Conjugate of antibody and functional substance or salt thereof, and antibody derivative and compound or salts thereof to be used in producing conjugate or salt thereof

Abstract

The present invention provides a conjugate of an antibody and a functional substance, said conjugate having excellent desired properties, or a salt thereof. More particularly, the present invention provides a conjugate of an antibody and a functional substance, said conjugate containing a structural unit represented by formula (1) [wherein: Ig represents an immunoglobulin unit containing two heavy chains and two light chains and bonded to LA adjacent to Ig via thiol groups in the side chains of a plurality of cysteine residues in the two heavy chains and two light chains; HG represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS represents a divalent group containing a cleavable moiety; ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group constitutes a π-electron conjugated system together with the aforesaid cleavable moiety); V represents an oxygen atom, a sulfur atom or an amino(NH) group; LA and LB independently represent a divalent group; D represents a functional substance; and the average number n of the aforesaid bonds per immunoglobulin unit is 1.5 or more] or a salt thereof, and substances related thereto.

Description

Conjugates of antibodies and functional substances or salts thereof, and antibody derivatives and compounds or salts thereof used in the production thereof

[0001] The present invention relates to a conjugate of an antibody and a functional substance, or a salt thereof, as well as a compound or a salt thereof used in the production thereof.

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

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

[0004] Another example of such a functional group used in the production of ADCs is a thiol group. Antibodies have disulfide groups because the heavy chains and the heavy and light chains are linked by disulfide bonds. For example, an IgG antibody composed of two heavy chains and two light chains has four disulfide bonds because the heavy chains and the heavy and light chains are linked by four disulfide bonds. Such disulfide bonds can be cleaved with a reducing agent. For example, if all four disulfide bonds in an IgG are cleaved, an IgG antibody with eight thiol groups is generated. Even if all four interchain disulfide bonds are cleaved, the heavy and light chains of the antibody do not dissociate. This is because each chain can maintain the antibody's higher-order structure through non-covalent bonds. This non-covalent bond also maintains the antibody's properties (target binding ability). For example, trastuzumab deruxtecan (Patent Document 5), known as Enhertz®, is an ADC in which all four interchain disulfide bonds are cleaved (reduced) before the drug is conjugated, with a drug-antibody ratio (DAR) of 8. Such ADCs are known to function as antigen-specific drugs by maintaining their antibody properties.

[0005] In ADCs, antibodies and drugs are linked via a linker. Various linkers are available for ADCs. For example, in ADCs used as anticancer drugs, linkers containing a dipeptide consisting of valine and citrulline (Val-Cit: VC structure) are available as linkers that are stable in human plasma and have a structure that can be cleaved by a specific enzyme to release the drug within cancer cells. Linkers containing such dipeptides are stable in human plasma, as shown in (A) below. However, as shown in (B) below, the VC structure is recognized by cathepsin B in the lysosomes of human cancer cells, resulting in cleavage of the amide bond present on the carboxy-terminal side of citrulline. Therefore, ADCs having linkers containing such dipeptides can release drugs within human cancer cells and exert their pharmacological effects.

[0006]

[0007]

[0008] However, ADCs having linkers containing dipeptides such as those described above are unstable in mouse plasma (Non-Patent Documents 1 and 2). This is because mouse plasma contains Ces1c, a carboxylase that recognizes VC structures and cleaves the amide bond present on the carboxy-terminal side of citrulline, and linkers containing dipeptides such as those described above are cleaved by Ces1c in the plasma. Therefore, the pharmacokinetics of ADCs having linkers containing dipeptides such as those described above differ significantly between mice and humans. This makes it difficult to evaluate their efficacy in humans using mice.

[0009]

[0010] As described above, in order to improve the instability of ADCs having an "antibody-spacer-VC structure-spacer-drug" structure in mouse plasma, attempts have been made to stabilize the ADC by modifying the linker (i.e., spacer-VC structure-spacer), and from this perspective, ADCs in which an antibody and a drug or a mimic thereof are linked via the above linker have been reported. For example, the following has been reported as an ADC in which the linker is not in the main chain linking the antibody and drug or a mimic thereof, but in a side chain of the main chain (Patent Document 6).

[0011] Ab: antibody; Cbz: benzyloxycarbonyl; Val: valine residue; Cit: citrulline residue

[0012] The above describes an example of a peptide structure cleaved by proteases in vivo, namely, the VC structure, which is typically cleaved by cathepsin B. However, it is known that peptide structures other than the VC structure are also cleaved by cathepsin B, and that other peptide structures are also cleaved by in vivo enzymes other than cathepsin B.

[0013] Incidentally, Non-Patent Document 3 describes that the higher the hydrophobicity of an ADC, the faster its plasma clearance, and that the hydrophobicity of an ADC can be evaluated by HIC (Hydrophobic Interaction Chromatography)-HPLC.

[0014] International Publication No. 2018 / 199337 International Publication No. 2019 / 240288 International Publication No. 2019 / 240287 International Publication No. 2020 / 090979 International Publication No. 2014 / 057687 International Publication No. 2015 / 038426

[0015] Dorywalska et al. , Bioconjugate Chem. , 2015, 26(4), 650-659 Dorywalska et al. , Mol Cancer Ther. , 2016, 15 (5), 958-70 Lyon et al. , Nat Biotechnol. , 2015, 33(7), 733-5

[0016] An object of the present invention is to provide a conjugate of an antibody and a functional substance, or a salt thereof, which has excellent desired properties.

[0017] As a result of extensive research, the present inventors have found that conjugates containing a linker of a specific structure in the side chain of the main chain linking the antibody and the drug (or drug mimic) (a linker that enables cleavage of the cleavable site and release of the drug (active drug with steric hindrance by the antibody eliminated) by engaging the π-electron resonance system; see Figure 1 ), and in which the linker is linked to a thiol group generated by cleavage of disulfide bonds between the heavy chains and between the heavy and light chains in the antibody (immunoglobulin unit), have excellent properties. For example, such conjugates or salts thereof can have excellent clearance (long residence time in the body) and low aggregation rates (high monomer ratios). Furthermore, the use of such linkers allows the antibody to retain hydrophilic groups after cleavage. That is, before cleavage, the drug is linked to the hydrophilic group in the form of a conjugate with the antibody, thereby suppressing the hydrophobicity of the drug (masking by the hydrophilic group). After cleavage, the hydrophilic group dissociates from the drug, allowing the drug to exhibit its inherent hydrophobicity. Therefore, for example, when the drug contained in the conjugate is a hydrophobic compound that is expected to have cell permeability (the so-called bystander effect), the present invention, which uses such a linker, has the advantage of easily enabling the drug to exhibit cell permeability. Herein, an ADC having such a linker is sometimes referred to as an exo-ADC.

[0018] The present inventors have also succeeded in developing antibody derivatives and compounds useful for preparing such conjugates. The conjugates, antibody derivatives, and compounds of the present invention, represented by structures such as those of formulas (1) to (7), have the technical feature of sharing partial structural units excluding X and Y among the structural units represented by formula (5). The present inventors have succeeded in developing a series of inventions having such technical features, and have completed the present invention. The prior art does not describe or suggest the chemical structure of the conjugates of the present invention or the relationship between such chemical structure and the excellent properties described above. The prior art also does not describe or suggest the antibody derivatives and compounds of the present invention that can be used to prepare such conjugates.

[0019] That is, the present invention is as follows.

[0020] In a first embodiment, the present invention provides a compound represented by the following formula (1): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. Ａ and bonded to, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavable moiety), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represent a divalent group, D represents a functional substance, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0021] In certain embodiments, the structural unit represented by formula (1) is represented by the following formula (1′): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. １ HG represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS represents a divalent group containing a cleavable moiety; ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety); R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represent a divalent group, D represents a functional substance, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0022] In certain embodiments, the structural unit represented by formula (1) is represented by the following formula (1″): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. １ is bonded to R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, CS represents a divalent group which contains a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represent a divalent group, D represents a functional substance, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0023] In certain embodiments, the structural unit represented by formula (1) is represented by the following formula (1a): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ , D, and n are the same as those shown in formula (1), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0024] In certain embodiments, the structural unit represented by formula (1a) is represented by the following formula (1b): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ, D, and n are the same as those shown in formula (1), and L Ｃ , and W are the same as those shown in formula (1a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0025] In certain embodiments, the structural unit represented by formula (1b) is represented by the following formula (1c): wherein Ig, HG, ring A, D, and n are the same as those represented by formula (1), and R Ａ , and R Ｂ are the same as those shown in formula (1b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0026] In certain embodiments, the structural unit represented by formula (1c) is represented by the following formula (1d): wherein Ig, ring A, D, and n are the same as those represented by formula (1), and R Ａ , and R Ｂ are the same as those shown in formula (1b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (1c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１, and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0027] In a preferred embodiment, the conjugate may exhibit an aggregation rate of 2.6% or less when analyzed by size exclusion chromatography.

[0028] In a second embodiment, the present invention provides a compound represented by the following formula (2): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. Ａ and bonded to, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavable moiety), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; ２ represents a bioorthogonal functional group, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0029] In a specific embodiment, the structural unit represented by formula (2) is represented by the following formula (2'): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. １ HG represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS represents a divalent group containing a cleavage site by cathepsin B; ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site); R １ , and R ２each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group; ２ represents a bioorthogonal functional group, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0030] In certain embodiments, the structural unit represented by formula (2) is represented by the following formula (2″): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. １ is bonded to R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, CS represents a divalent group which contains a cleavage site cleavable by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group; ２ represents a bioorthogonal functional group, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0031] In certain embodiments, the structural unit represented by formula (2) is represented by the following formula (2a): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ , B ２ , and n are the same as those shown in formula (2), and L Ｃ represents a bond or a divalent group, X Ａ , and XＢ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0032] In certain embodiments, the structural unit represented by formula (2a) is represented by the following formula (2b): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ , B ２ , and n are the same as those shown in formula (2), and L Ｃ , and W are the same as those shown in formula (2a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0033] In certain embodiments, the structural unit represented by formula (2b) is represented by the following formula (2c): [In the formula, Ig, HG, ring A, B ２ , and n are the same as those shown in formula (2), and R Ａ , and R Ｂ are the same as those shown in formula (2b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0034] In certain embodiments, the structural unit represented by formula (2c) is represented by the following formula (2d): [In the formula, Ig, ring A, B ２ , and n are the same as those shown in formula (2), and R Ａ , and R Ｂ are the same as those shown in formula (2b), and R １ , R２ , L １ , and L ２ are the same as those shown in formula (2c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0035] In a third embodiment, the present invention provides a compound represented by the following formula (3): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; １ represents a bioorthogonal functional group, and D represents a functional substance.], or a salt thereof.

[0036] In certain embodiments, the compound of formula (3) has the following formula (3'): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavage site), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group; １represents a bioorthogonal functional group, and D represents a functional substance.

[0037] In certain embodiments, the compound of formula (3) has the following formula (3″): [In the formula, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, CS represents a divalent group which contains a cleavage site cleavable by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group; １ represents a bioorthogonal functional group, and D represents a functional substance.

[0038] In certain embodiments, the compound of formula (3) has the following formula (3a): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and D are the same as those shown in formula (3), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0039] In certain embodiments, the compound of formula (3a) has the following formula (3b): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and D are the same as those shown in formula (3), and L Ｃ, and W are the same as those shown in formula (3a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0040] In certain embodiments, the compound of formula (3b) has the following formula (3c): [In the formula, HG, ring A, B １ , and D are the same as those shown in formula (3), and R Ａ , and R Ｂ are the same as those shown in formula (3b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0041] In certain embodiments, the compound of formula (3c) has the following formula (3d): [In the formula, rings A, B １ , and D are the same as those shown in formula (3), and R Ａ , and R Ｂ are the same as those shown in formula (3b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (3c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0042] The present invention also provides a reagent for derivatizing an antibody, comprising a compound represented by the above formula (3) or a subordinate formula thereof, or a salt thereof.

[0043] In a fourth embodiment, the present invention provides a compound represented by the following formula (4): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; １ represents the first bioorthogonal functional group, B ２ represents a second bioorthogonal functional group.], or a salt thereof.

[0044] In certain embodiments, the compound represented by formula (4) has the following formula (4'): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group; １ represents the first bioorthogonal functional group, B ２ represents a second bioorthogonal functional group.

[0045] In certain embodiments, the compound represented by formula (4) has the following formula (4″): [In the formula, R ＨＧ１ , and RＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, CS represents a divalent group which contains a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group; １ represents the first bioorthogonal functional group, B ２ represents a second bioorthogonal functional group.

[0046] In certain embodiments, the compound of formula (4) has the following formula (4a): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and B ２ are the same as those shown in formula (4), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0047] In certain embodiments, the compound of formula (4a) has the following formula (4b): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and B ２ are the same as those shown in formula (4), and L Ｃ , and W are the same as those shown in formula (4a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂrepresents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0048] In certain embodiments, the compound of formula (4b) has the following formula (4c): [In the formula, HG, ring A, B １ , and B ２ are the same as those shown in formula (4), and R Ａ , and R Ｂ are the same as those shown in formula (4b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0049] In certain embodiments, the compound of formula (4c) has the following formula (4d): [In the formula, rings A, B １ , and B ２ are the same as those shown in formula (4), and R Ａ , and R Ｂ are the same as those shown in formula (4b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (4c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0050] The present invention also provides a derivatization reagent for an antibody or a functional substance, which comprises a compound represented by the above formula (4) or a subordinate formula thereof, or a salt thereof.

[0051] In a fifth embodiment, the present invention provides a compound of the following (A), (B), or (C) or a salt thereof: (A) A compound of the following formula (5): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ represents a divalent group, and X and Y each independently represent a monovalent group; (B) a compound or a salt thereof represented by the following formula (6): [In the formula, HG, CS, ring A, V, L Ａ , and X are the same as those shown in formula (5), and L Ｂ represents a divalent group, ２ represents a bioorthogonal functional group.] or a salt thereof; or (C) a compound having a bioorthogonal functional group represented by the following formula (7): [In the formula, HG, CS, ring A, V, L Ａ , and Y are the same as those shown in formula (5), １ represents a bioorthogonal functional group.] or a salt thereof.

[0052] In a specific embodiment, the compound (A) is represented by the following formula (5'): [wherein HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable moiety), and X and Y each independently represent a monovalent group.]

[0053] In a specific embodiment, the compound (A) has the following formula (5″): [In the formula, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, CS represents a divalent group which contains a cleavable site, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system together with the cleavable site), and X and Y each independently represent a monovalent group.

[0054] In a specific embodiment, the compound (B) is represented by the following formula (6'): wherein HG, CS, ring A, and X are the same as those represented by formula (5), and R ２ represents a hydrogen atom or a monovalent group, and L ２ represents a divalent group, ２ represents a bioorthogonal functional group.

[0055] In a specific embodiment, the compound (B) is represented by the following formula (6″): wherein CS, ring A, and X are the same as those represented by formula (5), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ２ represents a hydrogen atom or a monovalent group, and L ２ represents a divalent group, ２ represents a bioorthogonal functional group.

[0056] In a specific embodiment, the compound (C) is represented by the following formula (7'): wherein HG, CS, ring A, V, and Y are the same as those represented by formula (5), and R １ represents a hydrogen atom or a monovalent group, and L １represents a divalent group, １ represents a bioorthogonal functional group.

[0057] In a specific embodiment, the compound (C) is represented by the following formula (7″): wherein CS, ring A, V, and Y are the same as those represented by formula (5), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R １ represents a hydrogen atom or a monovalent group, and L １ represents a divalent group, １ represents a bioorthogonal functional group.

[0058] In certain embodiments, the compound (A), (B), or (C) may be a compound (A-1), (B-1), or (C-1) below, respectively: (A-1) Formula (5a): [In the formula, HG, ring A, V, L Ａ , X, and Y are the same as those shown in formula (5), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH); (B-1) a compound or a salt thereof represented by the following formula (6a): [In the formula, HG, ring A, V, L Ａ , and X are the same as those shown in formula (5), and L Ｃ , X Ａ , X Ｂ , and W are the same as those shown in formula (5a), and L Ｂ , and B ２ are the same as those represented by formula (6), respectively; or (C-1) a compound represented by the following formula (7a): [In the formula, HG, ring A, V, L Ａ , and Y are the same as those shown in formula (5), and L Ｃ , X Ａ , X Ｂ , and W are the same as those shown in formula (5a), and B １ is the same as that represented by formula (7): or a salt thereof.

[0059] In certain embodiments, the compound (A-1), (B-1), or (C-1) may be a compound (A-2), (B-2), or (C-2) below, respectively: (A-2) Formula (5b): [In the formula, HG, ring A, V, L Ａ , X, and Y are the same as those shown in formula (5), and L Ｃ , and W are the same as those shown in formula (5a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents a side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue, or a salt thereof; (B-2) a compound represented by the following formula (6b): [In the formula, HG, ring A, V, L Ａ , and X are the same as those shown in formula (5), and L Ｃ , and W are the same as those shown in formula (5a), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and L Ｂ , and B ２ are the same as those represented by formula (6), respectively; or (C-2) a compound represented by the following formula (7b): [In the formula, HG, ring A, V, L Ａ , and Y are the same as those shown in formula (5), and L Ｃ , X Ａ , XＢ , and W are the same as those shown in formula (5a), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and B １ is the same as that represented by formula (7): or a salt thereof.

[0060] In certain embodiments, the compounds (A-2), (B-2), or (C-2) may be the compounds (A-3), (B-3), or (C-3) below, respectively: (A-3) Formula (5c): wherein HG, ring A, X, and Y are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those represented by formula (5b), respectively; (B-3) a compound represented by the following formula (6c): wherein HG, ring A, and X are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and B ２ are the same as those shown in equation (6), and R ２ represents a hydrogen atom or a monovalent group, and L ２ represents a divalent group. ], or a salt thereof; or (C-3) a compound represented by the following formula (7c): wherein HG, ring A, and Y are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and B １ is the same as that shown in equation (7), and R １ represents a hydrogen atom or a monovalent group, and L １ represents a divalent group, or a salt thereof.

[0061] In certain embodiments, the compounds (A-3), (B-3), or (C-3) may be the compounds (A-4), (B-4), or (C-4) below, respectively: (A-4) Formula (5d) below: wherein ring A, X, and Y are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ (B-4) a compound or a salt thereof represented by the following formula (6d): wherein ring A and X are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and L ＨＧ , R ＨＧ１ , and R ＨＧ２ is the same as that shown in formula (5d), and B ２ are the same as those shown in equation (6), and R ２ , and L ２ is the same as that represented by formula (6c), or a salt thereof; or (C-4) a compound represented by the following formula (7d): wherein ring A and Y are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and L ＨＧ , R ＨＧ１ , and R ＨＧ２ is the same as that shown in formula (5d), and B １ is the same as that shown in equation (7), and R １, and L １ is the same as that represented by formula (7c): or a salt thereof.

[0062] In a preferred embodiment, the immunoglobulin unit may be a human immunoglobulin unit.

[0063] In a preferred embodiment, the human immunoglobulin unit may be a human IgG antibody.

[0064] In a preferred embodiment, n may be between 2.0 and 8.0.

[0065] In a preferred embodiment, n may be between 6.0 and 8.0.

[0066] In a preferred embodiment, n may be between 7.0 and 8.0.

[0067] In a preferred embodiment, the hydrophilic group may be one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety.

[0068] In a preferred embodiment, the cleavage site may be an enzymatic cleavage site.

[0069] In a preferred embodiment, the enzyme may be cathepsin B.

[0070] In a preferred embodiment, the divalent group containing a cleavable moiety may satisfy the following conditions (i) to (iii): (i) the cleavable moiety is -CO-W- (wherein the hyphen (-) represents a bond, and W is an oxygen atom, a sulfur atom, or an amino group (NH), and is bonded to the divalent aromatic ring group); (ii) the bond between CO and W is the site that undergoes cleavage; (iii) the divalent aromatic ring group and W form a π-electron conjugated system.

[0071] In a preferred embodiment, ring A may be a phenylene group which may have a substituent.

[0072] In a preferred embodiment, the functional substance may be a drug, a labeling substance, or a stabilizer.

[0073] In a preferred embodiment, X Ａ represents a valine residue, a phenylalanine residue, a threonine residue, a leucine residue, or an alanine residue; X Ｂ may represent a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0074] In a preferred embodiment, a divalent group (-L ＨＧ -) is represented by the following formula (a): -(C(R ＨＧ ) ２ ) ｎ１ -(C=O) ｎ２ - (NR ＨＧ ) ｎ３ -(C(R ＨＧ ) ２ ) ｎ４ (a) wherein a plurality of R ＨＧ each independently represent a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, n1 is an integer of 0 to 3, n2 is an integer of 0 or 1, n3 is an integer of 0 or 1, and n4 is an integer of 0 to 3.

[0075] In a preferred embodiment, the divalent group represented by formula (a) is represented by the following formula (a1), (a2), or (a3): (a1) -(C(R ＨＧ ) ２ ) -; (a2) -(C(R ＨＧ ) ２ )-(C=O)-(NR ＨＧ )-(C(R ＨＧ ) ２ )-; or (a3) ​​-(C=O)-(C(R ＨＧ ) ２ ) ２ -; [wherein a plurality of R ＨＧ each independently represents a hydrogen atom, a hydrophilic group, or an alkyl group having 1 to 6 carbon atoms and containing a hydrophilic group.

[0076] In a preferred embodiment, the hydrophilic groups may each independently be a carboxylic acid group, a sulfonic acid group, or a hydroxyl group.

[0077] In a preferred embodiment, the hydrophilic group may be a carboxylic acid group.

[0078] In a preferred embodiment, B ２ The bioorthogonal functional group represented by may be a furan residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.

[0079] The conjugates or salts thereof of the present invention can have excellent properties such as a long residence time in the body and a high monomer ratio (low aggregation rate). The antibody derivatives and compounds or salts thereof, and reagents of the present invention are useful, for example, as synthetic intermediates in the production of the above-mentioned conjugates.

[0080] FIG. 1 shows an example of the release of a drug (an active drug in which steric hindrance caused by an antibody has been resolved) through cleavage of the cleavable site and engagement of the π-electron resonance system. The conjugates, antibody derivatives, and compounds of the present invention are designed to enable such release (see also the explanation of the action of cathepsin B in the Background Art section, as appropriate). The antibody can retain its hydrophilic group even after cleavage. That is, before cleavage, the drug is linked to a hydrophilic group in the form of a conjugate with the antibody, thereby suppressing the hydrophobicity of the drug (masking by the hydrophilic group). After cleavage, the hydrophilic group dissociates from the drug, allowing the drug to exhibit its inherent hydrophobicity. FIG. 2 shows the interrelationships between the conjugate of the present invention represented by formula (1), the antibody derivative of the present invention represented by formula (2), and the compounds of the present invention represented by formulas (3) to (7). These substances share partial structural units, excluding X and Y, of the structural unit represented by formula (5). Furthermore, these substances can be synthesized according to the scheme shown in FIG. 2. Accordingly, the present invention provides a series of inventions relating synthetic intermediates and final synthetic products. Figure 3 is a diagram showing an outline of the synthesis of the conjugate of the present invention represented by formula (1), the antibody derivative of the present invention represented by formula (2), and the compounds of the present invention represented by formulas (3) and (4). Figure 4 is a diagram showing an example of the synthesis of the compounds of the present invention represented by formulas (4) to (7). Figure 5 is a diagram showing an example of the synthesis of the compounds of formulas (4c) to (7c), which are preferred examples of the compounds of the present invention represented by formulas (4) to (7). DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide

[0081] 1. Definitions of General Terms In the present invention, the term "antibody" is as follows. Furthermore, the term "immunoglobulin unit" corresponds to a bivalent monomer unit that is the basic component of such antibodies, and is a unit containing two heavy chains and two light chains. Therefore, the definitions, examples, and preferred examples of the origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, and location of cysteine ​​residues of the immunoglobulin unit are the same as those of the antibody described below.

[0082] The origin of the antibody is not particularly limited, and may be derived from animals such as mammals and birds (e.g., chickens). Preferably, the immunoglobulin unit is derived from a mammal. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pets (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, more preferably humans.

[0083] The antibody may be a polyclonal or monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent 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 with specific glycosylation (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated 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, full-length antibodies or antibody fragments containing the variable region and CH1 and CH2 domains can be used as monoclonal antibodies, with full-length antibodies being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0084] Any antigen can be used as the antigen for the antibody. Examples of such antigens include proteins (including oligopeptides and polypeptides, and may also be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low-molecular-weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0085] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0086] (1) Cancer region PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesotheli n, CD138, c-Met, Ang2, VEGF- A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, maccrophase inhibitory factor, CD74, Notch1, Notch 2. Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM- CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEG F, RSPO, LIV-1, SLITRK6, Nect in-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissu 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, C D166, ROR1, GITR, BCMA, TBA, LAG- 3. EphA2, TIM-3, CD-200, EGFRvIII , CD16A, CD32B, PIGF, Axl, MICA / B , Thomsen-Friedenreich, CD39, CD 37、CD73、CLEC12A、Lgr3、トランスフェリンReceptor, TGFβ, IL-17, 5T4, RTK, Immune Supplier Protein, NaPi2b, ルイス blood type B antigen, A34, Lysil-Oxidase , DLK-1, TROP-2, α9インテグリン, TAG-72 (CA72-4), CD70,

[0087] (2) Autoimmune diseases / inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS , Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP (Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74 , CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT

[0088] (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

[0089] (4) Infectious diseases: Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0090] (5) Genetic and rare diseases: Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0091] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0092] (7) Bone / orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0093] (8) Blood diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0094] (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, Edoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

[0095] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, and ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, and daratumumab). , ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (if no IgG subtype is mentioned, IgG1 is implied).

