Regioselective conjugate of antibody and functional substance or salt thereof, and antibody derivative and compound for use in production thereof or salt thereof

Abstract

To provide a conjugate of an antibody and a functional substance or a salt thereof, in which the bonding ratio between the antibody and the functional substance is controlled within a specific range and which is superior in desired properties.SOLUTION: The present invention provides a regioselective conjugate of an antibody and a functional substance or a salt thereof, comprising a structural unit represented by the following formula (I'), where D denotes a functional substance and r is 1.5 to 2.5.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

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

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

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

[0005]

Chemical formula

[0006]

Chemical formula

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

[0008]

Chemical formula

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

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

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

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

Non-Patent Literature

[0013]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Summary of the Invention

Problems to be Solved by the Invention

[0014] An object of the present invention is to provide a conjugate of an antibody and a functional substance or a salt thereof that has excellent properties while controlling the binding ratio of the antibody and the functional substance within a specific range. The conjugate of an antibody and a functional substance or a salt thereof is excellent in properties while controlling the binding ratio of the antibody and the functional substance within a specific range.

Means for Solving the Problems

[0015] As a result of intensive studies, the present inventors have found that a site-selective conjugate containing a linker having a specific structure in the side chain of the main chain connecting an antibody and a drug (or drug mimetic) and having an average ratio of binding between the immunoglobulin unit and the functional substance (functional substance / immunoglobulin unit) within a desired range (1.5 to 2.5) has excellent properties. For example, such a site-selective conjugate or a salt thereof has excellent clearance (long in vivo residence time), low aggregation rate (high monomer ratio), high cleavability by cathepsin B (high ability to release the functional substance in human cells), and high stability in mouse plasma. Such a site-selective conjugate or a salt thereof has a hydrophilic group at or near the tip of the side chain that is easily exposed on the surface of the conjugate molecule, so that the hydrophilicity of the whole molecule can be efficiently improved, and it can exhibit excellent properties as described above.

[0016] The present inventors have also succeeded in developing antibody derivatives and compounds useful for producing such site-selective conjugates. The site-selective conjugates, antibody derivatives, and compounds of the present invention represented by the structures of formulas (I) to (VII) have the technical feature of sharing a partial structural unit excluding X and Y among the structural units represented by formula (V). The present inventors have succeeded in developing a series of inventions having such technical features and have completed the present invention. The prior art does not describe or suggest the chemical structure of the site-selective conjugate of the present invention and the relationship between such a chemical structure and the excellent properties as described above. The prior art also does not describe or suggest the antibody derivatives and compounds of the present invention that can be used for producing such site-selective conjugates.

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

[0018] In a first embodiment, the present invention provides the following formula (I): [Chemical formula] [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and is site-selectively bound to L1 adjacent to Ig via an amino group in the side chain of a lysine residue in the two heavy chains, and is bound, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R A represents the side chain of a valine residue, R B represents the side chain of a citrulline residue or an alanine residue, ring A represents a divalent aromatic ring group which may have a substituent, R1 and R2 each independently represent a hydrogen atom or a monovalent group, L1 and L2 each independently represent a divalent group, D represents a functional substance, The average ratio r of said binding per two heavy chains is 1.5 to 2.5. Provided is a site-selective conjugate of an antibody and a functional substance or a salt thereof, which comprises a structural unit represented by 〕.

[0019] In a specific embodiment, the structural unit represented by formula (I) is the following formula (I’):

Chemical formula

[0020] In a second embodiment, the present invention provides the following formula (II):

Chemical formula

[0021] In a specific embodiment, the structural unit represented by formula (II) is the following formula (II’):

Chemical formula

[0022] In a third embodiment, the present invention provides the following formula (III):

Chemical formula

[0023] In a specific embodiment, the compound represented by formula (III) is the following formula (III’):

Chemical formula

[0024] In a fourth embodiment, the present invention provides a reagent for derivatizing an antibody, which comprises the compound according to the third embodiment or a salt thereof.

[0025] In a fifth embodiment, the present invention provides the following formula (IV):

[0026]

Chemical formula

[0027] In a specific embodiment, the compound represented by formula (IV) is the following formula (IV’):

Chemical formula

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

[0029] In the seventh embodiment, the present invention provides a compound represented by the following formula (V):

Chemical formula

[0030] In a specific embodiment, the compound represented by the following formula (V) is a compound represented by the following formula (V’):

Chemical formula

[0031] In the eighth embodiment, the present invention provides a compound having a bioorthogonal functional group represented by the following formula (VI):

Chemical formula

[0032] In a specific embodiment, the compound represented by formula (VI) is the following formula (VI’):

Chemical formula

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

[0034] In a specific embodiment, the compound represented by formula (VII) is the following formula (VII’): [Chemical formula] 〔In the formula, R A , R B , ring A, and Y are each the same as those represented by formula (VII), L HG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 , and R HG2 are each independently a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, At least one hydrophilic group is contained at one or more sites selected from the group consisting of L HG , R HG1 , and R HG2 and is included in one or more sites selected from the group consisting of, R1 represents a hydrogen atom or a monovalent group, L1 represents a divalent group, B1 represents a bioorthogonal functional group. It may be a compound represented by 〕.

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

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

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

[0038] In a preferred embodiment, the site-selective binding may be achieved by an amide bond formed by the amino group in the side chain of the lysine residue and the carbonyl group in L1.

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

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

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

[0042] In a preferred embodiment, the functional substance may be a medicine, a labeling substance, or a stabilizer.

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

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

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

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

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

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

Advantages of the Invention

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

Brief Description of the Drawings

[0050]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

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

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

[0053] The type of antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE), or an antibody with a valence of four or more (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 with a predetermined sugar chain added (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of the isotype of monoclonal antibodies 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, more preferably a human IgG full-length monoclonal antibody.

[0054] Any antigen can be used as the antigen of the antibody. For example, such antigens include proteins [including oligopeptides and polypeptides. It may be a protein modified with a biomolecule such as a sugar (e.g., glycoprotein)], sugar chains, nucleic acids, and low molecular weight compounds. Preferably, the antibody may be an antibody using a protein as an 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.

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

[0056] (1) Cancer region PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, 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-cadherin, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2, CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transferrin Receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lewis Blood Group B Antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 Integrin, TAG-72(CA72-4), CD70

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

[0058] (3) Neurological diseases CGRP, CD20, β-Amyloid, β-Amyloid Protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO(Ig Domain Containing1), α-Synuclein, Extracellular tau, CD52, Insulin Receptor, tau Protein, TDP-43, SOD1, TauC3, JC Virus

[0059] (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

[0060] (5) Hereditary and rare diseases Amyloid AL, SEMA4D (CD100), Insulin receptor, ANGPTL3, IL4, IL13, FGF23, Adrenocorticotropic hormone, Transthyretin, Huntingtin

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

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

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

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

[0065] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, sirukumab, dinutuximab, olaratumab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, penpulizumab (IgG4), mepolizumab, elotuzumab, daratumumab, ikesekiizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, lirilumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesvacumab, brodalumab (IgG2), orlaratumab) (when not referring to the IgG subtype, it indicates IgG1).

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

[0067] According to the present invention, specific lysine residues in the heavy chain in the immunoglobulin unit constituting the antibody (e.g., lysine residues at positions 246 / 248, 288 / 290, or 317) can be site-specifically modified (see, for example, International Publication No. 2018 / 199337, International Publication No. 2019 / 240288, International Publication No. 2019 / 240287, and International Publication No. 2020 / 090979). As used herein, "site-specific" or "site-selectivity" means that, although a specific amino acid residue in an antibody is not unevenly distributed in a specific region, a predetermined structural unit capable of binding to the specific amino acid residue in the antibody is unevenly distributed in a specific region of the antibody. Therefore, expressions related to site-selectivity such as "having site-specifically", "site-specific binding", and "binding with site-selectivity" refer to the retention rate or binding rate of a predetermined structural unit in a target region containing one or more specific amino acid residues being significantly higher than the retention rate or binding rate of the structural unit in a non-target region containing a plurality of amino acid residues of the same type as the specific amino acid residue in the target region. Such site-selectivity may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%.

[0068] In the present invention, as long as specific lysine residues in the heavy chain of an antibody are site-specifically modified, specific amino acid residues at other positions may be further site-specifically modified. For example, methods for site-specifically modifying specific amino acid residues at predetermined positions in an antibody are described in International Publication No. WO2018 / 199337, International Publication No. WO2019 / 240288, International Publication No. WO2019 / 240287, and International Publication No. WO2020 / 090979. Such specific amino acid residues include amino acid residues having side chains that are easy to modify (e.g., amino group, carboxy group, amide group, hydroxy group, thiol group) (e.g., lysine residue, aspartic acid residue, glutamic acid residue, asparagine residue, glutamine residue, threonine residue, serine residue, tyrosine residue, cysteine residue), preferably lysine residues having side chains containing an amino group, tyrosine residues having side chains containing a hydroxy group, serine residues, and threonine residues, or cysteine residues having side chains containing a thiol group, and more preferably lysine residues (i.e., among the lysine residues at positions 246 / 248, 288 / 290, and 317, two lysine residues may be site-specifically double-modified, and three lysine residues may be site-specifically triple-modified).

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

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

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

[0072] (monovalent hydrocarbon group and related terms) Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

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

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

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

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

[0077] As the monovalent chain hydrocarbon group, alkyl is preferable.

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

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

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

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

[0082] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferable.

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

[0084] As the monovalent aromatic hydrocarbon group, phenyl is preferable.

[0085] Among these, as the monovalent hydrocarbon group, alkyl, cycloalkyl, and aryl are preferable.

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

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

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

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

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

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

[0092] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. As the arylene, an arylene having 6 to 14 carbon atoms is preferable, an arylene having 6 to 10 carbon atoms is more preferable, and an arylene having 6 carbon atoms is particularly preferable. Examples of the arylene include phenylene, naphthylene, and anthracenylene. 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.

[0093] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic ring preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent aromatic heterocyclic group include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a non-aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent non-aromatic heterocyclic group include pyrrolidionediy, pyrrolinedionediyl, oxirandiyl, aziridinediyl, azetidinediyl, oxetandiy, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolandiyl, 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.

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

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

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

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

[0098] Aralkyl means arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above. Preferred aralkyl has 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0099] Preferably, the substituent may be as follows: (i) Halogen atom; (ii) Alkyl, phenyl, or naphthyl having 1 to 12 carbon atoms; (iii) Aralkyl having 3 to 15 carbon atoms; (iv) 5- or 6-membered heterocycle; (v) R a -O-, R a -C(=O)-, R a -O-C(=O)-, or R a -C(=O)-O-(R a represents a hydrogen atom or alkyl having 1 to 12 carbon atoms.); (vi) NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O-, or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms.); or (vii) The same group as that listed in the above (vii).

