WO2024210154A1 - 抗体および機能性物質のコンジュゲート、抗体誘導体ならびに化合物、またはそれらの塩 - Google Patents

抗体および機能性物質のコンジュゲート、抗体誘導体ならびに化合物、またはそれらの塩 Download PDF

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WO2024210154A1
WO2024210154A1 PCT/JP2024/013787 JP2024013787W WO2024210154A1 WO 2024210154 A1 WO2024210154 A1 WO 2024210154A1 JP 2024013787 W JP2024013787 W JP 2024013787W WO 2024210154 A1 WO2024210154 A1 WO 2024210154A1
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group
divalent
salt
represented
hydrophilic
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French (fr)
Japanese (ja)
Inventor
豊 松田
友博 渡部
友博 藤井
ショーン チェ スンヨン
レディ マンダパティ アッピ
バッタライ ディーパック
マルコタン トーマス
マクファーレン ジェリー
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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Priority to EP24784938.3A priority Critical patent/EP4692115A1/en
Priority to JP2025513163A priority patent/JPWO2024210154A1/ja
Priority to CN202480023913.7A priority patent/CN120917041A/zh
Priority to KR1020257036865A priority patent/KR20250172615A/ko
Priority to AU2024243661A priority patent/AU2024243661A1/en
Publication of WO2024210154A1 publication Critical patent/WO2024210154A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to a conjugate of an antibody and a functional substance or a salt thereof, as well as an antibody derivative and a compound or a salt thereof used in the production thereof.
  • ADCs antibody drug conjugates
  • 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.
  • a representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)), which was jointly developed by Immunogene and Roche.
  • ADCs are made by binding functional groups in the side chains of specific amino acid residues present in antibodies to drugs.
  • An example of such a functional group used in making 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).
  • Antibodies have disulfide groups because the heavy chains and the heavy and light chains are linked by disulfide bonds.
  • an IgG antibody consisting of two heavy chains and two light chains has four disulfide groups because the heavy chains and the heavy and light chains are linked by four disulfide bonds.
  • Such disulfide bonds can be cleaved by a reducing agent. For example, when all four disulfide bonds in an IgG are cleaved, an IgG antibody having eight thiol groups is generated. Even when all four interchain disulfide bonds are cleaved, the heavy and light chains of the antibody do not dissociate.
  • trastuzumab deruxtecan Patent Document 5
  • Enherz® is an ADC with a drug-antibody ratio (DAR) of 8, in which all four interchain disulfide bonds are cleaved (reduced) before the drug is conjugated.
  • DAR drug-antibody ratio
  • Such ADCs are known to function as antigen-specific drugs by maintaining their antibody properties.
  • ADCs the antibody and the drug are linked via a linker.
  • linkers there are various types of linkers in ADCs.
  • a linker containing a dipeptide consisting of valine-citrulline (Val-Cit: VC structure) that is stable in human plasma and has a structure that can be cleaved by a specific enzyme to release the drug in cancer cells.
  • a dipeptide-containing linker is stable in human plasma as shown in (A) below, as shown in (B) below, the VC structure is recognized by cathepsin B in the lysosomes in human cancer cells, and the amide bond present on the carboxy terminal side of citrulline is cleaved. Therefore, an ADC having such a dipeptide-containing linker can release the drug in human cancer cells and exert its medicinal effect.
  • 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 the VC structure and cleaves the amide bond present on the carboxy-terminal side of citrulline, and the linkers containing dipeptides such as those described above are cleaved in the plasma by Ces1c. Therefore, the pharmacokinetics of ADCs having linkers containing dipeptides such as those described above are significantly different between mice and humans. This causes the problem that it is difficult to evaluate the efficacy of drugs in humans when used in mice.
  • 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.
  • the object of the present invention is to provide a conjugate or a salt thereof of an antibody and a functional substance that has excellent desired properties.
  • the present invention is as follows.
  • the present invention relates to a compound represented by the following formula (1):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS represents a divalent group containing a cleavable site;
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • D represents a functional substance;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the structural unit represented by formula (1) is represented by the following formula (1′):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • D represents a functional substance;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the structural unit represented by formula (1′) is represented by the following formula (1′′):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • R 1 and R 2 each independently represent a hydrogen atom or a monovalent group;
  • L 1 and L 2 each independently represent a divalent group;
  • D represents a functional substance;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the conjugate may exhibit an aggregation rate of 2.6% or less when analyzed by size exclusion chromatography.
  • the functional group in the side chain of the specific amino acid residue may be an amino group in the side chain of a lysine residue, and n may be 1.5 to 2.5, in which case L A may have a carbonyl group, and the bond may be achieved by an amide bond through the bond between the amino group in the side chain of the lysine residue and the carbonyl group in L A.
  • the lysine residue may be present at positions 246/248, 288/290, or 317 according to Eu numbering.
  • the structural unit has the following formula (I): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS1 represents a divalent group containing a cleavable site; V1 represents an oxygen atom, a sulfur atom, or an amino group (NH); L A1 and L B1 each independently represent a divalent group; D1 represents a functional substance; The average number of bonds per immunoglobulin unit, r, is 1.5 to 2.5.
  • the modifying moiety represented by formula (I) is represented by the following formula (I'): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), V1 represents an oxygen atom, a sulfur atom, or an amino group (NH); L A1 and L B1 each independently represent a divalent group; D1 represents a functional
  • the modifying moiety represented by formula (I') has the following formula (I''): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), R 11 and R 21 each independently represent a hydrogen atom or a monovalent group; L 11 and L 21 each independently represent a divalent group; D1 represents a functional substance; The
  • the functional group in the side chain of the specific amino acid residue may be a thiol group in the side chain of a cysteine residue, and n may be 2.0 to 8.0. n may be preferably 6.0 to 8.0, more preferably 7.0 to 8.0.
  • the present invention relates to a compound represented by the following formula (2):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS represents a divalent group containing a cleavable site;
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • B2 represents a bioorthogonal functional group; wherein the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the structural unit represented by formula (2) is represented by the following formula (2'):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • B2 represents a bioorthogonal functional group;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the structural unit represented by formula (2′) is represented by the following formula (2′′):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • R 1 and R 2 each independently represent a hydrogen atom or a monovalent group;
  • L 1 and L 2 each independently represent a divalent group;
  • B2 represents a bioorthogonal functional group;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the functional group in the side chain of the specific amino acid residue may be an amino group in the side chain of a lysine residue, and n may be 1.5 to 2.5, in which case L A may have a carbonyl group, and the bond may be achieved by an amide bond through the bond between the amino group in the side chain of the lysine residue and the carbonyl group in L A.
  • the lysine residue may be present at positions 246/248, 288/290, or 317 according to Eu numbering.
  • the structural unit has the following formula (II): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS1 represents a divalent group containing a cleavable site; V1 represents an oxygen atom, a sulfur atom, or an amino group (NH); L A1 and L B1 each independently represent a divalent group; B 21 represents a bioorthogonal functional group; The average number of bonds per immunoglobulin unit, r, is 1.5 to 2.5.
  • the modifying moiety represented by formula (II) is represented by the following formula (II'): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), V1 represents an oxygen atom, a sulfur atom, or an amino group (NH); L A1 and L B1 each independently represent a divalent group; B 21 represents a divalent group;
  • the modifying moiety represented by formula (II') is represented by the following formula (II''): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), R 11 and R 21 each independently represent a hydrogen atom or a monovalent group; L 11 and L 21 each independently represent a divalent group; B 21 represents a
  • the functional group in the side chain of the specific amino acid residue may be a thiol group in the side chain of a cysteine residue, and n may be 2.0 to 8.0. n may be preferably 6.0 to 8.0, more preferably 7.0 to 8.0.
  • 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B1 represents a bioorthogonal functional group; and D represents a functional substance.], or a salt thereof.
  • the structural unit represented by formula (3′) is 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); R 1 and R 2 each independently represent a hydrogen atom or a monovalent group; L 1 and L 2 each independently represent a divalent group; B1 represents a bioorthogonal functional group; D represents a functional substance.
  • the present invention also provides an antibody derivatization reagent, which contains a compound represented by the above formula (3') or a subordinate formula thereof, or a salt thereof.
  • 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B1 represents a first bioorthogonal functional group; and B1 represents a second bioorthogonal functional group.], or a salt thereof, comprising a first bioorthogonal functional group and a second bioorthogonal functional group.
  • formula (4') wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS' represents a divalent group containing a
  • the structural unit represented by formula (4') is 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); R 1 and R 2 each independently represent a hydrogen atom or a monovalent group; L 1 and L 2 each independently represent a divalent group; B1 represents a first bioorthogonal functional group; B2 represents a second bioorthogonal functional group.
  • the present invention also provides a derivatization reagent for an antibody or a functional substance, comprising a compound represented by the above formula (4') or a subordinate formula thereof, or a salt thereof.
  • the present invention provides a compound of the following formula (I'), (II'), or (III') or a salt thereof:
  • the compound (I'), (II'), or (III') may be a compound (I'', (II'', or (III'') below, respectively.
  • (I′′) 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); X and Y each independently represent a monovalent group; (II′′) The following formula (6′′): [Wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS' represents a divalent group containing a cleavable site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic aromatic
  • the immunoglobulin unit may be a human immunoglobulin unit.
  • the human immunoglobulin unit may be a human IgG antibody.
  • the monovalent group (HG-) containing a hydrophilic group is represented by the following formula (A): N(R HG1 )(R HG2 )-L HG- (A) [Wherein, LHG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 and R HG2 each independently represent a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; At least one hydrophilic group is contained in one or more sites selected from the group consisting of L HG , R HG1 , and R HG2 .
  • 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 —; [Wherein, Each of the multiple R HG independently represents a hydrogen atom, a hydrophilic group, or an alkyl group having 1 to 6 carbon atoms and containing a hydrophilic group.
  • the hydrophilic group may be one or more groups selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a sulfamide group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety.
  • the hydrophilic group may be one or more groups selected from the group consisting of a sulfonic acid group, a sulfamide group, a hydroxyl group, a polyethylene glycol group, a polysarcosine group, and a sugar moiety.
  • the cleavable site may be an enzymatic cleavable site.
  • the enzyme may be one or more enzymes selected from the group consisting of cathepsin B, plasmin, legumain, and caspase.
  • the enzyme may be one or more enzymes selected from the group consisting of plasmin, legumain, and caspase.
  • the enzymatic cleavage site may include VC, VA, AA, GGFG (SEQ ID NO: 2), FK, VK, AK, GC, VLK, NN, or DDVD (SEQ ID NO: 3).
  • the enzymatic cleavage site may include AA, GGFG (SEQ ID NO: 2), FK, VK, AK, GC, VLK, NN, or DDVD (SEQ ID NO: 3).
  • the monovalent group containing a hydrophilic group may contain one or more amino acid residues containing a carboxylic acid group in the side chain.
  • the building block of HG-CS may contain one or more amino acid residues containing a carboxylic acid group in the side chain and a peptide moiety containing an enzymatic cleavage site.
  • the peptide portion may include EVC, EEVC (SEQ ID NO: 4), EEEEVC (SEQ ID NO: 5), DVC, EVA, EAA, EGGFG (SEQ ID NO: 6), EFK, EEFK (SEQ ID NO: 7), EEVK (SEQ ID NO: 8), EEAK (SEQ ID NO: 9), EGC, EEVLK (SEQ ID NO: 10), ENN, EDDVD (SEQ ID NO: 11), ⁇ -Ala-VC, EGC, or EEGC (SEQ ID NO: 12).
  • the peptide portion may include EAA, EGGFG (SEQ ID NO: 6), EFK, EEFK (SEQ ID NO: 7), EEVK (SEQ ID NO: 8), EEAK (SEQ ID NO: 9), EGC, EEVLK (SEQ ID NO: 10), ENN, EDDVD (SEQ ID NO: 11), ⁇ -Ala-VC, or EEGC (SEQ ID NO: 12).
  • the cleavable site may be cleavable under acidic or reducing conditions.
  • the cleavable site may be a disulfide bond.
  • L A may be a divalent group having a side chain containing a hydrophilic group or a functional substance.
  • the functional substance may be a medicine, a labeling substance, or a stabilizer.
  • the bioorthogonal functional group may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.
  • ring A and/or ring A1 may be a phenylene group which may have a substituent.
  • the conjugate of the present invention or a salt thereof 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 of the present invention or their salts, and reagents are useful, for example, as synthetic intermediates in the production of the above-described conjugates.
  • FIG. 1 is a diagram showing an example of the release of a drug (an active drug in which the steric hindrance caused by an antibody has been eliminated) by cleavage of a cleavable site and linkage of a ⁇ -electron resonance system.
  • a drug an active drug in which the steric hindrance caused by an antibody has been eliminated
  • the conjugate, antibody derivative, and compound of the present invention can be designed to enable such release (see also Background Art as appropriate).