[0096] An immunoglobulin unit (e.g., a bivalent antibody such as IgG) consisting of two heavy chains and two light chains has four disulfide bonds between the heavy chains and between the heavy and light chains. When a reducing agent is applied sufficiently to such an immunoglobulin unit, eight thiol groups are generated from the four disulfide bonds. Any reducing agent capable of cleaving disulfide bonds to generate thiol groups can be used, including, for example, tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Incidentally, even when all four disulfide bonds are reduced, the heavy and light chains of the antibody do not dissociate. This is because the non-covalent bonds between each chain allow the antibody's higher-order structure to be maintained. These non-covalent bonds also maintain the antibody's properties (targeting and avidity). For example, trastuzumab deruxtecan, known as Enhertz®, is an ADC with a DAR of 8, in which all four interchain disulfide bonds are reduced (cleaved) to generate thiol groups to which drugs are attached. Such ADCs are known to function as antigen-specific drugs by maintaining their antibody properties.

[0097] In the present invention, an immunoglobulin unit comprising two heavy chains and two light chains can be bound to a modifying group (e.g., LA or L1, described below) adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues (multiple cysteine ​​residues in the two heavy chains and two light chains contained in the immunoglobulin unit) generated by treating the immunoglobulin unit with a reducing agent. The number corresponding to the multiple residues is, for example, 2 or more (e.g., 2 to 8), preferably 3 or more (e.g., 3 to 8), more preferably 4 or more (e.g., 4 to 8), even more preferably 5 or more (e.g., 5 to 8), and particularly preferably 6 or more (e.g., 6 to 8), 7 or more (e.g., 7 to 8), or 8.

[0098] In the present invention, as long as the above-mentioned cysteine ​​residues in the immunoglobulin unit or antibody are modified, specific amino acid residues at other positions may also be regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in the immunoglobulin unit or antibody are described in WO 2018 / 199337, WO 2019 / 240288, WO 2019 / 240287, and WO 2020 / 090979. Such specific amino acid residues may be amino acid residues having a side chain that is easily modified (e.g., an amino group, a carboxy group, an amide group, a hydroxy group) (e.g., a lysine residue, an aspartic acid residue, a glutamic acid residue, an asparagine residue, a glutamine residue, a threonine residue, a serine residue, a tyrosine residue) having a side chain that is easily modified (e.g., an amino group, a carboxy group, an amide group, a hydroxy group), etc. Preferred are lysine residues having a side chain containing an amino group, tyrosine residues having a side chain containing a hydroxy group, serine residues, and threonine residues, and more preferably lysine residues (e.g., lysine residues at positions 246 / 248, 288 / 290, and 317).

[0099] (Halogen Atom) Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0100] (Monovalent Group) Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

[0101] The monovalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) substituents described below.

[0102] (Monovalent Hydrocarbon Group and Related Terms) Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0103] A monovalent chain hydrocarbon group refers to a hydrocarbon group consisting only of a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0104] The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms. When the alkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0105] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. When the alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0106] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. When the alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0107] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0108] The monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not have to be composed only of alicyclic hydrocarbons, and may contain a chain structure as part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0109] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. When the cycloalkyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0110] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 or 6 carbon atoms. When the cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0111] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 or 6 carbon atoms. When the cycloalkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0112] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0113] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed of only aromatic rings, and it may contain a chain structure or an alicyclic hydrocarbon as part of the ring, and the aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, an aryl group having 6 to 12 carbon atoms is preferred, an aryl group having 6 to 10 carbon atoms is more preferred, and an aryl group having 6 carbon atoms is even more preferred. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms mentioned above. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0114] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0115] Among these, alkyl, cycloalkyl and aryl are preferred as the monovalent hydrocarbon group.

[0116] (Monovalent heterocyclic group and related terms) A monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic group preferably contains one or more atoms selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably contains one or more atoms selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom.

[0117] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0118] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0119] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0120] (Divalent Group) The divalent group is a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, —C(═O)—, —C(═S)—, —NR ７ -, -C(=O)-NR ７ -, -NR ７ -C(=O)-, -C(=S)-NR ７ -, -NR ７ -C(=S)-, -O-, -S-, -(OR ８ ) ｍ -, and -(S-R ８ ) ｍ１ -, or a group having a main chain structure containing two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2 or 3). ７ represents a hydrogen atom or a substituent to be described later. ８represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. m1 is an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, and particularly preferably an integer of 1 to 3.

[0121] The divalent linear hydrocarbon group is a linear alkylene, linear alkenylene, or linear alkynylene. The linear alkylene is a linear alkylene having 1 to 6 carbon atoms, preferably a linear alkylene having 1 to 4 carbon atoms. Examples of linear alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of the straight-chain alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. As the divalent straight-chain hydrocarbon group, a straight-chain alkylene is preferred.

[0122] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene.

[0123] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocycle 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. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is 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, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

[0124] Preferably, the divalent group is alkylene, arylene, —C(═O)—, —NR ７ -, -C(=O)-NR ７ -, -NR ７ -C(=O)-, -O-, and -(O-R ８ ) ｍ or a divalent group having a main chain structure containing one group selected from the group consisting of alkylene, arylene, —C(═O)—, —NR ７ -, -C(=O)-NR ７ -, -NR ７ -C(=O)-, -O-, and -(O-R ８ ) ｍ１ - is a divalent group having a main chain structure containing two or more groups selected from the group consisting of R ７ is a hydrogen atom or alkyl, R ８ is alkylene or arylene, and m1 may be an integer of 1 to 5 (ie, 1, 2, 3, 4, or 5). The alkylene, arylene, and alkyl are as defined above.

[0125] The main chain structure in the divalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, still more preferably 1 to 5, and particularly preferably 1 to 3) substituents described below.

[0126] (Substituents) Examples of the substituents include: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) an aralkyl; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or a monovalent hydrocarbon group; or (vi) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ- (R ｂ and R ｃ are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, and a carboxyl group.

[0127] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, and monovalent heterocyclic group in the above substituents are the same as those described above.

[0128] Aralkyl refers to arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0129] Preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) an aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl having 1 to 12 carbon atoms; (vi) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (vii) the same groups as those enumerated in (vii) above.

[0130] More preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 12 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (iv) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0131] Even more preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 6 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; (iv) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0132] Particularly preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 4 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; (iv) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0133] (Hydrophilic Group) A hydrophilic group is a group that can make a structural unit represented by formulas (1) to (7) or a subordinate formula thereof more hydrophilic. By having a hydrophilic group at a predetermined position in the structural unit, the properties of the conjugate can be further improved. Examples of such hydrophilic groups include a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety. The conjugate may contain one or more hydrophilic groups (e.g., 1, 2, 3, 4, or 5).

[0134] The polyethylene glycol (PEG) group is —(CH ２ -CH ２ -O-) ｋ１- is a divalent group represented by the formula: When the conjugate has a polyethylene glycol group, the conjugate may have a monovalent group in which one bond of the polyethylene glycol group is bonded to a hydrogen atom or a monovalent group (e.g., a monovalent hydrocarbon group). k1 may be, for example, an integer of 3 or greater, preferably an integer of 4 or greater, more preferably an integer of 5 or greater, and even more preferably an integer of 6 or greater. k1 may also be an integer of 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k1 may be an integer from 3 to 15, preferably an integer from 4 to 12, more preferably an integer from 5 to 10, and even more preferably an integer from 4 to 9.

[0135] The polysarcosine group is -(NCH ３ -CH ２ -CO-) ｋ２ - is a divalent group represented by the formula: -. A polysarcosine group can be used as a substitute for PEG. k2 may be, for example, an integer of 3 or greater, preferably an integer of 4 or greater, more preferably an integer of 5 or greater, and even more preferably an integer of 6 or greater. k2 may also be an integer of 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k2 may be an integer from 3 to 15, preferably an integer from 4 to 12, more preferably an integer from 5 to 10, and even more preferably an integer from 4 to 9.

[0136] The sugar moiety can be a monosaccharide, an oligosaccharide (e.g., a disaccharide, trisaccharide, tetrasaccharide, pentasaccharide), or a polysaccharide. The sugar moiety can include an aldose or a ketose, or a combination thereof. The sugar moiety can be a monosaccharide such as ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, or fructose, or an amino sugar (e.g., glucosamine), or an oligosaccharide or polysaccharide containing such a monosaccharide.

[0137] In certain embodiments, the sugar moiety may be a low-molecular-weight hydrophilic group. A low-molecular-weight hydrophilic group refers to a hydrophilic group having a molecular weight of 1,500 or less. The molecular weight of the low-molecular-weight hydrophilic group may preferably be 1,200 or less, 1,000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less. Examples of low-molecular-weight hydrophilic groups include carboxylic acid groups, sulfonic acid groups, hydroxyl groups, polyethylene glycol groups, polysarcosine groups, and sugar moieties (e.g., monosaccharides, oligosaccharides) that satisfy the above molecular weights.

[0138] (Bio-orthogonal functional group) Bio-orthogonal functional groups are groups that do not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bio-orthogonal functional groups are well known in the art (e.g., Sharpless K.B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

[0139] In the present invention, a bioorthogonal functional group for a protein is used as the bioorthogonal functional group. This is because the antibody to be derivatized with the reagent of the present invention is a protein. The bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 naturally occurring amino acid residues that make up proteins, or that reacts slowly with the side chains, but reacts with the desired functional group. The 20 naturally occurring amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Among these 20 naturally occurring amino acids, glycine has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine have hydrocarbon side chains (i.e., do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms). These amino acids are inert to normal reactions. Therefore, the bioorthogonal functional group for proteins is a group that does not react or reacts slowly with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, in addition to the side chains of these amino acids that have side chains that are inert to normal reactions, but reacts with the desired functional group.

[0140] Examples of such bioorthogonal functional groups include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, it is sufficient that they have a vinylene (ethenylene) moiety, which is the smallest unit having a double bond between carbon atoms; the same applies below), alkyne residues (in other words, it is sufficient that they have an ethynylene moiety, which is the smallest unit having a triple bond between carbon atoms; the same applies below), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position; the same applies below), isonitrile residues, sydnone residues, and selenium residues.

[0141] More specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of: [where R １ａ , single or multiple R １ｂ and one or more R １ｃ are the same or different and are the above-mentioned substituents or electron-withdrawing groups, and * is a bond.

[0142] Examples of the electron-withdrawing group include a halogen atom, an alkyl substituted with a halogen atom (e.g., trifluoromethyl), a boronic acid residue, mesyl, tosyl, triflate, nitro, cyano, a phenyl group, and a keto group (e.g., acyl), and a halogen atom, a boronic acid residue, mesyl, tosyl, and triflate are preferred.

[0143] In certain embodiments, the bioorthogonal functional group may be protected. An optionally protected bioorthogonal functional group refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. An unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above. A protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of the protecting group. The cleavage of the protecting group can be carried out by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of an amide bond). Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli selected from the group consisting of light, or (c) leaving the linker when using a cleavable linker containing a self-cleaving cleavable moiety. Such protecting groups and their cleavage conditions are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, J.M., Journal of American Chemical Society. Society. 132, 17928 (2010); Thompson, D. H. , Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G. , Journal of Controlled Release, 95, 291 (2004)).

[0144] Protected bioorthogonal functional groups include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

[0145] More specifically, the protected bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of: [wherein the wavy line perpendicular to the bond indicates the cleavage site, and ２ａ are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents, and * is a bond.

[0146] Preferably, the optionally protected bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0147] (Functional Substance) The functional substance is not particularly limited as long as it is a substance that imparts any function to an antibody, and examples thereof include drugs, labeling substances, affinity substances, transport substances, and stabilizers. Preferably, the functional substance is a drug, labeling substance, affinity substance, or transport substance, or may be a drug or labeling substance. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked.

[0148] The drug may be a drug for any disease, such as cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases / inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infection, viral infection), genetic / rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone / orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0149] More specifically, the drug may be an anticancer drug. Examples of anticancer drugs include chemotherapeutic agents, toxins, radioisotopes, and substances containing the same. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioactive isotopes of hydrogen atoms (e.g., ３ H), radioactive isotopes of carbon atoms (e.g., １４ C), radioactive isotopes of phosphorus atoms (e.g., ３２ P), radioactive isotopes of sulfur atoms (e.g., 35 Ｓ ), radioactive isotopes of yttrium (e.g., ９０ Y), radioactive isotopes of technetium (e.g., ９９ｍ Tc), radioactive isotopes of indium (e.g., １１１ In), radioactive isotopes of iodine atoms (e.g., １２３ I, １２５ I, １２９ I, １３１ I), radioactive isotopes of samarium (e.g., １５３ Sm), radioactive isotopes of rhenium (e.g., １８６ Re), radioactive isotopes of astatine (e.g., ２１１ At), radioactive isotopes of bismuth (e.g., ２１２ More specifically, examples of the drug include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0150] A labeling substance is a substance that enables detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2′-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), and substances containing the same.

[0151] An affinity substance is a substance that has affinity for a target. Examples of affinity substances include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary chains to target nucleic acids. The affinity substance is preferably an affinity protein or affinity peptide, and more preferably an antibody. The species of animals from which antibodies used as functional substances are derived are the same as those described above.

[0152] The type of antibody used as the functional substance may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent 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 with specific glycosylation (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated 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. Examples of antibodies used as the functional substance include full-length antibodies and fragments thereof (fragment antibodies). The antibody fragment may be any fragment that maintains binding to a desired antigen, and may be, for example, Fab, Fab', F(ab') ２ , scFv.

[0153] The antigenicity of the antibody used as the functional substance may be the same as or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, preferably different. Furthermore, the origin of the antibody used as the functional substance may be the same as or different from the origin of the immunoglobulin unit, preferably different. Therefore, the antibody used as the functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom. The antibody used as the functional substance may also be an IgG1, IgG2, IgG3, or IgG4 antibody mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom.

[0154] The transporter is a substance capable of transporting a compound. Preferred transporters are substances capable of encapsulating a compound in a protein shell (e.g., multimers) (e.g., ferritin such as human ferritin, virus particles, virus-like particles).

[0155] Stabilizers are substances that enable antibody stabilization, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0156] The functional substance may also be a peptide, a protein, a nucleic acid, a low-molecular-weight organic compound, a sugar chain, a lipid, a high-molecular-weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Examples of nucleic acids also include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of low-molecular-weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.

[0157] In certain embodiments, the functional substance may be a substance having an aromatic ring, such as monomethylauristatin (e.g., monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF)), or exatecan.

[0158] (Salts) In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Salts with inorganic acids include, for example, salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Salts with organic acids include, for example, 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. Salts with inorganic bases include, for example, salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Salts with organic bases include, for example, 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 a salt with an organic acid (e.g., trifluoroacetic acid).

[0159] 2. Conjugate or Salt thereof The present invention relates to a conjugate or salt thereof represented by the following formula (1): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. Ａand bonded to, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavable moiety, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavable moiety), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represent a divalent group, D represents a functional substance, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0160] In Formula (1) and other formulae presented in connection with the present invention, a hyphen (-) indicates that the two units (e.g., atoms, groups) on either side of it are covalently bonded. When there is no unit on one side of the hyphen (-), the hyphen (-) indicates a bond.

[0161] The antibody comprises an immunoglobulin unit as described above. Examples of such antibodies include IgG antibodies, IgD antibodies, and IgE antibodies, which comprise immunoglobulin units comprising two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy and light chains; IgA antibodies, which comprise immunoglobulin units comprising four heavy chains and four light chains and having disulfide bonds between the heavy chains and between the heavy and light chains; and IgM antibodies, which comprise immunoglobulin units comprising eight heavy chains and eight light chains and having disulfide bonds between the heavy chains and between the heavy and light chains. IgG antibodies (e.g., IgG1, IgG2, IgG3, IgG4) are preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0162] Antibody and linker site (L Ａ) can be achieved by bonding between a thiol group in the side chain of a cysteine ​​residue in the antibody and an atom or group capable of bonding thereto (e.g., a maleimide residue, a carbonyl group, a thiol group).

[0163] In formula (1), HG represents a hydrophilic group or a monovalent group containing a hydrophilic group. The hydrophilic group and the monovalent group are as described above. Preferably, HG may represent a monovalent group containing a hydrophilic group.

[0164] A cleavable site is one that can be cleaved in an appropriate environment (eg, intracellular or extracellular). Examples of the cleavable site include an enzyme-cleavable site (e.g., U.S. Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999); The FEBS Journal 287:1936-1969 (2020)), an acid-cleavable site (a site that can be cleaved at a local acidic site present in the body) (e.g., U.S. Pat. Nos. 5,622,929, 5,122,368, 5,824,805; The FEBS Journal 287:1936-1969 (2020)), a blood glutathione-cleavable site (e.g., U.S. Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999); The FEBS Journal 287:1936-1969 (2020)), and an acid-cleavable site (a site that can be cleaved at a local acidic site present in the body) (e.g., U.S. Pat. Nos. 5,622,929, 5,122,368, 5,824,805; The FEBS Journal 287:1936-1969 (2020)). 287:1936-1969 (2020)). The cleavable site may be self-immolative (e.g., WO 02 / 083180, WO 04 / 043493, WO 05 / 112919).

[0165] Preferably, the cleavage site is an enzymatic cleavage site. Examples of enzymatic cleavage sites include cleavage sites (typically cleavable peptide sites) by intracellular proteases (e.g., proteases present in lysosomes or endosomes) and extracellular proteases (e.g., secretory proteases). More preferably, the cleavage site is a cleavage site by an intracellular protease (e.g., a protease present in lysosomes or endosomes). Even more preferably, the cleavage site is a cleavage site by a protease present in lysosomes. Particularly preferably, the cleavage site is a cleavage site by cathepsin B.

[0166] In certain embodiments, the divalent group containing a cleavable site may satisfy the following conditions (i) to (iii): (i) the cleavable site is -CO-W- (wherein the hyphen (-) represents a bond, and W is an oxygen atom, a sulfur atom, or an amino group (NH), and is bonded to the divalent aromatic ring group); (ii) the bond between CO and W is the site that undergoes cleavage; (iii) the divalent aromatic ring group forms a π-electron conjugated system with W. When the divalent group containing a cleavable site by cathepsin B satisfies the above conditions (i) to (iii), the cleavage of the bond between CO and W works in conjunction with the π-electron conjugated system in the divalent aromatic ring group in ring A (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavable site) and V, thereby efficiently cleaving the bond between V and the tertiary carbon atom located under ring A. W is preferably an oxygen atom or an amino group (NH), more preferably an amino group (NH). Thus, the cleavable site is preferably an amide bond site.

[0167] The cleavage site may be a cleavage site comprising a peptide. The cleavage site comprising a peptide may be a cleavage site consisting of a cleavable peptide cleaved by an enzyme. The cleavage site comprising a peptide may also further comprise a peptide not involved in cleavage in addition to the cleavable peptide cleaved by the enzyme. Even if the cleavage site comprising a peptide further comprises a peptide not involved in cleavage, it can be cleaved as long as it comprises a cleavable peptide cleaved by the enzyme.

[0168] The amino acid residues constituting the peptide at the cleavable site may be any amino acid residue. Examples of such any amino acid residues include α-amino acid residues, β-amino acid residues, and γ-amino acid residues, with α-amino acid residues being preferred. Examples of α-amino acid residues include alanine residues, asparagine residues, cysteine ​​residues, glutamine residues, glycine residues, isoleucine residues, leucine residues, methionine residues, phenylalanine residues, proline residues, serine residues, threonine residues, tryptophan residues, tyrosine residues, valine residues, aspartic acid residues, glutamic acid residues, arginine residues, histidine residues, lysine residues, and citrulline residues. Such any amino acid residues may also be L-amino acid residues or D-amino acid residues, with L-amino acid residues being preferred. Preferably, the any amino acid residue may be an L-α-amino acid residue (e.g., the L-form of the specific α-amino acid residues described above) or a glycine residue.

[0169] In certain embodiments, the peptide-containing cleavage site may comprise a cleavage peptide comprised of amino acid residues selected from the group consisting of valine, phenylalanine, threonine, leucine, citrulline, alanine, glutamic acid, glutamine, lysine, arginine, and methionine residues, and combinations thereof.

[0170] The number of amino acid residues constituting the peptide at the cleavable site may be 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more. The number of amino acid residues constituting the peptide at the cleavable site may also be 20 or less, 15 or less, 12 or less, 10 or less, 8 or less, 6 or less, or 4 or less. More specifically, the number of amino acid residues constituting the peptide may be 2 to 20, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4, or 3 to 20, 3 to 15, 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 3 to 4, or 4 to 20, 4 to 15, 4 to 12, 4 to 10, 4 to 8, or 4 to 6.

[0171] In certain embodiments, the number of amino acid residues constituting the peptide at the cleavable site may be 2. The number of amino acid residues constituting the peptide at the cleavable site may also be 3 or 4. Alternatively, the number of amino acid residues constituting the cleavable peptide may be 2. The number of amino acid residues constituting the cleavable peptide may also be 3 or 4.

[0172] In certain embodiments, the divalent group containing the cleavable site, represented by CS, is -L Ｃ -CS'-W- (wherein the hyphen (-) represents a bond, L Ｃ represents a bond or a divalent group, CS' represents a cleavable site, and W represents an oxygen atom, a sulfur atom, or an amino group (NH). The definition, examples, and preferred examples of the cleavable site represented by CS' are the same as those of the cleavable site described above. W represents an oxygen atom, a sulfur atom, or an amino group (NH), preferably an oxygen atom or an amino group (NH), more preferably an amino group (NH). The same applies to CS (divalent groups containing a cleavable site) in other formulas.

[0173] Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavable site). The divalent aromatic ring group is the above-mentioned arylene or divalent aromatic heterocycle. The position of the divalent aromatic ring group to which two adjacent atoms (a carbon atom and an adjacent atom in CS) are bonded is not particularly limited, as long as cleavage between V and the carbon atom adjacent thereto occurs due to π-electron conjugation when the cleavable site is cleaved (see Figure 1). Such a position is common technical knowledge in the field and can be easily determined by a person skilled in the art depending on factors such as the type of cleavable site and the divalent aromatic ring group.

[0174] Preferably, ring A may be a divalent monocyclic aromatic ring group which may have a substituent. The divalent aromatic ring group is a phenylene group or a divalent monocyclic aromatic heterocyclic group.

[0175] More preferably, ring A may be a divalent 6-membered aromatic ring group. Examples of the 6-membered aromatic ring group include the various groups described above. In this case, the position of the divalent 6-membered aromatic ring group where two adjacent atoms are bonded is the ortho position or the para position, preferably the para position.

[0176] Even more preferably, ring A may be a phenylene group which may have a substituent. In this case, the positions of the phenylene group to which two adjacent atoms are bonded are ortho- or para-positions, preferably para-positions.

[0177] The substituents in the optionally substituted divalent aromatic ring group are as described above. Such substituents may be the electron-withdrawing groups described above.

[0178] V represents an oxygen atom, a sulfur atom, or an amino group (NH). V is preferably an oxygen atom or a sulfur atom, and more preferably an oxygen atom.

[0179] L Ａ is a divalent group that can link Ig and the carbon atom adjacent to the ring A. Ｂ is a divalent group capable of linking D and V.

[0180] In certain embodiments, L Ａ , and L ＢEach divalent group represented by may contain a moiety generated by the reaction of two bioorthogonal functional groups that can react with each other. Combinations of two bioorthogonal functional groups that can react with each other are well known, so those skilled in the art can appropriately select such combinations and appropriately design a divalent group containing a moiety generated by the reaction of two bioorthogonal functional groups that can react with each other. Examples of combinations of bioorthogonal functional groups that can react with each other include a combination of a thiol residue and a maleimide residue, a combination of a furan residue and a maleimide residue, a combination of a thiol residue and a halocarbonyl residue (in which the halogen is replaced by a thiol by a substitution reaction), a combination of an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with a substituent as described above) and an azide residue, a combination of a tetrazine residue and an alkene residue, a combination of a tetrazine residue and an alkyne residue, and a combination of a thiol residue and another thiol residue (disulfide bond). Thus, the moiety may be a group formed by the reaction of a thiol residue with a maleimide residue, a group formed by the reaction of a furan residue with a maleimide residue, a group formed by the reaction of a thiol residue with a halocarbonyl residue, a group formed by the reaction of an alkyne residue with an azide residue, or a group formed by the reaction of a tetrazine residue with an alkene residue, or a disulfide group formed by the combination of a thiol residue with another thiol residue.

[0181] In certain embodiments, the moiety can be a divalent group represented by any one of the following structural formulas: (Here, the white and black circles represent bonds.)

[0182] L Ａ In the formula, when the bond indicated by the white circle is bonded to an atom present on the Ig binding site side, the bond indicated by the black circle may be bonded to an atom present on the carbon atom side adjacent to ring A, and when the bond indicated by the white circle is bonded to an atom present on the carbon atom side adjacent to ring A, the bond indicated by the black circle may be bonded to an atom present on the Ig binding site side.

[0183] L ＢIn the case where the bond represented by the white circle is bonded to an atom present on the V side, the bond represented by the black circle may be bonded to an atom present on the bonding portion side of the functional substance (D), and when the bond represented by the white circle is bonded to an atom present on the bonding portion side of the functional substance (D), the bond represented by the black circle may be bonded to an atom present on the V side.

