Compounds or their salts, and antibodies obtained therefrom.

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, an antibody obtained thereby, and the like.

Background Art

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

[0003] The heterogeneity of ADCs such as T-DM1 has been a problem since the beginning of their development. That is, since a small molecule drug is randomly reacted with about 70 to 80 lysine residues in an antibody, the drug-antibody ratio (DAR) and the conjugation position are not constant. Usually, in such a random conjugation method, the DAR ranges from 0 to 8, and it has been found that a plurality of antibody drugs with different drug binding numbers are generated. In recent years, it has been reported that when the drug binding number and binding position of an ADC are changed, the pharmacokinetics, the drug release rate, and the effect change. From these facts, it is required to control the number and position of the conjugated drugs in next-generation ADCs. If the number and position are constant, it is considered that problems such as expected efficacy, variations in conjugated drugs, and lot-to-lot differences, so-called regulation problems, are solved.

[0004] While regioselective modification methods for antibodies are being studied worldwide, most of these methods involve genetic engineering or enzymatic modification. Regarding genetic engineering, although regioselectivity and number selectivity can be controlled, 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 ADCs). Furthermore, the long time required to construct antibody expression systems is also a problem.

[0005] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed, enabling regioselective modification of antibodies using chemical synthesis techniques (Patent Document 1). This method successfully modifies antibodies regioselectively 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 ADCs produced by this method, the antibody and drug are linked via a linker containing the peptide portion. The peptide portion has potential immunogenicity and is easily hydrolyzed in the blood. Therefore, there is room for improvement in ADCs produced by this method because they contain a peptide portion in the linker.

[0006] As an improvement to the above-mentioned C-CAP method, a technique has been reported that allows for the preparation of antibodies that do not contain the peptide moiety as a linker and that selectively possess a functional substance (e.g., a drug) by a chemical synthesis method using a predetermined compound containing an affinity peptide (Patent Documents 2-6). Avoiding the use of linkers containing the peptide moiety is desirable in clinical applications. These techniques propose multiple positions in the antibody that can be selectively modified with drugs, corresponding to various amino acid residues in the CH2 and CH3 domains (e.g., lysine residues, tyrosine residues, serine residues, and threonine residues). However, selectively modifying an antibody with a functional substance and controlling the binding ratio between the antibody and the functional substance to a desired range is not always easy. [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 [Overview of the project] [Problems that the invention aims to solve]

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

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

[0010] The inventors have also found that the above-mentioned azide group-containing compounds facilitate regioselective modification of different lysine residues in the heavy chain of immunoglobulin units, and therefore the azide group-containing compounds are highly versatile for regioselective modification of immunoglobulin units. For example, by using an azide group-containing compound having an affinity peptide with a certain amino acid sequence, it is possible to regioselectively modify the lysine residues at positions 246 / 248 of the human IgG heavy chain while highly controlling the average binding ratio between the immunoglobulin unit and the azide group-containing modifying group to the desired range. Furthermore, by using an azide group-containing compound having an affinity peptide with a different type of amino acid sequence, it is possible to regioselectively modify the lysine residues at positions 288 / 290 of the human IgG heavy chain while highly controlling the average binding ratio between the immunoglobulin unit and the azide group-containing modifying group to the desired range.

[0011] The inventors have further discovered that antibodies prepared using compounds represented by formula (I) or salts thereof, in which lysine residues in the heavy chain of immunoglobulin units are modified with azide group-containing modifying groups, and in which the average binding ratio of immunoglobulin units to azide group-containing modifying groups (number of azide group-containing modifying groups / immunoglobulin units) is highly controlled within a desired range (1.0 to 3.0), exhibit excellent stability, thus completing the present invention.

[0012] In other words, the present invention provides compounds or salts thereof as shown below, 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 (where 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 (where 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)RA-(RB-)N (where RA and RB 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 atom. [3] A compound or salt of [1] or [2], wherein the above immunoglobulin unit is a human immunoglobulin unit. [4] Any of the compounds [1] to [3] or a salt thereof, wherein the above immunoglobulin unit is human IgG. [5] A compound or salt thereof of any of [1] to [4], wherein the main chain portion in M, consisting of 3 to 5 carbon atoms, contains a linear alkylene, a ring-constituting carbon atom, or a combination thereof. [6] Any compound or salt thereof from [1] to [5], wherein the main chain linking M and Y has 6 to 20 atoms. [7] Any compound from [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 the lysine residue in the affinity peptide. [8] The affinity peptide is any of the compounds [1] to [7] or a salt thereof, comprising the amino acid sequence (A) below: (A)(X0-3)aC-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X0-3)b (SEQ ID NO: 1) Here, (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 residue, glycine residue, leucine residue, proline residue, arginine residue, valine residue, asparagine residue, glutamic acid residue, or phenylalanine residue. Xaa2 is a tyrosine residue, tryptophan residue, histidine residue, or phenylalanine residue. Xaa3 is a histidine residue, a phenylalanine residue, a tyrosine residue, a tryptophan 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. [9] The compound or a salt thereof according to [8], wherein the affinity peptide containing the amino acid sequence of (A) above contains the amino acid sequence of (1) below: (1) RGNCAYHKGQIIWCTYH (SEQ ID NO: 2).

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

[11] The compound or a salt thereof according to

[10] , wherein the affinity peptide containing the amino acid sequence of (B) above contains 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 (SEQ ID NO: 6).

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

[11] or a salt thereof, in which two thiol groups in the side chains of two cysteine residues (C) in the above-mentioned affinity peptide may be linked by a disulfide bond or via a linker. A reagent for antibody derivatization, comprising a compound represented by the formula (I) or a salt thereof.

[0013] The present invention also provides an antibody intermediate or a salt thereof. An antibody intermediate or a salt thereof, comprising a structural unit represented by the formula (II). The antibody intermediate or a salt thereof according to [1], wherein the antibody is a human antibody. The antibody intermediate or a salt thereof according to [1] or [2], wherein the antibody is human IgG. The antibody intermediate or a salt thereof according to any one of [1] to [3], wherein the above-mentioned 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. The antibody intermediate or a salt thereof according to any one of [1] to [4], wherein the above-mentioned lysine residue has positional selectivity for the lysine residue at position 246 / 248 of the human IgG heavy chain according to EU numbering. The antibody intermediate or a salt thereof according to any one of [1] to [4], wherein the above-mentioned lysine residue has positional selectivity for the lysine residue at position 288 / 290 of the human IgG heavy chain according to EU numbering. The antibody intermediate or a salt thereof according to any one of [1] to [6], wherein the above-mentioned average ratio r is 1.5 to 2.5. The antibody intermediate or a salt thereof according to any one of [1] to [7], wherein the main chain portion consisting of 3 to 5 carbon atoms in M is a linear alkylene, or a ring-constituting carbon atom, or a portion containing a combination thereof. The antibody intermediate or a salt thereof according to any one of [4] to [8], wherein the above-mentioned lysine residue is present 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.

[10] The antibody intermediate or salt of [9], wherein the two positions are at positions 246 / 248 and 288 / 290.

[11] Any antibody intermediate or salt of [1] to

[10] , wherein the main chain linking M and Y has 6 to 20 atoms.

[12] Any antibody intermediate or salt of [1] to

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

[13] Any antibody intermediate or salt thereof from [1] to

[12] , wherein the affinity peptide contains the amino acid sequence (A) below: (A)(X0-3)aC-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X0-3)b (SEQ ID NO: 1) Here, (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 residue, glycine residue, leucine residue, proline residue, arginine residue, valine residue, asparagine residue, glutamic acid residue, or phenylalanine residue. Xaa2 is a tyrosine residue, tryptophan residue, histidine residue, or phenylalanine residue. Xaa3 consists of a histidine residue, a phenylalanine residue, a tyrosine residue, and tryptophan. A residue, an arginine residue, or a glycine residue, Xaa4 is a lysine residue, Xaa5 is a glycine residue, serine residue, asparagine residue, glutamine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, tyrosine residue, tryptophan residue, histidine residue, threonine residue, leucine residue, alanine residue, valine residue, isoleucine residue, or arginine residue. Xaa6 is a glutamine residue, glutamic acid residue, asparagine residue, aspartic acid residue, proline residue, glycine residue, arginine residue, phenylalanine residue, or histidine residue.

[14] An affinity peptide containing the amino acid sequence of (A) above, comprising the amino acid sequence of (1) below, is an antibody intermediate of

[13] or a salt thereof: (1)RGNCAYHKGQIIWCTYH(Sequence ID 2).

[15] Any antibody intermediate or salt thereof from [1] to

[12] , wherein the affinity peptide contains the amino acid sequence of (B) below: (B)PNLNEEQRNARIRSI (Sequence ID 3).

[16] The affinity peptide containing the amino acid sequence of (B) above contains an amino acid sequence selected from the group consisting of (1) to (3) below, the antibody intermediate of

[15] or a salt thereof: (1) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC(Sequence ID 4); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (Sequence ID 5; or (3) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC(Sequence ID 6).

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

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

[0014] The present invention also provides azide group-introduced antibody derivatives or salts thereof. [1] An azide group-introduced antibody derivative or a salt thereof, comprising a structural unit represented by formula (III). [2] An azide group-introduced antibody derivative or a salt thereof of [1], wherein the monovalent group represented by T is an optionally substituted hydroxyamino group. [3] An azide group-modified antibody derivative or salt of [1] or [2], wherein the antibody is a human antibody. [4] An azide group-modified antibody derivative or salt of any of [1] to [3], wherein the antibody is human IgG. [5] A salt of any of the azide group-introduced antibody derivatives of [1] to [4], wherein the main chain portion in M, consisting of 3 to 5 carbon atoms, contains a linear alkylene, a ring-constituting carbon atom, or a combination thereof. [6] An azide group-introduced antibody derivative or salt thereof of any of [1] to [5], wherein the number of atoms in the main chain linking M and Y is 6 to 20. [7] An azide group-introduced antibody derivative or salt of any of [1] to [6], wherein the above-mentioned lysine residue is located 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. [8] An azide group-introduced antibody derivative or salt of any of [1] to [7], wherein the lysine residue has regioselectivity for the lysine residue at positions 246 / 248 of the human IgG heavy chain according to EU numbering. [9] The lysine residue has regioselectivity for the lysine residue at positions 288 / 290 of the human IgG heavy chain according to EU numbering, and is an azide group of any of [1] to [7]. A derivative of an introduced antibody or a salt thereof.

[10] An azide group-introduced antibody derivative or salt thereof from any of [1] to [9], wherein the above average ratio r is 1.5 to 2.5.

[11] An azide group-introduced antibody derivative or salt of any 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 the human IgG heavy chain according to EU numbering.

[12] An azide group-transformed antibody derivative or salt thereof of

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

[0015] The present invention also provides antibody and functional substance conjugates or salts thereof. [1] Conjugates or salts thereof of antibodies and functional substances, comprising a structural unit represented by formula (IV). [2] A conjugate of [1] or a salt thereof, wherein the antibody is a human antibody. [3] A conjugate or salt of [1] or [2], wherein the antibody is human IgG. [4] A conjugate or salt of any of [1] to [3], wherein the main chain portion in M, consisting of 3 to 5 carbon atoms, contains a linear alkylene, a ring-forming carbon atom, or a combination thereof. [5] A conjugate or salt of any of [1] to [4], wherein the main chain linking M and Y has 6 to 20 atoms. [6] A conjugate or salt of any of [1] to [5], wherein the above lysine residue is located 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. [7] A conjugate or salt of any of [1] to [6], wherein the lysine residue has regioselectivity for the lysine residue at positions 246 / 248 of the human IgG heavy chain according to EU numbering. [8] A conjugate or salt of any of [1] to [7], wherein the lysine residue has regioselectivity for the lysine residue at positions 288 / 290 of the human IgG heavy chain according to EU numbering. [9] Any of the conjugates or salts of [1] to [8], wherein the average ratio r is between 1.5 and 2.5.

[10] A conjugate or salt of any of [6] to [9], wherein the lysine residues are located 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.

[11] A conjugate of

[10] or a salt thereof, wherein the two positions are at positions 246 / 248 and 288 / 290.

