Conjugates of antibodies and functional substances or their salts, and compounds or salts used in their manufacture.

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] This invention relates to conjugates of antibodies and functional substances or salts thereof, as well as compounds or salts thereof used in their production. [Background technology]

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

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

[0004] In ADCs, antibodies and drugs are linked via a linker. Various types of linkers exist in ADCs. For example, in ADCs used as anticancer drugs, there are linkers containing a valine-citrulline dipeptide (Val-Cit:VC structure) that are stable in human plasma and have a structure that can be cleaved by specific enzymes to release the drug in cancer cells. As shown in (A) below, such a dipeptide-containing linker is stable in human plasma, but as shown in (B) below, the VC structure is recognized by cathepsin B in lysosomes within human cancer cells, and the amide bond at the carboxyl terminus of citrulline is cleaved. Therefore, ADCs having such a dipeptide-containing linker can release the drug in human cancer cells and exert their therapeutic effect.

[0005] [ka]

[0006] [ka]

[0007] However, ADCs having a linker containing the dipeptide described above are found in mouse plasma. It is unstable (Non-Patent Documents 1 and 2). This is because mouse plasma contains Ces1c, a carboxylase that recognizes the VC structure and cleaves the amide bond located at the carboxyl terminus of citrulline. As a result, the linker containing the aforementioned dipeptide is cleaved in the plasma by Ces1c. Therefore, the pharmacokinetics of ADCs containing such a dipeptide differ significantly between mice and humans. Consequently, it is difficult to evaluate the efficacy of drugs in mice compared to those in humans.

[0008] [ka]

[0009] As mentioned above, in order to improve the instability of ADCs having the structure "antibody-spacer-VC structure-spacer-drug" in mouse plasma, attempts are being made to stabilize ADCs by modifying the linker (i.e., spacer-VC structure-spacer).

[0010] For example, Patent Document 5 and Non-Patent Document 3 disclose how to stabilize the linker by introducing a hydrophilic group (PEG) into the spacer present between the VC structure and the drug (near the cleavage site by Ces1c).

[0011] On the other hand, techniques have also been reported to stabilize linkers by introducing specific groups into the spacer between the antibody and the VC structure. An example of a linker stabilized by such a technique is the linker described below 1) to 4), characterized by containing a structure in which at least one α-amino acid residue (i.e., X, or NH-C(R)-CO) is linked by an amide bond to the antibody-side position (the amino group of V) of the VC structure (i.e., a structure represented as 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 Literature 4); 3) Linkers containing a structure represented by NH-C(R)-CO-Val-Cit (where R represents a side chain having a hydrophilic group such as PEG) (Patent Documents 7-9); 4) A linker containing a structure represented by NH-C(R)-CO-Val-Cit (where R represents a side chain having a hydrophilic group such as a sulfonic acid group or sugar) (Patent Document 10); and 5) A linker containing a structure represented as NH-C(R)-CO-Val-Cit (where R indicates a side chain having a cyclodextrin) (Patent Document 11).

[0012] Furthermore, as another example of a linker in which a specific group is introduced into the spacer present between the antibody and the VC structure, the linkers described in 6) to 8) below have been reported: 6) A linker containing a structure represented by C(M)-CO-Val-Cit (wherein C(M), C represents a carbon atom, M represents a stability-regulating group including a side chain such as an aromatic ring group, and CO represents a carbonyl group linked to the amino group of a valine residue to form an amide bond) (Patent Document 12); 7) A highly controlled special PEG linker containing PEG in both the main chain and side chain of the spacer present between the antibody and the VC structure (Non-Patent Literature 5); and 8) C(R i The structure is )-NH, which is the VC analog structure (CO-R ii C(R) is linked to the antibody side position by an amide bond. i )-NH-CO-R ii -CO-Cit(here, R i R indicates a thiophenyl group. ii A linker (Patent Document 13) containing a structure represented by a cyclobutyl ring.

[0013] Incidentally, Non-Patent Document 6 states that the higher the hydrophobicity of the ADC, the faster the plasma clearance, and that the hydrophobicity of the 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] U.S. 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] U.S. 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] Japanese Patent Publication No. 2016-050204 [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 [Overview of the project] [Problems that the invention aims to solve]

[0016] The object of the present invention is to provide an antibody and functional substance conjugate or salt thereof that exhibits desirable 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 diligent research, the inventors selected a lysine residue in the heavy chain of an immunoglobulin unit as the modification site for the antibody, and a specific structural unit [i.e., CO-(N-R1)nX-CONH-CH2(-R, as described later]. A )-CONH-CH2(-R B We have found that by using a linker containing )-CO to link immunoglobulin units with functional substances, it is possible to easily produce a conjugate or salt thereof represented by formula (I) that exhibits desirable properties while highly controlling the average binding ratio of immunoglobulin units to functional substances (functional substance / immunoglobulin units) within a desired range (1.5 to 2.5). For example, such a conjugate or salt thereof can possess 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 cleavage by cathepsin B, and high stability in mouse plasma.

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

[0019] For example, the conjugates represented by formulas (I-1) and (I-2) have a structural unit in which a tertiary amide-type structure having a hydrophilic group is linked to a VC structure, in a linker for linking an antibody and a functional substance. However, the above-mentioned prior art neither teaches nor suggests the conjugate of the present invention, which includes a linker having such a structural unit.

[0020] Furthermore, the conjugate represented by formula (I-3) has a structural unit in which a γ-glutamic acid residue is linked to a VC structure within the linker for linking the antibody and the functional substance. However, the above-mentioned prior art neither teaches nor suggests a conjugate containing a linker having such a structural unit. In fact, while Patent Documents 6-11 disclose a linker containing a structural unit in which at least one α-amino acid residue (i.e., an α-type structure) is linked to a VC structure, they neither teach nor suggest a linker containing a structural unit in which a γ-glutamic acid residue is linked to a VC structure. Patent Documents 12, 13, and Non-Patent Document 5 also neither teach nor suggest a linker containing a structural unit in which a γ-glutamic acid residue is linked to a VC structure.

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

[0022]

Chemical formula

[0023] 〔Here, the white circle and the black circle indicate 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 of the white circle is bonded to L4, the bond of the black circle is bonded to L3.〕 The conjugate or its salt according to 〔7〕 or 〔8〕, which is a divalent group represented by any one of the structural formulas. 〔10〕The conjugate or its salt according to any one of 〔1〕~〔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 its salt 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 or its salt according to 〔10〕, wherein the structural unit represented by formula (I-1), (I-2), (I-3), or (I-4) is a structural unit represented by formula (I-1a’), (I-1b’), (I-1c’), (I-2’), (I-3a’), (I-3b’), (I-4’), (I-4b’), or (I-4c’).

[13] A structural unit represented by formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), (I-3b'), or (I-4') is defined as formula (I-1a'- 1) Conjugates or salts of

[12] , which are structural units represented by (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 has the following structural formula:

[0024] [ka]

[0025] (Here, the black and white circles indicate joining hands, The black circled bond is attached to the carbonyl group adjacent to L2. The white circle is connected to D. E indicates an electron-withdrawing group. n² is an integer from 1 to 4. A conjugate or salt of any of [1] to

[13] , which is a divalent base represented by ).

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

[16] A compound of

[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] A compound represented by formula (II-1), (II-2), or (II-3) is a compound of

[15] or

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

[18] A compound represented by formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), (II-3b'), (II-4b'), or (II-4c') is any of the compounds in

[15] to

[17] or a salt thereof, represented by 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 has the following structural formula:

[0026] [ka]

[0027] (Here, the black and white circles indicate joining hands, The black circled bond is attached to the carbonyl group adjacent to L2. The white circle's connecting hand is connected to D. E indicates an electron-withdrawing group. n2 is an integer from 1 to 4. ) A compound or salt of any of

[15] to

[18] that is a divalent group represented by: A derivatization reagent for antibodies comprising any of the compounds

[20] ,

[15] , to

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

[0028] The conjugate or salt of the present invention can exhibit desirable properties while highly controlling the average ratio of immunoglobulin units to functional substances (functional substance / immunoglobulin units) within a desired range (1.5 to 2.5). The compounds or salts thereof, and reagents of the present invention, are useful as synthetic intermediates in the production of the above-mentioned conjugates. [Modes for carrying out the invention]

[0029] 1. Definitions of general terms In this invention, the term "antibody" is defined as follows. The term "immunoglobulin unit" corresponds to the divalent monomer unit that is the basic building block of such an antibody, and is a unit that includes two heavy chains and two light chains. Therefore, the definition, examples, and preferred examples of immunoglobulin units, including their origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, lysine residue position, and regioselectivity, are the same as those for antibodies described below.

[0030] The origin of the antibodies is not particularly limited and may be derived from animals such as mammals or birds (e.g., chickens). Preferably, the immunoglobulin units are derived from mammals. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g., dogs, cats), livestock (e.g., cattle, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, and more preferably humans.

