Antibody-drug conjugate precursors and synthetic intermediates thereof

Abstract

Provided are: a method for improving the stability of an antibody-drug conjugate in a living body; and a conjugate precursor for obtaining a stabilized antibody-drug conjugate. Specifically, the present invention provides an antibody-drug conjugate precursor represented by general formula (I): Z-L-D (I) or a salt thereof, a synthetic intermediate thereof or a salt thereof, and the like. [In the formula, Z is a reactive group capable of reacting with a functional group present in an antibody or a modified antibody, D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of an antitumor drug molecule, an analog thereof, or a derivative thereof, L is a linker linking Z and D, and n is an integer from 1 to 2. And at least one of D and L has at least one lactone group as a substituent or protecting group at an arbitrary position].

Description

Technical Field

[0001] This invention relates to antibody-drug conjugate precursors (hereinafter also referred to as conjugate precursors) and their synthetic intermediates (hereinafter also referred to as conjugate precursor synthetic intermediates) that are useful for the synthesis of "antibody-drug conjugates" (ADCs) used as antitumor drugs. Background Technology

[0002] Antibody-drug conjugates (ADCs) that bind a cytotoxic drug payload to an antibody that binds to an antigen that can be expressed on the surface of cancer cells and internalized in the cell can be expected to "selectively deliver the drug to cancer cells, release / accumulate the drug within the cancer cells, and kill the cancer cells" (Non-Patent Literature 1-Non-Patent Literature 2). For example, Adcetris (brentuximab vedotin) (Patent Literature 1), which binds a methylaurestatin E to an anti-CD30 antibody, has been approved as a treatment for Hodgkin's lymphoma and undifferentiated large cell lymphoma; Kadcyla (trastuzumab emtansine) (Patent Literature 2), which binds emtansine to an anti-HER2 antibody, has been approved as a treatment for HER2-positive metastatic breast cancer; Enhertu (trastuzumab decratecan) (Patent Literature 2), which binds delutec to an anti-HER2 antibody, has been approved as a treatment for HER2-positive metastatic breast cancer. Trodelvy (goxatozumab), an SN-38 antibody, has been approved as a treatment for TROP-2-positive metastatic breast cancer; Blenrep (mabetuzumab), an anti-CD269 (BCMA) antibody bound to MMAF, has been approved as a treatment for multiple myeloma; Tivdak (teisosuzumab), an anti-CD142 antibody bound to methylaurestatin E, has been approved as a treatment for metastatic cervical cancer; and Zynlonta (ronutuximab), an anti-CD19 antibody bound to pyrrolobenzodiazepine dimer, has been approved as a treatment for B-cell lymphoma (Nonpatent Literature 3–10). In addition, many novel ADCs are under clinical development as anti-tumor drugs (Nonpatent Literature 11–14).

[0003] To ensure optimal antitumor effects, ADCs need to be stable in vivo (in the blood) after administration. However, generally, when the DAR (drug-to-antibody ratio) increases, the ADC's hydrophobicity increases, leading to aggregation and deterioration in pharmacokinetics, thus weakening its efficacy. For example, among ADCs using MMAE (methylaurestatin E) as the payload, there are reports of ADCs exhibiting the highest stability and efficacy in vivo when the DAR value is around 3-4, while those with a DAR value above this range become unstable and their efficacy weakens (Non-Patent Literature 15). To address this issue, research has been actively conducted to increase the hydrophilicity of ADCs (Patent Literature 3, Non-Patent Literatures 16-19).

[0004] In order to obtain such a “hydrophilic ADC that is stable in blood”, a “conjugate precursor consisting of a linker and an antitumor drug” is needed for conjugation with the desired antibody. This conjugate precursor must be sufficiently hydrophilic.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: WO2003043583

[0008] Patent Document 2: Japanese Patent Application Publication No. 2016-196484

[0009] Patent Document 3: WO2016001485

[0010] Non-patent literature

[0011] Non-patent literature 1: Current Opin. Chem. Biol., 2010, 14, 529-537

[0012] Non-patent literature 2: Expert Opin. Biol. Ther., 2004, 4, 1445-1452

[0013] Non-patent literature 3: Biotechnol Lett., 2016, 38, 1655-1664.

[0014] Non-patent literature 4: Chem Pharm Bull., 2019, 67, 173-185.

[0015] Non-patent literature 5: N Engl J Med., 2012, 367, 1783-1791

[0016] Non-patent literature 6: N Engl J Med., 2018, 378, 331-344.

[0017] Non-patent literature 7: Expert Opin. Biol. Ther., 2020, 8, 871-875

[0018] Non-patent literature 8: Drugs Today, 2021, 57, 653-663

[0019] Non-patent literature 9: Drugs, 2021, 81, 2141-2147

[0020] Non-patent literature 10: Drugs, 2021, 81, 1229-1233

[0021] Non-patent literature 11: Clin Cancer Res., 2019, 25, 5441-5448.

[0022] Non-patent literature 12: Molecules. 2020, 25, 4764

[0023] Non-patent literature 13: Methods Mol Biol., 2020, 2078, 1-22

[0024] Non-patent literature 14: Nat. Rev. Drug Discov., 2017, 16, 315

[0025] Non-patent literature 15: Clin Cancer Res., 2004, 10, 7063-7070

[0026] Non-patent literature 16: Mol Cancer Ther., 2017, 16, 116-123

[0027] Non-patent literature 17: Antibodies (Basel)., 2018, 7, 15

[0028] Non-patent literature 18: Nat. Biotech., 2015, 33, 733-735.

[0029] Non-Patent Literature 19: Separations 2019, 6(1), 1 Summary of the Invention

[0030] The problem the invention aims to solve

[0031] One object of the present invention is to provide a method for improving the stability of ADCs in vivo, and a conjugate precursor for obtaining stable ADCs.

[0032] To improve the stability of existing ADCs in vivo and develop more useful antitumor drugs, the inventors conducted in-depth research on conjugate precursors. As a result, they created conjugate precursors in which the antitumor drug or linker constituting the conjugate precursor has one or more lactone groups as substituents or protecting groups. ADCs synthesized using the conjugate precursors of this invention exhibit higher hydrophilicity and stability in the blood after administration compared to ADCs without lactone groups, and demonstrate potent antitumor activity as ADCs in both in vitro and in vivo experiments.

[0033] As mentioned above, numerous attempts have been made to introduce hydrophilic groups into conjugate precursors in order to obtain ADCs that are stable in vivo, but so far there has been no report on the "conjugate precursor with lactone group" of the present invention.

[0034] The present invention provides the following [1] to

[26] . [1]

[0036] An antibody-drug conjugate precursor (hereinafter also referred to as "conjugate precursor (I)") or a salt thereof, represented by the following general formula (I),

[0037] ZLD (I)

[0038] [In the formula, Z is a reactive group (hereinafter also referred to as "reactive group") capable of reacting with functional groups (hereinafter also denoted as "antibody functional groups") present in antibodies or modified antibodies (hereinafter collectively referred to as "antibodies").