[0016] The present invention also provides a method for producing antibody intermediates, azide group-modified antibody derivatives, 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 immunoglobulin unit to produce an antibody intermediate or a salt thereof containing a structural unit represented by formula (II); and (2) A method for producing an azide group-introduced antibody derivative or a salt thereof, comprising subjecting the above antibody intermediate or a salt thereof 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). [3] (1) Reacting a compound represented by formula (I) or a salt thereof with an antibody containing the above immunoglobulin unit to produce an antibody intermediate or a salt thereof containing a structural unit represented by formula (II); and (2) Subjecting the above antibody intermediate or a salt thereof 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 (3) A method for producing an antibody and functional substance conjugate 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 an antibody and functional substance conjugate or a salt thereof containing a structural unit represented by formula (IV). [4] A method for producing an azide-modified 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-modified 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-modified antibody derivative or a salt thereof containing a structural unit represented by formula (III); and (2) A method for producing an antibody and functional substance conjugate 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 an antibody and functional substance conjugate or a salt thereof containing a structural unit represented by formula (IV). [6] A method for producing an antibody and functional substance conjugate or 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 an antibody and functional substance conjugate or salt thereof, which contains a structural unit represented by formula (IV). [Effects of the Invention]

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

[0018] [Figure 1] Figure 1 is a schematic diagram (part 1) illustrating 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 of the immunoglobulin unit via an 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 (in the figure, the amino group in the side chain of the lysine residue) via an activated carbonyl group having a leaving group (X) to produce an antibody intermediate or a salt thereof. [Figure 2] Figure 2 is a schematic diagram (part 2) illustrating the concept of modification of an immunoglobulin unit by the compound of the present invention represented by formula (I) or a salt thereof. Cleavage of the thioester group generates an azide group-introduced antibody derivative or a salt thereof. [Figure 3] Figure 3 is a schematic diagram (part 3) illustrating the concept of modification of immunoglobulin units by the compound of the present invention represented by formula (I) or a salt thereof. The reaction of the azide group in the azide-introduced antibody derivative or a salt thereof with the functional substance (Z) generates an antibody and functional substance conjugate or a salt thereof. Such a reaction is known as the Strain-Promoted Azide-Alkyne Cyclization (SPAAC) reaction. [Figure 4] Figure 4 shows an overview of one embodiment of the present invention. [Modes for carrying out the invention]

[0019] 1. Definitions of general terms In this invention, the term "antibody" is defined as follows. The term "immunoglobulin unit" corresponds to the divalent monomer unit that is the basic building block of such an antibody, and is a unit that includes two heavy chains and two light chains. Therefore, the definition, examples, and preferred examples of immunoglobulin units, including their origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, lysine residue position, and regioselectivity, are the same as those for antibodies described below.

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

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

[0022] Any antigen can be used as the antigen for the antibody. For example, such antigens include proteins (including oligopeptides and polypeptides; proteins modified with biomolecules such as sugars (e.g., glycoproteins)), glycans, nucleic acids, and small molecule compounds. Preferably, the antibody may be an antibody that uses a protein as its antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0023] More specifically, the antigen protein of the antibody may be a disease target protein. Examples of disease target proteins 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, Transfeline Receiver, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lysil B Antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 Integrin, 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 diseases CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO( Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

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

[0028] (5) Hereditary 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) Blood disorders 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) and certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, allentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab) Examples include ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab, and specific human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, laxibakumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesitumumab, brodalumab (IgG2), olaratumab) (whereas the IgG subtype is not mentioned, it is assumed to be IgG1).

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

[0035] According to the present invention, specific lysine residues in the heavy chain of an antibody (e.g., lysine residues at positions 246 / 248 or 288 / 290) can be modified regioselectively. In this specification, "regioselectivity" means that, even though specific amino acid residues are not concentrated in specific regions of the antibody, a predetermined structural unit capable of binding to a specific amino acid residue in the antibody is concentrated in a specific region of the antibody. Therefore, expressions related to regioselectivity such as "regioselectively present," "regioselective binding," and "regioselective binding" mean that the possession or binding rate of a predetermined structural unit in a target region containing one or more specific amino acid residues is significantly higher than the possession or binding rate of the same structural unit in a non-target region containing multiple amino acid residues of the same type as the specific amino acid residue in the target region. Such regioselectivity is 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, specific lysine residues 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 readily hydrolyzed in the blood. Therefore, avoiding the use of a linker containing a peptide portion is desirable in clinical applications.

[0036] In this invention, as long as a specific lysine residue in the heavy chain of the antibody is regioselectively modified, specific amino acid residues at other positions may be further regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in International Publications 2018 / 199337, 2019 / 240288, 2019 / 240287, and 2020 / 090979. Such specific amino acid residues can be amino acid residues having easily modifiable side chains (e.g., amino group, carboxyl group, amide group, hydroxyl group, thiol group) (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine ​​residues), but preferably lysine residues having a side chain containing an amino group, tyrosine residues having a side chain containing a hydroxyl group, serine residues, and threonine residues, or cysteine ​​residues having a side chain containing a thiol group, and more preferably lysine residues (i.e., two of the lysine residues at positions 246 / 248, 288 / 290, and 317 may be regioselectively double-modified, or three lysine residues may be regioselectively triple-modified). More preferably, lysine residues located 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 located at 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. As for salts with inorganic acids Examples of salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as salts with ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or an organic acid (e.g., trifluoroacetic acid).

[0038] 2. Compounds or their salts 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 exhibits an affinity peptide having a binding region to the CH2 domain in an immunoglobulin unit containing two heavy chains and two light chains. M represents a trivalent group that connects the carbon atoms in the adjacent C=O and C=W with a main chain portion consisting of 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 indicates an azide group. La indicates a bond or a divalent group. Lb indicates a bond or a divalent group.

[0041] In formula (I) and other formulas presented in connection with the present invention, a hyphen (-) indicates that the two units on either side are covalently bonded. Thus, in formula (I), X is covalently bonded to the carbon atoms constituting the carbonyl group, M is covalently bonded to the carbon atoms constituting the carbonyl group, the carbon atoms constituting C=W, and Lb, S is covalently bonded to the carbon atoms 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 the amino group. Those skilled in the art can appropriately define such a leaving group. Examples of such leaving groups include: (a) RS (where 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 (where 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)RA-(RB-)N (where RA and RB 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 atom.

[0043] Preferably, the leaving group represented by X may be one of the following: (a) RS (where 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 (where 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)RA-(RB-)N (where RA and RB 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 one of the following: (a) RS (where 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 (where 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] More preferably, the leaving group represented by X may be one of the following: (a) RS (where 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 preferred, the leaving group represented by X may be one of the following: (a')RS (where R represents a monovalent aromatic hydrocarbon group which may have substituents (e.g., phenyl), and S represents a sulfur atom).

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

[0048] Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0049] A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure, and whose main chain does not contain a cyclic structure. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched.

[0050] As alkyl groups, alkyl groups having 1 to 12 carbon atoms are preferred, alkyl groups having 1 to 6 carbon atoms are more preferred, and alkyl groups having 1 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkyl groups 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] As the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0052] As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0053] Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0054] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.

[0055] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0056] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0057] As cycloalkynyls, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, and cyclo Examples include pentinyl and cyclohexynyl.

[0058] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0059] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0060] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0061] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups.

[0062] A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. A monovalent heterocyclic group is either a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heterocyclic group preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0063] As monovalent aromatic heterocyclic groups, those having 1 to 15 carbon atoms are preferred, those having 1 to 9 carbon atoms are more preferred, and those having 1 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazonal, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0064] As monovalent non-aromatic heterocyclic groups, non-aromatic heterocyclic groups having 2 to 15 carbon atoms are preferred, non-aromatic heterocyclic groups having 2 to 9 carbon atoms are more preferred, and non-aromatic heterocyclic groups having 2 to 6 carbon atoms are even more preferred. The number of carbon atoms in the above carbon atoms does not include the number of carbon atoms of substituents. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinil, azetidinil, oxetanil, thietanil, pyrrolidinil, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinil, oxazolidinil, piperidinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinil, dihydrooxazinil, tetrahydrooxazinil, dihydropyrimidinil, and tetrahydropyrimidinil.

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

[0066] The number of substituents in the "optionally substituted monovalent hydrocarbon group" and "optionally substituted monovalent heterocyclic group" 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: (i) halogen atom; (ii) Monovalent hydrocarbon group; (iii) monovalent heterocyclic groups; (iv) Aralkir; (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, representing a hydrogen atom or a monovalent hydrocarbon group); or (vii) Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

[0067] The definitions, examples, and preferred examples of halogen atoms, monovalent hydrocarbon groups, and monovalent heterocyclic groups in the above substituents are the same as those for monovalent hydrocarbon groups and monovalent heterocyclic groups described in R, RA, and RB above, respectively.

[0068] An aralkyl refers to an arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyls are as described above. Preferred aralkyls have 3 to 15 carbon atoms. Examples of such aralkyls include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0069] Preferably, the substituents may be: (i) halogen atom; (ii) Alkyl, phenyl, or naphthyl atoms having 1 to 12 carbon atoms; (iii) Aralkyl groups with 3 to 15 carbon atoms; (iv) A complex ring with 5 or 6 members; (v) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra represents a hydrogen atom or an alkyl group with 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 represent a hydrogen atom or an alkyl group with 1 to 12 carbon atoms); or (vii) The same base as those listed in (vii) above.

[0070] More preferably, the substituents may be: (i) halogen atom; (ii) Alkyl atoms having 1 to 12 carbon atoms; (iii) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra represents a hydrogen atom or an alkyl group with 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, representing a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (v) The same base as those listed in (vii) above.

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

[0072] Particularly preferred, the substituents may be: (i) halogen atom; (ii) Alkyl atoms having 1 to 4 carbon atoms; (iii) Ra-O-, Ra-C(=O)-, Ra-OC(=O)-, or Ra-C(=O)-O- (Ra represents a hydrogen atom or an alkyl group with 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, representing a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); or (v) The same base as those listed in (vii) above.

[0073] The affinity peptide represented by Y has a binding domain to the CH2 domain in an immunoglobulin unit comprising two heavy chains and two light chains. Any peptide having a binding domain to the CH2 domain in an immunoglobulin unit can be used as the affinity peptide. The affinity peptide contains an amino acid residue (e.g., lysine residue, proline residue, tryptophan residue, tyrosine residue, serine residue, threonine residue) containing a side chain with a portion (e.g., amino group, hydroxyl group) that can bind to a carbonyl group (C=O) adjacent to Y, and may form an amide bond with a carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue. Preferably, the affinity peptide contains a lysine residue and may form an amide bond with a carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue. Examples of such affinity peptides include those disclosed in International Publication Nos. 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979, as well as various affinity peptides disclosed in the literature cited in these international publications.

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

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

[0076] Affinity peptides containing the amino acid sequence (A) above, or its analog amino acid sequence, have a binding domain at the same site in the CH2 domain of the immunoglobulin unit, as can be understood from their conserved portion. Therefore, compounds having affinity peptides containing the analog amino acid sequence above, like compounds having affinity peptides containing the amino acid sequence (A) above, can bind to the same site in the CH2 domain of the immunoglobulin unit, regioselectively modifying the lysine residue at positions 246 / 248 of the human IgG heavy chain, while highly controlling the average binding ratio between the immunoglobulin unit and the azide group-containing modifying group to a desired range. The affinity peptides preferably contain the amino acid sequence (A) above, and more preferably contain the amino acid sequence (1) below: (1)RGNCAYHKGQIIWCTYH(Sequence ID 2).

[0077] In another embodiment, the affinity peptide may include the amino acid sequence (B) below, or an analog amino acid sequence thereof: (B)PNLNEEQRNARIRSI (Sequence ID 3).

[0078] Furthermore, examples of the amino acid sequence of (B) above, or its analog amino acid sequence, include the following: (1) The amino acid sequences of (a) to (d) described in International Publication No. 2018 / 199337 (e.g., the amino acid sequences of sequence numbers 61 to 72 and 92 described in International Publication No. 2018 / 199337); (2) The amino acid sequences of (a) to (b) described in International Publication No. 2019 / 240288 (e.g., the amino acid sequences of Sequence IDs 5 to 8, 11 to 14, 37 to 45, 97, 98, and 100 described in International Publication No. 2019 / 240288); (3) The amino acid sequences of (1) to (9) described in International Publication No. 2020 / 090979 (e.g., the amino acid sequences of sequence numbers 5 to 10, 22, 23, and 51 to 53 described in International Publication No. 2020 / 090979).

[0079] Affinity peptides containing the amino acid sequence of (B) above, or its analog amino acid sequence, have a binding domain at the same site in the CH2 domain of the immunoglobulin unit, as can be understood from their conserved portion. Therefore, compounds having affinity peptides containing the analog amino acid sequence above, like compounds having affinity peptides containing the amino acid sequence of (B) above, can bind to the same site in the CH2 domain of the immunoglobulin unit, regioselectively modifying the lysine residue at positions 288 / 290 of the human IgG heavy chain, while highly controlling the average binding ratio between the immunoglobulin unit and the azide group-containing modifying group to a desired range. The affinity peptides preferably contain the amino acid sequence of (B) above, and more preferably contain any of the following amino acid sequences (1) to (3): (1) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC(Sequence ID 4); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (Sequence ID 5; or (3) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC(Sequence ID 6).