[0031] The 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 predetermined glycan has been added (e.g., antibodies modified to have a glycan-binding consensus sequence such as an N-linked glycan-binding consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, as the monoclonal antibody, a full-length antibody or an antibody fragment containing a variable region and CH1 and CH2 domains can be used, but a full-length antibody is preferred. The antibody is preferably a human IgG monoclonal antibody, and more preferably a human full-length IgG monoclonal antibody.

[0032] Any antigen can be used as the antigen for the antibody. For example, such antigens include proteins (including oligopeptides and polypeptides; proteins modified with biomolecules such as sugars (e.g., glycoproteins)), glycans, nucleic acids, and small molecule compounds. Preferably, the antibody may be an antibody that uses a protein as its antigen. 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 antigen protein of the antibody may be a disease target protein. Examples of disease target proteins 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, 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) Infectious diseases Clostridium Difficile toxin B, cytomegalovirus, RSV, 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) Hereditary 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) Blood disorders 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) and certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, allentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab) Examples include ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab, and specific human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, laxibakumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesitumumab, brodalumab (IgG2), olaratumab) (whereas the IgG subtype is not mentioned, it is assumed to be IgG1).

[0044] The positions of amino acid residues in antibodies and the positions of constant regions in the heavy chain (e.g., the CH2 domain) follow EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, when targeting human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the human IgG CH2 domain, the lysine residue at position 248 corresponds to the 18th amino acid residue in the human IgG CH2 domain, the lysine residue at position 288 corresponds to the 58th amino acid residue in the human IgG CH2 domain, the lysine residue at position 290 corresponds to the 60th amino acid residue in the human IgG CH2 domain, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the human IgG CH2 domain. The notation 246 / 248 indicates that the target is the lysine residue at position 246 or 248. The notation 288 / 290 indicates that the target is the lysine residue at position 288 or 290.

[0045] According to the present invention, specific lysine residues in the heavy chain of an immunoglobulin unit constituting an antibody (e.g., lysine residues at positions 246 / 248, 288 / 290, or 317) can be regioselectively modified (see, for example, International Publication Nos. 2018 / 199337, 2019 / 240288, 2019 / 240287, and 2020 / 090979). In this specification, "regioselective" means that, even though specific amino acid residues are not eclectically distributed in a particular region of the antibody, a predetermined structural unit capable of binding to a specific amino acid residue in the antibody is eclectically distributed in a particular region of the antibody. Therefore, expressions related to regioselectivity, such as "possessing regioselectively," "regioselective binding," and "regioselective binding," mean that the possession or binding rate of a predetermined structural unit in a target region containing one or more specific amino acid residues is significantly higher than the possession or binding rate of the same structural unit in a non-target region containing multiple amino acid residues of the same type as the specific amino acid residues in the target region. Such regioselectivity 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 this invention, as long as a specific lysine residue in the heavy chain of the antibody is regioselectively modified... Furthermore, specific amino acid residues at other positions may be further modified in a regioselective manner. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in International Publications 2018 / 199337, 2019 / 240288, 2019 / 240287, and 2020 / 090979. Such specific amino acid residues can be amino acid residues having easily modifiable side chains (e.g., amino group, carboxyl group, amide group, hydroxyl group, thiol group) (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine ​​residues), but preferably lysine residues having a side chain containing an amino group, tyrosine residues having a side chain containing a hydroxyl group, serine residues, and threonine residues, or cysteine ​​residues having a side chain containing a thiol group, and more preferably lysine residues (i.e., two lysine residues among the lysine residues at positions 246 / 248, 288 / 290, and 317 may be regioselectively double-modified, or three lysine residues may be regioselectively triple-modified).

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

[0048] (Monovalent base) Examples of monovalent groups include monovalent hydrocarbon groups and monovalent heterocyclic groups.

[0049] The monovalent group may be substituted with one or more substituents (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) as described below.

[0050] (Monovalent hydrocarbon group, and related terminology) Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0051] A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure, and whose main chain does not contain a cyclic structure. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched.

[0052] As alkyls, alkyls having 1 to 12 carbon atoms are preferred, alkyls having 1 to 6 carbon atoms are more preferred, and alkyls having 1 to 4 carbon atoms are even more preferred. If the alkyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon number. Examples of alkyls 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] As the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. If the alkenyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0054] As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. If the alkynyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0055] Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0056] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.

[0057] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0058] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkenyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0059] As the cycloalkynyl, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkynyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0060] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0061] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. If the monovalent aromatic hydrocarbon group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0062] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0063] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups.

[0064] (Monovalent heterocyclic groups and related terminology) A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. Monovalent heterocyclic groups include monovalent aromatic heterocyclic groups and monovalent non-aromatic heterocyclic groups. The heteroatoms constituting the group preferably include one or more selected from the group consisting of oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen atoms, sulfur atoms, and nitrogen atoms.

[0065] As monovalent aromatic heterocyclic groups, those having 1 to 15 carbon atoms are preferred, those having 1 to 9 carbon atoms are more preferred, and those having 1 to 6 carbon atoms are even more preferred. If the monovalent aromatic heterocyclic group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazonal, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0066] As monovalent non-aromatic heterocyclic groups, non-aromatic heterocyclic groups having 2 to 15 carbon atoms are preferred, non-aromatic heterocyclic groups having 2 to 9 carbon atoms are more preferred, and non-aromatic heterocyclic groups having 2 to 6 carbon atoms are even more preferred. If the monovalent non-aromatic heterocyclic group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinil, azetidinil, oxetanil, thietanil, pyrrolidinil, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinil, oxazolidinil, piperidinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinil, dihydrooxazinil, tetrahydrooxazinil, dihydropyrimidinil, and tetrahydropyrimidinil.

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

[0068] (Divalent base) Divalent groups include divalent linear hydrocarbon groups, divalent cyclic hydrocarbon groups, divalent heterocyclic groups, -C(=O)-, -C(=S)-, -NR7-, -C(=O)-NR7-, -NR7-C(=O)-, -C(=S)-NR7-, -NR7-C(=S)-, -O-, -S-, and -(O-R8). m -, and -(S-R8) m1 -A group having a main chain structure comprising one group selected from the group consisting of -, or 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 is an integer from 1 to 10, preferably an integer from 1 to 8, more preferably an integer from 1 to 6, even more preferably an integer from 1 to 5, and particularly preferably an integer from 1 to 3.

[0069] Divalent linear hydrocarbon groups are linear alkylenes, linear alkenylenes, or linear alkylenes. The linear alkylenes are linear alkylenes having 1 to 6 carbon atoms, with linear alkylenes having 1 to 4 carbon atoms being preferred. Examples of linear alkylenes include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. Linear alkenylenes are linear alkenylenes having 2 to 6 carbon atoms, with linear alkenylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkenylenes include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, and linear alkynylene having 2 to 4 carbon atoms is preferred. Examples of linear alkynylenes include ethynylene, n- Examples include propynylene, n-butynylene, n-pentynylene, and n-hexynylene. As the divalent linear hydrocarbon group, linear alkylenes are preferred.

[0070] Divalent cyclic hydrocarbon groups are arylenes or divalent non-aromatic cyclic hydrocarbon groups. As for the arylene, arylene having 6 to 14 carbon atoms is preferred, arylene having 6 to 10 carbon atoms is more preferred, and arylene having 6 carbon atoms is particularly preferred. Examples of arylene include phenylene, naphthylene, and anthracenylene. As for the divalent non-aromatic cyclic hydrocarbon group, a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms is preferred, a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms is more preferred, and a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms is particularly preferred. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. Arylene is preferred as the divalent cyclic hydrocarbon group.

[0071] A divalent heterocyclic group is either a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms. As for the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As for the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxylandiyl, aziridindiyl, azetidinediyl, oxetanediyl, thietandiyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

[0072] Preferably, the divalent group is alkylene, arylene, -C(=O)-, -NR7-, -C(=O)-NR7-, -NR7-C(=O)-, -O-, and -(O-R8) m -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 -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 between 1 and 5 (i.e., 1, 2, 3, 4, or 5). Alkylenes, arylenes, and alkyls are the same as those described above.

[0073] The main chain structure at the divalent group may be substituted with one or more substituents (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) described later.

[0074] (substituent) Examples of substituents include: (i) halogen atom; (ii) Monovalent hydrocarbon group; (iii) monovalent heterocyclic groups; (iv) Aralkir; (v)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This 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 This represents a hydrogen atom or a monovalent hydrocarbon group, either identical or distinct. (vii) Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

[0075] The definitions, examples, and preferred examples of halogen atoms, monovalent hydrocarbon groups, and monovalent heterocyclic groups in the above substituents are the same as those described above.