  • the antibody can retain a hydrophilic group even after cleavage.
  • Fig. 2 is a diagram showing 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 among the structural units represented by formula (5). In addition, these substances can be synthesized according to the scheme shown in Fig. 2.
  • FIG. 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).
  • FIG. 4 is a diagram showing an example of the synthesis outline of the compounds represented by formulas (4) to (7).
  • 5 is a diagram showing an example of the synthesis outline of the compounds represented by formulas (4") to (7"), which are preferred examples of the compounds represented by formulas (4) to (7).
  • DIPEA N,N-diisopropylethylamine
  • DMF N,N-dimethylformamide
  • the term "antibody” is as follows.
  • the term "immunoglobulin unit” corresponds to a bivalent monomer unit that is a basic component of such an antibody, and is a unit containing two heavy chains and two light chains. Therefore, the definitions, examples, and preferred examples of the origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, and position of cysteine residues of the immunoglobulin unit are the same as those of the antibody described below.
  • the origin of the antibody is not particularly limited, and may be derived from animals such as mammals and birds (e.g., chickens).
  • the immunoglobulin unit is derived from a mammal.
  • mammals include, for example, primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pet animals (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, and more preferably humans.
  • the type of antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent or higher antibody (e.g., IgA antibody, IgM antibody).
  • the antibody is a monoclonal antibody.
  • monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies with a specific glycan added thereto (e.g., antibodies modified to have a glycan binding consensus sequence such as an N-glycan binding consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins.
  • isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY.
  • IgG e.g., IgG1, IgG2, IgG3, IgG4
  • IgM e.g., IgA, IgD, IgE, and IgY.
  • full-length antibodies or antibody fragments containing the variable region and the 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.
  • any antigen can be used as the antigen for the antibody.
  • antigens include proteins (including oligopeptides and polypeptides, and may be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low molecular weight compounds.
  • the antibody may be an antibody whose antigen is a protein.
  • proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.
  • the protein that is the antigen of the antibody may be a disease target protein.
  • diseases target proteins include the following:
  • Neurological diseases CGRP, CD20, ⁇ -amyloid, ⁇ -amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), ⁇ -synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus
  • monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab, , ikesekizumab (IgG4), reslizumab (IgG4)
  • An immunoglobulin unit (e.g., a bivalent antibody such as IgG) consisting 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.
  • a reducing agent is sufficiently applied to such an immunoglobulin unit, eight thiol groups are generated from the four disulfide bonds.
  • Any reducing agent that can cleave disulfide bonds to generate thiol groups can be used as the reducing agent, and examples of such reducing agents include tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, and ⁇ -mercaptoethanol.
  • trastuzumab deruxtecan known as Enhertz®
  • Enhertz® is an ADC with a DAR of 8 in which a drug is attached to the thiol group generated by reducing (cleaving) all four interchain disulfide bonds.
  • ADCs are known to function as antigen-specific drugs by maintaining their antibody properties.
  • specific lysine residues e.g., lysine residues at positions 246/248, 288/290, or 317) in the heavy chain of the immunoglobulin unit constituting the antibody can be regioselectively modified (see, e.g., WO 2018/199337, WO 2019/240288, WO 2019/240287, and WO 2020/090979).
  • regioselective or “regioselectivity” refers to a situation in which a specific structural unit capable of binding to a specific amino acid residue in an antibody is concentrated in a specific region in the antibody, even though a specific amino acid residue is not concentrated in a specific region in the antibody.
  • expressions related to position selectivity such as “having a position-selective property,” “position-selective binding,” and “binding with position selectivity,” mean that the retention or binding rate of a given structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues that are the same type as the specific amino acid residue in the target region.
  • position selectivity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or even 100%.
  • an immunoglobulin unit containing two heavy chains and two light chains can be bound to a modifying group adjacent to Ig (e.g., LA, L1, described below) via a thiol group in the side chain of a plurality of cysteine residues (a plurality of cysteine residues in the two heavy chains and two light chains contained in the immunoglobulin unit) generated by the action of a reducing agent on the immunoglobulin unit.
  • a modifying group adjacent to Ig e.g., LA, L1, described below
  • the number corresponding to the plurality 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), particularly preferably 6 or more (e.g., 6 to 8), 7 or more (e.g., 7 to 8), or 8.
  • specific amino acid residues at other positions may be further regioselectively modified.
  • various methods are known for regioselectively modifying specific amino acid residues at predetermined positions in the immunoglobulin unit or antibody.
  • amino acid residues having side chains that are easily modified e.g., amino group, carboxy group, amide group, hydroxy group
  • amino acid residues having side chains that are easily modified e.g., amino group, carboxy group, amide group, hydroxy group
  • amino acid residues having side chains that are easily modified e.g., amino group, carboxy group, amide group, hydroxy group
  • amino acid residues having side chains that are easily modified e.g., amino group, carboxy group, amide group, hydroxy group
  • amino acid residues having side chains that are easily modified e.g., amino group, carboxy group, amide group, hydroxy group
  • aspartic acid residues e.g., glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues
  • tyrosine residues, serine residues, and threonine residues having side chains containing hydroxy groups being preferred.
  • Halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.
  • the monovalent group may be substituted with one or more (e.g., 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.
  • Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.
  • a monovalent chain-like hydrocarbon group means a hydrocarbon group that is composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of monovalent chain-like hydrocarbon groups include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl, and alkynyl may be either linear or branched.
  • an alkyl having 1 to 12 carbon atoms is preferred, an alkyl having 1 to 6 carbon atoms is more preferred, and an alkyl having 1 to 4 carbon atoms is even more preferred.
  • the alkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • alkyl having 1 to 12 carbon atoms examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.
  • alkenyl an alkenyl having 2 to 12 carbon atoms is preferable, an alkenyl having 2 to 6 carbon atoms is more preferable, and an alkenyl having 2 to 4 carbon atoms is even more preferable.
  • the alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.
  • alkynyl an alkynyl having 2 to 12 carbon atoms is preferable, an alkynyl having 2 to 6 carbon atoms is more preferable, and an alkynyl having 2 to 4 carbon atoms is even more preferable.
  • the alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.
  • alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.
  • alkyl is preferred.
  • a monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not have to be composed only of alicyclic hydrocarbons, and may contain a chain structure as part of it.
  • Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.
  • Cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms.
  • the 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.
  • cycloalkenyl a cycloalkenyl having 3 to 12 carbon atoms is preferable, a cycloalkenyl having 3 to 6 carbon atoms is more preferable, and a cycloalkenyl having 5 to 6 carbon atoms is even more preferable.
  • the cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.
  • the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.
  • cycloalkynyl As the cycloalkynyl, cycloalkynyl having 3 to 12 carbon atoms is preferable, cycloalkynyl having 3 to 6 carbon atoms is more preferable, and cycloalkynyl having 5 to 6 carbon atoms is even more preferable.
  • 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.
  • Cycloalkyl is preferred as the monovalent alicyclic hydrocarbon group.
  • a monovalent aromatic hydrocarbon group means 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 it, and the aromatic ring may be either monocyclic or polycyclic.
  • the monovalent aromatic hydrocarbon group an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is even more preferable.
  • the 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 having 6 to 12 carbon atoms include phenyl and naphthyl.
  • Phenyl is preferred as the monovalent aromatic hydrocarbon group.
  • alkyl, cycloalkyl, and aryl are preferred as monovalent hydrocarbon groups.
  • the monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound.
  • the monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group.
  • the heteroatom constituting the heterocyclic group it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom.
  • an aromatic heterocyclic group having 1 to 15 carbon atoms is preferable, an aromatic heterocyclic group having 1 to 9 carbon atoms is more preferable, and an aromatic heterocyclic group having 1 to 6 carbon atoms is even more preferable.
  • the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.
  • Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.
  • a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is even more preferable.
  • the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.
  • Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.
  • 5- or 6-membered heterocyclic groups are preferred as monovalent heterocyclic groups.
  • R 7 represents a hydrogen atom or a substituent described later.
  • R8 represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group.
  • m1 represents 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.
  • the divalent straight chain hydrocarbon group is a straight chain alkylene, a straight chain alkenylene, or a straight chain alkynylene.
  • the straight-chain alkylene is a straight-chain alkylene having 1 to 6 carbon atoms, and preferably a straight-chain alkylene having 1 to 4 carbon atoms. Examples of the straight-chain alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene.
  • the linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, and preferably a linear alkenylene having 2 to 4 carbon atoms.
  • linear alkenylene examples include ethylenylene, n-propenylene, n-butenylene, n-pentenylene, and n-hexenylene.
  • the straight-chain alkynylene is a straight-chain alkynylene having 2 to 6 carbon atoms, and preferably a straight-chain alkynylene having 2 to 4 carbon atoms.
  • Examples of the straight-chain alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene.
  • the divalent linear hydrocarbon group is preferably a linear alkylene group.
  • the divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group.
  • the arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of the arylene include phenylene, naphthylene, and anthracenylene.
  • the divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms.
  • Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.
  • the divalent cyclic hydrocarbon group is preferably an arylene group.
  • the divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group.
  • the heteroatom constituting the heterocycle it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and it is more preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom.
  • the divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms.
  • divalent aromatic heterocyclic group examples 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.
  • a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferable.
  • 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,
  • R 7 is a hydrogen atom or an alkyl group;
  • R 8 is alkylene or arylene;
  • m1 may be an integer from 1 to 5 (ie, 1, 2, 3, 4, or 5).
  • the main chain structure in the divalent group may be substituted with one or more (e.g., 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.
  • Substituents include the following: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) 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) Nitro, sulfate, sulfonate, cyano, and carboxyl groups
  • Aralkyl refers to arylalkyl.
  • the definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above.
  • aralkyl aralkyl having 3 to 15 carbon atoms is preferred. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.
  • the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v) R a -O-, R a -C( ⁇ O)-, R a -O-C( ⁇ O)-, or R a -C( ⁇ O)-O- (R a is a hydrogen atom or an alkyl group 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 are a hydrogen atom or an alkyl group having 1 to 12 carbon
  • the substituents may be: (i) a halogen atom; (ii) 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 each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (v) the same groups as those enumerated in (vii) above.
  • the substituents may be: (i) a halogen atom; (ii) 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 each represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); or (v) the same groups as those enumerated in (vii) above.
  • the substituents may be: (i) a halogen atom; (ii) 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 each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); or (v) the same groups as those enumerated in (vii) above.
  • hydrophilic group is a group that can make the structural unit represented by formulas (1) to (7) or its subordinate formulae more hydrophilic. By having a hydrophilic group at a predetermined site in the structural unit, the properties of the conjugate can be further improved.
  • hydrophilic groups include a carboxylic acid group, a sulfonic acid group, a sulfamide 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).
  • a polyethylene glycol (PEG) group is a divalent group represented by -(CH 2 -CH 2 -O-) k1 -.
  • 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 more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more.
  • k1 may also be an integer of 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k1 may be an integer from 3 to 15, preferably an integer of 4 to 12, more preferably an integer of 5 to 10, and even more preferably an integer of 4 to 9.
  • the polysarcosine group is a divalent group represented by -(NCH 3 -CH 2 -CO-) k2 -.
  • the polysarcosine group can be used as an alternative to PEG.
  • k2 may be, for example, an integer of 3 or more, preferably an integer of 4 or more, more preferably an integer of 5 or more, and even more preferably an integer of 6 or more.
  • k2 may also be an integer of 15 or less, preferably an integer of 12 or less, more preferably an integer of 10 or less, and even more preferably an integer of 9 or less. More specifically, k2 may be an integer of 3 to 15, preferably an integer of 4 to 12, more preferably an integer of 5 to 10, and even more preferably an integer of 4 to 9.
  • the sugar moiety is a monosaccharide, an oligosaccharide (e.g., disaccharide, trisaccharide, tetrasaccharide, pentasaccharide), or a polysaccharide.
  • the sugar moiety can include an aldose or 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.
  • the sugar moiety may be a low molecular weight hydrophilic group.
  • a low molecular weight hydrophilic group refers to a hydrophilic group having a molecular weight of 1500 or less.
  • the molecular weight of the low molecular weight hydrophilic group may preferably be 1200 or less, 1000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less.
  • low molecular weight hydrophilic groups include carboxylic acid groups, sulfonic acid groups, and hydroxyl groups, as well as polyethylene glycol groups, polysarcosine groups, and sugar moieties (e.g., monosaccharides, oligosaccharides) that satisfy the above molecular weights.
  • the monovalent group containing a hydrophilic group is a monovalent group containing a hydrophilic group as described above.
  • the monovalent group is as described above.