[0184] In a preferred embodiment, L Ａ The divalent group represented by the formula: １ -N (-R １ )-CO-. １ represents a divalent group, and R １ represents a hydrogen atom or a monovalent group, and L １ The hyphen on the left side of and the hyphen on the right side of CO represent a bond. １ The bond of is bonded to Ig, and the bond of CO is bonded to the carbon atom adjacent to ring A.

[0185] In another preferred embodiment, L Ｂ The divalent group represented by the formula: ２ -N (-R ２ )-CO-. ２ represents a divalent group, and R ２ represents a hydrogen atom or a monovalent group, and L ２ The hyphen on the left side of and the hyphen on the right side of CO represent a bond. ２ is bonded to the functional substance (D), and the bond of CO is bonded to V.

[0186] In certain embodiments, L １ , and L ２ Each divalent group represented by the formula may contain a moiety formed by the reaction of two bioorthogonal functional groups capable of reacting with each other. １ , and L ２ The moiety formed by the reaction of two bioorthogonal functional groups capable of reacting with each other may be L Ａ , and L ＢThe divalent group represented by the formula (I) may contain a moiety similar to that formed by the reaction of two bioorthogonal functional groups capable of reacting with each other, as described above.

[0187] R １ , and R ２ R each independently represents a hydrogen atom or a monovalent group. The monovalent group is as described above. １ , and R ２ The monovalent group in is preferably a monovalent hydrocarbon group which may have a substituent, more preferably an alkyl which may have a substituent, and even more preferably an alkyl. As the alkyl, those described above are preferred.

[0188] In certain embodiments, R １ , and R ２ may be a protecting group for an amino group. Examples of such a protecting group include alkylcarbonyl groups (acyl groups) (e.g., acetyl groups, propoxy groups, butoxycarbonyl groups such as tert-butoxycarbonyl groups), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl groups), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl groups).

[0189] In a preferred embodiment, R １ , and R ２ are each independently a hydrogen atom or a protecting group for an amino group. １ , and R ２ may each be a hydrogen atom.

[0190] The functional substance represented by D is as described above.

[0191] n represents the average number of such bonds per immunoglobulin unit comprising two heavy chains and two light chains, and is 1.5 or greater. Such an average number may be, for example, 2.0 or greater, preferably 4.0 or greater, more preferably 6.0 or greater, even more preferably 7.0 or greater, particularly preferably 7.5 or greater, or 7.8 or greater. Such an average number may also be 8.0 or less. More specifically, such an average number may be preferably 2.0 to 8.0, more preferably 4.0 to 8.0, even more preferably 6.0 to 8.0, particularly preferably 7.0 to 8.0, 7.5 to 8.0, 7.8 to 8.0, or 8.0.

[0192] In certain embodiments, the conjugate or salt thereof of the present invention has the desired property of being less prone to aggregation and can be identified by its aggregation rate. More specifically, the aggregation rate of the conjugate or salt thereof of the present invention may be 5% or less. This is because, according to the present invention, antibody aggregation is easily avoided. The aggregation rate is preferably 4.8% or less, more preferably 4.6% or less, even more preferably 4.4% or less, particularly preferably 4.2% or less, 4.0% or less, 3.8% or less, 3.6% or less, 3.4% or less, 3.2% or less, 3.0% or less, 2.8% or less, or 2.6% or less. The aggregation rate of the antibody can be measured by size exclusion chromatography (SEC)-HPLC (see the Examples and ChemistrySelect, 2020, 5, 8435-8439).

[0193] In a preferred embodiment, the conjugate or salt thereof of the present invention may have an aggregation rate of 2.6% or less. The aggregation rate may also be 2.4% or less, 2.2% or less, 2.0% or less, 1.8% or less, 1.6% or less, 1.4% or less, 1.2% or less, 1.0% or less, or 0.8% or less.

[0194] Preferably, the structural unit represented by formula (1) is represented by the following formula (1′): wherein Ig, HG, CS, ring A, D, and n are the same as those represented by formula (1), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L１ , and L ２ each independently represents a divalent group. ], or the following formula (1″): wherein Ig, CS, ring A, D, and n are the same as those represented by formula (1), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0195] R ＨＧ１ , and R ＨＧ２ The definition, examples and preferred examples of the hydrophilic group or the monovalent group which may contain a hydrophilic group, represented by the formula: are the same as those described above.

[0196] L ＨＧ The definition, examples and preferred examples of the divalent group represented by the formula (I) which may contain a hydrophilic group are the same as those described above.

[0197] R １ , and R ２ The definitions, examples and preferred examples of the monovalent group represented by the formula １ , and R ２ is the same as the monovalent group represented by the following formula:

[0198] L １ , and L ２ The definitions, examples and preferred examples of the divalent group represented by the formula １ , and L ２ This is the same as the divalent group represented by the following formula:

[0199] Preferably, the structural unit represented by formula (1) is represented by the following formula (1a): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ, D, and n are the same as those shown in formula (1), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0200] L Ｃ represents a bond or a divalent group. The divalent group is as defined above.

[0201] X Ａ , and X Ｂ Each independently represents an amino acid residue, and therefore, X represented by formula (1a) Ａ -X Ｂ The structural unit corresponds to a dipeptide.

[0202] W is preferably an oxygen atom or an amino group (NH), and more preferably an amino group (NH).

[0203] X represented by formula (1a) Ａ -X Ｂ The structural unit of -W is X Ａ -X Ｂ and W. Those skilled in the art will readily appreciate that X can be cleaved between W and W to correspond to the cleavage sites by enzymes (e.g., intracellular proteases such as cathepsin B), acids, or blood glutathione, or to the self-immolative cleavage sites described above. Ａ and X Ｂ For example, the amino acid residue represented by X Ａ teeth 、 It may be a valine residue, a phenylalanine residue, a threonine residue, a leucine residue, or an alanine residue. Ｂ may be a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue. Ａ and X Ｂ The configuration of the amino acid residues in may be either L- or D-configuration, with L-configuration being preferred.Ａ teeth 、 is a valine residue, and X Ｂ is a citrulline residue or an alanine residue.

[0204] Preferably, the structural unit represented by formula (1a) is a structural unit represented by the following formula (1b): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ , D, and n are the same as those shown in formula (1), and L Ｃ , and W are the same as those shown in formula (1a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0205] R Ａ is the side chain of a valine residue (i.e., -CH(CH ３ ) ２ ) or R Ａ R may be the side chain of a phenylalanine, threonine, leucine, or alanine residue. Ａ The configuration of the amino acid residues in may be either L- or D-configuration, with the L-configuration being preferred.

[0206] R Ｂ is the side chain of the citrulline residue (i.e., -CH ２ CH ２ CH ２ NHCONH ２ ), or the side chain of an alanine residue (i.e., —CH ３ ) or R Ｂ R may be the side chain of a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue. Ｂ The configuration of the amino acid residues in may be either L- or D-configuration, with the L-configuration being preferred.

[0207] RＡ and R Ｂ The combination of Ａ is the side chain of a valine residue, and R Ｂ is preferably the side chain of a citrulline residue or an alanine residue. Ａ and R Ｂ Other preferred examples of combinations of are: Ａ is the side chain of a valine residue, and R Ｂ is a side chain of a glutamic acid residue, a lysine residue, an arginine residue, or a threonine residue; (b) R Ａ is the side chain of a phenylalanine residue, and R Ｂ is a side chain of a lysine residue, an arginine residue, or a glutamine residue; (c) R Ａ is the side chain of a threonine residue, and R Ｂ is a side chain of a threonine residue or a methionine residue; (d) R Ａ is the side chain of a leucine residue, and R Ｂ is the side chain of a glutamic acid residue; and (e) R Ａ is the side chain of an alanine residue, and R Ｂ is the side chain of an alanine residue.

[0208] Preferably, the structural unit represented by formula (1b) is a structural unit represented by the following formula (1c): wherein Ig, HG, ring A, D, and n are the same as those represented by formula (1), and R Ａ , and R Ｂ are the same as those shown in formula (1b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0209] R １ , and R ２ The definitions, examples and preferred examples of the monovalent group represented by the formula １ , and R ２is the same as the monovalent group represented by the following formula:

[0210] L １ , and L ２ The definitions, examples and preferred examples of the divalent group represented by the formula １ , and L ２ This is the same as the divalent group represented by the following formula:

[0211] Preferably, the structural unit represented by formula (1c) is a structural unit represented by the following formula (1d): wherein Ig, ring A, D, and n are the same as those represented by formula (1), and R Ａ , and R Ｂ are the same as those shown in formula (1b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (1c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0212] In formula (1d), L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group. The hydrophilic group and the divalent group are as described above. The divalent group which may contain a hydrophilic group is L ＨＧ It may be contained in the main chain connecting the nitrogen atom and carbon atom adjacent to the main chain or in a side chain of the main chain, and is preferably contained in a side chain of the main chain.

[0213] Preferably, the divalent group (-L ＨＧ -) may be a divalent group represented by the following formula (a): -(C(R ＨＧ ) ２ ) ｎ１ -(C=O)ｎ２ - (NR ＨＧ ) ｎ３ -(C(R ＨＧ ) ２ ) ｎ４ - (a) (The hyphens (-) at both ends indicate bonds.)

[0214] In formula (a), a plurality of R ＨＧ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group that may contain a hydrophilic group. The hydrophilic group and the monovalent group are as described above.

[0215] n1 is an integer of 0 to 3, preferably an integer of 0 to 2, and more preferably an integer of 0 or 1.

[0216] n2 is an integer of 0 or 1.

[0217] n3 is an integer of 0 or 1.

[0218] n4 is an integer of 0 to 3, preferably an integer of 0 to 2, and more preferably an integer of 0 or 1.

[0219] Even more preferably, the divalent group (-L ＨＧ -) may be a divalent group represented by the following formula (a1), (a2), or (a3): (a1) -(C(R ＨＧ ) ２ ) -; (a2) -(C(R ＨＧ ) ２ )-(C=O)-(NR ＨＧ )-(C(R ＨＧ ) ２ )-; or (a3) ​​-(C=O)-(C(R ＨＧ ) ２ ) ２ -.

[0220] In formula (a1), (a2), or (a3), a plurality of R ＨＧ are each independently a hydrogen atom, a hydrophilic group, or an alkyl group having 1 to 6 carbon atoms and containing a hydrophilic group. The hydrophilic group and the alkyl group having 1 to 6 carbon atoms are as described above.

[0221] In formula (1d), R ＨＧ１, and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group that may contain a hydrophilic group. The hydrophilic group and the monovalent group are as described above.

[0222] In certain embodiments, R ＨＧ１ , and R ＨＧ２ The monovalent group represented by the formula (I) which may contain a hydrophilic group may be a protecting group for an amino group. Examples of the protecting group for an amino group include R １ , and R ２ For example, R ＨＧ１ , and R ＨＧ２ One of the groups may be a hydrogen atom and the other may be a protecting group for an amino group.

[0223] Or, R ＨＧ１ , and R ＨＧ２ One of the groups may be a hydrogen atom, and the other may be a monovalent group containing a hydrophilic group. Examples of the monovalent group containing a hydrophilic group include an alkyl group containing a hydrophilic group, a carboxyl group containing a hydrophilic group, an alkylcarbonyl group containing a hydrophilic group (e.g., the groups described above), an alkyloxycarbonyl group containing a hydrophilic group, and an oxycarbonyl group containing a hydrophilic group.

[0224] In formula (1d), at least one hydrophilic group is L ＨＧ , R ＨＧ１ , and R ＨＧ２ The hydrophilic group is contained in one or more sites selected from the group consisting of: Examples of sites containing at least one hydrophilic group and combinations thereof include the following: (i) L ＨＧ alone; (ii) R ＨＧ１ alone; (iii) R ＨＧ２ alone; (iv) L ＨＧ and L ＨＧ１ (v) a combination of L ＨＧ and L ＨＧ２ (vi) a combination of L ＨＧ１ and L ＨＧ２ and (vii) L ＨＧ , L ＨＧ１ and L ＨＧ２ A combination of these L ＨＧPart, R ＨＧ１ Part, R ＨＧ２ Each of the moieties may contain one hydrophilic group, or may contain two or more hydrophilic groups.

[0225] The definitions, examples, and preferred examples of the symbols and elements in the symbols (e.g., specific elements such as cleavable sites or specific formulae) described in the above series of formulae apply similarly to the other formulae.

[0226] The conjugate of the present invention or a salt thereof is useful, for example, as a pharmaceutical or a reagent (eg, a diagnostic agent, a research reagent).

[0227] The conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate of the present invention or a salt thereof, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; flavorings such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene. The conjugate of the present invention or a salt thereof may also have any modification that provides stability (eg, PEGylation).

[0228] Suitable formulations for oral administration include solutions in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or 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 and emulsified in a suitable dispersion medium.

[0229] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0230] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc., but can be set appropriately.

[0231] In one embodiment, the conjugate or salt thereof of the present invention can be prepared by reacting an antibody derivative or salt thereof having a bioorthogonal functional group with a functional substance (Figure 3). Such a reaction can proceed through a reaction between the bioorthogonal functional group in the antibody derivative and the functional substance.

[0232] When the functional substance has a functional group that is easily reactive with the bioorthogonal functional group, the functional group of the functional substance can be appropriately reacted with the bioorthogonal functional group in the antibody derivative. The functional group that is easily reactive with the bioorthogonal functional group may vary depending on the specific type of bioorthogonal functional group. Those skilled in the art can appropriately select an appropriate functional group as a functional group that is easily reactive with the bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Examples of functional groups that readily react with bioorthogonal functional groups include, for example, alkyne residues when the bioorthogonal functional group is an azide residue; maleimide residues and disulfide residues when the bioorthogonal functional group is a thiol residue; hydrazine residues when the bioorthogonal functional group is an aldehyde residue or ketone residue; azide residues when the bioorthogonal functional group is a norbornene residue; and alkyne residues when the bioorthogonal functional group is a tetrazine residue. Of course, the above combinations of bioorthogonal functional groups and functional groups that readily react with them can be interchanged. Therefore, if the first example in the above combination is interchanged, a combination of an alkyne residue as the bioorthogonal functional group and an azide residue as the functional group that readily reacts with the bioorthogonal functional group can be used.

[0233] If the functional substance does not have a functional group that is easily reactive with the bioorthogonal functional group in the antibody derivative, the drug may be derivatized to have such a functional group. Derivatization is common knowledge in the art (e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be performed using any crosslinking agent. Alternatively, derivatization may be performed using a specific linker having the desired functional group. In the present invention, a derivatized functional substance is simply referred to as a "functional substance" because it is merely a type of functional substance.

[0234] The above reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in an appropriate reaction system, such as a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain 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 a reaction, see, for example, G. J. L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G. J. L. Bernardes et al., Chem. Asian. J. , 4, 630 (2009); B. G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0235] In another embodiment, the conjugate or salt thereof of the present invention can be produced by reacting a compound or salt thereof having a bioorthogonal functional group and a functional substance with a starting antibody having an Ig (immunoglobulin unit) (Figure 3). Such a starting antibody can be prepared by contacting an antibody containing an immunoglobulin unit (comprising four disulfide bonds) consisting of two heavy chains and two light chains with a reducing agent to generate an antibody containing a thiol group. Any reducing agent capable of cleaving disulfide bonds to generate a thiol group can be used, including, for example, tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. In this embodiment, the bioorthogonal functional group possessed by the compound is preferably a group capable of efficiently reacting with a thiol group (e.g., a maleimide residue, a halocarbonyl group, or a thiol group). The reaction between the compound having a bioorthogonal functional group and a functional substance or its salt and the starting antibody can be appropriately carried out under the above-mentioned conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds).

[0236] The production of the conjugate or its salt can be confirmed, for example, by reversed-phase HPLC under reducing conditions or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. The conjugate or its salt can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, high-performance liquid chromatography, affinity chromatography).

[0237] 3. Antibody derivatives or salts thereof The present invention also relates to antibody derivatives or salts thereof represented by the following formula (2): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and two light chains, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues in the two heavy chains and the two light chains. Ａand bonded to, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; ２ represents a bioorthogonal functional group, and the average number n of bonds per immunoglobulin unit is 1.5 or more.

[0238] In formula (2), Ig, HG, CS, ring A, V, L Ａ , L Ｂ , and n are as described above in formula (1). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1).

[0239] B ２ The bioorthogonal functional group represented by is as described above.

[0240] In certain embodiments, B ２ The bioorthogonal functional group represented by B may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. ２ The bioorthogonal functional group represented by the formula (I) may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. These bioorthogonal functional groups are preferred because of their excellent reaction efficiency and versatility.

[0241] Preferably, the structural unit represented by formula (2) is represented by the following formula (2'): [In the formula, Ig, HG, CS, ring A, B ２ , and n are the same as those shown in formula (2), and R １ , and R２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group. ], or the following formula (2″): [In the formula, Ig, CS, ring A, B ２ , and n are the same as those shown in formula (2), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0242] In these formulas, R ＨＧ１ , R ＨＧ２ , L ＨＧ , R １ , R ２ , L １ , and L ２ are as described above. Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above.

[0243] Preferably, the structural unit represented by formula (2) is represented by the following formula (2a): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ , B ２ , and n are the same as those shown in formula (2), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0244] In formula (2a), L Ｃ , XＡ , X Ｂ , and W are as described above in formula (1a). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1a).

[0245] Preferably, the structural unit represented by formula (2a) is a structural unit represented by the following formula (2b): [In the formula, Ig, HG, ring A, V, L Ａ , L Ｂ , B ２ , and n are the same as those shown in formula (2), and L Ｃ , and W are the same as those shown in formula (2a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0246] In formula (2b), R Ａ , and R Ｂ are as described above in formula (1b). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1b).

[0247] Preferably, the structural unit represented by formula (2b) is a structural unit represented by the following formula (2c): [In the formula, Ig, HG, ring A, B ２ , and n are the same as those shown in formula (2), and R Ａ , and R Ｂ are the same as those shown in formula (2b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0248] In formula (2c), R １ , R ２ , L １ , and L ２ are as described above in formula (1c). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1c).

[0249] Preferably, the structural unit represented by formula (2c) is a structural unit represented by the following formula (2d): [In the formula, Ig, ring A, B ２ , and n are the same as those shown in formula (2), and R Ａ , and R Ｂ are the same as those shown in formula (2b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (2c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0250] In formula (2d), L ＨＧ , R ＨＧ１ , and R ＨＧ２ are as described above in formula (1d). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1d).

[0251] The antibody derivative of the present invention or a salt thereof is useful, for example, as an intermediate for producing the conjugate of the present invention or a salt thereof.

[0252] The antibody derivative or its salt of the present invention can be produced, for example, by reacting a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or its salt with a starting antibody having an Ig (immunoglobulin unit) (Figure 3). The starting antibody is the same as that described above.

[0253] The reaction between the compound having the first bioorthogonal functional group and the second bioorthogonal functional group or a salt thereof and the starting antibody can be appropriately carried out under the above-mentioned conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds).

[0254] The production of the antibody derivative or its salt can be confirmed by the same method as described for the conjugate of the present invention. The antibody derivative or its salt can be appropriately purified by any of the purification methods described for the conjugate of the present invention.

[0255] 4. A compound having a bioorthogonal functional group and a functional substance, or a salt thereof The present invention also relates to a compound having the following formula (3): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; １ represents a bioorthogonal functional group, and D represents a functional substance.], or a salt thereof.

[0256] In formula (3), HG, CS, ring A, V, L Ａ , L Ｂ , and D are as described above in formula (1). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1).

[0257] B １represents a bioorthogonal functional group. The bioorthogonal functional group is as described above. Preferably, the bioorthogonal functional group is a group that can efficiently react with a thiol group (e.g., a maleimide residue, a halocarbonyl group, or a thiol group).

[0258] Preferably, the structural unit represented by formula (3) is represented by the following formula (3'): [In the formula, HG, CS, ring A, B １ , and D are the same as those shown in formula (3), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group. ], or the following formula (3″): [In the formula, CS, ring A, B １ , and D are the same as those shown in formula (3), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0259] In these formulas, R ＨＧ１ , R ＨＧ２ , L ＨＧ , R １ , R ２ , L １ , and L ２ are as described above. Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above.

[0260] Preferably, the compound represented by formula (3) is represented by the following formula (3a): [In the formula, HG, ring A, V, L Ａ , LＢ , B １ , and D are the same as those shown in formula (3), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0261] In formula (3a), L Ｃ , X Ａ , X Ｂ , and W are as described above in formula (1a). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1a).

[0262] Preferably, the compound represented by formula (3a) is represented by the following formula (3b): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and D are the same as those shown in formula (3), and L Ｃ , and W are the same as those shown in formula (3a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0263] In formula (3b), R Ａ , and R Ｂ are as described above in formula (1b). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1b).

[0264] Preferably, the compound represented by formula (3b) is represented by the following formula (3c): [In the formula, HG, ring A, B １, and D are the same as those shown in formula (3), and R Ａ , and R Ｂ are the same as those shown in formula (3b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0265] In formula (3c), R １ , R ２ , L １ , and L ２ are as described above in formula (1c). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1c).

[0266] Preferably, the compound represented by formula (3c) is represented by the following formula (3d): [In the formula, rings A, B １ , and D are the same as those shown in formula (3), and R Ａ , and R Ｂ are the same as those shown in formula (3b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (3c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0267] In formula (3d), L ＨＧ , R ＨＧ１ , and R ＨＧ２are as described above in formula (1d). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1d).

[0268] The compound of the present invention represented by formula (3) or a salt thereof is useful, for example, as an intermediate for producing the conjugate of the present invention. The compound of the present invention represented by formula (3) or a salt thereof is also useful, for example, for derivatizing any substance such as a biomolecule (e.g., a protein such as an antibody, a sugar, a nucleic acid, or a lipid).

[0269] A compound or a salt thereof having a bioorthogonal functional group and a functional substance can be produced, for example, by reacting a compound or a salt thereof having a first bioorthogonal functional group and a second bioorthogonal functional group with a functional substance (Figure 3). Details of the functional substance are as described above.

[0270] The reaction of a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof with a functional substance can be carried out in an appropriate reaction system, such as an organic solvent system or an aqueous solution (e.g., a buffer solution), at an appropriate temperature (e.g., about 15 to 200°C). The reaction system may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. Of course, such a reaction can also be carried out under the mild conditions described above.

[0271] The production of a compound or its salt having a bioorthogonal functional group and a functional substance can be confirmed by, for example, NMR, HPLC, or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Such a compound or its salt can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, high performance liquid chromatography, affinity chromatography).

[0272] 5. A compound having a first bioorthogonal functional group and a second bioorthogonal functional group, or a salt thereof The present invention also relates to a compound having the following formula (4): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; １ represents the first bioorthogonal functional group, B ２ represents a second bioorthogonal functional group.], or a salt thereof.

[0273] In formula (4), HG, CS, ring A, V, L Ａ , L Ｂ , and D are as described above in formula (1). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1).

[0274] B １ represents a first bioorthogonal functional group. The first bioorthogonal functional group is the same as that described above for the bioorthogonal functional group. Preferably, the bioorthogonal functional group is a group that can efficiently react with a thiol group (e.g., a maleimide residue, a halocarbonyl group, or a thiol group).

[0275] B ２ represents a second bioorthogonal functional group. The second bioorthogonal functional group is the same as described above for the bioorthogonal functional group.

[0276] In certain embodiments, B ２ The bioorthogonal functional group represented by B may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. ２The bioorthogonal functional group represented by the formula (I) may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue. These bioorthogonal functional groups are preferred because of their excellent reaction efficiency and versatility.

[0277] Preferably, the second bioorthogonal functional group may be a bioorthogonal functional group that does not react with the first bioorthogonal functional group or has low reactivity with the first bioorthogonal functional group. In this case, intermolecular reactions of the compound represented by formula (4) or its salt can be suppressed. Therefore, the first and second bioorthogonal functional groups can be used in combinations that do not react with each other or have low reactivity with each other. Such combinations of bioorthogonal functional groups are well known in the art. For example, examples of such combinations for preferred bioorthogonal functional groups, such as maleimide residues, thiol residues, furan residues, halocarbonyl residues, alkene residues, alkyne residues, azide residues, and tetrazine residues, are as follows:

[0278]

[0279] Preferably, the structural unit represented by formula (4) is represented by the following formula (4'): [In the formula, HG, CS, ring A, B １ , and B ２ are the same as those shown in formula (4), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group. ], or the following formula (4″): [In the formula, CS, ring A, B １ , and B ２ are the same as those shown in formula (4), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R１ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0280] In these formulas, R ＨＧ１ , R ＨＧ２ , L ＨＧ , R １ , R ２ , L １ , and L ２ are as described above. Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above.

[0281] Preferably, the compound represented by formula (4) is represented by the following formula (4a): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and B ２ are the same as those shown in formula (4), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0282] In formula (4a), L Ｃ , X Ａ , X Ｂ , and W are as described above in formula (1a). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1a).

[0283] Preferably, the compound represented by formula (4a) is a compound represented by the following formula (4b): [In the formula, HG, ring A, V, L Ａ , L Ｂ , B １ , and B ２ are the same as those shown in formula (4), and LＣ , and W are the same as those shown in formula (4a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0284] In formula (4b), R Ａ , and R Ｂ are as described above in formula (1b). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1b).

[0285] Preferably, the compound represented by formula (4b) is represented by the following formula (4c): [In the formula, HG, ring A, B １ , and B ２ are the same as those shown in formula (4), and R Ａ , and R Ｂ are the same as those shown in formula (4b), and R １ , and R ２ each independently represents a hydrogen atom or a monovalent group; L １ , and L ２ each independently represents a divalent group.