[0076] An aralkyl refers to an arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyls are as described above. Preferred aralkyls have 3 to 15 carbon atoms. Examples of such aralkyls include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0077] Preferably, the substituents may be: (i) halogen atom; (ii) alkyl, phenyl, or naphthyl atoms having 1 to 12 carbon atoms; (iii) Aralkyl groups with 3 to 15 carbon atoms; (iv) A complex ring with 5 or 6 members; (v)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 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 This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 12 carbon atoms. (vii) The same base as those listed in (vii) above.

[0078] More preferably, the substituents may be: (i) halogen atom; (ii) Alkyl atoms 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 This represents a hydrogen atom or an alkyl group with 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 This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 12 carbon atoms. (v) The same base as those listed in (vii) above.

[0079] More preferably, the substituents may be: (i) halogen atom; (ii) Alkyl atoms 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 This represents a hydrogen atom or an alkyl group with 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 cThis represents, either identical or different, a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. (v) The same base as those listed in (vii) above.

[0080] Particularly preferred, the substituents may be: (i) halogen atom; (ii) Alkyl atoms 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 This represents a hydrogen atom or an alkyl group with 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 This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. (v) The same base as those listed in (vii) above.

[0081] (hydrophilic group) A hydrophilic group is a group that can make a structural unit represented by formula (I) or a formula of a sub-concept more hydrophilic. By having a hydrophilic group at a predetermined site in the structural unit, the conjugate can be made more stable in mouse plasma. Examples of such hydrophilic groups include carboxylic acid groups, sulfonic acid groups, hydroxyl groups, polyethylene glycol groups, polysarcosine groups, and sugar moieties. One or more hydrophilic groups may be present in the conjugate (e.g., one, two, three, four, or five).

[0082] The polyethylene glycol group is -(CH2-CH2-O-) k1It is a divalent group represented by -. When the conjugate has a polyethylene glycol group, the conjugate may have a monovalent group in which one bonding hand 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 It is a divalent group represented by -. 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 is a monosaccharide, an oligosaccharide (e.g., disaccharide, trisaccharide, tetrasaccharide, pentasaccharide), or a polysaccharide. The sugar moiety can include an aldose or a 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 containing such monosaccharides.

[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 with a molecular weight of 1500 or less. The molecular weight of the low molecular weight hydrophilic group may preferably be 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, hydroxyl groups, and polyethylene glycol groups, polysarcosine groups, and sugar moieties (e.g., monosaccharides, oligosaccharides) that satisfy the above molecular weight requirements.

[0086] (Biocorthogonal functional groups) Bioorthogonal functional groups are groups that do not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bioorthogonal functional groups are well known in the art (see, for example, 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 this invention, a bioorthogonal functional group for proteins is used as the bioorthogonal functional group. This is because the thiol-transferred antibody to be derivatized by the reagent of this invention is a protein. A bioorthogonal functional group for proteins is a group that does not react with the side chains of the 20 natural amino acid residues that make up proteins, or reacts slowly with those side chains, but reacts with the desired functional group. The 20 natural amino acids that make up proteins 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). Of these 20 naturally occurring amino acids, glycine, which lacks a side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., their side chains do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inert to normal reactions. Therefore, bioorthogonal functional groups for proteins are those that do not react with the side chains of these amino acids, which have side chains that are inert to normal reactions, as well as those that react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, or react slowly, but do react 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, any residue having a vinylene (ethenylene) moiety, which is the smallest unit with an intercarbon double bond; the same applies below), alkyne residues (in other words, any residue having an ethynylene moiety, which is the smallest unit with an intercarbon triple bond; 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, and α-halocarbonyl residues (e.g., having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position). Carbonyl residues (the same applies below), isonitrile residues, cydonone residues, and selenium residues are examples.

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

[0090] [ka]

[0091] [Here, R 1a , one or more R 1b , and one or more R 1c These are identical or different substituents or electron-withdrawing groups as described above. • is a joint.

[0092] Examples of electron-withdrawing groups include halogen atoms, alkyl groups substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl groups, keto groups (e.g., acyl), and alkyloxy groups, with halogen atoms, boronic acid residues, mesyl, tosyl, and triflate being preferred.

[0093] Bioorthogonal functional groups may be protected. Bioorthogonal functional groups that may be protected refer to unprotected or protected bioorthogonal functional groups. Unprotected bioorthogonal functional groups correspond to the bioorthogonal functional groups described above. Protected bioorthogonal functional groups are groups that generate a bioorthogonal functional group by cleaving a protecting group. Cleavage of the protecting group can be performed by specific treatments under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli selected from the group consisting of light, or (c) standing up with a cleavage linker containing a self-degrading cleavageable moiety. Such protecting groups and their cleavage conditions are common technical knowledge in the field (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] Examples of protected bioorthogonal functional groups include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinaldiol residues.

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

[0096] [ka]

[0097] [Here, the wavy lines perpendicular to the joint indicate the cutting points.] One or more R 2a These are selected from the group consisting of hydrogen atoms or the substituents described above, either identical or different. • is a joint.

[0098] Preferably, the bioorthogonal functional group, which may be protected, is an unprotected bioorthogonal functional group.

[0099] (Functional substance) Functional substances are not particularly limited as long as they are substances that confer any desired function to an antibody, and include, for example, drugs, labeling substances, and stabilizers, but are preferably drugs or labeling substances. Functional substances 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 any drug for any disease. Such diseases include, for example, cancer (e.g., lung cancer, stomach cancer, colorectal 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 and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), neurological diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), and infectious diseases (e.g., bacterial infections). Diseases, viral infections, genetic and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), diseases in the orthopedic field (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, digestive organ diseases) can be mentioned. The drug may be a preventive or therapeutic agent for the disease or an agent for alleviating side effects.

[0101] More specifically, the drug may be an anticancer agent. Examples of anticancer agents include, for example, chemotherapeutic agents, toxins, radioisotopes or substances containing them. Examples of chemotherapeutic agents include, for example, DNA-damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include, for example, bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include, for example, 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), radioisotopes of rhenium (e.g., 186 Re), radioisotopes of astatine (e.g., 211 At), radioisotopes of bismuth (e.g., 212Bi) is one example. More specifically, drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calichemycin, duocalmycin, eribulin, anthracycline, dmDNA31, and tubulisin.

[0102] Labeling substances are substances that enable the detection of targets (e.g., tissues, cells, materials). 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 mentioned above), or substances containing them.

[0103] Stabilizers are substances that enable the stabilization of antibodies. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0104] Functional substances may also be peptides, proteins, nucleic acids, small organic compounds, glycans, lipids, high molecular weight polymers, metals (e.g., gold), and chelators. Examples of peptides include cell membrane permeable peptides, blood-brain barrier permeable peptides, and peptide pharmaceuticals. 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. Other examples of nucleic acids include RNA interference-inducible nucleic acids (e.g., siRNA), aptamers, and antisenses. Examples of small organic compounds include proteolytic 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. As for salts with inorganic acids Examples of salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as salts with ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or an organic acid (e.g., trifluoroacetic acid).

[0106] 2. Conjugate or its salt The present invention provides antibody and functional substance conjugates or salts thereof, comprising a structural unit represented by the following formula (I), wherein at least one hydrophilic group is present in the structural unit.

[0107] [ka]

[0108] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and it regioselectively forms an amide bond with an adjacent carbonyl group via the amino groups in the side chains of the lysine residues 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 substituents, where the divalent group has 1 to 3 carbon atoms forming a main chain portion that links two atoms adjacent to X, and the substituent is a monovalent group which may include a hydrophilic group. D indicates a functional substance. R A This indicates the side chain of the valine residue, R B This indicates a side chain of a citrulline residue or an alanine residue. n is 0 or 1, where if n is 1, the substituent in X may, together with R1, 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 formulas presented in connection with the present invention, the hyphen (-) indicates that the two units (e.g., atoms, groups) on either side are covalently bonded.

[0110] The antibody contains the above-mentioned immunoglobulin units. For example, an antibody may contain two such units. Examples include IgG antibodies, IgD antibodies, and IgE antibodies, which contain immunoglobulin units comprising a heavy chain and two light chains, with disulfide bonds between the heavy chains and between the heavy chains and the light chains; IgA antibodies, which contain four heavy chains and four light chains, with disulfide bonds between the heavy chains and between the heavy chains and the light chains and the ibuglobulin units; and IgM antibodies, which contain eight heavy chains and eight light chains, with disulfide bonds between the heavy chains and between the heavy chains and the light chains and the ibuglobulin units. However, IgG antibodies (e.g., IgG1, IgG2, IgG3, IgG4) are preferred. The antibodies are preferably human IgG monoclonal antibodies, and more preferably human full-length IgG monoclonal antibodies.