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

[0081] [ka]

[0082] The dashed lines in the carbonyl group-containing linker shown above represent the bond portion with the thiol group. This linker is more stable to reduction reactions and the like than a normal disulfide bond. Such peptides can be prepared, for example, by the method described in International Publication No. 2016 / 186206.

[0083] The amino acids constituting the affinity peptide may be either L-forms or D-forms, but L-forms are preferred (in the examples, all amino acid residues constituting the peptide are L-forms). The affinity peptide may be modified with a crosslinking agent to link a specific amino acid residue to a compound of formula (I) or a salt thereof. Examples of such specific amino acid residues include lysine residues, aspartic acid residues, and glutamic acid residues, but lysine residues are preferred. Examples of crosslinking agents include DSG (disuccinimidyl Examples of crosslinking agents include those containing two or more succinimidyl groups, such as glutarate (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate); those containing two or more imido acid portions, such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2HCl), and DMS (dimethyl suberimidate·2HCl); and those having SS bonds, such as DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., International Publication No. 2016 / 186206).

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

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

[0086] The main chain portion, consisting of 3 to 5 carbon atoms, is composed of a chain-like structure, a cyclic structure, or a combination thereof. If the main chain is a chain-like 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-like structure. On the other hand, if the main chain is a structure that includes a cyclic structure, the number of atoms in the main chain can be determined by counting a predetermined number of atoms that constitute the cyclic structure as the number of atoms in the main chain. Specifically, the number of atoms in the main chain in a cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (see, for example, the bolded paths in (a) to (d) below). If the main chain is a structure that includes a combination of chain-like and cyclic structures, the number of atoms in the main chain can be determined by adding the number of atoms in the chain-like 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 coupling. In case (a), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 2. In case (b), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 3. In case (c), since both paths are the shortest paths (equidistances), the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4. In case (d), since the condensation site is the shortest path, the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4.

[0089] The main chain portion, consisting of 3 to 5 carbon atoms, has a first carbon atom adjacent to M (carbon in C=O). Since it connects the elementary atom (C) to the second carbon atom adjacent to M (the carbon atom in C=W), it can be understood as a divalent group in relation to these first and second carbon atoms. On the other hand, M is a trivalent group that bonds not only to these 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 the main chain portion (a divalent group) consisting of 3 to 5 carbon atoms. Here, the main chain portion (a 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) Divalent cyclic hydrocarbon groups, and divalent groups consisting of one or two divalent linear hydrocarbon groups having 1 to 4 carbon atoms linked together; (3) Divalent cyclic hydrocarbon groups; and (4) A divalent group formed by the linkage of two divalent cyclic hydrocarbon groups (i.e., a divalent bicyclo structure).

[0090] Linear hydrocarbon groups with 3 to 5 carbon atoms are linear alkylenes, linear alkenylenes, or linear alkynylenes. Straight-chain alkylenes with 3 to 5 carbon atoms are n-propylene, n-butylene, or n-pentylene. Straight-chain alkenylenes with 3 to 5 carbon atoms are n-propynylene, n-butenylene, or n-pentenylene. Straight-chain alkynylenes with 3 to 5 carbon atoms are n-propynylene, n-butynylene, or n-pentynylene. As a divalent linear hydrocarbon group having 3 to 5 carbon atoms, linear alkylenes having 3 to 5 carbon atoms are preferred.

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

[0092] Divalent linear hydrocarbon groups with 1 to 4 carbon atoms are linear alkylenes with 1 to 4 carbon atoms, linear alkenylenes with 2 to 4 carbon atoms, or linear alkynylenes with 2 to 4 carbon atoms. Straight-chain alkylenes with 1 to 4 carbon atoms include methylene, ethylene, n-propylene, or n-butylene. Straight-chain alkenylenes with 1 to 4 carbon atoms are ethyleneylene, n-propynylene, or n-butenylene. Straight-chain alkynylenes with 1 to 4 carbon atoms are ethynylene, n-propynylene, or n-butynylene. As for divalent linear hydrocarbon groups with 1 to 4 carbon atoms, linear hydrocarbon groups with 1 to 4 carbon atoms Chelen is preferred.

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

[0094] In certain embodiments, the main chain portion of M consisting of 3 to 5 carbon atoms may include a linear alkylene, a ring-forming carbon atom, or a combination thereof. Therefore, the main chain portion (divalent group) consisting of 3 to 5 carbon atoms may consist of the following: (1') A straight-chain alkylene with 3 to 5 carbon atoms; (2') Divalent cyclic hydrocarbon groups, and divalent groups consisting of one or two linear alkylenes with 1 to 4 carbon atoms linked together; (3')divalent cyclic hydrocarbon group; and (4') A divalent group formed by the linking of two divalent cyclic hydrocarbon groups (i.e., a divalent bicyclo structure).

[0095] The main chain portion (divalent group) consisting of 3 to 5 carbon atoms is preferably (1') to (3') among (1') to (4') above, 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) consisting of 3 to 5 carbon atoms, represented by the following formula (i) or (ii):

[0096] [ka]

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

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

[0099] [ka]

[0100] [During the ceremony, m represents an integer from 0 to 4. k represents an integer between 0 and 4. Ph indicates phenylene. b1 represents a bond to a carbon atom in the C=O region adjacent to M. b2 represents a bond to the carbon atom in the C=W bond adjacent to M. Ph can bond to two linear alkylenes at the meta, ortho, or para positions. When the main chain portion (divalent group) consisting of 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 in the main chain portion (divalent group) consisting of 3 to 5 carbon atoms represented by formula (ii').

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

[0102] [ka]

[0103] [During the ceremony, m represents an integer between 0 and 2. k represents an integer between 0 and 2. b1 represents a bond to a carbon atom in the C=O region adjacent to M. b2 represents a bond to the carbon atom in the C=W bond adjacent to M. When the main chain portion (divalent group) consisting of 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 in the ortho position relative to the two alkylenes) in the main chain portion (divalent group) consisting of 3 to 5 carbon atoms represented by formula (ii'').

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

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

[0106] La is present in antibody intermediates produced by the compound represented by formula (I), but not in azide-modified antibody derivatives produced from the antibody intermediate, nor in conjugates of antibodies and functional substances. Therefore, the stability of La is unlikely to be a problem in antibodies produced by the compound represented by formula (I).

[0107] Lb is a portion of antibody intermediates, azide-modified antibody derivatives, and conjugates of antibodies and functional substances produced by the compound represented by formula (I). In addition, Lb acts as a linker that connects the antibody and the azide group in azide-modified antibody derivatives.

[0108] The main chain of 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- (where Rd represents a hydrogen atom or substituent), -O-, -S-, or 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 number of atoms constituting the main chain of the divalent group represented by La and Lb is preferably 1 to 17, more preferably 1 to 15. Furthermore, the lower limit of the number of atoms constituting the main chain of 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 for the number of atoms constituting the main chain in a divalent group represented by La and Lb may be 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less. If the main chain has a cyclic structure, the number of atoms in the main chain of the cyclic structure can be determined as described above.

[0109] Divalent linear hydrocarbon groups are linear alkylenes, linear alkenylenes, or linear alkylenes. The linear alkylenes are linear alkylenes having 1 to 6 carbon atoms, with linear alkylenes having 1 to 4 carbon atoms being preferred. Examples of linear alkylenes include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. Linear alkenylenes are linear alkenylenes having 2 to 6 carbon atoms, with linear alkenylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkenylenes include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. Linear alkylenes are linear alkylenes having 2 to 6 carbon atoms, with linear alkylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkylenes include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. As the divalent linear hydrocarbon group, linear alkylenes are preferred.

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

[0111] A divalent heterocyclic group is either a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms. As for the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As for the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxylandiyl, aziridindiyl, azetidinediyl, oxetanediyl, thietandiyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. A divalent aromatic heterocyclic group is preferred as the divalent 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 the lysine residue in the affinity peptide.

[0113] In certain embodiments, the main chain of the divalent group represented by Lb may not contain a cyclic structure, for ease of synthesis and other reasons. Therefore, the main chain of the divalent group represented by Lb may consist of a divalent linear hydrocarbon group, -C(=O)-, -C(=S)-, -NRd- (where Rd represents a hydrogen atom or substituent), -O-, -S-, or a combination of two or more of these (e.g., 2, 3, 4, or 5). Preferably, from the viewpoint of ensuring stability in 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 of 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 substituents, preferably 1 to 3, and more preferably 1 or 2 substituents. Furthermore, the Rd attached to the main chain in the first and second linkers are substituents. Examples of such substituents include: (i') Halogen atom; (ii') Monovalent hydrocarbon group; (iii') Aralkir; (iv') 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, representing a hydrogen atom or a monovalent hydrocarbon group). (vii') Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

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

[0116] Preferably, the substituents may be: (i') Halogen atom; (ii') Alkyl, phenyl, or naphthyl atoms having 1 to 12 carbon atoms; (iii') Aralkyl groups with 3 to 15 carbon atoms; (iv') A complex ring with 5 or 6 members; (v')Re-O-, Re-C(=O)-, Re-OC(=O)-, or Re-C(=O)-O- (Re represents a hydrogen atom or an alkyl group with 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, representing a hydrogen atom or an alkyl group with 1 to 12 carbon atoms); or (vii') The same base as those listed above (vii').

[0117] More preferably, the substituents may be: (i') Halogen atom; (ii') Alkyl atoms with 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 with 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, representing a hydrogen atom or an alkyl group with 1 to 12 carbon atoms); or (v') The same base as those listed above (vii').

[0118] More preferably, the substituents may be: (i') Halogen atom; (ii') Alkyl atoms with 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 with 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, representing a hydrogen atom or an alkyl group with 1 to 6 carbon atoms); or (v') The same base as those listed above (vii').

[0119] Particularly preferred, the substituents may be: (i') Halogen atom; (ii') Alkyl atoms with 1 to 4 carbon atoms; (iii') Re-O-, or Re-C(=O)- (Re represents a hydrogen atom or an alkyl group with 1 to 4 carbon atoms). (iv')NRfRg-(Rf and Rg are the same or different, representing a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); or (v') The same base as those listed above (vii').

[0120] In certain embodiments, the compound represented by formula (I) can be defined by the number of atoms in 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 in the main chain linking M and Y may be 6 to 20. Compounds represented by formula (I) having such a number of atoms in the main chain linking M and Y are easy to synthesize. Furthermore, it facilitates highly precise control of the average binding ratio between the antibody and the azide group-containing modifying group within a desired range, and offers excellent versatility for regioselective modification of immunoglobulin units. The number of atoms in the main chain linking M and Y may also be 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, or 8-10.

[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 salt thereof in which the Y portion of the compound or salt thereof represented by formula (I) is substituted with a leaving group (preferably a leaving group with a higher ability to leave than X, or a hydroxyl group), and then reacting the synthesized compound or salt thereof with an affinity peptide. For example, such a reaction can be carried out at a suitable temperature (e.g., about 15 to 200°C) in a suitable reaction system (e.g., in an organic solvent, an aqueous solution, or a mixed solvent). 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 the Y portion is substituted with a leaving group may be the compound represented by formula (I') or a salt thereof.

[0123] [ka]

[0124] [During the ceremony, X represents a leaving group, X' indicates a leaving group with a higher ability to leave than leaving group X. M represents a trivalent group that connects the carbon atoms in the adjacent C=O and C=W with a main chain portion consisting of 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 indicates an azide group. La indicates a bond or a divalent group. Lb indicates 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 that has a higher ability to leave than the leaving group X, and examples include the pentafluorophenyloxy group, the tetrafluorophenyloxy group, the p-nitrophenyloxy group, and the 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 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 its salt is useful, for example, as a synthetic intermediate for efficiently producing the compound represented by formula (I) or its salt.

[0128] The compound represented by formula (I') or its salt can be prepared from the compound represented by formula (I'') or its salt.

[0129] [ka]

[0130] [During the ceremony, X represents a leaving group, OH indicates a hydroxyl group. M represents a trivalent group that connects the carbon atoms in the adjacent C=O and C=W with a main chain portion consisting of 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 indicates an azide group. La indicates a bond or a divalent group. Lb indicates 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 or sulfur atom), La (bond, or divalent group), and Lb (bond, or divalent group) in formula (I'') are the same as those in the formula above.

[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 carboxyl group modifying reagents include pentafluorophenylating reagents (e.g., pentafluorophenyl trifluoroacetate), tetrafluorophenylating reagents (e.g., tetrafluorophenyl trifluoroacetate), p-nitrophenylating reagents (e.g., p-nitrophenyl trifluoroacetate), and N-succinimidylating reagents (e.g., N-succinimidyl trifluoroacetate). For example, such reactions can be carried out in a suitable organic solvent system (e.g., an organic solvent containing alkyl halides such as CH2Cl2 (e.g., methyl halides) and amines such as triethylamine) at a suitable 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 its salt is useful as a synthetic intermediate for efficiently producing, for example, the compound represented by formula (I') or its salt. Alternatively, the compound represented by (I'') or its salt is also useful as a synthetic intermediate for directly and efficiently producing the compound represented by formula (I) or its salt.