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

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

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

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

[0104] The polyethylene glycol (PEG) 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 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k1 may be an integer of 3 to 15, preferably an integer of 4 to 12, more preferably an integer of 5 to 10, and even more preferably an integer of 4 to 9.

[0105] The polysarcosine group is -(NCH3-CH2-CO-) k2 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 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k2 may be an integer of 3 to 15, preferably an integer of 4 to 12, more preferably an integer of 5 to 10, and even more preferably an integer of 4 to 9.

[0106] The sugar moiety is a monosaccharide, an oligosaccharide (e.g., disaccharide, trisaccharide, tetrasaccharide, pentasaccharide), or a polysaccharide. The sugar moiety can include aldoses or ketoses, or combinations 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.

[0107] In certain embodiments, the sugar moiety may be a low molecular weight hydrophilic group. A low molecular weight hydrophilic group refers to a hydrophilic group having a molecular weight of 1500 or less. The molecular weight of the low molecular weight hydrophilic group may 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 the low molecular weight hydrophilic group include a carboxylic acid group, a sulfonic acid group, a hydroxyl group, and a polyethylene glycol group, a polysarcosine group, and a sugar moiety (e.g., monosaccharide, oligosaccharide) that satisfy the above molecular weight.

[0108] (Bioorthogonal functional group) A bioorthogonal functional group refers to a group that does not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphoric acids) or has a slow reaction rate with biological components, but selectively reacts with components other than biological components. Bioorthogonal functional groups are well known in the art (see, for example, Sharpless K.B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

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

[0110] Examples of such bioorthogonal functional groups include an azide residue, an aldehyde residue, a thiol residue, an alkene residue (in other words, it suffices to have a vinylene (ethenylene) moiety which is the smallest unit having a carbon-carbon double bond; the same applies hereinafter), an alkyne residue (in other words, it suffices to have an ethynylene moiety which is the smallest unit having a carbon-carbon triple bond; the same applies hereinafter), a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an α-halocarbonyl residue (e.g., a carbonyl residue having a fluorine atom, a chlorine atom, a bromine atom or an iodine atom at the α-position; the same applies hereinafter), an isonitrile residue, a sydnone residue, and a selenium residue. Specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following.

[0111] Here,

Chemical formula

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

[0113] In certain embodiments, the bioorthogonal functional group may be protected. A bioorthogonal functional group that may be protected refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. The unprotected bioorthogonal functional group corresponds to the bioorthogonal functional groups described above. The protected bioorthogonal functional group is a group that generates a bioorthogonal functional group upon cleavage of the protecting group. Cleavage of the protecting group can be carried out by specific treatment under conditions (mild conditions) that do not cause protein denaturation and degradation (e.g., cleavage of amide bonds). Such specific treatment includes, for example, (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) leaving it for a while when using a cleavable linker containing a self-degradable cleavable moiety. Such protecting groups and their cleavage conditions are common general knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Jounal of American Chemical Society. 132, 1500 (2010).; Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004).; DeSimone, J.M., Journal of American Chemical Society. 132, 17928 (2010); Th ompson, D.H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R.G., Journal of Controlled Release, 95, 291 (2004)).

[0114] Examples of the protected bioorthogonal functional group include a disulfide residue, an ester residue, an acetal residue, a ketal residue, an imine residue, and a vicinal diol residue.

[0115] More specifically, the protected bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following. [Chemical formula] Here, the wavy line orthogonal to the bond indicates the cleavage site, single or multiple R 2a are the same or different and are selected from the group consisting of a hydrogen atom or the above-described substituents, · represents a bond. ]

[0116] Preferably, the bioorthogonal functional group that may be protected is an unprotected bioorthogonal functional group.

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

[0118] The drug may be a drug for any disease. Such diseases include, for example, cancer (e.g., lung cancer, gastric 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 diseases and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cerebrovascular diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, myotonic dystrophy), 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). The drug may be a preventive or therapeutic drug for the disease or a drug for alleviating side effects.

[0119] More specifically, the drug may be an anticancer agent. Examples of anticancer agents include chemotherapy agents, toxins, radioisotopes or substances containing them. Examples of chemotherapy agents include 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 bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioisotopes of hydrogen atoms (e.g., 3 H), radioisotopes of carbon atoms (e.g., 14 C), radioisotopes of phosphorus atoms (e.g., 32 P), radioisotopes of sulfur atoms (e.g., 35 S ), radioisotopes of yttrium (e.g., 90 Y), radioisotopes of technetium (e.g., 99m Tc), radioisotopes of indium (e.g., 111 In), radioisotopes of iodine atoms (e.g., 123 I, 125 I, 129 I, 131I), Radioactive isotopes of samarium (e.g., 153 Sm), radioactive isotopes of rhenium (e.g., 186 Re), radioactive isotopes of astatine (e.g., 211 At), radioactive isotopes of bismuth (e.g., 212 Bi). More specifically, as drugs, auristatins (MMAE, MMAF), maytansines (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, tubulysin are included.

[0120] The labeling substance is a substance that enables the detection of a target (e.g., tissue, cell, substance). Examples of the labeling substance include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamer), 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), radioactive isotopes (e.g., those described above), or substances containing them.

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

[0122] The type of antibody used as the functional substance may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE), or an antibody with a valence of 4 or more (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 sugar chain is added (e.g., an antibody modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of the isotype of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Examples of antibodies used as the functional substance include full-length antibodies and fragments thereof (fragment antibodies). The fragment antibody only needs to maintain the binding ability to the desired antigen, and examples include Fab, Fab’, F(ab’)2, and scFv.

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

[0124] The substance for transport is a substance having the ability to transport a compound. As the substance for transport, a substance that can encapsulate a compound in a protein outer shell (e.g., a multimer) (e.g., ferritin such as human ferritin, virus particles, virus-like particles) is preferred.

[0125] A stabilizer is a substance that enables the stabilization of an antibody. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0126] The functional substance may also be a peptide, protein, nucleic acid, low-molecular-weight organic compound, sugar chain, lipid, high-molecular polymer, metal (e.g., gold), or chelator. 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. Examples of nucleic acids also include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of low-molecular-weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.

[0127] In certain embodiments, the functional substance may be a substance having an aromatic ring. Examples of substances having an aromatic ring include monomethylauristatin [e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF)], or Exatecan.

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

[0129] 2. Site-selective conjugate or a salt thereof The present invention provides a site-selective conjugate of an antibody and a functional substance or a salt thereof, which contains the structural unit represented by the above formula (I). The site-selectivity of the conjugate of the present invention is as described above.

[0130] In formula (I) and other formulas presented in connection with the present invention, -(hyphen) indicates that the two units (e.g., atoms, groups) present on both sides are covalently bonded. The two units (e.g., atoms, groups) present on both sides are covalently bonded.

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

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

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

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

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

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

[0137] Ring A represents a divalent aromatic ring group which may have a substituent. The divalent aromatic ring group is the arylene or divalent aromatic heterocyclic ring as described above. The position of the divalent aromatic ring group where two adjacent atoms (carbon atom and nitrogen atom) are bonded, when the amide bond present on the carboxy terminal side of citrulline is cleaved by cathepsin B, is not particularly limited as long as cleavage occurs between the oxygen atom and the carbonyl group in -O-(C=O)- due to the conjugation of π electrons (for example, refer to the cleavage reaction shown in "(B) Explanation in lysosomes in human cancer cells" in the background art). Such a position is common general knowledge in the art and can be easily determined by those skilled in the art according to factors such as the type of the divalent aromatic ring group. The cleavage reaction shown in "(B) Explanation in lysosomes in human cancer cells" in the background art is referred to, and it is not particularly limited. Such a position is common general knowledge in the art and can be easily determined by those skilled in the art according to factors such as the type of the divalent aromatic ring group.

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

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

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

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

[0142] R1 and R2 each independently represent a hydrogen atom or a monovalent group. The monovalent group is as described above. The monovalent groups for R1 and R2 are preferably monovalent hydrocarbon groups which may have substituents, more preferably alkyls which may have substituents, and even more preferably alkyls. As the alkyl, those described above are preferred.

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

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

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

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

[0147] In certain embodiments, the above moiety may be a divalent group represented by any one of the following structural formulas. [Chemical formula] [Here, the white circle and the black circle indicate bonds.]

[0148] In L1, when the bond of the white circle is bonded to an atom on the Ig-binding portion side, the bond of the black circle may be bonded to an atom on the nitrogen atom (N) binding portion side in "N-R1", When the binding hand of the white circle is bound to an atom existing on the nitrogen atom (N) binding part side in "N-R1", the binding hand of the black circle may be bound to an atom existing on the Ig binding part side.

[0149] In L2, when the binding hand of the white circle is bound to an atom existing on the nitrogen atom (N) binding part side in "N-R2", the binding hand of the black circle may be bound to an atom existing on the functional substance (D) binding part side. When the binding hand of the white circle is bound to an atom existing on the functional substance (D) binding part side, the binding hand of the black circle may be bound to an atom existing on the nitrogen atom (N) binding part side in "N-R2".

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0165] In a specific embodiment, the monovalent group which may contain a hydrophilic group represented by R HG1 , and R HG2 may be a protecting group for an amino group. Examples of the protecting group for an amino group include those described above for R1 and R2. For example, one of R HG1 , and R HG2 may be a hydrogen atom and the other may be a protecting group for an amino group.

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

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

[0168] The symbols described in the above series of formulas, and the definitions, examples, and preferred examples of the elements in the symbols (e.g., specific elements such as cleavable moieties, or specific formulas) are the same for other formulas.

[0169] The site-selective conjugate of the present invention or a salt thereof is useful, for example, as a medicine or a reagent (e.g., a diagnostic agent, a research reagent).

[0170] The conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate of the present invention or a salt thereof, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc., binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc., disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, etc., lubricants such as magnesium stearate, aerosil, talc, sodium lauryl sulfate, etc., fragrances such as citric acid, menthol, glycylglycine ammonium salt, glycine, orange powder, etc., preservatives such as sodium benzoate, sodium bisulfite, methyl paraben, propyl paraben, etc., stabilizers such as citric acid, sodium citrate, acetic acid, etc., suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc., dispersing agents such as surfactants, diluents such as water, physiological saline, orange juice, etc., base waxes such as cacao butter, polyethylene glycol, white kerosene, etc., but are not limited thereto. The conjugate of the present invention or a salt thereof may also have any modification (e.g., PEGylation) to achieve stability.

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

[0172] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Examples of pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, tonicity agents, and the like. Aqueous and non-aqueous sterile suspension solutions are also included, which may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, preservatives, and the like.

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

[0174] In one embodiment, the site-selective conjugate of the present invention or a salt thereof can be produced by reacting an antibody derivative having a site-selective biocompatible functional group or a salt thereof with a functional substance (Figure 2). Such a reaction can be carried out by the reaction between the biocompatible functional group in the antibody derivative and the functional substance.

