt thereof: 【Chemistry 12】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); 【Chemistry 13】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); 【Chemistry 14】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); 【Chemistry 15】 [During the ceremony, Ig, L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); L １ａ , and L １ｂ each independently represents a bond or a divalent group, n1 is an integer from 3 to 20; 【Chemistry 16】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); 【Chemistry 17】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); 【Chemistry 18】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); 【Chemistry 19】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I); or 【Chemistry 20】 [During the ceremony, Ig, L １ , L ２ ,D.,R. Ａ , R Ｂ , r is the same as in formula (I).

2. The conjugate or salt thereof according to claim 1, wherein r is 1.9 to 2.

1.

3. L ３ , and L ４ Each independently represents -(C(R) ２ ) ｍ The conjugate or salt thereof according to claim 1 ,

4. L ２ has the following structural formula: 【Chemistry 21】 (Here, black and white circles indicate bonds, The bond in the black circle is L ２ is attached to the carbonyl group adjacent to The bond marked with a white circle is bonded to D. E represents an electron withdrawing group; and n2 is an integer of 1 to 4. The conjugate or salt thereof according to any one of claims 1 to 3, comprising a divalent group represented by the following formula:

5. The conjugate or a salt thereof according to claim 1 , wherein the functional substance is a drug, a labeling substance, or a stabilizer.

6. The conjugate or salt thereof according to claim 1 , wherein the functional substance is a peptide, a protein, a nucleic acid, or a low molecular weight organic compound.

7. The following formula (II-1), (II-2), or (II-3): 【Chemical 22】 [During the ceremony, L ２ represents a divalent group, L ３ is -(C(R) ２ ) ｍ -,-(OC(R) ２ -C(R) ２ ) ｍ - and - (C(R) ２ -C(R) ２ -O) ｍ represents a divalent group, including a divalent group selected from the group consisting of -, -, and combinations thereof; R １ represents a monovalent group containing a hydrophilic group, D represents a functional substance; R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, B 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; 【Chemistry 23】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); R ２ represents a monovalent group containing a hydrophilic group; or 【Chemistry 24】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); R ３ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a monovalent group containing a hydrophilic group, R ４ represents a monovalent group containing a hydrophilic group; The compound represented by formula (II-1), (II-2), or (II-3) is represented by the following formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), or (II-3b'): 【Chemistry 25】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); L ５ represents a bond or a divalent group, HG represents a hydrophilic group. 【Chemistry 26】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); Two L's ５ each independently represents a bond or a divalent group, Each of the two HGs independently represents a hydrophilic group. 【Chemical 27】 [During the ceremony, L ２ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); L ５ represents a bond or a divalent group, HG represents a hydrophilic group; L ３ａ , and L ３ｂ each independently represents a bond or a divalent group, HG′ represents a divalent hydrophilic group. 【Chemistry 28】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); L ５ represents a bond or a divalent group, HG represents a hydrophilic group. 【Chemical 29】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); L ５ represents a bond or a divalent group, HG represents a hydrophilic group; or 【Chemistry 30】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); HG represents a hydrophilic group. The compound represented by formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), or (II-3b') is a compound represented by the following formula (II-1a'-1), (II-1a'-2), (II-1b'-1), (II-1c'-1), (II-2'-1), (II-2'-2), (II-3a'-1), (II-3a'-2), or (II-3b'-1) or a salt thereof: 【Chemistry 31】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1). 【Chemistry 32】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1). 【Chemical 33】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1). 【Chemical 34】 [During the ceremony, L ２ ,D.,R. Ａ , R Ｂ and B are the same as in formula (II-1); L ３ａ , and L ３ｂ each independently represents a bond or a divalent group, n1 is an integer from 3 to 20; 【Chemistry 35】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1). 【Chemical 36】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1). 【Chemical 37】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1). 【Chemical 38】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1); or 【Chemical 39】 [During the ceremony, L ２ , L ３ ,D.,R. Ａ , R Ｂ and B is the same as in formula (II-1).

8. L ２ has the following structural formula: 【Chemistry 40】 (Here, black and white circles indicate bonds, The bond in the black circle is L ２ is attached to the carbonyl group adjacent to The bond marked with a white circle is bonded to D. E represents an electron withdrawing group; and n2 is an integer of 1 to 4. The compound according to claim 7, or a salt thereof.

9. The compound or salt thereof according to claim 7, wherein the functional substance is a drug, a labeling substance, or a stabilizer.

10. 8. The compound or salt thereof according to claim 7, wherein the functional substance is a peptide, a protein, a nucleic acid, or a low molecular weight organic compound.

11. A reagent for derivatizing an antibody, comprising the compound according to any one of claims 7 to 10 or a salt thereof.

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