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

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

[0113] A divalent group in X is a divalent group having 1 to 3 carbon atoms that form a main chain portion connecting two atoms adjacent to X. Such a main chain portion consists of a chain structure, a cyclic structure, or a combination thereof. If the main chain portion is a chain structure that does not include 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, if the main chain portion is a structure that includes a cyclic structure, the number of carbon atoms in the main chain portion can be determined by counting a predetermined number of carbon atoms that make up the cyclic structure as the number of carbon atoms in the main chain portion. Specifically, the number of carbon atoms in the main chain portion in a cyclic structure can be determined by counting the number of carbon atoms in the shortest path connecting two bonds in the cyclic structure (see, for example, the bolded paths in (a) to (d) below). If the main chain is a structure that includes a combination of chain and cyclic structures, the number of atoms in the main chain can be determined by adding the number of atoms in the chain structure that does not include a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure.

[0114] [ka]

[0115] • is a coupling. In case (a), the shortest path is the bolded path, so the number of atoms in the divalent cyclic structure that can be counted as the number of carbon atoms in the main chain is 2. In case (b), the shortest path is the bolded path, so the number of atoms in the divalent cyclic structure that can be counted as the number of carbon atoms in the main chain is 3. In case (c), since both paths are the shortest paths (equidistances), the number of carbon atoms in the divalent cyclic structure that can be counted as the number of carbon atoms in the main chain is 4 (therefore, in this invention, such a structure is excluded from the divalent group in X). In case (d), since the condensation site path is the shortest path, the number of carbon atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4 (therefore, in this invention, such a structure is excluded from the divalent groups in X).

[0116] The divalent group in X can be selected from the above-mentioned divalent groups such that its 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) Divalent cyclic hydrocarbon groups; and (3) Divalent cyclic hydrocarbon groups, and divalent groups formed by linking one or two divalent linear hydrocarbon groups having one or two carbon atoms.

[0118] Divalent linear hydrocarbon groups having 1 to 3 carbon atoms include linear alkylenes with 1 to 3 carbon atoms (e.g., methylene, ethylene, n-propylene), linear alkenylenes with 2 or 3 carbon atoms (e.g., ethenylene, propenylene), or linear alkylylenes with 2 or 3 carbon atoms (e.g., ethynylene, n-propynylene). Among divalent linear hydrocarbon groups having 1 to 4 carbon atoms, linear alkylenes with 1 to 3 carbon atoms are preferred.

[0119] The divalent cyclic hydrocarbon group is either 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 can be set to 3 to 5 as described above. As the arylene, arylene having 6 to 10 carbon atoms is preferred, and arylene having 6 carbon atoms is more preferred. Examples of arylene include phenylene and naphthylene. As the divalent non-aromatic cyclic hydrocarbon group, a divalent non-aromatic cyclic hydrocarbon group which is monocyclic or polycyclic and has 3 to 12 carbon atoms is preferable, a divalent non-aromatic cyclic hydrocarbon group which is monocyclic or polycyclic and has 4 to 10 carbon atoms is more preferable, and a divalent non-aromatic cyclic hydrocarbon group which is monocyclic and has 5 to 8 carbon atoms is particularly preferable. Examples of the divalent non-aromatic cyclic hydrocarbon group include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. As the divalent cyclic hydrocarbon group, arylene is preferable.

[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 preferable.

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

[0122] Regarding the substituent which 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 those described above.

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

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

[0126] n is either 0 or 1. If n is 0, there is no N-R1 unit, and X is directly bonded to two carbonyl groups. If n is 1, the substituents on X may, together with R1, form a ring which may contain hydrophilic groups. Such a ring contains the nitrogen atom linked to R1 as a ring constituent atom. Such a ring is preferably a 5-membered or 6-membered ring. Such a ring may also be an aromatic or aromatic ring, and the aromatic ring (e.g., pyrrolidine, piperazine) are preferred.

[0127] r represents the average ratio of the amide bonds per two heavy chains, and is between 1.5 and 2.5. Such an average ratio may be preferably 1.6 or higher, more preferably 1.7 or higher, even more preferably 1.8 or higher, and particularly preferably 1.9 or higher. Such an average ratio may also be preferably 2.4 or lower, more preferably 2.3 or lower, even more preferably 2.2 or lower, and particularly preferably 2.1 or lower. 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] Each structural unit represented by formula (I) contains at least one hydrophilic group (e.g., one, two, or three). Therefore, at least one of the following is hydrophilic: a monovalent group which may contain a hydrophilic group, represented by R1; a monovalent group which may contain a hydrophilic group, representing a substituent in a divalent group which may have a substituent, represented by X; and a ring which may contain a hydrophilic group when the substituent in X is combined with R1.

[0129] In certain embodiments, L1 may represent a divalent group represented by the following formula (i). -L4-Y-L3- (i) [During the ceremony, L3 and L4 are independently -(C(R)2) m -,-(OC(R)2-C(R)2) m-, and -(C(R)2-C(R)2-O) m - and a divalent group selected from the group consisting of combinations thereof, R is independently a hydrogen atom, an alkyl group with 1 to 6 carbon atoms, an alkenyl group with 2 to 6 carbon atoms, or an alkynyl group with 2 to 6 carbon atoms. m is an integer between 0 and 20. Y is a divalent group produced by the reaction of two bioorthogonal functional groups that can react with each other.

[0130] The alkyl groups with 1 to 6 carbon atoms, alkenyl groups with 2 to 6 carbon atoms, or alkynyl groups with 2 to 6 carbon atoms represented by R are the same as those described above.

[0131] m is an integer between 0 and 20. m may preferably be 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 preferably be 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. Furthermore, m can be set to a different integer for the two-valent bases shown in L3 and L4.

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

[0133] Y is a divalent group produced by the reaction of two mutually reactive bioorthogonal functional groups. Since combinations of two mutually reactive bioorthogonal functional groups are well known, those skilled in the art can appropriately select such combinations to appropriately set the divalent group produced by the reaction of two mutually reactive bioorthogonal functional groups. Examples of mutually reactive bioorthogonal functional group combinations include combinations of thiol residues and maleimide residues, combinations of furan residues and maleimide residues, combinations of thiol residues and halocarbonyl residues (where the halogen is replaced by a thiol by substitution reaction), combinations of alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with substituents as described above) and azide residues, combinations of tetrazine residues and alkene residues, and combinations of tetrazine residues and alkyne residues. Examples include combinations of a thiol residue and another thiol residue (disulfide bond). Therefore, 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 a 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] [Here, the white and black circles indicate joining moves.] If the white circle connection is connected to L3, then the black circle connection is connected to L4. If the white circle indicates a connection to L4, then the black circle indicates a connection 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 can be excellent in performance as a conjugate among the structural units represented by the above formula (I) (see Examples).

[0138]

Chemical formula

[0139] 〔In the formula, Ig, L1, L2, D, R A , R B , r are the same as those in formula (I),〕 R1 represents a monovalent group containing a hydrophilic group.〕

[0140]

Chemical formula

[0141] 〔In the formula, Ig, L1, L2, D, R A , R B , r are the same as those in formula (I), R2 represents a monovalent group containing a hydrophilic group.〕

[0142]

Chemical formula

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

[0144]

Chemical formula

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

[0146] In the above formulas (I-1), (I-2), (I-3), or (I-4), the hydrophilic group The monovalent groups and alkyl groups with 1 to 6 carbon atoms are the same as those described above.

[0147] In a preferred embodiment, the structural unit represented by formula (I) may be a structural unit represented by formula (I-1), (I-2), or (I-3). Such structural units may exhibit superior conjugate performance among the structural units represented by formula (I) (see Examples).

[0148] In one embodiment, in formula (I-3) above, 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 formula (I-3) above, R3 and R4 may each independently represent a monovalent group containing a hydrophilic group.

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

[0151] [ka]

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

[0153] [ka]

[0154] [During the ceremony, Ig, L1, L2, D, R A , R B , r is the same as in equation (I), The two L5 groups each independently represent a bonded or divalent group. The two HG groups each independently exhibit hydrophilicity.

[0155] [ka]

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

[0157] [ka]

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

[0159] [ka]

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

[0161] [ka]

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

[0163] [ka]

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

[0165] [ka]

[0166] [During the ceremony, Ig, L2, D, R A , R B , r is the same as in equation (I), L5 indicates a bond or a divalent group. HG indicates a hydrophilic group. L 1a , and L 1b Each of these independently represents a bond or a divalent group. HG' indicates a divalent hydrophilic group.

[0167] [ka]

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

[0169] In formulas (I-1a') to (I-4c'), the definitions, examples, and preferred examples of divalent groups and hydrophilic groups are the same as described above. Examples of divalent hydrophilic groups include polyethylene glycol groups, polysarcosine groups, and sugar moieties, as described above for hydrophilic groups, with polyethylene glycol groups and polysarcosine groups being preferred, and polyethylene glycol groups being more preferred. The electron-withdrawing group is the same as described above. The bond position of the electron-withdrawing group to the phenyl ring is ortho, meta, or para relative to the adjacent amino group (NH), with ortho or para being preferred, and ortho being preferred. n2 may preferably be 1 or 2. The definitions, examples, and preferred examples of divalent hydrophilic groups, electron-withdrawing groups and their bond positions, as well as n2, are the same for other formulas.