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

[0176] When the functional substance does not have a functional group that easily reacts with the bioorthogonal functional group in the antibody derivative, the drug may be derivatized to have such a functional group. Derivatization is common general knowledge in the art (e.g., WO 2004 / 010957, US 2006 / 0074008 A1, US 2005 / 0238649 A1). For example, derivatization may be performed using any crosslinking agent. Alternatively, derivatization may be performed using a specific linker having a desired functional group. In the present invention, since the derivatized functional substance is merely a kind of functional substance, it is simply referred to as "functional substance".

[0177] The above reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation and decomposition of proteins (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 solution. The pH of the buffer solution is, for example, 5 - 9, preferably 5.5 - 8.5, more preferably 6.0 - 8.0. The buffer solution 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 of such reactions, see, for example, G.J.L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G.J.L. Bernardes et al., Chem. Asian J. , 4, 630 (2009); B.G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate Chem., 25, 825 (2014).

[0178] In another embodiment, the site - selective conjugate of the present invention or a salt thereof can be produced by reacting a compound having a bio - orthogonal functional group and a functional substance or a salt thereof with a starting antibody having Ig (immunoglobulin unit) (Figure 2).

[0179] The above - mentioned starting antibody contains a lysine residue selectively modified with a bio - orthogonal functional group. The bio - orthogonal functional group in the starting antibody can be selected so as to be able to react with the bio - orthogonal functional group in the compound having a bio - orthogonal functional group and a functional substance. As the bio - orthogonal functional group in the starting antibody, various bio - orthogonal functional groups described above can be used. From the viewpoint of high versatility, etc., the bio - orthogonal functional group in the starting antibody may be a reimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.

[0180] In a specific embodiment, the above - mentioned starting antibody has the following formula (VIII): [Chemical formula] [wherein, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and an amide bond is regioselectively formed between the amino group in the side chain of the lysine residue in the two heavy chains and the carbonyl group adjacent to Ig. L is a divalent group selected from the group consisting of -(C(R)2) m -, -(O-C(R)2-C(R)2) m -, and -(C(R)2-C(R)2-O) m - and is a divalent group selected from the group consisting of. Each R is independently a hydrogen atom, alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms, or alkynyl having 2 to 6 carbon atoms. m is an integer from 0 to 10. B is a bioorthogonal functional group capable of reacting with the bioorthogonal functional group represented by B1. The average ratio r of the above-mentioned binding per two heavy chains is 1.5 to 2.5.]. It may contain an immunoglobulin unit represented by the above formula (VIII). The definitions, examples, and preferred examples of Ig and r in the above formula (VIII) are the same as those described above.

[0181] m is preferably an integer of 1 or more, more preferably an integer of 2 or more, 3 or more, 4 or more, or 5 or more. m may also be preferably an integer of 9 or less, more preferably an integer of 8 or less, 7 or less, or 6 or less. In a specific case, m may be an integer from 1 to 8 (preferably an integer from 2 to 6).

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

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

[0184] Confirmation of the production of the site-selective conjugate or a salt thereof depends on the specific raw materials and the molecular weight of the product, and can be carried out, for example, by reverse-phase HPLC under reducing conditions or by mass spectrometry. Confirmation of site selectivity can be carried out, for example, by peptide mapping. Peptide mapping can be carried out, for example, by treatment with a protease (e.g., trypsin, chymotrypsin,) and mass spectrometry. As the protease, an endoprotease is preferred. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. The conjugate or a salt thereof can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, high performance liquid chromatography, affinity chromatography). Confirmation of the production of the site-selective conjugate or a salt thereof depends on the specific raw materials and the molecular weight of the product, and can be carried out, for example, by reverse-phase HPLC under reducing conditions or by mass spectrometry. Confirmation of site selectivity can be carried out, for example, by peptide mapping. Peptide mapping can be carried out, for example, by treatment with a protease (e.g., trypsin, chymotrypsin,) and mass spectrometry. As the protease, an endoprotease is preferred. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. The conjugate or a salt thereof can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, high performance liquid chromatography, affinity chromatography).

[0185] 3. Antibody derivative or a salt thereof The present invention also provides an antibody derivative having a bioorthogonal functional group site-selectively or a salt thereof, which contains the structural unit represented by the above formula (II). The site selectivity of the antibody derivative of the present invention is as described above.

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

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

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

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

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

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

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

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

[0194] Confirmation of the production of the antibody derivative or a salt thereof can be carried out in the same manner as the method described for the site-selective conjugate of the present invention (the same applies to confirmation of site-selectivity). The antibody derivative or its salt can be appropriately purified by any purification method as described for the site-selective conjugate of the present invention.

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

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

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

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

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

[0200] In formula (III’), L HG , R HG1 , and R HG2is as described above in formula (I’). Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (I’).

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

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

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

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

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

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

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

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

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

[0210] [Table 1]

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

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

[0213] The compound of the present invention represented by formula (IV) or a salt thereof is useful, for example, as an antibody derivative of the present invention and as an intermediate for the production of the compound of the present invention represented by formula (III). The compound of the present invention represented by formula (IV) or a salt thereof is also useful, for example, for derivatizing any substance such as biomolecules (e.g., proteins such as antibodies, saccharides, nucleic acids, lipids) and functional substances.

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

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

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

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

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

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

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

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

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

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

[0224] The compound represented by formula (V) or a salt thereof is, for example, the following formula (V-1):

Chemical formula

Chemical formula

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

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

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

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

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

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

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

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

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

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

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

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

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

[0238] In formula (VII’), L HG , R HG1 , and R HG2is as described above in formula (I’). Therefore, the definitions, examples and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (I’).

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

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

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

Examples

[0242] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Chemical formula

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

Chemical formula

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

[0262] 11H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 5.92 (brs, 1H), 5.36 (brs, 2H), 5.01 (s, 1H), 4.34 - 4.29 (m, 1H), 4.26 - 4.20 (m, 1H), 4.14 - 4.10 (m, 1H), 3.53 (s, 3H), 3.00 - 2.83 (m, 2H), 2.18 - 2.13 (m, 2H), 1.94 - 1.89 (m, 2H), 1.84 - 1.23 (m, 17H), 0.79 (d, J = 6.8 Hz, 3H), 0.75 (d, J = 6.8 Hz, 3H).

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

[0264] (1 - 1 - 2) Synthesis of Pyrene (3)

Chemical Structure

[0265] (28.5 mg) of the alcohol (2) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (430 μL), stirred for 5 minutes under ice-cooling, and then bis(4-nitrophenyl) carbonate (26.6 mg, 85.7 μmol) and N,N-diisopropylethylamine (11.1 μL, 64.4 μmol) were added, followed by stirring at room temperature for 1.5 hours. When the reaction was monitored by LCMS, residual raw material was observed. Therefore, bis(4-nitrophenyl) carbonate (13.3 mg, 42.9 μmol) and N,N-diisopropylethylamine (5.54 μL, 32.2 μmol) were added, and the mixture was stirred at room temperature for 5 hours. Then, it was ice-cooled, and Sarcosin-Pyrene (64.9 mg, 215 μmol), 1-hydroxybenzotriazole (8.7 mg, 64 μmol), and N,N-diisopropylethylamine (57.2 μL, 333 μmol) were added, followed by stirring at room temperature for 16 hours. After the reaction, it was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and the above pyrene (3) (26.1 mg, 26.3 μmol) was obtained by lyophilization.

[0266] 1 H NMR (400 MHz, DMSO-d6) δ 10.09 - 10.07 (m, 1H), 8.63 - 8.60 (m, 1H), 8.33 - 7.65 (m, 13H), 7.60 - 7.57 (m, 2H), 7.37 - 7.32 (m, 2H), 5.94 - 5.91 (m, 1H), 5.72 - 5.70 (m, 1H), 5.37 (brs, 2H), 4.98 - 4.96 (m, 1H), 4.93 - 4.00 (m, 4H), 3.85 - 3.75 (m, 1H), 3.56 - 3.55 (m, 3H), 2.97 - 2.87 (m, 5H), 2.17 - 2.13 (m, 2H), 1.93 - 1.17 (m, 19H), 0.80 - 0.73 (m, 6H).

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

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

Chemical formula

[0269] Pyrene (3) (10.8 mg, 10.9 μmol) was dissolved in tetrahydrofuran (700 μL) and water (400 μL), stirred for 5 minutes under ice-cooling, then 1 M aqueous lithium hydroxide solution (109 μL, 109 μmol) was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, it was adjusted to about pH 6 using 0.1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and the above pyrene (4) (4.5 mg, 4.6 μmol) was obtained by lyophilization.

[0270] 1 H NMR (400 MHz, DMSO-d6) δ 13.05 (brs, 1H), 10.08 - 10.05 (m, 1H), 8.62 - 8.59 (m, 1H), 8.33 - 7.56 (m, 15H), 7.37 - 7.35 (m, 2H), 5.92 (brs, 1H), 5.61 - 5.60 (m, 1H), 5.36 (brs, 2H), 4.98 - 4.03 (m, 5H), 3.88 - 3.74 (m, 1H), 2.99 - 2.83 (m, 5H), 2.15 - 2.13 (m, 2H), 1.93 - 1.14 (m, 19H), 0.84 - 0.73 (m, 6H).

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

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

Chemical Structure

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

[0274] 1 H NMR (400 MHz, DMSO-d6) δ 10.02 - 9.99 (m, 1H), 8.85 - 7.88 (m, 14H), 7.67 - 7.65 (m, 1H), 7.60 - 7.51 (m, 2H), 7.33 - 7.27 (m, 2H), 6.91 - 6.87 (m, 2H), 5.92 - 5.91 (m, 1H), 5.63 - 5.62 (m, 1H), 5.36 (brs, 2H), 5.07 - 4.92 (m, 2H), 4.35 - 3.76 (m, 5H), 3.18 - 3.14 (m, 1H), 2.99 - 2.83 (m, 7H), 2.17 - 2.13 (m, 2H), 1.95 - 1.89 (m, 1H), 1.85 - 1.72 (m, 4H), 1.66 - 1.45 (m, 3H), 1.40 - 1.17 (m, 15H), 1.05 - 1.01 (m, 2H), 0.83 - 0.73 (m, 6H).

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

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

Chemical Structure

[0277] To pyrene (5) (2.2 mg, 1.9 μmol), 1,4-dioxane (380 μL) and 4 M hydrogen chloride / dioxane solution (95 μL, 380 μmol) were sequentially added, and the mixture was stirred at room temperature for 4 hours. After cooling in ice, N,N-diisopropylethylamine (71.8 μL, 418 μmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by 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 mimic (1) (2.1 mg, 1.9 μmol).