[0286] In formula (4c), R １ , R ２ , L １ , and L ２ are as described above in formula (1c). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1c).

[0287] Preferably, the compound represented by formula (4c) is represented by the following formula (4d): [In the formula, rings A, B １ , and B ２are the same as those shown in formula (4), and R Ａ , and R Ｂ are the same as those shown in formula (4b), and R １ , R ２ , L １ , and L ２ are the same as those shown in formula (4c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0288] In formula (4d), L ＨＧ , R ＨＧ１ , and R ＨＧ２ are as described above in formula (1d). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1d).

[0289] The compound of the present invention represented by formula (4) or a salt thereof is useful, for example, as an intermediate for producing the antibody derivative of the present invention and the compound of the present invention represented by formula (3). The compound of the present invention represented by formula (4) or a salt thereof is also useful, for example, for derivatizing any substance such as a biomolecule (e.g., a protein such as an antibody, a sugar, a nucleic acid, or a lipid) and a functional substance.

[0290] In one embodiment, the compound or salt thereof having a first bioorthogonal functional group and a second bioorthogonal functional group is a compound of formula (6) or a salt thereof having a bioorthogonal functional group, １ Suitable compounds having the formula (e.g., B １ -L １ -NH-R １ It can be produced by reacting with a compound represented by the formula (Figures 4 and 5). １ , L１ , and R １ The definition, examples, and preferred examples of are as described above.

[0291] In another embodiment, the compound or salt thereof having the first bioorthogonal functional group and the second bioorthogonal functional group is a compound of formula (7) or a salt thereof having a bioorthogonal functional group, L Ｂ -B ２ (e.g., bis(4-nitrophenyl)carbonate and N,N-diisopropylethylamine (DIPEA), followed by reaction with a suitable compound having B ２ -L ２ -NH-R ２ (Figures 4 and 5). ２ , L Ｂ , and R ２ The definition, examples, and preferred examples of are as described above.

[0292] The above reaction can be carried out in an appropriate reaction system, such as an organic solvent system or an aqueous solution (e.g., a buffer solution), at an appropriate temperature (e.g., about 15 to 200°C). The reaction system may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. Of course, such a reaction can also be carried out under the mild conditions described above.

[0293] The production of a compound or a salt thereof having a first bioorthogonal functional group and a second bioorthogonal functional group can be confirmed by, for example, NMR, HPLC, or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Such a compound or a salt thereof can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, high performance liquid chromatography, affinity chromatography).

[0294] 6. A Series of Compounds or Salts Thereof (1) Compounds or Salts Thereof The present invention also provides compounds or salts thereof represented by the following formula (5): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ represents a divalent group, and X and Y each independently represent a monovalent group, or a salt thereof.

[0295] In formula (5), HG, CS, ring A, V, and L Ａ are as described above in formula (1). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1).

[0296] X and Y each independently represent a monovalent group. The monovalent group is as described above.

[0297] Preferably, the structural unit represented by formula (5) is represented by the following formula (5'): [wherein HG, CS, ring A, X, and Y are the same as those shown in formula (5)], or the following formula (5″): wherein CS, ring A, X, and Y are the same as those represented by formula (5), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group.

[0298] In these formulas, R ＨＧ１ , R ＨＧ２ , and L ＨＧ are as described above. Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above.

[0299] Preferably, the compound represented by formula (5) is represented by the following formula (5a): [In the formula, HG, ring A, V, L Ａ , X, and Y are the same as those shown in formula (5), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0300] In formula (5a), L Ｃ , X Ａ , X Ｂ , and W are as described above in formula (1a). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1a).

[0301] Preferably, the compound represented by formula (5a) is represented by the following formula (5b): [In the formula, HG, ring A, V, L Ａ , X, and Y are the same as those shown in formula (5), and L Ｃ , and W are the same as those shown in formula (5a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0302] In formula (5b), R Ａ , and R Ｂ are as described above in formula (1b). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1b).

[0303] Preferably, the compound represented by formula (5b) is represented by the following formula (5c): wherein HG, ring A, X, and Y are the same as those represented by formula (5), and RＡ , and R Ｂ are the same as those represented by formula (5b).

[0304] Preferably, the compound represented by formula (5c) is represented by the following formula (5d): wherein ring A, X, and Y are the same as those represented by formula (5), and R Ａ , and R Ｂ are the same as those shown in formula (5b), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0305] In formula (5d), L ＨＧ , R ＨＧ１ , and R ＨＧ２ are as described above in formula (1d). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1d).

[0306] The compound represented by formula (5) or a salt thereof is useful, for example, as a synthetic intermediate for the conjugate of the present invention, the antibody derivative, and other compounds of the present invention.

[0307] The compound represented by formula (5) or a salt thereof is, for example, a compound represented by the following formula (5-1): [wherein HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, and CS′ represents a reactive divalent group containing a cleavable site] or a salt thereof, [wherein ring A represents a divalent aromatic ring group which may have a substituent and which is capable of reacting with CS', and X and Y each independently represent a monovalent group] or a salt thereof. Ａ The definitions, examples, and preferred examples of V, X, and Y are as described above. Such a reaction can be carried out under the same conditions as those described above for the preparation of a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof.

[0308] Preferably, the compound represented by formula (5-1) is represented by the following formula (5-1'): [wherein HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, R Ａ indicates the side chain of a valine residue, R Ｂ represents the side chain of a citrulline residue or an alanine residue.

[0309] Preferably, the compound represented by formula (5-2) is represented by the following formula (5-2'): [wherein ring A represents a divalent aromatic ring group which may have a substituent, and X and Y each independently represent a monovalent group.]

[0310] (2) A compound having a bioorthogonal functional group represented by formula (6) or a salt thereof The present invention also relates to a compound having a bioorthogonal functional group represented by formula (6): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ , and L Ｂ each independently represents a divalent group; X represents a monovalent group; B ２represents a bioorthogonal functional group.] or a salt thereof.

[0311] In formula (6), HG, CS, ring A, V, L Ａ , and L Ｂ are as described above in formula (1). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1).

[0312] The monovalent group represented by X is as described above.

[0313] B ２ The bioorthogonal functional group represented by is as described above.

[0314] Preferably, the compound represented by formula (6) is represented by the following formula (6'): wherein HG, CS, ring A, and X are the same as those represented by formula (5), and R ２ represents a hydrogen atom or a monovalent group, and L ２ represents a divalent group, ２ represents a bioorthogonal functional group. ], or the following formula (6″): wherein CS, ring A, and X are the same as those represented by formula (5), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ２ represents a hydrogen atom or a monovalent group, and L ２ represents a divalent group, ２ represents a bioorthogonal functional group.

[0315] In these formulas, R ＨＧ１ , R ＨＧ２ , L ＨＧ , R ２ , L ２ , and B ２are as described above. Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above.

[0316] Preferably, the compound represented by formula (6) is represented by the following formula (6a): [In the formula, HG, ring A, V, L Ａ , X, L Ｂ , and B ２ are the same as those shown in equation (6), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0317] In formula (6a), L Ｃ , X Ａ , X Ｂ , and W are as described above in formula (1a). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1a).

[0318] Preferably, the compound represented by formula (6a) is represented by the following formula (6b): [In the formula, HG, ring A, V, L Ａ , X, L Ｂ , and B ２ are the same as those shown in equation (6), and L Ｃ , and W are the same as those shown in formula (6a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0319] In formula (6b), R Ａ , and R Ｂare as described above in formula (1b). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1b).

[0320] Preferably, the compound represented by formula (6b) is represented by the following formula (6c): wherein HG, ring A, X, and B ２ are the same as those shown in equation (6), and R Ａ , and R Ｂ are the same as those shown in formula (6b), and R ２ represents a hydrogen atom or a monovalent group, and L ２ represents a divalent group.

[0321] In formula (6c), R ２ , and L ２ are as described above in formula (1c). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1c).

[0322] Preferably, the compound represented by formula (6c) is represented by the following formula (6d): wherein rings A, X, and B ２ are the same as those shown in equation (6), and R Ａ , and R Ｂ are the same as those shown in formula (6b), and R ２ , and L ２ is the same as that shown in formula (6c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0323] In formula (6d), L ＨＧ , R ＨＧ１ , and R ＨＧ２ are as described above in formula (1d). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1d).

[0324] The compound represented by formula (6) or a salt thereof is useful, for example, as a synthetic intermediate for the conjugate of the present invention, antibody derivatives, and specific compounds of the present invention. Such a compound or a salt thereof is also useful, for example, for derivatizing functional substances.

[0325] The compound represented by formula (6) or a salt thereof can be prepared by, for example, reacting a compound represented by formula (5) or a salt thereof with L Ｂ -B ２ (e.g., bis(4-nitrophenyl)carbonate and N,N-diisopropylethylamine (DIPEA), followed by reaction with a suitable compound having B ２ -L ２ -NH-R ２ (Figures 4 and 5). ２ , L ２ , and R ２ The definitions, examples, and preferred examples are as described above. Such a reaction can be carried out under the same reaction conditions as those described above for the preparation of a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof.

[0326] (3) A compound having a bioorthogonal functional group represented by formula (7) or a salt thereof The present invention also relates to a compound having a bioorthogonal functional group represented by formula (7): [wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS represents a divalent group containing a cleavage site by cathepsin B, ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a π-electron conjugated system with the cleavage site), V represents an oxygen atom, a sulfur atom, or an amino group (NH), and L Ａ represents a divalent group, Y represents a monovalent group, B１ represents a bioorthogonal functional group.] or a salt thereof.

[0327] In formula (7), HG, CS, ring A, V, and L Ａ are as described above in formula (1). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1).

[0328] The monovalent group represented by Y is as described above.

[0329] B １ The bioorthogonal functional group represented by is as described above.

[0330] Preferably, the compound represented by formula (7) is represented by the following formula (7'): wherein HG, CS, ring A, V, and Y are the same as those represented by formula (5), and R １ represents a hydrogen atom or a monovalent group, and L １ represents a divalent group, １ represents a bioorthogonal functional group. ], or the following formula (7″): wherein CS, ring A, V, and Y are the same as those represented by formula (5), and R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R １ represents a hydrogen atom or a monovalent group, and L １ represents a divalent group, １ represents a bioorthogonal functional group.

[0331] In these formulas, R ＨＧ１ , R ＨＧ２ , L ＨＧ , R １ , L １ , and B １are as described above. Therefore, the definitions, examples and preferred examples of these elements and other elements related to the elements are the same as those described above.

[0332] Preferably, the compound represented by formula (7) is represented by the following formula (7a): [In the formula, HG, ring A, V, L Ａ , Y, and B １ are the same as those shown in equation (7), and L Ｃ represents a bond or a divalent group, X Ａ , and X Ｂ each independently represents an amino acid residue, and W represents an oxygen atom, a sulfur atom, or an amino group (NH).

[0333] In formula (7a), L Ｃ , X Ａ , X Ｂ , and W are as described above in formula (1a). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1a).

[0334] Preferably, the compound represented by formula (7a) is represented by the following formula (7b): [In the formula, HG, ring A, V, L Ａ , Y, and B １ are the same as those shown in equation (7), and L Ｃ , X Ａ , X Ｂ , and W are the same as those shown in formula (7a), and R Ａ represents the side chain of a valine, phenylalanine, threonine, leucine, or alanine residue; R Ｂ represents the side chain of a citrulline residue, an alanine residue, a glutamic acid residue, a glutamine residue, a lysine residue, an arginine residue, a threonine residue, or a methionine residue.

[0335] In formula (7b), R Ａ , and R Ｂare as described above in formula (1b). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to these elements are the same as those described above in formula (1b).

[0336] Preferably, the compound represented by formula (7b) is represented by the following formula (7c): wherein HG, ring A, Y, and B １ are the same as those shown in equation (7), and R Ａ , and R Ｂ are the same as those shown in formula (7b), and R １ represents a hydrogen atom or a monovalent group, and L １ represents a divalent group.

[0337] In formula (7c), R １ , and L １ are as described above in formula (1c). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1c).

[0338] Preferably, the compound represented by formula (7c) is represented by the following formula (7d): wherein rings A, Y, and B １ are the same as those shown in equation (7), and R Ａ , and R Ｂ are the same as those shown in formula (7b), and R １ , and L １ is the same as that shown in formula (7c), and L ＨＧ represents a bond or a divalent group which may contain a hydrophilic group, R ＨＧ１ , and R ＨＧ２ each independently represents a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group, and at least one hydrophilic group is ＨＧ , R ＨＧ１ , and R ＨＧ２ and the amino acid sequence is contained in one or more positions selected from the group consisting of:

[0339] In formula (7d), L ＨＧ , R ＨＧ１ , and R ＨＧ２ are as described above in formula (1d). Therefore, the definitions, examples, and preferred examples of these elements and other elements related to the elements are the same as those described above in formula (1d).

[0340] The compound represented by formula (7) or a salt thereof is useful, for example, as a synthetic intermediate for the conjugate of the present invention, antibody derivatives, and specific compounds of the present invention. Such a compound or a salt thereof is also useful for derivatizing any substance, such as a biomolecule (e.g., a protein such as an antibody, a sugar, a nucleic acid, or a lipid).

[0341] The compound represented by formula (7) or a salt thereof can be prepared by, for example, reacting the compound represented by formula (7) or a salt thereof with B １ Suitable compounds having the formula (e.g., B １ -L １ -NH-R １ It can be produced by reacting with a compound represented by the formula (Figures 4 and 5). １ , L １ , and R １ The definitions, examples, and preferred examples are as described above. Such a reaction can be carried out under the same reaction conditions as those described above for the preparation of a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof.

[0342] The production of the compound represented by formula (5), (6), or (7) or a salt thereof can be confirmed by, for example, NMR, HPLC, or mass spectrometry, although this depends on the specific raw materials and the molecular weight of the product. Such a compound or a salt thereof can be appropriately purified by any purification method such as chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, high-performance liquid chromatography, affinity chromatography).

[0343] 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.

[0344] The following peptides in Example 1 were all prepared in the same manner: Ac-Glu(OtBu)-Val-Cit-OH, Z-Glu(OtBu)-Val-Cit-OH, Ac-Glu(OtBu)-Glu(OtBu)-Val-Cit-OH, and SCA(OtBu)-Glu(OtBu)-Val-Cit-OH. SCA(OtBu) stands for mono-tert-butyl succinate and succinic acid. Solid-phase peptide synthesis using Cl-TCP(Cl) ProTide Resin (CEM) by the Fmoc method was used to prepare peptides capped at the N-terminus with an acetyl group and at the N-terminus with succinic acid. These peptides were then cleaved from the resin while the amino acid side chains remained protected by overnight stirring in a 20% HFIP / dichloromethane solution. The resin was removed by filtration, and the solution was concentrated and purified by preparative HPLC to obtain the peptide product.

[0345] Ac-Glu(OtBu)-Val-Cit-OH

[0346] １ H NMR (400 MHz, DMSO-d ６ ) δ12.50 (brs, 1H), 8.22 (d, J=7.2Hz, 1H), 8.05 (d, J=8.0Hz, 1H), 7.69 (d, J=8.8Hz, 1H), 5. 95-5.93 (m, 1H), 5.38 (brs, 2H), 4.33-4.27 (m, 1H), 4.22-4.18 (m, 1H), 4.15-4.10 (m, 1H), 2.96-2.95 (m, 2H), 2.25-2.19 (m, 2H), 2.00-1.93 (m, 1H), 1.89-1.80 (m, 4H), 1.73-1.66 (m , 2H), 1.61-1.51 (m, 1H), 1.46-1.34 (m, 11H), 0.86 (d, J = 6.8Hz, 3H), 0.83 (d, J = 6.8Hz, 3H).

[0347] MS (ESI) m / z: 502.30 [M+H] ＋

[0348] Z-Glu(OtBu)-Val-Cit-OH

[0349] １ H NMR(400 MHz,DMSO-d ６ )δ12.50(brs,1H),7.93-7.37(m,8H),6.05-6.00(m,1H),5.44(brs,2H),5.08(s,2H),4.17-3.81(m,3H),3.00-2.90(m,2H),2.31-2.27(m,2H),2.10-1.34(m,16H),0.89-0.83(m,6H).

[0350] MS(ESI)m / z:594.30[M+H] ＋

[0351] Ac-Glu(OtBu)-Glu(OtBu)-Val-Cit-OH

[0352] １ H NMR(400 MHz,DMSO-d ６ )δ12.50(brs,1H),8.21(d,J=7.2Hz,1H),8.08(d,J=8.0Hz,1H),8.04(d,J=8.0Hz,1H),7.68(d,J=8.0Hz,1H),5.95(brs,1H),5.37(brs,2H),4.32-4.19(m,3H),4.16-4.11(m,1H),2.98-2.94(m,2H),2.27-2.13(m,4H),2.00-1.79(m,5H),1.76-1.52(m,5H),1.42-1.36(m,20H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.8Hz,3H).

[0353] MS(ESI)m / z:687.35[M+H] ＋

[0354] SCA(OtBu)-Glu(OtBu)-Val-Cit-OH

[0355] １ H NMR(400 MHz,DMSO-d ６)δ 12.70 (brs, 1H), 8.21 (d, J = 7.2 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 5.96 (brs, 1H), 5.25 (brs, 2H), 4.35 - 4.30 (m, 1H), 4.21 - 4.18 (m, 1H), 4.15 - 4.10 (m, 1H), 2.98 - 2.94 (m, 2H), 2.40 - 2.28 (m, 4H), 2.24 - 2.18 (m, 2H), 2.01 - 1.93 (m, 1H), 1.90 - 1.81 (m, 1H), 1.73 - 1.63 (m, 2H), 1.61 - 1.51 (m, 1H), 1.40 - 1.31 (m, 20H), 0.86 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H).

[0356] MS (ESI) m / z: 616.30 [M + H] ＋

[0357] Ac-Glu(OtBu)-Val-Ala-OH

[0358] １ H NMR (400 MHz, DMSO-d ６ )δ 12.50 (brs, 1H), 8.25 (d, J = 6.8 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H), 4.33 - 4.27 (m, 1H), 4.22 - 4.16 (m, 2H), 2.23 - 2.18 (m, 2H), 1.99 - 1.94 (m, 1H), 1.89 - 1.80 (m, 4H), 1.73 - 1.65 (m, 1H), 1.39 (s, 9H), 1.27 (d, J = 7.2 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H).

[0359] MS (ESI) m / z: 416.20 [M + H] ＋

[0360] Example 1: Synthesis of Linker-payload mimic (1-1) Synthesis of Linker-payload mimic (1) Linker-payload mimic (1) was synthesized as follows.

[0361] (1-1-1) Synthesis of alcohol (2)

[0362] Ac-Glu(OtBu)-Val-Cit-OH (19.9 mg, 39.7 μmol) was dissolved in N,N-dimethylformamide (400 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (18.1 mg, 47.6 μmol) and 2,4,6-trimethylpyridine (6.27 μL, 47.6 μmol) were added, followed by stirring at room temperature for 10 minutes. Subsequently, methyl 4-aminomandelate (8.63 mg, 47.6 μmol) was added, and the mixture was stirred at room temperature for 21.5 hours, followed by purification by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (2) (28.5 mg, quant).

[0363] １ H NMR (400 MHz, DMSO-d ６ ) δ9.95 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 5.92 (brs, 1H), 5.36 (brs, 2H), 5.01 (s, 1H), 4.34-4.29 (m, 1H), 4.26-4.20 (m, 1H), 4.14-4.10 (m, 1H), 3.53 (s, 3H), 3.00-2.83 (m, 2H), 2.18-2.13 (m, 2H), 1.94-1.89 (m, 2H), 1.84-1.23 (m, 17H), 0.79 (d, J = 6.8Hz, 3H), 0.75 (d, J = 6.8Hz, 3H).

[0364] MS (ESI) m / z: 665.30 [M+H] ＋

[0365] (1-1-2) Synthesis of pyrene (3)

[0366] The alcohol (2) (28.5 mg) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (430 μL) and stirred for 5 minutes under ice-cooling. Then, bis(4-nitrophenyl)carbonate (26.6 mg, 85.7 μmol) and N,N-diisopropylethylamine (11.1 μL, 64.4 μmol) were added, and the mixture was stirred at room temperature for 1.5 hours. The reaction was monitored by LCMS, and it was found that the starting material remained. Therefore, additional bis(4-nitrophenyl)carbonate (13.3 mg, 42.9 μmol) and N,N-diisopropylethylamine (5.54 μL, 32.2 μmol) were added, and the mixture was stirred at room temperature for 5 hours. The mixture was then cooled on ice, and sarcosin-pyrene (64.9 mg, 215 μmol), 1-hydroxybenzotriazole (8.7 mg, 64 μmol), and N,N-diisopropylethylamine (57.2 μL, 333 μmol) were added, followed by stirring at room temperature for 16 hours. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the pyrene (3) (26.1 mg, 26.3 μmol).

[0367] １ H NMR (400 MHz, DMSO-d ６ ) δ10.09-10.07 (m, 1H), 8.63-8.60 (m, 1H), 8.33-7.65 (m, 13H), 7.60-7.57 (m , 2H), 7.37-7.32 (m, 2H), 5.94-5.91 (m, 1H), 5.72-5.70 (m, 1H), 5.37 (brs, 2H) , 4.98-4.96 (m, 1H), 4.93-4.00 (m, 4H), 3.85-3.75 (m, 1H), 3.56-3.55 (m, 3H), 2.97-2.87 (m, 5H), 2.17-2.13 (m, 2H), 1.93-1.17 (m, 19H), 0.80-0.73 (m, 6H).

[0368] MS (ESI) m / z: 993.40 [M+H] ＋

[0369] (1-1-3) Synthesis of pyrene (4)

[0370] Pyrene (3) (10.8 mg, 10.9 μmol) was dissolved in tetrahydrofuran (700 μL) and water (400 μL) and stirred for 5 minutes under ice cooling. Then, 1 M aqueous lithium hydroxide solution (109 μL, 109 μmol) was added and stirred at room temperature for 1 hour. After the reaction was completed, the pH was adjusted to approximately 6 using 0.1 M hydrochloric acid and purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain pyrene (4) (4.5 mg, 4.6 μmol).

[0371] １ H NMR (400 MHz, DMSO-d ６ ) δ13.05 (brs, 1H), 10.08-10.05 (m, 1H), 8.62-8.59 (m, 1H), 8.33-7.56 (m, 15H), 7.37-7.35 (m, 2H), 5.92 (brs, 1H), 5.61-5.60 (m, 1H) , 5.36 (brs, 2H), 4.98-4.03 (m, 5H), 3.88-3.74 (m, 1H), 2.99-2.83 (m, 5H), 2.15-2.13 (m, 2H), 1.93-1.14 (m, 19H), 0.84-0.73 (m, 6H).

[0372] MS (ESI) m / z: 979.40 [M+H] ＋

[0373] (1-1-4) Synthesis of pyrene (5)

[0374] Pyrene (4) (3.7 mg, 3.8 μmol) was dissolved in N,N-dimethylformamide (400 μL) and then cooled on ice. N,N-diisopropylethylamine (1.9 μL, 11 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.9 mg, 5.6 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (1.3 mg, 5.7 μmol) was added, and the mixture was returned to room temperature and stirred for 2 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain pyrene (5) (1.3 mg, 1.1 μmol).

[0375] １ H NMR (400 MHz, DMSO-d ６ ) δ10.02-9.99 (m, 1H), 8.85-7.88 (m, 14H), 7.67-7.65 (m, 1H), 7.60-7.51 (m, 2H), 7.33-7.27 (m, 2H), 6.91-6.87 (m, 2H), 5.92-5.91 (m, 1H), 5.63-5.62 (m, 1H), 5.36 (brs, 2H), 5.07-4.92 (m, 2H), 4.35-3.76 (m, 5H), 3.18-3.14 (m, 1H), 2.99-2.83 (m, 7H), 2.17-2.13 (m, 2H), 1.95-1.89 (m, 1H), 1.85-1.72 (m, 4H), 1.66-1.45 (m, 3H), 1.40-1.17 (m, 15H), 1.05-1.01 (m, 2H), 0.83-0.73 (m, 6H).

[0376] MS (ESI) m / z: 1143.45 [M+H] ＋

[0377] (1-1-5) Synthesis of Linker-payload mimic (1)

[0378] To pyrene (5) (2.2 mg, 1.9 μmol), 1,4-dioxane (380 μL) and 4 M hydrogen chloride / dioxane solution (95 μL, 380 μmol) were added sequentially, and the mixture was stirred at room temperature for 4 hours. After cooling on ice, N,N-diisopropylethylamine (71.8 μL, 418 μmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain linker-payload mimic (1) (2.1 mg, 1.9 μmol).

[0379] １ H NMR (400 MHz, DMSO-d ６) δ12.06 (brs, 1H), 10.03-10.00 (m, 1H), 8.84-7.88 (m, 14H), 7.67-7.64 (m, 1H), 7.56-7.51 (m, 2H), 7 .33-7.27 (m, 2H), 6.90-6.87 (m, 2H), 5.92-5.90 (m, 1H), 5.63-5.61 (m, 1H), 5.36 (brs, 2H), 5.08-4.9 6 (m, 2H), 4.35-3.76 (m, 5H), 3.18-3.14 (m, 1H), 2.97-2.83 (m, 7H), 2.20-2.16 (m, 2H), 1.93-1.88 (m, 1H), 1.81-1.78 (m, 4H), 1.69-1.53 ​​(m, 3H), 1.35-1.17 (m, 6H), 1.08-1.01 (m, 2H), 0.81-0.73 (m, 6H).