[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 equation (I).

[0174] [ka]

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

[0176] In another specific 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 equation (I).

[0179] [ka]

[0180] [During the ceremony, Ig, L2, D, R A , R B , r is the same as in equation (I), L 1a , and L 1b Each of these independently represents a bond or a divalent group. n1 is an integer between 3 and 20.

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

[0182] In yet another specific 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 equation (I).

[0185] [ka]

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

[0187] In yet another specific 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 equation (I).

[0190] [ka]

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

[0192] In yet another specific 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 equation (I).

[0195] In yet another specific 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 equation (I).

[0198] In yet another specific 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 equation (I), L 1a , and L 1b Each of these independently represents a bond or a divalent group. n1 represents an integer between 3 and 20.

[0201] In yet another specific 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 equation (I), L2' indicates 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 equation (I), L2' indicates a bond or a divalent group.

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

[0207] [ka]

[0208] (Here, the black and white circles indicate joining hands, The black circled bond is attached to the carbonyl group adjacent to L2. The white circle is connected to D. E indicates an electron-withdrawing group. n² is an integer between 1 and 4. The electron-withdrawing group and its bonding position, as well as the definition, examples, and preferred examples of n2, are the same as those described above.

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

[0210] In certain embodiments, the conjugate or salt of the present invention can be identified by its aggregation rate because it has the desired property of being less prone to aggregation. More specifically, the aggregation rate of the conjugate or salt of the present invention may be 5% or less, as this makes it easier to avoid antibody aggregation. 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 or salt of the present invention is represented by 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 substituents, where the divalent group has 1 to 3 carbon atoms forming a main chain portion that links two atoms adjacent to X, and the substituent is a monovalent group which may include a hydrophilic group. B exhibits bioorthogonal functional groups, D indicates a functional substance. R A This indicates the side chain of the valine residue, R B This indicates a side chain of a citrulline residue or an alanine residue. n is 0 or 1, where if n is 1, the substituent in X may, together with R1, form a ring which may contain a hydrophilic group. The compound represented by ] or a salt thereof, It can be produced by reacting an immunoglobulin unit containing two heavy chains and two light chains with a starting antibody (where the two heavy chains contain lysine residues modified with regioselectively orthogonal functional groups). In formula (II) above, L2, L3, R1, X, D, R A , R B The definitions, examples, and preferred examples for , and n are the same as those described above for formula (I).

[0214] The definition, examples, and preferred examples of bioorthogonal functional groups shown in 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 can be selected to be reactive with the bioorthogonal functional groups (e.g., the bioorthogonal functional group represented by B') in the raw material antibody described later.

[0216] In preferred embodiments, 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) or a salt thereof. Such compounds or salts thereof are useful as synthetic intermediates in the production of conjugates that can exhibit superior performance among the compounds or salts thereof represented by formula (II) above (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 indicates a functional substance. R A This indicates the side chain of the valine residue, R B This indicates a side chain of a citrulline residue or an alanine residue. B exhibits bioorthogonal functional groups.

[0219] [ka]

[0220] [During the ceremony, L2, L3, D, R A , R B , and B are the same as in equation (II-1), R2 represents a monovalent group that 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 equation (II-1), R3 represents a monovalent group containing a hydrogen atom, an alkyl group with 1 to 6 carbon atoms, or 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 equation (II-1), R1 represents a hydrogen atom or an alkyl group with 1 to 6 carbon atoms. R6 represents a monovalent group containing a hydrophilic group.

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

[0226] In one embodiment, in formula (II-3) above, 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 formula (II-3) above, R3 and R4 may each independently represent a monovalent group containing a hydrophilic group.

[0228] In preferred embodiments, the compound represented by formula (II) or a salt thereof may be the compound represented by formula (II-1), (II-2), or (II-3) or a salt thereof. Such compounds or salts thereof are useful as synthetic intermediates in the production of conjugates that exhibit superior performance among the compounds or salts thereof represented by formula (II) (see Examples).

[0229] In a more preferred embodiment, the compound or salt represented by formula (II-1), (II-2), (II-3), or (II-4) above may be a compound or salt 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 compounds or salts are useful as synthetic intermediates in the production of conjugates that can exhibit superior performance among the compounds or salts represented by formula (II) above (see Examples).

[0230] [ka]

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

[0232] [ka]

[0233] [During the ceremony, L2, L3, D, R A , R B , and B are the same as in equation (II-1), The two L5 groups each independently represent a bonded or divalent group. The two HG groups each independently exhibit hydrophilicity.

[0234] [ka]

[0235] [During the ceremony, L2, D, R A , R B , and B are the same as in equation (II-1), L5 indicates a bond or a divalent group. HG indicates a hydrophilic group. L 3a , and L 3b Each of these independently represents a bond or a divalent group. HG' indicates a divalent hydrophilic group.

[0236] [ka]

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

[0238] [ka]

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

[0240] [ka]

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

[0242] [ka]

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

[0244] [ka]

[0245] [During the ceremony, L2, D, R A , R B , and B are the same as in equation (II-1), L5 indicates a bond or a divalent group. HG indicates a hydrophilic group. L 3a , and L 3b Each of these independently represents a bond or a divalent group. HG' indicates a divalent hydrophilic group.

[0246] [ka]

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

[0248] In formulas (II-1a') to (II-4c'), the definitions, examples, and preferred examples of structural units represented by divalent groups and hydrophilic groups are the same as those described above. The definitions, examples, and preferred examples of divalent hydrophilic groups, electron-withdrawing groups and their bonding positions, as well as the definition of 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 as follows:

[0251] [ka]

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

[0253] [ka]

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

[0255] In another specific embodiment, the compound represented by formula (II-1b') may be as follows:

[0256] [ka]

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

[0258] In another specific embodiment, the compound represented by formula (II-1c') may be as follows:

[0259] [ka]

[0260] [During the ceremony, L2, D, R A , R B , and B are the same as in equation (II-1), L 3a , and L 3b Each of these independently represents a bond or a divalent group. n1 is an integer between 3 and 20.

[0261] L 3a , and L 3b The definition, examples, and preferred examples of the divalent group shown, and n1, are the same as those described above (and so on).

[0262] In another specific embodiment, the compound represented by formula (II-2') may be as follows:

[0263] [ka]

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

[0265] [ka]

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

[0267] In yet another specific embodiment, the compound represented by formula (II-3a') may be as follows:

[0268] [ka]

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

[0270] [ka]

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

[0272] In yet another specific embodiment, the compound represented by formula (II-3b') may be as follows:

[0273] [ka]

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

[0275] In yet another specific embodiment, the compound represented by formula (II-4a') may be as follows:

[0276] [ka]

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

[0278] In yet another specific embodiment, the compound represented by formula (II-4b') may be as follows:

[0279] [ka]

[0280] [During the ceremony, L2, D, R A , R B, and B are the same as in equation (II-1), L 3a , and L 3b Each of these independently represents a bond or a divalent group. n1 is an integer between 3 and 20.

[0281] In yet another specific embodiment, the compound represented by formula (II-4c') may be as follows:

[0282] [ka]

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

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

[0287] [ka]

[0288] (Here, the black and white circles indicate joining hands, The black circled bond is attached to the carbonyl group adjacent to L2. The white circle is connected to D. E indicates an electron-withdrawing group. n² is an integer between 1 and 4. The electron-withdrawing group and its bonding position, as well as the definition, examples, and preferred examples of n2, are the same as those described above.

[0289] In a more preferred embodiment, the compound represented by formula (II) or a salt thereof may be a structural unit represented by formula (II-1a'), (II-1b'), (II-2'), (II-3a'), (II-3b'), (II-4b'), or (II-4c'), or by formulas corresponding to sub-concepts of these formulas. Such compounds or salts thereof are useful as synthetic intermediates in the production of conjugates that may have superior performance among the compounds or salts of formula (II) above (see Examples).

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

[0291] In a particular embodiment, the raw material antibody is of the following formula (III):

[0292] [ka]

[0293] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and it regioselectively forms an amide bond with an adjacent carbonyl group via the amino groups in the side chains of the lysine residues 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 -A divalent group selected from the group consisting of, R is independently a hydrogen atom, an alkyl group with 1 to 6 carbon atoms, an alkenyl group with 2 to 6 carbon atoms, or an alkynyl group with 2 to 6 carbon atoms. m is an integer between 0 and 10. B' is a bioorthogonal functional group that can react 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. It may also contain immunoglobulin units represented by [ ]. The definitions, examples, and preferred examples of Ig, L4, R, m, and r in formula (III) above are the same as those described above.