[0039] D represents an antitumor drug residue (hereinafter also referred to as "antitumor drug residue") obtained by removing one hydrogen atom or one hydroxyl group from any position in an antitumor drug molecule, its analogue, or its derivatives.

[0040] L is the connector that links Z and D.

[0041] At least one of D and L has one or more lactone groups as substituents or protecting groups at any position. [2]

[0043] According to the coupling precursor (I) or its salt as described in [1], wherein Z is maleimide (formula (i) below), α-halomethyl carbonyl (formula (ii) below), ethynyl phosphonamidate (formula (iii) below), carboxyl, active ester of carboxyl, thiol, hydroxyl, amino, alkynyl, cycloalkynyl, azacyclic alkynyl, or azide (-N3 group).

[0044] Formula (i)

[0045]

[0046] Formula (ii)

[0047] Hal-CH2-C(=O)-*

[0048] Equation (iii)

[0049]

[0050] In the above formula, * represents the bonding site with L, Hal represents a fluorine atom, chlorine atom, bromine atom, or iodine atom, and R... 16 [Methyl, ethyl, or -CH2CH2OCH2CH2OH group] [3]

[0052] According to the coupling precursor (I) or its salt as described in [1] or [2], wherein Z is maleimide (formula (i) above), α-halomethyl carbonyl (formula (ii) above), ethynylphosphonic amide ester (formula (iii) above), carboxyl, active ester of carboxyl, alkynyl, cycloalkynyl, azacyclic alkynyl, or azide (-N3). [4]

[0054] The coupling precursor (I) or its salt according to any one of [1] to [3], wherein Z is a maleimide group (formula (i) above), an α-halomethyl carbonyl group (formula (ii) above), a carboxyl group, or an active ester of a carboxyl group. [5]

[0056] According to any one of [1] to [4], the conjugate precursor (I) or its salt, wherein D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of: camptothecin; auristatin E (AE) or monomethylauristatin E (MMAE); maytansine; PBD (pyrrolebenzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; cazithromycin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytosine; vincristine; vinblastine; doxorubicin; sulphurin 10; superdox; ciprofloxacin; cardiol (CS-940); or analogues of these antitumor drugs; or the above-mentioned antitumor drug molecules or their analogues or derivatives; [6]

[0058] According to any one of [1] to [5], the conjugate precursor (I) or its salt, wherein D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of: camptothecin; auristatin E (AE) or monomethylauristatin E (MMAE); maytansine; PBD (pyrrole benzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; cazithromycin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytosine; vincristine; vinblastine; doxorubicin; sulphurin 10; superdox; or analogues of these antitumor drugs; or the above-mentioned antitumor drug molecules or their analogues or derivatives; [7]

[0060] According to any one of [1] to [6], the conjugate precursor (I) or its salt, wherein D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of: camptothecin; auristatin E (AE) or monomethylauristatin E (MMAE); maytansine; PBD (pyrrole benzodiazepine) dimer; eribulin; or analogues of these antitumor drugs; or derivatives of the aforementioned antitumor drug molecules or their analogues. [8]

[0062] The conjugate precursor (I) or its salt according to any one of [1] to [7], wherein D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of: an antitumor drug or an analogue of such antitumor drugs that has been phosphorylated at least one hydroxyl group present in the molecule; or a derivative thereof of the aforementioned antitumor drug molecule or its analogue. [9]

[0064] The coupling precursor (I) or its salt according to any one of [1] to [8], wherein L is an optionally substituted alkylene group;

[0065] One or more methylene groups in the chain of the alkylene group can be independently selected from...

[0066] -C(R 1 (R) 2 )-;-O-;-N(R 3 )-;-N(R 3 )-N(R 3 )-;-S-;-Se-;-Si(R 4 (R) 5 )-;-SS-;-Se-Se-;-SOm-;-SeOn-;-C(=C(R 6 (R) 7))-;-C(=O)-;-C(=S)-;-C(=N(R 8 ))-;-C(=N-OR 9 )-;-C(=NN(R 10 (R) 11 ))-;-P(=O)(R 12 )-;-P(=O)(OR 13 )-;-OP(=O)(R 12 -O-;-OP(=O)(OR 13 )-O-;-C(R 14 =; =C(R) 14 )-;-C(R 14 )=C(R 14 )-;-N=;=N-;-C≡C-;-(OC(R 1 (R) 2 )-C(R 1 (R) 2 )) 1-30 -;-(C(R 1 (R) 2 )-C(R 1 (R) 2 )-0) 1-30 -; optionally substituted alkenyl group; optionally substituted ynyl group; optionally substituted cycloalkyl group; optionally substituted cycloalkenyl group; optionally substituted cycloynyl group; optionally substituted azeotropic ynyl group; optionally substituted aryl group; optionally substituted heteroaryl group; and optionally substituted heterocyclic group, replacing one or more divalent groups in the group consisting of ;

[0067] R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 and R 14 Each of the following groups is independently selected from the group consisting of hydrogen atom, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted azaheteroylynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclic groups, R 3 When the alkyl group is alkyl, it may optionally form a cyclic structure together with the alkyl group in the adjacent methylene group.

[0068] m and n are each an independent integer from 0 to 2.

[10]

[0070] According to the coupling precursor (I) or its salt as described in [9], wherein L is an optionally substituted alkylene group;

[0071] One or more methylene groups in the chain of the alkylene group are replaced by one or more divalent groups independently selected from the following chemical formula group.

[0072] Chemical formula group:

[0073] -OC(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0074] -SC(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0075] -N(H or C1-C4 alkyl)-;

[0076] -N(H or C1-C4 alkyl)-OC(H or C1-C4 alkyl)(C(H or C1-C4 alkyl)-;

[0077] -C(H or C1-C4 alkyl)-SO 0-2 -C(H or C1-C4 alkyl)-;

[0078] -C(H or C1-C4 alkyl)-SSC(H or C1-C4 alkyl)-;

[0079] -C(H or C1-C4 alkyl)=C(H or C1-C4 alkyl)-;

[0080] -C(H or C1-C4 alkyl)=N-;

[0081] -C(H or C1-C4 alkyl) = NO-;

[0082] -C(H or C1-C4 alkyl)=NN(H or C1-C4 alkyl)-;

[0083] -C(=C(H or C1-C4 alkyl)(H or C1-C4 alkyl))-;

[0084] -C(=NO(H or C1-C4 alkyl))-;

[0085] -C(=NN(H or C1-C4 alkyl)(H or C1-C4 alkyl))-;

[0086] -O-Si(C1-C4 alkylCH3)(C1-C4 alkyl)-O-;

