Compound or salt thereof, and antibody obtained using the same

Abstract

To control the bonding ratio between an antibody and a modifying group within a desired range.SOLUTION: There is provided an azido group-containing compound represented by the following formula (I), or a salt thereof, as well as an antibody or a salt thereof that can be prepared using such a compound or salt.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a compound or a salt thereof, and an antibody obtained therefrom, etc. [Background technology]

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

[0003] Heterogeneity has been an issue for ADCs, including T-DM1, since their development. In other words, small molecular weight drugs are randomly reacted with the 70-80 lysine residues in the antibody, so the drug antibody ratio (DAR) and conjugation position are not constant. Normally, when this random conjugation method is used, the DAR is in the range of 0-8, and it is known that multiple antibody drugs with different numbers of drugs bound are produced. In recent years, it has been reported that changing the number and binding position of drugs bound to ADCs changes the pharmacokinetics, drug release rate, and efficacy. For these reasons, it is required to control the number and position of drugs to be conjugated in next-generation ADCs. It is believed that if the number and position are constant, the expected efficacy, variation in conjugated drugs, lot differences, so-called regulation problems will be solved.

[0004] Site-selective modification of antibodies is being researched around the world, but most of the research is based on genetic engineering techniques or enzymes. Although genetic engineering modification techniques can control site selectivity and number selectivity, problems have been pointed out, such as a decrease in the expression efficiency of the antibody itself (a decrease in the total yield when preparing ADC). Another problem is that it takes a long time to establish an antibody expression system.

[0005] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed, which enables site-selective modification of antibodies by chemical synthesis (Patent Document 1). This method has been successful in site-selective modification of antibodies by reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide. However, in the ADCs produced by this method, the antibody and the drug are linked via a linker containing a peptide moiety. The peptide moiety has potential immunogenicity and is easily hydrolyzed in blood. Therefore, there is room for improvement in the ADCs produced by this method in that the linker contains a peptide moiety.

[0006] As an improvement method of the above-mentioned C-CAP method, a technology has been reported that can prepare an antibody having a functional substance (e.g., a drug) regioselectively without a peptide moiety as a linker by a chemical synthesis method using a specific compound containing an affinity peptide (Patent Documents 2 to 6). Avoiding the use of a linker containing a peptide moiety is desirable in clinical applications. In these technologies, a number of positions corresponding to various amino acid residues (e.g., lysine residues, tyrosine residues, serine residues, and threonine residues) in the CH2 and CH3 domains have been proposed as positions of amino acid residues in an antibody that can be regioselectively modified with a drug. However, it is not necessarily easy to regioselectively modify an antibody with a functional substance and control the binding ratio between the antibody and the functional substance within a desired range. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 186206 [Patent Document 2] International Publication No. 2018 / 199337 [Patent Document 3] International Publication No. 2019 / 240287 [Patent Document 4] International Publication No. 2019 / 240288 [Patent Document 5] International Publication No. 2020 / 009165 [Patent Document 6] International Publication No. 2020 / 090979 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to control the binding ratio between the antibody and the modifying group within a desired range. [Means for solving the problem]

[0009] As a result of extensive investigations, the present inventors have found that by selecting a lysine residue in a heavy chain in an immunoglobulin unit as the modification position of an antibody and using a compound represented by formula (I) (azide group-containing compound) as a compound that enables site-specific modification of the lysine residue, it becomes easy to highly control the average ratio of bonds between immunoglobulin units and azide group-containing modifying groups (azide group-containing modifying group / immunoglobulin unit) within the desired range (1.0 to 3.0).

[0010] The present inventors have also found that the azide group-containing compound is highly versatile in site-selective modification of immunoglobulin units, since the azide group-containing compound facilitates site-selective modification of different lysine residues in the heavy chain of the immunoglobulin unit. For example, by using an azide group-containing compound having an affinity peptide with a certain amino acid sequence, the average ratio of the bonds between the immunoglobulin units and the azide group-containing modifying group can be highly controlled within the desired range while site-selectively modifying the lysine residues at positions 246 / 248 of the human IgG heavy chain. In addition, by using an azide group-containing compound having an affinity peptide with a different amino acid sequence, the average ratio of the bonds between the immunoglobulin units and the azide group-containing modifying group can be highly controlled within the desired range while site-selectively modifying the lysine residues at positions 288 / 290 of the human IgG heavy chain.

[0011] The present inventors further discovered that an antibody which can be prepared by using a compound represented by formula (I) or a salt thereof, in which lysine residues in the heavy chain in an immunoglobulin unit are modified with an azide group-containing modifying group and the average ratio of bonds between immunoglobulin units and azide group-containing modifying groups (number of azide group-containing modifying groups / immunoglobulin unit) is highly controlled within a desired range (1.0 to 3.0), exhibits excellent stability, and thus completed the present invention.

[0012] That is, the present invention provides the following compounds or salts thereof, or reagents for antibody derivatization containing them: [1] A compound represented by formula (I) or a salt thereof. [2] The compound of [1] or a salt thereof, wherein the leaving group is selected from the following: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom); or (c) R-A-(R-B-)N (wherein R and R each independently represent a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom); or (d) Halogen atoms. [3] The compound or salt thereof according to [1] or [2], wherein the immunoglobulin unit is a human immunoglobulin unit. [4] The compound or salt thereof according to any one of [1] to [3], wherein the immunoglobulin unit is human IgG. [5] A compound according to any one of [1] to [4], or a salt thereof, wherein the main chain portion having 3 to 5 carbon atoms in M ​​is a straight-chain alkylene, or a portion containing ring carbon atoms, or a combination thereof. [6] Any of the compounds according to [1] to [5], or salts thereof, wherein the main chain connecting M and Y has 6 to 20 atoms. [7] Any of the compounds according to [1] to [6] or a salt thereof, wherein the carbonyl group adjacent to La forms an amide bond with the amino group in the side chain of a lysine residue in the affinity peptide. [8] The compound according to any one of [1] to [7] or a salt thereof, wherein the affinity peptide comprises the following amino acid sequence (A): (A)(X0-3)aC-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X0-3)b (SEQ ID NO: 1) Where: (X0-3)a is none or 1 to 3 consecutive identical or different amino acid residues (other than lysine and cysteine ​​residues); (X0-3)b is none or 1 to 3 consecutive identical or different amino acid residues (other than lysine and cysteine ​​residues); Xaa1 is an alanine, glycine, leucine, proline, arginine, valine, asparagine, glutamic acid, or phenylalanine residue; Xaa2 is a tyrosine, tryptophan, histidine, or phenylalanine residue; Xaa3 is a histidine, phenylalanine, tyrosine, tryptophan, arginine, or glycine residue; Xaa4 is a lysine residue, Xaa5 is a glycine residue, a serine residue, an asparagine residue, a glutamine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a histidine residue, a threonine residue, a leucine residue, an alanine residue, a valine residue, an isoleucine residue, or an arginine residue; Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, an aspartic acid residue, a proline residue, a glycine residue, an arginine residue, a phenylalanine residue, or a histidine residue. [9] The compound or a salt thereof according to [8], wherein the affinity peptide comprising the amino acid sequence of (A) above comprises the amino acid sequence of (1) below: (1) RGNCAYHKGQIIWCTYH (sequence number 2).

[10] The compound according to any one of [1] to [7], or a salt thereof, wherein the affinity peptide comprises the following amino acid sequence (B): (B) PNLNEEQRNARIRSI (SEQ ID NO:3).

[11] The compound or a salt thereof according to

[10] , wherein the affinity peptide comprising the amino acid sequence of (B) above comprises an amino acid sequence selected from the group consisting of the following (1) to (3): (1) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 4); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5; or (3) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (sequence number 6).

[12] The N-terminal and C-terminal amino acid residues in the affinity peptide may be protected, and The compound or salt thereof according to any one of [8] to

[11] , wherein the two thiol groups in the side chains of the two cysteine ​​residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.

[13] A reagent for derivatizing an antibody, comprising a compound represented by formula (I) or a salt thereof.

[0013] The present invention also provides an antibody intermediate or a salt thereof. [1] An antibody intermediate or a salt thereof, which comprises a structural unit represented by formula (II): [2] The antibody intermediate or a salt thereof according to [1], wherein the antibody is a human antibody. [3] The antibody intermediate or a salt thereof according to [1] or [2], wherein the antibody is a human IgG. [4] The antibody intermediate or a salt thereof according to any one of [1] to [3], wherein the lysine residue is located at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering. [5] The antibody intermediate or a salt thereof according to any one of [1] to [4], wherein the lysine residue has regioselectivity for the lysine residue at position 246 / 248 of a human IgG heavy chain according to EU numbering. [6] The antibody intermediate or a salt thereof according to any one of [1] to [4], wherein the lysine residue has regioselectivity for the lysine residue at position 288 / 290 of a human IgG heavy chain according to EU numbering. [7] The antibody intermediate or a salt thereof according to any one of [1] to [6], wherein the average ratio r is 1.5 to 2.5. [8] The antibody intermediate or salt thereof according to any one of [1] to [7], wherein the main chain portion having 3 to 5 carbon atoms in M ​​is a moiety containing a straight-chain alkylene, a ring-constituting carbon atom, or a combination thereof. [9] The antibody intermediate or a salt thereof according to any one of [4] to [8], wherein the lysine residues are located at two positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering.

[10] The antibody intermediate or a salt thereof according to [9], wherein the two positions are positions 246 / 248 and 288 / 290.

[11] The antibody intermediate according to any one of [1] to

[10] , or a salt thereof, wherein the main chain connecting M and Y has 6 to 20 atoms.

[12] An antibody intermediate or a salt thereof according to any one of [1] to

[11] , wherein the carbonyl group adjacent to La forms an amide bond with the amino group in the side chain of a lysine residue in the affinity peptide.

[13] The antibody intermediate or a salt thereof according to any one of [1] to

[12] , wherein the affinity peptide comprises the following amino acid sequence (A): (A)(X0-3)aC-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X0-3)b (SEQ ID NO: 1) Where: (X0-3)a is none or 1 to 3 consecutive identical or different amino acid residues (other than lysine and cysteine ​​residues); (X0-3)b is none or 1 to 3 consecutive identical or different amino acid residues (other than lysine and cysteine ​​residues); Xaa1 is an alanine, glycine, leucine, proline, arginine, valine, asparagine, glutamic acid, or phenylalanine residue; Xaa2 is a tyrosine, tryptophan, histidine, or phenylalanine residue; Xaa3 is a histidine residue, a phenylalanine residue, a tyrosine residue, or a tryptophan residue. residue, an arginine residue, or a glycine residue; Xaa4 is a lysine residue, Xaa5 is a glycine residue, a serine residue, an asparagine residue, a glutamine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a histidine residue, a threonine residue, a leucine residue, an alanine residue, a valine residue, an isoleucine residue, or an arginine residue; Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, an aspartic acid residue, a proline residue, a glycine residue, an arginine residue, a phenylalanine residue, or a histidine residue.

[14] The antibody intermediate or a salt thereof according to

[13] , wherein the affinity peptide comprising the amino acid sequence of (A) above comprises the amino acid sequence of (1) below: (1) RGNCAYHKGQIIWCTYH (sequence number 2).

[15] The antibody intermediate or a salt thereof according to any one of [1] to

[12] , wherein the affinity peptide comprises the following amino acid sequence (B): (B) PNLNEEQRNARIRSI (SEQ ID NO:3).

[16] The antibody intermediate or a salt thereof according to

[15] , wherein the affinity peptide comprising the amino acid sequence of (B) above comprises an amino acid sequence selected from the group consisting of the following (1) to (3): (1) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 4); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5; or (3) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (sequence number 6).

[17] The N-terminal and C-terminal amino acid residues in the affinity peptide may be protected; and The antibody intermediate or a salt thereof according to any one of [1] to

[16] , wherein the two thiol groups in the side chains of the two cysteine ​​residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.

[0014] The present invention also provides an azide group-introduced antibody derivative or a salt thereof. [1] An azide group-introduced antibody derivative or a salt thereof, which comprises a structural unit represented by formula (III): [2] The azide group-introduced antibody derivative of [1], wherein the monovalent group represented by T is an optionally substituted hydroxyamino group, or a salt thereof. [3] The azido group-introduced antibody derivative or a salt thereof according to [1] or [2], wherein the antibody is a human antibody. [4] The azide group-introduced antibody derivative or a salt thereof according to any one of [1] to [3], wherein the antibody is human IgG. [5] The azide group-introduced antibody derivative or a salt thereof according to any one of [1] to [4], wherein the main chain portion having 3 to 5 carbon atoms in M ​​is a portion containing a straight-chain alkylene, a ring-constituting carbon atom, or a combination thereof. [6] The azido group-introduced antibody derivative according to any one of [1] to [5], or a salt thereof, wherein the main chain linking M and Y has 6 to 20 atoms. [7] The azide group-introduced antibody derivative or a salt thereof according to any one of [1] to [6], wherein the lysine residue is located at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering. [8] The azide group-introduced antibody derivative or a salt thereof according to any one of [1] to [7], wherein the lysine residue has regioselectivity for the lysine residue at position 246 / 248 of a human IgG heavy chain according to EU numbering. [9] An azide group according to any one of [1] to [7], wherein the lysine residue has a positional preference for the lysine residue at position 288 / 290 of the human IgG heavy chain according to EU numbering. An introduced antibody derivative or a salt thereof.

[10] The azido group-introduced antibody derivative or a salt thereof according to any one of [1] to [9], wherein the average ratio r is 1.5 to 2.5.

[11] The azide group-introduced antibody derivative or a salt thereof according to any one of [7] to

[10] , wherein the lysine residues are located at two positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering.

[12] The azido group-introduced antibody derivative or a salt thereof according to

[11] , wherein the two positions are positions 246 / 248 and 288 / 290.

[0015] The present invention also provides a conjugate of an antibody and a functional substance, or a salt thereof. [1] A conjugate of an antibody and a functional substance, which comprises a structural unit represented by formula (IV), or a salt thereof. [2] The conjugate or salt thereof according to [1], wherein the antibody is a human antibody. [3] The conjugate according to [1] or [2], or a salt thereof, wherein the antibody is human IgG. [4] The conjugate according to any one of [1] to [3], or a salt thereof, wherein the main chain portion having 3 to 5 carbon atoms in M ​​is a straight-chain alkylene, or a portion containing ring carbon atoms, or a combination thereof. [5] The conjugate according to any one of [1] to [4] or a salt thereof, wherein the main chain connecting M and Y has 6 to 20 atoms. [6] The conjugate according to any one of [1] to [5] or a salt thereof, wherein the lysine residue is located at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering. [7] The conjugate according to any one of [1] to [6] or a salt thereof, wherein the lysine residue has positional selectivity for the lysine residue at position 246 / 248 of a human IgG heavy chain according to EU numbering. [8] The conjugate according to any one of [1] to [7] or a salt thereof, wherein the lysine residue has regioselectivity for the lysine residue at position 288 / 290 of a human IgG heavy chain according to EU numbering. [9] The conjugate or a salt thereof according to any one of [1] to [8], wherein the average ratio r is 1.5 to 2.5.

[10] The conjugate according to any one of [6] to [9], or a salt thereof, wherein the lysine residues are present at two positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering.

[11] The conjugate or a salt thereof according to

[10] , wherein the two positions are positions 246 / 248 and 288 / 290.

[0016] The present invention also provides methods for producing antibody intermediates, antibody derivatives with azide groups introduced therein, or conjugates of antibodies and functional substances, or salts thereof. [1] A method for producing an antibody intermediate or a salt thereof, comprising reacting a compound represented by formula (I) or a salt thereof with an antibody containing the above-mentioned immunoglobulin unit to produce an antibody intermediate or a salt thereof containing a structural unit represented by formula (II). [2] (1) reacting a compound represented by formula (I) or a salt thereof with an antibody containing the above-mentioned immunoglobulin unit to produce an antibody intermediate containing a structural unit represented by formula (II) or a salt thereof; and (2) A method for producing an azide group-introduced antibody derivative or a salt thereof, comprising subjecting the above-mentioned antibody intermediate or a salt thereof to a thioester cleavage reaction to produce an azide group-introduced antibody derivative or a salt thereof, which comprises a structural unit represented by formula (III). [3] (1) reacting a compound represented by formula (I) or a salt thereof with an antibody containing the above-mentioned immunoglobulin unit to produce an antibody intermediate containing a structural unit represented by formula (II) or a salt thereof; and (2) subjecting the antibody intermediate or its salt to a thioester cleavage reaction to produce an azide group-introduced antibody derivative or its salt, which comprises a structural unit represented by formula (III); and (3) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting the above-mentioned azide group-introduced antibody derivative or a salt thereof with a target substance represented by formula (V) to produce a conjugate of an antibody and a functional substance or a salt thereof, which contains a structural unit represented by formula (IV). [4] A method for producing an azide group-introduced antibody derivative or a salt thereof, comprising subjecting an antibody intermediate or a salt thereof containing a structural unit represented by formula (II) to a thioester cleavage reaction to produce an azide group-introduced antibody derivative or a salt thereof containing a structural unit represented by formula (III). [5] (1) subjecting an antibody intermediate or a salt thereof containing a structural unit represented by formula (II) to a thioester cleavage reaction to produce an azide group-introduced antibody derivative or a salt thereof containing a structural unit represented by formula (III); and (2) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting the above-mentioned azide group-introduced antibody derivative or a salt thereof with a target substance represented by formula (V) to produce a conjugate of an antibody and a functional substance or a salt thereof, which contains a structural unit represented by formula (IV). [6] A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting an azide group-introduced antibody derivative or a salt thereof, which contains a structural unit represented by formula (III), with a target substance represented by formula (V) to produce a conjugate of an antibody and a functional substance or a salt thereof, which contains a structural unit represented by formula (IV). Effect of the Invention

[0017] The compound represented by formula (I) or a salt thereof can highly modify lysine residues in the heavy chain in the immunoglobulin unit so that the average ratio of bonds between the immunoglobulin unit and the azide group-containing modifying group (number of azide group-containing modifying groups / immunoglobulin unit) falls within a desired range (1.0 to 3.0). The compound represented by formula (I) or a salt thereof also has the advantage of being highly versatile in the site-selective modification of lysine residues at different positions in the heavy chain in the immunoglobulin unit. Therefore, the compound represented by formula (I) or a salt thereof is useful as a reagent for antibody derivatization. Furthermore, the compound represented by formula (I) or a salt thereof can provide an antibody intermediate represented by formula (II) or a salt thereof in which lysine residues in the heavy chain in the immunoglobulin unit are specifically modified with affinity peptide-containing groups and the average ratio of bonds between the immunoglobulin units and the azide group-containing modifying groups (number of azide group-containing modifying groups / immunoglobulin unit) is highly controlled within a desired range. Furthermore, an antibody produced using the antibody intermediate represented by formula (II) or a salt thereof as a raw material can inherit the desired properties (e.g., average binding ratio, regioselectivity) of the antibody intermediate or its salt. Thus, according to the present invention, it is possible to provide an azide group-introduced antibody derivative represented by formula (III) or a salt thereof, and a conjugate of an antibody and a functional substance represented by formula (IV) or a salt thereof, which have the desired properties as described above. Furthermore, the above-mentioned antibody of the present invention has excellent stability. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram (part 1) showing the concept of modification of an immunoglobulin unit by the compound of the present invention represented by formula (I) or a salt thereof. First, the compound of the present invention represented by formula (I) or a salt thereof associates with the CH2 domain in the immunoglobulin unit via the affinity peptide (Y). Next, the compound of the present invention represented by formula (I) or a salt thereof reacts with the side chain of a specific amino acid residue in the CH2 domain (the amino group in the side chain of a lysine residue in the figure) via an activated carbonyl group having a leaving group (X) to generate an antibody intermediate or a salt thereof. [Diagram 2] 2 is a schematic diagram (part 2) showing the concept of modification of an immunoglobulin unit with the compound of the present invention represented by formula (I) or a salt thereof. An azide group-introduced antibody derivative or a salt thereof is generated by cleavage of the thioester group. [Diagram 3] 3 is a schematic diagram (part 3) showing the concept of modification of an immunoglobulin unit with the compound of the present invention represented by formula (I) or a salt thereof. A conjugate of an antibody and a functional substance or a salt thereof is produced by reaction of the azide group in the azide group-introduced antibody derivative or a salt thereof with a functional substance (Z). Such a reaction is known as a strain-promoted azide-alkyne cyclization (SPAAC) reaction. [Figure 4] FIG. 4 is a diagram showing an overview of one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] 1. Definitions of General Terms In the present invention, the term "antibody" is as follows. The term "immunoglobulin unit" corresponds to a bivalent monomer unit that is a basic component of such an antibody, and is a unit containing two heavy chains and two light chains. Therefore, the definitions, examples, and preferred examples of the origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of lysine residue, and position selectivity of the immunoglobulin unit are the same as those of the antibody described below.