[0294] The bioorthogonal functional group indicated by B' is the same as the bioorthogonal functional group described above for the raw material antibody.

[0295] The above reaction can be carried out as appropriate under mild conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds). For example, such a reaction can be carried out at room temperature (e.g., about 15-30°C) in a suitable reaction system, such as a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, and more preferably 6.0-8.0. The buffer may contain a suitable 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 on such reactions, see, for example, GJLBernardes et al., Chem. Rev., 115, 2174 (2015); GJLBernardes et al., Chem. Asian. J., 4, 630 (2009); BGDavies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0296] The formation of the conjugate or its salt can be confirmed by reverse-phase HPLC under reducing conditions or by mass spectrometry, depending on the specific molecular weights of the raw materials and products. The conjugate or its salt can be purified as appropriate by any method, such as chromatography (e.g., affinity chromatography).

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

[0298] The conjugate or salt thereof of the present invention may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate or salt thereof of the present invention, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, 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, glycyrrhizine ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersants such as surfactants; diluents such as water, physiological saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and kerosene. The conjugate or salt of the present invention may also have any modifications (e.g., PEGylation) that provide stability.

[0299] Suitable formulations for oral administration include liquid preparations in which an effective amount of ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of ligand as a solid or granule; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution of an effective amount of the active ingredient is dispersed in a suitable dispersion medium and emulsified.

[0300] Pharmaceutical compositions are suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Suitable pharmaceutical compositions for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Also, aqueous and non-aqueous sterile suspensions are examples, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0301] The dosage of a pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the target animal species, the target animal's drug tolerance, body weight, age, etc., but can be set as appropriate.

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

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

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

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

[0306] Details of the raw material antibody or its salt derivatized by the reagent of the present invention, and the conjugate or its salt obtained by the derivatization reaction of the raw material antibody or its salt, are as described above.

[0307] The reagents of the present invention may be provided in the form of compositions further comprising other components. Such other components include, for example, solutions and stabilizers (e.g., antioxidants, preservatives). As the solution, aqueous solutions are preferred. Examples of aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline) and buffer solutions (e.g., aqueous phosphoric acid solution, Tris-hydrochloride buffer, carbonic acid-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), but buffer solutions are preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagents 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 Pyrene(2) (73.7 mg, 0.104 mmol) was dissolved in N,N-dimethylformamide (5 mL), and known 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. The mixture was stirred at room temperature for 2 hours, then diethylamine (2 mL, 18.95 mmol) was added and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the mixture was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then 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·H2O (11.1 mg, 0.025 mmol) was dissolved in dimethylformamide (1 mL), and 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. The mixture was stirred at room temperature for 2.5 hours, then diethylamine (0.2 mL, 1.91 mmol) was added and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the mixture was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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), and 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. The mixture was stirred at room temperature for 3 hours, and 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. The mixture was stirred at room temperature for 1.5 hours. Subsequently, 4-dimethylaminopyridine (0.5 mg, 0.004 mmol) was added, and the mixture was stirred for a further 2.5 hours. After concentration under reduced pressure, the mixture was purified by column chromatography (dichloromethane:methanol = 9:1). The product was then collected in a flurry of cells. The solution was recovered and concentrated under reduced pressure to obtain the above-mentioned 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 mL of 1,4-dioxane and 1 mL of hydrogen chloride-1,4-dioxane solution, and the mixture was stirred in an ice bath for 2 hours. Dimethylformamide (1 mL) was added, and the mixture was raised to room temperature and then concentrated under reduced pressure. The solution was then purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain 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), and 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. The mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the solution was purified by column chromatography (hexane:ethyl acetate = 4:1). The fraction containing the product was recovered 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, and after concentration under reduced pressure, maleimide (7) (244.6 mg, 0.458 mmol) was obtained by freeze-drying.

[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), and 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. 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 recovered and concentrated under reduced pressure to obtain 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. Then 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain 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 (300 mg, 1.420 mmol) was dissolved in dimethylformamide (2 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate (647.8 mg, 1.704 mmol), iminodiacetic acid (189.0 mg, 1.420 mmol), N,N-diisopropylethylamine (0.29 mL, 1.704 mmol), and 4-dimethylaminopyridine (34.7 mg, 0.284 mmol) were added, and the mixture was stirred for 1 hour. After concentration under reduced pressure, the mixture was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain maleimide (10) (65.9 mg, 0.202 mmol).

[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), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (54.0 mg, 0.142 mmol) was added. The mixture was stirred for 1 hour, and then a dimethylformamide solution (0.3 mL) of 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. The mixture was stirred for a further 3 hours. After purification by reverse-phase preparative chromatography, the fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 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), and 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. To the reaction mixture, the known product Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) (150 mg, 0 1 mL of 0.13 mmol DMF solution was added, and the mixture was stirred for a further 30 minutes. After the reaction was complete, 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 a 1:1 ratio of water to THF (8 mL) and stirred at room temperature for 15 minutes. After the reaction was complete, 1 M aqueous hydrochloric acid was carefully added to the reaction solution to adjust the pH to 5-6. This solution was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), and 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 a further 5 minutes. After the reaction was complete, 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 fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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 complete, 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), and 4-methylmorpholine (0.082 mL, 0.074 mmol) and succinic anhydride (7.4 mg, 0.074 mmol) were added. The mixture was stirred at room temperature for 1 hour, and the reaction solution was diluted with a 1:1 water:acetonitrile solution (containing 0.05 v / v% formic acid). The solution was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), and 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 a further 2 minutes. After the reaction was complete, 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain 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 (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,​​​​​​​​​​​​​​​​​​​​​​​​​​

[0391] Maleimide (10) (8 mg, 0.0245 mmol) was dissolved in dimethylformamide (0.2 mL), and 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. To the reaction mixture, the known Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) (13.8 mg, 0.0123 mmol) and N,N-diisopropylethylamine (0.00 86 mL (0.0491 mmol) was added and the mixture was stirred for a further 2 hours. After the reaction was complete, 1 M hydrochloric acid aqueous solution 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain 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), and 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. To the reaction mixture, methoxy-PEG8-amine (4.7 mg, 0.0122 mmol) and N,N-diisopropylethylamine (0.0035 mL, 0.0204 mmol) were added, and the mixture was stirred for a further 10 minutes. After the reaction was complete, 1 M hydrochloric acid aqueous solution 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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 for 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), stirred at 40°C for 24 hours, and 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 - 1.68(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 subsided, THF (2 mL) and Boc₂O (416 mg, 1.91 mmol) were added, and the mixture was stirred at room temperature for 72 hours. After removing the THF by vacuum concentration, the reaction solution was adjusted to approximately 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. Further HATU (26.2 mg, 0.0689 mmol) and DIPEA (21.8 μL, 0.125 mmol) were added to the reaction solution and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted in a 1:1 water:acetonitrile solution, and purified by reverse-phase preparative chromatography. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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), then trifluoroacetic acid (1 mL) was added, and the mixture was stirred overnight at room temperature. 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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). Compound (38) (35.5 mg, 0.112 mmol) was dissolved in DMF (1.5 mL) in a separate flask, and this solution was added to the aforementioned 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 a separate 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 aforementioned 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 approximately pH 4 with 1N hydrochloric acid, diluted in a 1:1 water:acetonitrile solution, and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 1:1 with water:acetonitrile solution (containing 0.05% formic acid) and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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) The 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 the mixture was 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 fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), then bis(4-nitrophenyl) carbonate (27.3 mg, 0.0879 mmol) and DIPEA (11.4 μL, 0.0657 mmol) were added, and the mixture was 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 the mixture was stirred at room temperature for 20 hours. Diethylamine (93.0 μL, 0.882 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, and acetonitrile was removed by concentration under reduced pressure. By removing the residue and performing freeze-drying, compound (48) (1.9 mg, 0.0021 mmol) was obtained.

[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), 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 the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), then 4N hydrogen chloride dioxane solution (315 μL, 1.26 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was cooled to 0°C, DIPEA (238 μL, 1.39 mmol) was added, and the mixture was stirred at room temperature for 1 minute. After concentrating the reaction solution, it was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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) The 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 colidine (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 the mixture was stirred at room temperature for 24 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), then bis(4-nitrophenyl) carbonate (12.6 mg, 0.0426 mmol) and DIPEA (5.4 μL, 0.0318 mmol) were added, and the mixture was stirred at room temperature for 4 hours. Next, bis(4-nitrophenyl) carbonate (6.5 mg, 0.0213 mmol) and DIPEA (2.7 μL, 0.016 mmol) were added, and the mixture was 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 the mixture was stirred at room temperature for 16 hours. Diethylamine (29.7 μL, 0.284 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried. By performing this 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), 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 the mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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), then 4N hydrogen chloride dioxane solution (181 μL, 724 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was cooled to 0°C, DIPEA (136 μL, 798 mmol) was added, and the mixture was stirred at room temperature for 1 minute. After concentrating the reaction solution, it was diluted with a 1:1 water:acetonitrile solution and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried 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 as follows: It was synthesized in one step from commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) and a 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), and a dimethylformamide solution (0.5 mL) of N,N-diisopropylethylamine (0.025 mL, 0.142 mmol) and a known sarcosine-pyrene (WO2018218004A1) (7.6 mg, 0.025 mmol) was added. The mixture was stirred for 17 hours. After purification by reverse-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: Linker-payload synthesis (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 for Linker-Payload(29) were as follows:

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

[0489] Example 3: Synthesis of ADC mimic (3-1) Synthesis of ADC mimic In the following comparative examples and examples, the antibody derivative (thiol-modified trastuzumab) described in Examples 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used as the thiol-modified 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 follows EU numbering).