[0087] -C(H or C1-C4 alkyl)-N(C1-C4 alkyl)-CH2-;

[0088] -C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-C(=O)-C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0089] -C(=O)-O-;

[0090] -C(=O)-S-;

[0091] -C(=O)-N(H or C1-C4 alkyl)-;

[0092] -OC(=O)-O-;

[0093] -N(H or C1-C4 alkyl)-C(=O)-N(H or C1-C4 alkyl)-;

[0094] -C(=O)-N(H or C1-C4 alkyl)-C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0095] -C(=O)-(CH2) 1-10 -(O-CH2CH2) 1-20 -O-(CH2) 1-10 -C(=O)-;

[0096] -C(=O)-(CH2) 1-10 -(O-CH2CH2) 1-20 -O-(CH2) 1-10 -CH2-NH-;

[0097] -NH-CH2-(CH2) 1-10 (O-CH2CH2) 1-20 -O-(CH2) 1-10 -CH2-NH-;

[0098] -C(=O)-N(C1-C4 alkyl)-CH2CH2-N(C1-C4 alkyl)-C(=O)-;

[0099]

[0100] [In the formula, * represents the bonding site with the adjacent group];

[0101] -P(=O)(OH)-O-;

[0102] -OP(=O)(OH)-O-;

[0103] -OP(=S)(OH)-O-;

[0104] -OP(=O)(OH)-S-;

[0105] -OP(=O)(OH)-OP(=O)(OH)-O-;

[0106] -OP(=O)(OH)-OP(=O)(OH)-OP(=O)(OH)-O-;

[0107] -CH(CH2-NH2)-;

[0108] -CH(CH2-NH(C1-C4 alkyl))-;

[0109] -CH(CH2-N(C1-C4 alkyl)2)-;

[0110] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 0-20 -C1-C4 alkyl)-;

[0111] -CH(CH2-NH-C(=O)-(CH2) 0-20 -lactone group)-;

[0112] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-lactone group)-;

[0113] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-NH-lactone group)-;

[0114] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-NH-C(=O)-lactone group)-;

[0115] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-lactone group)-;

[0116] -CH(CH2-NH-(CH2) 1-20 -(O) 0-1 -(P(=O)(OH)2)-;

[0117] -CH(CH2-NH-C(=O)-(CH2) 1-20-(P(=O)(OH)2)-;

[0118] -CH(CH2-NH-C(=O)-(CH2) 1-20 -C(=O)-NH-(CH2) 1-10 -(P(=O)(OH)2)-;

[0119] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-(P(=O)(OH)2)-;

[0120] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -(P(=O)(OH)2)-;

[0121] -CH(CH2-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-;

[0122] -CH(CH2-NH-C(=O)-(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-;

[0123] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-;

[0124] -(cis)-CH=CH-P(=O)(O-CH2CH3)-NH-(phenylene)-C(=O)-;

[0125] -C(=O)-(cyclohexylene);

[0126] -(succinimide group)-;

[0127] -Ser-;

[0128] -Cys-;

[0129] -Ama-:

[0130] -Asp-;

[0131] -Glu-;

[0132] -Orn-;

[0133] -Lys-;

[0134] -ValLys-;

[0135] -ValCit-;

[0136] -ValAla-;

[0137] -GlyGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0138] -PheLys-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0139] -ValLys-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0140] -ValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0141] -ValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0142] -AspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0143] -LysAspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0144] -GluValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0145] -LysValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0146] -SerValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0147] -AspValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0148] -GluValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0149] -LysValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0150] -SerValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0151] -GlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0152] -AspGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0153] -GluGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0154] -LysGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0155] -SerGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0156] -AspAspAspAspAsp-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0157] -GlyGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0158] -PheLys-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0159] -ValLys-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0160] -ValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0161] -ValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0162] -AspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0163] -GluValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0164] -LysValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0165] -SerValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0166] -AspValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0167] -GluValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0168] -LysValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0169] -SerValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0170] -GlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0171] -AspGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O-);

[0172] -GluGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0173] -LysGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0174] -SerGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0175] -AspAspAspAspAsp-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0176] -GlyGlyPheGly-C(=O)-NH-CH2-;

[0177] -AspGlyGlyPheGly-C(=O)-NH-CH2-;

[0178] (Among the aforementioned amino acid residues and amino acid residues in peptides,

[0179] The carboxyl groups of the side chains of Ama, Asp, and Glu may optionally form (alkyl) esters, (aminoalkyl) esters, ((N-alkylamino)alkyl) esters, ((N,N-dialkylamino)alkyl) esters, ((trialkylammonium)alkyl) esters, (lactone) esters, ((lactone)alkyl) esters, ((phosphoryl)alkyl) esters, or ((bisphosphoryl)alkyl) esters; or may optionally form unsubstituted amides, alkylamides, dialkylamides, ((N-alkylamino)alkyl)amides, ((N,N-dialkylamino)alkyl)amides, ((trialkylammonium)alkyl)amides, (lactone)amides, ((lactone)alkyl)amides, (phosphoryl)amides, ((phosphoryl)alkyl)amides, or ((bisphosphoryl)alkyl)amides.

[0180] The amino groups in the side chains of Lys and Orn can have one or two alkyl, alkylcarbonyl, (aminoalkyl)carbonyl, ((N-alkylamino)alkyl)carbonyl, ((N,N-dialkylamino)alkyl)carbonyl, ((trialkylammonium)alkyl)carbonyl, lactone, (lactone)alkyl, (lactone)carbonyl, (lactone)alkyl)carbonyl, phosphoryl, (phosphoryl)alkyl, (phosphoryl)alkyl, (phosphoryl)alkyl)carbonyl, (bisphosphoryl)alkyl, or (bisphosphoryl)alkyl)carbonyl as substituents.

[0181] The hydroxyl group on the side chain of Ser and the thiol group on the side chain of Cys can have alkyl, alkylcarbonyl, aminoalkyl, (aminoalkyl)carbonyl, (N-alkylamino)alkyl, ((N-alkylamino)alkyl)carbonyl, (N,N-dialkylamino)alkyl, ((N,N-dialkylamino)alkyl)carbonyl, (trialkylammonium)alkyl, ((trialkylammonium)alkyl)carbonyl, lactone, (lactone)alkyl, (lactone)carbonyl, ((lactone)alkyl)carbonyl, phosphoryl, (phosphoryl)alkyl, ((phosphoryl)alkyl)carbonyl, (bisphosphoryl)alkyl, or ((bisphosphoryl)alkyl)carbonyl as substituents.

[0182] And the following formula: 1,2,3-triazolyl.

[0183]

[0184]

[0185] [In the formula, * represents the bonding site with the adjacent group.]

[11]

[0187] The coupling precursor (I) or its salt as described in [9], wherein L is any divalent group selected from the following group of chemical formulas.