[0134] The compound represented by formula (I'') can be obtained by various synthesis methods shown in the examples.

[0135] The formation of the above-mentioned series of compounds or their salts can be confirmed by methods such as electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific molecular weights of the raw materials and products. Such compounds or their salts can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above 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 represents an immunoglobulin unit containing two heavy chains and two light chains, and forms an amide bond with an adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. Y represents an affinity peptide having a binding region to the CH2 domain in the immunoglobulin unit. M represents a trivalent group that connects the carbon atoms in the adjacent C=O and C=W with a main chain portion consisting of 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 indicates an azide group. La indicates a bond or a divalent group. Lb indicates 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 or sulfur atom), La (bond or divalent group), and Lb (bond or divalent group) in formula (II) are the same as those in the formula above.

[0140] In formula (II), the average ratio (r) of the above amide bonds per two heavy chains represents 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 higher, more preferably 1.7 or higher, even more preferably 1.8 or higher, and particularly preferably 1.9 or higher. Such an average ratio may also be preferably 2.4 or lower, more preferably 2.3 or lower, even more preferably 2.2 or lower, and particularly preferably 2.1 or lower. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1. Lysine residues having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to Ig may be modified to show an average ratio of the above values ​​at one desired position in the two heavy chains (for example, 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). Furthermore, if each of the two heavy chains is modified at two or more positions, lysine residues having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to Ig may be modified to show an average ratio of the above values ​​at each of the two desired positions in the two heavy chains (for example, 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 of the present invention can be obtained by reacting the compound or salt of the present invention with an antibody containing the above-mentioned immunoglobulin unit. In the reaction, first, the compound or salt of the present invention is mixed with the antibody. This allows the compound or salt of the present invention to associate with the antibody via an affinity peptide having affinity for the antibody. Next, after the association with the antibody, the carbonyl group (XC=O) having a leaving group (X) can regioselectively react with the amino group in the side chain of the lysine residue in the heavy chain of the antibody. Through this reaction, the amino group and the carbon atom of the carbonyl group bond, and the leaving group (X) is removed from the carbonyl group, thereby obtaining the antibody intermediate or salt of the present invention. The molar ratio of the compound or salt of the present invention to the antibody in the reaction (compound or salt of the present invention / antibody) is not particularly limited as it varies depending on factors such as the type of compound or salt 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 reactions can be carried out appropriately under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds). For example, such reactions can be carried out appropriately The reaction can be carried out at room temperature (e.g., about 15-30°C) in a reaction system, such as a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, and more preferably 6.0-8.0. The buffer may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, GJLBernardes et al., Chem. Rev., 115, 2174 (2015); GJLBernardes et al., Chem. Asian. J., 4, 630 (2009); BGDavies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0143] The formation of antibody intermediates or their salts can be confirmed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, depending on the specific molecular weight of the raw materials and products. Regioselectivity can be confirmed by peptide mapping, for example. Peptide mapping can be performed by protease treatment and mass spectrometry, for example. Endoproteases are preferred as proteases. 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 by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The antibody intermediates or their salts can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0144] 4. Azide group-modified antibody derivatives or their salts The present invention provides an azide group-introduced antibody derivative or a salt thereof, comprising a structural unit represented by the following formula (III).

[0145] [ka]

[0146] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and forms an amide bond with an adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. M represents a trivalent group that connects the carbon atoms in the adjacent C=O and C=W with a main chain portion consisting of 3 to 5 carbon atoms. O represents an oxygen atom. T indicates a monovalent group. W represents an oxygen atom or a sulfur atom. N3 indicates an azide group. Lb indicates 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 or sulfur atom), and Lb (bond, or divalent group) in formula (III) are the same as those in the formula above.

[0148] T represents a monovalent group. T can be generated by a cleavage reaction between the carbon atom and the sulfur atom in the substructure "C(=W)-S" of the compound represented by formula (II). Therefore, an appropriate monovalent group can be used for T depending on the type of cleavage reaction.

[0149] In certain embodiments, the monovalent group represented by T may be a substituted hydroxyamino group. A substituted hydroxyamino group can be represented by the following formula (α).

[0150] [ka]

[0151] [During the ceremony, R1 and R2 represent, either identically or differently, a hydrogen atom or an optionally substituted monovalent hydrocarbon group. The definitions, examples, and preferred examples of monovalent hydrocarbon groups and substituents are as described above.

[0152] In formula (III), the average ratio (r) of the above amide bonds per two heavy chains represents 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 higher, more preferably 1.7 or higher, even more preferably 1.8 or higher, and particularly preferably 1.9 or higher. Such an average ratio may also be preferably 2.4 or lower, more preferably 2.3 or lower, even more preferably 2.2 or lower, and particularly preferably 2.1 or lower. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1. Lysine residues having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to Ig may be modified at one desired position in the two heavy chains (for example, 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) to show the average ratio of the above values. Also, if each of the two heavy chains is modified at two or more positions, lysine residues having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to Ig may be modified at two or more desired positions in the two heavy chains (for example, 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) to show the average ratio of the above values. One position selected from the group; preferably, at each position in positions 246 / 248 and 288 / 290, it may be modified to show the average ratio of the above 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 that do not cause denaturation or degradation of proteins (immunoglobulin / antibody) (e.g., cleavage of amide bonds) (mild conditions as described above). More specifically, the thioester can be cleaved by stirring for an appropriate time (e.g., 1 hour) in a hydroxylamine hydrochloride solution in the range of pH 4.0 to 8.0 and 10 mM to 10 M (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160).

[0154] Confirmation of the formation of azide-modified antibody derivatives or their salts can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw materials and molecular weight of the product. Confirmation of regioselectivity can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Confirmation of the number of thiol groups introduced can be performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Azide-modified antibody derivatives or their salts can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0155] 5. Conjugates of antibodies and functional substances or their salts The present invention provides antibody and functional substance conjugates or salts thereof, comprising a structural unit represented by the following formula (IV).

[0156] [ka]

[0157] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and two The lysine residue in the heavy chain forms an amide bond with the carbonyl group adjacent to Ig via the amino group in the side chain. M represents a trivalent group that connects the carbon atoms in the adjacent C=O and C=W with a main chain portion consisting of 3 to 5 carbon atoms. O represents an oxygen atom. T indicates a monovalent group. W represents an oxygen atom or a sulfur atom. N represents a nitrogen atom. Lb indicates a bond or a divalent group. Z indicates a functional substance. L indicates a bond or a divalent group. Ring A shows a ring fused with 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 or sulfur atom), and Lb (bond, or divalent group) in formula (IV) are the same as those in the formula above.

[0159] Ring A represents a ring fused with a triazole ring. The components of ring A do not include the triazole ring itself that is fused, but include the part of the double bond between the carbon atoms shared with triazole. Therefore, it can be said that ring A is a ring having a double bond between carbon atoms.

[0160] Ring A is a monocyclic ring or a condensed ring of a monocyclic ring and another ring. Ring A may have a substituent. As the monocyclic ring, a homocyclic ring or a heterocyclic ring 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 monocyclic ring is a homocyclic ring or a heterocyclic ring containing one or more selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom. As the monocyclic ring, a 5- to 12-membered monocyclic ring is preferable, a 6- to 10-membered monocyclic ring is more preferable, and a 7- to 9-membered monocyclic ring is even more preferable. As the monocyclic ring, a non-aromatic monocyclic ring is preferable.

[0161] When ring A is a condensed ring, examples of the other ring condensed with the monocyclic ring include cycloalkane, arene, and heterocyclic ring.

[0162] As the cycloalkane condensed with the monocyclic ring, a cycloalkane having 3 to 24 carbon atoms is preferable, a cycloalkane having 6 to 18 carbon atoms is more preferable, a cycloalkane having 3 to 14 carbon atoms is even more preferable, and a cycloalkane having 3 to 10 carbon atoms is even more preferably. The carbon atom number of the substituent is not included in the above carbon atom number. Examples of the cycloalkane include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane.

[0163] As the arene condensed with the monocyclic ring, an arene having 6 to 24 carbon atoms is preferable, an arene having 6 to 18 carbon atoms is more preferable, an arene having 6 to 14 carbon atoms is even more preferable, and an arene having 6 to 10 carbon atoms is even more preferably. The carbon atom number of the substituent is not included in the above carbon atom number. Examples of the arene include benzene, naphthalene, and anthracene.

[0164] The heterocyclic ring fused to the monocyclic ring is an aromatic heterocyclic ring or a non-aromatic heterocyclic ring. The heteroatom constituting the heterocyclic ring preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom. atom and one or more selected from the group consisting of nitrogen atoms.

[0165] The aromatic heterocyclic ring fused to the monocyclic ring is preferably an aromatic heterocyclic ring having 3 to 21 carbon atoms, more preferably an aromatic heterocyclic ring having 3 to 15 carbon atoms, still more preferably an aromatic heterocyclic ring having 3 to 9 carbon atoms, and still more preferably an aromatic heterocyclic ring having 3 to 6 carbon atoms. The carbon atoms of the substituents are not included in the above carbon atom numbers. More specifically, examples of the aromatic heterocyclic ring include pyrene, pyrrole, furan, thiophene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrroline, piperidine, triazole, purine, anthraquinone, carbazole, fluorene, quinoline, and isoquinoline.

[0166] The non-aromatic heterocyclic ring fused to the monocyclic ring is preferably a non-aromatic heterocyclic ring having 3 to 21 carbon atoms, more preferably a non-aromatic heterocyclic ring having 3 to 15 carbon atoms, still more preferably a non-aromatic heterocyclic ring having 3 to 9 carbon atoms, and still more preferably a non-aromatic heterocyclic ring having 3 to 6 carbon atoms. The carbon atoms of the substituents are not included in the above carbon atom numbers. More specifically, examples of the non-aromatic heterocyclic ring 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 ring A may have are the same as those described above in (i) to (vii), and the preferred range is also the same. The number of 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 monoring, or a fused ring of a 7- to 9-membered monoring and another ring (see, e.g., Org. Biomol. Chem. 2013, 11, 6439, Angew. Chem. Int. Ed. 2015, 54, 1190). For example, such a ring A may be a ring of formula (i') to (vii') described in International Publication No. 2017 / 191817.

[0169] In formula (IV), the divalent group represented by L is a linear, branched, or cyclic group, or a combination thereof, that connects the functional substance (Z) to ring A. The main chain of the divalent group represented by L may consist of, for example, a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR- (where R represents a hydrogen atom or substituent), -O-, -S-, or a combination of two or more of these (e.g., 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 substituents. Such substituents are the same as those described in (i) to (vii) above, and the preferred ranges are also the same. The number of substituents is, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2. L can be bonded to any ring-constituting atom in ring A that is different from the carbon atom shared with the triazole.

[0170] Functional substances are not particularly limited as long as they are substances that confer any desired function to an antibody, and include, for example, drugs, labeling substances, and stabilizers, but are preferably drugs or labeling substances. Functional substances may also be a single functional substance or a substance in which two or more functional substances are linked together.

[0171] The drug may be a drug for any disease. Examples of such diseases include: Examples include cancer (e.g., lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumors, melanoma), autoimmune and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), neurological diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood disorders (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, digestive system diseases). The drug may be a preventative or therapeutic agent for a disease, or a drug to alleviate side effects.

[0172] More specifically, the drugs are anticancer agents. Examples of anticancer agents include chemotherapeutic agents, toxins, radioisotopes, or substances containing them. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., lysine). Examples of radioactive isotopes include the radioactive isotopes of hydrogen (e.g., 3H), carbon (e.g., 14C), phosphorus (e.g., 32P), sulfur (e.g., 35S), yttrium (e.g., 90Y), technetium (e.g., 99mTc), indium (e.g., 111In), iodine (e.g., 123I, 125I, 129I, 131I), samarium (e.g., 153Sm), and rhenium. Examples include sex isotopes (e.g., 186Re), radioactive isotopes of astatine (e.g., 211At), and radioactive isotopes of bismuth (e.g., 212Bi). More specifically, examples of drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calichemycin, duocalmycin, eribulin, anthracyclines, dmDNA31, and tubulsin.

[0173] Labeling substances are substances that enable the detection of targets (e.g., tissues, cells, materials). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those mentioned above), or substances containing them.

[0174] Stabilizers are substances that enable the stabilization of antibodies. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0175] Functional substances may also be peptides, proteins, nucleic acids, small organic compounds, glycans, lipids, high molecular weight polymers, metals (e.g., gold), and chelators. Examples of peptides include cell membrane permeable peptides, blood-brain barrier permeable peptides, and peptide pharmaceuticals. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Other examples of nucleic acids include RNA interference-inducible nucleic acids (e.g., siRNA), aptamers, and antisenses. Examples of small organic compounds include proteolytic chimeric molecules, dyes, and photodegradable compounds.