[0278] 1 H NMR (400 MHz, DMSO-d6) δ 12.06 (brs, 1H), 10.03 - 10.00 (m, 1H), 8.84 - 7.88 (m, 14H), 7.67 - 7.64 (m, 1H), 7.56 - 7.51 (m, 2H), 7.33 - 7.27 (m, 2H), 6.90 - 6.87 (m, 2H), 5.92 - 5.90 (m, 1H), 5.63 - 5.61 (m, 1H), 5.36 (brs, 2H), 5.08 - 4.96 (m, 2H), 4.35 - 3.76 (m, 5H), 3.18 - 3.14 (m, 1H), 2.97 - 2.83 (m, 7H), 2.20 - 2.16 (m, 2H), 1.93 - 1.88 (m, 1H), 1.81 - 1.78 (m, 4H), 1.69 - 1.53 (m, 3H), 1.35 - 1.17 (m, 6H), 1.08 - 1.01 (m, 2H), 0.81 - 0.73 (m, 6H).

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

[0280] (1 - 2) Synthesis of Linker-payload mimic (6) Linker-payload mimic (6) was synthesized as follows.

Chemical Structure

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

[0282] Dissolve Z-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 84.3 μmol) in N,N-dimethylformamide (1.5 mL), add 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (38.4 mg, 101 μmol) and 2,4,6-trimethylpyridine (13.3 μL, 101 μmol), and stir at room temperature for 10 minutes. Subsequently, add methyl 4-aminomandelate (18.3 mg, 101 μmol), stir at room temperature for 16 hours, and then purify by reverse-phase preparative chromatography. Collect the fraction containing the product, remove acetonitrile by concentration under reduced pressure, and obtain the above alcohol (7) (49.1 mg, 64.9 μmol) by lyophilization.

[0283] 1 H NMR (400 MHz, DMSO-d6) δ 10.04 - 9.95 (m, 1H), 8.40 - 7.28 (m, 12H), 6.00 - 5.97 (m, 1H), 5.43 (brs, 2H), 5.08 - 4.97 (m, 3H), 4.43 - 4.37 (m, 1H), 4.24 - 4.20 (m, 1H), 4.16 - 4.05 (m, 1H), 3.60 - 3.59 (m, 3H), 3.04 - 2.91 (m, 2H), 2.26 - 2.20 (m, 2H), 2.03 - 1.22 (m, 16H), 0.88 - 0.78 (m, 6H).

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

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

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

[0287] 1 H NMR (400 MHz, DMSO-d6) δ 10.11 - 9.97 (m, 1H), 8.64 - 8.60 (m, 1H), 8.36 - 7.95 (m, 11H), 7.82 - 7.74 (m, 1H), 7.65 - 7.57 (m, 2H), 7.49 - 7.19 (m, 8H), 5.91 - 5.90 (m, 1H), 5.72 - 5.70 (m, 1H), 5.36 (brs, 2H), 4.98 - 4.86 (m, 4H), 4.41 - 4.00 (m, 3H), 3.85―3.75 (m, 1H), 3.56 - 3.54 (m, 3H), 3.00 - 2.82 (m, 5H), 2.19 - 2.12 (m, 2H), 1.92 - 1.17 (m, 16H), 0.80 - 0.73 (m, 6H).

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

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

Chemical Structure

[0290] Pyrene (8) (44.8 mg, 41.3 μmol) was dissolved in tetrahydrofuran (3.75 mL) and water (1.25 mL), stirred for 5 minutes under ice-cooling, then lithium hydroxide monohydrate (8.7 mg, 210 μmol) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, it was adjusted to about pH 6 using 0.1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and the above pyrene (9) (18.4 mg, 17.2 μmol) was obtained by lyophilization.

[0291] 1 1H NMR (400 MHz, DMSO-d6) δ 13.02 (brs, 1H), 10.09 - 9.97 (m, 1H), 8.62 - 8.59 (m, 1H), 8.32 - 7.78 (m, 12H), 7.63 - 7.44 (m, 3H), 7.37 - 7.35 (m, 2H), 7.29 - 7.20 (m, 5H), 5.91 (brs, 1H), 5.61 - 5.60 (m, 1H), 5.36 (brs, 2H), 4.98 - 4.86 (m, 4H), 4.45 - 4.30 (m, 1H), 4.24 - 4.15 (m, 1H), 4.07 - 4.02 (m, 1H), 3.84 - 3.74 (m, 1H), 2.97 - 2.82 (m, 5H), 2.19 - 2.12 (m, 2H), 1.93 - 1.11 (m, 16H), 0.80 - 0.72 (m, 6H).

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

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

Chemical formula

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

[0295] 1 H NMR (400 MHz, DMSO-d6) δ 10.11 - 10.01 (m, 1H), 8.91 - 8.63 (m, 1H), 8.40 - 7.96 (m, 12H), 7.72 - 7.27 (m, 11H), 6.97 - 6.94 (m, 2H), 5.99 (brs, 1H), 5.71 - 5.69 (m, 1H), 5.43 (brs, 2H), 5.14 - 4.96 (m, 4H), 4.49 - 4.40 (m, 1H), 4.30 - 4.23 (m, 1H), 4.16 - 3.83 (m, 3H), 3.25 - 3.22 (m, 1H), 3.02 - 2.90 (m, 7H), 2.26 - 2.22 (m, 2H), 2.01 - 1.98 (m, 1H), 1.92 - 1.84 (m, 1H), 1.77 - 1.66 (m, 2H), 1.65 - 1.04 (m, 16H), 1.12 - 1.04 (m, 2H), 0.88 - 0.80 (m, 6H).

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

[0297] (1 - 2 - 5) Linker-payload mimic (6) synthesis

Chemical Structure

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

[0299] 1 H NMR (400 MHz, DMSO-d6) δ 12.05 (brs, 1H), 10.04 - 9.94 (m, 1H), 8.84 - 8.57 (m, 1H), 8.32 - 7.89 (m, 12H), 7.72 - 7.20 (m, 11H), 6.90 - 6.86 (m, 2H), 5.90 (brs, 1H), 5.64 - 5.62 (m, 1H), 5.36 (brs, 2H), 5.07 - 4.88 (m, 4H), 4.42 - 4.29 (m, 1H), 4.21 - 4.15 (m, 1H), 4.08 - 3.76 (m, 3H), 3.18 - 3.14 (m, 1H), 2.95 - 2.82 (m, 7H), 2.21 - 2.16 (m, 2H), 1.92 - 0.97 (m, 13H), 0.82 - 0.74 (m, 6H).

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

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

Chemical formula

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

Chemical formula

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

[0304] 1 H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.26 - 7.88 (m, 3H), 7.68 - 7.60 (m, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 6.00 - 5.97 (m, 1H), 5.43 (brs, 2H), 5.08 (s, 1H), 4.40 - 4.37 (m, 1H), 4.32 - 4.19 (m, 3H), 3.60 (s, 3H), 3.09 - 2.90 (m, 2H), 2.25 - 2.18 (m, 4H), 2.03 - 1.53 (m, 10H), 1.46 - 1.36 (m, 20H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H).

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

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

Chemical Structure

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

[0308] 1 H NMR (400 MHz, DMSO-d6) δ 10.14 - 10.12 (m, 1H), 8.69 - 8.67 (m, 1H), 8.40 - 7.82 (m, 13H), 7.76 - 7.72 (m, 1H), 7.68 - 7.65 (m, 2H), 7.44 - 7.39 (m, 2H), 5.99 (brs, 1H), 5.79 (d, J = 6.4 Hz, 1H), 5.44 (brs, 2H), 5.05 - 4.97 (m, 2H), 4.44 - 4.08 (m, 4H), 3.93 - 3.80 (m, 1H), 3.63 - 3.62 (m, 3H), 3.05 - 2.92 (m, 5H), 2.25 - 2.14 (m, 4H), 2.00 - 1.28 (m, 30H), 0.87 - 0.80 (m, 6H).

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

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

Chemical Structure

[0311] Pyrene (13) (30.7 mg, 26.1 μmol) was dissolved in tetrahydrofuran (2.25 mL) and water (0.75 mL), stirred for 5 minutes under ice-cooling, then lithium hydroxide monohydrate (5.5 mg, 0.13 mmol) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, it was adjusted to about pH 6 using 0.1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and the above pyrene (14) (17.2 mg, 14.8 μmol) was obtained by freeze-drying.

[0312] 1 H NMR (400 MHz, DMSO-d6) δ 13.06 (brs, 1H), 10.13 - 10.10 (m, 1H), 8.69 - 8.66 (m, 1H), 8.40 - 7.85 (m, 13H), 7.77 - 7.73 (m, 1H), 7.67 - 7.64 (m, 2H), 7.44 - 7.42 (m, 2H), 5.99 (brs, 1H), 5.68 - 5.67 (m, 1H), 5.44 (brs, 2H), 5.05 - 4.96 (m, 2H), 4.46 - 4.10 (m, 4H), 3.92 - 3.81 (m, 1H), 3.05 - 2.90 (m, 5H), 2.25 - 2.14 (m, 4H), 2.03 - 1.29 (m, 30H), 0.88 - 0.80 (m, 6H).

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

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

Chemical Structure

[0315] Pyrene (14) (14.7 mg, 12.6 μmol) was dissolved in N,N-dimethylformamide (1.0 mL), cooled in ice, and N,N-diisopropylethylamine (4.29 μL, 25.2 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (9.8 mg, 19 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.1 mg, 19 μmol) was added, the mixture was returned to room temperature, and stirred for 3.5 h. After completion of the reaction, it 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 (15) (7.0 mg, 9.2 μmol).

[0316] 1 H NMR (400 MHz, DMSO-d6) δ 10.08 - 10.05 (m, 1H), 8.92 - 7.95 (m, 15H), 7.75 - 7.73 (m, 1H), 7.63 - 7.59 (m, 2H), 7.40 - 7.34 (m, 2H), 6.97 - 6.94 (m, 2H), 6.00 - 5.98 (m, 1H), 5.70 - 5.69 (m, 1H), 5.43 (brs, 2H), 5.14 - 5.01 (m, 2H), 4.45 - 4.38 (m, 1H), 4.32 - 3.83 (m, 5H), 3.25 - 3.20 (m, 1H), 3.10 - 2.90 (m, 7H), 2.25 - 2.18 (m, 4H), 2.08 - 1.24 (m, 34H), 1.12 - 1.04 (m, 2H), 0.88 - 0.81 (m, 6H).

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

[0318] (1 - 3 - 5)Linker-payload mimic (11) synthesis

Chemical Structure

[0319] To pyrene (15) (6.1 mg, 4.6 μmol), 1,4-dioxane (920 μL) and 4 M hydrogen chloride / dioxane solution (230 μL, 918 μmol) were sequentially added, and the mixture was stirred at room temperature for 4 hours. After cooling in ice, N,N-diisopropylethylamine (172 μL, 1.10 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by 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 mimic (11) (3.7 mg, 3.0 μmol).