[0020] The origin of the antibody is not particularly limited, and may be derived from animals such as mammals and birds (e.g., chickens). Preferably, the immunoglobulin unit is derived from a mammal. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pet animals (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, more preferably humans.

[0021] The type of antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a specific glycosylation has been added (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylation consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, full-length antibodies or antibody fragments containing the variable region and the CH1 domain and CH2 domain can be used as monoclonal antibodies, with full-length antibodies being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0022] Any antigen can be used as the antigen of the antibody. For example, such antigens include proteins (including oligopeptides and polypeptides, and may be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low molecular weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0023] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of the disease target protein include the following:

[0024] (1) Oncology PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2. NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1(CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfelin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Luis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 inhibitory protein, TAG-72 (CA72-4), CD70,

[0025] (2) Autoimmune diseases and inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP(Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT

[0026] (3) Neurological disorders 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

[0027] (4) Infectious disease Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0028] (5) Genetic and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0029] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0030] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0031] (8) Hematological diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0032] (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

[0033] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab), , ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (if no IgG subtype is mentioned, it refers to IgG1).

[0034] The positions of amino acid residues in the antibody and the positions of the constant region of the heavy chain (e.g., CH2 domain) follow EU numbering (http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, in the case of human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the CH2 region of human IgG, the lysine residue at position 248 corresponds to the 18th amino acid residue in the CH2 region of human IgG, the lysine residue at position 288 corresponds to the 58th amino acid residue in the CH2 region of human IgG, the lysine residue at position 290 corresponds to the 60th amino acid residue in the CH2 region of human IgG, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the CH2 region of human IgG. The designation 246 / 248 indicates that the lysine residue at position 246 or 248 is the target. The designation 288 / 290 indicates that the lysine residue at position 288 or 290 is the target.

[0035] According to the present invention, specific lysine residues (e.g., lysine residues at positions 246 / 248 or 288 / 290) in the heavy chain of an antibody can be modified regioselectively. As used herein, "regioselective" or "regioselectivity" refers to a situation in which a specific structural unit capable of binding to a specific amino acid residue in an antibody is distributed in a specific region of the antibody, even though the specific amino acid residue is not distributed in a specific region of the antibody. Thus, expressions related to regioselectivity, such as "having a regioselective property," "binding regioselectively," and "binding with regioselectivity," mean that the retention or binding rate of a specific structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues that are the same type as the specific amino acid residue in the target region. Such position selectivity may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%. According to the present invention, a specific lysine residue in the heavy chain of an antibody can be regioselectively modified without using a linker containing a peptide. The peptide portion has potential immunogenicity and is easily hydrolyzed in blood. Therefore, avoiding the use of a linker containing a peptide portion is desirable in clinical applications.

[0036] In the present invention, as long as a specific lysine residue in the heavy chain of an antibody is regioselectively modified, a specific amino acid residue at another position may be further regioselectively modified.For example, a method for regioselectively modifying a specific amino acid residue at a predetermined position in an antibody is described in WO 2018 / 199337, WO 2019 / 240288, WO 2019 / 240287, and WO 2020 / 090979. Such specific amino acid residues may be amino acid residues having a side chain that is easily modified (e.g., an amino group, a carboxy group, an amide group, a hydroxy group, a thiol group) (e.g., a lysine residue, an aspartic acid residue, a glutamic acid residue, an asparagine residue, a glutamine residue, a threonine residue, a serine residue, a tyrosine residue, a cysteine ​​residue) and preferably a lysine residue having a side chain containing an amino group, a tyrosine residue, a serine residue, or a threonine residue having a side chain containing a hydroxy group, or a cysteine ​​residue having a side chain containing a thiol group, and more preferably a lysine residue (i.e., of the lysine residues at positions 246 / 248, 288 / 290, and 317, two lysine residues may be site-selectively doubly modified, and three lysine residues may be site-selectively triple modified). Even more preferably, lysine residues at two positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering may be regioselectively modified. Particularly preferably, lysine residues at two positions, 246 / 248 and 288 / 290, may be regioselectively modified.

[0037] In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Salts with inorganic acids include, for example, salts with inorganic acids, salts with organic bases, and salts with amino acids. Examples of the salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of the salts with inorganic bases include salts with alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium and magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of the salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of the salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, and ornithine) and acidic amino acids (e.g., aspartic acid and glutamic acid). The salt is preferably a salt with an inorganic acid (eg, hydrogen chloride) or a salt with an organic acid (eg, trifluoroacetic acid).

[0038] 2. Compound or salt thereof The present invention provides a compound represented by the following formula (I) or a salt thereof:

[0039] [ka]

[0040] [During the ceremony, X represents a leaving group, Y represents an affinity peptide having a binding region in the CH2 domain of an immunoglobulin unit comprising two heavy chains and two light chains; M represents a trivalent group connecting a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom or a sulfur atom; N3 represents an azide group; La represents a bond or a divalent group; Lb represents a bond or a divalent group.

[0041] In formula (I) and other formulas presented in connection with the present invention, - (hyphen) indicates that the two units on either side of it are covalently bonded. Thus, in formula (I), X is covalently bonded to the carbon atom constituting the carbonyl group, M is covalently bonded to the carbon atom constituting the carbonyl group, the carbon atom constituting C=W, and Lb, S is covalently bonded to the carbon atom constituting C=W and La, La is covalently bonded to S and Y, Lb is covalently bonded to M and N3, Y is covalently bonded to La, and N3 is covalently bonded to Lb.

[0042] The leaving group represented by X is a group that can be eliminated by a reaction between the carbon atom of the carbonyl group adjacent to X and an amino group. Those skilled in the art can appropriately select such a leaving group. Examples of such leaving groups include the following: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom); (c) R-A-(R-B-)N (wherein R and R each independently represent a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom); or (d) Halogen atoms.

[0043] Preferably, the leaving group represented by X may be: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom); or (c) R-A-(R-B-)N (wherein R and R each independently represent a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom).

[0044] More preferably, the leaving group represented by X may be: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); or (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom).

[0045] Even more preferably, the leaving group represented by X may be: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom).

[0046] Particularly preferably, the leaving group represented by X may be: (a') RS (wherein R represents a monovalent aromatic hydrocarbon group (e.g., phenyl) which may have a substituent, and S represents a sulfur atom).

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

[0048] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0049] The monovalent chain-like hydrocarbon group means a hydrocarbon group that is composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain-like hydrocarbon group include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0050] The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of the substituent. Examples of the alkyl group 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.

[0051] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0052] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0053] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0054] The monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it is not necessary to be composed only of alicyclic hydrocarbons, and it may contain a chain structure as a part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0055] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0056] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0057] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, and cycloalkyl. pentynyl, and cyclohexynyl.

[0058] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0059] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it is not necessary to be composed of only an aromatic ring, and it may contain a chain structure or an alicyclic hydrocarbon as a part thereof, and the aromatic ring may be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is even more preferable. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

[0060] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0061] Among these, the monovalent hydrocarbon group is preferably an alkyl group, a cycloalkyl group, or an aryl group.

[0062] The monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocyclic group, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom.

[0063] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0064] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0065] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0066] The number of "substituents" in the "monovalent hydrocarbon group which may be substituted" and the "monovalent heterocyclic group which may be substituted" represented by R, RA, and RB above may be, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2. Examples of such substituents include the following: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) aralkyl; (v) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra represents a hydrogen atom or a monovalent hydrocarbon group); (vi) NRbRc-, NRbRc-C(=O)-, NRbRc-C(=O)-O-, or Rb-C(=O)-NRc- (Rb and Rc are the same or different and each represents a hydrogen atom or a monovalent hydrocarbon group); or (vii) Nitro, sulfate, sulfonate, cyano, and carboxyl groups.

[0067] 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 of the monovalent hydrocarbon group and monovalent heterocyclic group described above for R, R, and R, respectively.

[0068] The aralkyl refers to an arylalkyl. The definitions, examples and preferred examples of aryl and alkyl in the arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such an aralkyl include benzoyl, phenethyl, naphthylmethyl and naphthylethyl.

[0069] Preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra is a hydrogen atom or an alkyl having 1 to 12 carbon atoms; (vi) NRbRc-, NRbRc-C(=O)-, NRbRc-C(=O)-O-, or Rb-C(=O)-NRc- (Rb and Rc are the same or different and are a hydrogen atom or an alkyl having 1 to 12 carbon atoms; or (vii) The same groups as those enumerated in (vii) above.

[0070] More preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms; (iii) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra is a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); (iv) NRbRc-, NRbRc-C(=O)-, NRbRc-C(=O)-O-, or Rb-C(=O)-NRc- (Rb and Rc are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (v) The same groups as those listed in (vii) above.

[0071] Even more preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 6 carbon atoms; (iii) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); (iv) NRbRc-, NRbRc-C(=O)-, NRbRc-C(=O)-O-, or Rb-C(=O)-NRc- (Rb and Rc are the same or different and each represents a hydrogen atom). or alkyl having 1 to 6 carbon atoms; (v) The same groups as those listed in (vii) above.

[0072] Particularly preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 4 carbon atoms; (iii) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); (iv) NRbRc-, NRbRc-C(=O)-, NRbRc-C(=O)-O-, or Rb-C(=O)-NRc- (Rb and Rc are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); or (v) The same groups as those listed in (vii) above.

[0073] The affinity peptide represented by Y has a binding region in the CH2 domain of an immunoglobulin unit including two heavy chains and two light chains. Any peptide having a binding region in the CH2 domain of an immunoglobulin unit can be used as the affinity peptide. The affinity peptide includes an amino acid residue (e.g., lysine residue, proline residue, tryptophan residue, tyrosine residue, serine residue, threonine residue) having a side chain including a moiety (e.g., amino group, hydroxyl group) capable of binding to a carbonyl group (C=O) adjacent to Y, and may form an amide bond with the carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue. Preferably, the affinity peptide includes a lysine residue, and may form an amide bond with the carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue. Examples of such affinity peptides that can be used include the affinity peptides disclosed in International Publication No. 2016 / 186206, International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, and International Publication No. 2020 / 090979, as well as the various affinity peptides disclosed in the documents cited in these international publications.

[0074] In one embodiment, the affinity peptide may comprise the amino acid sequence of (A) below, or an analogue thereof: (A)(X0-3)aC-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X0-3)b (SEQ ID NO: 1).

[0075] Furthermore, examples of the amino acid sequence of (A) above or its analogue amino acid sequence include the following: (1) Amino acid sequences represented by formulas (I) to (V) described in WO 2016 / 186206 (e.g., amino acid sequences of SEQ ID NOs: 1 to 17, 36, and 37 described in WO 2016 / 186206); (2) Amino acid sequences represented by formulas (i), (i-1), (i-1') and (i-1''), (i-2), (ii) to (viii) described in WO 2018 / 199337 (e.g., amino acid sequences of SEQ ID NOs: 20 to 60, 73 to 91, and 94 to 105 described in WO 2018 / 199337); (3) Amino acid sequences represented by formulas (1-1) to (1-9) and (2-1) described in WO 2019 / 240287 (e.g., amino acid sequences of SEQ ID NOs: 5 and 8 to 92 described in WO 2019 / 240287); (4) Amino acid sequences represented by formulas (1-1) to (1-9) and (2-1) described in WO 2019 / 240288 (e.g., amino acid sequences of SEQ ID NOs: 15 to 24, 46 to 96, 99, and 108 described in WO 2019 / 240288); (5) Amino acids (10) to (11) described in International Publication No. 2020 / 090979 Sequence (e.g., the amino acid sequences of SEQ ID NOs: 24 and 25 described in WO 2020 / 090979).

[0076] An affinity peptide containing the amino acid sequence of (A) above or an analogue thereof has a binding region at the same site in the CH2 domain of an immunoglobulin unit, as can be seen from the conserved portion. Thus, like a compound having an affinity peptide containing the amino acid sequence of (A) above, a compound having an affinity peptide containing the analogue amino acid sequence binds to the same site in the CH2 domain of an immunoglobulin unit, and can site-selectively modify the lysine residues at positions 246 / 248 of the human IgG heavy chain, while highly controlling the average ratio of binding between the immunoglobulin unit and the azide group-containing modifying group within a desired range. The affinity peptide preferably contains the amino acid sequence of (A) above, and more preferably contains the amino acid sequence of (1) below: (1) RGNCAYHKGQIIWCTYH (sequence number 2).

[0077] In another embodiment, the affinity peptide may comprise the amino acid sequence of (B) below, or an analogue thereof: (B) PNLNEEQRNARIRSI (SEQ ID NO:3).

[0078] Furthermore, examples of the amino acid sequence of (B) above or its analogue amino acid sequence include the following: (1) Amino acid sequences (a) to (d) described in WO 2018 / 199337 (e.g., amino acid sequences of SEQ ID NOs: 61 to 72, 92 described in WO 2018 / 199337); (2) Amino acid sequences (a) to (b) described in WO 2019 / 240288 (e.g., amino acid sequences of SEQ ID NOs: 5 to 8, 11 to 14, 37 to 45, 97, 98, and 100 described in WO 2019 / 240288); (3) The amino acid sequences of (1) to (9) described in WO 2020 / 090979 (e.g., the amino acid sequences of SEQ ID NOs: 5 to 10, 22, 23, and 51 to 53 described in WO 2020 / 090979).

[0079] An affinity peptide containing the amino acid sequence of (B) above or an analogue thereof has a binding region at the same site in the CH2 domain of an immunoglobulin unit, as can be seen from the conserved portion. Thus, like a compound having an affinity peptide containing the amino acid sequence of (B) above, a compound having an affinity peptide containing the analogue amino acid sequence binds to the same site in the CH2 domain of an immunoglobulin unit, and can site-selectively modify the lysine residues at positions 288 / 290 of the human IgG heavy chain, while highly controlling the average ratio of binding between the immunoglobulin unit and the azide group-containing modifying group within a desired range. The affinity peptide preferably contains the amino acid sequence of (B) above, and more preferably contains any of the following amino acid sequences (1) to (3): (1) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 4); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5; or (3) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (sequence number 6).

[0080] At least two cysteine ​​residues spaced apart in each amino acid sequence of the affinity peptide can form a cyclic peptide through a disulfide bond. Alternatively, the thiol groups in the two cysteine ​​residues in the peptide can be linked by a linker (e.g., a carbonyl group-containing linker as shown below).

[0081] [ka]

[0082] The broken line portion of the carbonyl group-containing linker shown above means the bond portion with the thiol group. The linker is more stable against reduction reactions and the like than a normal disulfide bond. Such a peptide can be prepared, for example, by the method described in WO 2016 / 186206.

[0083] The amino acids constituting the affinity peptide may be either L- or D-form, but L-form is preferred (in the examples, all amino acid residues constituting the peptide are L-form). The affinity peptide may be linked to the compound of formula (I) or a salt thereof by modifying a specific amino acid residue with a crosslinking agent. Examples of such specific amino acid residues include lysine residues, aspartic acid residues, and glutamic acid residues, with lysine residues being preferred. Examples of crosslinking agents include disuccinimidyl stearate (DSG), Examples of the crosslinking agent include crosslinkers containing preferably two or more succinimidyl groups, such as DSS (disuccinimidyl suberate), DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2 HCl), and DMS (dimethyl suberimidate·2 HCl), which contain preferably two or more imide acid moieties, as well as crosslinkers having an SS bond, such as DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., WO 2016 / 186206).

[0084] The above affinity peptide may have its amino and carboxy groups at its terminals protected. Examples of the protecting group for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). The protecting group for the N-terminal amino group is preferably an acetyl group. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid may have a cyclic structure of pyroglutamic acid. When the N-terminal amino acid is glutamine, the protected N-terminal glutamine may have a cyclic structure of pyroglutamic acid. Examples of the protecting group for the C-terminal carboxy group include groups capable of forming esters or amides. Examples of the group capable of forming an ester or amide include an alkyloxy group (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), an aryloxy group (e.g., phenyloxy, naphthyloxy), an aralkyloxy group (e.g., benzyloxy), and an amino group. As a protecting group for the C-terminal carboxy group, an amino group is preferred.

[0085] M is a carbon atom in C=O and a carbon atom in C=W adjacent to M, that is, the first carbon atom (carbon atom in C=O) adjacent to M and the second carbon atom (carbon atom in C=W) adjacent to M. It represents a trivalent group linking M and the first and second carbon atoms adjacent to M through a main chain portion having 3 to 5 carbon atoms. Therefore, the main chain portion linking the first and second carbon atoms adjacent to M does not contain a heteroatom. The use of a compound having such a structure can be expected to provide the following advantages. First, the use of the above compound makes it easy to highly control the average bond ratio between the antibody and the azide group-containing modifying group within a desired range (1.5 to 2.5). In the present invention, the average bond ratio between the antibody and a predetermined group (e.g., the azide group-containing modifying group) can be confirmed by analyzing MS analysis data using a DAR calculator (software from Agilent). Secondly, the above compounds can be used to regioselectively modify different lysine residues in the heavy chain of the immunoglobulin unit. For example, the affinity peptide having a certain amino acid sequence can be used as Y to regioselectively modify the lysine residues at positions 246 / 248 of the human IgG heavy chain. The affinity peptide having a different amino acid sequence can be used as Y to regioselectively modify the lysine residues at positions 288 / 290 of the human IgG heavy chain. Third, the antibodies prepared by using the above compounds can exhibit excellent stability.

[0086] The main chain portion having 3 to 5 carbon atoms is composed of a chain structure, a cyclic structure, or a structure including a combination of these. When the main chain is a chain structure that does not include a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure. On the other hand, when the main chain is a structure that includes a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms constituting the cyclic structure as the number of atoms in the main chain. Specifically, the number of atoms in the main chain in the cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (for example, see the bold paths in (a) to (d) below). When the main chain is a structure that includes a combination of a chain structure and a cyclic structure, the number of atoms in the main chain can be determined by adding the number of atoms in the chain structure that does not include a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure.

[0087] [ka]

[0088] · is a bond. In the case of (a), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 2. In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 3. In the case of (c), both paths are the shortest paths (equidistant), so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4. In the case of (d), since the path of the condensation site is the shortest path, the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.

[0089] The main chain portion consisting of 3 to 5 carbon atoms is the first carbon atom adjacent to M (the carbon atom in C=O). Since M connects the first carbon atom (atom in C=W) to the second carbon atom adjacent to M (carbon atom in C=W), it can be understood as a divalent group in relation to the first and second carbon atoms. On the other hand, M is a trivalent group that bonds not only to the first and second carbon atoms but also to Lb. Therefore, M can be expressed as a trivalent group obtained by removing one hydrogen atom from a main chain portion (divalent group) consisting of 3 to 5 carbon atoms. Here, the main chain portion (divalent group) consisting of 3 to 5 carbon atoms may be composed of the following: (1) a divalent linear hydrocarbon group having 3 to 5 carbon atoms; (2) A divalent group in which a divalent cyclic hydrocarbon group and a divalent linear hydrocarbon group (one or two) having 1 to 4 carbon atoms are linked together; (3) a divalent cyclic hydrocarbon group; and (4) A divalent group in which two divalent cyclic hydrocarbon groups are linked (i.e., a divalent bicyclo structure).