[0490] [ka]

[0491] (In the above structure, the NH-CH2-CH2-CH2-CH2- extending from the antibody heavy chain corresponds to the side chain of the lysine residue, and the thiol-containing group HS-CH2-CH2-C(=O) is attached to the amino group in the side chain of this lysine residue.)

[0492] To a 20 μM solution (pH 7.4 PBS buffer) of the thiol-introduced antibody, 10 equivalents of a 1.25 mM DMF solution of the Linker-payload mimic synthesized in Comparative Example 1 and Example 1 were added. After standing at room temperature for 2 hours, the solution was purified using NAP-5 Columns (GE Healthcare) to obtain the ADC mimic.

[0493] ADC mimic 1 with 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, where two Linker-payload mimic(1) molecules were introduced into the reaction product.

[0494] [ka]

[0495] Similarly, ADC mimic 2 with the following structure was synthesized from the Linker-payload mimic(5) of Examples 1-2 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and a peak was observed at 150803, where two Linker-payload mimic(5) molecules were introduced into the reaction product.

[0496] [ka]

[0497] Similarly, ADC mimic 3 with the following structure was synthesized from the Linker-payload mimic(9) of Examples 1-3 and a thiol-containing antibody. ESI-TOFMS analysis was performed, and a peak at 150620 was observed in the reaction product, where two Linker-payload mimic(9) molecules were introduced.

[0498] [ka]

[0499] Similarly, ADC mimic 4 with 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 a peak was observed at 150244, in which two Linker-payload mimic(28) molecules were introduced.

[0500] [ka]

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

[0502] [Table 1]

[0503] Example 4: Synthesis of ADC (4-1) ADC synthesis To a 20 μM solution (pH 7.4 PBS buffer) of the thiol-introduced antibody, 10 equivalents of a 1.25 mM DMF solution of the Linker-payload mimic synthesized in Comparative Example 2 and Example 1 were added. After standing at room temperature for 2 hours, the solution was purified using NAP-5 Columns (GE Healthcare) to obtain 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 revealed a peak at 151230, where two Linker-payload(11) molecules were introduced.

[0505] [ka]

[0506] ADC2 with the following structure was synthesized from the Linker-payload(12) synthesized in Example 2-2 and a thiol-containing antibody. ESI-TOFMS analysis revealed a peak at 151443, where two Linker-payload(12) molecules were introduced.

[0507] [ka]

[0508] ADC3 with the following structure was synthesized from Linker-payload(18) synthesized in Example 2-3 and a thiol-containing antibody. ESI-TOFMS analysis revealed a peak at 152173, where two Linker-payload(18) molecules 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 revealed a peak at 151270, where two Linker-payload(19) molecules were introduced.

[0511] [ka]

[0512] ADC5 with the following structure was synthesized from the Linker-payload(20) synthesized in Example 2-5 and a thiol-containing antibody. ESI-TOFMS analysis revealed a peak at 152005, where two Linker-payload(20) molecules 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 revealed a peak at 151492, where two Linker-payload(21) molecules were introduced.

[0515] [ka]

[0516] ADC7 with 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 a peak was confirmed at 151295, where two Linker-payload(29) molecules were introduced into the reaction product.

[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 revealed a peak at 151091, where two MC-VC-MMAE molecules were introduced.

[0519] [ka]

[0520] Similarly, ADC mimic 9 with the following structure was synthesized from Linker-payload(32) and thiol-containing antibody as described in Examples 2-7. ESI-TOFMS analysis revealed a peak at 152261, where two Linker-payload(32) molecules 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 previously reported (WO2019 / 240287A1), and it was confirmed that the DAR was 2.

[0523] [Table 2]

[0524] Example 5: Evaluation of the hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC) HIC-HPLC analysis was performed according to a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732). The measurements were performed under the following conditions. The hydrophobicity of ADC can be evaluated by the retention time of ADC in the HIC chromatogram.

[0525] Measurement system: Chromaster (registered trademark) (manufactured by Hitachi, Ltd.) Column: Tosoh Biobuthyl NPR 2.5μm 4.6×35mm column, manufactured by Tosoh Bioscience Co., Ltd. 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 (280nm)

[0526] [Table 3]

[0527] Example 6: Evaluation of aggregation rates of ADC and ADC mimic by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to a previously reported study (ChemistrySelect, 2020, 5, 8435-8439). The measurements were performed under the following conditions.

[0528] Measurement system: 1260 HPLC system (manufactured by Agilent) Column: Agilent AdvanceBio SEC 300Å 2.7μm, 4.6mm × 150mm Flow rate: 0.25mL / min Eluent: 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride aqueous solution (pH 6.8), 10% v / v isopropanol Detector: UV (280nm)

[0529] [Table 4]

[0530] Example 7: Evaluation of ADC mimic using the enzyme cathepsin B The cathepsin B cleavage ability of various ADC mimics was evaluated by analyzing the amount of fluorescent molecules detached from the ADC mimic, as described below.

[0531] (7-1) Cathepsin B cleavage test The following procedure was performed in accordance with a previously reported study (Nature Communications 2018, 9, 2512). To 180 μL of MES buffer (10 mM MES, 40 μM DTT, pH 5.0), ADC mimic was added to a concentration of 1 mg / mL, and then 30 μL was dispensed into six Eppendorf tubes. Three of the six samples were immediately mixed with 100 μL of acetonitrile at 0°C, stirred by vortexing, and then centrifuged to obtain precipitates. The supernatant was collected and analyzed by HPLC. The remaining three tubes were incubated at 37°C for 6 hours. To each sample, 100 μL of acetonitrile was added, stirred by vortexing, and then centrifuged to obtain precipitates. The supernatant was collected and analyzed by HPLC.

[0532] (7-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis The measurement involved measuring the fluorescence molecular weight lost from the ADC mimic using liquid chromatography / fluorescence detection. Three samples prepared in Example 7-1, to which acetonitrile was immediately added at 0°C, were designated as the 0-hour sample, and three samples incubated at 37°C for 6 hours as described in Example 7-1 were designated as the 6-hour sample. The difference in fluorescence intensity between the 6-hour and 0-hour samples was analyzed.

[0533] Using Pyrene, the correlation between the area of ​​fluorescence intensity measured by HPLC and the concentration was calculated. Using this formula, the difference in fluorescence intensity for each ADC mimic was converted to concentration. The elimination rate was calculated as the percentage of the aforementioned difference in fluorescence intensity, with the concentration at time 0 set to 100%.

[0534] [Table 5]

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

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

[0537] (8-1) Cathepsin B cleavage test The procedure was carried out in accordance with the previously reported method (Nature Communications 2018, 9, 2512).

[0538] (8-2) Analysis of the amount of detached payload using HPLC analysis The amount of payload detached from the ADC was measured using liquid chromatography-mass spectrometry (including tandem mass spectrometry). Three samples in Example 8-1 to which acetonitrile was immediately added at 0°C were designated as the 0-hour sample, and three samples in Example 8-1 incubated at 37°C for 6 hours were designated as the 6-hour sample. The MS intensities of the payload detected from the 6-hour and 0-hour samples were calculated by extracting ion chromatograms, and the differences between them were analyzed.

[0539] Separately, MMAE was used to calculate the correlation between the TIC area and concentration obtained by HPLC. Using this calculation formula, the TIC of the fluorescence intensity of each ADC was converted to concentration. The elimination rate was calculated as the percentage difference in the aforementioned ion chromatograms, with the concentration on Day 0 set to 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 released its payload immediately.

[0542] Example 9: Evaluation of ADC mimicry using mouse plasma (9-1) Plasma stability test of ADC mimic 500 μL of mouse plasma (Charles River) was mixed with ADC mimic to a concentration of 0.1 mg / mL, and then sterile filtered. This solution was dispensed into six Eppendorf tubes in 50 μL portions. Three of the six samples were stored in an incubator set to 37°C for 4 days. The remaining three were stored in a freezer at -80°C for the same period. 100 μL of acetonitrile was added to each sample, and the mixture was vortexed and then centrifuged to obtain a precipitate. The resulting supernatant was collected and analyzed by HPLC.