[0176] If a functional substance does not have a desired functional group (e.g., a functional group readily linkable to ring A or L), the functional substance may be derivatized to have such a functional group. Derivatization is common technical knowledge in the art (e.g., International Publication 2004 / 010957, U.S. Patent Application Publication 2006 / 0074008, U.S. Patent Application Publication 2005 / 0238649). For example, derivatization may be carried out using any crosslinking agent. Alternatively, derivatization may be carried out using a specific linker having the desired functional group. For example, such a linker may be capable of separating the functional substance and the antibody by linker cleavage in a suitable environment (e.g., intracellular or extracellular). 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. Patent No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), and linkers that can be cleaved at locally acidic sites present in the body (e.g., U.S. Patent Nos. 5,622,929, 5,122,368; 5,824,805). Linkers may also be self-immolative (e.g., International Publication No. 02 / 083180, International Publication No. 04 / 043493, International Publication No. 05 / 112919). In this invention, derivatized functional substances are also simply referred to as "functional substances."

[0177] In formula (IV), the average ratio (r) of the above amide bonds per two heavy chains represents 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 higher, more preferably 1.7 or higher, even more preferably 1.8 or higher, and particularly preferably 1.9 or higher. Such an average ratio may also be preferably 2.4 or lower, more preferably 2.3 or lower, even more preferably 2.2 or lower, and particularly preferably 2.1 or lower. More specifically, such an average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1. Lysine residues having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to Ig may be modified to show an average ratio of the above values ​​at one desired position in the two heavy chains (for example, 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). Furthermore, if each of the two heavy chains is modified at two or more positions, lysine residues having a side chain containing an amino group forming an amide bond with a carbonyl group adjacent to Ig may be modified to show an average ratio of the above values ​​at each of the two desired positions in the two heavy chains (for example, 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 or salt thereof of the present invention can be obtained by reacting the azide group-introduced antibody derivative or salt thereof of the present invention with a functional substance. Such a reaction can be carried out under conditions that do not cause denaturation or degradation of proteins (immunoglobulins / antibodies) (e.g., cleavage of amide bonds) (mild conditions as described above). As the functional substance, a derivative having a cycloalkyne group that can react 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 with triple bonds between carbon atoms. Z indicates a functional substance. L indicates a bond or a divalent group.

[0181] Ring A' represents a ring having a triple bond between carbon atoms. Ring A' is a monoring or a fused ring of a monoring and another ring. Ring A' may have substituents. As a monoring, an allotropic ring or a heteroring containing one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms is preferred. More preferably, the monoring is an allotropic ring or a heteroring containing one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms. As a monoring, a 7-10 membered monoring is preferred, and a 7-9 membered monoring is more preferred. As a monoring, a non-aromatic monoring is preferred.

[0182] If ring A' is a fused ring, other rings that can be fused with the monoring include, for example, cycloalkanes, arenes, and heterorings. The definitions, examples, and preferred examples of cycloalkanes, arenes, and heterorings are the same as those for the other rings in a fused ring for ring A'.

[0183] The substituents that ring A’ may have are the same as the substituents (i) to (vii) described above, and the preferred ranges are also the same. The number of substituents is, for example, 1 to 5, preferably 1 to 3, 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. The reaction between such a ring A’ and an azide group is also known as the 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, as such a ring A, rings of formula (i'') to (vii'') described in International Publication No. 2017 / 191817 may be used.

[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 since it varies depending on factors such as the type of the azide group-introduced antibody derivative or its salt, the type of the functional substance, and the reaction time, etc., but is, for example, 2 or more, preferably 3 or more, more preferably 5 or more. 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 with respect to the azide group-introduced antibody derivative or its salt can be used.

[0186] Confirmation of the formation of the conjugate or its salt is based on the molecular weights of its specific raw materials and products Depending on the method, this can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. Regioselectivity can be confirmed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. Endoproteases are preferred as proteases. 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, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The conjugate or its salt can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above, and affinity chromatography).

[0187] 6.Applications The compounds or salts of the present invention can highly control the average ratio of binding between immunoglobulin units and azide group-containing modifying groups (azide group-containing modifying group / immunoglobulin unit) to a desired range (1.0 to 3.0). The compounds or salts of the present invention can also regioselectively modify lysine residues in the heavy chain of immunoglobulin units. Accordingly, the present invention provides antibody derivatization reagents comprising the compounds or salts of the present invention.

[0188] The reagents of the present invention may be provided in the form of compositions further comprising other components. Such other components include, for example, solutions and stabilizers (e.g., antioxidants, preservatives). As the solution, aqueous solutions are preferred. Examples of aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline) and buffer solutions (e.g., aqueous phosphoric acid solution, Tris-hydrochloride buffer, carbonic acid-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), but buffer solutions are preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagents of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

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

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

[0191] The conjugate or salt thereof of the present invention may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate or salt thereof of the present invention, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, etc. Examples of binders include polycellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants include starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants include magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances include citric acid, menthol, glycyrrhizine ammonium salt, glycine, and orange powder; preservatives include sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers include citric acid, sodium citrate, and acetic acid; suspending agents include methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersants include surfactants; diluents include water, physiological saline, and orange juice; and base waxes include cocoa butter, polyethylene glycol, and kerosene. The conjugate or salt of the present invention may also have any modifications (e.g., PEGylation) to achieve stability.

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

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

[0194] The dosage of a pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the target animal species, the target animal's drug tolerance, body weight, age, etc., but can be set as appropriate. [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 reagent for IgG1 modification (1-1) Synthesis of Modified 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. Dissolve in 4 mL of (0.4 mL), add Dimethyl-5-Aminoisophthalate (977 mg, 4.67 mmol) and DMAP (1.7 g, 14.0 mmol), and stir at 50°C for 1 hour. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 1 / 1), concentrate the reaction mixture. Elute with a mixed solution of hexane and ethyl acetate, and each fraction was examined 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 (2) (1.08 g, 4.59 mmol).

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

[0200] [ka]

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

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

[0203] [ka]

[0204] 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 the mixture was stirred at room temperature for 2 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the organic layer was extracted with ethyl acetate and 2 M HCl aqueous solution, and the organic layer was concentrated. Elution was performed 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 same as the amino acid sequence of Sequence ID No. 2.

[0208] The peptide consisting of the amino acid sequence of Sequence ID No. X1 was synthesized in solid phase according to the method described in International Publication No. 2018 / 199337 (the same applies to peptide synthesis below). Ac-RGNCAYHKGQIIWCTYH-NH2 (Sequence ID No. 2, 50.0 mg, 19.6 μmol, where the 4th and 14th cysteine ​​molecules each form intramolecular disulfide bonds) 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 mixture, and the mixture was stirred for a further 2 hours. After confirming the reaction by LC-MS, the product was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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 Modified Reagent (1)

[0211] [ka]

[0212] All of the above amino acid sequences are the same as the amino acid sequence of Sequence 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 a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above-mentioned modified 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), and NBS (783 mg, 4.4 mmol) and benzoyl peroxide (40 mg, 0.17 mmol) were added. The mixture was stirred at 90°C for 20 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the reaction mixture was concentrated. It was dissolved in a hexane / ethyl acetate = 1 / 1 mixed solvent and filtered. The crystals were collected and vacuum dried to obtain 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 linker intermediate (7) (300 mg, 1.04 mmol) synthesized in (1-2-1) was added and the mixture was stirred at 60°C for 3 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the organic layer was extracted with ethyl acetate and water and concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was examined 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 (8) (268 mg, 1.08 mmol).

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

[0222] [ka]

[0223] The compound synthesized in (1-2-2) (268 mg, 1.08 mmol) was dissolved in methanol (5.4 mL) and THF (5 mL), and 1 M NaOH aqueous solution was added and the mixture was stirred at room temperature for 2 hours. After confirming the reaction by MS, 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. Vacuum drying was performed to obtain linker intermediate (9) (227 mg, 1.00 mmol).

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

[0225] [ka]

[0226] 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 the mixture was stirred at room temperature for 20 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the organic layer was extracted with ethyl acetate and 1 M HCl aqueous solution and concentrated. Elution was performed 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 (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 same as the amino acid sequence of Sequence 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 a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the modification 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 100 mL of 1 M NaOH aqueous solution, and DMF was added and the mixture was stirred at 110°C for 20 hours. After confirming the reaction by LC / MS, the reaction mixture 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. Vacuum drying was performed to obtain 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, thionyl chloride (21.4 mL, 288 mmol) was added at 0°C, and the mixture was stirred at room temperature for 3 hours. After confirming the reaction by LC / MS, the mixture was extracted with ethyl acetate and sodium bicarbonate solution, and the organic layer was concentrated to obtain 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), and 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 by 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 recovered, the organic solvent was removed by concentrating under reduced pressure, and then vacuum drying was performed 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, the organic layer was concentrated, and vacuum-dried to obtain 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 1 M 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 solution was acidified with 2 M HCl aqueous solution, extracted with ethyl acetate and 2 M HCl aqueous solution, and the organic layer was concentrated. Vacuum drying was performed to obtain linker intermediate (16) (637.8 m). (g, 2.56 mmol).

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

[0248] [ka]

[0249] 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 the mixture was stirred at room temperature for 20 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 3 / 1), the organic layer was extracted with ethyl acetate and water, and concentrated. Elution was performed 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 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 same as the amino acid sequence of Sequence ID No. 2.

[0253] (5) Peptide intermediate synthesized in (1-1-4) (15.0 mg, 6.93 μmol, (However, the 4th and 14th cysteines each form disulfide bonds intramolecularly.) The linker intermediate (17) (40.2 mg, 69.3 μmol) synthesized in (1-3-6) was 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 mixture was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the modification 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) and N-methylmorpholine (873 μL, 8.39 mmol) were added, and the mixture was stirred at 0°C for 30 minutes. Then, hydrazine hydrate (1.36 g, 6.71 mmol) dissolved in 1 M NaOH aqueous solution (4 mL) was added, and the mixture was stirred at room temperature for 3 hours. After concentrating under reduced pressure, 1 M NaOH aqueous solution was added to adjust the pH of the system to pH 10, and after washing with ethyl acetate, 1 M HCl aqueous solution was added to the aqueous layer to adjust the pH of the system to 3.0, and ethyl acetate was added for washing. Sodium sulfate was added to the resulting ethyl acetate solution. Sodium sulfate was removed by filtration, and the solution was purified by concentrated column chromatography under reduced pressure (dichloromethane:methanol = 10:1). The fraction containing the product was recovered 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), and thiophenol (627 μL, 6.15 mmol), benzotriazole-1-yloxy (3.49 g, 6.71 mmol), and DIPEA (1.42 mL, 8.39 mmol) were added. The mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate = 4:1). The fraction containing the product was recovered 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. After that, the mixture was concentrated under reduced pressure to remove the dichloromethane, and water was added and 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] Linker intermediate (21) (100 mg, 0.274 mmol) was dissolved in dichloromethane (3 mL), and (40.6 μL, 0.280 mmol), benzotriazole-1-yloxy (150 mg, 0.288 mmol), and DIPEA (70.1 μL, 0.412 mmol) were added. The mixture was stirred at room temperature for 2 hours. The pH of the system was adjusted to 3 by adding 1 M aqueous HCl, and the mixture was diluted with dichloromethane. After washing with water and saline solution, sodium sulfate was added. After removing the 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 recovered and concentrated under reduced pressure to obtain 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. The mixture was then concentrated under reduced pressure to remove the dichloromethane, water was added, and the mixture was freeze-dried. The mixture was then purified by column chromatography (dichloromethane:methanol = 10:1). The fraction containing the product was recovered 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 same as the amino acid sequence of Sequence ID No. 2.