[0090] The straight chain hydrocarbon group having 3 to 5 carbon atoms is a straight chain alkylene group having 3 to 5 carbon atoms, a straight chain alkenylene group having 3 to 5 carbon atoms, or a straight chain alkynylene group having 3 to 5 carbon atoms. The straight chain alkylene having 3 to 5 carbon atoms is n-propylene, n-butylene, or n-pentylene. The linear alkenylene having 3 to 5 carbon atoms is n-propenylene, n-butenylene, or n-pentenylene. The straight-chain alkynylene having 3 to 5 carbon atoms is n-propynylene, n-butynylene, or n-pentynylene. As the divalent linear hydrocarbon group having 3 to 5 carbon atoms, a linear alkylene group having 3 to 5 carbon atoms is preferable.

[0091] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. By appropriately setting two bonds in such a divalent cyclic hydrocarbon group, the number of atoms constituting the main chain as described above can be set to 3 to 5. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of the arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene group.

[0092] The divalent linear hydrocarbon group having 1 to 4 carbon atoms is a linear alkylene group having 1 to 4 carbon atoms, a linear alkenylene group having 2 to 4 carbon atoms, or a linear alkynylene group having 2 to 4 carbon atoms. The straight chain alkylene having 1 to 4 carbon atoms is methylene, ethylene, n-propylene, or n-butylene. The straight-chain alkenylene having 1 to 4 carbon atoms is ethylenylene, n-propenylene, or n-butenylene. The straight-chain alkynylene having 1 to 4 carbon atoms is ethynylene, n-propynylene, or n-butynylene. The divalent linear hydrocarbon group having 1 to 4 carbon atoms is preferably a linear alkyl group having 1 to 4 carbon atoms. Kiren is preferred.

[0093] Of the main chain portion (divalent group) having 3 to 5 carbon atoms, (1) to (3) are preferred among the above (1) to (4), and (1) and (2) are more preferred.

[0094] In certain embodiments, the main chain portion of M having 3 to 5 carbon atoms may be a straight chain alkylene, or a moiety containing ring carbon atoms, or a combination thereof. Thus, the main chain portion (divalent group) having 3 to 5 carbon atoms may be composed of the following: (1') linear alkylene having 3 to 5 carbon atoms; (2') a divalent group formed by linking a divalent cyclic hydrocarbon group and a linear alkylene group (one or two) having 1 to 4 carbon atoms; (3') a divalent cyclic hydrocarbon group; and (4') A divalent group in which two divalent cyclic hydrocarbon groups are linked together (i.e., a divalent bicyclo structure).

[0095] Of the above (1') to (4'), the main chain portion (divalent group) having 3 to 5 carbon atoms is preferably (1') to (3'), and more preferably (1') and (2'). Therefore, M may be a trivalent group obtained by removing one hydrogen atom from the main chain portion (divalent group) having 3 to 5 carbon atoms, represented by the following formula (i) or (ii):

[0096] [ka]

[0097] [During the ceremony, n represents an integer of 3 to 5; m represents an integer of 0 to 4; k represents an integer of 0 to 4; Ring B represents a divalent cyclic hydrocarbon group; b1 represents a bond to the carbon atom in C=O adjacent to M, b2 represents a bond to the carbon atom in C=W adjacent to M. The divalent cyclic hydrocarbon group represented by ring B is the same as that described above. When the main chain portion (divalent group) having 3 to 5 carbon atoms is represented by formula (ii), M is preferably a trivalent group obtained by removing one hydrogen atom from ring B in the main chain portion (divalent group) having 3 to 5 carbon atoms represented by formula (ii).

[0098] Preferably, the main chain portion (divalent group) having 3 to 5 carbon atoms and represented by the above formula (ii) may be represented by the following formula (ii').

[0099] [ka]

[0100] [During the ceremony, m represents an integer of 0 to 4; k represents an integer of 0 to 4; Ph represents phenylene; b1 represents a bond to the carbon atom in C=O adjacent to M, b2 represents a bond to the carbon atom in C=W adjacent to M. Ph can be bonded to two straight-chain alkylenes at the meta, ortho, or para positions. When the main chain portion (divalent group) having 3 to 5 carbon atoms is represented by formula (ii'), M is preferably a trivalent group in which one hydrogen atom has been removed from the phenylene in the main chain portion (divalent group) having 3 to 5 carbon atoms represented by formula (ii').

[0101] More preferably, the main chain portion (divalent group) having 3 to 5 carbon atoms and represented by the above formula (ii) may be represented by the following formula (ii″).

[0102] [ka]

[0103] [During the ceremony, m represents an integer of 0 to 2; k represents an integer of 0 to 2; b1 represents a bond to the carbon atom in C=O adjacent to M, b2 represents a bond to the carbon atom in C=W adjacent to M. When the main chain portion (divalent group) having 3 to 5 carbon atoms is represented by formula (ii''), M is preferably a trivalent group obtained by removing one hydrogen atom from the phenylene (preferably the carbon atom at the ortho position to the two alkylenes) in the main chain portion (divalent group) having 3 to 5 carbon atoms represented by formula (ii'').

[0104] W represents an oxygen atom or a sulfur atom, and preferably represents an oxygen atom.

[0105] La and Lb each independently represent a bond or a divalent group.

[0106] La is a moiety that is included in the antibody intermediate produced by the compound represented by formula (I), but is not included in the azide group-introduced antibody derivative produced from the antibody intermediate, or in the conjugate of the antibody and the functional substance. Therefore, in the antibody produced by the compound represented by formula (I), the stability of La is unlikely to be a problem.

[0107] Lb is a moiety contained in an antibody intermediate produced by the compound represented by formula (I), an azide group-introduced antibody derivative, and a conjugate of an antibody and a functional substance, and acts as a linker linking an antibody and an azide group in the azide group-introduced antibody derivative.

[0108] The main chain in the divalent group represented by La and Lb is preferably composed of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NRd- (Rd represents a hydrogen atom or a substituent), -O-, -S-, or a group consisting of a combination of two or more of these (for example, 2 to 15, preferably 2 to 10, more preferably 2 to 8, even more preferably 2 to 6, and particularly preferably 2, 3, 4, or 5). The number of atoms constituting the main chain in the divalent group represented by La and Lb is preferably 1 to 17, more preferably 1 to 15. The lower limit of the number of atoms constituting the main chain in the divalent group represented by La and Lb may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more. The upper limit of the number of atoms constituting the main chain in the divalent groups represented by La and Lb may be 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less. When the main chain is a structure containing a cyclic structure, the number of atoms in the main chain in the cyclic structure can be determined as described above.

[0109] The divalent straight chain hydrocarbon group is a straight chain alkylene, a straight chain alkenylene, or a straight chain alkynylene. The straight-chain alkylene is a straight-chain alkylene having 1 to 6 carbon atoms, and preferably a straight-chain alkylene having 1 to 4 carbon atoms. Examples of the straight-chain alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, and preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethylenylene, n-propenylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, and preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of the linear alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. The divalent linear hydrocarbon group is preferably a linear alkylene group.

[0110] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of the arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene group.

[0111] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent aromatic heterocyclic group include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferable. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. The divalent heterocyclic group is preferably a divalent aromatic heterocyclic group.

[0112] In certain embodiments, the carbonyl group adjacent to La may form an amide bond with the amino group in the side chain of a lysine residue in the affinity peptide.

[0113] In a specific embodiment, the main chain in the divalent group represented by Lb may not contain a cyclic structure from the viewpoint of ease of synthesis, etc. Therefore, the main chain in the divalent group represented by Lb may be composed of a divalent linear hydrocarbon group, -C(=O)-, -C(=S)-, -NRd- (Rd represents a hydrogen atom or a substituent), -O-, -S-, or a group consisting of a combination of two or more of these (e.g., 2, 3, 4, or 5). Preferably, from the viewpoint of ensuring the stability of Lb, -C(=O)-NRd-, -C(=O)-O-, -C(=O)-S-, -C(=S)-NRd-, -C(=S)-O-, and -C(=S)-S- are excluded from the above combinations. The number of atoms constituting the main chain in the divalent group represented by Lb is preferably 1 to 12, more preferably 1 to 10.

[0114] The groups constituting the main chain in the divalent groups represented by La and Lb may have, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2, substituents. In addition, the above Rd attached to the main chain in the first linker and the second linker corresponds to a substituent. Examples of such substituents include the following: (i') a halogen atom; (ii') a monovalent hydrocarbon group; (iii') aralkyl; (iv') a monovalent heterocyclic group; (v') Re-O-, Re-C(=O)-, Re-OC(=O)-, or Re-C(=O)-O- (Re represents a hydrogen atom or a monovalent hydrocarbon group); or (vi') NRfRg-, NRfRg-C(=O)-, NRfRg-C(=O)-O-, or Rf-C(=O)-NRg- (Rf and Rg are the same or different and each represents a hydrogen atom or a monovalent hydrocarbon group); (vii') A nitro group, a sulfate group, a sulfonate group, a cyano group, and a carboxyl group.

[0115] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, aralkyl, and monovalent heterocyclic group in the above substituents are the same as those of R, R, and R, and the halogen atom, monovalent hydrocarbon group, aralkyl, and monovalent heterocyclic group described in the above (i) to (iv), respectively.

[0116] Preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii') aralkyl having 3 to 15 carbon atoms; (iv') a 5- or 6-membered heterocycle; (v') Re-O-, Re-C(=O)-, Re-OC(=O)-, or Re-C(=O)-O- (Re represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); (vi') NRfRg-, NRfRg-C(=O)-, NRfRg-C(=O)-O-, or Rf-C(=O)-NRg- (Rf and Rg are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (vii') The same groups as those enumerated in (vii') above.

[0117] More preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 12 carbon atoms; (iii') Re-O-, Re-C(=O)-, Re-OC(=O)-, or Re-C(=O)-O- (Re represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); (iv') NRfRg-, NRfRg-C(=O)-, NRfRg-C(=O)-O-, or Rf-C(=O)-NRg- (Rf and Rg are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (v') The same groups as those enumerated in (vii') above.

[0118] Even more preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 6 carbon atoms; (iii') Re-O-, Re-C(=O)-, Re-OC(=O)-, or Re-C(=O)-O- (Re represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); (iv') NRfRg-, NRfRg-C(=O)-, NRfRg-C(=O)-O-, or Rf-C(=O)-NRg- (Rf and Rg are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); or (v') The same groups as those enumerated in (vii') above.

[0119] Particularly preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 4 carbon atoms; (iii') Re-O- or Re-C(=O)- (Re is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); (iv') NRfRg- (Rf and Rg are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); or (v') The same groups as those enumerated in (vii') above.

[0120] In a specific embodiment, the compound represented by formula (I) can be defined by the number of atoms of the main chain linking M and Y. The main chain linking M and Y corresponds to the main chain "CS-La-C" in the structure "C(=W)-S-La-C(=O)" linking M and Y. The number of atoms of the main chain linking M and Y may be 6 to 20. The compound represented by formula (I) having such a number of atoms of the main chain linking M and Y is easy to synthesize. In addition, it is easy to highly control the average ratio of the bond between the antibody and the azide group-containing modifying group within a desired range, and is excellent in versatility for site-selective modification of immunoglobulin units. The number of atoms in the main chain connecting M and Y may also be 6 to 19, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 8 to 19, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, or 8 to 10.

[0121] The compound of the present invention represented by formula (I) or a salt thereof can be obtained, for example, by synthesizing a compound or a salt thereof in which the Y moiety in the compound represented by formula (I) or a salt thereof is substituted with a leaving group (preferably a leaving group having a higher leaving ability than X, or hydroxy), and then reacting the synthesized compound or a salt thereof with an affinity peptide. For example, such a reaction can be carried out in a suitable reaction system (e.g., an organic solvent or an aqueous solution, or a mixed solvent thereof) at a suitable temperature (e.g., about 15 to 200°C). The reaction system 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.

[0122] Preferably, the compound represented by formula (I) or a salt thereof in which Y is substituted with a leaving group may be a compound represented by the following formula (I') or a salt thereof.

[0123] [ka]

[0124] [During the ceremony, X represents a leaving group, X' represents a leaving group having a higher leaving ability than the leaving group X; M represents a trivalent group connecting a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom or a sulfur atom; N3 represents an azide group; La represents a bond or a divalent group; Lb represents a bond or a divalent group.

[0125] The leaving group represented by X′, which has a higher ability to leave than the leaving group X, is not particularly limited as long as it is a leaving group which has a higher ability to leave than the leaving group X, and examples thereof include a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, and an N-succinimidyloxy group.

[0126] The definitions, examples, and preferred examples of symbols, terms, and expressions such as X (leaving group), M (trivalent group), W (oxygen atom or sulfur atom), La (bond or divalent group), and Lb (bond or divalent group) in formula (I') are the same as those in the above formula.

[0127] The compound represented by formula (I') or a salt thereof is useful, for example, as a synthetic intermediate for efficiently producing the compound represented by formula (I) or a salt thereof.

[0128] The compound represented by formula (I') or a salt thereof can be produced from a compound represented by the following formula (I'') or a salt thereof.

[0129] [ka]

[0130] [During the ceremony, X represents a leaving group, OH represents a hydroxy group; M represents a trivalent group connecting a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom or a sulfur atom; N3 represents an azide group; La represents a bond or a divalent group; Lb represents a bond or a divalent group.

[0131] The definitions, examples, and preferred examples of symbols, terms, and expressions such as X (leaving group), M (trivalent group), W (oxygen atom or sulfur atom), La (bond or divalent group), and Lb (bond or divalent group) in formula (I'') are the same as those in the above formula.

[0132] The compound represented by formula (I') or a salt thereof can be obtained by reacting the compound represented by formula (I'') or a salt thereof with a carboxyl group modifying reagent. Examples of the carboxyl group modifying reagent include pentafluorophenylation reagents (e.g., pentafluorophenyl trifluoroacetate), tetrafluorophenylation reagents (e.g., tetrafluorophenyl trifluoroacetate), paranitrophenylation reagents (e.g., paranitrophenyl trifluoroacetate), and N-succinimidylation reagents (e.g., N-succinimidyl trifluoroacetate). For example, such a reaction can be carried out in a suitable organic solvent system (e.g., an organic solvent containing an alkyl halide (e.g., methyl halide) such as CH2Cl2, and an amine such as triethylamine) at an appropriate temperature (e.g., about -10 to 30°C). 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.

[0133] The compound represented by formula (I″) or a salt thereof is useful, for example, as a synthetic intermediate for efficiently producing the compound represented by formula (I′) or a salt thereof. Alternatively, the compound represented by formula (I″) or a salt thereof is also useful as a synthetic intermediate for directly and efficiently producing the compound represented by formula (I) or a salt thereof.

[0134] The compounds of formula (I'') can be obtained by various synthetic methods as illustrated in the Examples.

[0135] The production of the above-mentioned series of compounds or their salts can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Such compounds or their salts can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0136] 3.Antibody intermediate or its salt The present invention provides an antibody intermediate or a salt thereof comprising a structural unit represented by the following formula (II):

[0137] [ka]

[0138] [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and forms an amide bond between the amino groups in the side chains of the lysine residues in the two heavy chains and the carbonyl groups adjacent to the Ig; Y represents an affinity peptide having a binding region in the CH2 domain of the immunoglobulin unit, M represents a trivalent group connecting a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom or a sulfur atom; N3 represents an azide group; La represents a bond or a divalent group; Lb represents a bond or a divalent group; The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.0.

[0139] The immunoglobulin unit represented by Ig is as described above. The definitions, examples, and preferred examples of symbols, terms, and expressions such as Y (affinity peptide), M (trivalent group), W (oxygen atom or sulfur atom), La (bond or divalent group), and Lb (bond or divalent group) in formula (II) are the same as those in the above formula.

[0140] In formula (II), the average ratio (r) of the amide bonds per two heavy chains indicates the average ratio of bonds between immunoglobulin units and azide group-containing modifying groups (number of azide group-containing modifying groups / immunoglobulin units). Such an average ratio is 1.0 to 3.0, and preferably 1.5 to 2.5. Furthermore, such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1. A lysine residue having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to an Ig may be modified to show an average ratio of the above-mentioned values ​​at one desired position in the two heavy chains (e.g., one position selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering). When each of the two heavy chains is modified at two or more positions, a lysine residue having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to an Ig may be modified to show an average ratio of the above-mentioned values ​​at each of two or more desired positions in the two heavy chains (e.g., one position selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering; preferably positions 246 / 248 and 288 / 290).

[0141] The antibody intermediate or salt thereof of the present invention can be obtained by reacting the compound or salt thereof of the present invention with an antibody containing the above immunoglobulin unit. In the reaction, the compound or salt thereof of the present invention is first mixed with the antibody. This allows the compound or salt thereof to associate with the antibody via an affinity peptide having affinity with the antibody. Next, after the association of the antibody, the carbonyl group (XC=O) having a leaving group (X) can react regioselectively with the amino group in the side chain of the lysine residue in the heavy chain of the antibody. By such a reaction, the amino group is bonded to the carbon atom of the carbonyl group, and the leaving group (X) is eliminated from the carbonyl group, thereby obtaining the antibody intermediate or salt thereof of the present invention. In the reaction, the molar ratio of the compound or salt thereof of the present invention to the antibody (compound or salt thereof of the present invention / antibody) is not particularly limited because it varies depending on factors such as the compound or salt thereof of the present invention and the type of antibody, but is, for example, 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 50, and particularly preferably 6 to 30.

[0142] Such a reaction can be carried out under mild conditions that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). The reaction can be carried out in a reaction system, for example, a buffer solution, at room temperature (for example, about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain 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, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0143] The production of the antibody intermediate or its salt can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, depending on the specific raw material and the molecular weight of the product. The position selectivity can be confirmed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease treatment and mass spectrometry. The protease is preferably an endoprotease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of affinity peptides introduced can be confirmed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The antibody intermediate or its salt can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0144] 4. Azide group-introduced antibody derivative or its salt The present invention provides an azide group-introduced antibody derivative or a salt thereof, which contains a structural unit represented by the following formula (III):

[0145] [ka]

[0146] [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and forms an amide bond between the amino groups in the side chains of the lysine residues in the two heavy chains and the carbonyl groups adjacent to the Ig; M represents a trivalent group connecting a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms; O represents an oxygen atom; T represents a monovalent group; W represents an oxygen atom or a sulfur atom; N3 represents an azide group; Lb represents a bond or a divalent group; The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.0.

[0147] The immunoglobulin unit represented by Ig is as described above. The definitions, examples, and preferred examples of symbols, terms, and expressions such as M (trivalent group), W (oxygen atom or sulfur atom), and Lb (bond or divalent group) in formula (III) are the same as those in the above formula.

[0148] T represents a monovalent group. T can be generated by a cleavage reaction between a carbon atom and a sulfur atom in the partial structure "C(=W)-S" in the compound represented by formula (II). Therefore, as T, an appropriate monovalent group can be adopted according to the type of cleavage reaction.

[0149] In certain embodiments, the monovalent group represented by T may be an optionally substituted hydroxyamino group. The optionally substituted hydroxyamino group may be represented by the following formula (α):

[0150] [ka]

[0151] [During the ceremony, R1 and R2 are the same or different and each represents a hydrogen atom or an optionally substituted monovalent hydrocarbon group. The definitions, examples, and preferred examples of the monovalent hydrocarbon group and the substituent are as described above.