  • the monovalent group containing a hydrophilic group is represented by the following formula (A): N(R HG1 )(R HG2 )-L HG- (A) [Wherein, LHG represents a bond or a divalent group which may contain a hydrophilic group, R HG1 and R HG2 each independently represent a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group; At least one hydrophilic group is contained in one or more sites selected from the group consisting of L HG , R HG1 , and R HG2 .
  • the hyphen (-) located at the right terminal represents a bond.
  • LHG represents a bond or a divalent group which may contain a hydrophilic group.
  • RHG1 and RHG2 are each independently a hydrogen atom or a monovalent group which does not contain a hydrophilic group
  • LHG is a divalent group which contains a hydrophilic group.
  • at least one of RHG1 and RHG2 is a hydrophilic group or a monovalent group which contains a hydrophilic group
  • LHG may be a divalent group which may contain a hydrophilic group, but is also preferably a divalent group which does not contain a hydrophilic group.
  • R HG1 and R HG2 each independently represent a hydrogen atom, a hydrophilic group, or a monovalent group which may contain a hydrophilic group.
  • the hydrophilic group and the monovalent group are as described above.
  • the monovalent groups which may contain a hydrophilic group, represented by R HG1 and R HG2 may be a protecting group for an amino group.
  • R HG1 and R HG2 may be a hydrogen atom, and the other may be a protecting group for an amino group.
  • Examples of the protecting group for an amino group include an alkylcarbonyl group (acyl group) (e.g., an acetyl group, a propoxy group, a butoxycarbonyl group such as a tert-butoxycarbonyl group), an alkyloxycarbonyl group (e.g., a fluorenylmethoxycarbonyl group), an aryloxycarbonyl group, and an arylalkyl(aralkyl)oxycarbonyl group (e.g., a benzyloxycarbonyl group).
  • acyl group e.g., an acetyl group, a propoxy group, a butoxycarbonyl group such as a tert-butoxycarbonyl group
  • an alkyloxycarbonyl group e.g., a fluorenylmethoxycarbonyl group
  • an aryloxycarbonyl group e.g., a benzyloxycarbonyl group
  • one of R HG1 and R HG2 may be a hydrogen atom, and the other may be a monovalent group containing a hydrophilic group.
  • 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, an alkyloxycarbonyl group containing a hydrophilic group, and an oxycarbonyl group containing a hydrophilic group.
  • At least one hydrophilic group is contained in one or more moieties selected from the group consisting of L HG , R HG1 , and R HG2 .
  • moieties containing at least one hydrophilic group and combinations thereof include the following: (i) LHG alone; (ii) RHG1 alone; (iii) RHG2 alone; (iv) a combination of LHG and LHG1 ; (v) a combination of LHG and LHG2 ; (vi) a combination of LHG1 and LHG2 ; and (vii) a combination of LHG , LHG1 and LHG2 .
  • Each of these LHG moieties, RHG1 moieties, and RHG2 moieties may contain one hydrophilic group, or may contain two or more hydrophilic groups.
  • the divalent group which may contain a hydrophilic group is a divalent group which may contain a hydrophilic group as described above.
  • the divalent group is as described above.
  • the hyphens (-) disposed at both ends represent bonds.
  • each of the multiple RHGs 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.
  • n1 is an integer from 0 to 3, preferably an integer from 0 to 2, and more preferably an integer of 0 or 1.
  • n2 is an integer of 0 or 1.
  • n3 is an integer of 0 or 1.
  • n4 is an integer from 0 to 3, preferably an integer from 0 to 2, and more preferably an integer of 0 or 1.
  • the divalent group which may contain a hydrophilic group may be a divalent group 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 —.
  • each of the multiple RHGs independently represents 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.
  • the cleavable site is a site that can be cleaved in an appropriate environment (e.g., inside or outside a cell).
  • 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)
  • a cleavable site under acidic conditions a site that can be cleaved at a local acidic site present in the body
  • the cleavable site may be self-immolative (e.g., WO 02/083180, WO 04/043493, WO 05/112919).
  • the cleavable site may be a cleavable site containing a peptide.
  • the cleavable site containing a peptide may be a cleavable site consisting of a cleavable peptide that is cleaved by an enzyme.
  • the cleavable site containing a peptide may also contain, in addition to the cleavable peptide that is cleaved by the enzyme, a peptide that is not involved in the cleavage.
  • the cleavable site containing a peptide further contains a peptide that is not involved in the cleavage, it can be cleaved as long as it contains a cleavable peptide that is cleaved by the enzyme.
  • the amino acid residues constituting the peptide at the cleavable site may be any amino acid residue.
  • any amino acid residues include ⁇ -amino acid residues, ⁇ -amino acid residues, and ⁇ -amino acid residues, with ⁇ -amino acid residues being preferred.
  • ⁇ -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.
  • the any amino acid residue may be an L- ⁇ -amino acid residue (e.g., the L-form of the specific ⁇ -amino acid residues described above) or a glycine residue.
  • the peptide-containing cleavage site may include a cleavage peptide composed of amino acid residues selected from the group consisting of valine residues, phenylalanine residues, threonine residues, leucine residues, citrulline residues, alanine residues, glutamic acid residues, glutamine residues, lysine residues, arginine residues, and methionine residues, and combinations thereof.
  • 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 2-20, 2-15, 2-12, 2-10, 2-8, 2-6, or 2-4, or 3-20, 3-15, 3-12, 3-10, 3-8, 3-6, or 3-4, or 4-20, 4-15, 4-12, 4-10, 4-8, or 4-6.
  • 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.
  • 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.
  • the cleavage site may be an enzyme cleavage site.
  • enzyme 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).
  • the cleavage site is a cleavage site by an intracellular protease (e.g., a protease present in lysosomes or endosomes).
  • the cleavage site is a cleavage site by a protease present in lysosomes.
  • the cleavage site is a cleavage site by cathepsin B, plasmin, legumain, and caspase.
  • the cleavage sites by cathepsin B, plasmin, legumain, and caspase may be cleavage sites including VC, VA, AA, GGFG (SEQ ID NO: 2), FK, VK, AK, GC, VLK, NN, or DDVD (SEQ ID NO: 3).
  • the peptide sites of VC, VA, AA, GGFG (SEQ ID NO: 2), FK, VK, AK, and GC can be cleaved by cathepsin B.
  • the peptide site of VLK is known to be cleaved by interstitial plasmin.
  • the peptide site of NN is known to be cleaved by legumain.
  • the peptide site of DDVD is known to be cleaved by caspase.
  • Examples of such cleavage sites include EVC, EEVC (SEQ ID NO: 4), EEEEVC (SEQ ID NO: 5), DVC, EVA, EAA, EGGFG (SEQ ID NO: 6), EFK, EEFK (SEQ ID NO: 7), EEVK (SEQ ID NO: 8), EEAK (SEQ ID NO: 9), EGC, EEVLK (SEQ ID NO: 10), ENN, EDDVD (SEQ ID NO: 11), ⁇ -Ala-VC, EGC, and EEGC (SEQ ID NO: 12).
  • the cleavable site may be a site cleavable under acidic or reducing conditions.
  • cleavable sites under acidic conditions include alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, ⁇ -thiopropionate residues, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, and 2-(diphenylphosphino)benzoate residues.
  • cleavable sites under reducing conditions include disulfide residues, alkoxyalkyl residues, and azo residues.
  • the divalent group containing a cleavable site when it is adjacent to an optionally substituted divalent aromatic ring group (e.g., ring A, ring A1), it 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 and W form a ⁇ -electron conjugated system.
  • 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
  • cleavage of the bond between CO and W is coordinated with the ⁇ -electron conjugated system in the divalent aromatic ring group in ring A (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system with the cleavable site) and V, thereby efficiently cleaving the bond between the tertiary carbon atom connected to ring A and V (FIG. 1).
  • W is preferably an oxygen atom or an amino group (NH), more preferably an amino group (NH).
  • the cleavable site is preferably an amide bond site.
  • the divalent group containing a cleavable site represented by CS may be -L CSa -CS a -W csa - [wherein the hyphen (-) represents a bond, L CSa represents a bond or a divalent group, CS a represents a cleavable site, and W cs represents an oxygen atom, a sulfur atom, or an amino group (NH). ].
  • the definition, examples, and preferred examples of the divalent group represented by L CSa are the same as those of the divalent group described above.
  • the definition, examples, and preferred examples of the cleavable site represented by CS a are the same as those of the cleavable site described above.
  • W csa represents an oxygen atom, a sulfur atom, or an amino group (NH), preferably represents an oxygen atom or an amino group (NH), and more preferably represents an amino group (NH).
  • the divalent group containing a cleavable site represented by CS may be -L CSb -CS b -W csb - [wherein the hyphen (-) represents a bond, L CSb represents a bond or a divalent group, CS b represents a cleavable site, and W csb represents a bond or a divalent group. ].
  • the definition, examples, and preferred examples of the divalent group represented by L CSb and W csb are the same as those of the divalent group described above.
  • the definition, examples, and preferred examples of the cleavable site represented by CS b are the same as those of the cleavable site described above.
  • CS b is preferably a disulfide bond
  • W csa is preferably an alkylene (e.g., methylene) as a divalent group.
  • Bio-orthogonal functional groups refers to a group that does not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates) or reacts slowly with biological components, but selectively reacts with components other than biological components.
  • Bioorthogonal functional groups are well known in the art (see, 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)).
  • a bioorthogonal functional group for a protein is used as the bioorthogonal functional group. This is because the antibody to be derivatized by the reagent of the present invention is a protein.
  • the bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 natural amino acid residues that make up a protein, or reacts slowly with the side chains, but reacts with the target functional group.
  • the 20 natural amino acids that make up a protein are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K).
  • glycine which has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, which have a hydrocarbon side chain (i.e., the side chain does not contain a heteroatom selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inactive against normal reactions.
  • the bioorthogonal functional group for a protein 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 inactive against normal reactions, but reacts with the functional group of interest.
  • bioorthogonal functional groups include, for example, azide residues, aldehyde residues, thiol residues, alkene residues (in other words, it is sufficient if 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 if 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, ⁇ -halocarbonyl residues (e.g
  • the bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [Where: R 1a , one or more R 1b , and one or more R 1c are the same or different and are the above-mentioned substituents or electron-withdrawing groups; ⁇ is a bond.
  • electron-withdrawing groups include halogen atoms, alkyl substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl groups, and keto groups (e.g., acyl), with halogen atoms, boronic acid residues, mesyl, tosyl, and triflate being preferred.
  • the bioorthogonal functional group may be protected.
  • the bioorthogonal functional group that may be protected refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group.
  • the unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above.
  • the 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 performed 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 a specific treatment include (a) treatment with one or more substances selected from the group consisting of an acidic substance, a basic substance, a reducing agent, an oxidizing agent, and an enzyme, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) leaving the product when a cleavable linker containing a self-degrading cleavable portion is used.
  • a specific treatment include (a) treatment with one or more substances selected from the group consisting of an acidic substance, a basic substance, a reducing agent, an oxidizing agent, and an enzyme, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) leaving the product when a cleavable linker containing a self-degrading cleavable portion is used.
  • Such protecting groups and their cleavage conditions are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner,
  • protected bioorthogonal functional groups include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.
  • the protected bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; One or more R 2a are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents; ⁇ is a bond.
  • the bioorthogonal functional group which may be protected, is an unprotected bioorthogonal functional group.
  • the functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred.
  • the functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked together.
  • the drug may be a drug for any disease.
  • diseases include 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 tumors, and melanoma), autoimmune and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, and systemic lupus erythematosus), cranial and neurological diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections and viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarth
  • the drug may be an anticancer drug.
  • the anticancer drug include chemotherapeutic agents, toxins, radioisotopes, or substances containing the same.
  • the chemotherapeutic agent include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds.
  • the toxin include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin).
  • radioisotope examples include a radioisotope of a hydrogen atom (e.g., 3H ), a radioisotope of a carbon atom (e.g., 14C ), a radioisotope of a phosphorus atom (e.g., 32P ), a radioisotope of a sulfur atom (e.g., 35S ), a radioisotope of yttrium (e.g., 90Y ), a radioisotope of technetium (e.g., 99mTc ), a radioisotope of indium (e.g., 111In ), a radioisotope of an iodine atom (e.g., 123I , 125I , 129I , 131I ), a radioisotope of samarium (e.g., 153Sm ), a radioisotope of rhenium (e.g.,
  • the drug includes auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogues, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, tubulysin, and exatecan.
  • auristatins MMAE, MMAF
  • maytansine DM1, DM4
  • PBD pyrrolobenzodiazepine
  • IGN camptothecin analogues
  • calicheamicin calicheamicin
  • duocarmycin duocarmycin
  • eribulin eribulin
  • anthracyclines dmDNA31
  • tubulysin tubulysin, and exatecan.
  • a labeling substance is a substance that allows for the detection of a target (e.g., tissue, cell, substance).
  • labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those mentioned above), or substances containing them.