[0188] Chemical formula group:

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] [In the above formula, * represents the bonding site with the reactive group Z, and # represents the bonding site with the antitumor drug residue D.]

[12]

[0196] According to any one of [1] to

[11] , the coupling precursor (I) or its salt, wherein L has at any position one of the following groups as a substituent or protecting group: lactone group, phosphoryl group (-P(=O)(OH)2); phosphoryl alkyl carbonyl group (-C(=O)-alkylene-P(=O)(OH)2); bisphosphonyl methyl group (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino group; monoalkyl amino group; dialkyl amino group; cyclic amino group in which the two alkyl groups of the dialkyl amino group together with the nitrogen atom to which they are bonded form a ring; and trialkylammonium group. When the total number of such groups is multiple, they may be the same or different.

[13]

[0198] The coupling precursor (I) or its salt is selected from the group consisting of the following examples.

[0199] Example 1

[0200]

[0201] Example 2

[0202]

[0203] Example 3

[0204]

[0205] Example 4

[0206]

[0207] Example 5

[0208]

[0209] Example 6

[0210]

[0211] Example 7

[0212]

[0213] Example 8

[0214]

[0215] Example 9

[0216]

[0217] Example 10

[0218]

[0219] Example 11

[0220]

[0221] Example 12

[0222]

[0223] Example 13

[0224]

[0225] Example 14

[0226]

[0227] Example 15

[0228]

[0229] Example 16

[0230]

[0231] Example 17

[0232]

[0233] Example 18

[0234]

[0235] Example 19

[0236]

[0237] Example 20

[0238]

[0239] Example 21

[0240]

[0241] Example 22

[0242]

[0243] Example 23

[0244]

[0245] Example 24

[0246]

[0247] Example 25

[0248]

[0249] Example 26

[0250]

[0251] Example 27

[0252]

[0253] Example 28

[0254]

[0255] Example 29

[0256]

[14]

[0258] A synthetic intermediate (II) of a coupling precursor (I) represented by the following general formula (II) (hereinafter also referred to as coupling precursor synthetic intermediate (II)), or a salt thereof:

[0259] Z 1 -L 1 -D 1 (II)

[0260] [In the formula, Z] 1 For the reactive groups defined above [1], or

[0261] A joining group (hereinafter also referred to as "joining group") used to join the reactive group or the antitumor drug residue defined above [1] to the linker, wherein the joining group is optionally a protector protected by the protecting group (hereinafter also referred to as "protector").

[0262] D 1 These are residues of antitumor drugs, or conjugating groups, or their protective forms.

[0263] Z 1 When D is a reactive group, 1 As a bonding group,

[0264] L 1 To make Z 1 and D 1 Connector

[0265] D 1 When it is an anti-tumor drug residue, D 1 and L 1 At least one of them has one or more lactone groups as substituents or protecting groups at any position.

[0266] D 1 When it is a bonding group,

[0267] L 1 The amino group has one or more substituents or protecting groups selected from lactone group; phosphoryl group (-P(=O)(OH)2); phosphoryl alkyl carbonyl group (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl group (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino group; monoalkyl amino group; dialkyl amino group; cyclic amino group in which the two alkyl groups of the dialkyl amino group form a ring with the nitrogen atom to which they are bonded; and trialkylammonium group as substituents or protecting groups, and when the total number of such groups is greater than or equal to, they may be the same or different.

[0268]

[15] Synthesize intermediate (II) or a salt thereof according to the coupling precursor described in

[14] , wherein,

[0269] The conjugating group is hydroxyl, nitro, cyano, amide, oxo, azide, amino, optionally a monoalkylamino group with a substituent, imino (including -N=, imino of cyclic amines), carboxyl, phosphoryl, alkynyl, cycloalkynyl, azacyclic alkynyl, or mercapto, or a protecting group thereof.

[0270]

[16] Synthesizing intermediate (II) or a salt thereof according to the coupling precursor described in

[14] or

[15] , wherein,

[0271] The conjugating group is hydroxyl, nitro, cyano, oxo, azide, amino, optionally a monoalkylamino group with a substituent, imino (including -N=, imino of cyclic amines), carboxyl, phosphoryl, alkynyl, cycloalkynyl, azacyclic alkynyl, or mercapto, or a protecting group thereof.

[0272]

[17] The coupling precursor synthesis intermediate (II) or a salt thereof according to any one of

[14] to

[16] , wherein the conjugating group is hydroxyl, nitro, oxo, azide, amino, optionally a monoalkylamino, imino (including -N=, imino of cyclic amines), carboxyl, phosphoryl, alkynyl, cycloalkynyl, azacyclic alkynyl, or mercapto, or a protecting form thereof.

[0273]

[18] The coupling precursor synthesis intermediate (II) or its salt according to any one of

[14] to

[17] , wherein the conjugating group is hydroxyl, nitro, oxo, azide, amino, optionally a monoalkylamino, imino (including -N=, imino of cyclic amines), carboxyl, phosphoryl, alkynyl, cycloalkynyl, or azacyclic alkynyl, or a protecting form thereof.

[0274]

[19] The coupling precursor synthesis intermediate (II) or a salt thereof according to any one of

[14] to

[18] , wherein L 1 For optional substituted alkylene groups,

[0275] One or more methylene groups in the chain of the alkylene group are replaced by one or more divalent groups selected from any one of the preceding [9] to

[11] .

[0276]

[20] The coupling precursor synthesis intermediate (II) or its salt according to any one of

[14] to

[19] , wherein Z 1 D is a group selected from any of the bonding groups described in any one of

[15] to

[18] above. 1 It is a group selected from any one of the antitumor drug residues mentioned in [5] to [8] above.

[0277]

[21] The coupling precursor synthesis intermediate (II) or its salt according to any one of

[14] to

[19] , wherein Z 1 and D 1 Each is independently a group selected from any of the aforementioned

[15] to

[18] .

[0278]

[22] The coupling precursor synthesis intermediate (II) or a salt thereof according to any one of

[14] to

[19] , wherein Z 1 D is a group selected from the reactive groups mentioned in any one of [2] to [4] above. 1 It is a group selected from any of the bonding groups mentioned in

[15] to

[18] above.

[0279]

[23] The coupling precursor synthesis intermediate (II) or a salt thereof according to any one of

[14] to

[20] , wherein Z 1The group is selected from any of the bonding groups mentioned in

[15] to

[18] above.

[0280] D 1 The group is selected from any of the antitumor drug residues mentioned above [5] to [8].

[0281] D 1 and L 1 At least one of them has one or more lactone groups as substituents or protecting groups at any position, and independently of this,

[0282] D 1 and L 1 At least one of the following groups has, at any position, a group selected from phosphoryl (-P(=O)(OH)2); phosphorylalkylcarbonyl (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino; monoalkylamino; dialkylamino; a cyclic amino group in which the two alkyl groups of the dialkylamino group together with the nitrogen atom to which they are bonded form a ring; and trialkylammonium as a substituent or protecting group, wherein when the total number of such groups is greater than or equal to, they may be identical or different.