[0320] 1 H NMR (400 MHz, DMSO-d6) δ 12.04 (brs, 2H), 10.01 - 9.98 (m, 1H), 8.83 - 7.89 (m, 15H), 7.71 - 7.69 (m, 1H), 7.56 - 7.51 (m, 2H), 7.33 - 7.26 (m, 2H), 6.90 - 6.87 (m, 2H), 5.91 - 5.90 (m, 1H), 5.64 - 5.62 (m, 1H), 5.36 (brs, 2H), 5.08 - 4.92 (m, 2H), 4.35 - 4.32 (m, 1H), 4.25 - 3.76 (m, 5H), 3.18 - 3.14 (m, 1H), 2.99 - 2.82 (m, 7H), 2.21 - 2.15 (m, 4H), 1.93 - 1.17 (m, 16H), 1.05 - 1.01 (m, 2H), 0.82 - 0.74 (m, 6H).

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

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

Chemical formula

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

Chemical formula

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

[0325] 1 H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.13 (d, J = 7.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.99 (brs, 1H), 5.43 (brs, 2H), 5.08 (s, 1H), 4.41 - 4.30 (m, 2H), 4.21 - 4.17 (m, 1H), 3.60 (s, 3H), 3.04 - 2.95 (m, 2H), 2.44 - 2.15 (m, 6H), 2.03 - 1.95 (m, 1H), 1.92 - 1.83 (m, 1H), 1.74 - 1.58 (m, 3H), 1.46 - 1.34 (m, 20H), 0.86 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H).

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

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

Chemical formula

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

[0329] 1 H NMR (400 MHz, DMSO-d6) δ 10.15 - 10.13 (m, 1H), 8.70 - 8.67 (m, 1H), 8.40 - 8.03 (m, 11H), 7.90 - 7.86 (m, 1H), 7.75 - 7.72 (m, 1H), 7.68 - 7.64 (m, 2H), 7.44 - 7.39 (m, 2H), 5.99 (brs, 1H), 5.79 (d, J = 6.8 Hz, 1H), 5.44 (brs, 2H), 5.05 - 4.98 (m, 2H), 4.40 - 4.08 (m, 3H), 3.93 - 3.80 (m, 1H), 3.63 - 3.62 (m, 3H), 3.05 - 2.90 (m, 5H), 2.44 - 2.15 (m, 6H), 2.02 - 1.98 (m, 1H), 1.91 - 1.85 (m, 1H), 1.75 - 1.55 (m, 3H), 1.50 - 1.30 (m, 20H), 0.88 - 0.80 (m, 6H).

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

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

Chem.

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

[0333] 1 H NMR (400 MHz, DMSO-d6) δ 13.06 (brs, 1H), 10.14 - 10.11 (m, 1H), 8.68 - 8.66 (m, 1H), 8.40 - 8.01 (m, 11H), 7.89 - 7.85 (m, 1H), 7.75 - 7.72 (m, 1H), 7.67 - 7.64 (m, 2H), 7.44 - 7.41 (m, 2H), 5.99 (brs, 1H), 5.68 - 5.67 (m, 1H), 5.44 (brs, 2H), 5.05 - 4.93 (m, 2H), 4.44 - 4.10 (m, 3H), 3.92 - 3.81 (m, 1H), 3.05 - 2.94 (m, 5H), 2.43 - 2.15 (m, 6H), 2.02 - 1.97 (m, 1H), 1.88 - 1.85 (m, 1H), 1.77 - 1.66 (m, 3H), 1.61 - 1.30 (m, 20H), 0.88 - 0.80 (m, 6H).

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

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

Chem.

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

[0337] 1 H NMR (400 MHz, DMSO-d6) δ 10.01 - 9.98 (m, 1H), 8.85 - 7.88 (m, 14H), 7.68 - 7.65 (m, 1H), 7.56 - 7.51 (m, 2H), 7.33 - 7.27 (m, 2H), 6.90 - 6.87 (m, 2H), 5.92 - 5.90 (m, 1H), 5.63 - 5.62 (m, 1H), 5.36 (brs, 2H), 5.08 - 4.92 (m, 2H), 4.36 - 3.76 (m, 5H), 3.18 - 3.14 (m, 1H), 3.00 - 2.83 (m, 7H), 2.36 - 2.08 (m, 6H), 1.95 - 1.89 (m, 1H), 1.84 - 1.78 (m, 1H), 1.68 - 1.50 (m, 3H), 1.42 - 1.17 (m, 24H), 1.07 - 0.97 (m, 2H), 0.81 - 0.74 (m, 6H).

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

[0339] (1 - 4 - 5) Linker-payload mimic (16) synthesis

Chemical Structure

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

[0341] 1 H NMR (400 MHz, DMSO-d6) δ 12.02 (brs, 2H), 10.04 - 9.98 (m, 1H), 8.85 - 7.89 (m, 14H), 7.65 - 7.62 (m, 1H), 7.56 - 7.52 (m, 2H), 7.33 - 7.27 (m, 2H), 6.90 - 6.87 (m, 2H), 5.92 (brs, 1H), 5.63 - 5.62 (m, 1H), 5.37 (brs, 2H), 5.08 - 4.93 (m, 2H), 4.36 - 3.76 (m, 5H), 3.18 - 3.14 (m, 1H), 3.02 - 2.80 (m, 7H), 2.38 - 2.16 (m, 6H), 1.96 - 1.77 (m, 2H), 1.70 - 1.17 (m, 9H), 1.06 - 0.98 (m, 2H), 0.81 - 0.74 (m, 6H).

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

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

Chemical Structure

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

Chemical Structure

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

[0346] 1 H NMR (400 MHz, DMSO-d6) δ 9.98 - 9.89 (m, 1H), 8.32 - 8.18 (m, 1H), 8.07 - 7.85 (m, 1H), 7.73 - 7.59 (m, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.08 (s, 1H), 4.45 - 4.34 (m, 1H), 4.32 - 4.27 (m, 1H), 4.20 - 4.09 (m, 1H), 3.59 (s, 3H), 2.24 - 2.20 (m, 2H), 2.01 - 1.96 (m, 1H), 1.90 - 1.80 (m, 4H), 1.72 - 1.67 (m, 1H), 1.39 - 1.36 (m, 9H), 1.30 (d, J = 7.2 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H).

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

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

Chemical formula

[0349] Alcohol (22) (17.5 mg, 30.2 μmol) was dissolved in N,N-dimethylformamide (174 μL) and stirred for 5 minutes under ice-cooling. After that, bis(4-nitrophenyl) carbonate (18.9 mg, 60.8 μmol) and N,N-diisopropylethylamine (7.80 μL, 45.4 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. When LCMS was checked, since the raw material remained, bis(4-nitrophenyl) carbonate (9.5 mg, 31 μmol) and N,N-diisopropylethylamine (3.90 μL, 22.7 μmol) were added, and the mixture was stirred at room temperature for 1 hour. Then, it was ice-cooled, and Sarcosin-Pyrene (36.2 mg, 120 μmol), 1-hydroxybenzotriazole (6.3 mg, 47 μmol), and N,N-diisopropylethylamine (40.3 μL, 235 μmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, it was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and the above pyrene (23) (12.9 mg, 14.2 μmol) was obtained by lyophilization.

[0350] 1 H NMR (400 MHz, DMSO-d6) δ 10.05 - 10.02 (m, 1H), 8.61 - 8.60 (m, 1H), 8.30 - 7.78 (m, 12H), 7.67 - 7.64 (m, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.37 - 7.33 (m, 2H), 5.72 - 5.70 (m, 1H), 4.98 - 4.89 (m, 2H), 4.34 - 4.00 (m, 3H), 3.85 - 3.75 (m, 1H), 3.56 - 3.55 (m, 3H), 2.93 - 2.87 (m, 3H), 2.17 - 2.13 (m, 2H), 1.94 - 1.91 (m, 1H), 1.85 - 1.75 (m, 4H), 1.65 - 1.61 (m, 1H), 1.31 - 1.23 (m, 12H), 0.80 - 0.73 (m, 6H).

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

[0352] (1-5-3) Synthesis of Pyrene (24) [Chemical Structure]

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

[0354] 1 H NMR (400 MHz, DMSO-d6) δ 13.05 (brs, 1H), 10.04 - 9.91 (m, 1H), 8.63 - 8.60 (m, 1H), 8.33 - 7.77 (m, 12H), 7.69 - 7.55 (m, 3H), 7.37 - 7.35 (m, 2H), 5.61 - 5.60 (m, 1H), 4.98 - 4.86 (m, 2H), 4.26 - 4.03 (m, 3H), 3.84 - 3.74 (m, 1H), 2.93 - 2.87 (m, 3H), 2.18 - 2.13 (m, 2H), 1.95 - 1.92 (m, 1H), 1.85 - 1.77 (m, 4H), 1.67 - 1.62 (m, 1H), 1.31 - 1.24 (m, 12H), 0.81 - 0.75 (m, 6H).

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

[0356] (1-5-4) Synthesis of Pyrene (25) [Chemical Structure]

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

[0358] 1 H NMR (400 MHz, DMSO-d6) δ 9.98 - 9.95 (m, 1H), 8.83 - 7.77 (m, 14H), 7.67 - 7.65 (m, 1H), 7.56 - 7.50 (m, 2H), 7.34 - 7.27 (m, 2H), 6.89 - 6.86 (m, 2H), 5.63 - 5.61 (m, 1H), 5.06 - 4.93 (m, 2H), 4.37 - 3.76 (m, 5H), 3.17 - 3.14 (m, 1H), 2.95 - 2.83 (m, 5H), 2.17 - 2.10 (m, 2H), 1.95 - 1.89 (m, 1H), 1.85 - 1.75 (m, 4H), 1.65 - 1.62 (m, 1H), 1.31 - 1.17 (m, 16H), 1.02 - 1.00 (m, 2H), 0.81 - 0.74 (m, 6H).

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

[0360] (1 - 5 - 5) Linker - payload mimic (21) synthesis

Chemical Structure

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

[0362] 1 H NMR (400 MHz, DMSO-d6) δ 11.99 (brs, 1H), 9.98 - 9.96 (m, 1H), 8.84 - 7.88 (m, 14H), 7.66 - 7.63 (m, 1H), 7.54 - 7.50 (m, 2H), 7.34 - 7.27 (m, 2H), 6.90 - 6.87 (m, 2H), 5.63 - 5.61 (m, 1H), 5.07 - 4.93 (m, 2H), 4.36 - 3.76 (m, 5H), 3.17 - 3.14 (m, 1H), 2.95 - 2.83 (m, 5H), 2.20 - 2.16 (m, 2H), 1.95 - 1.90 (m, 1H), 1.88 - 1.76 (m, 4H), 1.67 - 1.62 (m, 1H), 1.32 - 1.17 (m, 7H), 1.02 - 1.01 (m, 2H), 0.81 - 0.74 (m, 6H).