  • enzymes e.g., peroxidase, alkaline phosphatase, luciferase, ⁇ -gal
  • Stabilizers are substances that allow for the stabilization of antibodies.
  • stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.
  • 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.
  • peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs.
  • proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies.
  • nucleic acids include DNA, RNA, and artificial nucleic acids.
  • nucleic acids examples include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense.
  • RNA interference-inducing nucleic acids e.g., siRNA
  • aptamers e.g., aptamers
  • antisense e.g., antisense
  • low molecular weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.
  • salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid.
  • salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
  • salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium.
  • salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine.
  • 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).
  • conjugate or Salt thereof The present invention relates to a conjugate or salt thereof represented by the following formula (1):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS represents a divalent group containing a cleavable site;
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • D represents a functional substance;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • a hyphen (-) indicates that the two units (e.g., atoms, groups) on either side of it are covalently bonded. If there is no unit on one side of the hyphen (-), the hyphen (-) indicates that it is a bond.
  • the antibody contains an immunoglobulin unit as described above.
  • examples of such antibodies include IgG antibodies, IgD antibodies, and IgE antibodies that contain one immunoglobulin unit that contains two heavy chains and two light chains and has disulfide bonds between the heavy chains and between the heavy and light chains, IgA antibodies that contain two of the above immunoglobulin units, and IgM antibodies that contain four of the above immunoglobulin units, with IgG antibodies (e.g., IgG1, IgG2, IgG3, IgG4) being preferred.
  • the antibody is preferably a human IgG monoclonal antibody, and more preferably a full-length human IgG monoclonal antibody.
  • the bond between the antibody and the linker site ( LA ) can be achieved via a functional group in the side chain of a specific amino acid residue in the Ig.
  • the functional group in the side chain of a specific amino acid residue in the Ig include an amino group in the side chain of a lysine residue in the Ig and a thiol group in the side chain of a cysteine residue in the Ig.
  • the bond can be achieved by a bond between the amino group in the side chain of the lysine residue and an atom or group (e.g., a carbonyl group) that can be bonded thereto.
  • the bond can be achieved by a bond between the thiol group in the side chain of the cysteine residue and an atom or group (e.g., a maleimide residue, a carbonyl group, a thiol group) that can be bonded thereto.
  • an atom or group e.g., a maleimide residue, a carbonyl group, a thiol group
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group.
  • the hydrophilic group and the monovalent group are as described above.
  • HG may represent a monovalent group containing a hydrophilic group.
  • CS represents a divalent group containing a cleavable site.
  • the cleavable site and the divalent group are as described above.
  • 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.
  • the divalent group represented by L A is a divalent group capable of linking Ig and a carbon atom adjacent to ring A.
  • the divalent group represented by L B is a divalent group capable of linking D and V.
  • the divalent groups represented by L A and L B may each contain a moiety generated by the reaction of two bioorthogonal functional groups capable of reacting with each other. Since combinations of two bioorthogonal functional groups capable of reacting with each other are well known, a person skilled in the art can appropriately select such combinations to appropriately set a divalent group containing a moiety generated by the reaction of two bioorthogonal functional groups capable of reacting with each other.
  • Examples of combinations of bioorthogonal functional groups capable of reacting with each other include 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 (a 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 by a substituent as described above) and an azide residue, a combination of a tetrazine residue and an alkene residue, a combination of a tetrazine residue and an alkyne residue, and a combination of a thiol residue and another thiol residue (disulfide bond).
  • 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.
  • the moiety may be a divalent group represented by any one of the following structural formulas: (Here, the white and black circles represent bonds.)
  • the bond represented by the black circle may be bonded to an atom present on the carbon atom side adjacent to ring A
  • the bond indicated by the open circle is bonded to an atom present on the carbon atom side adjacent to ring A
  • the bond indicated by the closed circle may be bonded to an atom present on the Ig binding portion side.
  • the bond represented by the black circle when 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), 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.
  • the divalent group represented by L A may be -L 1 -N(-R 1 )-CO-.
  • L 1 represents a divalent group
  • R 1 represents a hydrogen atom or a monovalent group
  • the hyphen on the left side of L 1 and the hyphen on the right side of CO represent bonds.
  • the bond of L 1 is bonded to Ig
  • the bond of CO is bonded to the carbon atom adjacent to ring A.
  • the divalent group represented by L B may be -L 2 -N(-R 2 )-CO-.
  • L 2 represents a divalent group
  • R 2 represents a hydrogen atom or a monovalent group
  • the hyphen on the left side of L 2 and the hyphen on the right side of CO represent bonds.
  • the bond of L 2 is bonded to the functional substance (D), and the bond of CO is bonded to V.
  • the divalent groups represented by L 1 and L 2 may each contain a moiety formed by the reaction of two bioorthogonal functional groups capable of reacting with each other.
  • the moiety formed by the reaction of two bioorthogonal functional groups capable of reacting with each other that may be contained in the divalent groups represented by L 1 and L 2 is similar to the moiety formed by the reaction of two bioorthogonal functional groups capable of reacting with each other that may be contained in the divalent groups represented by L A and L B as described above.
  • R 1 and R 2 each independently represent a hydrogen atom or a monovalent group.
  • the monovalent group is as described above.
  • the monovalent group in R 1 and R 2 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.
  • the alkyl the one described above is preferable.
  • the monovalent groups represented by R 1 and R 2 may be a protecting group for an amino group.
  • protecting groups include alkylcarbonyl groups (acyl groups) (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl groups), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl groups), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl groups).
  • alkylcarbonyl groups acyl groups
  • alkyloxycarbonyl groups e.g., fluorenylmethoxycarbonyl groups
  • aryloxycarbonyl groups e.g., fluorenylmethoxycarbonyl groups
  • aryloxycarbonyl groups e.g., benzyloxycarbonyl groups
  • R 1 and R 2 each independently represent a hydrogen atom or a protecting group for an amino group.
  • R 1 and R 2 each may be a hydrogen atom.
  • the functional substance represented by D is as described above.
  • n represents the average number of such bonds per immunoglobulin unit containing two heavy chains and two light chains, and is 1.5 or more.
  • such average number may be 1.6 or more, preferably 1.7 or more, more preferably 1.8 or more, even more preferably 1.9 or more.
  • Such average number may also be 2.4 or less, preferably 2.3 or less, more preferably 2.2 or less, even more preferably 2.1 or less. Particularly preferably, such average number may be 2.0.
  • such average number may be, for example, 2.0 or more, preferably 4.0 or more, more preferably 6.0 or more, even more preferably 7.0 or more, particularly preferably 7.5 or more, or 7.8 or more.
  • Such average number may also be 8.0 or less. More specifically, such 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.
  • L A may be a divalent group having a side chain containing a hydrophilic group or a functional substance.
  • the side chain containing a hydrophilic group or a functional substance may be a side chain containing a hydrophilic group and a functional substance.
  • the side chain containing a hydrophilic group and a functional substance may be a side chain containing (i) a hydrophilic group, and (ii) a functional substance, and (iii) a cleavable site between the hydrophilic group and the functional substance.
  • the number of such side chains contained in the divalent group is not particularly limited, and may be, for example, 1 to 4, 1 to 3, 1 or 2, or 1.
  • (i) the hydrophilic group, and (ii) the functional material, and (iii) the side chain comprising a cleavable site between the hydrophilic group and the functional material are represented by the following formula ( ⁇ ): [Wherein, A wavy hyphen (-) represents a bond to the main chain in L A that connects Ig to a carbon atom in formula (1), HG ⁇ represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS ⁇ represents a divalent group containing a cleavable site; V ⁇ represents an oxygen atom, a sulfur atom, or an amino group (NH); L A ⁇ and L B ⁇ each independently represent a divalent group; D ⁇ represents a functional substance.
  • HG ⁇ represents a hydrophilic group or a monovalent group containing a hydrophilic group.
  • the hydrophilic group and the monovalent group are as described above.
  • the hydrophilic group represented by HG ⁇ or the monovalent group containing a hydrophilic group may be the same as or different from the hydrophilic group represented by HG or the monovalent group containing a hydrophilic group.
  • CS ⁇ represents a divalent group containing a cleavable site.
  • the cleavable site and the divalent group are as described above.
  • the divalent group containing a cleavable site represented by CS ⁇ may be the same as or different from the divalent group containing a cleavable site represented by CS.
  • V ⁇ represents an oxygen atom, a sulfur atom, or an amino group (NH).
  • the type of V ⁇ may be the same as or different from the type of V.
  • V ⁇ may be a sulfur atom or an amino group (NH).
  • V ⁇ is preferably an oxygen atom or a sulfur atom, and more preferably an oxygen atom.
  • the divalent group represented by L A ⁇ is a divalent group having a bond to the main chain in L A that connects Ig and a carbon atom.
  • the divalent group represented by L A ⁇ may be the same as or different from the divalent group represented by L A.
  • the divalent group represented by L B ⁇ is a divalent group that can link D ⁇ and V ⁇ .
  • the divalent group represented by L B ⁇ may be the same as or different from the divalent group represented by L B.
  • the divalent groups represented by L A ⁇ and L B ⁇ may contain a moiety generated by the reaction of two bioorthogonal functional groups that can react with each other, similar to the divalent groups represented by L A and L B , respectively.
  • D ⁇ represents a functional substance.
  • the functional substance is as described above.
  • the functional substance represented by D ⁇ may be the same as or different from the functional substance represented by D.
  • the present invention also relates to a compound represented by the following formula (1'):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • D represents a functional substance;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • Ig, HG, V, L.sub.A , L.sub.B , D, and n are the same as those in formula (1).
  • CS' represents a divalent group containing a cleavable moiety.
  • the cleavable moiety and the divalent group are as described above.
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -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, so long as cleavage occurs between V and the adjacent carbon atom due to ⁇ -electron conjugation when the cleavable site is cleaved (see FIG. 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 the cleavable site and the divalent aromatic ring group.
  • 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.
  • ring A may be a divalent six-membered aromatic ring group.
  • the six-membered aromatic ring group include the various groups described above.
  • the position of the divalent six-membered aromatic ring group to which the two adjacent atoms are bonded is the ortho position or the para position, and preferably the para position.
  • ring A may be a phenylene group which may have a substituent.
  • the position of the phenylene group to which the two adjacent atoms are bonded is the ortho position or the para position, preferably the para position.
  • substituents in the optionally substituted divalent aromatic ring group are as described above.
  • Such a substituent may be an electron-withdrawing group as described above.
  • L A may be a divalent group having a side chain containing a hydrophilic group or a functional substance.
  • the side chain containing a hydrophilic group or a functional substance may be a side chain containing a hydrophilic group and a functional substance.
  • the side chain containing a hydrophilic group and a functional substance may be a side chain containing (i) a hydrophilic group, and (ii) a functional substance, and (iii) a cleavable site between the hydrophilic group and the functional substance.
  • the number of such side chains contained in the divalent group is not particularly limited, and may be, for example, 1 to 4, 1 to 3, 1 or 2, or 1.
  • (i) the hydrophilic group, and (ii) the functional material, and (iii) the side chain comprising a cleavable site between the hydrophilic group and the functional material are represented by the following formula ( ⁇ ′): [Wherein, A wavy hyphen (-) represents a bond to the main chain in L A which connects Ig to the carbon atom in formula (1'), HG ⁇ represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS ⁇ ′ represents a divalent group containing a cleavable site; Ring A ⁇ represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), V ⁇ represents an oxygen atom, a sulfur atom, or an amino group (NH); L A ⁇ and L B ⁇ each independently represent a divalent group; D ⁇ represents a functional substance.
  • HG ⁇ , V ⁇ , L A ⁇ , L B ⁇ , and D ⁇ in formula ( ⁇ ') are the same as those shown in formula ( ⁇ ).
  • CS ⁇ ' represents a divalent group containing a cleavable site.
  • the cleavable site and the divalent group are as described above.
  • the divalent group containing a cleavable site represented by CS ⁇ ' may be the same as or different from the divalent group containing a cleavable site represented by CS'.
  • Ring A ⁇ represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system with the cleavable site).
  • the divalent aromatic ring group which may have a substituent is as described above.
  • the divalent aromatic ring group which may have a substituent represented by ring A ⁇ may be the same as or different from the divalent aromatic ring group which may have a substituent represented by ring A.
  • the present invention also relates to a compound represented by the following formula (1′′):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • R 1 and R 2 each independently represent a hydrogen atom or a monovalent group;
  • L 1 and L 2 each independently represent a divalent group;
  • D represents a functional substance;
  • the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • R 1 and R 2 each independently represent a hydrogen atom or a monovalent group.
  • the monovalent group is as described above.
  • the monovalent group in R 1 and R 2 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.
  • the alkyl the one described above is preferable.
  • the monovalent groups represented by R 1 and R 2 may be a protecting group for an amino group.