[0380] MS (ESI) m / z: 1087.45 [M+H] ＋

[0381] (1-2) Synthesis of Linker-payload mimic (6) Linker-payload mimic (6) was synthesized as follows.

[0382] (1-2-1) Synthesis of alcohol (7)

[0383] Z-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 84.3 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (38.4 mg, 101 μmol) and 2,4,6-trimethylpyridine (13.3 μL, 101 μmol) were added. The mixture was stirred at room temperature for 10 minutes. Subsequently, methyl 4-aminomandelate (18.3 mg, 101 μmol) was added, and the mixture was stirred at room temperature for 16 hours. The mixture was then purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the above alcohol (7) (49.1 mg, 64.9 μmol).

[0384] １H NMR (400 MHz, DMSO-d ６ ) δ10.04-9.95 (m, 1H), 8.40-7.28 (m, 12H), 6.00-5.97 (m, 1H), 5.43 (brs, 2H), 5.08-4.97 (m, 3H), 4.43-4.37 (m, 1H), 4.24-4.2 0 (m, 1H), 4.16-4.05 (m, 1H), 3.60-3.59 (m, 3H), 3.04-2.91 (m, 2H), 2.26-2.20 (m, 2H), 2.03-1.22 (m, 16H), 0.88-0.78 (m, 6H).

[0385] MS (ESI) m / z: 757.30 [M+H] ＋

[0386] (1-2-2) Synthesis of pyrene (8)

[0387] Alcohol (7) (44.6 mg, 58.9 μmol) was dissolved in N,N-dimethylformamide (650 μL) and stirred for 5 minutes under ice cooling. After that, bis(4-nitrophenyl)carbonate (53.8 mg, 177 μmol) and N,N-diisopropylethylamine (22.5 μL, 133 μmol) were added and stirred at room temperature for 4 hours. After that, the mixture was ice-cooled, and sarcosin-pyrene (89.1 mg, 295 μmol), 1-hydroxybenzotriazole (11.9 mg, 88.4 μmol), and N,N-diisopropylethylamine (77.7 μL, 457 μmol) were added and stirred at room temperature for 18 hours. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the pyrene (8) (49.2 mg, 45.3 μmol).

[0388] １H NMR (400MHz, DMSO-d6) δ10.11-9.97 (m, 1H), 8.64-8.60 (m, 1H), 8.36-7.95 (m, 11H), 7.8 2-7.74 (m, 1H), 7.65-7.57 (m, 2H), 7.49-7.19 (m, 8H), 5.91-5.90 (m, 1H), 5.72-5.70 (m, 1H), 5.36 (brs, 2H), 4.98-4.86 (m, 4H), 4.41-4.00 (m, 3H), 3.85-3.75 (m, 1H), 3.56-3.5 4 (m, 3H), 3.00-2.82 (m, 5H), 2.19-2.12 (m, 2H), 1.92-1.17 (m, 16H), 0.80-0.73 (m, 6H).

[0389] MS (ESI) m / z: 1085.45 [M+H] ＋

[0390] (1-2-3) Synthesis of pyrene (9)

[0391] Pyrene (8) (44.8 mg, 41.3 μmol) was dissolved in tetrahydrofuran (3.75 mL) and water (1.25 mL) and stirred for 5 minutes under ice cooling. Lithium hydroxide monohydrate (8.7 mg, 210 μmol) was then added and stirred at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to approximately 6 using 0.1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (9) (18.4 mg, 17.2 μmol).

[0392] １ H NMR (400 MHz, DMSO-d ６) δ13.02 (brs, 1H), 10.09-9.97 (m, 1H), 8.62-8.59 (m, 1H), 8.32-7.78 (m, 12H), 7.63-7.44 (m, 3H), 7.37-7.35 (m, 2H), 7.29-7.20 (m, 5H), 5.91 (brs, 1H), 5.61-5.60 (m, 1H), 5.36 (br s, 2H), 4.98-4.86 (m, 4H), 4.45-4.30 (m, 1H), 4.24-4.15 (m, 1H), 4.07-4.02 (m, 1H), 3.84- 3.74 (m, 1H), 2.97-2.82 (m, 5H), 2.19-2.12 (m, 2H), 1.93-1.11 (m, 16H), 0.80-0.72 (m, 6H).

[0393] MS (ESI) m / z: 1071.45 [M+H] ＋

[0394] (1-2-4) Synthesis of pyrene (10)

[0395] Pyrene (9) (15.6 mg, 14.6 μmol) was dissolved in N,N-dimethylformamide (1.0 mL) and then cooled on ice. N,N-diisopropylethylamine (5.0 μL, 29 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (11.4 mg, 21.9 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.8 mg, 22 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain pyrene (10) (15.4 mg, 12.5 μmol).

[0396] １ H NMR (400 MHz, DMSO-d ６) δ10.11-10.01 (m, 1H), 8.91-8.63 (m, 1H), 8.40-7.96 (m, 12H), 7.72-7.27 (m, 11H), 6.97-6.94 (m, 2H) ), 5.99 (brs, 1H), 5.71-5.69 (m, 1H), 5.43 (brs, 2H), 5.14-4.96 (m, 4H), 4.49-4.40 (m, 1H), 4.30-4.2 3 (m, 1H), 4.16-3.83 (m, 3H), 3.25-3.22 (m, 1H), 3.02-2.90 (m, 7H), 2.26-2.22 (m, 2H), 2.01-1.98 (m, 1H), 1.92-1.84 (m, 1H), 1.77-1.66 (m, 2H), 1.65-1.04 (m, 16H), 1.12-1.04 (m, 2H), 0.88-0.80 (m, 6H).

[0397] MS (ESI) m / z: 1235.50 [M+H] ＋

[0398] (1-2-5) Synthesis of Linker-payload mimic (6)

[0399] 1,4-Dioxane (2.0 mL) and 4 M hydrogen chloride / dioxane solution (490 μL, 1.96 mmol) were added sequentially to pyrene (10) (12.1 mg, 9.79 μmol), and the mixture was stirred at room temperature for 4 hours. After cooling on ice, N,N-diisopropylethylamine (366 μL, 2.15 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain linker-payload mimic (6) (6.0 mg, 5.1 μmol).

[0400] １ H NMR (400 MHz, DMSO-d ６) δ12.05 (brs, 1H), 10.04-9.94 (m, 1H), 8.84-8.57 (m, 1H), 8.32-7.89 (m, 12H), 7.72- 7.20 (m, 11H), 6.90-6.86 (m, 2H), 5.90 (brs, 1H), 5.64-5.62 (m, 1H), 5.36 (brs, 2H), 5. 07-4.88 (m, 4H), 4.42-4.29 (m, 1H), 4.21-4.15 (m, 1H), 4.08-3.76 (m, 3H), 3.18-3.14 (m, 1H), 2.95-2.82 (m, 7H), 2.21-2.16 (m, 2H), 1.92-0.97 (m, 13H), 0.82-0.74 (m, 6H).

[0401] MS (ESI) m / z: 1179.50 [M+H] ＋

[0402] (1-3) Synthesis of Linker-payload mimic (11) Linker-payload mimic (11) was synthesized as follows.

[0403] (1-3-1) Synthesis of alcohol (12)

[0404] Ac-Glu(t-Bu)-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 72.8 μmol) was dissolved in N,N-dimethylformamide (800 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (33.2 mg, 87.4 μmol) and 2,4,6-trimethylpyridine (11.5 μL, 87.4 μmol) were added. The mixture was stirred at room temperature for 10 minutes. Subsequently, methyl 4-aminomandelate (15.8 mg, 87.4 μmol) was added, and the mixture was stirred at room temperature for 16 hours. The mixture was then purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the above alcohol (12) (54.0 mg, 63.5 μmol).

[0405] １ H NMR (400 MHz, DMSO-d ６) δ10.00 (s, 1H), 8.26-7.88 (m, 3H), 7.68-7.60 (m, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 6.00-5.97 (m, 1H), 5.43 (brs, 2H), 5.08 (s, 1H), 4.40-4.37 (m, 1H), 4.32-4.19 (m, 3H), 3.60 (s, 3H), 3.09-2. 90 (m, 2H), 2.25-2.18 (m, 4H), 2.03-1.53 ​​(m, 10H), 1.46-1.36 (m, 20H), 0.86 (d, J = 6.8Hz, 3H), 0.82 (d, J = 6.8Hz, 3H).

[0406] MS (ESI) m / z: 850.40 [M+H] ＋

[0407] (1-3-2) Synthesis of pyrene (13)

[0408] Alcohol (12) (50.3 mg, 59.2 μmol) was dissolved in N,N-dimethylformamide (650 μL) and stirred for 5 minutes under ice cooling. Then, bis(4-nitrophenyl)carbonate (54.0 mg, 178 μmol) and N,N-diisopropylethylamine (22.7 μL, 133 μmol) were added and the mixture was stirred at room temperature for 5 hours. The mixture was then ice-cooled, and sarcosin-pyrene (89.5 mg, 296 μmol), 1-hydroxybenzotriazole (12.0 mg, 88.8 μmol), and N,N-diisopropylethylamine (78.1 μL, 459 μmol) were added, followed by stirring at room temperature for 18 hours. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the pyrene (13) (34.0 mg, 28.9 μmol).

[0409] １ H NMR (400 MHz, DMSO-d ６) δ10.14-10.12 (m, 1H), 8.69-8.67 (m, 1H), 8.40-7.82 (m, 13H), 7.76-7.72 (m, 1H) , 7.68-7.65 (m, 2H), 7.44-7.39 (m, 2H), 5.99 (brs, 1H), 5.79 (d, J = 6.4Hz, 1H), 5.44 (brs, 2H), 5.05-4.97 (m, 2H), 4.44-4.08 (m, 4H), 3.93-3.80 (m, 1H), 3.63-3.62 (m, 3H), 3.05-2.92 (m, 5H), 2.25-2.14 (m, 4H), 2.00-1.28 (m, 30H), 0.87-0.80 (m, 6H).

[0410] MS (ESI) m / z: 1178.50 [M+H] ＋

[0411] (1-3-3) Synthesis of pyrene (14)

[0412] Pyrene (13) (30.7 mg, 26.1 μmol) was dissolved in tetrahydrofuran (2.25 mL) and water (0.75 mL) and stirred for 5 minutes under ice cooling. Lithium hydroxide monohydrate (5.5 mg, 0.13 mmol) was then added and stirred at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to approximately 6 using 0.1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (14) (17.2 mg, 14.8 μmol).

[0413] １ H NMR (400 MHz, DMSO-d ６) δ13.06 (brs, 1H), 10.13-10.10 (m, 1H), 8.69-8.66 (m, 1H), 8.40-7.85 (m, 13H), 7.77-7.73 (m, 1H), 7.67-7.64 (m, 2H), 7.44-7.42 (m, 2H), 5.99 (brs, 1H), 5.68-5. 67 (m, 1H), 5.44 (brs, 2H), 5.05-4.96 (m, 2H), 4.46-4.10 (m, 4H), 3.92-3.81 (m, 1 H), 3.05-2.90 (m, 5H), 2.25-2.14 (m, 4H), 2.03-1.29 (m, 30H), 0.88-0.80 (m, 6H).

[0414] MS (ESI) m / z: 1164.55 [M+H] ＋

[0415] (1-3-4) Synthesis of pyrene (15)

[0416] Pyrene (14) (14.7 mg, 12.6 μmol) was dissolved in N,N-dimethylformamide (1.0 mL) and then cooled on ice. N,N-diisopropylethylamine (4.29 μL, 25.2 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (9.8 mg, 19 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.1 mg, 19 μmol) was added, and the mixture was returned to room temperature and stirred for 3.5 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain pyrene (15) (7.0 mg, 9.2 μmol).

[0417] １ H NMR (400 MHz, DMSO-d ６) δ10.08-10.05 (m, 1H), 8.92-7.95 (m, 15H), 7.75-7.73 (m, 1H), 7.63-7.59 (m, 2H), 7.4 0-7.34 (m, 2H), 6.97-6.94 (m, 2H), 6.00-5.98 (m, 1H), 5.70-5.69 (m, 1H), 5.43 (brs, 2H) , 5.14-5.01 (m, 2H), 4.45-4.38 (m, 1H), 4.32-3.83 (m, 5H), 3.25-3.20 (m, 1H), 3.10-2.9 0 (m, 7H), 2.25-2.18 (m, 4H), 2.08-1.24 (m, 34H), 1.12-1.04 (m, 2H), 0.88-0.81 (m, 6H).

[0418] MS (ESI) m / z: 1328.60 [M+H] ＋

[0419] (1-3-5) Synthesis of Linker-payload mimic (11)

[0420] 1,4-Dioxane (920 μL) and 4 M hydrogen chloride / dioxane solution (230 μL, 918 μmol) were added sequentially to pyrene (15) (6.1 mg, 4.6 μmol), and the mixture was stirred at room temperature for 4 hours. After cooling on ice, N,N-diisopropylethylamine (172 μL, 1.10 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain linker-payload mimic (11) (3.7 mg, 3.0 μmol).

[0421] １ H NMR (400 MHz, DMSO-d ６) δ12.04 (brs, 2H), 10.01-9.98 (m, 1H), 8.83-7.89 (m, 15H), 7.71-7.69 (m, 1H), 7.56-7.51 ( m, 2H), 7.33-7.26 (m, 2H), 6.90-6.87 (m, 2H), 5.91-5.90 (m, 1H), 5.64-5.62 (m, 1H), 5.36 (b rs, 2H), 5.08-4.92 (m, 2H), 4.35-4.32 (m, 1H), 4.25-3.76 (m, 5H), 3.18-3.14 (m, 1H), 2.99- 2.82 (m, 7H), 2.21-2.15 (m, 4H), 1.93-1.17 (m, 16H), 1.05-1.01 (m, 2H), 0.82-0.74 (m, 6H).

[0422] MS (ESI) m / z: 1216.45 [M+H] ＋

[0423] (1-4) Synthesis of Linker-payload mimic (16) Linker-payload mimic (16) was synthesized as follows.

[0424] (1-4-1) Synthesis of alcohol (17)

[0425] SCA(t-Bu)-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 81.2 μmol) was dissolved in N,N-dimethylformamide (890 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (37.0 mg, 97.4 μmol) and 2,4,6-trimethylpyridine (12.8 μL, 97.4 μmol) were added and stirred at room temperature for 10 minutes. Subsequently, methyl 4-aminomandelate (17.6 mg, 97.4 μmol) was added and stirred at room temperature for 16 hours, followed by purification by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the above alcohol (17) (60.4 mg, 77.5 μmol).

[0426] １ H NMR (400 MHz, DMSO-d６ ) δ10.01 (s, 1H), 8.13 (d, J = 7.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.99 (brs, 1H), 5.43 (brs, 2H), 5.08 (s, 1H), 4.41-4.30 (m, 2H), 4.21-4.17 (m, 1H), 3.60 (s, 3H), 3.04-2.95 (m, 2H), 2.44-2.1 5 (m, 6H), 2.03-1.95 (m, 1H), 1.92-1.83 (m, 1H), 1.74-1.58 (m, 3H), 1.46-1.34 (m, 20H), 0.86 (d, J = 6.8Hz, 3H), 0.83 (d, J = 6.8Hz, 3H).

[0427] MS (ESI) m / z: 779.40 [M+H] ＋

[0428] (1-4-2) Synthesis of pyrene (18)

[0429] Alcohol (17) (58.0 mg, 74.5 μmol) was dissolved in N,N-dimethylformamide (820 μL) and stirred for 5 minutes under ice cooling. After that, bis(4-nitrophenyl)carbonate (68.0 mg, 223 μmol) and N,N-diisopropylethylamine (28.5 μL, 168 μmol) were added and stirred at room temperature for 6 hours. After that, the mixture was ice-cooled, and sarcosin-pyrene (113 mg, 373 μmol), 1-hydroxybenzotriazole (15.1 mg, 112 μmol), and N,N-diisopropylethylamine (98.3 μL, 578 μmol) were added, followed by stirring at room temperature for 17 hours. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the pyrene (18) (37.1 mg, 33.5 μmol).

[0430] １ H NMR (400 MHz, DMSO-d ６) δ10.15-10.13 (m, 1H), 8.70-8.67 (m, 1H), 8.40-8.03 (m, 11H), 7.90-7.86 (m, 1H), 7.75-7.72 (m, 1H) , 7.68-7.64 (m, 2H), 7.44-7.39 (m, 2H), 5.99 (brs, 1H), 5.79 (d, J=6.8Hz, 1H), 5.44 (brs, 2H), 5.05-4. 98 (m, 2H), 4.40-4.08 (m, 3H), 3.93-3.80 (m, 1H), 3.63-3.62 (m, 3H), 3.05-2.90 (m, 5H), 2.44-2.15 (m, 6H), 2.02-1.98 (m, 1H), 1.91-1.85 (m, 1H), 1.75-1.55 (m, 3H), 1.50-1.30 (m, 20H), 0.88-0.80 (m, 6H).

[0431] MS (ESI) m / z: 1107.55 [M+H] ＋

[0432] (1-4-3) Synthesis of pyrene (19)

[0433] Pyrene (18) (17.4 mg, 15.7 μmol) was dissolved in tetrahydrofuran (675 μL) and water (225 μL) and stirred for 5 minutes under ice-cooling. After that, 1 M aqueous lithium hydroxide solution (86.4 μL, 86.4 μmol) was added and the mixture was stirred under ice-cooling for 5 hours. After the reaction was completed, the pH was adjusted to approximately 6 using 0.1 M hydrochloric acid and purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (19) (17.2 mg, 15.7 μmol).

[0434] １ H NMR (400 MHz, DMSO-d ６) δ13.06 (brs, 1H), 10.14-10.11 (m, 1H), 8.68-8.66 (m, 1H), 8.40-8.01 (m, 11H), 7.89-7.85 (m, 1H), 7.75-7.72 (m, 1H), 7.67-7.64 (m, 2H), 7.44-7.41 (m, 2H), 5.99 (brs, 1H), 5.68-5.67 (m, 1H), 5.44 (br s, 2H), 5.05-4.93 (m, 2H), 4.44-4.10 (m, 3H), 3.92-3.81 (m, 1H), 3.05-2.94 (m, 5H), 2.43-2.15 (m, 6 H), 2.02-1.97 (m, 1H), 1.88-1.85 (m, 1H), 1.77-1.66 (m, 3H), 1.61-1.30 (m, 20H), 0.88-0.80 (m, 6H).

[0435] MS (ESI) m / z: 1093.50 [M+H] ＋

[0436] (1-4-4) Synthesis of pyrene (20)

[0437] Pyrene (19) (16.3 mg, 14.9 μmol) was dissolved in N,N-dimethylformamide (1.0 mL) and then cooled on ice. N,N-diisopropylethylamine (5.1 μL, 30 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (11.7 mg, 22.4 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (4.9 mg, 22 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain pyrene (20) (12.1 mg, 9.62 μmol).

[0438] １ H NMR (400 MHz, DMSO-d ６) δ10.01-9.98 (m, 1H), 8.85-7.88 (m, 14H), 7.68-7.65 (m, 1H), 7.56-7.51 (m, 2H), 7.33-7.27 (m, 2H), 6.90-6.87 (m, 2H), 5.92-5.90 (m, 1H), 5.63-5.62 (m, 1H), 5.36 (brs, 2H), 5.08-4.92 (m, 2H), 4.36-3.76 (m, 5H), 3.18-3.14 (m, 1H), 3.00-2.83 (m, 7H), 2.36-2.08 (m, 6H), 1.95-1.89 (m, 1H), 1.84-1.78 (m, 1H), 1.68-1.50 (m, 3H), 1.42-1.17 (m, 24H), 1.07-0.97 (m, 2H), 0.81-0.74 (m, 6H).

[0439] MS (ESI) m / z: 1257.55 [M+H] ＋

[0440] (1-4-5) Synthesis of Linker-payload mimic (16)

[0441] Ethyl acetate (1.7 mL) and 4 M hydrogen chloride / ethyl acetate (2.09 mL, 8.36 mmol) were added sequentially to pyrene (20) (10.5 mg, 8.35 μmol), and the mixture was stirred at room temperature for 4.5 hours. After cooling on ice, N,N-diisopropylethylamine (781 μL, 4.59 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reversed-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain linker-payload mimic (16) (7.5 mg, 6.6 μmol).

[0442] １ H NMR (400 MHz, DMSO-d ６) δ12.02 (brs, 2H), 10.04-9.98 (m, 1H), 8.85-7.89 (m, 14H), 7.65-7.62 (m, 1H), 7.56-7.52 (m, 2H), 7.33-7.27 (m, 2H), 6.90-6.87 (m, 2H), 5.92 (brs, 1H), 5.63-5.62 (m, 1H), 5.37 (br s, 2H), 5.08-4.93 (m, 2H), 4.36-3.76 (m, 5H), 3.18-3.14 (m, 1H), 3.02-2.80 (m, 7H), 2.38- 2.16 (m, 6H), 1.96-1.77 (m, 2H), 1.70-1.17 (m, 9H), 1.06-0.98 (m, 2H), 0.81-0.74 (m, 6H).

[0443] MS (ESI) m / z: 1145.45 [M+H] ＋

[0444] (1-5) Synthesis of Linker-payload mimic (21) Linker-payload mimic (21) was synthesized as follows.

[0445] (1-5-1) Synthesis of alcohol (22)

[0446] Ac-Glu(OtBu)-Val-Ala-OH (20.9 mg, 50.3 μmol) was dissolved in N,N-dimethylformamide (700 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (29.0 mg, 76.3 μmol) and 2,4,6-trimethylpyridine (10.1 μL, 76.7 μmol) were added. The mixture was stirred at room temperature for 12 minutes. Subsequently, methyl 4-aminomandelate (11.0 mg, 60.7 μmol) was added, and the mixture was stirred at room temperature for 18 hours. The mixture was then purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the above alcohol (22) (20.3 mg, 35.1 μmol).

[0447] １ H NMR (400 MHz, DMSO-d ６) δ9.98-9.89 (m, 1H), 8.32-8.18 (m, 1H), 8.07-7.85 (m, 1H), 7.73-7.59 (m, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 5.08 (s, 1H), 4.45-4.34 (m, 1H), 4.32-4.27 (m, 1H), 4.20-4.09 (m, 1H), 3.59 (s, 3H), 2.24-2.20 (m, 2H), 2.01-1.96 (m, 1H) ), 1.90-1.80 (m, 4H), 1.72-1.67 (m, 1H), 1.39-1.36 (m, 9H), 1.30 (d, J = 7.2Hz, 3H), 0.86 (d, J = 6.8Hz, 3H), 0.82 (d, J = 6.8Hz, 3H).

[0448] MS (ESI) m / z: 579.30 [M+H] ＋

[0449] (1-5-2) Synthesis of pyrene (23)

[0450] Alcohol (22) (17.5 mg, 30.2 μmol) was dissolved in N,N-dimethylformamide (174 μL) and stirred for 5 minutes under ice cooling. Then, bis(4-nitrophenyl)carbonate (18.9 mg, 60.8 μmol) and N,N-diisopropylethylamine (7.80 μL, 45.4 μmol) were added, and the mixture was stirred at room temperature for 30 minutes. LCMS analysis showed that the starting material remained, so bis(4-nitrophenyl)carbonate (9.5 mg, 31 μmol) and N,N-diisopropylethylamine (3.90 μL, 22.7 μmol) were added, and the mixture was stirred at room temperature for 1 hour. The mixture was then cooled on ice, and sarcosin-pyrene (36.2 mg, 120 μmol), 1-hydroxybenzotriazole (6.3 mg, 47 μmol), and N,N-diisopropylethylamine (40.3 μL, 235 μmol) were added, followed by stirring at room temperature for 1 hour. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the pyrene (23) (12.9 mg, 14.2 μmol).

[0451] １H NMR (400 MHz, DMSO-d ６ ) δ10.05-10.02 (m, 1H), 8.61-8.60 (m, 1H), 8.30-7.78 (m, 12H), 7.67-7.64 (m, 1H), 7.57 ( d, J=8.8Hz, 2H), 7.37-7.33 (m, 2H), 5.72-5.70 (m, 1H), 4.98-4.89 (m, 2H), 4.34-4.00 (m, 3H), 3.85-3.75 (m, 1H), 3.56-3.55 (m, 3H), 2.93-2.87 (m, 3H), 2.17-2.13 (m, 2H), 1.94-1 91 (m, 1H), 1.85-1.75 (m, 4H), 1.65-1.61 (m, 1H), 1.31-1.23 (m, 12H), 0.80-0.73 (m, 6H).

[0452] MS (ESI) m / z: 907.35 [M+H] ＋

[0453] (1-5-3) Synthesis of pyrene (24)

[0454] Pyrene (23) (11.8 mg, 13.0 μmol) was dissolved in tetrahydrofuran (800 μL) and water (400 μL) and then cooled on ice. 1 M aqueous lithium hydroxide solution (65 μL, 65 μmol) was added and stirred for 30 minutes. After the reaction was completed, the pH was adjusted to approximately 6 using 0.1 M hydrochloric acid and purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (24) (8.9 mg, 10.0 μmol).