[0543] (9-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis The measurement involved measuring the fluorescence molecular weight lost from the ADC mimic using liquid chromatography / fluorescence detection. Three samples stored in a freezer in Example 9-1 were designated as Day=0, and three samples stored at 37°C in Example 9-1 were designated as Day=4. The difference in fluorescence intensity between Day=4 and Day=0 was analyzed.

[0544] The rate of detachment of fluorescent molecules was calculated according to Example 7-2. The results were evaluated based on the rate of fluorescent molecule detachment, as shown in the table below.

[0545] [Table 7]

[0546] Example 10: Evaluation of ADCs using mouse plasma (10-1) Plasma stability test of ADCs 500 μL of mouse plasma (Charles River) was mixed with ADC mimic to a concentration of 0.1 mg / mL, and then sterile filtered. This solution was dispensed into six Eppendorf tubes in 50 μL portions. Three of the six samples were stored in an incubator set to 37°C for 4 days. The remaining three were stored in a freezer at -80°C for the same period. 100 μL of acetonitrile was added to each sample, and the mixture was vortexed and then centrifuged to obtain a precipitate. The resulting supernatant was collected and analyzed by HPLC.

[0547] (10-2) Analysis of the amount of lost payload using HPLC analysis The amount of payload detached from the ADC was measured using liquid chromatography-mass spectrometry (including tandem mass spectrometry). Three samples from Example 10-1, to which acetonitrile was immediately added at 0°C, were designated as the 0-hour sample, and three samples from Example 8-1, incubated at 37°C for 6 hours, were designated as the 6-hour sample. The MS intensities of the payload detected in the 6-hour and 0-hour samples were calculated by extracting ion chromatograms, and the difference between them was analyzed. The payload detachment rate was calculated according to Example 8-2.

[0548] [Table 8]

Claims

1. Formula (I) below: 【Chemistry 1】 [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and it regioselectively forms an amide bond with an adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. The immunoglobulin unit is the human immunoglobulin unit. L １ However, the following equation (i): -L ４ -Y-L ３ - (i) [During the ceremony, L ３ and L ４ each independently represents a divalent group selected from the group consisting of -(C(R) ２ ), -(O-C(R) ｍ -C(R) ２ ), -(C(R) ２ -C(R) ｍ ), and -(C(R) ２ -C(R) ２ -O), ｍ and combinations thereof. R is 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 between 0 and 20. Y has the following structural formula: 【Chemistry 2】 [Here, the white and black circles indicate joining moves.] The white circle is connected by an L ３ When joined, the black circle represents the L-shaped joint. ４ It is connected, The white circle is connected by an L ４ When joined, the black circle represents the L-shaped joint. ３ It is a divalent group represented by one of the following structural formulas: It is bonded to . It is a divalent group represented by . L ２ This indicates a divalent group, R １ This indicates a monovalent group which may contain a hydrophilic group. X represents a divalent group which may have substituents, where the divalent group has 1 to 3 carbon atoms forming a main chain portion that links two atoms adjacent to X, and the substituent is a monovalent group which may include a hydrophilic group. D indicates a functional substance. R Ａ This indicates the side chain of the valine residue, R Ｂ This indicates a side chain of a citrulline residue or an alanine residue. n is either 0 or 1, where if n is 1, the substituent in X is R １ Together with it, it may form a ring that may contain hydrophilic groups. The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.

5. The structure includes a structural unit represented by ] and at least one hydrophilic group is present in the structural unit. Lysine residues are located at positions 246 / 248, 288 / 290, or 317 according to Eu numbering. Conjugates of antibodies and functional substances or salts thereof, The structural unit represented by formula (I) is the structural unit represented by the following formulas (I-1), (I-2), or (I-3): 【Transformation 3】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I),] R １ This indicates 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 equation (I), R ２ This indicates a monovalent group containing a hydrophilic group. ]; or 【Transformation 5】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I), R ３ This represents a monovalent group containing a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydrophilic group. R ４ This indicates a monovalent group containing a hydrophilic group. A structural unit represented by formula (I-1), (I-2), or (I-3) is a structural unit represented by the following formulas: (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), or (I-3b'): 【Transformation 6】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. 【Transformation 7】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I), Two L ５ Each of these independently represents a bond or a divalent group. The two HG groups each independently exhibit hydrophilicity. 【Transformation 8】 [During the ceremony, Ig, L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. L １ａ , and L １ｂ Each of these independently represents a bond or a divalent group. HG' indicates 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 equation (I), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. 【Chemistry 10】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I), L ５ This indicates a bond or a divalent group. HG indicates 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 equation (I), HG indicates a hydrophilic group. A structural unit represented by formula (I-1a'), (I-1b'), (I-1c'), (I-2'), (I-3a'), or (I-3b') is represented by the following formulas: (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). Conjugates of antibodies and functional substances, or their salts, which are structural units: 【Chemistry 12】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I). 【Chemistry 13】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I). 【Chemistry 14】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I). 【Chemistry 15】 [During the ceremony, Ig, L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I), L １ａ , and L １ｂ Each of these independently represents a bond or a divalent group. n1 is an integer between 3 and 20. 【Chemistry 16】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (I). 【Chemistry 17】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as 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 equation (I). ]; or 【Chemistry 20】 [During the ceremony, Ig, L １ , L ２ , D, R Ａ , R Ｂ , r is the same as in equation (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 of these is independently -(C(R) ２ ) ｍ - A conjugate or salt thereof according to claim 1, comprising -

4. L ２ However, the following structural formula: 【Chemistry 21】 (Here, the black and white circles indicate joining moves, The black circle is the joint, L ２ It is bonded to the adjacent carbonyl group, The white circle is connected to D. E indicates an electron-withdrawing group. A conjugate or salt thereof according to any one of claims 1 to 3, comprising a divalent group represented by n2 (where n2 is an integer from 1 to 4).

5. The conjugate or 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, protein, nucleic acid, or low molecular weight organic compound.

7. The following equations (II-1), (II-2), or (II-3): 【Chemistry 22】 [During the ceremony, L ２ This indicates 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 - and a divalent group selected from the group consisting of combinations thereof, R １ This indicates a monovalent group containing a hydrophilic group, D indicates a functional substance. R Ａ This indicates the side chain of the valine residue, R Ｂ This indicates a side chain of a citrulline residue or an alanine residue. B exhibits bioorthogonal functional groups, The bioorthogonal functional groups are maleimide residues, thiol residues, furan residues, halocarbonyl residues, alkene residues, alkyne residues, azide residues, or tetrazine residues. 【Chemistry 23】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1), R ２ This indicates 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 equation (II-1), R ３ This represents a hydrogen atom, or a monovalent group containing an alkyl group with 1 to 6 carbon atoms, or a hydrophilic group. R ４ This represents a monovalent group containing a hydrophilic group. A compound or salt thereof represented by ] A compound represented by formula (II-1), (II-2), or (II-3) is represented by the following formulas (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 equation (II-1), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. 【Chemistry 26】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1), Two L ５ Each of these independently represents a bond or a divalent group. The two HG groups each independently exhibit hydrophilicity. 【Chemistry 27】 [During the ceremony, L ２ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. L ３ａ , and L ３ｂ Each of these independently represents a bond or a divalent group. HG' indicates 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 equation (II-1), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. 【Chemistry 29】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1), L ５ This indicates a bond or a divalent group. HG indicates a hydrophilic group. ]; or 【Transformation 30】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1), HG indicates a hydrophilic group. A compound represented by formula (II-1a'), (II-1b'), (II-1c'), (II-2'), (II-3a'), or (II-3b') is a compound or salt thereof represented by the following formulas: (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): 【Chemistry 31】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). 【Chemistry 32】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). 【Transformation 33】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). 【Transformation 34】 [During the ceremony, L ２ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1), L ３ａ , and L ３ｂ Each of these independently represents a bond or a divalent group. n1 is an integer between 3 and 20. 【Chemistry 35】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). 【Transformation 36】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). 【Chemistry 37】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). 【Transformation 38】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1). ]; or 【Chemistry 39】 [During the ceremony, L ２ , L ３ , D, R Ａ , R Ｂ , and B are the same as in equation (II-1).

8. L ２ However, the following structural formula: 【Chemistry 40】 (Here, the black and white circles indicate joining moves, The black circle is the joint, L ２ It is bonded to the adjacent carbonyl group, The white circle is connected to D. E indicates an electron-withdrawing group. The compound or salt thereof according to claim 7, comprising a divalent group represented by n2 (where n2 is an integer from 1 to 4).

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

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

11. A reagent for derivatization of antibodies comprising a compound or a salt thereof according to any one of claims 7 to 10.

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