  • protecting groups include alkylcarbonyl groups (acyl groups) (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl groups), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl groups), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl groups).
  • alkylcarbonyl groups acyl groups
  • alkyloxycarbonyl groups e.g., fluorenylmethoxycarbonyl groups
  • aryloxycarbonyl groups e.g., fluorenylmethoxycarbonyl groups
  • aryloxycarbonyl groups e.g., benzyloxycarbonyl groups
  • R 1 and R 2 each independently represent a hydrogen atom or a protecting group for an amino group.
  • R 1 and R 2 each may be a hydrogen atom.
  • the divalent group represented by L 1 is a divalent group that can link Ig and a nitrogen atom.
  • the divalent group represented by L 2 is a divalent group that can link D and a nitrogen atom.
  • the divalent groups represented by L 1 and L 2 may each contain a moiety generated by the reaction of two bioorthogonal functional groups that can react with each other.
  • the divalent groups represented by L 1 and L 2 may contain a moiety generated by the reaction of two bioorthogonal functional groups that can react with each other, which is similar to the moiety generated by the reaction of two bioorthogonal functional groups that can react with each other as described above, which may be contained in the divalent groups represented by L A and L B.
  • the conjugate of an antibody and a functional substance or a salt thereof may be one in which a lysine residue of the antibody is modified.
  • the functional group in the side chain of the specific amino acid residue may be an amino group in the side chain of the lysine residue, and n may be 1.5 to 2.5.
  • L A has a carbonyl group, and the bond is achieved by an amide bond formed by the bond between the amino group in the side chain of the lysine residue and the carbonyl group in L A.
  • the lysine residue to be modified is a lysine residue located at positions 246/248, 288/290 or 317 according to Eu numbering, preferably a lysine residue located at positions 246/248 or 288/290 according to Eu numbering.
  • the structural unit represented by the above formula (1) is a structural unit represented by the following formula (I): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS1 represents a divalent group containing a cleavable site; V1 represents an oxygen atom, a sulfur atom, or an amino group (NH); L A1 and L B1 each independently represent a divalent group; D1 represents a functional substance; The average number of bonds per immunoglobulin unit, r, is between 1.5 and 2.5.
  • the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue located at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue in formula (1) having an amino group to which L A is bonded).
  • the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue located at position 246/248, or 288/290 according to Eu numbering (wherein the lysine residue is different from the lysine residue in formula (1) having an amino group to which L A is bonded).
  • HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group.
  • the hydrophilic group or the monovalent group containing a hydrophilic group is as described above.
  • the hydrophilic group or the monovalent group containing a hydrophilic group represented by HG 1 may be the same as or different from the hydrophilic group or the monovalent group containing a hydrophilic group represented by HG in the above formula (1).
  • CS 1 represents a divalent group containing a cleavable site.
  • the divalent group containing a cleavable site is as described above.
  • the divalent group containing a cleavable site represented by CS 1 may be the same as or different from the divalent group containing a cleavable site represented by CS in the above formula (1).
  • V1 represents an oxygen atom, a sulfur atom, or an amino group (NH).
  • the type of V1 may be the same as or different from the type of V.
  • V1 when V is an oxygen atom, V1 may be a sulfur atom or an amino group (NH).
  • V1 is preferably an oxygen atom or a sulfur atom, and more preferably an oxygen atom.
  • L A1 and L B1 each independently represent a divalent group.
  • the divalent groups represented by L A1 and L B1 may be the same as or different from the divalent groups represented by L A and L B , respectively.
  • the divalent groups represented by L A1 and L B1 may contain a moiety generated by the reaction of two bioorthogonal functional groups capable of reacting with each other, similar to the divalent groups represented by L A and L B , respectively.
  • D1 represents a functional substance.
  • the functional substance is as described above.
  • the functional substance represented by D1 may be the same as or different from the functional substance represented by D in the above formula (1).
  • the average number r of bonds per immunoglobulin unit indicates the average number of bonds per immunoglobulin unit containing two heavy chains and two light chains and is between 1.5 and 2.5. Such an average number may be 1.6 or more, 1.7 or more, 1.8 or more, or 1.9 or more. Such an average number may also be 2.4 or less, 2.3 or less, 2.2 or less, or 2.1 or less. Particularly preferably, such an average number may be 2.0.
  • the structural unit represented by the above formula (1') is a structural unit represented by the following formula (I'): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), V1 represents an oxygen atom, a sulfur atom,
  • CS 1 ' represents a divalent group containing a cleavable site.
  • the cleavable site and the divalent group are as described above.
  • the functional substance represented by D 1 may be the same as or different from the functional substance represented by D in the above formula (1 ').
  • Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system with the cleavable site).
  • the divalent aromatic ring group which may have a substituent is as described above.
  • the divalent aromatic ring group which may have a substituent and is represented by ring A1 may be the same as or different from the divalent aromatic ring group which may have a substituent and is represented by ring A in formula (1') above.
  • the structural unit represented by the above formula (1′′) is a structural unit represented by the following formula (I′′): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), R 11 and R 21 each independently represent a hydrogen atom or
  • R 11 and R 21 each independently represent a hydrogen atom or a monovalent group.
  • the monovalent group is as described above.
  • the hydrogen atom or monovalent group represented by R 11 and R 21 may be the same as or different from those represented by R 1 and R 2 in the above formula (1 '').
  • L 11 and L 21 each independently represent a divalent group.
  • the divalent group is as described above.
  • the divalent group represented by L 11 and L 21 may be the same as or different from the divalent group represented by L 1 and L 2 in the above formula (1 '').
  • the conjugate of an antibody and a functional substance or a salt thereof may be one in which a cysteine residue in the antibody (generally, an immunoglobulin unit contains eight cysteine residues) is modified.
  • the functional group in the side chain of the specific amino acid residue may be a thiol group in the side chain of the cysteine residue, and n may be 2.0 to 8.0. n may preferably be 3.0 or more, 3.5 or more, 4.0 or more, 4.5 or more, 5.0 or more, 5.5 or more, 6.0 or more, 6.5 or more, 7.0 or more, or 7.5 or more.
  • the conjugate or salt thereof of the present invention has the desired property of being less likely to aggregate, and can therefore be characterized 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).
  • the conjugate of the present invention or its salt is useful, for example, as a medicine or a reagent (e.g., a diagnostic agent or a research reagent).
  • a medicine or a reagent e.g., a diagnostic agent or a research reagent.
  • the conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition.
  • a pharmaceutical composition may contain, in addition to the conjugate of the present invention or a salt thereof, a pharma- ceutically acceptable carrier.
  • medicamentously 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 hydrogen carbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin am
  • Preferred formulations for oral administration include liquid preparations in which an effective amount of the ligand is dissolved in a diluent such as water, saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granules; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.
  • a diluent such as water, saline, or orange juice
  • suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium
  • emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a
  • compositions suitable for 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.
  • 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. of the subject, but can be set appropriately.
  • the conjugate or salt thereof of the present invention can be produced by reacting an antibody derivative or salt thereof having a bioorthogonal functional group with a functional substance ( Figure 3). Such a reaction can proceed by a reaction between the bioorthogonal functional group in the antibody derivative and the functional substance.
  • the functional group of the functional substance can be appropriately reacted with the bioorthogonal functional group in the antibody derivative.
  • the functional group that easily reacts with the bioorthogonal functional group may vary depending on the specific type of the bioorthogonal functional group.
  • a person skilled in the art can appropriately select an appropriate functional group as a functional group that easily reacts with the bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195).
  • Examples of functional groups that easily react with bioorthogonal functional groups include, but are not limited to, 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 a 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.
  • the above combinations of bioorthogonal functional groups and functional groups that easily 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 easily reacts with the bioorthogonal functional group can be used.
  • 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. 2006/0074008, U.S. 2005/0238649).
  • derivatization may be performed using any crosslinking agent.
  • derivatization may be performed using a specific linker that has the desired functional group.
  • the derivatized functional substance is simply referred to as a "functional substance" since it is merely one type of functional substance.
  • 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).
  • 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 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. For details of such a reaction, see, for example, G. J. L.
  • the conjugate or salt of the present invention can be produced by reacting a compound or salt having a bioorthogonal functional group and a functional substance with a starting antibody having an Ig (immunoglobulin unit) ( Figure 3).
  • a compound or salt having a bioorthogonal functional group and a functional substance with a starting antibody having an Ig (immunoglobulin unit) ( Figure 3).
  • an antibody having an Ig (immunoglobulin unit) can be used as the starting antibody.
  • an antibody containing an immunoglobulin unit composed of two heavy chains and two light chains
  • an antibody containing a thiol group can be prepared as the starting antibody by contacting the antibody with a reducing agent to generate an antibody containing a thiol group.
  • a reducing agent any reducing agent that can cleave a disulfide bond to generate a thiol group can be used, and examples of the reducing agent include tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, and ⁇ -mercaptoethanol.
  • the bioorthogonal functional group possessed by the compound is preferably a group capable of efficiently reacting with a thiol group (e.g., maleimide residue, halocarbonyl group, thiol group).
  • a thiol group e.g., maleimide residue, halocarbonyl group, thiol group.
  • the reaction between the compound having a bioorthogonal functional group and a functional substance or its salt and the above-mentioned raw 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).
  • the production of the conjugate or its salt can be confirmed by, for example, reverse-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, reverse-phase column chromatography, high-performance liquid chromatography, affinity chromatography).
  • Antibody Derivative or Salt thereof also relates to an antibody derivative or a salt thereof represented by the following formula (2):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS represents a divalent group containing a cleavable site;
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • B2 represents a bioorthogonal functional group; wherein the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • the bioorthogonal functional group represented by B2 is as described above.
  • the bioorthogonal functional group represented by B2 may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.
  • the bioorthogonal functional group represented by B2 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 they have excellent reaction efficiency and are highly versatile.
  • L A may be a divalent group having a side chain containing a hydrophilic group or a functional substance.
  • the side chain containing a hydrophilic group or a functional substance may be a side chain containing a hydrophilic group and a functional substance.
  • the side chain containing a hydrophilic group and a functional substance may be a side chain containing (i) a hydrophilic group, and (ii) a functional substance, and (iii) a cleavable site between the hydrophilic group and the functional substance.
  • the number of such side chains contained in the divalent group is not particularly limited, and may be, for example, 1 to 4, 1 to 3, 1 or 2, or 1.
  • (i) the hydrophilic group, and (ii) the functional material, and (iii) the side chain comprising a cleavable site between the hydrophilic group and the functional material are represented by the following formula ( ⁇ ): [Wherein, A wavy hyphen (-) represents a bond to the main chain in L A that connects Ig to a carbon atom in formula (1), HG ⁇ represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS ⁇ represents a divalent group containing a cleavable site; 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 2 ⁇ represents a bioorthogonal functional group.
  • HG ⁇ represents a hydrophilic group or a monovalent group containing a hydrophilic group.
  • the hydrophilic group and the monovalent group are as described above.
  • the hydrophilic group represented by HG ⁇ or the monovalent group containing a hydrophilic group may be the same as or different from the hydrophilic group represented by HG or the monovalent group containing a hydrophilic group.
  • CS ⁇ represents a divalent group containing a cleavable site.
  • the cleavable site and the divalent group are as described above.
  • the divalent group containing a cleavable site represented by CS ⁇ may be the same as or different from the divalent group containing a cleavable site represented by CS.
  • V ⁇ represents an oxygen atom, a sulfur atom, or an amino group (NH).
  • the type of V ⁇ may be the same as or different from the type of V.
  • V ⁇ may be a sulfur atom or an amino group (NH).
  • V ⁇ is preferably an oxygen atom or a sulfur atom, and more preferably an oxygen atom.
  • the divalent group represented by L A ⁇ is a divalent group having a bond to the main chain in L A that links Ig and a carbon atom.
  • the divalent group represented by L A ⁇ may be the same as or different from the divalent group represented by L A.
  • the divalent group represented by L B ⁇ is a divalent group that can link D ⁇ and V ⁇ .
  • the divalent group represented by L B ⁇ may be the same as or different from the divalent group represented by L B.
  • the divalent groups represented by L A ⁇ and L B ⁇ may contain a moiety generated by the reaction of two bioorthogonal functional groups that can react with each other, similar to the divalent groups represented by L A and L B , respectively.
  • B 2 ⁇ represents a functional substance.
  • the functional substance is as described above.
  • the functional substance represented by D ⁇ may be the same as or different from the functional substance represented by D.
  • the present invention also relates to a compound represented by the following formula (2'):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • V represents an oxygen atom, a sulfur atom, or an amino group (NH);
  • L A and L B each independently represent a divalent group;
  • B2 represents a bioorthogonal functional group; wherein the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • L A may be a divalent group having a side chain containing a hydrophilic group or a functional substance.