[0455] １ H NMR (400 MHz, DMSO-d ６) δ13.05 (brs, 1H), 10.04-9.91 (m, 1H), 8.63-8.60 (m, 1H), 8.33-7.77 (m, 12H), 7. 69-7.55 (m, 3H), 7.37-7.35 (m, 2H), 5.61-5.60 (m, 1H), 4.98-4.86 (m, 2H), 4.26-4. 03 (m, 3H), 3.84-3.74 (m, 1H), 2.93-2.87 (m, 3H), 2.18-2.13 (m, 2H), 1.95-1.92 (m, 1H), 1.85-1.77 (m, 4H), 1.67-1.62 (m, 1H), 1.31-1.24 (m, 12H), 0.81-0.75 (m, 6H).

[0456] MS (ESI) m / z: 893.35 [M+H] ＋

[0457] (1-5-4) Synthesis of pyrene (25)

[0458] Pyrene (24) (6.7 mg, 7.5 μmol) was dissolved in N,N-dimethylformamide (400 μL) and then cooled on ice. N,N-diisopropylethylamine (3.9 μL, 22 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (5.9 mg, 11 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (2.4 mg, 11 μmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain pyrene (25) (4.1 mg, 3.9 μmol).

[0459] １ H NMR (400 MHz, DMSO-d ６) δ9.98-9.95 (m, 1H), 8.83-7.77 (m, 14H), 7.67-7.65 (m, 1H), 7.56-7.50 (m, 2H), 7.34-7 .27 (m, 2H), 6.89-6.86 (m, 2H), 5.63-5.61 (m, 1H), 5.06-4.93 (m, 2H), 4.37-3.76 (m, 5H) , 3.17-3.14 (m, 1H), 2.95-2.83 (m, 5H), 2.17-2.10 (m, 2H), 1.95-1.89 (m, 1H), 1.85-1.7 5 (m, 4H), 1.65-1.62 (m, 1H), 1.31-1.17 (m, 16H), 1.02-1.00 (m, 2H), 0.81-0.74 (m, 6H).

[0460] MS (ESI) m / z: 1057.45 [M+H] ＋

[0461] (1-5-5) Synthesis of Linker-payload mimic (21)

[0462] To pyrene (25) (2.4 mg, 2.3 μmol), 1,4-dioxane (454 μL) and 4 M hydrogen chloride / dioxane solution (568 μL, 2.27 mmol) were added sequentially, and the mixture was stirred at room temperature for 4 hours. N,N-dimethylformamide (300 μL) was added, and then N,N-diisopropylethylamine (214 μL, 1.25 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was purified by reverse-phase preparative chromatography, and the fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain linker-payload mimic (21) (1.1 mg, 1.1 μmol).

[0463] １ H NMR (400 MHz, DMSO-d ６) δ11.99 (brs, 1H), 9.98-9.96 (m, 1H), 8.84-7.88 (m, 14H), 7.66-7.63 (m, 1H), 7.54-7.50 (m , 2H), 7.34-7.27 (m, 2H), 6.90-6.87 (m, 2H), 5.63-5.61 (m, 1H), 5.07-4.93 (m, 2H), 4.36-3. 76 (m, 5H), 3.17-3.14 (m, 1H), 2.95-2.83 (m, 5H), 2.20-2.16 (m, 2H), 1.95-1.90 (m, 1H), 1.8 8-1.76 (m, 4H), 1.67-1.62 (m, 1H), 1.32-1.17 (m, 7H), 1.02-1.01 (m, 2H), 0.81-0.74 (m, 6H).

[0464] MS (ESI) m / z: 999.35 [MH] ―

[0465] Comparative Example 1: Synthesis of Linker-Payload (1-1) Synthesis of Linker-Payload (26) Linker-Payload (26) was synthesized as follows.

[0466] Linker-Payload (26) was synthesized according to the following scheme.

[0467] The results of MS analysis of Linker-Payload (26) were as follows:

[0468] MS (ESI) m / z: 1050.55 [M+H] ＋

[0469] Example 2: Synthesis of ADC mimic (2-1) Synthesis of ADC mimic In the following comparative examples and examples, the antibody derivative (thiol group-introduced trastuzumab) described in Examples 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used as the thiol group-introduced antibody. This antibody derivative has the following structure, in which a thiol group has been regioselectively introduced into trastuzumab (humanized IgG1 antibody) via the amino group in the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue follows EU numbering). (In the above structure, the NH-CH extending from the antibody heavy chain ２ -CH ２ -CH ２ -CH ２ - corresponds to the side chain of a lysine residue, and the thiol-containing group HS-CH is connected to the amino group in the side chain of this lysine residue. ２ -CH ２ -C(=O) is added. Since no modifications at other lysine residues were detected by peptide mapping, it is believed that the regioselectivity at positions 246 and 248 of the antibody heavy chain is 100%.

[0470] To a buffer (PBS buffer, pH 7.4) solution (20 μM) of the thiol group-introduced antibody was added 10 equivalents of a DMF solution (10 mM) of the linker-payload mimic synthesized in Example 1, and the mixture was allowed to stand at room temperature for 2 hours. After that, the mixture was purified using NAP-5 Columns (manufactured by GE Healthcare) to obtain an ADC mimic.

[0471] ADC mimic 1 having the following structure was synthesized from the linker-payload mimic (1) synthesized in Example 1-1 and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 150350, where two linker-payload mimics (1) were introduced.

[0472] Similarly, ADC mimic 2 having the following structure was synthesized from Linker-payload mimic (6) of Example 1-2 and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 150535, where two Linker-payload mimics (6) had been introduced.

[0473] Similarly, ADC mimic 3 having the following structure was synthesized from Linker-payload mimic (11) of Example 1-3 and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had a peak at 150609, where two Linker-payload mimics (11) were introduced.

[0474] Similarly, ADC mimic 4 having the following structure was synthesized from Linker-payload mimic (16) of Example 1-4 and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 150466, where two Linker-payload mimics (16) had been introduced.

[0475] Similarly, ADC mimic 5 having the following structure was synthesized from Linker-payload mimic (26) of Comparative Example 1 and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 150276, where two Linker-payload mimics (26) had been introduced.

[0476] (2-2) DAR Analysis of ADC Mimic ESI-TOFMS analysis of the ADC mimic synthesized in Example 2-1 was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2.

[0477]

[0478] Example 3: Evaluation of hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC) HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). Measurement was performed under the following conditions. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram.

[0479] Measurement system: Chromaster (registered trademark) (manufactured by Hitachi) Column: Tosoh Biobutyl NPR 2.5 μm 4.6 × 35 mm column manufactured by Tosoh Biosciences Gradient: Linear gradient of eluent A / B Flow rate: 0.8 mL / min Eluent A: 1.1 M (NH ４ ) ２ SO ４ , 25 mM Na ２ HPO ４ / NaH ２ P.O. ４ (pH 6.0) Eluent B: 25mM Na２ HPO ４ / NaH ２ P.O. ４ (pH 6.0, 25 v / v% isopropanol added) Detector: UV (280 nm)

[0480]

[0481] As a result, it was confirmed that the ADC mimics synthesized in Examples 1-1, 1-2, 1-3, and 1-4 tended to have a short retention time, indicating high hydrophilicity. Therefore, it was confirmed that the ADC mimics synthesized in Examples 1-1, 1-2, 1-3, and 1-4 are preferable ADCs because they are thought to have slow plasma clearance and long retention times in the body.

[0482] Example 4: Evaluation of aggregation rate of ADC and ADC mimic by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to a previous report (Chemistry Select, 2020, 5, 8435-8439). The measurement was performed under the following conditions.

[0483] Measurement system: 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300 Å 2.7 μm, 4.6 mm × 150 mm Flow rate: 0.25 mL / min Eluent: 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride aqueous solution (pH 6.8), 10% v / v isopropanol Detector: UV (280 nm)

[0484]

[0485] Example 5: Evaluation of ADC mimics using the enzyme cathepsin B The cleavage ability of various ADC mimics with cathepsin B was evaluated by analyzing the amount of fluorescent molecules cleaved from the ADC mimic as described below.

[0486] (5-1) Cathepsin B Cleavage Test This test was performed as described previously (Nature Communications 2018, 9, 2512) as follows. ADC mimic was added to 180 μL of MES buffer (10 mM MES, 40 μM DTT, pH 5.0) to a concentration of 1 mg / mL, and then 30 μL of the solution was dispensed into six Eppendorf tubes. Three of the six samples were immediately added with 100 μL of acetonitrile at 0°C, vortexed, and then centrifuged to obtain a precipitate. The resulting supernatant was collected and analyzed by HPLC. The remaining three samples were incubated at 37°C for 6 hours. Acetonitrile (100 μL each) was added to each sample, vortexed, and then centrifuged to obtain a precipitate. The resulting supernatant was collected and analyzed by HPLC.

[0487] (5-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis The amount of fluorescent molecules detached from the ADC mimic was measured using liquid chromatography / fluorescence detection. Three samples to which acetonitrile was immediately added at 0°C in Example 7-1 were designated as time 0, and three samples incubated at 37°C for 6 hours as described in Example 7-1 were designated as time 6, and the difference in fluorescence intensity between the 6-hour samples and the 0-hour samples was analyzed.

[0488] Separately, Pyrene was used to calculate the correlation between the area of ​​fluorescence intensity measured by HPLC and the concentration. Using this calculation formula, the difference in fluorescence intensity for each ADC mimic was converted to concentration. The dropout rate was calculated as the percentage of the difference in fluorescence intensity, with the concentration at time 0 taken as 100%.

[0489]

[0490] As shown in Table 4, the synthesized ADC mimics were found to have sufficient cathepsin B cleavage activity.

[0491] Example 6: Evaluation of ADC mimic using mouse plasma (6-1) Plasma stability test of ADC mimic ADC mimic was added to 500 μL of mouse plasma (Charles River) to a concentration of 0.1 mg / mL, followed by sterile filtration. 50 μL of this solution was dispensed into six Eppendorf tubes. Three of the six samples were stored in an incubator set at 37°C for four days. The remaining three were similarly stored in a freezer at -80°C for four days. 100 μL of acetonitrile was added to each sample, and the mixture was vortexed and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0492] (6-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis The amount of fluorescent molecules detached from the ADC mimic was measured using liquid chromatography / fluorescence detection. The three samples stored in the freezer in Example 9-1 were designated as Day = 0, and the three samples stored at 37°C in Example 9-1 were designated as Day = 4, and the difference in fluorescence intensity between Day = 4 and Day = 0 was analyzed.

[0493] The loss rate of fluorescent molecules was calculated in accordance with Example 5-2. The loss rate of fluorescent molecules was evaluated based on the results shown in the table below.

[0494]

[0495] As a result, compared with the ADC mimic synthesized in Comparative Example 1, the ADC mimics synthesized in Examples 1-1 and 1-2 exhibited stability three times or more times higher, and the ADC mimics synthesized in Examples 1-3 and 1-4 exhibited stability ten times or more times higher.

[0496] Example 7: Synthesis of ADC mimics Example 7-1: Synthesis of ADC mimic 6 To 3 mg / mL of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) in PBSE buffer (50 mM phosphate buffered saline (PBS), 10 mM EDTA, pH = 7.4), a PBSE solution of a reducing agent (TCEP) (2 mM) was added in an amount of 8 equivalents relative to the antibody, and the mixture was incubated at 37°C for 1 hour. Dimethylacetamide (DMA) (6% v / v) and a 10 mM DMA solution of various linker-payload mimics (1) (20 equivalents relative to the antibody) were sequentially added to the resulting reaction mixture, and the mixture was gently stirred at 20°C for 1 hour. An excess amount of N-acetylcysteine ​​was added to the reaction mixture, and the mixture was stirred for 20 minutes at 25° C. The final mixture was purified using a NAP-5 desalting column (GE Healthcare) and eluted with formulation buffer (20 mM histidine, 5 wt % trehalose added, pH 5.2) to obtain ADC mimic 6.

[0497] Example 7-2: Synthesis of ADC mimic 7 According to Example 7-1, ADC mimic 7 was obtained from Linker-payload mimic (11).

[0498] Example 7-3: Synthesis of ADC mimic 8 According to Example 7-1, ADC mimic 8 was obtained from Linker-payload mimic (21).

[0499] Example 7-4: Synthesis of ADC mimic 9 According to Example 7-1, ADC mimic 9 was obtained by reacting Linker-payload mimic (1) with the anti-CD20 IgG antibody rituximab (Roche).

[0500] Example 7-5: Synthesis of ADC mimic 10 According to Example 7-1, ADC mimic 10 was obtained by reacting Linker-payload mimic (11) with anti-CD20 IgG antibody rituximab (Roche).

[0501] Example 7-6: Synthesis of ADC mimic 11 According to Example 7-1, ADC mimic 11 was obtained by reacting Linker-payload mimic (21) with anti-CD20 IgG antibody rituximab (Roche).

[0502] Example 7-7: Synthesis of ADC mimic 12 According to Example 7-1, ADC mimic 12 was obtained by reacting Linker-payload mimic (1) with the anti-TNF-α IgG antibody infliximab (Centocor).

[0503] Example 7-8: Synthesis of ADC mimic 13 According to Example 7-1, the linker-payload mimic (11) was reacted with the anti-TNF-α IgG antibody infliximab (Centocor) to obtain ADC mimic 13.

[0504] Example 7-9: Synthesis of ADC mimic 14 According to Example 7-1, ADC mimic 14 was obtained by reacting linker-payload mimic (21) with the anti-TNF-α IgG antibody infliximab (Centocor).

[0505] Comparative Example 2: Synthesis of ADC mimic Comparative Example 2-1: Synthesis of linker-payload mimic (30) Linker-payload mimic (30) was synthesized in one step from commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) and known sarcosine-pyrene (WO2018218004A1).

[0506] Commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) (15.5 mg, 0.021 mmol) was dissolved in dichloromethane (1 mL), N,N-diisopropylethylamine (0.025 mL, 0.142 mmol), known sarcosine-pyrene (WO2018218004A1) (7.6 mg, 0.025 mmol), dimethylformamide solution (0.5 mL) was added, and the mixture was stirred for 17 hours. After purification by reversed-phase preparative chromatography, the fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain Linker-payload mimic (30) (7.3 mg, 0.008 mmol).

[0507] １ H NMR (400 MHz, DMSO-d6) δ9.98 (s, 1H), 8.34 (d, J = 9.2Hz, 2H), 8.32-8.23 (m, 4H), 8.16 (s, 2H), 8.10-8.00 (m, 4H), 7.80 (d , J=8.8Hz, 1H), 7.59 (d, J=8.4Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 6.99 (s, 2H), 5.96 (m, 1H), 5.40 (s, 2H), 5.01 (s, 2H) ), 4.95 (d, J = 6.0Hz, 2H), 4.38 (m, 1H), 4.19 (m, 1H), 3.03-2.92 (m, 3H), 2.67 (m, 1H), 2.33 (m, 1H), 2.20-2.07 (m, 2H), 1.97 (m, 1H), 1.67 (m, 1H), 1.59 (m, 1H), 1.51-1.45 (m, 6H), 1.26-1.15 (m, 3H), 0.83 (dd, J = 12.8, 6.8Hz, 6H)

[0508] MS (ESI) m / z: 901.45 [M+H] ＋

[0509] Comparative Example 2-2: Synthesis of ADC mimics 15 to 17 According to Example 7-1, ADC mimic 15 was obtained from trastuzumab, ADC mimic 16 from rituximab, and ADC mimic 17 from infliximab by reacting with linker-payload mimic (30).

[0510] Comparative Example 2-3: Synthesis of Linker-payload mimic (31)

[0511] Comparative Example 2-3-1: Synthesis of pyrene (32)

[0512] Fmoc-Val-Cit-PAB-PNP (CAS No: 863971-53-2, 121.2 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (5 mL), and a known (described in WO2018218004A1) sarcosine-pyrene (59.2 mg, 0.196 mmol), N,N-diisopropylethylamine (39 μL, 0.227 mmol), and 4-dimethylaminopyridine (3.7 mg, 0.03 mmol) were added. The mixture was stirred at room temperature for 2 hours, and then diethylamine (2 mL, 18.95 mmol) was added and the mixture was stirred at room temperature for 1.5 hours. The mixture was concentrated under reduced pressure and then purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the pyrene (32) (73.7 mg, 0.104 mmol).

[0513] １ H NMR (400MHz, DMSO-d6) δ10.21 (s, 1H), 8.68 (s, 1H), 8.43-7.93 (m, 12H), 7.60 (t, J = 7.1Hz, 2H), 7.31 (m, 2H), 6.04 (s, 1H), 5.48 (s, 2H), 5.02 (d, J = 16.1Hz, 4H), 4.5 4 (s, 1H), 3.96 (s, 2H), 3.66 (s, 2H), 2.92 (d, J = 6.1Hz, 3H), 2.08 (q, J = 6.6Hz, 1H) , 1.80-1.56 (m, 2H), 1.46 (s, 2H), 1.21-1.13 (m, 1H), 0.94 (dt, J=6.8, 3.0Hz, 6H).

[0514] MS (ESI) m / z: 708.80 [M+H] ＋

[0515] Comparative Example 2-3-2: Synthesis of pyrene (33)

[0516] Fmoc-Glu(OtBu)-OH・H ２ O (11.1 mg, 0.025 mmol) was dissolved in dimethylformamide (1 mL), pyrene (32) (17.3 mg, 0.024 mmol), 1-hydroxy-7-azabenzotriazole (5.1 mg, 0.037 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.3 mg, 0.038 mmol), and triethylamine (7.1 μL, 0.51 mmol) were added, and the mixture was stirred at room temperature for 2.5 hours. After that, diethylamine (0.2 mL, 1.91 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain pyrene (33) (15.6 mg, 0.017 mmol).

[0517] １ H NMR (400MHz, Chloroform-d) δ10.87 (s, 1H), 8.68 (m, 1H), 8.41-7.97 (m, 13H), 7.60-7.58 (m, 2H) , 7.30 (m, 2H), 6.00 (t, J = 6.2Hz, 1H), 5.45 (s, 2H), 5.05-4.99 (m, 4H), 4.46 (m, 1H), 4.26 (m, 1H), 3 .. 96 (s, 2H), 3.88 (m, 1H), 3.07 (m, 1H), 2.96 (m, 1H), 2.93 (d, J = 5.6Hz, 3H), 2.68 (t, J = 1.8Hz, 1H), 2.34-2.30 (m, 3H), 2.03 (m, 1H), 1.93-1.90 (m, 2H), 1.30 (s, 9H), 1.15 (s, 1H), 0.92-0.86 (m, 6H).

[0518] MS (ESI) m / z: 893.45 [M+H] ＋

[0519] Comparative Example 2-3-3: Synthesis of pyrene (34)

[0520] Pyrene (33) (15.6 mg, 0.017 mmol) was dissolved in dimethylformamide (1.5 mL), 6-maleimidohexanoic acid (3.7 mg, 0.018 mmol), 1-hydroxy-7-azabenzotriazole (3.5 mg, 0.025 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.8 mg, 0.025 mmol), and triethylamine (4.8 μL, 0.34 mmol) were added, and the mixture was stirred at room temperature for 3 hours. 6-maleimidohexanoic acid (1.8 mg, 0.009 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 mg, 0.012 mmol), and triethylamine (2.4 μL, 0.17 mmol) were then added, and the mixture was stirred at room temperature for 1.5 hours. 4-Dimethylaminopyridine (0.5 mg, 0.004 mmol) was then added, and the mixture was further stirred for 2.5 hours. After concentration under reduced pressure, the mixture was purified by column chromatography (dichloromethane:methanol=9:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain the pyrene (34) (8.0 mg, 0.007 mmol).

[0521] １ H NMR (400MHz, DMSO-d6) δ10.02 (s, 1H), 8.65 (m, 1H), 8.40-7.96 (m, 9H), 7.69-7.53 (m, 3H), 7.28 (m, 2H), 7 .00-6.98 (m, 2H), 5.97 (m, 1H), 5.41 (s, 2H), 5.04-4.98 (m, 4H), 4.56 (t, 5.4Hz, 1H), 4.41-4, 27 (m, 2H), 4 , 21-4.15 (m, 2H), 3.95 (s, 2H), 2.91 (d, J = 5.6Hz, 3H), 2.22-2.15 (m, 2H), 2.10-2.07 (m, 2H), 1.69 (m, 1H) , 1.59 (m, 1H), 1.50-1.44 (m, 6H), 1.46 (d, J = 2.0Hz, 9H), 1.35-1.15 (m, 4H), 0.82 (dd, J = 14.8, 6.8Hz, 6H)

[0522] MS (ESI) m / z: 1086.60 [M+H] ＋

[0523] Comparative Example 2-3-4: Synthesis of Linker-payload mimic (31)

[0524] Pyrene (4) (9.7 mg, 0.0089 mmol) was dissolved in 1,4-dioxane (1 mL) and a hydrogen chloride / 1,4-dioxane solution (1 mL), and the mixture was stirred in an ice-water bath for 2 hours. Dimethylformamide (1 mL) was added, and the mixture was allowed to warm to room temperature and then concentrated under reduced pressure. The mixture was then purified by reverse-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain linker-payload mimic (31) (2.0 mg, 0.002 mmol).

[0525] １ H NMR (400MHz, DMSO-d6) δ10.03 (s, 1H), 8.67 (m, 1H), 8.40-7.96 (m, 9H), 7.69-7.57 (m, 3H), 7.2 8 (m, 2H), 6.98 (s, 2H), 5.98 (m, 1H), 5.87 (m, 1H), 5.42 (s, 2H), 5.05-4.97 (m, 4H), 4.4 (m, 1H), 4,29 (m, 1H), 4.19 (m, 1H), 3.95 (s, 2H), 2.90 (d, J = 5.2Hz, 3H), 2.22-2.17 (m, 2H), 2.11-2.09 ( m, 2H), 1.71-1.59 (m, 3H), 1.49-1.44 (m, 6H), 1.30-1.15 (m, 4H), 0.82 (dd, J = 15.0, 6.6Hz, 6H)

[0526] MS (ESI) m / z: 1030.50 [M+H] ＋

[0527] Comparative Example 2-4: Synthesis of ADC mimics 18 to 20 According to Example 7-1, linker-payload mimic (31) was reacted to obtain ADC mimic 18 from trastuzumab, ADC mimic 19 from rituximab, and ADC mimic 20 from infliximab.

[0528] Example 8: DAR analysis of ADC mimics DAR analysis of the ADC mimics synthesized in Example 7 and Comparative Example 2 was performed by HIC-HPLC analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). The measurement was performed under the following conditions.

[0529] Measurement system: Chromaster (registered trademark) (manufactured by Hitachi) Column: Tosoh Biobutyl NPR 2.5 μm 4.6 × 35 mm column manufactured by Tosoh Biosciences Gradient: Linear gradient of eluent A / B Flow rate: 0.8 mL / min Eluent A: 1.1 M (NH ４ ) ２ SO ４ , 25 mM Na ２ HPO ４ / NaH ２ P.O. ４ (pH 6.0) Eluent B: 25mM Na ２ HPO ４ / NaH ２ P.O. ４ (pH 6.0, 25 v / v% isopropanol added) Detector: UV (280 nm)

[0530]

[0531] Example 9: Evaluation of hydrophobicity of ADC mimic by hydrophobic column chromatography (HIC-HPLC) HIC-HPLC analysis was performed using the conditions of Example 8. The measurement was carried out under the following conditions. The hydrophobicity of the ADC mimic can be evaluated based on the retention time of the ADC in the HIC chromatogram.

[0532]

[0533] Compared with the linear ADCs ADC mimic 15 and ADC mimic 18, the exo-ADCs ADC mimics 6, 7, and 8 have earlier retention times in the HIC chromatogram, indicating that they are more hydrophilic ADC mimics.

[0534]

[0535] Compared with the linear ADCs ADC mimic 16 and ADC mimic 19, the exo-type ADCs ADC mimics 9, 10, and 11 have earlier retention times in the HIC chromatogram, indicating that they are more hydrophilic ADC mimics.

[0536]

[0537] Compared with the linear ADCs ADC mimic 17 and ADC mimic 20, the exo-ADCs ADC mimics 12, 13, and 14 have earlier retention times in the HIC chromatogram, indicating that they are more hydrophilic ADC mimics.

[0538] As a result, it was confirmed that the ADC mimics synthesized in Examples 7-1, 7-2, 7-3, 7-4, 7-5, 7-6, 7-7, 7-8, and 7-9 tended to have short retention times, indicating high hydrophilicity. Therefore, it was confirmed that the ADC mimics synthesized in Examples 7-1, 7-2, 7-3, 7-4, 7-5, 7-6, 7-7, 7-8, and 7-9 are preferable ADCs because they are thought to have slow plasma clearance and long retention times in the body.

[0539] Example 10: Evaluation of aggregation rate of ADC and ADC mimic by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to a previous report (Chemistry Select, 2020, 5, 8435-8439). The measurement was performed under the following conditions.

[0540] Measurement system: 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300 Å 2.7 μm, 4.6 mm × 150 mm Flow rate: 0.25 mL / min Eluent: 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride aqueous solution (pH 6.8), 10% v / v isopropanol Detector: UV (280 nm)

[0541]

[0542]

[0543]

[0544] As a result, it was confirmed that the ADC mimics synthesized in Examples 7-1, 7-2, 7-3, 7-4, 7-5, 7-6, 7-7, 7-8, and 7-9 tended to have a low aggregation rate and were more stable. Therefore, it was confirmed that the ADC mimics synthesized in Examples 7-1, 7-2, 7-3, 7-4, 7-5, 7-6, 7-7, 7-8, and 7-9 were preferable ADCs.