[0152] In formula (III), the average ratio (r) of the amide bonds per two heavy chains indicates the average ratio of bonds between immunoglobulin units and azide group-containing modifying groups (number of azide group-containing modifying groups / immunoglobulin units). Such an average ratio is 1.0 to 3.0, and preferably 1.5 to 2.5. Furthermore, such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1. The lysine residue having a side chain containing an amino group forming an amide bond with the carbonyl group adjacent to the Ig may be modified at one desired position in the two heavy chains (e.g., one position selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering) so as to show an average ratio of the above-mentioned values. When each of the two heavy chains is modified at two or more positions, the lysine residue having a side chain containing an amino group forming an amide bond with the carbonyl group adjacent to the Ig may be modified at two or more desired positions in the two heavy chains (e.g., one position selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering). Preferably, each of the positions in the sequence (one position selected from the group consisting of: positions 246 / 248, and positions 288 / 290) may be modified to exhibit an average ratio of such values.

[0153] The azide group-introduced antibody derivative or a salt thereof of the present invention can be obtained by subjecting the antibody intermediate or a salt thereof of the present invention to a thioester cleavage reaction. The thioester cleavage reaction can be carried out under conditions (mild conditions as described above) that do not cause denaturation or decomposition (e.g., cleavage of amide bonds) of proteins (immunoglobulins / antibodies). More specifically, the thioester can be cleaved by stirring for an appropriate time (e.g., 1 hour) in a hydroxylamine hydrochloride solution having a pH of 4.0 to 8.0 and a concentration in the range of 10 mM to 10 M (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160.).

[0154] The production of the azide group-introduced antibody derivative or its salt can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw material and the molecular weight of the product. The position selectivity can be confirmed by, for example, peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. The protease is preferably an endoprotease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of thiol groups introduced can be confirmed by, for example, electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The azide group-introduced antibody derivative or its salt can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0155] 5. Conjugate of antibody and functional substance or its salt The present invention provides a conjugate of an antibody and a functional substance, which comprises a structural unit represented by the following formula (IV), or a salt thereof:

[0156] [ka]

[0157] [During the ceremony, Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and The amino group in the side chain of the lysine residue in the heavy chain of forms an amide bond with the carbonyl group adjacent to Ig, M represents a trivalent group connecting a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms; O represents an oxygen atom; T represents a monovalent group; W represents an oxygen atom or a sulfur atom; N represents a nitrogen atom; Lb represents a bond or a divalent group; Z represents a functional substance; L represents a bond or a divalent group; Ring A represents a ring fused to a triazole ring; The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.0.

[0158] The immunoglobulin unit represented by Ig is as described above. The definitions, examples, and preferred examples of symbols, terms, and expressions such as M (trivalent group), W (oxygen atom or sulfur atom), and Lb (bond or divalent group) in formula (IV) are the same as those in the above formula.

[0159] Ring A represents a ring fused to a triazole ring. The constituent portion of ring A does not include the fused triazole ring itself, but includes the double bond between the carbon atoms shared with the triazole. Therefore, ring A can be said to be a ring having a double bond between carbon atoms.

[0160] Ring A is a monocycle or a condensed ring of a monocycle and another ring. Ring A may have a substituent. The monocycle is preferably a homocycle or a heterocycle containing 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. More preferably, the monocycle is a homocycle or a heterocycle containing one or more selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom. The monocycle is preferably a 5- to 12-membered monocycle, more preferably a 6- to 10-membered monocycle, and even more preferably a 7- to 9-membered monocycle. The monocycle is preferably a non-aromatic monocycle.

[0161] When ring A is a fused ring, other rings fused to the monocycle include, for example, cycloalkanes, arenes, and heterocycles.

[0162] The cycloalkane condensed with the monocycle is preferably a cycloalkane having 3 to 24 carbon atoms, more preferably a cycloalkane having 6 to 18 carbon atoms, even more preferably a cycloalkane having 3 to 14 carbon atoms, and even more preferably a cycloalkane having 3 to 10 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkane include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane.

[0163] The arene condensed with the monocycle is preferably an arene having 6 to 24 carbon atoms, more preferably an arene having 6 to 18 carbon atoms, even more preferably an arene having 6 to 14 carbon atoms, and even more preferably an arene having 6 to 10 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the arene include benzene, naphthalene, and anthracene.

[0164] The heterocycle condensed with the monocycle is an aromatic heterocycle or a non-aromatic heterocycle. The heterocycle preferably contains at least one heteroatom 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. It is more preferable that the alkyl group contains one or more selected from the group consisting of a nitrogen atom and a silyl atom.

[0165] The aromatic heterocycle condensed with the monocycle is preferably an aromatic heterocycle having 3 to 21 carbon atoms, more preferably an aromatic heterocycle having 3 to 15 carbon atoms, even more preferably an aromatic heterocycle having 3 to 9 carbon atoms, and even more preferably an aromatic heterocycle having 3 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. More specifically, examples of the aromatic heterocycle include pyrene, pyrrole, furan, thiophene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrroline, piperidine, triazole, purine, anthraquinone, carbazole, fluorene, quinoline, and isoquinoline.

[0166] The non-aromatic heterocycle condensed with the monocycle is preferably a non-aromatic heterocycle having 3 to 21 carbon atoms, more preferably a non-aromatic heterocycle having 3 to 15 carbon atoms, even more preferably a non-aromatic heterocycle having 3 to 9 carbon atoms, and even more preferably a non-aromatic heterocycle having 3 to 6 carbon atoms. The number of carbon atoms of the substituent is not included in the number of carbon atoms. More specifically, examples of the non-aromatic heterocycle include oxirane, aziridine, azetidine, oxetane, thietane, pyrrolidine, dihydrofuran, tetrahydrofuran, dioxolane, tetrahydrothiophene, imidazolidine, oxazolidine, piperidine, dihydropyran, tetrahydropyran, tetrahydrothiopyran, morpholine, thiomorpholine, piperazine, dihydrooxazine, tetrahydrooxazine, dihydropyrimidine, and tetrahydropyrimidine.

[0167] The substituents that the ring A may have are the same as the above-mentioned substituents (i) to (vii), and the preferred ranges are also the same. The number of the substituents is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

[0168] Preferably, ring A may be a 7- to 9-membered monocyclic ring or a condensed ring of a 7- to 9-membered monocyclic ring with another ring (see, for example, Org. Biomol. Chem. 2013, 11, 6439, Angew. Chem. Int. Ed. 2015, 54, 1190). For example, as such ring A, rings of formulas (i') to (vii') described in International Publication No. 2017 / 191817 can be used.

[0169] In formula (IV), the divalent group represented by L is a straight-chain, branched-chain, or cyclic group, or a group consisting of a combination thereof, which connects the functional substance (Z) and the ring A. The main chain in the divalent group represented by L may be composed of, for example, a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR- (R represents a hydrogen atom or a substituent), -O-, -S-, or a group consisting of a combination of two or more of these (for example, 2 to 15, preferably 2 to 10, more preferably 2 to 8, even more preferably 2 to 6, particularly preferably 2, 3, 4, or 5). The divalent group represented by L may have a substituent. Such a substituent is the same as the above-mentioned substituents (i) to (vii), and the preferred range is also the same. The number of the substituents is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2. L can be bonded to any ring atom in ring A other than the carbon atom shared with the triazole.

[0170] The functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked together.

[0171] The drug may be a drug for any disease. Such diseases include, for example, For example, cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases / inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infection, viral infection), genetic / rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone / orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0172] More specifically, the drug is an anticancer drug. Examples of the anticancer drug include chemotherapeutic agents, toxins, radioisotopes, or substances containing the same. Examples of the chemotherapeutic agent include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of the toxin include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioisotopes of hydrogen atoms (e.g., 3H), radioisotopes of carbon atoms (e.g., 14C), radioisotopes of phosphorus atoms (e.g., 32P), radioisotopes of sulfur atoms (e.g., 35S), radioisotopes of yttrium (e.g., 90Y), radioisotopes of technetium (e.g., 99mTc), radioisotopes of indium (e.g., 111In), radioisotopes of iodine atoms (e.g., 123I, 125I, 129I, 131I), radioisotopes of samarium (e.g., 153Sm), and radioisotopes of rhenium. Radioisotopes of bismuth (e.g., 186Re), astatine (e.g., 211At), and bismuth (e.g., 212Bi). More specifically, drugs include auristatins (MMAE, MMAF), maytansines (DM1, DM4), PBDs (pyrrolobenzodiazepines), IGNs, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0173] A labeling substance is a substance that allows detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2′-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), or substances containing the same.

[0174] Stabilizers are substances that allow for the stabilization of antibodies, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0175] The functional substance may also be a peptide, a protein, a nucleic acid, a low molecular weight organic compound, a sugar chain, a lipid, a high molecular weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Examples of nucleic acids also include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of low molecular weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.

[0176] If the functional substance does not have a desired functional group (e.g., a functional group that is easy to link to ring A or L), the functional substance may be derivatized to have such a functional group. Derivatization is common knowledge in the art (e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be performed using any crosslinking agent. Alternatively, derivatization may be performed using a specific linker having a desired functional group. For example, such a linker may be one that allows the functional substance and the antibody to be separated by cleavage of the linker in an appropriate environment (e.g., inside or outside a cell). Examples of such linkers include peptidyl linkers that are degraded by specific proteases (e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secretory proteases)) (e.g., U.S. Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), and linkers that can be cleaved at local acidic sites present in vivo (e.g., U.S. Pat. Nos. 5,622,929, 5,122,368, and 5,824,805). The linker may be self-immolative (e.g., WO 02 / 083180, WO 04 / 043493, and WO 05 / 112919). In the present invention, the derivatized functional substance is also simply referred to as a "functional substance."

[0177] In formula (IV), the average ratio (r) of the amide bonds per two heavy chains indicates the average ratio of bonds between immunoglobulin units and azide group-containing modifying groups (number of functional substance-containing groups / immunoglobulin units). Such an average ratio is 1.0 to 3.0, and preferably 1.5 to 2.5. Furthermore, such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1. A lysine residue having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to an Ig may be modified to show an average ratio of the above-mentioned values ​​at one desired position in the two heavy chains (e.g., one position selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering). When each of the two heavy chains is modified at two or more positions, a lysine residue having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to an Ig may be modified to show an average ratio of the above-mentioned values ​​at each of two or more desired positions in the two heavy chains (e.g., one position selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering; preferably positions 246 / 248 and 288 / 290).

[0178] The conjugate of the present invention or a salt thereof can be obtained by reacting the azide group-introduced antibody derivative of the present invention or a salt thereof with a functional substance. Such a reaction can be carried out under conditions (mild conditions as described above) that do not cause denaturation or decomposition (e.g., cleavage of amide bonds) of proteins (immunoglobulins / antibodies). As the functional substance, a substance derivatized to have a cycloalkyne group capable of reacting with an azide group under mild conditions can be used. Such a functional substance can be represented by the following formula (V).

[0179] [ka]

[0180] [During the ceremony, Ring A' represents a ring having a triple bond between carbon atoms; Z represents a functional substance; L represents a bond or a divalent group.

[0181] Ring A' represents a ring having a triple bond between carbon atoms. Ring A' is a monocycle or a condensed ring of a monocycle and another ring. Ring A' may have a substituent. As the monocycle, a homocycle or a heterocycle containing 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 is preferable. More preferably, the monocycle is a homocycle or a heterocycle containing one or more selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom. As the monocycle, a 7- to 10-membered monocycle is preferable, and a 7- to 9-membered monocycle is more preferable. As the monocycle, a non-aromatic monocycle is preferable.

[0182] When ring A' is a fused ring, the other ring fused with the monocycle may be, for example, a cycloalkane, an arene, or a heterocycle. The definitions, examples, and preferred examples of the cycloalkane, arene, and heterocycle are the same as those of the other rings in the fused ring with ring A'.

[0183] The substituents that the ring A' may have are the same as the above-mentioned substituents (i) to (vii), and the preferred ranges are also the same. The number of the substituents is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

[0184] Preferably, ring A' is a 7- to 9-membered monocyclic ring or a condensed ring of a 7- to 9-membered monocyclic ring and another ring. Such a reaction between ring A' and an azide group is also known as a strain-promoted azide-alkyne cyclization (SPAAC) reaction (e.g., Org.Biomol.Chem.2013,11,6439; Angew.Chem.Int.Ed.2015,54,1190; J.Am.Chem.Soc.2004,126,15046; J.Am.Chem.Soc.2008,130,11486; Chem.Commun.2010,46,97). For example, rings of formulas (i'') to (vii'') described in International Publication No. 2017 / 191817 may be used as such ring A.

[0185] In the reaction, the molar ratio of the functional substance to the azide group-introduced antibody derivative or its salt (functional substance / azide group-introduced antibody derivative or its salt) is not particularly limited as it varies depending on factors such as the type of the azide group-introduced antibody derivative or its salt and the functional substance, and the reaction time, but is, for example, at least 2, preferably at least 3, and more preferably at least 5. In order to sufficiently react the functional substance with the thiol group of the azide group-introduced antibody derivative in a short reaction time, a sufficient amount (e.g., an excess amount) of the functional substance can be used relative to the azide group-introduced antibody derivative or its salt.

[0186] The formation of the conjugate or its salt can be confirmed by determining the molecular weight of the specific raw material and the product. Although it depends on the type, it can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. The position selectivity can be confirmed by, for example, peptide mapping. Peptide mapping can be confirmed by, for example, protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. The protease is preferably an endoprotease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of functional substances introduced can be confirmed by, for example, electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The conjugate or a salt thereof can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0187] 6.Applications The compound of the present invention or a salt thereof can highly control the average ratio of bonds between immunoglobulin units and azide group-containing modifying groups (azide group-containing modifying groups / immunoglobulin units) to a desired range (1.0 to 3.0). The compound of the present invention or a salt thereof can also site-selectively modify lysine residues in the heavy chains in the immunoglobulin units. Thus, the present invention provides a reagent for antibody derivatization, comprising the compound of the present invention or a salt thereof.

[0188] The reagent of the present invention may be provided in the form of a composition further containing other components. Such other components include, for example, a solution and a stabilizer (e.g., an antioxidant, a preservative). The solution is preferably an aqueous solution. Examples of the aqueous solution include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), and buffer solutions (e.g., aqueous phosphate solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), with buffer solutions being preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[0189] The antibody intermediate of the present invention or a salt thereof, and the azide group-introduced antibody derivative of the present invention or a salt thereof are useful, for example, as intermediates for preparing a conjugate of an antibody and a functional substance or a salt thereof.

[0190] The conjugate or salt thereof of the present invention is useful, for example, as a medicine or a reagent (e.g., a diagnostic agent, a research reagent). In particular, the conjugate or salt thereof of the present invention, which is regioselectively modified with a functional substance and in which the average ratio of the antibody and the functional substance binding is highly controlled within a desired range (1.0 to 3.0), is useful as a medicine. It has been reported that changing the number and binding position of the drug bound to an antibody-drug conjugate (ADC) changes the pharmacokinetics, the release rate, and the effect of the drug. For these reasons, it is required to control the number and position of the drug to be conjugated in the next-generation ADC. It is believed that if the number and position are constant, the expected efficiency, the variation of the conjugated drug, the lot difference, so-called regulation problems will be solved. Therefore, the conjugate or salt thereof of the present invention can solve such regulation problems.

[0191] 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 a pharma- ceutical acceptable carrier in addition to the conjugate of the present invention or a salt thereof. Examples of the pharma- ceutical acceptable carrier include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, glycerol ... Examples of suitable additives include, but are not limited to, binders such as pyrulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch, disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate, lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben, stabilizers such as citric acid, sodium citrate, and acetic acid, suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate, dispersing agents such as surfactants, diluents such as water, saline, and orange juice, and base waxes such as cacao butter, polyethylene glycol, and white kerosene. The conjugate or salt of the present invention may also have any modification (e.g., PEGylation) that provides stability.

[0192] Formulations suitable for oral administration include liquid preparations in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granules; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0193] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0194] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to which the composition is administered, the drug tolerance, body weight, age, etc. of the subject, but can be determined appropriately. EXAMPLES

[0195] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0196] Example 1: Synthesis of IgG1 modifying reagents (1-1) Synthesis of Modification Reagent (1) (1-1-1) Synthesis of linker intermediate (2)

[0197] [ka]

[0198] 2-Azido-1,3-dimethylimidazolinium hexafluorophosphate (4.0 g, 14.0 mmol) was added to CH2Cl2 (23. The mixture was dissolved in 4 mL of dimethyl-5-aminoisophthalate, and then dimethyl-5-aminoisophthalate (977 mg, 4.67 mmol) and DMAP (1.7 g, 14.0 mmol) were added and stirred at 50°C for 1 hour. After confirming the reaction with LC / MS and TLC (hexane / ethyl acetate = 1 / 1), the reaction solution was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 2 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (2) (1.08 g, 4.59 mmol).

[0199] (1-1-2) Synthesis of linker intermediate (3)

[0200] [ka]

[0201] The linker intermediate 2 (1.08 g, 4.59 mmol) synthesized in (1-1-1) was dissolved in methanol (23 mL), and 1M NaOH aqueous solution (13.8 mL, 13.8 mmol) and THF (23 mL) were added and stirred at room temperature for 2 hours. After confirming the reaction by LC / MS, 2M The reaction solution was acidified with an aqueous HCl solution, then extracted with ethyl acetate and a 2M aqueous HCl solution, and the organic layer was concentrated and dried in vacuum to obtain linker intermediate 3 (953.2 mg, 4.60 mmol).

[0202] (1-1-3) Synthesis of linker (4)

[0203] [ka]

[0204] The linker intermediate 3 (200 mg, 0.97 mmol) synthesized in (1-1-2) was dissolved in DMF (4.85 mL), and pentafluorophenol (893 mg, 4.85 mmol), PyBOP (1.0 g, 1.94 mmol), DIPEA (990 μL, 5.82 mmol), and DMAP (11.9 mg, 0.097 mmol) were added and stirred at room temperature for 2 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), it was extracted with ethyl acetate and 2M HCl aqueous solution, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker (4) (173.4 mg, 0.32 mmol).

[0205] (1-1-4) Synthesis of peptide intermediate (5)

[0206] [ka]

[0207] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0208] The peptide consisting of the amino acid sequence of SEQ ID NO:X1 was synthesized in solid phase according to the method described in International Publication No. 2018 / 199337 (same as below for the synthesis of peptides). Ac-RGNCAYHKGQIIWCTYH-NH2 (SEQ ID NO:2, 50.0 mg, 19.6 μmol, except that the 4th and 14th cysteines each form a disulfide bond within the molecule) and N-Succinimidyl-3(Acetylthio)propionate (48.0 mg, 196 μmol) were dissolved in DMF (1.00 mL), triethylamine (8.2 μL, 58.8 μmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After confirming the reaction by LC-MS, 1.0 M NH2OH and 20 mM EDTA (2 mL) were added to the reaction solution and stirred for another 2 hours. After confirming the reaction by LC-MS, the product was dissolved in 0.05% trifluoroacetic acid aqueous solution, subjected to reversed-phase high performance liquid chromatography using octadodecyl-group chemically bonded silica gel as a packing material, eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain peptide intermediate (5) (29.1 mg, 13.4 μmol).

[0209] MS(ESI)m / z:z=3 727.10[M+3H]3+

[0210] (1-1-5) Synthesis of Modification Reagent (1)

[0211] [ka]

[0212] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0213] The peptide intermediate (5) (29.1 mg, 13.4 μmol) synthesized in (1-1-4) and the linker (4) (72.3 mg, 134 μmol) synthesized in (1-1-3) were dissolved in DMF (1.00 mL), triethylamine (5.6 μL, 40.2 μmol) and DMAP were added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC-MS, the mixture was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above-mentioned modification reagent (1) (9.0 mg, 3.55 μmol).

[0214] MS(ESI)m / z:z=3 845.55[M+3H]3+

[0215] (1-2) Synthesis of Modification Reagent (6) (1-2-1) Synthesis of linker intermediate (7)

[0216] [ka]

[0217] Dimethyl-5-methylisophthalate (830 mg, 4.0 mmol) was dissolved in acetonitrile (100 mL), NBS (783 mg, 4.4 mmol) and benzyl peroxide (40 mg, 0.17 mmol) were added, and the mixture was stirred at 90°C for 20 hours. After confirming the reaction with LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated. It was dissolved in a mixed solvent of hexane / ethyl acetate = 1 / 1 and filtered. The crystals were collected and dried in vacuum to obtain the linker intermediate (7) (630 mg, 2.19 mmol).