  • the side chain containing a hydrophilic group or a functional substance may be a side chain containing a hydrophilic group and a functional substance.
  • the side chain containing a hydrophilic group and a functional substance may be a side chain containing (i) a hydrophilic group, and (ii) a functional substance, and (iii) a cleavable site between the hydrophilic group and the functional substance.
  • the number of such side chains contained in the divalent group is not particularly limited, and may be, for example, 1 to 4, 1 to 3, 1 or 2, or 1.
  • (i) the hydrophilic group, and (ii) the functional material, and (iii) the side chain comprising a cleavable site between the hydrophilic group and the functional material has the following formula ( ⁇ ′): [Wherein, A wavy hyphen (-) represents a bond to the main chain in L A which connects Ig to the carbon atom in formula (1'), HG ⁇ represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS ⁇ ' represents a divalent group containing a cleavable site; Ring A ⁇ represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), 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 2 ⁇ represents a bioorthogonal functional group.
  • CS ⁇ ' represents a divalent group containing a cleavable site.
  • the cleavable site and the divalent group are as described above.
  • the divalent group containing a cleavable site represented by CS ⁇ ' may be the same as or different from the divalent group containing a cleavable site represented by CS'.
  • Ring A ⁇ represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system with the cleavable site).
  • the divalent aromatic ring group which may have a substituent is as described above.
  • the divalent aromatic ring group which may have a substituent represented by ring A ⁇ may be the same as or different from the divalent aromatic ring group which may have a substituent represented by ring A.
  • the present invention also relates to a compound represented by the following formula (2′′):
  • Ig refers to an immunoglobulin unit that contains two heavy chains and two light chains, and is linked to adjacent L A via functional groups in the side chains of specific amino acid residues in Ig;
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group,
  • CS' represents a divalent group containing a cleavable site;
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site);
  • R 1 and R 2 each independently represent a hydrogen atom or a monovalent group;
  • L 1 and L 2 each independently represent a divalent group;
  • B2 represents a bioorthogonal functional group; wherein the average number n of bonds per immunoglobulin unit is 1.5 or more.
  • Ig, HG, B 2 and n in formula (2'') are each the same as those shown in formula (1).
  • CS' and ring A in formula (2'') are each the same as those shown in formula (1').
  • R 1 , R 2 , L 1 and L 2 in formula (2'') are each the same as those shown 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.
  • the antibody derivative or salt thereof may have a modified lysine residue in the antibody.
  • the functional group in the side chain of the specific amino acid residue may be an amino group in the side chain of the lysine residue, and n may be 1.5 to 2.5.
  • L A has a carbonyl group, and the bond is achieved by an amide bond formed by the bond between the amino group in the side chain of the lysine residue and the carbonyl group in L A.
  • the modified lysine residue is a lysine residue located at positions 246/248, 288/290 or 317 according to Eu numbering, preferably a lysine residue located at positions 246/248 or 288/290 according to Eu numbering.
  • the structural unit represented by the above formula (2) is a structural unit represented by the following formula (II): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS1 represents a divalent group containing a cleavable site; V1 represents an oxygen atom, a sulfur atom, or an amino group (NH); L A1 and L B1 each independently represent a divalent group; B 21 represents a bioorthogonal functional group; The average number of bonds per immunoglobulin unit, r, is 1.5 to 2.5.
  • B 21 represents a bioorthogonal functional group.
  • the bioorthogonal functional group is as described above.
  • the bioorthogonal functional group represented by B 21 may be the same as or different from the bioorthogonal functional group represented by B 21 in the above formula (2).
  • the structural unit represented by the above formula (2') is a structural unit represented by the following formula (II'): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), V1 represents an oxygen atom, a sulfur atom
  • the structural unit represented by the above formula (2′′) is a structural unit represented by the following formula (II′′): [Wherein, the wavy hyphen (-) is bonded to an amino group in the side chain of a lysine residue at position 246/248, 288/290, or 317 according to Eu numbering (wherein the lysine residue is different from the lysine residue having the amino group to which L A is bonded in formula (1)); HG 1 represents a hydrophilic group or a monovalent group containing a hydrophilic group; CS 1 ' represents a divalent group containing a cleavable site; Ring A1 represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site), R 11 and R 21 each independently represent a hydrogen atom
  • the antibody derivative or salt thereof of the present invention is useful, for example, as an intermediate for producing the conjugate or salt thereof of the present invention.
  • 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.
  • reaction between the compound having the first and second bioorthogonal functional groups or their salts 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).
  • the production of the antibody derivative or its salt can be confirmed in the same manner as described for the conjugate of the present invention.
  • the antibody derivative or its salt can be appropriately purified by any purification method such as those described for the conjugate of the present invention.
  • a compound having a bioorthogonal functional group and a functional substance, or a salt thereof 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 cleavable site; V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B1 represents a bioorthogonal functional group; and D represents a functional substance.], or a salt thereof.
  • B1 represents a bioorthogonal functional group.
  • the bioorthogonal functional group is as described above.
  • the present invention also relates to 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B1 represents a bioorthogonal functional group; and D represents a functional substance.], or a salt thereof.
  • 3' 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 site; Ring A represents a divalent aromatic ring group
  • HG, CS', ring A, V, L A , L B , and D are as described above in formula (1').
  • B 1 is as described above in formula (3).
  • the present invention also relates to 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); R 1 and R 2 each independently represent a hydrogen atom or a monovalent group; L 1 and L 2 each independently represent a divalent group; B1 represents a bioorthogonal functional group; and D represents a functional substance.], or a salt thereof.
  • HG, CS′, ring A, R 1 , R 2 , L 1 , L 2 , and D are as described above in formula (1′′).
  • B 1 is as described above in formula (3).
  • the compounds represented by formula (3), formula (3'), and formula (3") or salts thereof are useful, for example, as intermediates for producing the conjugates of the present invention.
  • the compounds represented by formula (3), formula (3'), and formula (3") or salts thereof are 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).
  • the compound or its salt having a bioorthogonal functional group and a functional substance can be produced, for example, by reacting a compound or its salt 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.
  • the reaction of the compound having the first and second bioorthogonal functional groups or a salt thereof with the 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) system, 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.
  • 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.
  • 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).
  • a compound having a first bioorthogonal functional group and a second bioorthogonal functional group, or a salt thereof 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 cleavable site; V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B1 represents a first bioorthogonal functional group; and B1 represents a second bioorthogonal functional group.], or a salt thereof.
  • B2 represents a second bioorthogonal functional group.
  • the second bioorthogonal functional group is the same as described above for the bioorthogonal functional group.
  • the bioorthogonal functional group represented by B2 may be a maleimide residue, a thiol residue, a furan residue, a halocarbonyl residue, an alkene residue, an alkyne residue, an azide residue, or a tetrazine residue.
  • the bioorthogonal functional group represented by B2 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 they have excellent reaction efficiency and are highly versatile.
  • 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.
  • the intermolecular reaction 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 a combination that does not react with each other or has low reactivity with each other.
  • Such combinations of bioorthogonal functional groups are well known in the art.
  • examples of such combinations for the preferred bioorthogonal functional groups maleimide residues, thiol residues, furan residues, halocarbonyl residues, alkene residues, alkyne residues, azide residues, and tetrazine residues, are as follows:
  • the present invention also relates to 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; B1 represents a first bioorthogonal functional group; and B1 represents a second bioorthogonal functional group.], or a salt thereof.
  • formula (4') wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS' represents a divalent group containing a cleavable site; Ring A represents a divalent aromatic ring group which may have
  • HG, CS', ring A, V, L A , L B , and B 2 are as described above in formula (2').
  • B 1 is as described above in formula (4).
  • the present invention also relates to 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); R 1 and R 2 each independently represent a hydrogen atom or a monovalent group; L 1 and L 2 each independently represent a divalent group; B1 represents a first bioorthogonal functional group; and B1 represents a second bioorthogonal functional group.], or a salt thereof, comprising a first bioorthogonal functional group and a second bioorthogonal functional group.
  • HG, CS′, ring A, R 1 , R 2 , L A1 , L B1 , and B 2 are as described above in formula (2′′).
  • B 1 is as described above in formula (4).
  • the compounds represented by formulae (4), (4') and (4" or salts thereof are useful, for example, as intermediates for producing the antibody derivatives of the present invention and the compounds represented by formulae (3), (3') and (3").
  • the compounds represented by formulae (4), (4') and (4" or salts thereof are 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, a lipid), and a functional substance.
  • a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof can be prepared by reacting a compound of formula (6) having a bioorthogonal functional group or a salt thereof with a suitable compound having B 1 (e.g., a compound represented by B 1 -L 1 -NH-R 1 ) (FIGS. 4 and 5).
  • B 1 e.g., a compound represented by B 1 -L 1 -NH-R 1
  • a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof can be prepared by reacting a compound of formula (7) having a bioorthogonal functional group or a salt thereof with an appropriate compound having L B -B 2 (e.g., by reacting with bis(4-nitriphenyl)carbonate and N,N-diisopropylethylamine (DIPEA) and then reacting with a compound represented by B 2 -L 2 -NH-R 2 ) ( Figures 4 and 5).
  • DIPEA bis(4-nitriphenyl)carbonate and N,N-diisopropylethylamine
  • the above reaction can be carried out in a suitable reaction system, such as an organic solvent system or an aqueous solution (e.g., a buffer solution) system, at a suitable temperature (e.g., about 15 to 200°C).
  • a suitable reaction system such as an organic solvent system or an aqueous solution (e.g., a buffer solution) system, at a suitable temperature (e.g., about 15 to 200°C).
  • the reaction system may contain a suitable catalyst.
  • the reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.
  • such a reaction can also be carried out under the mild conditions described above.
  • 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 materials and the molecular weight of the product.
  • 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).
  • a Series of Compounds or Salts Thereof (1) Compound or Salt Thereof
  • the present invention also relates to a compound 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 site; V represents an oxygen atom, a sulfur atom, or an amino group (NH); Each L A independently represents a divalent group, X and Y each independently represent a monovalent group; or a salt thereof.
  • HG, CS, V, and LA 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).
  • X and Y each independently represent a monovalent group.
  • the monovalent group is as described above.
  • the present invention also relates to a compound 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); Each L A independently represents a divalent group, X and Y each independently represent a monovalent group; or a salt thereof.
  • the present invention also relates to a compound 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); X and Y each independently represent a monovalent group; or a salt thereof.
  • 5′′ a compound 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 site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); X and
  • 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, X and Y each independently represent a monovalent group.
  • HG, CS, V, L A , L B , and B 2 are as described above in formula (2).
  • X is the same as described above in formula (5).
  • the present invention also relates to a compound represented by the following formula (6'): [Wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS' represents a divalent group containing a cleavable site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A and L B each independently represent a divalent group; X represents a monovalent group; and B2 represents a bioorthogonal functional group.] or a salt thereof.
  • the compounds represented by formulas (6), (6'), and (6'') or salts thereof are useful, for example, as synthetic intermediates for the conjugates, antibody derivatives, and specific compounds of the present invention. Such compounds or salts thereof are also useful, for example, for derivatizing functional substances.
  • the compounds represented by formulas (6), (6'), and (6'') or salts thereof can be prepared, for example, by reacting the compounds represented by formulas (5), (5'), and (5'') or salts thereof with an appropriate compound having L B -B 2 (e.g., by reacting with bis(4-nitriphenyl) carbonate and N,N-diisopropylethylamine (DIPEA), and then reacting with a compound represented by B 2 -L 2 -NH-R 2) (FIGS. 4 and 5).
  • L B -B 2 e.g., by reacting with bis(4-nitriphenyl) carbonate and N,N-diisopropylethylamine (DIPEA), and then reacting with a compound represented by B 2 -L 2 -NH-R 2) (FIGS. 4 and 5).
  • DIPEA bis(4-nitriphenyl) carbonate and N,N-diisopropylethyl
  • a compound having a bioorthogonal functional group represented by formula (7) or a salt thereof 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 cleavable site; V represents an oxygen atom, a sulfur atom, or an amino group (NH); Each L A independently represents a divalent group, B1 represents a bioorthogonal functional group; and Y represents a monovalent group.], or a salt thereof.
  • HG, CS, V, L A and B 1 are as described above in formula (3).
  • Y is as described above in formula (5).
  • the present invention also relates to a compound represented by the following formula (7'): [Wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS' represents a divalent group containing a cleavable site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); L A represents a divalent group, B1 represents a bioorthogonal functional group; and Y represents a monovalent group.], or a salt thereof.