[0545] Example 11: Evaluation of ADC mimics using the enzyme cathepsin B The cleavage ability of various ADC mimics with cathepsin B was evaluated by analyzing the amount of fluorescent molecules cleaved from the ADC mimics as described below.

[0546] Example 11-1: Cathepsin B cleavage test This test was performed as described previously (Nature Communications 2018, 9, 2512) as follows. ADC mimic 7 was added to 180 μL of MES buffer (10 mM MES, 40 μM DTT, pH 5.0) to a concentration of 1 mg / mL, and then 30 μL of the mixture was dispensed into six Eppendorf tubes. Three of the six samples were immediately added with 100 μL of acetonitrile at 0°C, vortexed, and then centrifuged to obtain a precipitate. The resulting supernatant was collected and analyzed by HPLC. The remaining three samples were incubated at 37°C for 6 hours. A 100 μL of acetonitrile was added to each sample, vortexed, and then centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0547] Example 11-2: Analysis of the amount of detached fluorescent molecules using HPLC analysis The amount of fluorescent molecules detached from ADC mimic 7 was measured using liquid chromatography / fluorescence detection. Three samples to which acetonitrile was immediately added at 0°C in Example 11-1 were designated as time 0, and three samples incubated at 37°C for 6 hours as described in Example 11-1 were designated as time 6, and the difference in fluorescence intensity between the 6-hour samples and the 0-hour samples was analyzed.

[0548] Separately, Pyrene was used to calculate the correlation between the area of ​​fluorescence intensity measured by HPLC and the concentration. Using this calculation formula, the difference in fluorescence intensity for each ADC mimic was converted to concentration. The percentage of the difference in fluorescence intensity was calculated as the elimination rate, assuming the concentration at time 0 as 100%. The elimination rate of fluorescent molecules from ADC mimic 7 at 6 hours was 400%, indicating that ADC mimic 7 had sufficient cathepsin B cleavage.

[0549] Example 12: Synthesis of linker-payload (12-1) Synthesis of linker-payload (35) Linker-payload (35) was synthesized as follows.

[0550] (12-1-1) Synthesis of carbonate (36)

[0551] The alcohol (2) (105 mg, 0.158 mmol) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (2 mL) and stirred for 5 minutes under ice-cooling. Then, bis(4-nitrophenyl)carbonate (100 mg, 0.329 mmol) and N,N-diisopropylethylamine (83 μL, 0.48 mmol) were added, and the mixture was stirred for 19.5 hours at room temperature under a nitrogen atmosphere. After removing the N,N-dimethylformamide using an evaporator, the obtained crude product was dissolved in ethyl acetate (3 mL), and then diethyl ether (3 mL) was added. The resulting solution was filtered to remove any residue, and the organic solvent was removed using a vacuum pump to obtain carbonate (36) (102 mg, 0.123 mmol).

[0552] MS (ESI) m / z: 830.1 [M+H] ＋ ,852.1[M+Na] ＋

[0553] (12-1-2) Synthesis of compound (37)

[0554] Compound (36) (69 mg, 0.083 mmol) obtained in (12-1-1) was dissolved in N,N-dimethylformamide (3.5 mL), and 1-hydroxybenzotriazole (16 mg, 0.10 mmol) and commercially available monomethylauristatin E (MMAE, 61 mg, 0.085 mmol) were added at room temperature. Subsequently, diisopropylethylamine (29 μL, 0.17 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 22.5 hours. After removing the organic solvent using an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 37 (79 mg, 0.056 mmol).

[0555] MS (ESI) m / z: 1408.9 [M+H] ＋

[0556] (12-1-3) Synthesis of compound (38)

[0557] Compound (37) (97 mg, 0.069 mmol) obtained in (12-1-2) was dissolved in tetrahydrofuran (7 mL) and water (2 mL), and lithium hydroxide (1.0 M, 1.4 mL, 1.4 mmol) was added under ice-cooling, followed by stirring for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 6, and then acetonitrile:water = 1:1 was added, followed by purification by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 38 (70 mg, 0.050 mmol).

[0558] MS (ESI) m / z: 1394.7 [M+H] ＋

[0559] (12-1-4) Synthesis of compound (39)

[0560] Compound (38) (34 mg, 0.024 mmol) obtained in (12-1-3) was dissolved in N,N-dimethylformamide (3 mL) and then cooled on ice. N,N-diisopropylethylamine (25 μL, 0.14 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (20 mg, 0.038 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (8.7 mg, 0.040 mmol) was added, and the mixture was returned to room temperature and stirred for 20 hours. After completion of the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain compound (39) (29 mg, 0.019 mmol).

[0561] MS (ESI) m / z: 1558.9 [M+H] ＋

[0562] (12-1-5) Synthesis of Linker-payload (35)

[0563] Acetonitrile (500 μL) and 85 wt % aqueous phosphoric acid solution (0.50 mL, 7.3 mmol) were added sequentially to compound (39) (29 mg, 0.019 mmol) obtained in (12-1-4), and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, water (2 mL) was added, and the reaction mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain Linker-payload (35) (18 mg, 0.012 mmol).

[0564] MS (ESI) m / z: 1502.9 [M+H] ＋

[0565] (12-2) Synthesis of Linker-payload (40) Linker-payload (40) was synthesized as follows.

[0566] (12-2-1) Synthesis of compound (41)

[0567] Compound (38) (10 mg, 0.0072 mmol) obtained in (12-1-3) was dissolved in N,N-dimethylformamide (1 mL) and then cooled on ice. N,N-diisopropylethylamine (10 μL, 0.057 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (7.0 mg, 0.013 mmol) were added. Next, a solution of DBCO-hexylamine (4.7 mg, 0.015 mmol) in N,N-dimethylformamide (0.5 mL) was added, and the mixture was returned to room temperature and stirred for 20 hours. After completion of the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain compound (41) (5.8 mg, 0.0034 mmol).

[0568] MS (ESI) m / z: 1696.0 [M+H] ＋

[0569] (12-2-2) Synthesis of Linker-payload (40)

[0570] Acetonitrile (500 μL) and 85 wt % aqueous phosphoric acid solution (0.50 mL, 7.3 mmol) were added sequentially to compound (41) (13 mg, 0.0077 mmol) obtained in (12-2-1), and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, water (2 mL) was added, and the reaction solution was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain Linker-payload (40) (7.4 mg, 0.0045 mmol).

[0571] MS (ESI) m / z: 1638.9 [M+H] ＋

[0572] (12-3) Synthesis of Linker-payload (42) Linker-payload (42) was synthesized as follows.

[0573] (12-3-1) Synthesis of carbonate (43)

[0574] The alcohol (2) (140 mg, 0.165 mmol) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (4 mL) and stirred for 5 minutes under ice-cooling. Then, bis(4-nitrophenyl)carbonate (108 mg, 0.355 mmol) and N,N-diisopropylethylamine (100 μL, 0.574 mmol) were added, and the mixture was stirred for 18 hours at room temperature under a nitrogen atmosphere. After removing the organic solvent using an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and the mixture was lyophilized to obtain compound 43 (130 mg, 0.128 mmol).

[0575] MS (ESI) m / z: 1015.6 [M+H] ＋

[0576] (12-3-2) Synthesis of compound (44)

[0577] Compound (43) (85 mg, 0.084 mmol) obtained in (12-3-1) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (20 mg, 0.13 mmol) and commercially available monomethylauristatin E (MMAE, 63 mg, 0.088 mmol) were added at room temperature. Subsequently, diisopropylethylamine (75 μL, 0.43 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 23 hours. After removing the organic solvent using an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 44 (94 mg, 0.059 mmol).

[0578] MS (ESI) m / z: 1593.6 [M+H] ＋

[0579] (12-3-3) Synthesis of compound (45)

[0580] Compound (44) (94 mg, 0.059 mmol) obtained in (12-3-2) was dissolved in tetrahydrofuran (5 mL) and water (2 mL), and lithium hydroxide (1.0 M, 0.6 mL, 0.6 mmol) was added under ice-cooling, followed by stirring for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 5, and then acetonitrile:water = 1:1 was added, followed by purification by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 45 (77 mg, 0.049 mmol).

[0581] MS (ESI) m / z: 1579.7 [M+H] ＋

[0582] (12-3-4) Synthesis of compound (46)

[0583] Compound (45) (77 mg, 0.049 mmol) obtained in (12-3-3) was dissolved in N,N-dimethylformamide (3 mL) and then cooled on ice. N,N-diisopropylethylamine (50 μL, 0.29 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (87 mg, 0.17 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (35 mg, 0.16 mmol) was added, and the mixture was returned to room temperature and stirred for 20 hours. After completion of the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain compound (46) (65 mg, 0.037 mmol).

[0584] MS (ESI) m / z: 1744.7 [M+H] ＋

[0585] (12-3-5) Synthesis of Linker-payload (42)

[0586] Acetonitrile (2 mL) and 85 wt% aqueous phosphoric acid solution (1.00 mL, 14.6 mmol) were added sequentially to compound (46) (65 mg, 0.037 mmol) obtained in (12-3-4), and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, water (2 mL) was added, and the reaction solution was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain linker-payload (42) (49 mg, 0.030 mmol).

[0587] MS (ESI) m / z: 1631.6 [M+H] ＋

[0588] (12-4) Synthesis of Linker-payload (47) Linker-payload (47) was synthesized as follows.

[0589] (12-4-1) Synthesis of compound (48)

[0590] Compound (43) (67 mg, 0.066 mmol) obtained in (12-3-1) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (17 mg, 0.11 mmol) and commercially available Exatecan mesylate (CAS: 169869-90-3, 35 mg, 0.066 mmol) were added at room temperature. Subsequently, diisopropylethylamine (50 μL, 0.29 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. After removing the organic solvent using an evaporator, a solution of acetonitrile:water = 1:1 was added, and the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound 48 (57 mg, 0.043 mmol).

[0591] MS (ESI) m / z: 1311.7 [M+H] ＋

[0592] (12-4-2) Synthesis of compound (49)

[0593] Compound (48) (57 mg, 0.043 mmol) obtained in (12-4-1) was dissolved in tetrahydrofuran (3 mL) and water (1.5 mL), and lithium hydroxide (1.0 M, 0.5 mL, 0.5 mmol) was added under ice-cooling, followed by stirring for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 5, and then acetonitrile:water = 1:1 was added, followed by purification by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain compound 49 (45 mg, 0.035 mmol).

[0594] MS (ESI) m / z: 1297.6 [M+H] ＋

[0595] (12-4-3) Synthesis of compound (50)

[0596] Compound (49) (45 mg, 0.035 mmol) obtained in (12-4-2) was dissolved in N,N-dimethylformamide (3 mL) and then cooled on ice. N,N-diisopropylethylamine (30 μL, 0.17 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (55 mg, 0.11 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (22 mg, 0.10 mmol) was added, and the mixture was returned to room temperature and stirred for 18 hours. After completion of the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain compound (50) (22 mg, 0.015 mmol).

[0597] MS (ESI) m / z: 1462.7 [M+H] ＋

[0598] (12-3-5) Synthesis of Linker-payload (47)

[0599] Acetonitrile (1 mL) and 85 wt% aqueous phosphoric acid solution (1.0 mL, 14.6 mmol) were added sequentially to compound (50) (22 mg, 0.015 mmol) obtained in (12-4-3), and the mixture was stirred at room temperature for 1.5 hours. After completion of the reaction, water (1 mL) was added, and the reaction solution was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain linker-payload (47) (18.8 mg, 0.0139 mmol).

[0600] １ H NMR (300MHz; DMSO-d6) δ 10.04 (s, 1H), 8.10-8.05 (m, 4H), 7.80-7.77 (m, 2H), 7.59-7.55 (m, 2H), 7.35-7.30 (m, 3H), 6 99 (d, J=8.8Hz, 2H), 6.54-6.53 (m, 1H), 6.01 (brs, 1H), 5.74 (d, J=9.0Hz, 1H), 5.43 (brs, 4H) , 5.33-5.28 (m, 2H), 4.29-4.15 (m, 4H), 3.19-2.98 (m5H), 2.43-2.38 (m, 5H), 2.27-2.20 (m, 7 H), 1.95-1.83 (m, 8H), 1.73-1.62 (m, 4H), 1.42-1.30 (m, 7H), 1.23-1.13 (m, 3H), 0.84 (m, 9H).

[0601] MS (ESI) m / z: 1349.2 [M+H] ＋

[0602] (12-5) Synthesis of Linker-payload (125) The linker-payload (125) shown below was synthesized in the same manner as in the synthesis of linker-payload mimic (120), except that MMAE was used instead of sarcosine-pyrene.

[0603] MS (ESI) m / z: 1968.14 [M+H] ＋

[0604] (12-6) Synthesis of Linker-payload (126) The linker-payload (126) shown below was synthesized in the same manner as in the synthesis of linker-payload mimic (35), except that Exatecan mesylate was used instead of MMAE.

[0605] MS (ESI) m / z: 1220.50 [M+H] ＋

[0606] Example 13: Synthesis of ADC (13-1) Synthesis of ADC1 To 3 mg / mL of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) in PBSE buffer (50 mM phosphate buffered saline (PBS), 10 mM EDTA, pH = 7.4), a PBSE solution of a reducing agent (TCEP) (2 mM) was added in an amount of 8 equivalents relative to the antibody, and the mixture was incubated at 37°C for 1 hour. Dimethylacetamide (DMA) (6% v / v) and a 10 mM DMA solution of various Linker-payload 35 (20 equivalents relative to the antibody) were sequentially added to the resulting reaction mixture, and the mixture was gently stirred at 20°C for 1 hour. An excess amount of N-acetylcysteine ​​was added to the reaction mixture, and the mixture was stirred at 25°C for 20 minutes. The final mixture was purified using a NAP-5 desalting column (GE Healthcare) and eluted with a formulation buffer (20 mM histidine, 5 wt % trehalose added, pH 5.2) to obtain ADC mimic 1.

[0607] (13-2) Synthesis of ADC2 ADC2 was obtained from Linker-payload (42) according to (13-1).

[0608] (13-3) Synthesis of ADC3 ADC3 was obtained from Linker-payload (47) according to (13-1).

[0609] (13-4) Synthesis of ADC9 ADC9 was obtained from Linker-payload (125) according to (13-1).

[0610] (13-5) Synthesis of ADC10 ADC10 was obtained from Linker-payload (126) according to Example 13-1.

[0611] Example 14: HIC-HPLC analysis of ADC HIC-HPLC analysis was carried out using the conditions of Example 8.

[0612] Subsequently, the hydrophobicity of the ADC was evaluated using HIC-HPLC. Measurements were performed according to Example 9. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram. Trastuzumab, the raw material antibody, was used for comparison.

[0613] The exo-ADCs ADC1, 2, and 3 have retention times in the HIC chromatogram comparable to those of the starting antibody, indicating that they are more hydrophilic ADCs.

[0614] Example 15: Evaluation of aggregation rate of ADC by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was carried out according to Example 10.

[0615] As a result, it was confirmed that the ADCs synthesized in Examples 13-1, 13-2, and 13-3 tended to have a low aggregation rate and were more stable. Therefore, it was confirmed that the ADC synthesized in Example 13 was a preferable ADC.

[0616] Example 16: Synthesis of ADC (16-1) Synthesis of ADC4 In the following comparative examples and examples, the antibody derivative (thiol group-introduced trastuzumab) described in Example 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used as the thiol group-introduced antibody. This antibody derivative has the following structure, in which a thiol group has been regioselectively introduced into trastuzumab (a humanized IgG1 antibody) via the amino group in the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the positions of the lysine residues are in accordance with EU numbering). (In the above structure, the NH-CH extending from the antibody heavy chain ２ -CH２ -CH ２ -CH ２ - corresponds to the side chain of a lysine residue, and the thiol-containing group HS-CH is connected to the amino group in the side chain of this lysine residue. ２ -CH ２ -C(=O) is added. Since no modifications at other lysine residues were detected by peptide mapping, it is believed that the regioselectivity at positions 246 and 248 of the antibody heavy chain is 100%.

[0617] To a buffer (pH 7.4 PBS buffer) solution (20 μM) of the thiol group-introduced antibody, 10 equivalents of a DMF solution (10 mM) of the linker-payload (35) synthesized in Example 13-1 was added, and the mixture was left to stand at room temperature for 2 hours. After this, the mixture was purified using NAP-5 Columns (GE Healthcare) to obtain ADC 4. ESI-TOFMS analysis confirmed that the reaction product had peaks at 151414, where two linker-payloads (35) had been introduced.

[0618] (16-2) Synthesis of ADC5 According to (16-1), ADC5 was obtained from Linker-payload (42). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151673 where two Linker-payloads (42) were introduced.

[0619] (16-3) Synthesis of ADC6 According to (16-1), ADC6 was obtained from Linker-payload (47). ESI-TOFMS analysis confirmed that the reaction product had a peak at 151109, where two Linker-payloads (47) were introduced.

[0620] (16-4) Synthesis of ADC6 According to (16-1), ADC8 was obtained from Linker-payload (125). ESI-TOFMS analysis confirmed that the reaction product had peaks at 150 and 524, where two Linker-payloads (125) were introduced.

[0621] (16-5) Synthesis of ADC11 According to (16-1), ADC11 was obtained from Linker-payload (126). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 150615 where two Linker-payloads (126) were introduced.

[0622] Example 17: HIC-HPLC analysis of ADC HIC-HPLC analysis was carried out using the conditions in Example 8.

[0623] Subsequently, the hydrophobicity of the ADC was evaluated using HIC-HPLC. Measurements were performed according to Example 9. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram. Trastuzumab, the raw material antibody, was used for comparison.

[0624] The exo-ADCs ADCs 4, 5, and 6 have retention times in the HIC chromatogram comparable to those of the starting antibody, indicating that they are more hydrophilic ADCs.

[0625] Example 18: Evaluation of aggregation rate of ADC by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was carried out according to Example 10.

[0626] As a result, it was confirmed that the ADCs synthesized in (16-1), (16-2), and (16-3) tended to have a low aggregation rate and were more stable. Therefore, it was confirmed that the ADC synthesized in Example 16 was a preferable ADC.

[0627] Example 19: Synthesis of linker-payload mimic (19-1) Synthesis of linker-payload mimic (56) Linker-payload mimic (56) was synthesized as follows.

[0628] (19-1-1) Synthesis of alcohol (57)

[0629] Ac-Asp(OtBu)-Val-Cit-OH (51.7 mg, 103 μmol) was dissolved in N,N-dimethylformamide (520 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (46.9 mg, 123 μmol) and 2,4,6-trimethylpyridine (15.9 μL, 123 μmol) were added. The mixture was stirred at room temperature for 10 minutes. Subsequently, methyl 4-aminomandelate (22.3 mg, 123 μmol) was added, and the mixture was stirred at room temperature for 19 hours. The mixture was then purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the alcohol (57) (56.3 mg, 86.5 μmol).

[0630] １ H NMR (400 MHz, DMSO-d ６ ) δ9.99 (s, 1H), 8.27 (d, J = 8.4Hz, 1H), 8.17 (d, J = 8.4Hz, 1H), 7.59 (d, J = 8.8Hz, 1H), 7.56 (d, J = 8.8Hz, 2H), 7. 31 (d, J=8.8Hz, 2H), 5.98 (brs, 1H), 5.41 (brs, 2H), 5.08 (s, 1H), 4.64-4.59 (m, 1H), 4.39-4.34 (m, 1H), 4.22-4 .. 18 (m, 1H), 3.59 (s, 3H), 3.01-2.94 (m, 2H), 2.69-2.63 (m, 1H), 2.44-2.38 (m, 1H), 2.00-1.95 (m, 1H), 1.83 (s, 3H), 1.69-1.66 (m, 1H), 1.62-1.53 ​​(m, 1H), 1.43-1.34 (m, 11H), 0.84 (d, J = 6.8Hz, 3H), 0.79 (d, J = 6.8Hz, 3H).

[0631] MS (ESI) m / z: 651.35 [M+H] ＋

[0632] (19-1-2) Synthesis of pyrene (58)

[0633] Alcohol (57) (55.0 mg, 84.5 μmol) was dissolved in N,N-dimethylformamide (423 μL) and stirred for 5 minutes under ice cooling. Then, bis(4-nitrophenyl)carbonate (77.1 mg, 254 μmol) and N,N-diisopropylethylamine (32.3 μL, 190 μmol) were added and the mixture was stirred at room temperature for 2 hours. The mixture was then ice-cooled, and sarcosin-pyrene (76.7 mg, 254 μmol), 1-hydroxybenzotriazole (17.1 mg, 127 μmol), and N,N-diisopropylethylamine (53.9 μL, 317 μmol) were added, followed by stirring at room temperature for 1.5 hours. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (58) (60.9 mg, 62.2 μmol).

[0634] １ H NMR (400 MHz, DMSO-d ６ ) δ10.14-10.11 (m, 1H), 8.68-8.66 (m, 1H), 8.39-8.01 (m, 10H), 7.89-7.85 (m, 1H), 7.67-7.58 (m, 3H), 7.44- 7.39 (m, 2H), 5.98 (brs, 1H), 5.79-5.77 (m, 1H), 5.42 (brs, 2H), 5.04-4.97 (m, 2H), 4.65-4.59 (m, 1H), 4.39-4 .. 36 (m, 1H), 4.28-4.07 (m, 2H), 3.92-3.82 (m, 1H), 3.62-3.61 (m, 3H), 2.99-2.91 (m, 5H), 2.69-2.63 (m, 1H), 2 .44-2.38 (m, 1H), 2.01-1.97 (m, 1H), 1.83 (s, 3H), 1.70-1.60 (m, 2H), 1.44-1.30 (m, 11H), 0.86-0.77 (m, 6H).

[0635] MS (ESI) m / z: 979.45 [M+H] ＋

[0636] (19-1-3) Synthesis of pyrene (59)

[0637] Pyrene (58) (24.9 mg, 25.4 μmol) was dissolved in tetrahydrofuran (1.88 mL) and water (625 μL) and stirred for 5 minutes under ice cooling. Then, 1 M aqueous lithium hydroxide solution (30.5 μL, 30.5 μmol) was added and stirred at room temperature for 50 minutes. After the reaction was completed, the pH was adjusted to approximately 6 using 0.1 M hydrochloric acid and purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (59) (8.3 mg, 8.6 μmol).

[0638] １ H NMR (400 MHz, DMSO-d ６ ) δ13.06 (brs, 1H), 10.13-10.09 (m, 1H), 8.68-8.65 (m, 1H), 8.39-8.00 (m, 10H), 7.88-7.84 (m, 1H), 7.67-7. 59 (m, 3H), 7.44-7.41 (m, 2H), 5.98 (brs, 1H), 5.68-5.67 (m, 1H), 5.42 (brs, 2H), 5.04-4.93 (m, 2H), 4.64-4. 61 (m, 1H), 4.41-4.35 (m, 1H), 4.32-4.09 (m, 2H), 3.91-3.80 (m, 1H), 2.99-2.91 (m, 5H), 2.70-2.64 (m, 1H), 2 .44-2.38 (m, 1H), 2.01-1.98 (m, 1H), 1.83 (s, 3H), 1.70-1.57 (m, 2H), 1.45-1.30 (m, 11H), 0.86-0.77 (m, 6H).

[0639] MS (ESI) m / z: 965.45 [M+H] ＋

[0640] (19-1-4) Synthesis of pyrene (60)

[0641] Pyrene (59) (7.2 mg, 7.5 μmol) was dissolved in N,N-dimethylformamide (150 μL) and then cooled on ice. N,N-diisopropylethylamine (2.5 μL, 14.9 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (5.8 mg, 11.2 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (2.4 mg, 11.2 μmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain pyrene (60) (5.7 mg, 5.1 μmol).

[0642] １ H NMR (400 MHz, DMSO-d ６ ) δ10.07-10.04 (m, 1H), 8.91-8.88 (m, 1H), 8.65-7.95 (m, 12H), 7.62-7.58 (m, 3H), 7.40-7.36 (m, 2H), 6.96-6.94 (m, 2H), 5.97 (brs, 1H), 5.70-5.68 (m, 1H), 5.41 (brs, 2H), 5.14-5.00 (m, 2H), 4.65-4.61 (m, 1H), 4.42-4.37 (m, 1H) ), 4.25-4.19 (m, 1H), 4.15-3.82 (m, 2H), 3.30-3.21 (m, 2H), 3.04-2.89 (m, 7H), 2.70-2.64 (m, 1H), 2.44-2.38 (m, 1 H), 2.01-1.97 (m, 1H), 1.83 (s, 3H), 1.75-1.60 (m, 2H), 1.48-1.24 (m, 15H), 1.12-1.03 (m, 2H), 0.86-0.78 (m, 6H).

[0643] MS (ESI) m / z: 1129.50 [M+H] ＋

[0644] (19-1-5) Synthesis of Linker-payload mimic (56)

[0645] Acetonitrile (208 μL) was added to pyrene (60) (4.7 mg, 4.2 μmol), and 85% phosphoric acid solution (72.4 μL, 1.25 mmol) was added under ice cooling, followed by stirring at room temperature for 3 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain linker-payload mimic (56) (3.1 mg, 2.9 μmol).