[0283] Ac-RGNCAYHKGQIIWCTYH-NH2 (SEQ ID NO: 2, 30.9 mg, 14.9 μmol, where the 4th and 14th cysteine ​​groups are 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above-mentioned modified 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] 500 mg of tert-Butyl 3-bromo-5-nitrobenzoate (1.65 mmol) was dissolved in 8.25 mL of a 1,4-Dioxane / H2O = 4 / 1 mixture. 447 mg of 2-Ethoxycarbonylvinylboronic acid pinacol ester (1.98 mmol), 67.8 mg of Pd(dppf)Cl2 (0.083 mmol), and 438 mg of Na2CO3 (4.13 mmol) were added, and the mixture was stirred at 100°C for 2 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 10 / 1), the organic layer was extracted with ethyl acetate and water, and concentrated. Elution was performed with a hexane / ethyl acetate mixture, and each fraction was examined by TLC (hexane / ethyl acetate = 10 / 1). The fraction containing the product was recovered, 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), and Pd / C (5% Pd, 53% H2O) (5.0 mol%) was added. 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, the filtrate was concentrated, and then vacuum dried 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 CH2Cl2 (5.45 mL), and 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 mixture was concentrated. Elution was performed with a mixed solvent of hexane and ethyl acetate, and confirmed by TLC (hexane / ethyl acetate = 2 / 1). The fraction containing the product was recovered, 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 1 M NaOH aqueous solution (1.74 mL) and ethanol (4 mL) were added at 0°C and the mixture was stirred at room temperature for 1.5 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 2 / 1), 1 M HCl aqueous solution (2.61 mL) was added at 0°C to make the mixture acidic. The mixture was extracted with ethyl acetate and 1 M HCl aqueous solution, the organic layer was concentrated, and vacuum-dried 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. The mixture was stirred at room temperature for 1.5 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 4 / 1), the organic layer was extracted with ethyl acetate and water, and concentrated. Elution was performed 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 CH2Cl2 / TFA=1 / 1 mixed solvent (10 mL) 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 concentrating under reduced pressure, and the mixture was dried under 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 the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the organic layer was extracted with ethyl acetate and water, and concentrated. Elution was performed 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 CH2Cl2 / TFA=1 / 1 mixed solvent (10 mL) 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 concentrating under reduced pressure, and the mixture was dried under 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 same as the amino acid sequence of SEQ ID NO: 4.

[0319] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4, 330.0 mg, 7.06 μmol, where the 5th and 34th cysteine ​​groups each form intramolecular disulfide bonds), 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 mixture was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain modification 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] Dissolve 3-Iodo-5-Nitrobenzoic Acid (1.0 g, 3.41 mmol) in toluene (17 mL) and prepare N,N-Dimethylformamide Di-tertbutyl acetal (5 mL, 20.5 mmol) was added and the mixture was stirred at 80°C for 2 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate = 5 / 1), the reaction mixture was concentrated. Vacuum drying was performed 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 1,4-Dioxane / H2O=4 / 1 mixed solution (18 mL), and (E)-Ethoxyethene-2-boronicacid pinacol ester (851 mg, 4.3 mmol), Pd(dppf)Cl2 (146 mg, 0.179 mmol), and Na2CO3 (948 mg, 8.95 mmol) were added. The mixture was stirred at 100°C for 3 hours. After confirming the reaction by LC / MS and TLC (hexane / ethyl acetate=10 / 1), the organic layer was extracted with ethyl acetate and water, and concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was examined 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] Linker intermediate (35) 614 mg (2.09 mmol) mixed with THF (20 mL), 1 M The compound was dissolved in HCl aqueous solution (2.0 mL), 6 M HCl aqueous 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 reaction was extracted with ethyl acetate and 1 M HCl aqueous solution, and the organic layer was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was examined 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 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), and 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 the solution slightly acidic, and the mixture was extracted with ethyl acetate. The organic layer was concentrated, and the mixture was vacuum-dried 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 the mixture was stirred for 1.5 hours. 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 vacuum dried 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, and 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 by membrane filtration and the solvent was concentrated. Vacuum drying was performed 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 Dissolve (mg, 3.0 mmol) in CH2Cl2 (15.0 mL), add triethylamine (2.09 mL, 15.0 mmol) and pentafluorophenyltrifluoroacetic acid (1.02 mL, 6.0 mmol) at 0°C, and stir at 0°C for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 2 / 1), concentrate the reaction mixture. Elute with a mixed solution of hexane and ethyl acetate, and each fraction was examined 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 (40) (1.4 g, 3.51 mmol).

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

[0343] [ka]

[0344] Dissolve linker intermediate (39) (348.4 mg, 1.31 mmol) in DMF (6.55 mL), then add linker intermediate (40) (523 mg, 1.31 mmol), DIPEA (445 μL, 2.62 mmol), and DMAP (16.0 mg, 0.131 mmol). The mixture was then stirred at room temperature for 1 hour. After confirming the reaction by LC / MS, the organic layer was extracted with water and ethyl acetate and concentrated. The mixture was then vacuum dried to obtain 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), and 1 M 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 solution was made more acidic with 2 M HCl aqueous solution, extracted with ethyl acetate, and the organic layer was concentrated. The mixture was vacuum dried 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 the mixture was stirred at room temperature for 1 hour. After confirming the reaction by LC / MS and TLC (ethyl acetate), the organic layer was extracted with ethyl acetate and water, and the organic layer was concentrated. Elution was performed 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 CH2Cl2 / TFA = 1 / 1 mixed solvent 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 vacuum-dried 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), and 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 by LC / MS and TLC (ethyl acetate), the organic layer was extracted with ethyl acetate and water, and the organic layer was concentrated. Elution was performed 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 (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 CH2Cl2 / TFA=1 / 1 mixed solvent 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. Each fraction was examined 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 same as the amino acid sequence of SEQ ID NO: 4.

[0364] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4, 30.0 mg, 7.06 μmol, where the 4th and 14th cysteine ​​groups each form intramolecular disulfide bonds) 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 solution was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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 (608 mg, 2.89 mmol) was dissolved in dichloromethane (15 mL), and D-proline tert-butyl hydrochloride (1.32 g, 6.36 mmol), WSC·HCl (1.39 mg, 2.72 mmol), 1-hydroxy-7-azabenzotriazole (78.7 mg, 0.578 mmol), and triatylamine (2.00 mL, 14.5 mmol) were added. The mixture was stirred overnight at room temperature. After concentrating under reduced pressure, the solution was diluted with ethyl acetate, washed with water and saline solution, and then sodium sulfate was added. After removing sodium sulfate by iontophoresis, the solution was concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate = 1:1). The fraction containing the product was recovered and concentrated under reduced pressure to obtain the 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), and tris(2-carboxyethyl)phosphine (760 mg, 2.65 mmol) dissolved in water (5 mL) was added. The mixture was stirred overnight at room temperature, and the reaction solution was diluted with ethyl acetate. After washing with water and saline solution, sodium sulfate was added. Sodium sulfate was removed by filtration, and the mixture was concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate = 1:1). The fraction containing the product was recovered 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), benzotriazole-1-yloxy (89.9 mg, 0.173 mmol), and DIPEA (73.5 μL, 0.432 mmol) were added. The mixture was 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 fraction containing the product was recovered 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. After that, it was concentrated under reduced pressure to remove the dichloromethane, and water was added and 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), and pentafluorophenyltrifluoroacetic acid (23.4 mg, 0.137 mmol) and triethylamine (28.6 mL, 0.206 mmol) were added. The mixture was 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 fraction containing the product was recovered 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 Modified Reagent (47)

[0387] [ka]

[0388] All of the above amino acid sequences are the same as the amino acid sequence 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 cysteine ​​groups are intramolecularly disulfide-bonded) was dissolved in DMF (374 μL), and 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above-mentioned modification reagent (47) (7.76 mg, 1.62 μmol).

[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), and 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 triatylamine (2.33 mL, 16.8 mmol) were added. The mixture was stirred overnight at room temperature. After concentrating under reduced pressure, the solution was diluted with ethyl acetate, washed with water and saline, and then sodium sulfate was added. After removing the sodium sulfate by filtration, the solution was concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate = 1:1). The fraction containing the product was recovered and concentrated under reduced pressure to obtain 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] 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. The mixture was stirred overnight at room temperature, then the reaction mixture was diluted with ethyl acetate, washed with water and saline solution, and sodium sulfate was added. Filtration revealed sodium sulfate The pulp was removed, the mixture was concentrated under reduced pressure, and then purified by column chromatography (hexane:ethyl acetate = 1:1). The fraction containing the product was recovered and concentrated under reduced pressure to obtain 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), benzotriazole-1-yloxy (452 ​​mg, 0.868 mmol), and DIPEA (370 μL, 2.17 mmol) were added. The mixture was 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 fraction containing the product was recovered 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. After that, the mixture was concentrated under reduced pressure to remove the dichloromethane, and water was added and 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), and pentafluorophenyltrifluoroacetic acid (110 mL, 0.644 mmol) and triethylamine (134 μL, 0.966 mmol) were added. The mixture was stirred at room temperature for 3 hours. After concentration under reduced pressure, the mixture was purified by column chromatography (hexane:ethyl acetate = 1:1). The fraction containing the product was recovered 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 same as the amino acid sequence of SEQ ID NO: 4.

[0414] The peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4, 96.1 mg, 22.6 μmol, where the 5th and 35th cysteine ​​groups are intramolecularly disulfide-bonded) 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 reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized 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 same as the amino acid sequence of SEQ ID NO: 4.

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

[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 same as the amino acid sequence 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 cysteine ​​groups are intramolecularly disulfide-bonded) was dissolved in DMF (300 mL). Linker intermediate (62) (23.2 mg, 33.1 μmol) derived from L-proline tert-butyl hydrochloride using the same method as for linker intermediate (58) in Example (1-8-5), and triethylamine (1.40 μL, 9.93 μmol) were added. The mixture was stirred at room temperature for 3 hours and then purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above-mentioned 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 same as the amino acid sequence of SEQ ID NO: 5.

[0429] The peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 5, 100 mg, 23.4 μmol, where the 5th and 35th cysteine ​​groups are 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. The mixture was stirred at room temperature for 30 minutes and then purified by reverse-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain 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 same as the amino acid sequence of SEQ ID NO: 5.

[0434] The peptide of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 5, 101 mg, 23.6 μmol, where the 5th and 35th cysteine ​​groups are intramolecularly disulfide-bonded) was dissolved in DMF (1.01 mL), and 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 reverse-phase preparative chromatography. The fraction containing the product was collected, 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 same as those of Sequence ID No. 6.

[0439] Ac-MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH2 (SEQ ID NO: 6, 30.0 mg, 7.08 μmol, where the 3rd and 32nd cysteine ​​groups each form intramolecular disulfide bonds) 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 mixture was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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 Modified 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), and DIPEA (408 μL, 2.40 mmol) were added and the mixture was 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), and DIPEA (408 μL, 2.40 mmol) were 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 organic layer was extracted with ethyl acetate and water, and the organic layer was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was examined by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was recovered and concentrated under reduced pressure to remove the organic solvent and obtain the linker intermediate (67).

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

[0445] [ka]

[0446] The linker intermediate (67) was dissolved in a CH2Cl2 / TFA=1 / 1 mixed solvent (10.0 mL) 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. Elution was performed with a mixed solution of dichloromethane and methanol, and each fraction was examined by LC / MS. The fraction containing the product was recovered, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried. Interbody (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 Modified Reagent (66)

[0449] [ka]

[0450] All of the above amino acid sequences are the same as those of Sequence ID No. 6.

[0451] Ac-MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH2 (SEQ ID NO: 6, 25.0 mg, 6.2 μmol, where the 3rd and 32nd cysteine ​​groups each form intramolecular disulfide bonds) 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 a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was examined by LC-MS. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried 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 and analysis of the anti-HER2 antibody trastuzumab using a modification reagent containing affinity substances] (2-1) Modification of the anti-HER2 antibody trastuzumab using a modification reagent containing affinity substances (2-1-1) Modification reactions using modification reagents (1, 6, 11) The modification reaction of the anti-HER2 antibody trastuzumab was carried out using the modification reagent (1) synthesized in Example (1-1-4). Modification reagent (1) was dissolved in DMF to a 30 mM solution. 500 μg of the 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 30 mM peptide were added. 1.12 μL of reagent (10 equivalents relative to the antibody) was added and the mixture was stirred at 37°C for 1 hour to obtain the trastuzumab-peptide conjugate (69).

[0454] Using a similar method, an antibody modification reaction was carried out with the modifying reagent (6) to obtain the trastuzumab-peptide complex (70).

[0455] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (11) to obtain a trastuzumab-peptide complex (71).

[0456] (2-1-2) Modification reaction using modification reagent (18) The modification reaction of the anti-HER2 antibody trastuzumab was carried out using the modification reagent (18) synthesized in Example (1-4-6). 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 10 mM peptide reagent were added. The mixture was stirred at room temperature for 1 hour to obtain the trastuzumab-peptide conjugate (72).

[0457] (2-1-3) Modification reactions using modification reagents (24, 33, 65, 66) The modified 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 MHEPES buffer (pH 8.0), and 1.69 μL (10 equivalents relative to the antibody) of the 20 mM peptide reagent was added. The mixture was stirred at room temperature for 1 hour to obtain the trastuzumab-peptide conjugate (73).

[0458] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (33) to obtain a trastuzumab-peptide complex (74).

[0459] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (65) to obtain a trastuzumab-peptide complex (75).

[0460] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (66) to obtain a trastuzumab-peptide complex (76).

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

[0462] Using a similar method, an antibody modification reaction was performed with a modifying reagent (53) to obtain a trastuzumab-peptide complex (78).