  • HG represents a hydrophilic group or a monovalent group containing a hydrophilic group
  • CS' represents a divalent group containing a cleavable site
  • Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms
  • the present invention also relates to a compound represented by the following formula (7′′): [Wherein, HG represents a hydrophilic group or a monovalent group containing a hydrophilic group, CS' represents a divalent group containing a cleavable site; Ring A represents a divalent aromatic ring group which may have a substituent (wherein the divalent aromatic ring group forms a ⁇ -electron conjugated system together with the cleavable site); V represents an oxygen atom, a sulfur atom, or an amino group (NH); R 1 represents a hydrogen atom or a monovalent group; L 1 represents a divalent group; B1 represents a bioorthogonal functional group; and Y represents a monovalent group.], or a salt thereof.
  • the compounds represented by formulas (7), (7'), and (7'') or salts thereof are useful, for example, as synthetic intermediates for the conjugates, antibody derivatives, and certain compounds of the present invention. Such compounds or salts thereof are 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).
  • a biomolecule e.g., a protein such as an antibody, a sugar, a nucleic acid, or a lipid.
  • Compounds represented by formulas (7), (7'), and (7'') or salts thereof can be produced, for example, by reacting compounds represented by formulas (5), (5'), and (5'') or salts thereof with an appropriate compound having B 1 (e.g., a compound represented by B 1 -L 1 -NH-R 1 ) (FIGS. 4 and 5).
  • B 1 e.g., a compound represented by B 1 -L 1 -NH-R 1
  • Such reactions can be carried out under the same reaction conditions as those described above for the production of a compound having a first bioorthogonal functional group and a second bioorthogonal functional group or a salt thereof.
  • methyl 4-aminomandelate (8.63 mg, 47.6 ⁇ mol) was added, and the mixture was stirred at room temperature for 21.5 hours, and then 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 (2) (28.5 mg, quant).
  • the alcohol (2) (28.5 mg) obtained in (1-1-1) was dissolved in N,N-dimethylformamide (430 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (26.6 mg, 85.7 ⁇ mol) and N,N-diisopropylethylamine (11.1 ⁇ L, 64.4 ⁇ mol) were added and stirred at room temperature for 1.5 hours.
  • the reaction was tracked by LCMS and it was found that the raw materials remained, so bis(4-nitrophenyl)carbonate (13.3 mg, 42.9 ⁇ mol) and N,N-diisopropylethylamine (5.54 ⁇ L, 32.2 ⁇ mol) were added and stirred at room temperature for 5 hours.
  • Alcohol (7) (44.6 mg, 58.9 ⁇ mol) was dissolved in N,N-dimethylformamide (650 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (53.8 mg, 177 ⁇ mol) and N,N-diisopropylethylamine (22.5 ⁇ L, 133 ⁇ mol) were added and stirred at room temperature for 4 hours.
  • methyl 4-aminomandelate (15.8 mg, 87.4 ⁇ mol) was added and the mixture was stirred at room temperature for 16 hours, after which it 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 above alcohol (12) (54.0 mg, 63.5 ⁇ mol).
  • Alcohol (12) (50.3 mg, 59.2 ⁇ mol) was dissolved in N,N-dimethylformamide (650 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (54.0 mg, 178 ⁇ mol) and N,N-diisopropylethylamine (22.7 ⁇ L, 133 ⁇ mol) were added and stirred at room temperature for 5 hours.
  • Linker-payload mimic (16) was synthesized as follows.
  • Alcohol (17) (58.0 mg, 74.5 ⁇ mol) was dissolved in N,N-dimethylformamide (820 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (68.0 mg, 223 ⁇ mol) and N,N-diisopropylethylamine (28.5 ⁇ L, 168 ⁇ mol) were added and stirred at room temperature for 6 hours.
  • the reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove the acetonitrile, and then freeze-dried to obtain Linker-payload mimic (16) (7.5 mg, 6.6 ⁇ mol).
  • the reaction solution was purified by reversed-phase preparative chromatography, and the fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain Linker-payload mimic (21) (1.1 mg, 1.1 ⁇ mol).
  • Linker-Payload (26) was synthesized according to the following scheme.
  • Example 2 Synthesis of ADC mimic (2-1) Synthesis of ADC mimic
  • 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 is regioselectively introduced into trastuzumab (humanized IgG1 antibody) via the amino group of the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue is in accordance with EU numbering).
  • NH-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 - extending from the antibody heavy chain corresponds to the side chain of a lysine residue, and a thiol-containing group, HS-CH 2 -CH 2 -C( ⁇ O), is added to the amino group in the side chain of this lysine residue. Since no modification with other lysine residues was detected by peptide mapping method, it is considered that the position selectivity at positions 246 and 248 of the antibody heavy chain is 100%.
  • Example 2-1-1 Synthesis of ADC mimic 1
  • 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 was performed, and the reaction product was confirmed to have a peak at 150350 where two Linker-payload mimics (1) were introduced.
  • Example 2-1-2 Synthesis of ADC mimic 2 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 was performed, and the reaction product was confirmed to have peaks at 150535 where two Linker-payload mimics (6) were introduced.
  • Example 2-1-3 Synthesis of ADC mimic 3 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.
  • Example 2-1-4 Synthesis of ADC mimic 4 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) were introduced.
  • Comparative Example 2-1-5 Synthesis of ADC mimic 5 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) were introduced.
  • 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). The measurement was performed under the following conditions. The hydrophobicity of the ADC can be evaluated by the retention time of the ADC in the HIC chromatogram.
  • HIC-HPLC hydrophobic column chromatography
  • 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.
  • Measurement system 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300 ⁇ 2.7 ⁇ m, 4.6 mm ⁇ 150 mm Flow rate: 0.25 mL/min. Eluent: 100 mM sodium dihydrogen phosphate/sodium hydrogen phosphate, 250 mM sodium chloride in water (pH 6.8), 10% v/v isopropanol. Detector: UV (280 nm).
  • Example 5 Evaluation of ADC mimics using enzyme cathepsin B The cleavage ability of various ADC mimics with cathepsin B was evaluated by analyzing the amount of fluorescent molecules shed from the ADC mimic, as described below.
  • 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 of 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 being taken as 100%.
  • the synthesized ADC mimic was found to have sufficient cathepsin B cleavage.
  • Example 6 Evaluation of ADC mimic using mouse plasma (6-1) Plasma stability test of ADC mimic ADC mimic was added to 500 ⁇ L of mouse plasma (Charles River) to a concentration of 0.1 mg/mL, and then sterilized by filtration. This solution was dispensed into six Eppendorf tubes at 50 ⁇ L each. 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. Each sample was added with 100 ⁇ L of acetonitrile, stirred with a vortex, and centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.
  • the loss rate of fluorescent molecules was calculated according to Example 5-2.
  • the loss rate of fluorescent molecules was evaluated as shown in the table below.
  • the ADC mimics synthesized in Examples 2-1-1 and 2-1-2 were more than three times as stable as the ADC mimic synthesized in Comparative Example 1, and the ADC mimics synthesized in Examples 2-1-3 and 2-1-4 were more than ten times as stable.
  • Example 7 Synthesis of ADC mimic
  • 8 equivalents of a PBSE solution (2 mM) of a reducing agent (TCEP) relative to the antibody were added, and incubated at 37 ° C. for 1 hour.
  • DMA Dimethylacetamide
  • 10 mM DMA solution (20 equivalents relative to the antibody) of various Linker-payload mimics (1) were added in sequence to the obtained 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 6.
  • Example 7-2 Synthesis of ADC mimic 7 According to Example 7-1, ADC mimic 7 was obtained from Linker-payload mimic (11).
  • Example 7-3 Synthesis of ADC mimic 8 According to Example 7-1, ADC mimic 8 was obtained from Linker-payload mimic (21).
  • Example 7-4 Synthesis of ADC mimic 9 According to Example 7-1, the linker-payload mimic (1) was reacted with the anti-CD20 IgG antibody rituximab (Roche) to obtain ADC mimic 9.
  • Example 7-5 Synthesis of ADC mimic 10 According to Example 7-1, the linker-payload mimic (11) was reacted with the anti-CD20 IgG antibody rituximab (Roche) to obtain ADC mimic 10.
  • Example 7-6 Synthesis of ADC mimic 11 According to Example 7-1, the linker-payload mimic (21) was reacted with the anti-CD20 IgG antibody rituximab (Roche) to obtain ADC mimic 11.
  • Example 7-7 Synthesis of ADC mimic 12 According to Example 7-1, the linker-payload mimic (1) was reacted with the anti-TNF- ⁇ IgG antibody infliximab (Centocor) to obtain ADC mimic 12.
  • Example 7-8 Synthesis of ADC mimic 13 According to Example 7-1, the linker-payload mimic (11) was reacted with the anti-TNF- ⁇ IgG antibody infliximab (Centocor) to obtain ADC mimic 13.
  • Example 7-9 Synthesis of ADC mimic 14 According to Example 7-1, the linker-payload mimic (21) was reacted with the anti-TNF- ⁇ IgG antibody infliximab (Centocor) to obtain ADC mimic 14.
  • Comparative Example 2 Synthesis of ADC mimic Comparative Example 2-1: Synthesis of Linker-payload mimic (30) Linker-payload mimic (30) was synthesized as follows. It was synthesized in one step from commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) and known Sarcosine-pyrene (WO2018218004A1).
  • MC-VC-PAB-PNP (CAS No: 159857-81-5) (15.5 mg, 0.021 mmol) was dissolved in dichloromethane (1 mL), and N,N-diisopropylethylamine (0.025 mL, 0.142 mmol) and a solution of known sarcosine-pyrene (WO2018218004A1) (7.6 mg, 0.025 mmol) in dimethylformamide (0.5 mL) were added and stirred for 17 hours. After purification by reversed-phase preparative chromatography, the fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain Linker-payload mimic (30) (7.3 mg, 0.008 mmol).
  • Linker-payload mimic (30) (7.3 mg, 0.008 mmol).
  • Comparative Example 2-2 Synthesis of ADC mimics 15 to 17 According to Example 7-1, Linker-payload mimic (30) was reacted to obtain ADC mimic 15 from trastuzumab, ADC mimic 16 from rituximab, and ADC mimic 17 from infliximab.
  • 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 known (described in WO2018218004A1) sarcosine-pyrene (59.2 mg, 0.196 mmol), N,N-diisopropylethylamine (39 ⁇ L, 0.227 mmol), and 4-dimethylaminopyridine (3.7 mg, 0.03 mmol) were added and stirred at room temperature for 2 hours, after which diethylamine (2 mL, 18.95 mmol) was added and stirred at room temperature for 1.5 hours.
  • 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 ⁇ L, 0.51 mmol) were added, and the mixture was stirred at room temperature for 2.5 hours. Diethylamine (0.2 mL, 1.91 mmol) was then added, and the mixture was stirred at room temperature for 1.5 hours.
  • 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.
  • Example 8 DAR analysis of ADC mimics The 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.
  • 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 performed 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.
  • HIC-HPLC hydrophobic column chromatography
  • the exo-ADCs ADC mimics 12, 13, and 14 have faster retention times in the HIC chromatogram, indicating that they are more hydrophilic ADC mimics.
  • 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.
  • Measurement system 1260 HPLC system (Agilent) Column: Agilent AdvanceBio SEC 300 ⁇ 2.7 ⁇ m, 4.6 mm ⁇ 150 mm Flow rate: 0.25 mL/min. Eluent: 100 mM sodium dihydrogen phosphate/sodium hydrogen phosphate, 250 mM sodium chloride in water (pH 6.8), 10% v/v isopropanol. Detector: UV (280 nm).
  • Example 11 Evaluation of ADC mimics using enzyme cathepsin B The cleavage ability of various ADC mimics with cathepsin B was evaluated by analyzing the amount of fluorescent molecules shed from the ADC mimic, as described below.
  • Example 11-1 Cathepsin B cleavage test This was carried out as follows according to a previous report (Nature Communications 2018, 9, 2512). After adding ADC mimic 7 to 180 ⁇ L of MES buffer (10 mM MES, 40 ⁇ M DTT, pH 5.0) to a concentration of 1 mg/mL, 30 ⁇ L was dispensed into six Eppendorf tubes. Three of the six samples were immediately added with 100 ⁇ L of acetonitrile at 0 ° C., stirred with a vortex, and then centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis. The remaining three were incubated at 37 ° C. for 6 hours. A precipitate was obtained by adding 100 ⁇ L of acetonitrile to each sample, stirring with a vortex, and then centrifuging. The resulting supernatant solution was collected and subjected to HPLC analysis.
  • MES buffer 10 mM MES, 40 ⁇ M DTT
  • Example 11-2 Analysis of the amount of dropped fluorescent molecules using HPLC analysis Measurement was performed using liquid chromatography/fluorescence detection to measure the amount of fluorescent molecules dropped from ADC mimic 7. Three samples to which acetonitrile was immediately added at 0°C in Example 11-1 were designated as 0 hour samples, and three samples incubated at 37°C for 6 hours as described in Example 11-1 were designated as 6 hour samples, and the difference in fluorescence intensity between the 6 hour samples and the 0 hour samples was analyzed.