[0363] MS (ESI) m / z: 999.35 [M - H] ―

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

Chemical Structure

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

Chemical formula

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

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

[0368] Example 2: Synthesis of ADC mimic (2-1) Synthesis of ADC mimic In the following comparative examples and examples, as the thiol group-introduced antibody, an antibody derivative (thiol group-introduced trastuzumab) described in Example 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used. 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 heavy chain of the antibody (the position of the lysine residue follows EU numbering).

Chemical formula

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

[0370] From the Linker-payload mimic (1) synthesized in Example 1-1 and the thiol-containing antibody, ADC mimic 1 having the following structure was synthesized. ESI-TOFMS analysis was performed, and a peak was confirmed at 150350 where two Linker-payload mimics (1) were introduced in the reaction product.

Chemical formula

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

Chemical formula

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

Chemical formula

[0373] Similarly, ADC mimic 4 with the following structure was synthesized from the Linker-payload mimic (16) and thiol-containing antibody of Examples 1-4. ESI-TOFMS analysis was performed, and a peak was confirmed at 150466 where two Linker-payload mimics (16) were introduced in the reaction product.

Chemical formula

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

Chemical formula

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

[0376]

Table 2

[0377] Example 3: Evaluation of the hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC) According to the previous report (Anal.Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed. The measurement was carried out using the following conditions. The hydrophobicity of the ADC can be evaluated by the retention time of the ADC in the HIC chromatogram.

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

[0379]

Table 3

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

[0381] Example 4: Evaluation of Aggregation Rates of ADCs and ADC Mimics by Size Exclusion Chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to the previously reported method (ChemistrySelect, 2020, 5, 8435-8439). The measurement was carried out using the following conditions.

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

[0383]

Table 4

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

[0385] (5-1) Cathepsin B cleavage test Performed as follows according to the previously reported (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 dispensed into six Eppendorf tubes at 30 μL each. Of the six samples, three were immediately added with 100 μL of acetonitrile each at 0 °C, stirred by vortexing, and then centrifuged to obtain a precipitate. The resulting supernatant was collected and subjected to HPLC analysis. The remaining three were incubated at 37 °C for 6 hours. 100 μL of acetonitrile was added to each sample, stirred by vortexing, and then centrifuged to obtain a precipitate. The resulting supernatant was collected and subjected to HPLC analysis.

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

[0387] Separately, using Pyrene, the correlation between the fluorescence intensity area by HPLC and the concentration was calculated. Using the calculation formula, the difference in fluorescence intensity of each of the above ADC mimics was converted into concentration. When the concentration at 0 hours was set to 100%, the ratio of the difference in fluorescence intensity described above was calculated as the dropout rate.

[0388]

Table 5

[0389] As shown in Table 4, it was found that the synthesized ADC mimics had sufficient cathepsin B cleavage.

[0390] Example 6: Evaluation of ADC mimics using mouse plasma (6-1) Plasma stability test of ADC mimics To 500 μL of mouse plasma (manufactured by Charles River), ADC mimic was added to a concentration of 0.1 mg / mL, followed by sterile filtration. This solution was dispensed into six Eppendorf tubes at 50 μL each. Three of the six samples were stored in an incubator set at 37 °C for 4 days. The remaining three were stored in a -80 °C freezer for 4 days in the same manner. To each sample, 100 μL of acetonitrile was added, stirred by vortexing, and then centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

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

[0392] The calculation of the dropout rate of the fluorescent molecule was performed according to Example 5-2. The results evaluated the dropout rate of the fluorescent molecule as shown in the following table.

[0393]

Table 6

[0394] As a result, compared with the ADC mimic synthesized in Comparative Example 1, the ADC mimics synthesized in Examples 1-1 and 1-2 showed more than 3-fold stability, and the ADC mimics synthesized in Examples 1-3 and 1-4 showed more than 10-fold stability.

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

Chemical formula

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

Chemical formula

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

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

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

Chem.

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

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

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

Chem.

[0403] The compound (37) (97 mg, 0.069 mmol) obtained in (7-1-2) was dissolved in tetrahydrofuran (7 mL) and water (2 mL), and lithium hydroxide (1.0 M, 1.4 mL, 1.4 mmol) was added under ice-cooling, followed by stirring for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 6, and then acetonitrile:water = 1:1 was added, and the product 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 38 (70 mg, 0.050 mmol).

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

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

Chemical Structure

[0406] The compound (38) (34 mg, 0.024 mmol) obtained in (7-1-3) was dissolved in N,N-dimethylformamide (3 mL), cooled under ice-cooling, and N,N-diisopropylethylamine (25 μL, 0.14 mmol) and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (20 mg, 0.038 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (8.7 mg, 0.040 mmol) was added, and the mixture was returned to room temperature and stirred for 20 hours. After completion of the reaction, the product 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 (39) (29 mg, 0.019 mmol).

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

[0408] Synthesis of Linker-payload (35)

Chemical Structure

[0409] (7-1-4) The compound (39) (29 mg, 0.019 mmol) obtained was successively added with acetonitrile (500 μL) and 85 wt% aqueous phosphoric acid solution (0.50 mL, 7.3 mmol), and stirred at room temperature for 3 hours. After completion of the reaction, water (2 mL) was added, and the reaction solution was purified by reversed-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 (35) (18 mg, 0.012 mmol).

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

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

Chemical formula

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

Chemical formula

[0413] The compound (38) (10 mg, 0.0072 mmol) obtained in (7-1-3) was dissolved in N,N-dimethylformamide (1 mL), cooled on ice, and then N,N-diisopropylethylamine (10 μL, 0.057 mmol) and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (7.0 mg, 0.013 mmol) were added. Next, a solution of DBCO-hexylamine (4.7 mg, 0.015 mmol) in N,N-dimethylformamide (0.5 mL) was added, the mixture was returned to room temperature, and stirred for 20 hours. After completion of the reaction, it 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 compound (41) (5.8 mg, 0.0034 mmol).

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

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

Chemical formula

[0416] Acetonitrile (500 μL) and 85 wt% aqueous phosphoric acid solution (0.50 mL, 7.3 mmol) were sequentially added to the compound (41) (13 mg, 0.0077 mmol) obtained in (7-2-1), and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water (2 mL) was added, and the reaction solution 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 Linker-payload (40) (7.4 mg, 0.0045 mmol).

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

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

Chemical formula

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

Chemical formula

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

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

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

Chemical formula

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

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

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

Chemical formula

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

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

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

Chemical formula

[0429] The compound (45) (77 mg, 0.049 mmol) obtained in (7-3-3) was dissolved in N,N-dimethylformamide (3 mL), cooled on ice, and N,N-diisopropylethylamine (50 μL, 0.29 mmol) and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (87 mg, 0.17 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (35 mg, 0.16 mmol) was added, the mixture was returned to room temperature, and stirred for 20 hours. After completion of the reaction, it 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 compound (46) (65 mg, 0.037 mmol).

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

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

Chemical formula

[0432] To the compound (46) (65 mg, 0.037 mmol) obtained in (7-3-4), acetonitrile (2 mL) and 85 wt% aqueous phosphoric acid solution (1.00 mL, 14.6 mmol) were sequentially added, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, water (2 mL) was added, the reaction solution 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 Linker-payload (42) (49 mg, 0.030 mmol).

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

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

[0435] (7 - 4 - 1) Synthesis of Compound (48) [Chemistry]

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

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

[0438] (7 - 4 - 2) Synthesis of Compound (49) [Chemistry]

[0439] The compound (48) (57 mg, 0.043 mmol) obtained in (7-4-1) was dissolved in tetrahydrofuran (3 mL) and water (1.5 mL), and lithium hydroxide (1.0 M, 0.5 mL, 0.5 mmol) was added under ice-cooling, followed by stirring for 1 hour. Hydrochloric acid was added to the reaction solution, and after adjusting the pH to 5, acetonitrile:water = 1:1 was added, and the mixture was purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and lyophilization was performed to obtain compound 49 (45 mg, 0.035 mmol).

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

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

Chemical formula

[0442] The compound (49) (45 mg, 0.035 mmol) obtained in (7-4-2) was dissolved in N,N-dimethylformamide (3 mL), then cooled under ice, and N,N-diisopropylethylamine (30 μL, 0.17 mmol) and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (55 mg, 0.11 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (22 mg, 0.10 mmol) was added, the mixture was returned to room temperature, and stirring was continued for 18 hours. After completion of the reaction, 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 lyophilized to obtain compound (50) (22 mg, 0.015 mmol).

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

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

Chemical formula

[0445] (7-4-3) The compound (50) (22 mg, 0.015 mmol) obtained was successively added with acetonitrile (1 mL) and 85 wt% aqueous phosphoric acid solution (1.0 mL, 14.6 mmol), and stirred at room temperature for 1.5 hours. After completion of the reaction, water (1 mL) was added, and the reaction solution 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 Linker-payload (47) (18.8 mg, 0.0139 mmol).

[0446] 1 H NMR (300 MHz; DMSO-d6) δ 10.04 (s, 1H), 8.10 - 8.05 (m, 4H), 7.80 - 7.77 (m, 2H), 7.59 - 7.55 (m, 2H), 7.35 - 7.30 (m, 3H), 6.99 (d, J = 8.8 Hz, 2H), 6.54 - 6.53 (m, 1H), 6.01 (brs, 1H), 5.74 (d, J = 9.0 Hz, 1H), 5.43 (brs, 4H), 5.33 - 5.28 (m, 2H), 4.29 - 4.15 (m, 4H), 3.19 - 2.98 (m5H), 2.43 - 2.38 (m, 5H), 2.27 - 2.20 (m, 7H), 1.95 - 1.83 (m, 8H), 1.73 - 1.62 (m, 4H), 1.42 - 1.30 (m, 7H), 1.23 - 1.13 (m, 3H), 0.84 (m, 9H).

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

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

Chemical Structure

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

[0450] Synthesis of (7-6) Linker-payload(126) The following Linker-payload(126) was synthesized in the same manner using Exatecan mesylate instead of MMAE in the synthesis of Linker-payload mimic(35).

Chemical Structure

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

[0452] Example 8: Synthesis of ADC Synthesis of (8-1) ADC4 In the following comparative examples and examples, as the thiol group-introduced antibody, an antibody derivative (thiol group-introduced trastuzumab) described in Example 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used. This antibody derivative has the following structure in which a thiol group is site-selectively 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 heavy chain of the antibody (the position of the lysine residue follows EU numbering).