[0463] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (59) to obtain a trastuzumab-peptide complex (79).

[0464] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (61) to obtain a trastuzumab-peptide complex (80).

[0465] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (63) to obtain a trastuzumab-peptide complex (81).

[0466] Using a similar method, an antibody modification reaction was carried out with a modifying reagent (64) to obtain a trastuzumab-peptide complex (82).

[0467] (2-2) Analysis using ESI-TOFMS ESI-TOFMS analysis of the trastuzumab-peptide complex (69) was performed according to a previously reported procedure (International Publication No. 2019 / 240287 (WO2019 / 240287A1)). A peak was observed at 148222 for the starting material trastuzumab. For the reaction product, a peak was confirmed at 152915, where two modification reagents (1) were introduced. Subsequently, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 1. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 1 was 1.9. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 1.9) was confirmed.

[0468] [Table 1]

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

[0470] [Table 2]

[0471] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (71), and a peak was observed at 152973, where two modification reagents (11) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 3. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 3 was 1.8. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 1.8) was confirmed.

[0472] [Table 3]

[0473] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (72), and a peak was observed at 153023, where two modification reagents (18) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 4. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 4 was 1.8. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 1.8) was confirmed.

[0474] [Table 4]

[0475] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (73), and a peak was observed at 157267, where two modification reagents (24) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 5. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 5 was 1.7. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 1.7) was confirmed.

[0476] [Table 5]

[0477] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (74), and a peak was observed at 157619, where two modification reagents (33) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 6. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 6 was 1.5. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 1.5) was confirmed.

[0478] [Table 6]

[0479] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (75), and a peak was observed at 161051, where two modified reagents (65) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 7. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 7 was 2.0. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 2.0) was confirmed.

[0480] [Table 7]

[0481] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (76), and a peak was observed at 156802, where two modified reagents (66) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 8. The average peptide / antibody binding ratio calculated from the DAR peaks and %Area in Table 8 was 1.5. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 1.5) was confirmed.

[0482] [Table 8]

[0483] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (77), and a peak was observed at 157567, where two modification reagents (47) were introduced. Next, the peptide / antibody binding ratio was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 9. The DAR peaks and %Area in Table 9 were used to calculate the peptide / antibody binding ratio. The average peptide / antibody binding ratio was 2.0. Therefore, the formation of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0484] [Table 9]

[0485] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (78), and a peak was observed at 157540, where two modification reagents (53) were introduced. Next, the peptide / antibody binding ratio was 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 peaks and %Area in Table 10 was 2.0. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 2.0) was confirmed.

[0486] [Table 10]

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

[0488] [Table 11]

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

[0490] [Table 12]

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

[0492] [Table 13]

[0493] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (82), and a peak was observed at 157596, where two modification reagents (64) were introduced. Next, the peptide / antibody binding ratio was 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 peaks and %Area in Table 14 was 2.0. Therefore, the formation of an antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio of 2.0) 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 complex (69) under reduction conditions was performed according to a previously reported study (International Publication No. 2019 / 240287 (WO2019 / 240287A1)). The starting material trastuzumab showed heavy chain peaks at 50594 and 50755, and a light chain peak at 23439. The reaction products were 52917 and 53079, with a linker introduced into the heavy chain, and 23439, the same as the starting material, was confirmed in the light chain.

[0496] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (70) revealed that the reaction products were 52956 and 53118, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0497] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (71) revealed that the reaction products were 52971 and 53133, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0498] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (72) revealed that the reaction products were 52996 and 53157, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0499] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (73) revealed that the reaction products were 55091 and 55253, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0500] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (75) revealed that the reaction products were 54849 and 55010, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0501] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (76) revealed that the reaction products were 54888 and 55050, with a linker introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0502] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (77) revealed that the reaction products were 55268 and 55430, with a linker introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0503] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (78) revealed that the reaction products were 55254 and 55416, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[0504] Similarly, ESI-TOFMS analysis was performed on the trastuzumab-peptide complex (81), and the reaction The resulting compounds were identified as 55296 and 55458, which have a linker introduced into the heavy chain, and 23439, which is the same as the starting material, in the light chain.

[0505] Similarly, ESI-TOFMS analysis of the trastuzumab-peptide complex (82) revealed that the reaction products were 55281 and 55443, in which a linker was introduced into the heavy chain, and 23439, the same as the starting material, in the light chain.

[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 complex Using the trastuzumab-peptide conjugate (69), a linker cleavage reaction was performed with hydroxyamine hydrochloride according to a previously reported procedure (International Publication No. 2019 / 240287 (WO2019 / 240287A1)) to obtain an azide-introduced antibody (83).

[0507] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (70) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (84).

[0508] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (71) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (85).

[0509] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (72) was performed with hydroxyamine hydrochloride to obtain the azide-introduced antibody (86).

[0510] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (73) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (87).

[0511] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (74) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (88).

[0512] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (75) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (87).

[0513] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (76) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (89).

[0514] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (77) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (90).

[0515] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (78) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (90).

[0516] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (79) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (90).

[0517] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (80) was performed with hydroxyamine hydrochloride to obtain the azide-introduced antibody (90).

[0518] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (81) was performed with hydroxyamine hydrochloride to obtain an azide-introduced antibody (90).

[0519] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (82) was performed with hydroxyamine hydrochloride to obtain the azide-introduced antibody (90).

[0520] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (72) was performed with methoxyamine hydrochloride to obtain the azide-introduced antibody (91).

[0521] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (77) was performed with methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0522] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (78) was performed with methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0523] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (79) was performed with methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0524] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (80) was performed with methoxyamine hydrochloride to obtain the azide-introduced antibody (92).

[0525] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (81) was performed with methoxyamine hydrochloride to obtain an azide-introduced antibody (92).

[0526] Using a similar method, the linker cleavage reaction of the trastuzumab-peptide complex (82) was performed with methoxyamine hydrochloride to obtain the azide-introduced antibody (92).

[0527] (3-2) Analysis using ESI-TOFMS ESI-TOFMS analysis of the azide-introduced antibody (84) was performed according to a previously reported procedure (International Publication No. 2019 / 240287 (WO2019 / 240287A1)), and a peak was confirmed at 148657, where the linker was cleaved.

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

[0529] Similarly, ESI-TOFMS analysis was performed on the azide-introduced antibody (86), 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 progress in linker cleavage.

[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 progress in linker cleavage.

[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 derivative of trastuzumab with an arbitrary compound and ESI-TOFMS analysis of the product] (4-1) Conjugation of azide-introduced antibodies and fluorescent substances In accordance with previously reported findings (International Publication No. 2019 / 240287 (WO2019 / 240287A1) and International Publication No. 2019 / 240288 (WO2019 / 240288A1)), Carboxyrhodamine 110-PEG4-DBCO (manufactured by Broadpharm) was added to azide-introduced antibody (83) to obtain ADC mimic (93).

[0536] A similar method was used to obtain ADC mimic (94) from azide-introduced antibody (84).

[0537] A similar method was used to obtain an ADC mimic (95) from an azide-introduced antibody (85).

[0538] A similar method was used to obtain ADC mimic (96) from azide-introduced antibody (86).

[0539] A similar method was used to obtain ADC mimic (97) from azide-introduced antibody (87).

[0540] A similar method was used to obtain ADC mimic (98) from azide-introduced antibody (88).

[0541] A similar method was used to obtain ADC mimic (99) from azide-introduced antibody (89).

[0542] A similar method was used to obtain ADC mimic (100) from azide-introduced antibody (90).

[0543] A similar method was used to obtain ADC mimic (101) from azide-introduced antibody (91).

[0544] A similar method was used to obtain ADC mimic (102) from azide-introduced antibody (92).

[0545] Using a similar method, ADC(103) was obtained by reacting azide-introduced antibody (83) with DBCO-MMAE (manufactured by Abzena).

[0546] Using a similar method, ADC (104) was obtained by reacting azide-introduced antibody (86) with DBCO-MMAE (manufactured by Abzena).

[0547] Using a similar method, ADC (105) was obtained by reacting azide-introduced antibody (91) with DBCO-MMAE (manufactured by Abzena).

[0548] Based on the above, it has been confirmed that the compound or salt of the present invention is useful for producing antibody-drug conjugates with excellent DAR properties.

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

[0550] [ka]

[0551] All of the above amino acid sequences are the same as the amino acid sequence of Sequence ID No. 2.

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

[0553] [ka]

[0554] All of the above amino acid sequences are the same as the amino acid sequence of Sequence ID No. 2.

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

[0556] [ka]

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

[0558] (5-1) Plasma stability test 700 μL of rat plasma (Charles River) was mixed with ADC mimic to a concentration of 0.1 mg / mL, and then sterile filtered. This solution was dispensed into six Eppendorf tubes in 50 μL portions. Three of the six samples were stored in an incubator set to 37°C for 4 days. The remaining three were stored in a freezer at -80°C for the same period. 100 μL of acetonitrile was added to each sample, and the mixture was vortexed and then centrifuged to obtain a precipitate. The resulting supernatant was collected and analyzed by HPLC.

[0559] (5-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis The measurement involved measuring the fluorescence molecular weight lost from the ADC mimic using liquid chromatography / fluorescence detection. Three samples stored in a freezer in Example 5-1 were designated as Day=0, and three samples stored at 37°C in Example 5-1 were designated as Day=4. The difference in fluorescence intensity between Day=4 and Day=0 was analyzed.

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

[0561] Example: Increase in fluorescence intensity of fluorescent molecules detached from ADC mimic = [(Fluorescence intensity on Day 4) - (Fluorescence intensity on Day 0)]

[0562] The increase in fluorescence intensity of the fluorescent molecule that was removed from the control ADC mimic = [(Fluorescence intensity on Day 4) - (Fluorescence intensity on Day 0)]

[0563] Using Carboxyrhodamine 110-PEG4-DBCO, the correlation between the fluorescence intensity area measured by HPLC and the concentration was calculated. Using this calculation formula, the increase in fluorescence intensity for each ADC mimic was converted to concentration. The dropout rate was calculated as the percentage of the aforementioned increase in concentration relative to the Day 0 concentration (set as 100%).

[0564] [Table 15]

[0565] [Table 16]

[0566] [Table 17]

[0567] Comparative Examples 1 (107) and 2 (109) do not correspond to M (a trivalent group that links carbon atoms in C=O adjacent to M and carbon atoms in C=W with a main chain portion consisting of 3 to 5 carbon atoms). This is because Comparative Examples 1 and 2 are linked not by 3 to 5 carbon atoms, but by a main chain portion consisting of 2 carbon atoms (Comparative Example 1) and a main chain portion consisting of 2 carbon atoms and 1 nitrogen atom (Comparative Example 2).

[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 15-17).

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

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

[0571] (6-2) Analysis of the amount of detached payload using HPLC analysis The amount of detached payload was measured using liquid chromatography-mass spectrometry (including tandem mass spectrometry). Three samples stored in a freezer in Example 6-1 were designated as Day=0, and three samples stored at 37°C in Example 6-1 were designated as Day=4. The MS intensities of the detected payloads for 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) as previously reported (International Publication No. 2019 / 240287 (WO2019 / 240287A1)), ADC(103) to (105) synthesized in Example 4 were more stable, and the results were similar to those of the comparative study of the ADC mimic in Example 5.

[0573] [Example 6: Confirmation of regioselectivity of azide-introduced antibodies] Peptide mapping was performed on the trastuzumab azide transderm obtained in (4-1) using the following procedure.

[0574] (6-1) Peptide mapping of trastuzumab azide derivative (86) Following a previous report (International Publication No. 2019 / 240288 (WO2019 / 240288A)), peptide mapping was performed on the trastuzumab azide transderm (86). Analysis using LC-MS / MS revealed a peptide fragment consisting of 33 amino acids, THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR, which includes azide modification of lysine residues due to trypsin digestion of trastuzumab. Furthermore, analysis using BioPharma Finder showed that modification of the lysine residue at position 246 or 248 in the EU numbering occurred with high selectivity. These results indicate that in the trastuzumab azide transderm (86) obtained in (4-1) above, site-selective conjugation occurs on the antibody heavy chain, specifically at Lys246 and Lys248 in the EU numbering.

[0575] (6-2) Peptide mapping of trastuzumab azide derivative (91) Peptide mapping was performed on the trastuzumab azide transmodifier (91) obtained in (4-1) in the same manner as in (6-1). Analysis using LC-MS / MS revealed a peptide fragment consisting of 33 amino acids, THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR, which includes azide modification of lysine residues due to trypsin digestion of trastuzumab. Furthermore, analysis using BioPharma Finder showed that modification of the lysine residue at position 246 or 248 in the EU numbering occurred with high selectivity.