[0218] (1-2-2) Synthesis of linker intermediate (8)

[0219] [ka]

[0220] Sodium azide (204 mg, 3.13 mmol) was dissolved in DMF (2.0 mL), and the linker intermediate (7) (300 mg, 1.04 mmol) synthesized in (1-2-1) was added and stirred at 60°C for 3 hours. After confirming the reaction with LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (8) (268 mg, 1.08 mmol).

[0221] (1-2-3) Synthesis of linker intermediate (9)

[0222] [ka]

[0223] The compound (268 mg, 1.08 mmol) synthesized in (1-2-2) was dissolved in methanol (5.4 mL) and THF (5 mL), and 1M NaOH aqueous solution was added and stirred at room temperature for 2 hours. After confirming the reaction with MS, the mixture was acidified with 2M HCl aqueous solution, extracted with ethyl acetate and 2M HCl aqueous solution, and the organic layer was concentrated. The product was dried in vacuum to obtain the linker intermediate (9) (227 mg, 1.00 mmol).

[0224] (1-2-4) Synthesis of linker intermediate (10)

[0225] [ka]

[0226] The linker intermediate (9) (227 mg, 1.00 mmol) synthesized in (1-2-3) was dissolved in DMF (5.0 mL), and pentafluorophenol (920 mg, 5.00 mmol), PyBOP (1.3 g, 2.5 mmol), DIPEA (1.02 mL, 6.0 mmol), and DMAP (12.2 mg, 0.1 mmol) were added and stirred at room temperature for 20 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the mixture was extracted with ethyl acetate and 1M HCl aqueous solution, and the organic layer was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the linker intermediate (10) (395.7 mg, 0.72 mmol).

[0227] (1-2-5) Synthesis of Modification Reagent (6)

[0228] [ka]

[0229] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0230] The peptide intermediate (5) (30.3 mg, 13.9 μmol) synthesized in (1-1-4) and the linker intermediate (10) (76.9 mg, 139 μmol) synthesized in (1-2-4) were dissolved in DMF (1.00 mL), triethylamine (5.8 μL, 41.7 μmol) and DMAP were added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC-MS, the mixture was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as a packing material, and eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the modified reagent (6) (14.4 mg, 5.65 μmol).

[0231] MS(ESI)m / z:z=3 850.20[M+3H]3+

[0232] (1-3) Synthesis of Modification Reagent (11) (1-3-1) Synthesis of linker intermediate (12)

[0233] [ka]

[0234] 5-Methyl-1,3-benzenediacetonitrile (5.0 g, 29.4 mmol) was dissolved in 1 M NaOH aqueous solution (100 mL), DMF was added, and the mixture was stirred at 110°C for 20 hours. After confirming the reaction by LC / MS, the reaction solution was concentrated. The mixture was acidified with 2 M HCl aqueous solution, extracted with ethyl acetate and 2 M HCl aqueous solution, and the organic layer was concentrated. The mixture was dried in vacuum to obtain the linker intermediate (12) (6.0 g, 28.8 mmol).

[0235] (1-3-2) Synthesis of linker intermediate (13)

[0236] [ka]

[0237] The linker intermediate (12) (6.0 g, 28.8 mmol) synthesized in (1-3-1) was dissolved in methanol, and thionyl chloride (21.4 mL, 288 mmol) was added at 0°C and stirred at room temperature for 3 hours. After confirming the reaction by LC / MS, the mixture was extracted with ethyl acetate and sodium bicarbonate water, and the organic layer was concentrated to obtain the linker intermediate (13).

[0238] (1-3-3) Synthesis of linker intermediate (14)

[0239] [ka]

[0240] The compound synthesized in (1-3-2) was dissolved in carbon tetrachloride (28.8 mL), N-Bromosuccinimide (5.6 g, 31.6 mmol) and Benzoylperoxide (157 mg, 0.49 mmol) were added, and the mixture was stirred at 85 ° C for 2 hours. Then, N-Bromosuccinimide (5.6 g, 31.6 mmol) was added and the mixture was stirred at 85 ° C for 30 minutes. After confirming the reaction with LC / MS and TLC (hexane / ethyl acetate = 3 / 1), the crystals were removed and the organic solvent was removed by concentrating under reduced pressure. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then dried in vacuum to obtain the linker intermediate (14) (870 mg, 2.76 mmol).

[0241] (1-3-4) Synthesis of linker intermediate (15)

[0242] [ka]

[0243] The linker intermediate (14) (870 mg, 2.76 mmol) synthesized in (1-3-3) was dissolved in DMF (5.0 mL), sodium azide (540 mg, 8.28 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 3 / 1), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated and dried in vacuum to obtain the linker intermediate (15).

[0244] (1-3-5) Synthesis of linker intermediate (16)

[0245] [ka]

[0246] The linker intermediate (16) synthesized in (1-3-4) was dissolved in THF (3.0 mL), and 1M NaOH aqueous solution (7.0 mL) was added at 0°C and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS, the mixture was acidified with 2M HCl aqueous solution, extracted with ethyl acetate and 2M HCl aqueous solution, and the organic layer was concentrated. The mixture was dried in vacuum to obtain the linker intermediate (16) (637.8 m g, 2.56 mmol).

[0247] (1-3-6) Synthesis of linker intermediate (17)

[0248] [ka]

[0249] The linker intermediate (16) (237 mg, 0.95 mmol) synthesized in (1-3-5) was dissolved in DMF (4.75 mL), and pentafluorophenol (874 mg, 4.75 mmol), PyBOP (1.24 g, 2.38 mmol), DIPEA (969 μL, 5.70 mmol), and DAMP (11.6 mg, 0.095 mmol) were added and stirred at room temperature for 20 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 3 / 1), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the linker intermediate (17) (300 mg, 0.52 mmol).

[0250] (1-3-7) Synthesis of Modification Reagent (11)

[0251] [ka]

[0252] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0253] The peptide intermediate (5) synthesized in (1-1-4) (15.0 mg, 6.93 μmol, (Note that the 4th and 14th cysteines each form a disulfide bond within the molecule) and the linker intermediate (17) (40.2 mg, 69.3 μmol) synthesized in (1-3-6) were dissolved in DMF (1.00 mL), triethylamine (2.89 μL, 20.79 μmol) and DMAP were added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC-MS, the product was dissolved in 0.05% aqueous trifluoroacetic acid solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the modified reagent (11) (1.44 mg, 0.56 μmol).

[0254] MS(ESI)m / z:z=3 854.30[M+3H]3+

[0255] (1-4) Synthesis of Modification Reagent (18) (1-4-1) Synthesis of linker intermediate (19)

[0256] [ka]

[0257] 5-Azidopentanoic acid (800 mg, 5.59 mmol) was dissolved in THF (14 mL), isobutyl chloroformate (808 μL, 6.15 mmol), N-methylmorpholine (873 μL, 8.39 mmol) were added, and the mixture was stirred at 0 ° C for 30 minutes. Hydrazine hydrate (1.36 g, 6.71 mmol) dissolved in 1M NaOH aqueous solution (4 mL) was then added and stirred at room temperature for 3 hours. After concentration under reduced pressure, 1M NaOH aqueous solution was added, the pH of the system was adjusted to pH 10, and the mixture was washed with ethyl acetate. 1M HCl aqueous solution was added to the aqueous layer, the pH of the system was adjusted to 3.0, ethyl acetate was added for washing, and sodium sulfate was added to the obtained ethyl acetate solution. Sodium sulfate was removed by filtration, and the mixture was purified by vacuum concentration column chromatography (dichloromethane:methanol = 10:1). The fraction containing the product was collected and concentrated under reduced pressure to obtain the linker intermediate (19).

[0258] 1H NMR(400MHz,Chloroform-d)δ6.29(d,J=7.7Hz,1H),4.56(td,J=8.0,4.9Hz,1H),3.32(t,J=6.6Hz,2H),2.53 -2.38(m,3H),2.36-2.16(m,3H),2.12(s,2H),1.96(dq,J=14.7,7.6Hz,1H),1.84-1.59(m,4H),1.50(s,9H).

[0259] MS(ESI)m / z:329[M+H]+

[0260] (1-4-2) Synthesis of linker intermediate (20)

[0261] [ka]

[0262] Linker intermediate (19) (2.41 g, 5.59 mmol) was dissolved in dichloromethane (28 mL), thiophenol (627 μL, 6.15 mmol), benzotriazol-1-yloxy (3.49 g, 6.71 mmol), and DIPEA (1.42 mL, 8.39 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate=4:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (20) (2.20 g, 5.23 mmol).

[0263] 1H NMR(400MHz,Chloroform-d)δ7.43(s,5H),6.10(d,J=7.8Hz,1H),4.55(td,J=7.7,4.9Hz,1H),3.31(t,J=6.7Hz,2H),2.87-2.63( m,2H),2.28(dd,J=8.7,5.9Hz,2H),2.16-1.98(m,1H),1.83-1.58(m,4H),1.50(s,9H),1.37-1.22(m,2H),0.91(t,J=6.7Hz,1H).

[0264] MS(ESI)m / z:421[M+H]+

[0265] (1-4-3) Synthesis of linker intermediate (21)

[0266] [ka]

[0267] Linker intermediate (20) (2.20 g, 5.23 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to remove dichloromethane, and water was added and the mixture was freeze-dried to obtain linker intermediate (21) (1.98 g, 5.43 mmol).

[0268] 1H NMR(400MHz,Chloroform-d)δ7.44(s,J=6.3,4.6,2.4Hz,5H),6.76(s,1H),4.62(td,J=7.5,4.9Hz,1H),3.31(t,J=6.6Hz,2H),2.88(qt,J=16.8 ,6.8Hz,2H),2.33(dt,J=12.4,6.8Hz,3H),2.18(dq,J=14.4,7.4Hz,1H),1.74(dq,J=11.8,7.5,6.9Hz,2H),1.63(ddd,J=17.7,10.5,4.8Hz,2H).

[0269] MS(ESI)m / z:365[M+H]+

[0270] (1-4-4) Synthesis of linker intermediate (22)

[0271] [ka]

[0272] The linker intermediate (21) (100 mg, 0.274 mmol) was dissolved in dichloromethane (3 mL), and (40.6 μL, 0.280 mmol), benzotriazol-1-yloxy (150 mg, 0.288 mmol), and DIPEA (70.1 μL, 0.412 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The pH of the system was adjusted to 3 by adding 1M HCl aqueous solution, and dichloromethane was added for dilution. After washing with water and saline, sodium sulfate was added. After removing sodium sulfate by filtration, the mixture was concentrated under reduced pressure and purified by column chromatography (hexane: ethyl acetate = 4:1). The fraction containing the product was collected and concentrated under reduced pressure to obtain the linker intermediate (22) (84.7 mg, 0.171 mmol).

[0273] 1H NMR(400MHz,Chloroform-d)δ7.50-7.38(m,5H),6.33(d,J=8.4Hz,1H),4.78(tdd,J=7.8,4.6,3.0Hz,1H),3.70-3.54(m,2 H),3.32(dt,J=9.1,6.7Hz,2H),2.96-2.67(m,2H),2.30(pd,J=7.1,4.5Hz,2H),1.85-1.60(m,6H),1.49(d,J=2.8Hz,9H).

[0274] MS(ESI)m / z:495[M+H]+

[0275] (1-4-5) Synthesis of linker intermediate (23)

[0276] [ka]

[0277] Linker intermediate (22) (84.7 mg, 0.171 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added, and the mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure to remove dichloromethane, water was added, and the mixture was lyophilized, followed by purification by column chromatography (dichloromethane:methanol=10:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (23) (46.8 mg, 0.107 mmol).

[0278] 1H NMR(400MHz,Methanol-d4)δ7.44(dq,J=2.3,1.5Hz,5H),4.69-4.57(m,1H),3.79-3.67(m,2H), 3.40-3.30(m,2H),2.89-2.71(m,2H),2.44-2.23(m,4H),2.08-1.95(m,1H),1.82-1.61(m,4H).

[0279] MS(ESI)m / z:439[M+H]+

[0280] (1-4-6) Synthesis of Modification Reagent (18)

[0281] [ka]

[0282] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0283] Ac-RGNCAYHKGQIIWCTYH-NH2 (SEQ ID NO:2, 30.9 mg, 14.9 μmol, where the 4th and 14th cysteines are each intramolecularly disulfide bonded) was dissolved in (468 μL), and linker intermediate (23) (46.8 mg, 0.107 mmol) and WSC·HCl (29.7 mg, 0.155 mmol) were added. The mixture was stirred at room temperature for 5 hours, and then eluted by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above modification reagent (18) (15.1 mg, 6.02 μmol).

[0284] (1-5) Synthesis of Modification Reagent (24) (1-5-1) Synthesis of linker intermediate (25)

[0285] [ka]

[0286] tert-Butyl 3-bromo-5-nitrobenzoate (500mg, 1.65mmol) was dissolved in a mixture of 1,4-Dioxane / H2O=4 / 1 (8.25mL), 2-Ethoxycarbonylvinylboronic acid pinacol ester (447mg, 1.98mmol), Pd(dppf)Cl2 (67.8mg, 0.083mmol), and Na2CO3 (438mg, 4.13mmol) were added and stirred at 100℃ for 2 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate=10 / 1), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated. The mixture was eluted with a mixture of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate=10 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the linker intermediate (25) (371.7 mg, 1.16 mmol).

[0287] 1H NMR (400MHz, Chloroform-d) δ=8.69(d,J=1.8,1H),8.43(t,J=2.0,1H),8.35(d,J=1.6,1H),7.67(d,J=16.0 ,1H),6.55(d,J=16.0,1H),4.23(q,J=7.1,2H),1.57(s,9H),1.36-1.22(m,3H).

[0288] (1-5-2) Synthesis of linker intermediate (26)

[0289] [ka]

[0290] Linker intermediate (25) (371.7 mg, 1.16 mmol) was dissolved in methanol (5.8 mL) and ethyl acetate (5.0 mL), Pd / C (5% Pd, 53% HO) (5.0 mol%) was added, and the mixture was stirred under hydrogen for 3 hours. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the mixture was filtered through a membrane filter, and the filtrate was concentrated and dried in vacuum to obtain linker intermediate (26) (320.5 mg, 1.09 mmol).

[0291] 1H NMR (400MHz, Chloroform-d) δ=7.24-7.11(m,1H),6.75(t,J=1.9,1H),4.14(q,J=7.1,2H), 2.90(t,J=7.8,2H),2.61(t,J=7.8,2H),1.58(s,9H),1.25(td,J=7.1,4.5,3H).

[0292] (1-5-3) Synthesis of linker intermediate (27)

[0293] [ka]

[0294] Linker intermediate (26) (320.5 g, 1.09 mmol) was dissolved in CHCl (5.45 mL), 2-Azido-1,3-dimethylimidazoliniumHexafluorophosphate (932 mg, 3.27 mmol) and DMAP (399 mg, 3.27 mmol) were added, and the mixture was stirred for 1.5 hours. After confirming the reaction by TLC (hexane / ethyl acetate = 2 / 1), the reaction solution was concentrated. The mixture was eluted with a mixed solvent of hexane and ethyl acetate, and confirmed by TLC (hexane / ethyl acetate = 2 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (27) (278 mg, 1.18 mmol).

[0295] 1H NMR (400MHz, Chloroform-d) δ=7.60(d,J=1.5,1H),7.50(t,J=1.8,1H),7.02(t,J=2.0,1H),4.14(q,J=7.1, 2H),2.98(t,J=7.7,2H),2.64(t,J=7.7,2H),1.60(s,10H),1.25(t,J=7.1,4H).

[0296] (1-5-4) Synthesis of linker intermediate (28)

[0297] [ka]

[0298] Linker intermediate (27) (278 mg, 1.18 mmol) was dissolved in THF (4.35 mL), and 1M NaOH aqueous solution (1.74 mL) and ethanol (4 mL) were added at 0°C and stirred at room temperature for 1.5 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 2 / 1), 1M HCl aqueous solution (2.61 mL) was added at 0°C to make it acidic. Extraction was performed with ethyl acetate and 1M HCl aqueous solution, and the organic layer was concentrated and dried in vacuum to obtain linker intermediate (28) (246.6 mg, 0.85 mmol).

[0299] 1H NMR (400MHz, Chloroform-d) δ=7.61(d,J=1.5,1H),7.52(t,J=1.8,1H),7.02(t,J=1.9,1H),3.00(t,J=7.7,2H),2.72(t,J=7.7,2H),1.60( s,8H).

[0300] (1-5-5) Synthesis of linker intermediate (29)

[0301] [ka]

[0302] Linker intermediate (28) (246.6 mg, 0.85 mmol) was dissolved in DMF (4.25 mL), and Benzenethiol (86.7 μL, 0.85 mmol), PyBOP (442 mg, 0.85 mmol), and DIPEA (218 μL, 1.28 mmol) were added and stirred at room temperature for 1.5 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 4 / 1), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 4 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (29) (250.9 mg, 0.65 mmol).

[0303] 1H NMR (400MHz, Chloroform-d) δ=7.53(d,J=1.6,1H),7.44(t,J=1.8,1H),7.34(p,J=3.6,5H),6.93(t,J=1.9,1H),3.04-2.80(m,4H),1.53(s,10H).

[0304] (1-5-6) Synthesis of linker intermediate (30)

[0305] [ka]

[0306] Linker intermediate (29) (250.9 mg, 0.65 mmol) was dissolved in a mixed solvent (10 mL) of CHCl / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 4 / 1), the organic solvent was removed by concentration under reduced pressure, and the mixture was dried in vacuum to obtain linker intermediate (30) (215.1 mg, 0.66 mmol).

[0307] 1H NMR (400MHz, Chloroform-d) δ=7.74(s,1H),7.64(d,J=1.8,1H),7.49-7.32(m,4H),7.10(d,J=2.0,1H),3.19-2.88(m,4H).

[0308] (1-5-7) Synthesis of linker intermediate (31)

[0309] [ka]

[0310] Linker intermediate (30) (215.1 mg, 0.66 mmol) was dissolved in DMF (3.3 mL), and TertButyl-2-Sulfanylacetate (97.8 mg, 0.66 mmol), PyBOP (343 mg, 0.66 mmol), and DIPEA (168 μL, 0.99 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction with LC / MS and TLC (hexane / ethyl acetate = 5 / 1), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (31) (247.8 mg, 0.54 mmol).

[0311] 1H NMR (400MHz, Chloroform-d) δ=7.60(d,J=1.6,1H),7.49(t,J=1.9,1H),7.42(p,J=3.7,4H),7.08(d, J=1.9,1H),3.82(d,J=3.1,2H),3.03(dd,J=21.1,6.9,4H),1.50(s,9H).

[0312] (1-5-8) Synthesis of linker intermediate (32)

[0313] [ka]

[0314] Linker intermediate (31) (247.8 mg, 0.54 mmol) was dissolved in a mixed solvent (10 mL) of CHCl / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the organic solvent was removed by concentration under reduced pressure, and the mixture was dried in vacuum to obtain linker intermediate (32) (227.3 mg, 0.56 mmol).

[0315] 1H NMR (400MHz, Chloroform-d) δ=7.70(d,J=1.6,1H),7.42(t,J=1.9,1H),7.41(p,J=3.7,4H),7.01(d,J=1.9,1H),3.67(d,J=3.1,2H),3.20(dd,J=21.1,6.9,4H). MS(ESI)m / z:z=1 402.29[M+H]+

[0316] (1-5-9) Synthesis of Modification Reagent (24)

[0317] [ka]

[0318] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:4.

[0319] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4, 330.0 mg, 7.06 μmol, where the 5th and 34th cysteines each form a disulfide bond within the molecule), linker intermediate (32) (29 mg, 70.6 μmol), and WSC·HCl (13.5 mg, 70.6 μmol) were dissolved in DMF (1.00 mL) and stirred at room temperature for 1 hour. After confirming the reaction by LC-MS, the product was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain modified reagent (24) (5.5 mg, 1.19 μmol).