Chemical Structure

[0453] To a solution of the thiol group-introduced antibody in buffer (pH 7.4 PBS buffer) (20 μM), 10 equivalents of a DMF solution (10 mM) of the Linker-payload (35) synthesized in Example 12-1 was added. After allowing to stand at room temperature for 2 hours, purification was performed using NAP-5 Columns (manufactured by GE Healthcare) to obtain ADC 4. ESI-TOFMS analysis was carried out, and a peak was confirmed at 151414 where two Linker-payloads (35) were introduced in the reaction product.

Chemical formula

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

Chemical formula

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

Chemical formula

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

Chemical formula

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

Chemical Formula

[0458] Example 9: HIC-HPLC Analysis of ADC Using the conditions of Example 3, HIC-HPLC analysis was performed.

Table 7

[0459] Subsequently, the hydrophobicity evaluation of ADC was performed using HIC-HPLC. The measurement was carried out according to Example 3. In the HIC chromatogram, the hydrophobicity degree of ADC can be evaluated by the retention time of ADC. The raw material antibody Trastuzumab was used as a comparison object.

Table 8

[0460] It can be seen that the exo-type ADCs ADC4, 5, and 6 have a retention time in the HIC chromatogram comparable to that of the raw material antibody, indicating that they are more hydrophilic ADCs.

[0461] Example 10: Evaluation of the Aggregation Rate of ADC by Size Exclusion Chromatography (SEC-HPLC) According to Example 4, SEC-HPLC analysis was performed.

Table 9

[0462] As a result, it was confirmed that the ADCs synthesized in (8-1), (8-2), and (8-3) tend to have a low aggregation rate, indicating higher stability. Therefore, it was confirmed that the ADC synthesized in Example 8 is a preferable ADC.

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

Chemical formula

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

Chemical formula

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

[0466] 11H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 5.98 (brs, 1H), 5.41 (brs, 2H), 5.08 (s, 1H), 4.64 - 4.59 (m, 1H), 4.39 - 4.34 (m, 1H), 4.22 - 4.18 (m, 1H), 3.59 (s, 3H), 3.01 - 2.94 (m, 2H), 2.69 - 2.63 (m, 1H), 2.44 - 2.38 (m, 1H), 2.00 - 1.95 (m, 1H), 1.83 (s, 3H), 1.69―1.66 (m, 1H), 1.62 - 1.53 (m, 1H), 1.43 - 1.34 (m, 11H), 0.84 (d, J = 6.8 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).

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

[0468] (11 - 1 - 2) Synthesis of Pyrene (58)

Chem.

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

[0470] 1 H NMR (400 MHz, DMSO-d6) δ 10.14 - 10.11 (m, 1H), 8.68 - 8.66 (m, 1H), 8.39 - 8.01 (m, 10H), 7.89 - 7.85 (m, 1H), 7.67 - 7.58 (m, 3H), 7.44 - 7.39 (m, 2H), 5.98 (brs, 1H), 5.79 - 5.77 (m, 1H), 5.42 (brs, 2H), 5.04 - 4.97 (m, 2H), 4.65 - 4.59 (m, 1H), 4.39 - 4.36 (m, 1H), 4.28 - 4.07 (m, 2H), 3.92 - 3.82 (m, 1H), 3.62 - 3.61 (m, 3H), 2.99 - 2.91 (m, 5H), 2.69 - 2.63 (m, 1H), 2.44 - 2.38 (m, 1H), 2.01 - 1.97 (m, 1H), 1.83 (s, 3H), 1.70 - 1.60 (m, 2H), 1.44 - 1.30 (m, 11H), 0.86 - 0.77 (m, 6H).

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

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

Chemical Structure

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

[0474] 1 H NMR (400 MHz, DMSO-d6) δ 13.06 (brs, 1H), 10.13 - 10.09 (m, 1H), 8.68 - 8.65 (m, 1H), 8.39 - 8.00 (m, 10H), 7.88 - 7.84 (m, 1H), 7.67 - 7.59 (m, 3H), 7.44 - 7.41 (m, 2H), 5.98 (brs, 1H), 5.68 - 5.67 (m, 1H), 5.42 (brs, 2H), 5.04 - 4.93 (m, 2H), 4.64 - 4.61 (m, 1H), 4.41 - 4.35 (m, 1H), 4.32 - 4.09 (m, 2H), 3.91 - 3.80 (m, 1H), 2.99 - 2.91 (m, 5H), 2.70 - 2.64 (m, 1H), 2.44 - 2.38 (m, 1H), 2.01 - 1.98 (m, 1H), 1.83 (s, 3H), 1.70 - 1.57 (m, 2H), 1.45 - 1.30 (m, 11H), 0.86 - 0.77 (m, 6H).

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

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

Chemical formula

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

[0478] 1 H NMR (400 MHz, DMSO-d6) δ 10.07 - 10.04 (m, 1H), 8.91 - 8.88 (m, 1H), 8.65 - 7.95 (m, 12H), 7.62 - 7.58 (m, 3H), 7.40 - 7.36 (m, 2H), 6.96 - 6.94 (m, 2H), 5.97 (brs, 1H), 5.70 - 5.68 (m, 1H), 5.41 (brs, 2H), 5.14 - 5.00 (m, 2H), 4.65 - 4.61 (m, 1H), 4.42 - 4.37 (m, 1H), 4.25 - 4.19 (m, 1H), 4.15 - 3.82 (m, 2H), 3.30 - 3.21 (m, 2H), 3.04 - 2.89 (m, 7H), 2.70 - 2.64 (m, 1H), 2.44 - 2.38 (m, 1H), 2.01 - 1.97 (m, 1H), 1.83 (s, 3H), 1.75 - 1.60 (m, 2H), 1.48 - 1.24 (m, 15H), 1.12 - 1.03 (m, 2H), 0.86 - 0.78 (m, 6H).

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

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

Chemical Structure

[0481] Acetonitrile (208 μL) was added to pyrene (60) (4.7 mg, 4.2 μmol), and 85% phosphoric acid solution (72.4 μL, 1.25 mmol) was added under ice-cooling. The mixture was stirred at room temperature for 3 hours. After completion of the reaction, it 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 Linker-payload mimic (56) (3.1 mg, 2.9 μmol).

[0482] 1 H NMR (400 MHz, DMSO-d6) δ 12.38 (brs, 1H), 10.05 - 10.01 (m, 1H), 8.91 - 8.88 (m, 1H), 8.65 - 7.94 (m, 12H), 7.63 - 7.58 (m, 3H), 7.40 - 7.34 (m, 2H), 6.96 - 6.93 (m, 2H), 6.00 (brs, 1H), 5.70 - 5.68 (m, 1H), 5.44 (brs, 2H), 5.15 - 4.99 (m, 2H), 4.64 - 4.60 (m, 1H), 4.43 - 4.37 (m, 1H), 4.25 - 4.22 (m, 1H), 4.15 - 3.82 (m, 2H), 3.30 - 3.21 (m, 2H), 3.04 - 2.89 (m, 7H), 2.73 - 2.69 (m, 1H), 2.46 - 2.44 (m, 1H), 2.04 - 1.96 (m, 1H), 1.84 - 1.83 (m, 3H), 1.77 - 1.59 (m, 2H), 1.44 - 1.04 (m, 8H), 0.86 - 0.77 (m, 6H).

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

[0484] (11 - 2) Synthesis of Linker-payload mimic (66) Linker-payload mimic (1) was synthesized as follows.

Chemical Structure

[0485] (11-2-1) Synthesis of Alcohol (67) [Chemical formula]

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

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

[0488] (11-2-2) Synthesis of Pyrene (68) [Chemical formula]

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

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

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

Chemical formula

[0492] Pyrene (68) (20.3 mg, 13.1 μmol) was dissolved in tetrahydrofuran (983 μL) and water (327 μL), and stirred for 5 minutes under ice-cooling. Then, 1 M aqueous lithium hydroxide solution (31.4 μL, 31.4 μmol) was added, and the mixture was stirred at room temperature for 2.5 hours. After completion of the reaction, it was adjusted to about pH 6 using 1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and the pyrene (69) (16.7 mg, 10.9 μmol) was obtained by lyophilization.

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

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

Chem.

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

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

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

Chem.

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

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

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

Chemical formula

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

Chemical formula

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

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

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

Chemical formula

[0505] Alcohol (96) (60.5 mg, 72.4 μmol) was dissolved in N,N-dimethylformamide (3.6 mL), then cooled on ice, and N,N-diisopropylethylamine (25 μL, 145 μmol) and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (56.5 mg, 109 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (23.7 mg, 109 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After completion of the reaction, it 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 alcohol (97) (68.0 mg, 72.4 μmol).

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

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

Chemical formula

[0508] Alcohol (97) (10.0 mg, 10.0 μmol) was dissolved in N,N-dimethylformamide (0.1 mL), then cooled on ice, and bis(4-nitrophenyl) carbonate (30.4 mg, 100 μmol) and N,N-diisopropylethylamine (3.8 μL, 22.5 μmol) were added, and the mixture was stirred at room temperature for 3 hours. After the reaction, it was purified by normal-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 compound (98) (5.6 mg, 4.8 μmol).

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

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

Chemical formula

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

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

[0513] (11-3-5) Synthesis of Linker-payload mimic (15) Linker-payload mimic (15) was synthesized in the same manner as in Example (1-3-5).

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

Chemical formula

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

Chemical formula

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

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

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

Chemical formula

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

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

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

Chemical formula

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

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

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

Chemical formula

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

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

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

[0528] (11-1)'s Linker-payload mimic (56) and a thiol-containing antibody were used to synthesize ADC mimic 22 with the following structure. ESI-TOFMS analysis was performed, and a peak was confirmed at 150322 where two Linker-payload mimics (56) were introduced into the reaction product.

Chemical Structure

[0529] Similarly, (11-2)'s Linker-payload mimic (66) and a thiol-containing antibody were used to synthesize ADC mimic 24 with the following structure. ESI-TOFMS analysis was performed, and a peak was confirmed at 151125 where two Linker-payload mimics (66) were introduced into the reaction product.

Chemical Structure

[0530] Similarly, (11-4)'s Linker-payload mimic (120) and a thiol-containing antibody were used to synthesize ADC mimic 33 with the following structure. ESI-TOFMS analysis was performed, and a peak was confirmed at 151279 where two Linker-payload mimics (120) were introduced into the reaction product.

Chemical Structure

[0531] DAR Analysis of (12-2) ADC Mimic ESI-TOFMS analysis of the ADC mimic synthesized in Example 12-1 was performed according to the previously reported (WO2019 / 240287A1), and it was confirmed that the DAR was 2.

Table 10

[0532] Example 13: Evaluation of the hydrophobicity of ADCs and ADC mimics by hydrophobic interaction column chromatography (HIC-HPLC) According to the previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed. The measurement was carried out using the following conditions. The hydrophobicity of ADCs can be evaluated by the retention time of ADCs in the HIC chromatogram.