[0576] (6-3) Peptide mapping of trastuzumab azide derivative (90) Following a previous report (International Publication No. 2019 / 240288 (WO2019 / 240288A)), peptide mapping was performed on the trastuzumab azide transderm (90). Analysis using LC-MS / MS revealed a peptide fragment, FNWYVDGVEVHNAKTKPR, consisting of 18 amino acid residues, including azide modification of lysine residues due to trypsin digestion of trastuzumab. Furthermore, analysis using BioPharma Finder showed that modification of lysine residues at positions 288 or 290 in the EU numbering occurred with high selectivity. These results indicate that in the trastuzumab azide transderm (90) obtained in (4-1) above, regioselective conjugation occurs on the antibody heavy chain, specifically at Lys288 and Lys290 in the EU numbering.

[0577] (6-4) Peptide mapping of trastuzumab azide derivative (92) The trastuzumab azid transderm obtained in (4-1) (92) was obtained in the same manner as in (6-1). Peptide mapping was performed. Analysis using LC-MS / MS revealed a peptide fragment, FNWYVDGVEVHNAKTKPR, consisting of 18 amino acid residues, including azide modification of lysine residues due to trypsin digestion of trastuzumab. Furthermore, analysis using BioPharma Finder showed that modification of lysine residues at positions 288 or 290 in the EU numbering occurred with high selectivity. These results indicate that in the trastuzumab azide transderm (92) obtained in (4-1) above, regioselective conjugation occurs on the antibody heavy chain, specifically at Lys288 and Lys290 in the EU numbering.

[0578] Example 7: Regioselective modification of multiple target regions of IgG1 Fc using IgG1 Fc affinity peptide reagents, and synthesis of antibody-drug conjugates. (7-1) Production of azide group-introduced antibody derivatives by regioselective modification of the anti-HER2 antibody trastuzumab followed by cleavage of the thioester group The azide-modified antibody (91) synthesized in Example (3-1) was replaced with 50 mM HEPES buffer (pH 8.2). To this solution, 10 equivalents of the modification reagent (64) synthesized in Example (1-14) were added relative to the antibody, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was replaced with 20 mM ammonium acetate buffer. The mass of the obtained antibody was measured by ESI-TOFMS, and 158,319 peaks were observed, indicating the introduction of two binding peptides into the azide-modified antibody synthesized in (3-1). These results confirm that the antibody obtained in this example (7-1) includes modifications to the lysine residues at positions 246 or 248 and 288 or 290 in the EU numbering on the two heavy chains of the antibody.

[0579] (7-2) Production of azide group-introduced antibody derivatives by regioselective modification of the anti-HER2 antibody trastuzumab followed by cleavage of the thioester group Using the method of Example (3-1), the linker cleavage reaction of the antibody-peptide conjugate obtained in Example (7-1) was performed with methoxyamine hydrochloride to obtain an azide-modified antibody. ESI-TOFMS analysis of the obtained azide antibody revealed peaks at 149 and 350, indicating that linker cleavage had progressed and four azide groups had been introduced to the antibody. These results confirm 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 in the EU numbering on the two heavy chains of the antibody.

[0580] (7-3) ADC synthesis with DAR=4 Using the same method as in Example (4-1), the azide-introduced antibody obtained in Example (7-2) was reacted with DBCO-MMAE (manufactured by Abzena) to obtain ADC. Therefore, it was confirmed that the antibody obtained in this example (7-2) contains four drugs (DAR=4) introduced into the side chains of the lysine residues at positions 246 or 248 and 288 or 290 in the EU numbering on the two heavy chains of the antibody.

Claims

1. Formula (I) below: 【Chemistry 1】 [During the ceremony, X represents a leaving group, The leaving group is R-S (where R represents a hydrogen atom or a potentially substituted monovalent phenyl group, and S represents a sulfur atom), Y represents an affinity peptide having a binding region to the CH2 domain in the antibody. The aforementioned antibody is human IgG, The affinity peptide comprises an amino acid sequence selected from the group consisting of (1) to (4) below: (1) RGNCAYHKGQIIWCTYH (Sequence ID 2); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (Sequence ID 4); (3) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (Sequence ID 5); and (4) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (Sequence ID 6), M represents a trivalent group that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion is a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a combination of a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. O represents an oxygen atom. S represents a sulfur atom. W represents an oxygen atom. N ３ This indicates an azide group, La indicates a divalent group. The main chain of the divalent group represented by La consists of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, a -C(=O)- group, or a combination of two or more of these groups. Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. It is composed of a group consisting of ), -O-, or two or more combinations thereof. A compound represented by ] or a salt thereof.

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

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 the lysine residue in the affinity peptide.

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

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

6. The following formula (I'): 【Chemistry 2】 [During the ceremony, X represents a leaving group, The leaving group is R-S (where R represents a hydrogen atom or a potentially substituted monovalent phenyl group, and S represents a sulfur atom), X' indicates a leaving group with a higher ability to leave than leaving group X. A leaving group with a higher ability to leave than the aforementioned leaving group X is a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a p-nitrophenyloxy group, or an N-succinimidyloxy group. M represents a trivalent group that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion is a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a combination of a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. O represents an oxygen atom. S represents a sulfur atom. W represents an oxygen atom. N ３ This indicates an azide group, La indicates a divalent group. The main chain of the divalent group represented by La consists of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, a -C(=O)- group, or a combination of two or more of these groups. Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. It is composed of a group consisting of ), -O-, or two or more combinations thereof. A compound represented by ] or a salt thereof.

7. The following equation (I''): 【Transformation 3】 [During the ceremony, X represents a leaving group, The leaving group is R-S (where R represents a hydrogen atom or a potentially substituted monovalent phenyl group, and S represents a sulfur atom), OH indicates a hydroxyl group. M represents a trivalent group that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion is a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a combination of a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. O represents an oxygen atom. S represents a sulfur atom. W represents an oxygen atom. N ３ This indicates an azide group, La indicates a divalent group. The main chain of the divalent group represented by La consists of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, a -C(=O)- group, or a combination of two or more of these groups. The main chain in the divalent group represented by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ -, where R ｄ represents a hydrogen atom.), -O-, or a group composed of a combination of two or more of these. A compound represented by ] or a salt thereof.

8. Formula (III): 【Chemistry 4】 [During the ceremony, Ig exhibits antibody properties and forms an amide bond with the adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. The aforementioned antibody is human IgG, The lysine residue is located 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 that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion is a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a combination of a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. O represents an oxygen atom. T represents a monovalent group, which may be a substituted hydroxyamino group. W represents an oxygen atom. N ３ This indicates an azide group, Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. The group consists of a ), -O-, or a combination of two or more of these. An azide group-introduced antibody derivative or a salt thereof, comprising a structural unit represented by [ ], wherein the average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0.

9. Formula (IV): 【Transformation 5】 [During the ceremony, Ig exhibits antibody properties and forms an amide bond with the adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. The aforementioned antibody is human IgG, The lysine residue is located 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 that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion is a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a combination of a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. O represents an oxygen atom. T represents a monovalent group, which may be a substituted hydroxyamino group. W represents an oxygen atom. N represents a nitrogen atom. Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. The group consists of a ), -O-, or a combination of two or more of these. Z indicates a functional substance. L indicates a divalent group. Ring A shows a ring fused with a triazole ring. Ring A is a monocycle or a fused ring of a monocycle and another ring, and includes a portion of the carbon-carbon double bond shared with the triazole. The monocycle is an allocyclic ring, or a heterocycle containing one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms. The other ring fused with the monoring is a cycloalkane, an arene, or a heteroring. The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0. Conjugates or salts of antibodies and functional substances containing the structural unit represented by ].

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

11. The azide-transformed 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 regioselectivity for the lysine residue at positions 246 / 248 of the human IgG heavy chain according to EU numbering.

12. The azide-transformed 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 regioselectivity for the lysine residue at position 288 / 290 of the human IgG heavy chain according to EU numbering.

13. An azide group-introduced antibody derivative or salt thereof according to any one of claims 8 or 10 to 12, wherein the average ratio r is 1.5 to 2.5, or a conjugate or salt thereof according to any one of claims 9 to 12.

14. An azide-introduced antibody derivative or 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 a conjugate or salt thereof according to any one of claims 11 to 13.

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

16. Formula (III): 【Transformation 6】 [During the ceremony, Ig exhibits antibody properties and forms an amide bond with the adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. The aforementioned antibody is human IgG, The lysine residue is located 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 that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion consists of a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. This is a combination of the above, O represents an oxygen atom. T represents a monovalent group, which may be a substituted hydroxyamino group. W represents an oxygen atom. N ３ This indicates an azide group, Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. The group consists of a ), -O-, or a combination of two or more of these. The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0. An azide group-introduced antibody derivative or a salt thereof containing a structural unit represented by the following formula (V): 【Transformation 7】 [During the ceremony, Ring A' represents a ring having triple bonds between carbon atoms. Z indicates a functional substance. L indicates a divalent group. It reacts with the target substance represented by ] Formula (IV): 【Transformation 8】 [During the ceremony, Ring A shows a ring fused to a triazole ring. Ring A is a monocycle or a fused ring of a monocycle and another ring, and includes a portion of the carbon-carbon double bond shared with the triazole. The monocycle is an allocyclic ring, or a heterocycle containing one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms. The other ring fused with the monoring is a cycloalkane, an arene, or a heteroring. N represents a nitrogen atom. Ig, M, O, T, W, Lb, and r are the same as those in formula (III) above. A method for producing an antibody and functional substance conjugate or a salt thereof, comprising generating an antibody and functional substance conjugate or a salt thereof containing a structural unit represented by ].

17. Formula (II) below: 【Chemistry 9】 [During the ceremony, Ig exhibits antibody properties and forms an amide bond with the adjacent carbonyl group via the amino groups in the side chains of the lysine residues in the two heavy chains. The aforementioned antibody is human IgG, The lysine residue is located 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 to the CH2 domain in the antibody, The affinity peptide comprises an amino acid sequence selected from the group consisting of (1) to (4) below: (1) RGNCAYHKGQIIWCTYH (Sequence ID 2); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (Sequence ID 4); (3) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (Sequence ID 5); and (4) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (Sequence ID 6), M represents a trivalent group that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion consists of a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. This is a combination of the above, O represents an oxygen atom. S represents a sulfur atom. W represents an oxygen atom. N ３ This indicates an azide group, La indicates a divalent group. The main chain of the divalent group represented by La consists of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, a -C(=O)- group, or a combination of two or more of these groups. Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. The group consists of a ), -O-, or a combination of two or more of these. The average ratio r of the amide bonds per two heavy chains is 1.0 to 3.

0. An antibody intermediate or a salt thereof containing the structural unit represented by ] is subjected to a thioester cleavage reaction. A method for producing an antibody and functional substance conjugate or salt thereof according to claim 16, further comprising generating an azide group-introduced antibody derivative or a salt thereof comprising a structural unit represented by formula (III) as described in claim 16.

18. Formula (I) below: 【Chemistry 10】 [During the ceremony, X represents a leaving group, The leaving group is R-S (where R represents a hydrogen atom or a potentially substituted monovalent phenyl group, and S represents a sulfur atom), Y represents an affinity peptide having a binding region to the CH2 domain in the antibody. The aforementioned antibody is human IgG, The affinity peptide comprises an amino acid sequence selected from the group consisting of (1) to (4) below: (1) RGNCAYHKGQIIWCTYH (Sequence ID 2); (2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (Sequence ID 4); (3) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (Sequence ID 5); and (4) MQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC (Sequence ID 6), M represents a trivalent group that connects a carbon atom in C=O adjacent to M with a carbon atom in C=W via a main chain portion consisting of 3 to 5 carbon atoms, wherein the main chain portion is a linear alkylene with 3 carbon atoms, a phenylene constituent carbon atom with 3 carbon atoms, or a combination of a linear alkylene with 1 or 2 carbon atoms and a phenylene constituent carbon atom with 3 carbon atoms. O represents an oxygen atom. S represents a sulfur atom. W represents an oxygen atom. N ３ This indicates an azide group, La indicates a divalent group. The main chain of the divalent group represented by La consists of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, a -C(=O)- group, or a combination of two or more of these groups. Lb indicates a divalent group. The main chain in the divalent group indicated by Lb is a divalent linear hydrocarbon group, -C(=O)-, -NR ｄ - (Here, R ｄ represents a hydrogen atom. It is composed of a group consisting of ), -O-, or two or more combinations thereof. The compound represented by ] or a salt thereof is reacted with the antibody, Formula (II) below: 【Chemistry 11】 [During the ceremony, Ig represents the antibody, Y, M, O, S, W, N ３ , La, and Lb are the same as those in formula (I) above, A method for producing an antibody and functional substance conjugate or salt thereof according to claim 17, further comprising producing an antibody intermediate or salt thereof containing a structural unit represented by formula (III) of claim 17, the average ratio r of the amide bonds per two heavy chains being 1.0 to 3.0.

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