[0320] MS(ESI)m / z:z=3 928.30[M+3H]3+

[0321] (1-6) Synthesis of Modification Reagent (33) (1-6-1) Synthesis of linker intermediate (34)

[0322] [ka]

[0323] 3-Iodo-5-nitrobenzoic acid (1.0 g, 3.41 mmol) was dissolved in toluene (17 mL) and N,N-dimethylformamide was added. Di-tertbutyl acetal (5 mL, 20.5 mmol) was added and stirred at 80° C. for 2 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated and dried in vacuum to obtain linker intermediate (34) (1.37 g, 3.92 mmol).

[0324] (1-6-2) Synthesis of linker intermediate (35)

[0325] [ka]

[0326] Linker intermediate (34) (1.25 g, 3.58 mmol) was dissolved in a mixed solution (18 mL) of 1,4-Dioxane / H2O = 4 / 1, (E)-Ethoxyethene-2-boronic acid pinacol ester (851 mg, 4.3 mmol), Pd(dppf)Cl2 (146 mg, 0.179 mmol), and Na2CO3 (948 mg, 8.95 mmol) were added and stirred at 100 ° C for 3 hours. After confirming the reaction with LC / MS and TLC (hexane / ethyl acetate = 10 / 1), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 10 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (35) (415 mg, 1.41 mmol).

[0327] (1-6-3) Synthesis of linker intermediate (36)

[0328] [ka]

[0329] The linker intermediate (35) (614 mg, 2.09 mmol) was dissolved in THF (20 mL), 1 M The product was dissolved in an aqueous HCl solution (2.0 mL), 6M aqueous HCl solution (1 mL) was added, and the mixture was stirred at 60°C for 20 hours. After confirming the reaction by TLC (hexane / ethyl acetate = 2 / 1), the mixture was extracted with ethyl acetate and 1M aqueous HCl solution, and the organic layer was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 2 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the linker intermediate (36) (426.4 mg, 1.61 mmol).

[0330] (1-6-4) Synthesis of linker intermediate (37)

[0331] [ka]

[0332] Linker intermediate (36) 426.4 mg, 1.61 mmol) was dissolved in tBuOH (16.1 mL), 2-methyl-2-butene (1.71 mL, 16.1 mmol), 0.85 M NaH2PO4 aqueous solution (11.4 mL, 9.66 mmol), and 0.5 M NaClO2 aqueous solution (16.1 mL, 8.05 mmol) were added, and the mixture was stirred at room temperature for 1.5 hours. After confirming the reaction by LC / MS, 1.0 M NaHSO3 aqueous solution (16.1 mL, 16.1 mmol) was added to stop the reaction. 1 M HCl aqueous solution was added to make it slightly acidic, and the mixture was extracted with ethyl acetate and the organic layer was concentrated. The mixture was dried in vacuum to obtain linker intermediate (37) (591.5 mg, 2.10 mmol).

[0333] (1-6-5) Synthesis of linker intermediate (38)

[0334] [ka]

[0335] Linker intermediate (37) (591.5 mg, 2.10 mmol) was dissolved in DMF, and iodomethane (392 μg, 6.30 mmol) and potassium carbonate (319 mg, 2.31 mmol) were added and stirred for 1.5 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 3 / 1), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was dried in vacuum to obtain linker intermediate (38) (413.5 mg, 1.40 mmol).

[0336] (1-6-6) Synthesis of linker intermediate (39)

[0337] [ka]

[0338] Linker intermediate (38) (413.5 mg, 1.40 mmol) was dissolved in methanol, Pd / C (317 mg, 0.07 mmol) was added, and the mixture was stirred under hydrogen for 1 hour. After confirming the reaction by LC / MS, Pd / C was removed using a membrane filter, and the solvent was concentrated. The mixture was dried in vacuum to obtain linker intermediate (39) (348.4 mg, 1.31 mmol).

[0339] (1-6-7) Synthesis of linker intermediate (40)

[0340] [ka]

[0341] 11-Azido-3,6,9-trioxaundecanoic acid(700 mg, 3.0 mmol) was dissolved in CHCl (15.0 mL), triethylamine (2.09 mL, 15.0 mmol) and pentafluorophenyl trifluoroacetic acid (1.02 mL, 6.0 mmol) were added at 0°C, and the mixture was stirred at 0°C for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 2 / 1), the reaction solution was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 2 / 1). The fractions containing the product were collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the linker intermediate (40) (1.4 g, 3.51 mmol).

[0342] (1-6-8) Synthesis of linker intermediate (41)

[0343] [ka]

[0344] Linker intermediate (39) (348.4 mg, 1.31 mmol) was dissolved in DMF (6.55 mL), and linker intermediate (40) (523 mg, 1.31 mmol), DIPEA (445 μL, 2.62 mmol), and DMAP (16.0 mg, 0.131 mmol) were added. The mixture was added and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS, the mixture was extracted with water and ethyl acetate, and the organic layer was concentrated. The mixture was dried in vacuum to obtain the linker intermediate (41) (429.4 mg, 0.89 mmol).

[0345] (1-6-9) Synthesis of linker intermediate (42)

[0346] [ka]

[0347] Linker intermediate (41) (429.4 mg, 0.89 mmol) was dissolved in THF (4.45 mL), 1M NaOH aqueous solution (2.67 mL, 2.67 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC / MS, the mixture was acidified with 2M HCl aqueous solution, extracted with ethyl acetate, and the organic layer was concentrated. The mixture was dried in vacuum to obtain linker intermediate (42) (412.5 mg, 0.88 mmol).

[0348] (1-6-10) Synthesis of linker intermediate (43)

[0349] [ka]

[0350] Linker intermediate (42) (412.5 mg, 0.88 mmol) was dissolved in DMF (4.40 mL), and Benzenethiol (89.7 μL, 0.88 mmol), PyBOP (458 mg, 0.88 mmol), and DIPEA (226 μL, 1.33 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction with LC / MS and TLC (ethyl acetate), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (ethyl acetate). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (43) (388.3 mg, 0.69 mmol).

[0351] (1-6-11) Synthesis of linker intermediate (44)

[0352] [ka]

[0353] Linker intermediate (43) (388.3 mg, 0.69 mmol) was dissolved in a mixed solvent of CHCl / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (ethyl acetate), the organic solvent was removed and the mixture was dried in vacuum to obtain linker intermediate (44) (382.7 mg, 0.76 mmol).

[0354] (1-6-12) Synthesis of linker intermediate (45)

[0355] [ka]

[0356] Linker intermediate (44) (382.7 mg, 0.76 mmol) was dissolved in DMF (3.8 mL), tBu-2-Sulfanylacetate (112.6 mL, 0.76 mmol), PyBOP (395 mg, 0.76 mmol), and DIPEA (194 μL, 1.14 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction with LC / MS and TLC (ethyl acetate), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (ethyl acetate). The fractions containing the product were collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (45) (350 mg, 0.55 mmol).

[0357] (1-6-13) Synthesis of linker intermediate (46)

[0358] [ka]

[0359] Linker intermediate (45) (350 mg, 0.55 mmol) was dissolved in a mixed solvent of CHCl / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS, the organic solvent was removed, and the mixture was eluted with a mixed solution of dichloromethane and methanol, and each fraction was confirmed by LC / MS. The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain linker intermediate (46) (130.8 mg, 0.23 mmol).

[0360] MS(ESI)m / z:z=1 577.83[M+H]+

[0361] (1-6-14) Synthesis of Modification Reagent (33)

[0362] [ka]

[0363] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:4.

[0364] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4, 30.0 mg, 7.06 μmol, where the 4th and 14th cysteines each form a disulfide bond within the molecule) and linker intermediate (46) 40.7 mg, 70.6 μmol) were dissolved in DMF (1.00 mL), WSC·HCl (13.5 mg, 70.6 μmol) and DIPEA (3.6 μL, 21.2 μmol) were added, and the mixture was stirred at room temperature for 2 hours. After confirming the reaction by LC-MS, the mixture was dissolved in 0.05% trifluoroacetic acid aqueous solution, and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the modified reagent (33) (4.1 mg, 0.85 μmol).

[0365] MS(ESI)m / z:z=3 802.50[M+3H]3+

[0366] (1-7) Synthesis of Modification Reagent (47) (1-7-1) Synthesis of linker intermediate (48)

[0367] [ka]

[0368] 3,3'-Dithiodipropionic acid (608mg, 2.89mmol) was dissolved in dichloromethane (15mL), D-proline tert-butyl hydrochloride (1.32g, 6.36mmol), WSC·HCl (1.39mg, 2.72mmol), 1-hydroxy-7-azabenzotriazole (78.7mg, 0.578mmol), and triethylamine (2.00mL, 14.5mmol) were added, and the mixture was stirred overnight at room temperature. After concentration under reduced pressure, the mixture was diluted with ethyl acetate, washed with water and brine, and sodium sulfate was added. Filtered. After removing sodium sulfate by filtration, the mixture was concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate=1:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (48) (1.06 g, 2.04 mmol).

[0369] 1H NMR(400MHz,Chloroform-d)δ4.40(dd,J=8.6,3.5Hz,2H),3.64-3.49(m,J=8.5,7.9,4H),3.08-2.86(m,4H),2.77( td,J=8.1,7.6,3.3Hz,4H),2.23-1.87(m,8H),1.68(s,2H),1.47(s,16H),1.40-1.24(m,2H),0.90(t,J=6.6Hz,2H). MS(ESI)m / z:517[M+H]+

[0370] (1-7-2) Synthesis of linker intermediate (49)

[0371] [ka]

[0372] Linker intermediate (48) (1.06 g, 2.04 mmol) was dissolved in DMF (5 mL), tris(2-carboxyethyl)phosphine (760 mg, 2.65 mmol) dissolved in water (5 mL) was added, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with ethyl acetate, washed with water and saline, and then sodium sulfate was added. The sodium sulfate was removed by filtration, concentrated under reduced pressure, and then purified by column chromatography (hexane:ethyl acetate = 1:1). The fraction containing the product was collected and concentrated under reduced pressure to obtain linker intermediate (49) (859 mg, 3.70 mmol).

[0373] 1H NMR(400MHz,Chloroform-d)δ4.42(dd,J=8.6,3.5Hz,1H),3.70-3.56(m,2H),3.52(dt,J=9.6,6.9Hz,1H) ,2.84(pd,J=11.1,9.7,6.0Hz,3H),2.75-2.43(m,3H),2.31-1.86(m,5H),1.86-1.64(m,3H),1.48(s,9H). MS(ESI)m / z:260[M+H]+

[0374] (1-7-3) Synthesis of linker intermediate (50)

[0375] [ka]

[0376] Linker intermediate (21) (52.4 mg, 0.144 mmol) was dissolved in dichloromethane (1 mL), and linker intermediate (49) (41.9 mg, 0.158 mmol), benzotriazol-1-yloxy (89.9 mg, 0.173 mmol), and DIPEA (73.5 μL, 0.432 mmol) were added and stirred at room temperature for 1 hour. After concentration under reduced pressure, the mixture was purified by column chromatography (hexane: ethyl acetate = 1:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (50) (38.7 mg, 0.0639 mmol).

[0377] 1H NMR(400MHz,Chloroform-d)δ7.49-7.37(m,5H),6.26(q,J=11.3,9.5Hz,1H),4.72(tt,J=8.1,3.9Hz,1H),4.39(dd,J=8.6,3.3Hz,1H),3.67-3.40( m,2H),3.31(t,J=6.6Hz,2H),3.20(dt,J=14.4,7.1Hz,2H),2.92-2.40(m, 4H),2.30(t,J=6.9,6.2Hz,3H),1.80-1.60(m,4H),1.48(d,J=2.1Hz,9H). MS(ESI)m / z:606[M+H]+

[0378] (1-7-4) Synthesis of linker intermediate (51)

[0379] [ka]

[0380] Linker intermediate (50) (38.7 mg, 0.0639 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to remove dichloromethane, and water was added and the mixture was freeze-dried to obtain linker intermediate (51) (39.8 mg, 0.0639 mmol).

[0381] 1H NMR(400MHz,Chloroform-d)δ7.85(s,3H),7.43(q,J=5.8,4.9Hz,5H),6.82(s,1H),4.80-4.48(m,2H),3.78-3.40(m, 2H),3.31(t,J=6.5Hz,2H),3.19(t,J=6.9Hz,2H),2.94-2.51(m,4H),2.41-2.22(m,4H),2.22-1.87(m,4H),1.68(dp,J =28.1,7.3Hz,4H).

[0382] (1-7-5) Synthesis of linker intermediate (52)

[0383] [ka]

[0384] Linker intermediate (51) (37.7 mg, 0.0686 mmol) was dissolved in dichloromethane (1 mL), pentafluorophenyl trifluoroacetic acid (23.4 mg, 0.137 mmol) and triethylamine (28.6 µL, 0.206 mmol) were added, and the mixture was stirred at room temperature for 1 h. After concentration under reduced pressure, the mixture was purified by column chromatography (hexane: ethyl acetate = 1:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (52) (37.4 mg, 0.0523 mmol).

[0385] MS(ESI)m / z:716[M+H]+

[0386] (1-7-6) Synthesis of Modification Reagent (47)

[0387] [ka]

[0388] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:4.

[0389] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-N The H2 peptide (SEQ ID NO: 4, 22.2 mg, 5.23 μmol, where the 5th and 35th cysteines are each intramolecularly disulfide bonded) was dissolved in DMF (374 μL), and the linker intermediate (52) (37.4 mg, 52.3 μmol) and triethylamine (2.2 μL, 0.0157 mmol) were added. The mixture was stirred at room temperature for 3 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above-mentioned modification reagent (47) (7.76 mg, 1.62 μmmol).

[0390] MS(ESI)m / z:z=3 1595[M+3H]3+,z=4 1196[M+4H]4+,z=5 957[M+5H]5+,z=6 798[M+6H]6+

[0391] (1-8) Synthesis of Modification Reagent (53) (1-8-1) Synthesis of linker intermediate (54)

[0392] [ka]

[0393] Dithioglycolic acid (613 mg, 3.36 mmol) was dissolved in dichloromethane (17 mL), D-proline tert-butyl hydrochloride (1.53 g, 7.40 mmol), WSC·HCl (1.61 mg, 8.40 mmol), 1-hydroxy-7-azabenzotriazole (91.4 mg, 0.672 mmol), and triethylamine (2.33 mL, 16.8 mmol) were added, and the mixture was stirred at room temperature overnight. After concentration under reduced pressure, ethyl acetate was added for dilution, and the mixture was washed with water and saline, and 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 = 1:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain the linker intermediate (54) (1.09 g, 2.23 mmol).

[0394] 1H NMR (400MHz, Chloroform-d) δ4.39(dt,J=8.3,3.6Hz,2H),3.79-3.60(m,8H),2.35-2.14(m,2H),2.14-1.85(m,6H),1.47(s,18H). MS(ESI)m / z:489[M+H]+

[0395] (1-8-2) Synthesis of linker intermediate (55)

[0396] [ka]

[0397] The linker intermediate (54) (1.09 g, 2.23 mmol) was dissolved in DMF (5 mL), and tris(2-carboxyethyl)phosphine (831 mg, 2.90 mmol) dissolved in water (5 mL) was added. After stirring overnight at room temperature, the reaction solution was diluted with ethyl acetate, washed with water and saline, and sodium sulfate was added. Sodium sulfate was removed by filtration. The mixture was concentrated under reduced pressure, and then purified by column chromatography (hexane:ethyl acetate=1:1). The fractions containing the product were collected and concentrated under reduced pressure to give the linker intermediate (55) (867 mg, 3.53 mmol).

[0398] 1H NMR(400MHz,Chloroform-d)δ4.39(ddd,J=14.0,8.4,3.1Hz,1H),3.77-3.53(m ,2H),3.39-3.24(m,2H),2.33-2.06(m,2H),2.06(m,2H),1.49(d,J=4.5Hz,9H). MS(ESI)m / z:246[M+H]+

[0399] (1-8-3) Synthesis of linker intermediate (56)

[0400] [ka]

[0401] Linker intermediate (21) (264 g, 0.723 mmol) was dissolved in dichloromethane (4 mL), and linker intermediate (55) (195 mg, 0.795 mmol), benzotriazol-1-yloxy (452 ​​mg, 0.868 mmol), and DIPEA (370 μL, 2.17 mmol) were added and stirred at room temperature for 1 hour. After concentration under reduced pressure, the mixture was purified by column chromatography (hexane: ethyl acetate = 1:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (56) (180 mg, 0.303 mmol).

[0402] 1H NMR(400MHz,Chloroform-d)δ7.44(p,J=3.5Hz,5H),6.52(m,1H),4.75(td,J=8.5,4.6Hz,1H),4.42(ddd,J=19.2,8.4,3.3Hz,1H),3.8 0-3.49(m,4H),3.39-3.26(m,2H),2.94-2.67(m,2H),2.39-2.27(m,2H),2.27-1.87(m,3H),1.87-1.57(m,8H),1.47(d,J=3.2Hz,9H). MS(ESI)m / z:592[M+H]+

[0403] (1-8-4) Synthesis of linker intermediate (57)

[0404] [ka]

[0405] Linker intermediate (56) (180 mg, 0.303 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to remove dichloromethane, and water was added and the mixture was freeze-dried to obtain linker intermediate (57) (172.6 mg, 0.322 mmol).

[0406] 1H NMR(400MHz,Chloroform-d)δ7.43(q,J=5.7,4.7Hz,5H),4.71(td,J=8.7,4.7Hz,1H),4.53(t,J=5.7Hz,1H),3.97- 3.47(m,4H),3.32(dt,J=12.9,6.5Hz,2H),2.82(qq,J=15.4,7.4,6.1Hz,2H),2.44-2.00(m,8H),1.87-1.59(m,4H). MS(ESI)m / z:536[M+H]+

[0407] (1-8-5) Synthesis of linker intermediate (58)

[0408] [ka]

[0409] Linker intermediate (57) (172.6 mg, 0.322 mmol) was dissolved in dichloromethane (1.6 mL), pentafluorophenyl trifluoroacetic acid (110 mL, 0.644 mmol) and triethylamine (134 μL, 0.966 mmol) were added, and the mixture was stirred at room temperature for 3 h. After concentration under reduced pressure, the mixture was purified by column chromatography (hexane: ethyl acetate = 1:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (58) (158.6 mg, 0.226 mmol).

[0410] 1H NMR(400MHz,Chloroform-d)δ7.47-7.36(m,5H),6.59-6.40(m,1H),4.92-4.78(m,1H),4.73(tt,J=7.6,3.5Hz ,1H),4.01-3.62(m,4H),3.31(qd,J=6.6,2.8Hz,2H),2.99-2.68(m,2H),2.50-2.09(m,8H),1.84-1.56(m,4H). MS(ESI)m / z:702[M+H]+

[0411] (1-8-6) Synthesis of Modification Reagent (53)

[0412] [ka]

[0413] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:4.

[0414] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 peptide (SEQ ID NO: 4, 96.1 mg, 22.6 μmol, where the 5th and 35th cysteines are disulfide-bonded intramolecularly.) was dissolved in DMF (1.59 mL), and linker intermediate (58) (159 mg, 226 μmol) and triethylamine (9.4 μL, 67.8 μmol) were added. The mixture was stirred at room temperature for 3 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above-mentioned modification reagent (53) (49.4 mg, 10.4 μmmol).

[0415] MS(ESI)m / z:z=3 1590[M+3H]3+,z=4 1193[M+4H]4+,z=5 954[M+5H]5+,z=6 795[M+6H]6+

[0416] (1-9) Synthesis of Modification Reagent (59)

[0417] [ka]

[0418] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:4.