[0533] Measurement system: Chromaster (registered trademark) (manufactured by Hitachi, Ltd.) Column: Tosoh Biobuthyl NPR 2.5 μm 4.6×35 mm column manufactured by Tosoh Bioscience, LLC Gradient: Linear gradient of eluent A / B Flow rate: 0.8 mL / min Eluent A: 1.1 M (NH4)2SO4, 25 mM Na2HPO4 / NaH2PO4 (pH 6.0) Eluent B: 25 mM Na2HPO4 / NaH2PO4 (pH 6.0, added with 25 v / v% isopropanol) Detector: UV (280 nm) [Table 11]

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

[0535] Example 14: Evaluation of the aggregation rate of ADCs and ADC mimics by size exclusion chromatography (SEC-HPLC) According to the previous report (ChemistrySelect, 2020, 5, 8435-8439), SEC-HPLC analysis was performed. The measurement was carried out using the following conditions.

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

Table 12

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

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

[0539] (15-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis Analysis and analysis were performed in the same manner as in Example 5.

Table 13

[0540] As shown in Table 12, it was found that the synthesized ADC mimic had sufficient cathepsin B cleavage.

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

[0542] (16-2) Analysis of the amount of shed fluorescent molecules using HPLC Analysis and analysis were performed in the same manner as in Example 6. [Table 14]

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

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

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

[0546] Example 18: Synthesis of ADC (18-1) Synthesis of linker intermediate (18-1-1) Synthesis of linker intermediate (115) [Chemical formula]

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

[0548] 1 H NMR (400 MHz, Chloroform-d) δ 6.29 (d, J = 7.7 Hz, 1H), 4.56 (td, J = 8.0, 4.9 Hz, 1H), 3.32 (t, J = 6.6 Hz, 2H), 2.53 - 2.38 (m, 3H), 2.36 - 2.16 (m, 3H), 2.12 (s, 2H), 1.96 (dq, J = 14.7, 7.6 Hz, 1H), 1.84 - 1.59 (m, 4H), 1.50 (s, 9H).

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

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

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

[0552] 1 H NMR (400 MHz, Chloroform-d) δ 7.43 (s, 5H), 6.10 (d, J = 7.8 Hz, 1H), 4.55 (td, J = 7.7, 4.9 Hz, 1H), 3.31 (t, J = 6.7 Hz, 2H), 2.87 - 2.63 (m, 2H), 2.28 (dd, J = 8.7, 5.9 Hz, 2H), 2.16 - 1.98 (m, 1H), 1.83 - 1.58 (m, 4H), 1.50 (s, 9H), 1.37 - 1.22 (m, 2H), 0.91 (t, J = 6.7 Hz, 1H).

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

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

Chemical formula

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

[0556] 11H NMR (400 MHz, Chloroform-d) δ 7.44 (s, J = 6.3, 4.6, 2.4 Hz, 5H), 6.76 (s, 1H), 4.62 (td, J = 7.5, 4.9 Hz, 1H), 3.31 (t, J = 6.6 Hz, 2H), 2.88 (qt, J = 16.8, 6.8 Hz, 2H), 2.33 (dt, J = 12.4, 6.8 Hz, 3H), 2.18 (dq, J = 14.4, 7.4 Hz, 1H), 1.74 (dq, J = 11.8, 7.5, 6.9 Hz, 2H), 1.63 (ddd, J = 17.7, 10.5, 4.8 Hz, 2H).

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

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

Chemical formula

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

[0560] 11H NMR (400 MHz, Chloroform-d) δ 7.50 - 7.38 (m, 5H), 6.33 (d, J = 8.4 Hz, 1H), 4.78 (tdd, J = 7.8, 4.6, 3.0 Hz, 1H), 3.70 - 3.54 (m, 2H), 3.32 (dt, J = 9.1, 6.7 Hz, 2H), 2.96 - 2.67 (m, 2H), 2.30 (pd, J = 7.1, 4.5 Hz, 2H), 1.85 - 1.60 (m, 6H), 1.49 (d, J = 2.8 Hz, 9H).

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

[0562] (18 - 1 - 5) Synthesis of Linker Intermediate (120)

Chemical Structure

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

[0564] 1 1H NMR (400 MHz, Methanol-d4) δ 7.44 (dq, J = 2.3, 1.5 Hz, 5H), 4.69 - 4.57 (m, 1H), 3.79 - 3.67 (m, 2H), 3.40 - 3.30 (m, 2H), 2.89 - 2.71 (m, 2H), 2.44 - 2.23 (m, 4H), 2.08 - 1.95 (m, 1H), 1.82 - 1.61 (m, 4H).

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

[0566] (18-2) Preparation of an affinity reagent (18) having an azide group

Chem.

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

[0568] (18-3) Introduction of two molecules of the peptide reagent into Trastuzumab

Chem.

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

[0570] (18-4) Synthesis of Trastuzumab (T-1) with an Azide Group Introduced

Chemical Structure

[0571] Using the method reported previously (WO2019 / 240287A1) as a reference, a methoxyamine solution was added to the antibody into which the modifying reagent (121) obtained in Example 18-3 was introduced, and the mixture was shaken at room temperature for 3 hours to carry out a cleavage reaction. As a result, an antibody with an azide group introduced was obtained. Analysis was performed by HIC-HPLC analysis according to the previously reported method (Anal.Chem., 2019, 91, 20, 12724-12732) to confirm that the azide group was introduced.

[0572] (18-5) Synthesis of ADC7 Linker-payload (40) was added to the above azide-introduced antibody to obtain ADC (7). ESI-TOFMS analysis was performed, and a peak was confirmed at 151276 into which 2 Linker-payloads (40) were introduced in the reaction product. Also, ESI-TOFMS analysis was performed according to the previously reported method (WO2019 / 240287A1) to confirm that the DAR was 2.

Chemical Structure

[0573] Example 19: Evaluation of ADC Using Mouse Plasma (19-1) Plasma Stability Test of ADC To 500 μL of mouse plasma (manufactured by Charles River), an ADC mimic was added to a concentration of 0.1 mg / mL, followed by sterile filtration. This solution was dispensed into six Eppendorf tubes at 50 μL each. Three of the six samples were stored in an incubator set at 37 °C for 4 days. The remaining three were stored in a -80 °C freezer for 4 days in the same manner. To each sample, 100 μL of acetonitrile was added, stirred by vortexing, and then centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0574] (19-2) Analysis of the amount of payload detached using HPLC The measurement was performed using liquid chromatography-mass spectrometry (including tandem mass spectrometry) to measure the amount of payload detached from the ADC. The samples stored in a -80 °C freezer for 4 days in Example 19-1 were used as the day 0 samples, and the three samples incubated at 37 °C for 4 days in Example 19-1 were used as the day 4 samples. The MS intensities of the payloads detected from the day 4 samples and the day 0 samples were calculated by extracted ion chromatogram, respectively, and the difference between them was analyzed.

[0575] Separately, using MMAE, the correlation between the area of the TIC by HPLC and the concentration was calculated. Using the calculated formula, the TIC of the fluorescence intensity of each ADC was converted to a concentration. The ratio of the difference in the aforementioned ion chromatogram when the concentration on Day 0 was set to 100% was calculated as the detachment rate.

Table 15

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

Claims

1. The following formula (I'): 【Chemistry 1】 [During the ceremony, Ig represents an antibody and is linked regioselectively to L1 adjacent to Ig via an amino group in the side chain of a lysine residue present at positions 246 / 248, 288 / 290, or 317 according to Eu numbering in the two heavy chains; R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, Ring A is a phenylene group which may have a substituent, R １ , and R ２ each independently represents a hydrogen atom or an alkyl group; L １ has a carbonyl group, and the site-selective bond with Ig is via the amino group in the side chain of the lysine residue, and L １ represents a divalent group achieved by an amide bond through the bonding of a carbonyl group in L ２ represents a divalent group, D represents a functional substance; the average ratio r of said bonds per two heavy chains is between 1.5 and 2.5; (-L ＨＧ -) is represented by the following formula (a1) or (a2): (a1) -CHR ＨＧ - <h2 style=";text-align:left;direction:ltr">(a22) -CHR<h2 style=";text-align:left;direction:ltr"> ＨＧ <h2 style=";text-align:left;direction:ltr"> -(C=O)-NH-CHR<h2 style=";text-align:left;direction:ltr"> ＨＧ <h2 style=";text-align:left;direction:ltr"> - [During the ceremony, R ＨＧ is -CH ２ -CH ２ R is a monovalent group represented by the formula: ＨＧ１ , and R ＨＧ２ are each independently a hydrogen atom, an acyl group, or a benzyloxycarbonyl group;

2. The regioselective conjugate or salt thereof according to claim 1 , wherein the antibody is an Fc region protein or an Fc region fusion protein.

3. The following formula (III'): 【Chemistry 2】 [During the ceremony, R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, Ring A is a phenylene group which may have a substituent, R １ , and R ２ each independently represents a hydrogen atom or an alkyl group; L １ , and L ２ each independently represents a divalent group, B １ represents a bioorthogonal functional group, D represents a functional substance; (-L ＨＧ -) is represented by the following formula (a1) or (a2): (a1) -CHR ＨＧ - <h2 style=";text-align:left;direction:ltr">(a22) -CHR<h2 style=";text-align:left;direction:ltr"> ＨＧ <h2 style=";text-align:left;direction:ltr"> -(C=O)-NH-CHR<h2 style=";text-align:left;direction:ltr"> ＨＧ <h2 style=";text-align:left;direction:ltr"> - [During the ceremony, R ＨＧ is -CH ２ -CH ２ -COOH; R ＨＧ１ , and R ＨＧ２ are each independently a hydrogen atom, an acyl group, or a benzyloxycarbonyl group, or a salt thereof.

4. The following formula (VII'): 【Chemistry 3】 [During the ceremony, R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline or alanine residue, Ring A represents a divalent aromatic ring group which may have a substituent, R １ represents a hydrogen atom or a monovalent group, L １ represents a divalent group, B １ represents a bioorthogonal functional group, (-L ＨＧ -) is represented by the following formula (a1) or (a2): (a1) -CHR ＨＧ - <h2 style=";text-align:left;direction:ltr">(a22) -CHR<h2 style=";text-align:left;direction:ltr"> ＨＧ <h2 style=";text-align:left;direction:ltr"> -(C=O)-NH-CHR<h2 style=";text-align:left;direction:ltr"> ＨＧ <h2 style=";text-align:left;direction:ltr"> - [During the ceremony, R ＨＧ is -CH ２ -CH ２ -COOH; R ＨＧ１ , and R ＨＧ２ are each independently a hydrogen atom, an acyl group, or a benzyloxycarbonyl group, or a salt thereof.

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