[0283]

[24] The coupling precursor synthesis intermediate (II) or a salt thereof according to any one of

[14] to

[19] ,

[21] and

[22] , wherein Z 1 The group is selected from the reactive groups described in any one of [2] to [4] above, or from the conjugating groups described in any one of

[15] to

[18] above.

[0284] D 1 The group is selected from any of the bonding groups described in

[15] to

[18] above.

[0285] L 1 It has one or more groups selected from lactone; phosphoryl (-P(=O)(OH)2); phosphorylalkyl carbonyl (-C(=O)-alkylene-P(=O)(OH)2); bisphosphonylmethyl (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino; monoalkylamino; dialkylamino; cyclic amino in which the dialkyl forms a ring; and trialkylammonium as substituents or protecting groups, and when the total number of such groups is greater than or equal to, they may be the same or different.

[0286]

[25] A coupling precursor synthesis intermediate (II) selected from the group consisting of the following embodiments, or a salt thereof:

[0287] Example 1-1

[0288]

[0289] Examples 1-2

[0290]

[0291] Examples 1-3

[0292]

[0293] Examples 1-6

[0294]

[0295] Examples 1-7

[0296]

[0297] Example 2-1

[0298]

[0299] Example 2-2

[0300]

[0301] Example 2-3

[0302]

[0303] Examples 2-4

[0304]

[0305] Examples 3-4

[0306]

[0307] Examples 3-5

[0308]

[0309] Examples 3-6

[0310]

[0311] Examples 3-7

[0312]

[0313] Example 4-2

[0314]

[0315] Example 5-1

[0316]

[0317] Example 6-1

[0318]

[0319] Example 6-2

[0320]

[0321] Example 6-3

[0322]

[0323] Example 6-4

[0324]

[0325] Example 6-5

[0326]

[0327] Example 7-4

[0328]

[0329] Example 7-5

[0330]

[0331] Example 9-1

[0332]

[0333] Example 9-2

[0334]

[0335] Example 10-1

[0336]

[0337] Example 10-2

[0338]

[0339] Examples 10-5

[0340]

[0341] Examples 10-6

[0342]

[0343] Example 11-1

[0344]

[0345] Example 12-1

[0346]

[0347] Example 13-1

[0348]

[0349] Example 14-1

[0350]

[0351] Example 15-1

[0352]

[0353] Example 15-2

[0354]

[0355] Example 15-3

[0356]

[0357] Example 15-4

[0358]

[0359] Example 16-2

[0360]

[0361] Example 17-1

[0362]

[0363] Example 17-2

[0364]

[0365] Example 17-3

[0366]

[0367] Example 17-4

[0368]

[0369] Example 18-1

[0370]

[0371] Example 19-3

[0372]

[0373] Example 19-4

[0374]

[0375] Example 19-5

[0376]

[0377] Example 19-6

[0378]

[0379] Example 19-7

[0380]

[0381] Examples 19-8

[0382]

[0383] Examples 19-9

[0384]

[0385] Examples 19-10

[0386]

[0387] Example 20-1

[0388]

[0389] Example 20-2

[0390]

[0391] Example 20-3

[0392]

[0393] Example 23-1

[0394]

[0395] Example 23-2

[0396]

[0397] Example 23-3

[0398]

[0399] Example 23-4

[0400]

[0401] Example 23-5

[0402]

[0403] Example 24-1

[0404]

[0405] Example 24-2

[0406]

[0407] Example 25-3

[0408]

[0409] Example 25-4

[0410]

[0411] Example 26-1

[0412]

[0413] Example 26-2

[0414]

[0415] Hereinafter, embodiments of the present invention will be described. It should be noted that embodiments formed by arbitrarily selecting and combining the following embodiments, as well as embodiments formed by arbitrarily combining the following embodiments with any of the embodiments or implementations described in this specification, are also included in the present invention.

[0416] [Method A1]

[0417] A coupling precursor (I) or a salt thereof, wherein, in general formula (I),

[0418] ZLD (I)

[0419] Z is a reactive group that can react with functional groups present in antibodies or modified antibodies.

[0420] [Method A2]

[0421] According to [method A1], the coupling precursor (I) or its salt, wherein Z is maleimide (formula (i) above), α-halomethyl carbonyl (formula (ii) above), ethynylphosphonic amide ester (formula (iii) above), carboxyl, active ester body of carboxyl, mercapto, hydroxyl, amino, alkynyl, cycloalkynyl, azacyclic alkynyl or azide (-N3 group).

[0422] [Method A3]

[0423] According to [method A1] or [method A2], the coupling precursor (I) or its salt, wherein Z is maleimide (formula (i) above), α-halomethyl carbonyl (formula (ii) above), ethynylphosphonic amide ester (formula (iii) above), carboxyl, active ester of carboxyl, alkynyl, cycloalkynyl, azacyclic alkynyl, or azide (-N3 group).

[0424] [Method A4]

[0425] The coupling precursor (I) or its salt according to any one of [Formula A1] to [Formula A3], wherein Z is a maleimide group (formula (i) above), an α-halomethyl carbonyl group (formula (ii) above), a carboxyl group, or an active ester of a carboxyl group.

[0426] [Method B1]

[0427] The conjugate precursor (I) or its salt according to any one of [method A1] to [method A4], wherein D is an antitumor drug residue (hereinafter also referred to as "antitumor drug residue") formed by removing one hydrogen atom or one hydroxyl group from any position of an antitumor drug molecule or its analogue or its derivative.

[0428] [Method B2]

[0429] The conjugate precursor (I) or its salt according to any one of [method A1] to [method A4] and [method B1], wherein D is: camptothecin; auristatin E (AE) or monomethylauristatin E (MMAE); maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; cazithromycin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytidine; vincristine; vinblastine; doxorubicin; salicylate; superdox; ciprofloxacin; cardiol (CS-940); or analogues of these antitumor drugs; or antitumor drug residues of the above-mentioned antitumor drug molecules or their analogues or derivatives.

[0430] [Method B3]

[0431] The conjugate precursor (I) or its salt according to any one of [method A1] to [method A4], [method B1] and [method B2], wherein D is: camptothecin; auristatin E (AE) or monomethylauristatin E (MMAE); maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; cazithromycin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytidine; vincristine; vinblastine; doxorubicin; sulphurin 10; superdox; or analogues of these antitumor drugs; or antitumor drug residues of the above-mentioned antitumor drug molecules or their analogues or derivatives.