[0419] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 peptide (SEQ ID NO: 4, 24.5 mg, 5.77 μmol, where the 5th and 35th cysteines are disulfide-bonded intramolecularly.) was dissolved in DMF (500 μL), and the linker intermediate (60) (41.3 mg, 57.7 μmol) derived from L-proline tert-butyl hydrochloride in the same manner as in the linker intermediate (52) of Example (1-7-5) and triethylamine (2.40 μL, 17.3 mmol) were added, stirred at room temperature for 3 hours, and purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the modification reagent (59) (13.4 mg, 2.80 μmmol).

[0420] MS(ESI)m / z:z=4 1196[M+4H]4+,z=5 957[M+5H]5+,z=6 798[M+6H]6+

[0421] (1-10) Synthesis of Modification Reagent (61)

[0422] [ka]

[0423] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:4.

[0424] The peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4, 14.0 mg, 3.31 μmol, where the 5th and 35th cysteines are disulfide-bonded intramolecularly.) was dissolved in DMF (300 mL), and the linker intermediate (62) (23.2 mg, 33.1 μmol) derived from L-proline tert-butyl hydrochloride in the same manner as in the linker intermediate (58) of Example (1-8-5) and triethylamine (1.40 μL, 9.93 μmol) were added, stirred at room temperature for 3 hours, and purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above modification reagent (61) (6.6 mg, 1.38 μmmol).

[0425] MS(ESI)m / z:z=3 1590[M+3H]3+,z=4 1193[M+4H]4+,z=6 795[M+6H]6+

[0426] (1-11) Synthesis of Modification Reagent (63)

[0427] [ka]

[0428] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:5.

[0429] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 peptide (SEQ ID NO: 5, 100 mg, 23.4 μmol, where the 5th and 35th cysteines are each intramolecularly disulfide bonded.) was dissolved in DMF (1002 μL), and linker intermediate (52) (50.3 mg, 70.2 μmol) and triethylamine (9.8 μL, 70.2 μmol) were added. After stirring at room temperature for 30 minutes, it was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above-mentioned modification reagent (63) (63.7 mg, 13.2 μmmol).

[0430] MS(ESI)m / z:z=3 1603[M+3H]3+,z=4 1203[M+4H]4+,z=5 963[M+5H]5+

[0431] (1-12) Synthesis of Modification Reagent (64)

[0432] [ka]

[0433] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:5.

[0434] The peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO:5, 101 mg, 23.6 μmol, where the 5th and 35th cysteines are disulfide-bonded intramolecularly) was dissolved in DMF (1.01 mL), and the linker intermediate (58) (50.0 mg, 71.3 μmol) and triethylamine (9.9 μL, 71.3 μmol) were added. The mixture was stirred at room temperature for 1.5 hours, and then purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above-mentioned modification reagent (64) (61.2 mg, 12.8 μmmol).

[0435] MS(ESI)m / z:z=3 1599[M+3H]3+,z=4 1200[M+4H]4+,z=5 960[M+5H]5+,z=6 800[M+6H]6+

[0436] (1-13) Synthesis of Modification Reagent (65)

[0437] [ka]

[0438] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:6.

[0439] Ac-MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH2 (SEQ ID NO: 6, 30.0 mg, 7.08 μmol, where the 3rd and 32nd cysteines each form a disulfide bond within the molecule) and linker intermediate (32) (28.4 mg, 70.8 μmol) were dissolved in DMF (1.00 mL), WSC·HCl (13.6 mg, 70.8 μmol) was added, and the mixture was stirred at room temperature for 2 hours. After confirming the reaction by LC-MS, the product was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above peptide (5.9 mg, 1.34 μmol).

[0440] MS(ESI)m / z:z=4 1098.20[M+3H]3+

[0441] (1-14) Synthesis of Modification Reagent (66) (1-14-1) Synthesis of linker intermediate (67)

[0442] [ka]

[0443] The linker intermediate (16) (400 mg, 1.60 mmol) synthesized in Example (1-3-5) was dissolved in DMF (8.0 mL), and Benzenethiol (163 μL, 1.60 mmol), PyBOP (833 mg, 1.60 mmol), DIPEA (408 μL, 2.40 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS, tBu-2-Sulfanylacetate (237 mg, 1.60 mmol), PyBOP (833 mg, 1.60 mmol), DIPEA (408 μL, 2.40 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 3 / 1), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, yielding the linker intermediate (67).

[0444] (1-14-2) Synthesis of linker intermediate (68)

[0445] [ka]

[0446] The linker intermediate (67) was dissolved in a mixed solvent (10.0 mL) of CH2Cl2 / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (dichloromethane / methanol = 10 / 1), the organic solvent was removed. The product was eluted with a mixed solution of dichloromethane and methanol, and each fraction was confirmed by LC / MS. The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, then vacuum dried to obtain the product in the linker. The intermediate (68) was obtained (162.9 mg, 0.39 mmol).

[0447] 1H NMR (400MHz, Chloroform-d) δ=7.39(s,5H),7.23(s,2H),7.19(s,1H),4.36(s,2H),3.92(d,J=8.5,4H),3.72(s,2H). MS(ESI)m / z:z=1 402.29[M+H]+

[0448] (1-14-3) Synthesis of Modification Reagent (66)

[0449] [ka]

[0450] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:6.

[0451] Ac-MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH2 (SEQ ID NO:6, 25.0 mg, 6.2 μmol, where the 3rd and 32nd cysteines each form a disulfide bond within the molecule) and linker intermediate (68) (25.0 mg, 62.0 μmol) were dissolved in DMF (1.00 mL), WSC·HCl (11.9 mg, 62.0 μmol) was added, and the mixture was stirred at room temperature for 2 hours. After confirming the reaction by LC-MS, the mixture was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the modified reagent (66) (8.60 mg, 1.95 μmol).

[0452] MS(ESI)m / z:z=4 1101.70[M+3H]3+

[0453] [Example 2: Modification of anti-HER2 antibody trastuzumab using a modification reagent having an affinity substance and its analysis] (2-1) Modification of anti-HER2 antibody trastuzumab using modification reagents with affinity substances (2-1-1) Modification reaction using modification reagents (1, 6, 11) The modification reagent (1) synthesized in Example (1-1-4) was used to carry out a modification reaction of the anti-HER2 antibody trastuzumab. The modification reagent (1) was dissolved in DMF to a concentration of 30 mM. 500 μg of IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in ammonium acetate buffer (pH 5.5), 6.88 μL of DMF (5.0 mg / mL), 30 mM peptide The reagent was added in an amount of 1.12 μL (10 equivalents relative to the antibody) and stirred at 37° C. for 1 hour to obtain a trastuzumab-peptide conjugate (69).

[0454] In a similar manner, antibody modification reaction was carried out using modification reagent (6) to obtain trastuzumab-peptide conjugate (70).

[0455] In a similar manner, antibody modification reaction was carried out using the modification reagent (11) to obtain a trastuzumab-peptide conjugate (71).

[0456] (2-1-2) Modification reaction using modification reagent (18) The modification reagent (18) synthesized in Example (1-4-6) was used to carry out a modification reaction of the anti-HER2 antibody trastuzumab. The modification reagent (18) was dissolved in DMF to a concentration of 10 mM. 500 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in ammonium acetate buffer (pH 5.5), and 6.88 μL (5.0 mg / mL) of DMF and 3.3 μL (10 equivalents relative to the antibody) of a 10 mM peptide reagent were added and stirred at room temperature for 1 hour to obtain a trastuzumab-peptide complex (72).

[0457] (2-1-3) Modification reaction using modification reagents (24, 33, 65, 66) The modification reagent (24) synthesized in Example (1-5-9) was dissolved in dimethyl sulfoxide to a concentration of 20 mM. 500 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 100 μL (5.0 mg / mL) or 200 μL (2.5 mg / mL) of 0.1 M HEPES buffer (pH 8.0), and 1.69 μL (10 equivalents relative to the antibody) of a 20 mM peptide reagent was added and stirred at room temperature for 1 hour to obtain a trastuzumab-peptide complex (73).

[0458] In a similar manner, antibody modification reaction was carried out using the modification reagent (33) to obtain a trastuzumab-peptide conjugate (74).

[0459] In a similar manner, antibody modification reaction was carried out using the modification reagent (65) to obtain a trastuzumab-peptide conjugate (75).

[0460] In a similar manner, antibody modification reaction was carried out using the modification reagent (66) to obtain a trastuzumab-peptide conjugate (76).

[0461] (2-1-4) Modification reaction using modification reagents (47, 53, 59, 61, 63, 64) The modification reagent (47) synthesized in Example (1-7-6) was dissolved in DMF to a concentration of 20 mM. 500 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 100 μL (5.0 mg / mL) of 50 mM HEPES buffer (pH 8.2), and 1.69 μL (10 equivalents relative to the antibody) of a 20 mM peptide reagent was added and stirred at room temperature for 1 hour to obtain a trastuzumab-peptide complex (77).

[0462] In a similar manner, antibody modification reaction was carried out using the modification reagent (53) to obtain a trastuzumab-peptide conjugate (78).

[0463] In a similar manner, antibody modification reaction was carried out using the modification reagent (59) to obtain a trastuzumab-peptide conjugate (79).

[0464] In a similar manner, antibody modification reaction was carried out using the modification reagent (61) to obtain a trastuzumab-peptide conjugate (80).

[0465] In a similar manner, antibody modification reaction was carried out using the modification reagent (63) to obtain a trastuzumab-peptide conjugate (81).

[0466] In a similar manner, antibody modification reaction was carried out using the modification reagent (64) to obtain a trastuzumab-peptide conjugate (82).

[0467] (2-2) Analysis by ESI-TOFMS ESI-TOFMS analysis of the trastuzumab-peptide conjugate (69) was performed as previously reported (International Publication No. WO2019 / 240287A1). A peak was observed at 148,222 for the raw material trastuzumab. A peak was confirmed for the reaction product at 152,915, where two units of the modification reagent (1) were introduced. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 1. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 1 was 1.9. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio of 1.9) represented by the following structural formula was confirmed.

[0468] [Table 1]

[0469] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (70) was performed, and a peak was confirmed at 152945 for the reaction product, where two units of the modification reagent (6) were introduced. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 2. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 2 was 1.7. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 1.7) represented by the following structural formula was confirmed.

[0470] [Table 2]

[0471] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (71) was performed, and a peak was confirmed at 152973, where two units of the modification reagent (11) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 3. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 3 was 1.8. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio of 1.8) represented by the following structural formula was confirmed.

[0472] [Table 3]

[0473] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (72) was performed, and a peak was confirmed at 153023, where two units of the modification reagent (18) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 4. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 4 was 1.8. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio of 1.8) represented by the following structural formula was confirmed.

[0474] [Table 4]

[0475] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (73) was performed, and a peak was confirmed at 157267 for the reaction product, where two units of the modification reagent (24) were introduced. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 5. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 5 was 1.7. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio of 1.7) represented by the following structural formula was confirmed.

[0476] [Table 5]

[0477] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (74) was performed, and a peak was confirmed at 157619, where two units of the modification reagent (33) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 6. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 6 was 1.5. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio of 1.5) represented by the following structural formula was confirmed.

[0478] [Table 6]

[0479] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (75) was performed, and a peak was confirmed at 161051, where two units of the modification reagent (65) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 7. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 7 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0480] [Table 7]

[0481] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (76) was performed, and a peak was confirmed at 156802, where two units of the modification reagent (66) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 8. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 8 was 1.5. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 1.5) represented by the following structural formula was confirmed.

[0482] [Table 8]

[0483] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (77) was performed, and the reaction product was confirmed to have a peak at 157567, where two units of the modification reagent (47) were introduced. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 9. The DAR peak and %Area in Table 9 were used to calculate the The average peptide / antibody binding ratio obtained was 2.0. Therefore, the production of the antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio: 2.0) was confirmed.

[0484] [Table 9]

[0485] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (78) was performed, and a peak was confirmed at 157540, where two units of the modification reagent (53) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using a DAR calculator (Agilent software), and the results are shown in Table 10. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 10 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0486] [Table 10]

[0487] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (79) was performed, and a peak was confirmed at 157563, where two units of the modification reagent (59) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using the DAR calculator (Agilent software), and the results are shown in Table 11. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 11 was 1.6. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 1.6) represented by the following structural formula was confirmed.

[0488] [Table 11]

[0489] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (80) was performed, and a peak was confirmed at 157543, where two units of the modification reagent (61) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using a DAR calculator (software from Agilent), and the results are shown in Table 12. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 12 was 1.6. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 1.6) represented by the following structural formula was confirmed.

[0490] [Table 12]

[0491] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (81) was performed, and a peak was confirmed at 152784, where two units of the modification reagent (63) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using a DAR calculator (software from Agilent), and the results are shown in Table 13. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 13 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0492] [Table 13]

[0493] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (82) was performed, and a peak was confirmed at 157596, where two units of the modification reagent (64) were introduced, for the reaction product. The peptide / antibody binding ratio was then confirmed using a DAR calculator (Agilent software), and the results are shown in Table 14. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 14 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0494] [Table 14]

[0495] (2-3) Confirmation of heavy chain selectivity by ESI-TOFMS under reducing conditions ESI-TOFMS analysis of the trastuzumab-peptide conjugate (69) under reducing conditions was performed as previously reported (International Publication No. WO2019 / 240287A1). The raw trastuzumab had heavy chain peaks at 50594 and 50755, and a light chain peak at 23439. The reaction products were confirmed to be 52917 and 53079, in which a linker was introduced to the heavy chain, and 23439, the same as the raw material, in the light chain.

[0496] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (70) was performed, and the reaction products were confirmed to be 52956 and 53118, in which a linker was introduced into the heavy chain, and 23439, which was the same as the raw material, in the light chain.

[0497] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (71) was performed, and the reaction products were confirmed to be 52971 and 53133, in which a linker was introduced into the heavy chain, and 23439, which was the same as the raw material, in the light chain.

[0498] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (72) was performed, and the reaction products were confirmed to be 52996 and 53157, which had a linker introduced into the heavy chain, and 23439, which had the same light chain as the raw material.

[0499] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (73) was performed, and the reaction products were confirmed to be 55091 and 55253, which had linkers introduced into the heavy chain, and 23439, which had the same light chain as the raw material.

[0500] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (75) was performed, and the reaction products were confirmed to be 54849 and 55010, in which a linker was introduced into the heavy chain, and 23439, the same as the raw material, in the light chain.

[0501] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (76) was performed, and the reaction products were confirmed to be 54888 and 55050, in which a linker was introduced into the heavy chain, and 23439, the same as the raw material, in the light chain.

[0502] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (77) was performed, and the reaction products were confirmed to be 55268 and 55430, in which a linker was introduced into the heavy chain, and 23439, which was the same as the raw material, in the light chain.

[0503] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (78) was performed, and the reaction products were confirmed to be 55254 and 55416, which had linkers introduced into the heavy chain, and 23439, which had the same light chain as the raw material.

[0504] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (81) was performed. The reaction products confirmed were 55296 and 55458, which had linkers introduced into the heavy chain, and 23439, which had the same light chain as the raw material.

[0505] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide conjugate (82) was performed, and the reaction products were confirmed to be 55281 and 55443, which had a linker introduced into the heavy chain, and 23439, which had the same light chain as the raw material.

[0506] [Example 3: Linker cleavage reaction of trastuzumab-affinity substance complex and its analysis by ESI-TOFMS] (3-1) Linker cleavage reaction of trastuzumab-affinity substance conjugate Using the trastuzumab-peptide conjugate (69), a linker cleavage reaction was carried out using hydroxyamine hydrochloride according to a previous report (International Publication No. WO2019 / 240287A1), to obtain an azide-introduced antibody (83).

[0507] In a similar manner, the linker of the trastuzumab-peptide conjugate (70) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (84).

[0508] In a similar manner, the linker of the trastuzumab-peptide conjugate (71) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (85).

[0509] In a similar manner, the linker of the trastuzumab-peptide conjugate (72) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (86).

[0510] In a similar manner, the linker of the trastuzumab-peptide conjugate (73) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (87).

[0511] In a similar manner, the linker of the trastuzumab-peptide conjugate (74) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (88).

[0512] In a similar manner, the linker of the trastuzumab-peptide conjugate (75) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (87).

[0513] In a similar manner, the linker of the trastuzumab-peptide conjugate (76) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (89).

[0514] Using a similar method, the linker of the trastuzumab-peptide conjugate (77) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (90).

[0515] In a similar manner, the linker of the trastuzumab-peptide conjugate (78) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (90).

[0516] In a similar manner, the linker of the trastuzumab-peptide conjugate (79) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (90).

[0517] In a similar manner, the linker of the trastuzumab-peptide conjugate (80) was cleaved using hydroxylamine hydrochloride to obtain an azide-introduced antibody (90).

[0518] In a similar manner, the linker of the trastuzumab-peptide conjugate (81) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (90).

[0519] In a similar manner, the linker of the trastuzumab-peptide conjugate (82) was cleaved using hydroxylamine hydrochloride to obtain the azide-introduced antibody (90).

[0520] In a similar manner, the linker of the trastuzumab-peptide conjugate (72) was cleaved using methoxyamine hydrochloride to obtain the azide-introduced antibody (91).

[0521] Using a similar method, the linker of the trastuzumab-peptide conjugate (77) was cleaved using methoxyamine hydrochloride to obtain the azide-introduced antibody (92).

[0522] In a similar manner, the linker of the trastuzumab-peptide conjugate (78) was cleaved using methoxyamine hydrochloride to obtain the azide-introduced antibody (92).

[0523] Using a similar method, the linker of the trastuzumab-peptide conjugate (79) was cleaved using methoxyamine hydrochloride to obtain the azide-introduced antibody (92).

[0524] In a similar manner, the linker of the trastuzumab-peptide conjugate (80) was cleaved using methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0525] In a similar manner, the linker of the trastuzumab-peptide conjugate (81) was cleaved using methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0526] In a similar manner, the linker of the trastuzumab-peptide conjugate (82) was cleaved using methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0527] (3-2) Analysis by ESI-TOFMS ESI-TOFMS analysis of the azide-introduced antibody (84) was performed as previously reported (International Publication No. WO2019 / 240287A1), and a peak was confirmed at 148657, where the linker was cleaved.

[0528] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (85) was performed, and a peak was confirmed at 148712, where the linker was cleaved.

[0529] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (86) was performed, and a peak was confirmed at 148755, indicating that linker cleavage had progressed.

[0530] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (87) was performed, and a peak was confirmed at 148679, indicating that linker cleavage had progressed.

[0531] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (89) was performed, and a peak was confirmed at 148706, where linker cleavage had progressed.

[0532] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (90) was performed, and a peak was confirmed at 148775, indicating that linker cleavage had progressed.

[0533] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (91) was performed, and a peak was confirmed at 148779, indicating that linker cleavage had progressed.

[0534] Similarly, ESI-TOFMS analysis of the azide-introduced antibody (92) was performed, and a peak was confirmed at 148790, indicating that linker cleavage had progressed.

[0535] [Example 4: Conjugation of a site-specific azide-introduced trastuzumab with any compound and ESI-TOFMS analysis of the product] (4-1) Conjugation of azide-introduced antibodies with fluorescent substances According to previous reports (International Publication No. WO2019 / 240287A1 and International Publication No. WO2019 / 240288A1), Carboxyrhodamine 110-PEG4-DBCO (Broadpharm) was added to the azide-introduced antibody (83) to obtain the ADC mimic (93).

[0536] Using a similar technique, an ADC mimic (94) was obtained from an azide-incorporated antibody (84).

[0537] Using a similar technique, an ADC mimic (95) was obtained from the azide-introduced antibody (85).

[0538] Using a similar technique, an ADC mimic (96) was obtained from an azide-incorporated antibody (86).

[0539] Using a similar technique, an ADC mimic (97) was obtained from an azide-incorporated antibody (87).

[0540] Using a similar technique, an ADC mimic (98) was obtained from an azide-introduced antibody (88).

[0541] Using a similar technique, an ADC mimic (99) was obtained from an azide-incorporated antibody (89).