[0646] １ H NMR (400 MHz, DMSO-d ６ ) δ12.38 (brs, 1H), 10.05-10.01 (m, 1H), 8.91-8.88 (m, 1H), 8.65-7.94 (m, 12H), 7.63-7.58 (m, 3H), 7.40-7.34 (m, 2H), 6.96-6.93 (m, 2H), 6.00 (brs, 1H), 5.70-5.68 (m, 1H), 5.44 (brs, 2H), 5.15-4.99 (m, 2H), 4.64-4.60 (m, 1H), 4 .. 43-4.37 (m, 1H), 4.25-4.22 (m, 1H), 4.15-3.82 (m, 2H), 3.30-3.21 (m, 2H), 3.04-2.89 (m, 7H), 2.73-2.69 (m, 1H), 2 .46-2.44 (m, 1H), 2.04-1.96 (m, 1H), 1.84-1.83 (m, 3H), 1.77-1.59 (m, 2H), 1.44-1.04 (m, 8H), 0.86-0.77 (m, 6H).

[0647] MS (ESI) m / z: 1073.50 [M+H] ＋

[0648] (19-3) Synthesis of Linker-payload mimic (66) Linker-payload mimic (1) was synthesized as follows.

[0649] (19-3-1) Synthesis of alcohol (67)

[0650] Ac-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Val-Cit-OH (50.2 mg, 47.3 μmol) was dissolved in N,N-dimethylformamide (237 μL), 1-[bis(dimethylamino)methylene]-1H-1,2,3,triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (21.6 mg, 56.8 μmol) and 2,4,6-trimethylpyridine (7.48 μL, 56.8 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, methyl 4-aminomandelate (10.3 mg, 56.8 μmol) was added, and the mixture was stirred at room temperature for 20 hours, followed by purification by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to give the above alcohol (67) (47.7 mg, 39.1 μmol).

[0651] MS (ESI) m / z: 1220.65 [M+H] ＋

[0652] (19-3-2) Synthesis of pyrene (68)

[0653] The alcohol (67) (46.3 mg, 37.9 μmol) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (380 μL) and stirred for 5 minutes under ice-cooling. Then, bis(4-nitrophenyl)carbonate (34.6 mg, 114 μmol) and N,N-diisopropylethylamine (14.5 μL, 85.3 μmol) were added and the mixture was stirred at room temperature for 2 hours. The mixture was then ice-cooled, and sarcosin-pyrene (34.5 mg, 114 μmol), 1-hydroxybenzotriazole (7.7 mg, 56.9 μmol), and N,N-diisopropylethylamine (24.2 μL, 142 μmol) were added, followed by stirring at room temperature for 2 hours. After the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (68) (37.1 mg, 24.0 μmol).

[0654] MS (ESI) m / z: 775.30 [M+H] ＋

[0655] (19-3-3) Synthesis of pyrene (69)

[0656] Pyrene (68) (20.3 mg, 13.1 μmol) was dissolved in tetrahydrofuran (983 μL) and water (327 μL) and stirred for 5 minutes under ice cooling. Then, 1 M aqueous lithium hydroxide solution (31.4 μL, 31.4 μmol) was added and stirred at room temperature for 2.5 hours. After the reaction was completed, the pH was adjusted to approximately 6 using 1 M hydrochloric acid and purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the pyrene (69) (16.7 mg, 10.9 μmol).

[0657] MS (ESI) m / z: 1534.85 [M+H] ＋

[0658] (19-3-4) Synthesis of pyrene (70)

[0659] Pyrene (69) (14.9 mg, 9.71 μmol) was dissolved in N,N-dimethylformamide (486 μL) and then cooled on ice. N,N-diisopropylethylamine (3.3 μL, 19.4 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (7.6 mg, 14.6 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (3.2 mg, 14.6 μmol) was added, and the mixture was returned to room temperature and stirred for 1 hour. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain pyrene (70) (13.1 mg, 7.71 μmol).

[0660] MS (ESI) m / z: 1698.90 [M+H] ＋

[0661] (19-3-5) Synthesis of Linker-payload mimic (66)

[0662] Acetonitrile (309 μL) was added to pyrene (70) (5.25 mg, 3.09 μmol), and 85% phosphoric acid solution (53.8 μL, 927 μmol) was added under ice-cooling, followed by stirring at room temperature for 25 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain linker-payload mimic (66) (3.7 mg, 2.51 μmol).

[0663] MS (ESI) m / z: 1474.00 [M+H] ＋

[0664] (19-3) Synthesis of Linker-payload mimic (11) Linker-payload mimic (11) was also synthesized via a route different from that of (1-3) as follows.

[0665] (19-3-1) Synthesis of alcohol (96)

[0666] The alcohol (12) (80.0 mg, 94.1 μmol) obtained in Example (1-3-1) was dissolved in tetrahydrofuran (7.0 mL) and water (2.35 mL) and stirred for 5 minutes under ice-cooling. Then, a 1 M aqueous solution of lithium hydroxide (226 μL, 226 μmol) was added and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to about 6 using 1 M hydrochloric acid and purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain the above alcohol (96) (61.5 mg, 73.6 μmol).

[0667] MS (ESI) m / z: 836.40 [M+H] ＋

[0668] (19-3-2) Synthesis of alcohol (97)

[0669] Alcohol (96) (60.5 mg, 72.4 μmol) was dissolved in N,N-dimethylformamide (3.6 mL) and then cooled on ice. N,N-diisopropylethylamine (25 μL, 145 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (56.5 mg, 109 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (23.7 mg, 109 μmol) was added, and the mixture was returned to room temperature and stirred for 3 hours. After completion of the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain alcohol (97) (68.0 mg, 72.4 μmol).

[0670] MS (ESI) m / z: 1000.50 [M+H] ＋

[0671] (19-3-3) Synthesis of compound (98)

[0672] Alcohol (97) (10.0 mg, 10.0 μmol) was dissolved in N,N-dimethylformamide (0.1 mL) and then cooled on ice. Bis(4-nitrophenyl)carbonate (30.4 mg, 100 μmol) and N,N-diisopropylethylamine (3.8 μL, 22.5 μmol) were added, and the mixture was stirred at room temperature for 3 hours. After the reaction, the mixture was purified by normal-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above compound (98) (5.6 mg, 4.8 μmol).

[0673] MS (ESI) m / z: 1163.50 [M+H] ＋

[0674] (19-3-4) Synthesis of pyrene (15)

[0675] Compound (98) (10.0 mg, 8.6 μmol) was dissolved in N,N-dimethylformamide (86 μL) and then cooled on ice. Sarcosin-pyrene (2.2 mg, 7.2 μmol), 1-hydroxybenzotriazole (1.5 mg, 11 μmol), and N,N-diisopropylethylamine (1.8 μL, 11 μmol) were added, and the mixture was stirred at room temperature for 4 hours. After the reaction, the mixture was purified by normal-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the pyrene (15) (3.0 mg, 2.3 μmol).

[0676] MS (ESI) m / z: 1328.60 [M+H] ＋

[0677] (19-3-5) Synthesis of Linker-payload mimic (11) Linker-payload mimic (11) was synthesized in the same manner as in Example (1-3-5).

[0678] (19-4) Synthesis of Linker-payload mimic (120) Linker-payload mimic (120) was synthesized as follows.

[0679] (19-4-1) Synthesis of compound (122)

[0680] 21-[(tert-butoxycarbonyl)amino]-4,7,10,13,16,19-hexaoxaheneicosanoic acid (121) (70.0 mg, 154 μmol) was dissolved in N,N-dimethylformamide (7.72 mL) and then cooled on ice. N,N-diisopropylethylamine (52.0 μL, 309 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (120 mg, 232 μmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (50.6 mg, 232 μmol) was added, and the mixture was returned to room temperature and stirred for 4 hours. After completion of the reaction, the mixture was purified by reverse-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain compound (122) (83.4 mg, 135 μmol).

[0681] MS (ESI) m / z: 618.50 [M+H] ＋

[0682] (19-4-2) Synthesis of compound (123)

[0683] Compound (122) (82.0 mg, 133 μmol) was dissolved in dichloromethane (13.3 mL) and trifluoroacetic acid (6.64 mL) and stirred at room temperature for 30 minutes. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by concentration under reduced pressure to obtain the compound (123) (71.8 mg, quant).

[0684] MS (ESI) m / z: 518.40 [M+H] ＋

[0685] (19-4-3) Synthesis of pyrene (124)

[0686] Pyrene (14) (16.0 mg, 14.0 μmol) was dissolved in N,N-dimethylformamide (690 μL) and then cooled on ice. N,N-diisopropylethylamine (9.3 μL, 55.0 μmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (11 mg, 21.0 μmol) were added. PEG6 (11.0 mg, 21.0 μmol) was then added, the mixture was returned to room temperature, and stirred for 2.5 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain pyrene (124) (11.2 mg, 6.73 μmol).

[0687] MS (ESI) m / z: 1664.80 [M+H] ＋

[0688] (19-4-4) Synthesis of Linker-payload mimic (120)

[0689] Acetonitrile (200 μL) was added to pyrene (124) (5.0 mg, 3.0 μmol), and 85% phosphoric acid solution (60.0 μL, 880 μmol) was added under ice cooling, followed by stirring at room temperature for 25 hours. After completion of the reaction, the product was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain Linker-payload mimic (120) (2.4 mg, 1.5 μmol).

[0690] MS (ESI) m / z: 1552.65 [M+H] ＋

[0691] Example 20: Synthesis of ADC mimic (20-1) Synthesis of ADC mimic In the following examples, ADC mimics were prepared in the same manner as in Example 2. ADC mimic 22 having the following structure was synthesized from linker-payload mimic (56) (19-1) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 150322, where two linker-payload mimics (56) had been introduced.

[0692] Similarly, ADC mimic 24 with the following structure was synthesized from linker-payload mimic (66) (19-2) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had peaks at 151125, where two linker-payload mimics (66) had been introduced.

[0693] Similarly, ADC mimic 33 with the following structure was synthesized from linker-payload mimic (120) (19-4) and a thiol-containing antibody. ESI-TOFMS analysis confirmed that the reaction product had a peak at 151279, where two linker-payload mimics (120) had been introduced.

[0694] (20-2) DAR analysis of ADC mimic ESI-TOFMS analysis of the ADC mimic synthesized in Example 20-1 was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2.

[0695] Example 21: Evaluation of hydrophobicity of ADC and ADC mimic by hydrophobic column chromatography (HIC-HPLC) HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). Measurement was performed under the following conditions. The hydrophobicity of the ADC can be evaluated based on the retention time of the ADC in the HIC chromatogram.

[0696] Measurement system: Chromaster (registered trademark) (manufactured by Hitachi) Column: Tosoh Biobutyl NPR 2.5 μm 4.6 × 35 mm column manufactured by Tosoh Biosciences Gradient: Linear gradient of eluent A / B Flow rate: 0.8 mL / min Eluent A: 1.1 M (NH ４ ) ２ SO ４ , 25 mM Na ２ HPO ４ / NaH ２ P.O. ４(pH 6.0) Eluent B: 25mM Na ２ HPO ４ / NaH ２ P.O. ４ (pH 6.0, 25 v / v% isopropanol added) Detector: UV (280 nm)

[0697] As a result, it was confirmed that the ADC mimics synthesized in Examples 19-1, 19-2, and 19-4 tended to have a short retention time, indicating high hydrophilicity. Therefore, the ADC mimics synthesized in Examples 19-1, 19-2, and 19-4 were confirmed to be preferable ADCs because they are thought to have slow plasma clearance and long retention times in the body.

[0698] Example 22: Evaluation of aggregation rate of ADC and ADC mimic by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was performed according to a previous report (Chemistry Select, 2020, 5, 8435-8439). The measurement was performed under the following conditions.

[0699] Measurement system: 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300 Å 2.7 μm, 4.6 mm × 150 mm Flow rate: 0.25 mL / min Eluent: 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride aqueous solution (pH 6.8), 10% v / v isopropanol Detector: UV (280 nm)

[0700] Example 23: Evaluation of ADC mimics using the enzyme cathepsin B The cleavage ability of various ADC mimics with cathepsin B was evaluated by analyzing the amount of fluorescent molecules cleaved from the ADC mimic as described below.

[0701] (23-1) Cathepsin B Cleavage Test The test was carried out in the same manner as in Example 5.

[0702] (23-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis. Analysis was carried out in the same manner as in Example 5.

[0703] As shown in Table 22, the synthesized ADC mimics were found to have sufficient cathepsin B cleavage.

[0704] Example 24: Evaluation of ADC mimic using mouse plasma (24-1) Plasma stability test of ADC mimic The test was carried out in the same manner as in Example 6.

[0705] (24-2) Analysis of the amount of detached fluorescent molecules using HPLC analysis. Analysis was carried out in the same manner as in Example 6.

[0706] As a result, compared with the ADC mimic synthesized in Comparative Example 1, the ADC mimic synthesized in Example 19-4 exhibited stability that was at least twice as high, and the ADC mimics synthesized in Examples 19-1 and 19-2 exhibited stability that was at least 10 times as high.

[0707] Example 25: Synthesis of linker-payload The NMR spectrum data of the linker-payload (42) synthesized in Example (12-3-5) was as follows.

[0708] １ H NMR (300MHz; DMSO-d ６ ) δ12.06 (brs, 2H), 10.05-10.07 (m, 1H), 8.47-8.28 (m, 1H), 8.29-8.19 (m, 1H), 8.13-8.04 (m, 3H), 7.91-7.56 (m, 5H), 7.39-7.17 (m, 7H), 6.99 (s, 2H), 5.99 (s, 1H), 5.86-5.67 (m, 1H), 5.43-5.35 (m, 3H), 4.77-4.16 (m, 6H), 4.00-3.98 (m , 2H), 3.80-3.76 (m, 1H), 3.52-3.18 (m, 12H), 3.01-2.73 (m, 9H), 2.26-2.14 (m, 6H), 2.12-2.09 (m, 2H), 1.97-1.90 (m , 2H), 1.84 (s, 6H), 1.76-1.69 (m, 5H), 1.43-1.35 (m, 10H), 1.23-1.14 (m, 3H), 1.01-0.96 (m, 7H), 0.83-0.68 (m, 24H).

[0709] Example 26: Synthesis of ADC (26-1-1) Synthesis of linker intermediate (115)

[0710] 5-Azidopentanoic acid (800 mg, 5.59 mmol) was dissolved in THF (14 mL), and isobutyl chloroformate (808 μL, 6.15 mmol) and N-methylmorpholine (873 μL, 8.39 mmol) were added. After stirring at 0°C for 30 minutes, hydrazine hydrate (1.36 g, 6.71 mmol) dissolved in 1M aqueous NaOH (4 mL) was added and stirred at room temperature for 3 hours. After concentration under reduced pressure, 1M aqueous NaOH was added, the pH of the system was adjusted to pH 10, and the mixture was washed with ethyl acetate. 1M aqueous HCl was added to the aqueous layer, the pH of the system was adjusted 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 mixture was purified by vacuum concentration column chromatography (dichloromethane:methanol = 10:1). The fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (115).

[0711] １ H 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).

[0712] MS (ESI) m / z: 329 [M+H] ＋

[0713] (26-1-2) Synthesis of linker intermediate (116)

[0714] Linker intermediate (116) (2.41 g, 5.59 mmol) was dissolved in dichloromethane (28 mL), and thiophenol (627 μL, 6.15 mmol), benzotriazol-1-yloxy (3.49 g, 6.71 mmol), and DIPEA (1.42 mL, 8.39 mmol) were added, followed by stirring at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by column chromatography (hexane:ethyl acetate=4:1). Fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (117) (2.20 g, 5.23 mmol).

[0715] １ H 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).

[0716] MS (ESI) m / z: 421 [M+H] ＋

[0717] (26-1-3) Synthesis of linker intermediate (118)

[0718] Linker intermediate (117) (2.20 g, 5.23 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to remove dichloromethane, and water was added thereto, followed by lyophilization to obtain linker intermediate (118) (1.98 g, 5.43 mmol).

[0719] １H NMR (400MHz, Chloroform-d) δ7.44 (s, J = 6.3, 4.6, 2.4Hz, 5H), 6.76 (s, 1 H), 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, 6.9 Hz, 2H), 1.63 (ddd, J = 17.7, 10.5, 4.8 Hz, 2H).

[0720] MS (ESI) m / z: 365 [M+H] ＋

[0721] (26-1-4) Synthesis of linker intermediate (119)

[0722] Linker intermediate (118) (100 mg, 0.274 mmol) was dissolved in dichloromethane (3 mL), and (40.6 μL, 0.280 mmol), benzotriazol-1-yloxy (150 mg, 0.288 mmol), and DIPEA (70.1 μL, 0.412 mmol) were added, followed by stirring 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 brine, 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 fractions containing the product were collected and concentrated under reduced pressure to obtain linker intermediate (119) (84.7 mg, 0.171 mmol).

[0723] １ H 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).

[0724] MS (ESI) m / z: 495 [M+H] ＋

[0725] (26-1-5) Synthesis of linker intermediate (120)

[0726] Linker intermediate (119) (84.7 mg, 0.171 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to remove the dichloromethane. Water was added and the mixture was lyophilized. The mixture was then purified by column chromatography (dichloromethane:methanol = 10:1). The fractions containing the product were collected and concentrated under reduced pressure to give linker intermediate (120) (46.8 mg, 0.107 mmol).

[0727] １ H 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).

[0728] MS (ESI) m / z: 439 [M+H] ＋

[0729] (26-2) Preparation of affinity reagent (18) having an azide group (The above amino acid sequence is the amino acid sequence of SEQ ID NO: 1.)

[0730] Ac-RGNCAYHKGQIIWCTYH-NH2 (SEQ ID NO: 1, 30.9 mg, 14.9 μmol, where the two cysteines at positions 4 and 14 are each intramolecularly disulfide-bonded) described previously (WO2019 / 240287A1) was dissolved in dimethylformamide (468 μL), and the linker intermediate (120) (46.8 mg, 0.107 mmol) synthesized in Example 6-1-5 and WSC.HCl (29.7 mg, 0.155 mmol) were added. The mixture was stirred at room temperature for 5 hours and then eluted by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above-mentioned modification reagent (121) (15.1 mg, 6.02 μmol).

[0731] (26-3) Introduction of two peptide molecules into trastuzumab (The above amino acid sequence is the amino acid sequence of SEQ ID NO: 1.)

[0732] Subsequently, using the peptide reagent (121) prepared in Example 26-2, conjugation was carried out on trastuzumab according to the method previously reported (WO2019 / 240287A1). As a result, an antibody into which the modified reagent (121) was introduced was obtained. DAR analysis of the antibody into which the peptide reagent (121) was introduced was performed by HIC-HPLC analysis according to a previously reported method (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two peptide reagents had been introduced.

[0733] (26-4) Synthesis of azido-group-introduced trastuzumab (T-1) (The above amino acid sequence is the amino acid sequence of SEQ ID NO: 1.)

[0734] A cleavage reaction was carried out on the antibody to which the modification reagent (121) obtained in Example 26-3 had been introduced, by adding a methoxyamine solution and shaking at room temperature for 3 hours, following a previously reported method (WO 2019 / 240287 A1). As a result, an antibody to which an azide group had been introduced was obtained. Analysis was carried out by HIC-HPLC analysis according to a previously reported method (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that an azide group had been introduced.

[0735] (26-5) Synthesis of ADC7 Linker-payload (40) was added to the above azide-introduced antibody to obtain ADC (7). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151276, where two Linker-payloads (40) had been introduced. Furthermore, ESI-TOFMS analysis was performed according to a previous report (WO2019 / 240287A1), and the DAR was confirmed to be 2.

[0736] Example 27: Evaluation of ADC using mouse plasma (27-1) Plasma stability test of ADC ADC mimic was added to 500 μL of mouse plasma (Charles River) to a concentration of 0.1 mg / mL, followed by sterile filtration. 50 μL of this solution was dispensed into six Eppendorf tubes. Three of the six samples were stored in an incubator set at 37°C for four days. The remaining three were similarly stored in a freezer at -80°C for four days. 100 μL of acetonitrile was added to each sample, and the mixture was vortexed and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0737] (27-2) Analysis of the amount of detached payload using HPLC analysis The amount of payload detached from the ADC was measured using liquid chromatography-mass spectrometry (including tandem mass spectrometry). The sample stored in the same manner as in Example 27-1 for 4 days in a -80°C freezer was designated as day 0, and the three samples incubated at 37°C for 4 days in Example 27-1 were designated as day 4. The MS intensities of the payload detected from the day 4 sample and the day 0 sample were calculated using extracted ion chromatograms, and the difference between them was analyzed.

[0738] Separately, using MMAE, the correlation between the area of ​​the TIC measured by HPLC and the concentration was calculated. The TIC of the fluorescence intensity of each ADC was converted to a concentration using this calculation formula. The dropout rate was calculated as the percentage of the difference in the ion chromatograms, with the concentration on Day 0 taken as 100%.

[0739] As a result, it was found that the ADCs synthesized in Examples 12-1, 12-2, and 12-3 had high stability.

[0740] Example 28: Evaluation of ADC using mouse plasma (28-1) Plasma stability test of ADC ADC mimic was added to 500 μL of mouse plasma (Charles River) to a concentration of 0.1 mg / mL, followed by sterile filtration. 50 μL of this solution was dispensed into six Eppendorf tubes. Three of the six samples were stored in an incubator set at 37°C for four days. The remaining three were similarly stored in a freezer at -80°C for four days. 100 μL of acetonitrile was added to each sample, and the mixture was vortexed and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0741] (28-2) Analysis of the amount of detached payload using HPLC analysis The amount of payload detached from the ADC was measured using liquid chromatography mass spectrometry (including tandem mass spectrometry). In Example 28-1, the sample stored in a freezer at -80°C for 4 days was designated as day 0, and in Example 28-1, three samples incubated at 37°C for 4 days were designated as day 4. The MS intensities of the payload detected from the day 4 sample and the day 0 sample were calculated using extracted ion chromatograms, and the difference between them was analyzed. The payload detachment rate was calculated according to Example 27-2.

[0742]

[0743] As a result, it was found that the ADCs synthesized in Examples 12-3 and 12-5 had high stability.

Claims

1. The following formula (1b): 【Chemistry 1】 [During the ceremony, Ig represents an antibody, and L is adjacent to Ig via thiol groups in the side chains of multiple cysteine ​​residues. Ａ It is coupled with HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, the hydrophilic group is one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polysarcosine group, and a sugar moiety; L C represents a bond or a divalent group; R A represents the side chain of a valine residue; R B represents the side chain of a citrulline residue or an alanine residue; W represents an oxygen atom, a sulfur atom, or an amino group (NH); Ring A represents an optionally substituted phenylene group, V represents an oxygen atom, a sulfur atom, or an amino group (NH); L Ａ , and L Ｂ each independently represents a divalent group, D represents a functional substance, wherein the average number n of bonds per antibody is 1.5 or more.

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

3. The conjugate or salt thereof according to claim 1, wherein the antibody is a human IgG antibody.

4. The conjugate or salt thereof according to claim 1, wherein n is 2.0 to 8.

0.

5. The conjugate or salt thereof according to claim 1, wherein n is 6.0 to 8.

0.

6. The conjugate or salt thereof according to claim 1, wherein n is 7.0 to 8.

0.

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

8. The structural unit represented by formula (1b) is represented by the following formula (1d): 【Chemistry 2】 [During the ceremony, Ig, R A , R B , ring A, D, and n are the same as those represented by formula (1b), respectively; R 1 and R 2 each independently represent a hydrogen atom or alkyl; L 1 and L 2 each independently represent a divalent group; (-L HG -) is represented by the following formula (a): -(C(RHG) 2) n1 -(C=O) n2 -(NR HG) n3 -(C(R HG) 2) n4 - (a) [During the ceremony, each of the multiple R HG s independently represents a hydrogen atom, a hydrophilic group, or a monovalent group that may contain a hydrophilic group; n1 is an integer from 0 to 3, n2 is an integer of 0 or 1, n3 is an integer of 0 or 1, n4 is an integer of 0 to 3. R HG1 and R HG2 each independently represent a hydrogen atom, a hydrophilic group, or a monovalent group that may contain a hydrophilic group; at least one hydrophilic group is included at one or more moieties selected from the group consisting of L HG , R HG1 , and R HG2 ; The conjugate or salt thereof according to claim 1, comprising a structural unit represented by the following formula: wherein the hydrophilic group is one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polysarcosine group, and a sugar moiety.

9. The divalent group represented by formula (a) is represented by the following formula (a1), (a2), or (a3): (a1) -(C(RHG) 2 )-; (a2) —(C(R HG ) 2 )—(C═O)—(NR HG )—(C(R HG ) 2 )—; or (a3) -(C=O)-(C(R HG ) 2 ) 2 -; [During the ceremony, The conjugate or salt thereof according to claim 8, wherein the plurality of R HG s each independently represent a hydrogen atom, a hydrophilic group, or an alkyl group having 1 to 6 carbon atoms and containing a hydrophilic group.

10. The conjugate or salt thereof according to any one of claims 1 to 9, wherein the hydrophilic groups are each independently a carboxylic acid group, a sulfonic acid group, or a hydroxyl group.

11. The conjugate or salt thereof according to claim 10, wherein the hydrophilic group is a carboxylic acid group.

12. The following formula (3b): 【Transformation 3】 [During the ceremony, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, the hydrophilic group is one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a hydroxyl group, a polysarcosine group, and a sugar moiety; L C represents a bond or a divalent group; R A represents the side chain of a valine residue; R B represents the side chain of a citrulline residue or an alanine residue; W represents an oxygen atom, a sulfur atom, or an amino group (NH); Ring A represents an optionally substituted phenylene group, V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B 1 represents a bioorthogonal functional group; the bioorthogonal functional group is a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue; and D represents a functional substance.] A compound or a salt thereof having a bioorthogonal functional group and a functional substance,

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