[0432] [Method B4]

[0433] The conjugate precursor (I) or its salt according to any one of [method A1] to [method A4] and [method B1] to [method B3], wherein D is: camptothecin; auristatin E (AE) or monomethylauristatin E (MMAE); maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; or an analogue of these antitumor drugs; or an antitumor drug residue of the above-mentioned antitumor drug molecule or its analogues or derivatives.

[0434] [Method B5]

[0435] The conjugate precursor (I) or its salt according to any one of [method A1] to [method A4] and [method B1] to [method B4], wherein D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of: an antitumor drug or an analogue of such antitumor drugs whose hydroxyl group is phosphorylated within the molecule; or the aforementioned antitumor drug molecule or its analogues or derivatives thereof.

[0436] [Method C1]

[0437] The coupling precursor (I) or its salt according to any one of [method A1] to [method A4] and [method B1] to [method B4], wherein,

[0438] L is an alkylene group that can be optionally substituted;

[0439] One or more methylene groups in the chain of this alkylene group can be independently selected from...

[0440] -C(R 1 (R) 2 )-;-O-;-N(R 3 )-;-N(R 3 )-N(R3 )-;-S-;-Se-;-Si(R 4 (R) 5 )-;-SS-;-Se-Se-;-SOm-;-SeOn-;-C(=C(R 6 (R) 7 ))-;-C(=O)-;-C(=S)-;-C(=N(R 8 ))-;-C(=N-OR 9 )-;-C(=NN(R 10 (R) 11 ))-;-P(=O)(R 12 )-;-P(=O)(OR 13 )-;-OP(=O)(R 12 -O-;-OP(=O)(OR 13 )-O-;-C(R 14 =; =C(R) 14 )-;-C(R 14 )=C(R 14 )-;-N=;=N-;-C≡C-;-(OC(R 1 (R) 2 )-C(R 1 (R) 2 )) 1-30 -;-(C(R 1 (R) 2 )-C(R 1 (R) 2 )-0) 1-30 -; substituted with one or more divalent groups from the group consisting of optionally substituted alkenyl groups, optionally substituted ynyl groups, optionally substituted cycloalkyl groups; optionally substituted cycloalkenyl groups; optionally substituted cycloalkynyl groups; optionally substituted azeotropic ynyl groups; optionally substituted aryl groups; optionally substituted heteroaryl groups; and optionally substituted heterocyclic groups.

[0441] R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 and R 14Each of the following groups is independently selected from the group consisting of hydrogen atom, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted azaheteroylynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclic groups, R 3 When the alkyl group is alkyl, it may optionally form a cyclic structure together with the alkyl group in the adjacent methylene group.

[0442] m and n are each an independent integer from 0 to 2.

[0443] [Method C2]

[0444] According to the coupling precursor (I) or its salt as described in [method C1], wherein,

[0445] L is an alkylene group that can be optionally substituted;

[0446] One or more methylene groups in the chain of the alkylene group are replaced by one or more divalent groups independently selected from the following chemical formula group.

[0447] Chemical formula group:

[0448] -OC(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0449] -SC(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0450] -N(H or C1-C4 alkyl)-;

[0451] -N(H or C1-C4 alkyl)-OC(H or C1-C4 alkyl)(C(H or C1-C4 alkyl)-;

[0452] -C(H or C1-C4 alkyl)-SO 0-2 -C(H or C1-C4 alkyl)-;

[0453] -C(H or C1-C4 alkyl)-SSC(H or C1-C4 alkyl)-;

[0454] -C(H or C1-C4 alkyl)=C(H or C1-C4 alkyl)-;

[0455] -C(H or C1-C4 alkyl)=N-;

[0456] -C(H or C1-C4 alkyl) = NO-;

[0457] -C(H or C1-C4 alkyl)=NN(H or C1-C4 alkyl)-;

[0458] -C(=C(H or C1-C4 alkyl)(H or C1-C4 alkyl))-;

[0459] -C(=NO(H or C1-C4 alkyl))-;

[0460] -C(=NN(H or C1-C4 alkyl)(H or C1-C4 alkyl))-;

[0461] -O-Si(C1-C4 alkylCH3)(C1-C4 alkyl)-O-;

[0462] -C(H or C1-C4 alkyl)-N(C1-C4 alkyl)-CH2-;

[0463] -C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-C(=O)-C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0464] -C(=O)-O-;

[0465] -C(=O)-S-;

[0466] -C(=O)-N(H or C1-C4 alkyl)-;

[0467] -OC(=O)-O-;

[0468] -N(H or C1-C4 alkyl)-C(=O)-N(H or C1-C4 alkyl)-;

[0469] -C(=O)-N(H or C1-C4 alkyl)-C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-;

[0470] -C(=O)-(CH2) 1-10 -(O-CH2CH2) 1-20 -O-(CH2) 1-10 -C(=O)-;

[0471] -C(=O)-(CH2) 1-10 -(O-CH2CH2) 1-20 -O-(CH2) 1-10 -CH2-NH-;

[0472] -NH-CH2-(CH2) 1-10 (O-CH2CH2) 1-20 -O-(CH2) 1-10 -CH2-NH-;

[0473] -C(=O)-N(C1-C4 alkyl)-CH2CH2-N(C1-C4 alkyl)-C(=O)-;

[0474]

[0475] [In the formula, * represents the bonding site with the adjacent group];

[0476] -P(=O)(OH)-O-;

[0477] -OP(=O)(OH)-O-;

[0478] -OP(=S)(OH)-O-;

[0479] -OP(=O)(OH)-S-;

[0480] -OP(=O)(OH)-OP(=O)(OH)-O-;

[0481] -OP(=O)(OH)-OP(=O)(OH)-OP(=O)(OH)-O-;

[0482] -CH(CH2-NH2)-;

[0483] -CH(CH2-NH(C1-C4 alkyl))-;

[0484] -CH(CH2-N(C1-C4 alkyl)2)-;

[0485] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 0-20 -C1-C4 alkyl)-;

[0486] -CH(CH2-NH-C(=O)-(CH2) 0-20 -lactone group)-;

[0487] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-lactone group)-;

[0488] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-NH-lactone group)-;

[0489] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-NH-C(=O)-lactone group)-;

[0490] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-lactone group)-;

[0491] -CH(CH2-NH-(CH2) 1-20 -(O) 0-1 -(P(=O)(OH)2)-;

[0492] -CH(CH2-NH-C(=O)-(CH2) 1-20 -(P(=O)(OH)2)-;

[0493] -CH(CH2-NH-C(=O)-(CH2) 1-20 -C(=O)-NH-(CH2) 1-10 -(P(=O)(OH)2)-;

[0494] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-(P(=O)(OH)2)-;

[0495] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -(P(=O)(OH)2)-;

[0496] -CH(CH2-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-;

[0497] -CH(CH2-NH-C(=O)-(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-;

[0498] -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-;