[0542] Using a similar method, an ADC mimic (100) was obtained from the azide-introduced antibody (90).

[0543] Using a similar technique, an ADC mimic (101) was obtained from the azide-introduced antibody (91).

[0544] Using a similar technique, an ADC mimic (102) was obtained from the azide-introduced antibody (92).

[0545] Using a similar method, the azide-introduced antibody (83) was reacted with DBCO-MMAE (Abzena) to obtain ADC (103).

[0546] Using a similar method, the azide-introduced antibody (86) was reacted with DBCO-MMAE (Abzena) to obtain ADC (104).

[0547] Using a similar method, the azide-introduced antibody (91) was reacted with DBCO-MMAE (Abzena) to obtain ADC (105).

[0548] From the above, it was confirmed that the compound of the present invention or a salt thereof is useful for producing an antibody-drug conjugate having excellent DAR.

[0549] (Reference Example 1) Synthesis of modification reagent (106) and derivatization into ADC mimic (107) The modification reagent (106) was synthesized as described below, and reacted with the anti-HER2 antibody trastuzumab according to Example 2. The linker was then cleaved according to Example 3, and the resulting reagent was reacted with Carboxyrhodamine 110-PEG4-DBCO (Broadpharm) according to Example 4 to obtain the ADC mimic (107).

[0550] [ka]

[0551] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0552] (Reference Example 2) Synthesis of modification reagent (108) and derivatization into ADC mimic (109) The modification reagent (108) was synthesized as described below, and reacted with the anti-HER2 antibody trastuzumab according to Example 2. The linker was then cleaved according to Example 3, and the resulting reagent was reacted with Carboxyrhodamine 110-PEG4-DBCO (Broadpharm) according to Example 4 to obtain the ADC mimic (109).

[0553] [ka]

[0554] All of the above amino acid sequences are the amino acid sequences of SEQ ID NO:2.

[0555] (Reference Example 3) Modification reaction using azide antibody (111) and its analysis The following azido antibody (111) described previously in International Publication No. 2019 / 240287 (WO2019 / 240287A1) was treated with carboxylamine according to Example 4. The ADC mimic (112) was obtained by reacting with 110-PEG4-DBCO (Broadpharm), and similarly, the ADC mimic (113) was obtained by reacting with DBCO-MMAE (Abzena).

[0556] [ka]

[0557] [Example 5: Evaluation of ADC mimics by stability test using rat plasma] The stability of various ADC mimics in blood was evaluated by analyzing the amount of fluorescent molecules that came off from the ADC mimics when they were incubated in rat blood, as described below.

[0558] (5-1) Plasma stability test ADC mimic was added to 700μL of rat plasma (Charles River) to a concentration of 0.1mg / mL, and then sterilized and filtered. This solution was dispensed into six Eppendorf tubes at 50μL each. Three of the six samples were stored in an incubator set at 37℃ for four days. The remaining three were similarly stored in a freezer at -80℃ for four days. Each sample was added with 100μL of acetonitrile, stirred with a vortex mixer, and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0559] (5-2) Analysis of the amount of shed fluorescent molecules using HPLC analysis The measurement was performed by using liquid chromatography / fluorescence detection to measure the molecular weight of the fluorescence dropped from the ADC mimic. The three samples stored in the freezer in Example 5-1 were taken as Day = 0, and the three samples stored at 37°C in Example 5-1 were taken as Day = 4, and the difference in fluorescence intensity between Day = 4 and Day = 0 was analyzed.

[0560] The results were evaluated as shown in the table below. The ADC mimic synthesized in Example 16(1) was used as a control to compare the fluorescence intensity of the Example compounds, and the ratio of the increase was calculated using the following formula. A lower ratio of the increase indicates higher stability in plasma.

[0561] Increase in fluorescence intensity of fluorescent molecules shed from the ADC mimic of the embodiment = [(fluorescence intensity on Day = 4) - (fluorescence intensity on Day = 0)]

[0562] Increase in fluorescence intensity of fluorescent molecules shed from control ADC mimic = [(fluorescence intensity on Day 4) - (fluorescence intensity on Day 0)]

[0563] Separately, Carboxyrhodamine 110-PEG4-DBCO was used to calculate the correlation between the area of ​​fluorescence intensity by HPLC and the concentration. Using this calculation formula, the increase in fluorescence intensity of each ADC mimic was converted to concentration. The dropout rate was calculated as the ratio of the increase in concentration to the concentration on Day 0, which was taken as 100%.

[0564] [Table 15]

[0565] [Table 16]

[0566] [Table 17]

[0567] Comparative Example 1 (107) and Comparative Example 2 (109) do not correspond to M (a trivalent group that connects a carbon atom in C=O adjacent to M and a carbon atom in C=W via a main chain portion having 3 to 5 carbon atoms). This is because Comparative Examples 1 and 2 connect via a main chain portion having 2 carbon atoms (Comparative Example 1) and a main chain portion having 2 carbon atoms and one nitrogen atom (Comparative Example 2), rather than 3 to 5 carbon atoms.

[0568] As a result, the ADC mimic synthesized in Example 3 was stable, while the ADC mimics synthesized in Comparative Examples 1 and 2 were unstable (Tables 15 to 17).

[0569] [Example 6: Evaluation of ADC by stability test using rat plasma] The blood stability of various ADCs was performed according to Example 5.

[0570] (6-1) Plasma stability test The ADCs synthesized in Example 4-1 and Comparative Example 2 were incubated using rat plasma in the same manner as in Example 5-1, and three samples stored in a freezer and three samples stored at 37°C were prepared for each ADC.

[0571] (6-2) Analysis of the amount of shed payload using HPLC analysis The amount of the dropped payload was measured using liquid chromatography mass spectrometry (including tandem mass spectrometry). The three samples stored in the freezer in Example 6-1 were taken as Day=0, and the three samples stored at 37°C in Example 6-1 were taken as Day=4. The MS intensities of the detected payloads on Day=4 and Day=0 were calculated using extracted ion chromatograms, and the difference between them was analyzed.

[0572] Compared to ADC (112) synthesized from azide antibody (109) previously described (International Publication No. WO2019 / 240287A1), ADCs (103) to (105) synthesized in Example 4 were stable, similar to the results of the comparative study of ADC mimics in Example 5.

[0573] [Example 6: Confirmation of position selectivity of azide-introduced antibody] Peptide mapping was performed on the trastuzumab azide derivative obtained in (4-1) using the following steps.

[0574] (6-1) Peptide mapping of trastuzumab azide derivative (86) Peptide mapping of the trastuzumab azide derivative (86) was performed as previously reported (International Publication No. WO2019 / 240288A). Analysis using LC-MS / MS revealed the presence of a peptide fragment consisting of 33 amino acid residues, THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR, which contains azide modification of lysine residues by trypsin digestion of trastuzumab. Analysis using BioPharma Finder showed that modification of lysine residues at positions 246 and 248 in the EU numbering system was highly selective. These results demonstrated that the trastuzumab azide derivative (86) obtained in (4-1) above was conjugated site-selectively to Lys246 and Lys248 (EU numbering) on ​​the heavy chain of the antibody.

[0575] (6-2) Peptide mapping of trastuzumab azide derivative (91) Peptide mapping of the trastuzumab azide derivative (91) obtained in (4-1) was performed in the same manner as in (6-1). Analysis using LC-MS / MS revealed the presence of a peptide fragment consisting of 33 amino acid residues, THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR, which contains azide modification of lysine residues by trypsin digestion of trastuzumab. Analysis using BioPharma Finder showed that modification of lysine residues at positions 246 and 248 in the EU numbering system was highly selective.

[0576] (6-3) Peptide mapping of trastuzumab azide derivative (90) Peptide mapping of the trastuzumab azide derivative (90) was performed as previously reported (International Publication No. WO2019 / 240288A). Analysis using LC-MS / MS revealed the presence of a peptide fragment of 18 amino acid residues, FNWYVDGVEVHNAKTKPR, which contains azide modification of lysine residues by trypsin digestion of trastuzumab. Analysis using BioPharma Finder showed that modification of lysine residues at positions 288 and 290 in the EU numbering system was highly selective. These results demonstrated that the trastuzumab azide derivative (90) obtained in (4-1) above was conjugated site-selectively to Lys288 and Lys290 (EU numbering) on ​​the heavy chain of the antibody.

[0577] (6-4) Peptide mapping of trastuzumab azide derivative (92) Trastuzumab azide derivative (92) obtained in (4-1) in the same manner as (6-1) Peptide mapping of trastuzumab was performed. Analysis using LC-MS / MS revealed the presence of a peptide fragment of FNWYVDGVEVHNAKTKPR, a peptide consisting of 18 amino acid residues containing azide modification of lysine residues, which was observed upon trypsin digestion of trastuzumab. Analysis using BioPharma Finder also showed that modification of lysine residues at positions 288 and 290 in the EU numbering system was highly selective. These results demonstrated that the trastuzumab azide derivative (92) obtained in (4-1) above was conjugated site-selectively to Lys288 and Lys290 (EU numbering) on ​​the antibody heavy chain.

[0578] Example 7: Site-selective modification of different target regions of IgG1 Fc with IgG1 Fc affinity peptide reagents and synthesis of antibody drug conjugates (7-1) Site-selective modification of anti-HER2 antibody trastuzumab followed by cleavage of the thioester group to produce an azide-introduced antibody derivative The azide-introduced antibody (91) synthesized in Example (3-1) was substituted with 50 mM HEPES buffer (pH 8.2). To this solution, 10 equivalents of the modification reagent (64) synthesized in Example (1-14) relative to the antibody were added and stirred at room temperature for 3 hours. The reaction solution was substituted with 20 mM ammonium acetate buffer. The mass of the obtained antibody was measured by ESI-TOFMS, and a peak of 158,319 was confirmed, which is the result of introducing two binding peptides into the azide-introduced antibody synthesized in (3-1). These results confirmed that the antibody obtained in this Example (7-1) contained modifications of the lysine residues at positions 246 or 248 and 288 or 290 (EU numbering) on ​​the two heavy chains of the antibody.

[0579] (7-2) Site-selective modification of anti-HER2 antibody trastuzumab followed by cleavage of the thioester group to produce an azide-introduced antibody derivative The antibody-peptide complex obtained in Example (7-1) was subjected to a linker cleavage reaction using methoxyamine hydrochloride in the same manner as in Example (3-1) to obtain an azide-introduced antibody. The obtained azide antibody was subjected to ESI-TOFMS analysis, and peaks were confirmed at 149,350, indicating that linker cleavage had progressed and four azide groups had been introduced into the antibody. From these results, it was confirmed that the antibody obtained in this Example (7-2) contains four azide groups (azide group / antibody binding ratio = 4) introduced into the side chains of the lysine residues at positions 246 or 248 and 288 or 290 (EU numbering) on ​​the two heavy chains of the antibody.

[0580] (7-3) Synthesis of ADC with DAR=4 In the same manner as in Example (4-1), the azide-introduced antibody obtained in Example (7-2) was reacted with DBCO-MMAE (Abzena) to obtain an ADC. Therefore, the antibody obtained in this Example (7-2) was confirmed to contain four drugs (DAR=4) introduced into the side chains of the lysine residues at positions 246 or 248 and 288 or 290 (EU numbering) on ​​the two heavy chains of the antibody.

Claims

1. The following formula (I): 【Chemistry 1】 [During the ceremony, X represents a leaving group; the leaving group is R-S (wherein R represents a hydrogen atom or an optionally substituted monovalent phenyl group, and S represents a sulfur atom); Y represents an affinity peptide having a binding region in the CH2 domain of an immunoglobulin unit comprising two heavy chains and two light chains; the immunoglobulin unit is human IgG, The affinity peptide comprises an amino acid sequence selected from the group consisting of the following (1) to (4): (1) RGNCAYHKGQIIWCTYH (SEQ ID NO: 2); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 4); (3) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); and (4) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (SEQ ID NO: 6), M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom; N ３ represents an azide group, La represents a divalent group consisting of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, or a group consisting of a combination of two or more thereof; Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; represents a divalent group consisting of --O-- or a combination of two or more of these; or a salt thereof.

2. 2. The compound or salt thereof according to claim 1, wherein the main chain moiety having 3 to 5 carbon atoms in M ​​is a linear alkylene having 3 carbon atoms.

3. 3. The compound or salt thereof according to claim 1 or 2, wherein the carbonyl group adjacent to La forms an amide bond with the amino group in the side chain of a lysine residue in the affinity peptide.

4. The N-terminal and C-terminal amino acid residues in the affinity peptide may be protected; and The compound or salt thereof according to any one of claims 1 to 3, wherein two thiol groups in the side chains of the two cysteine ​​residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.

5. A reagent for derivatizing an antibody, comprising the compound according to any one of claims 1 to 4 or a salt thereof.

6. The following formula (I'): 【Chemistry 2】 [During the ceremony, X represents a leaving group; the leaving group is R-S (wherein R represents a hydrogen atom or an optionally substituted monovalent phenyl group, and S represents a sulfur atom); X' represents a leaving group having a higher ability to leave than the leaving group X; The leaving group having a higher ability to leave than the leaving group X is a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimide group. is a midyloxy group, M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom; N ３ represents an azide group, La represents a divalent group consisting of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, or a group consisting of a combination of two or more thereof; Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; represents a divalent group consisting of --O-- or a combination of two or more of these; or a salt thereof.

7. The following formula (I″): 【Chemistry 3】 [During the ceremony, X represents a leaving group; the leaving group is R-S (wherein R represents a hydrogen atom or an optionally substituted monovalent phenyl group, and S represents a sulfur atom); OH represents a hydroxy group; M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom; N ３ represents an azide group, La represents a divalent group consisting of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, or a group consisting of a combination of two or more thereof; Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; represents a divalent group consisting of --O-- or a combination of two or more of these; or a salt thereof.

8. The following formula (III): 【Chemistry 4】 [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue in the two heavy chains; the immunoglobulin unit is human IgG, the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering; M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; T represents a monovalent group, which is an optionally substituted hydroxyamino group; W represents an oxygen atom; N ３ represents an azide group, Lb represents a divalent group, The divalent group is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; ), —O—, or a group consisting of a combination of two or more thereof; and the average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0.

9. The following formula (IV): 【Chemistry 5】 [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue in the two heavy chains; the immunoglobulin unit is human IgG, the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering; M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; T represents a monovalent group, which is an optionally substituted hydroxyamino group; W represents an oxygen atom; N represents a nitrogen atom; Lb represents a divalent group, The divalent group is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; ), —O—, or a group consisting of a combination of two or more thereof; Z represents a functional substance; L represents a divalent group; Ring A represents a ring fused to a triazole ring; Ring A is a monocyclic ring or a condensed ring of a monocyclic ring with another ring, and contains a double bond between carbon atoms shared with the triazole; The monocycle is a homocycle or a heterocycle containing one or more atoms 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; the other ring condensed with said monocyclic ring is a cycloalkane, an arene, or a heterocycle; The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0. A conjugate of an antibody and a functional substance, or a salt thereof, comprising a structural unit represented by the following formula:

10. The azide group-introduced antibody derivative or a salt thereof according to claim 8, wherein the main chain moiety having 3 to 5 carbon atoms in M ​​is a linear alkylene having 3 carbon atoms; or the conjugate or a salt thereof according to claim 9.

11. The azide group-introduced antibody derivative according to claim 8 or 10, or the conjugate or salt thereof according to claim 9 or 10, wherein the lysine residue has positional selectivity for the lysine residue at position 246 / 248 of the human IgG heavy chain according to EU numbering.

12. The azide group-introduced antibody derivative according to claim 8 or 10, or the conjugate or salt thereof according to claim 9 or 10, wherein the lysine residue has positional selectivity for the lysine residue at position 288 / 290 of the human IgG heavy chain according to EU numbering.

13. The azide group-introduced antibody derivative or a salt thereof according to any one of claims 8 or 10 to 12, or the conjugate or a salt thereof according to any one of claims 9 to 12, wherein the average ratio r is 1.5 to 2.

5.

14. The azide group-introduced antibody derivative or a salt thereof according to any one of claims 11 to 13, wherein the lysine residues are located at two positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering; or the conjugate or a salt thereof according to any one of claims 11 to 13.

15. The azide group-introduced antibody derivative or a salt thereof, or the conjugate or a salt thereof according to claim 14, wherein the two positions are positions 246 / 248 and 288 / 290.

16. The following formula (III): 【Chemistry 6】 [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue in the two heavy chains; the immunoglobulin unit is human IgG, the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering; M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; T represents a monovalent group, which is an optionally substituted hydroxyamino group; W represents an oxygen atom; N ３ represents an azide group, Lb represents a divalent group, The divalent group is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; ), —O—, or a group consisting of a combination of two or more thereof; The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0. 【Chemistry 7】 [During the ceremony, Ring A' represents a ring having a triple bond between carbon atoms; Z represents a functional substance; L represents a divalent group. The following formula (IV): 【Chemistry 8】 [During the ceremony, Ring A represents a ring fused to a triazole ring; Ring A is a monocyclic ring or a condensed ring of a monocyclic ring and another ring, and has a carbon atom shared with the triazole. including the portion of the double bond between atoms, The monocycle is a homocycle or a heterocycle containing one or more atoms 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; the other ring condensed with said monocyclic ring is a cycloalkane, an arene, or a heterocycle; N represents a nitrogen atom; Ig, M, O, T, W, Lb, and r are the same as those in formula (III); and Z and L are the same as those in formula (V).

17. The following formula (II): 【Chemistry 9】 [During the ceremony, Ig refers to an immunoglobulin unit that includes two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue in the two heavy chains; the immunoglobulin unit is human IgG, the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering; Y represents an affinity peptide having a binding region in the CH2 domain of the immunoglobulin unit, The affinity peptide comprises an amino acid sequence selected from the group consisting of the following (1) to (4): (1) RGNCAYHKGQIIWCTYH (SEQ ID NO: 2); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 4); (3) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); and (4) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (SEQ ID NO: 6), M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom; N ３ represents an azide group, La represents a divalent group consisting of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, or a group consisting of a combination of two or more thereof; Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom; represents a divalent group composed of —O—, or a group consisting of a combination of two or more thereof; the average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0. A method for producing a conjugate of an antibody and a functional substance or a salt thereof according to claim 16, further comprising producing a conjugate of an antibody and a functional substance or a salt thereof comprising a structural unit represented by formula (III) according to claim 16.

18. The following formula (I): 【Chemistry 10】 [During the ceremony, X represents a leaving group; the leaving group is R-S (wherein R represents a hydrogen atom or an optionally substituted monovalent phenyl group, and S represents a sulfur atom); Y is a CH2 domain in an immunoglobulin unit that contains two heavy chains and two light chains. The peptide has a binding region in the the immunoglobulin unit is human IgG, The affinity peptide comprises an amino acid sequence selected from the group consisting of the following (1) to (4): (1) RGNCAYHKGQIIWCTYH (SEQ ID NO: 2); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 4); (3) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); and (4) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (SEQ ID NO: 6), M represents a trivalent group linking a carbon atom in the C═O adjacent to M and a carbon atom in the C═W via a main chain portion having 3 to 5 carbon atoms, said main chain portion being a straight-chain alkylene having 3 carbon atoms, a carbon atom constituting a phenylene having 3 carbon atoms, or a combination of a straight-chain alkylene having 1 or 2 carbon atoms and a carbon atom constituting a phenylene having 3 carbon atoms; O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom; N ３ represents an azide group, La represents a divalent group consisting of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, or a group consisting of a combination of two or more thereof; Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (where R ｄ represents a hydrogen atom. ), —O—, or a divalent group consisting of a group consisting of a combination of two or more of these. ] or a salt thereof is reacted with an antibody containing the immunoglobulin unit, The following formula (II): 【Chemistry 11】 [During the ceremony, Ig denotes the immunoglobulin unit, Y, M, O, S, W, N ３ , La, and Lb are the same as those in formula (I); wherein the average ratio r of the amide bonds per two heavy chains is 1.0 to 3.0.

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