  • Linker-payload (47) was synthesized as follows.
  • Linker-payload (125) was synthesized in the same manner as in the synthesis of Linker-payload mimic (120), except that MMAE was used instead of sarcosine-pyrene.
  • Linker-payload (126) Synthesis of Linker-payload (126)
  • 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.
  • Example 13 Synthesis of ADC
  • DMA dimethylacetamide
  • 10 mM DMA solution 20 equivalents relative to the antibody
  • ADC 2 was obtained from Linker-payload (42).
  • Example 13-3 Synthesis of ADC 3 According to Example 13-1, ADC 3 was obtained from Linker-payload (47).
  • Example 13-4 Synthesis of ADC 9 According to Example 13-1, ADC 9 was obtained from Linker-payload (125).
  • Example 13-5 Synthesis of ADC 10 According to Example 13-1, ADC 10 was obtained from Linker-payload (126).
  • Example 13-6 Synthesis of ADC 17 According to Example 13-1, ADC 17 was obtained from Linker-payload (146).
  • Example 14 HIC-HPLC analysis of ADC HIC-HPLC analysis was carried out using the conditions of Example 8.
  • the hydrophobicity of the ADC was evaluated using HIC-HPLC.
  • the measurement was 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 which is the raw material antibody, was used for comparison.
  • exo-ADCs ADCs 1, 2, and 3 have retention times in the HIC chromatogram comparable to those of the raw antibody, indicating that they are more hydrophilic ADCs.
  • Example 15 Evaluation of aggregation rate of ADC by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was carried out according to Example 10.
  • Example 16 Synthesis of ADC (16-1) Synthesis of ADC 4
  • 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 is regioselectively introduced into trastuzumab (humanized IgG1 antibody) via the amino group of the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue is in accordance with EU numbering).
  • NH-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 - extending from the antibody heavy chain corresponds to the side chain of a lysine residue, and a thiol-containing group, HS-CH 2 -CH 2 -C( ⁇ O), is added to the amino group in the side chain of this lysine residue. Since no modification with other lysine residues was detected by peptide mapping method, it is considered that the position selectivity at positions 246 and 248 of the antibody heavy chain is 100%.
  • Example 16-2 Synthesis of ADC 5 According to Example 16-1, ADC 5 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.
  • Example 16-3 Synthesis of ADC 6 According to Example 16-1, ADC 6 was obtained from Linker-payload (47). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151109 where two Linker-payloads (47) were introduced.
  • Example 16-4 Synthesis of ADC 6 According to Example 16-1, ADC 8 was obtained from Linker-payload (125). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 150524 where two Linker-payloads (125) were introduced.
  • Example 16-5 Synthesis of ADC 11 According to Example 16-1, ADC 11 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.
  • Example 16-6 Synthesis of ADC 12 According to Example 16-1, ADC 12 was obtained from Linker-payload (130). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 152861 where two Linker-payloads (130) were introduced.
  • Example 16-7 Synthesis of ADC 13 According to Example 16-1, ADC 13 was obtained from Linker-payload (132). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have peaks at 151632 where two Linker-payloads (132) were introduced.
  • Example 16-8 Synthesis of ADC 14 According to Example 16-1, ADC 14 was obtained from Linker-payload (134). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151409 where two Linker-payloads (134) were introduced.
  • Example 16-9 Synthesis of ADC 15 According to Example 16-1, ADC 15 was obtained from Linker-payload (141). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 151095 where two Linker-payloads (141) were introduced.
  • Example 16-10 Synthesis of ADC 16 According to Example 16-1, ADC 16 was obtained from Linker-payload (146). ESI-TOFMS analysis was performed, and the reaction product was confirmed to have a peak at 150789 where two Linker-payloads (146) were introduced.
  • Example 17 HIC-HPLC analysis of ADC HIC-HPLC analysis was carried out using the conditions of Example 8.
  • the hydrophobicity of the ADC was evaluated using HIC-HPLC.
  • the measurement was 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 which is the raw material antibody, was used for comparison.
  • exo-ADCs ADCs 4, 5, 6, 12, 13, 14, 15, and 16 have retention times in the HIC chromatogram comparable to those of the raw antibody, indicating that they are more hydrophilic ADCs.
  • Example 18 Evaluation of aggregation rate of ADC by size exclusion chromatography (SEC-HPLC) SEC-HPLC analysis was carried out according to Example 10.
  • Linker-payload mimic (56) was synthesized as follows.
  • Alcohol (57) (55.0 mg, 84.5 ⁇ mol) was dissolved in N,N-dimethylformamide (423 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (77.1 mg, 254 ⁇ mol) and N,N-diisopropylethylamine (32.3 ⁇ L, 190 ⁇ mol) were added and stirred at room temperature for 2 hours.
  • Linker-payload mimic (61) Linker-payload mimic (1) was synthesized as follows.
  • methyl 4-aminomandelate (19.3 mg, 106 ⁇ mol) was added and the mixture was stirred at room temperature for 20 hours, after which it 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 above alcohol (62) (36.1 mg, 49.7 ⁇ mol).
  • methyl 4-aminomandelate (10.3 mg, 56.8 ⁇ mol) was added and the mixture was stirred at room temperature for 20 hours, and then 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 above alcohol (67) (47.7 mg, 39.1 ⁇ mol).
  • Linker-payload mimic (71) was synthesized as follows.
  • Alcohol (72) (40.1 mg, 51.2 ⁇ mol) was dissolved in N,N-dimethylformamide (512 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (46.7 mg, 153 ⁇ mol) and N,N-diisopropylethylamine (22.0 ⁇ L, 115 ⁇ mol) were added and stirred at room temperature for 2 hours and 20 minutes.
  • methyl 4-aminomandelate (13.9 mg, 77.6 ⁇ mol) was added and the mixture was stirred at room temperature for 25 hours, after which it 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 above alcohol (77) (53.6 mg, 55.3 ⁇ mol).
  • Alcohol (77) (52.3 mg, 54.0 ⁇ mol) was dissolved in N,N-dimethylformamide (540 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (49.2 mg, 162 ⁇ mol) and N,N-diisopropylethylamine (20.7 ⁇ L, 122 ⁇ mol) were added and stirred at room temperature for 3 hours.
  • Linker-payload mimic (81) was synthesized as follows.
  • methyl 4-aminomandelate (11.8 mg, 64.9 ⁇ mol) was added and the mixture was stirred at room temperature for 25 hours, after which it 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 above alcohol (82) (43.9 mg, 47.7 ⁇ mol).
  • methyl 4-aminomandelate (12.1 mg, 66.9 ⁇ mol) was added and the mixture was stirred at room temperature for 25 hours, and then 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 above alcohol (87) (41.3 mg, 46.2 ⁇ mol).
  • Alcohol (87) (40.3 mg, 45.1 ⁇ mol) was dissolved in N,N-dimethylformamide (451 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl) acid (41.2 mg, 135 ⁇ mol) and N,N-diisopropylethylamine (17.3 ⁇ L, 101 ⁇ mol) were added and stirred at room temperature for 2 hours and 10 minutes.
  • Linker-payload mimic (11) was also synthesized via a route different from that of (1-3) as follows.
  • Example (1-3-1) The alcohol (12) (80.0 mg, 94.1 ⁇ mol) 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, after which 1M aqueous lithium hydroxide solution (226 ⁇ L, 226 ⁇ mol) was added and stirred at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to about 6 using 1M hydrochloric acid and purified by reversed-phase preparative chromatography. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above alcohol (96) (61.5 mg, 73.6 ⁇ mol).
  • Alcohol (96) (60.5 mg, 72.4 ⁇ mol) was dissolved in N,N-dimethylformamide (3.6 mL) and cooled on ice. N,N-diisopropylethylamine (25 ⁇ L, 145 ⁇ mol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (56.5 mg, 109 ⁇ mol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (23.7 mg, 109 ⁇ mol) was added, the mixture was returned to room temperature, and stirred for 3 hours. After the reaction was completed, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain alcohol (97) (68.0 mg, 72.4 ⁇ mol).
  • the alcohol (97) (10.0 mg, 10.0 ⁇ mol) was dissolved in N,N-dimethylformamide (0.1 mL) and then cooled on ice.
  • Bis(4-nitrophenyl) carbonate (30.4 mg, 100 ⁇ mol) and N,N-diisopropylethylamine (3.8 ⁇ L, 22.5 ⁇ mol) were added and stirred at room temperature for 3 hours.
  • 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 the product was freeze-dried to obtain the above compound (98) (5.6 mg, 4.8 ⁇ mol).
  • methyl 4-aminomandelate (11.8 mg, 65.1 ⁇ mol) was added and the mixture was stirred at room temperature for 68 hours, and then 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 above alcohol (100) (31.9 mg, 30.8 ⁇ mol).
  • Alcohol (100) (30.5 mg, 29.5 ⁇ mol) was dissolved in N,N-dimethylformamide (169 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (27.4 mg, 90.1 ⁇ mol) and N,N-diisopropylethylamine (11.4 ⁇ L, 66.3 ⁇ mol) were added and stirred at room temperature for 2 hours.
  • Linker-payload mimic (104) was synthesized as follows.
  • methyl 4-aminomandelate (22.6 mg, 125 ⁇ mol) was added and the mixture was stirred at room temperature for 16.5 hours, and then 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 above alcohol (105) (110 mg, 98.5 ⁇ mol).
  • Linker-payload mimic (109) was synthesized as follows.
  • Alcohol (111) 48.4 mg, 43.8 ⁇ mol was dissolved in N,N-dimethylformamide (156 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (21.6 mg, 69.6 ⁇ mol) and N,N-diisopropylethylamine (8.8 ⁇ L, 51.2 ⁇ mol) were added and stirred at room temperature for 2 hours and 10 minutes.
  • methyl 4-aminomandelate (16.8 mg, 93.1 ⁇ mol) was added and the mixture was stirred at room temperature for 21 hours, after which it was 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 (116) (53.3 mg, 66.0 ⁇ mol).
  • Alcohol (116) (25.0 mg, 30.9 ⁇ mol) was dissolved in N,N-dimethylformamide (309 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (28.2 mg, 92.7 ⁇ mol) and N,N-diisopropylethylamine (11.8 ⁇ L, 69.5 ⁇ mol) were added and stirred at room temperature for 2 hours.
  • Linker-payload mimic (120) was synthesized as follows.
  • N-(5-aminopentyl)maleimide hydrochloride 50.6 mg, 232 ⁇ mol was added, the mixture was returned to room temperature, and stirred for 4 hours. After the reaction was completed, the mixture was purified by reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain compound (122) (83.4 mg, 135 ⁇ mol).
  • Alcohol (190) (135.7 mg, 211 ⁇ mol) was dissolved in THF (5.3 mL) and water (5.3 mL), 5% palladium carbon (9.6 mg, 2.1 ⁇ mol) was added, and the mixture was replaced with hydrogen and stirred at room temperature for 5 hours. After the reaction, the solids were removed by filtration, washed with water, and freeze-dried to obtain alcohol (191) (105.5 mg, 207 ⁇ mol).
  • Alcohol (191) (104.3 mg, 205 ⁇ mol) was dissolved in dimethyl sulfoxide (2.05 mL), N,N-diisopropylethylamine (105 ⁇ L, 615 ⁇ mol) and dimethylsulfamoyl chloride (32.7 ⁇ L, 308 ⁇ mol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was purified by reversed-phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain alcohol (127) (76.4 mg, 124 ⁇ mol).
  • Alcohol (127) (7.2 mg, 11.7 ⁇ mol) was dissolved in N,N-dimethylformamide (117 ⁇ L) and stirred for 5 minutes under ice cooling, after which bis(4-nitrophenyl)carbonate (10.7 mg, 35.1 ⁇ mol) and N,N-diisopropylethylamine (4.5 ⁇ L, 26.3 ⁇ mol) were added and stirred at room temperature for 1 hour.
  • Pyrene (128) (29.0 mg, 30.7 ⁇ mol) was dissolved in tetrahydrofuran (2.33 mL) and water (775 ⁇ L) and stirred for 5 minutes under ice cooling, after which 1 M lithium hydroxide aqueous solution (73.7 ⁇ L, 73.7 ⁇ mol) 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 reverse phase preparative chromatography. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain pyrene (129) (26.1 mg, 28.1 ⁇ mol).

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WO2026076215A1 (en) * 2024-10-03 2026-04-09 Solve Therapeutics, Inc. Conjugates and uses thereof
WO2026085106A1 (en) * 2024-10-15 2026-04-23 Solve Therapeutics, Inc. Conjugates and uses thereof

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026076215A1 (en) * 2024-10-03 2026-04-09 Solve Therapeutics, Inc. Conjugates and uses thereof
WO2026085106A1 (en) * 2024-10-15 2026-04-23 Solve Therapeutics, Inc. Conjugates and uses thereof

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