[0499] -N((CH2) 1-10-(O-CH2CH2) 1-20 -NH-C(=O)-(CH2) 1-10 -(D-glucose-6-O-yl)-;

[0500] -N((CH2) 1-10 -(O-CH2CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -(O-CH2CH2) 1-10 -NH-C(=O)-(CH2) 1-10 -(D-glucose-6-O-yl)-;

[0501] -N((CH2) 1-10 -(O-CH2CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -(O-CH2CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -(O-CH2CH2) 1-10 -NH-C(=O)-(CH2) 1-10 -(D-glucose-6-O-yl)-;

[0502] -(cis)-CH=CH-P(=O)(O-CH2CH3)-NH-(phenylene)-C(=O)-;

[0503] -C(=O)-(cyclohexylene);

[0504] -(succinimide group)-;

[0505] -Ser-;

[0506] -Cys-;

[0507] -Ama-:

[0508] -Asp-;

[0509] -Glu-;

[0510] -Orn-;

[0511] -Lys-;

[0512] -ValLys-;

[0513] -ValCit-;

[0514] -ValAla-;

[0515] -GlyGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0516] -PheLys-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0517] -ValLys-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0518] -ValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0519] -ValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0520] -AspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0521] -LysAspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0522] -GluValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0523] -LysValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0524] -SerValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0525] -AspValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0526] -GluValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0527] -LysValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0528] -SerValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0529] -GlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0530] -AspGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0531] -GluGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0532] -LysGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0533] -SerGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0534] -AspAspAspAspAsp-C(=O)-NH-(optionally substituted phenylene)-CH2-;

[0535] -GlyGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0536] -PheLys-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0537] -ValLys-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0538] -ValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0539] -ValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0540] -AspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0541] -GluValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0542] -LysValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0543] -SerValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0544] -AspValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0545] -GluValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0546] -LysValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0547] -SerValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0548] -GlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0549] -AspGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O-);

[0550] -GluGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0551] -LysGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0552] -SerGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0553] -AspAspAspAspAsp-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-;

[0554] -GlyGlyPheGly-C(=O)-NH-CH2-;

[0555] -AspGlyGlyPheGly-C(=O)-NH-CH2-;

[0556] (Among the aforementioned amino acid residues and amino acid residues in peptides,

[0557] The carboxyl groups of the side chains of Ama, Asp, and Glu may optionally form (alkyl) esters, (aminoalkyl) esters, ((N-alkylamino)alkyl) esters, ((N,N-dialkylamino)alkyl) esters, ((trialkylammonium)alkyl) esters, (lactone) esters, ((lactone)alkyl) esters, ((phosphoryl)alkyl) esters, or ((bisphosphoryl)alkyl) esters; or may optionally form unsubstituted amides, alkylamides, dialkylamides, ((N-alkylamino)alkyl)amides, ((N,N-dialkylamino)alkyl)amides, ((trialkylammonium)alkyl)amides, (lactone)amides, ((lactone)alkyl)amides, (phosphoryl)amides, ((phosphoryl)alkyl)amides, or ((bisphosphoryl)alkyl)amides.

[0558] The amino groups in the side chains of Lys and Orn can have one or two alkyl, alkylcarbonyl, (aminoalkyl)carbonyl, ((N-alkylamino)alkyl)carbonyl, ((N,N-dialkylamino)alkyl)carbonyl, ((trialkylammonium)alkyl)carbonyl, lactone, (lactone)alkyl, (lactone)carbonyl, (lactone)alkyl)carbonyl, phosphoryl, (phosphoryl)alkyl, (phosphoryl)alkyl, (phosphoryl)alkyl)carbonyl, (bisphosphoryl)alkyl, or (bisphosphoryl)alkyl)carbonyl as substituents.

[0559] The hydroxyl group on the side chain of Ser and the thiol group on the side chain of Cys can have alkyl, alkylcarbonyl, aminoalkyl, (aminoalkyl)carbonyl, (N-alkylamino)alkyl, ((N-alkylamino)alkyl)carbonyl, (N,N-dialkylamino)alkyl, ((N,N-dialkylamino)alkyl)carbonyl, (trialkylammonium)alkyl, ((trialkylammonium)alkyl)carbonyl, lactone, (lactone)alkyl, (lactone)carbonyl, ((lactone)alkyl)carbonyl, phosphoryl, (phosphoryl)alkyl, ((phosphoryl)alkyl)carbonyl, (bisphosphoryl)alkyl, or ((bisphosphoryl)alkyl)carbonyl as substituents.

[0560] And the following formula: 1,2,3-triazolyl.

[0561]

[0562]

[0563] [In the formula, * represents the bonding site with the adjacent group.]

[0564] [Method C3]

[0565] The coupling precursor (I) or its salt according to [method C1], wherein L contains one or more structures selected from the following.

[0566]

[0567] [In the formula, * represents the bonding site with the adjacent group.]

[0568] [Method C4]

[0569] The coupling precursor (I) or its salt according to [method C1], wherein L contains one or more structures selected from the following.

[0570]

[0571] [In the formula, * represents the bonding site with the adjacent group.]

[0572] [Method C5]

[0573] The coupling precursor (I) or its salt according to [method C3] or [method C4], wherein L further comprises one or more structures selected from the following.

[0574]

[0575] [In the formula, * represents the bonding site with the adjacent group.]

[0576] [Method C6]

[0577] The coupling precursor (I) or its salt according to [method C1], wherein L is any divalent group selected from the following group of chemical formulas.

[0578] Chemical formula group:

[0579]

[0580]

[0581]

[0582]

[0583] [In the above formula, * represents the bonding site with the reactive group Z, and # represents the bonding site with the antitumor drug residue D.]

[0584] [Method C7]

[0585] According to any one of [method A1] to [method A4], [method B1] to [method B5] and [method C1] to [method C6], the coupling precursor (I) or its salt thereof, wherein L has at any position one or more groups selected from lactone, phosphoryl (-P(=O)(OH)2); phosphoryl alkyl carbonyl (-C(=O)-alkylene-P(=O)(OH)2); bisphosphonyl methyl (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino; monoalkyl amino; dialkyl amino; a cyclic amino in which the two alkyl groups of the dialkyl amino form a ring together with the nitrogen atom to which they are bonded; and trialkylammonium as a substituent or protecting group, wherein when the total number of such groups is greater than or equal to, they may be identical or different.

[0586] [Method D1]

[0587] A synthetic intermediate (II) of a coupling precursor, or a salt thereof, wherein, in the above general formula (II):

[0588] Z 1 -L 1 -D 1 (II)

[0589] Z 1 The conjugating group is a hydroxyl, nitro, cyano, amide, oxo, azide, amino, optional monoalkylamino with substituents, imino (including -N=, imino of cyclic amines), carboxyl, phosphoryl, alkynyl, cycloalkynyl, azacyclic alkynyl, or mercapto; or a protecting group thereof.

[0590] [Method D2]

[0591] Synthetic intermediate (II) or its salt according to [method D1], wherein Z 1 The conjugating group is a hydroxyl, nitro, cyano, oxo, azide, amino, optionally a monoalkylamino with a substituent, imino (including -N=, imino of cyclic amines), carboxyl, phosphoryl, alkynyl, cycloalkynyl, azacyclic alkynyl, or mercapto; or a protecting group thereof.

[0592] [Method D3]

[0593] Synthetic intermediate (II) or its salt according to the coupling precursor as described in [Method D1] or [Method D2], wherein Z 1 The conjugating group is a hydroxyl, nitro, oxo, azide, amino, optionally a monoalkylamino group with a substituent, an imino (including -N=, imino of cyclic amines), a carboxyl, phosphoryl, alkynyl, cycloalkynyl, azacyclic alkynyl, or a mercapto; or a protecting group thereof.

[0594] [Method D4]

[0595] The coupling precursor synthesis intermediate (II) or its salt according to any one of [method D1] to [method D3], wherein Z 1 The conjugating group is a hydroxyl, nitro, oxo, azide, amino, optionally a monoalkylamino group with a substituent, an imino (including -N=, imino of cyclic amines), a carboxyl, phosphoryl, alkynyl, cycloalkynyl, or azacyclic alkynyl; or a protecting form thereof.

[0596] [Method E1]

[0597] The coupling precursor synthesis intermediate (II) or its salt according to any one of [methods D1] to [methods D4], wherein L 1 The substituted alkylene group is optionally replaced; one or more methylene groups in the chain of the alkylene group are replaced by one or more groups selected from the divalent groups described in [Scheme C1] to [Scheme C4] above.

[0598] [Method F1]

[0599] The coupling precursor synthesis intermediate (II) or its salt is prepared according to any one of [methods D1] to [methods D4] and [method E1], wherein Z 1 The group is selected from the bonding groups described in [methods D1] to [methods D4] above.

[0600] D 1 The group is selected from the antitumor drug residues described in [method B1] to [method B5] above.

[0601] [Method F2]

[0602] The coupling precursor synthesis intermediate (II) or its salt is prepared according to any one of [methods D1] to [methods D4] and [method E1], wherein Z 1 and D 1 Each group is independently selected from the bonding groups described in [Manner D1] to [Manner D4] above.

[0603] [Method F3]

[0604] The coupling precursor synthesis intermediate (II) or its salt is prepared according to any one of [methods D1] to [methods D4] and [method E1], wherein Z 1 The group is selected from the reactive groups described in [method A1] to [method A4] above.

[0605] D 1 It is a group selected from the bonding groups described in [Manner D1] to [Manner D4] above.

[0606] [Method G1]

[0607] According to any one of [methods D1] to [methods D4], [method E1], and [method F1], the coupling precursor intermediate (II) or its salt, wherein Z 1 The group is selected from the bonding groups described in [methods D1] to [methods D4] above.

[0608] D 1 The group is selected from the residues of the antitumor drugs described in [methods B1] to [methods B5] above.

[0609] D 1 and L 1 At least one of them has one or more lactone groups as substituents or protecting groups at any position, and independently of this,

[0610] D 1 and L 1 At least one of the following groups has, at any position, a group selected from phosphoryl (-P(=O)(OH)2); phosphorylalkylcarbonyl (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino; monoalkylamino; dialkylamino; a cyclic amino group in which the two alkyl groups of the dialkylamino group together with the nitrogen atom to which they are bonded form a ring; and trialkylammonium as a substituent or protecting group, wherein when the total number of such groups is greater than or equal to, they may be identical or different.

[0611] [Method G2]

[0612] According to any one of [methods D1] to [methods D4], [method E1], [method F2], and [method F3], the coupling precursor synthesis intermediate (II) or its salt, wherein Z 1 The group is selected from the reactive groups described in [method A1] to [method A4] above, or from the conjugating groups described in [method D1] to [method D4] above.

[0613] D 1 The group is selected from the bonding groups described in [methods D1] to [methods D4] above.

[0614] L 1The amino group has one or more of the following groups as substituents or protecting groups at any position: lactone; phosphoryl group (-P(=O)(OH)2); phosphoryl alkyl carbonyl group (-C(=O)-alkylene-P(=O)(OH)2); bisphosphonyl methyl group (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino group; monoalkyl amino group; dialkyl amino group; cyclic amino group in which the two alkyl groups of the dialkyl amino group together with the nitrogen atom to which they are bonded form a ring; and trialkylammonium group. When the total number of such groups is greater than or equal to, they may be the same or different.

[0615] Among the above divalent groups, the groups that are asymmetrical and the groups that can be asymmetrical are represented by L and L in general formulas (I) and (II), respectively. 1 When a portion of it is present, the divalent group can be oriented in any direction on the paper, left, right, up, or down. For example, -N(R 3 )-C(R 1 (R) 2 When - exists as part of L, it can be -N(R) 3 - On side B, -C(R) 1 (R) 2 - On the D side, or -C(R) 1 (R) 2 - On side B, -N(R) 3 - On the D side.

[0616] It should be noted that "three-letter units" are symbols used to represent the following amino acids, especially in this specification, representing the divalent residues (-NH-CH(R) of each amino acid). 15 )-C(=O)-). Where, R 15 The following are the amino acids represented by the following groups: hydrogen atom (Gly), methyl (Ala), isopropyl (Val), isobutyl (Leu), (butan-2-yl)yl (Ile), phenylmethyl (Phe), hydroxymethyl (Ser), mercaptomethyl (Cys), carboxymethyl (Asp), 2-carboxyethyl (Glu), 3-aminopropyl (Orn), 4-aminobutyl (Lys), 3-(ureo)propyl (Cit), 3-guanidinopropyl (Arg), or (1H-imidazol-4-yl)methyl (His) (the parentheses indicate the corresponding amino acid). Additionally, the above formula (-NH-CH(R) 15 In )-C(=O)-), R 15 The propyl group, together with the nitrogen atom of "-NH-" in the formula, forms a 5-membered ring, becoming a cyclic amino acid (Pro; proline).

[0617] Glycine

[0618] Ala; alanine

[0619] Valine;

[0620] Leu; Leucine

[0621] Ile; Isoleucine

[0622] Phe; Phenylalanine

[0623] Serine;

[0624] Cys; Cysteine

[0625] Asp; Aspartic acid

[0626] Glutamic acid

[0627] Ornithine; Ornithine

[0628] Lysine;

[0629] Cit; Citrulline

[0630] Arginine; Arginine

[0631] His; histidine

[0632] Proline;

[0633] It should be noted that in the coupling precursor represented by general formula (I) and the intermediate for synthesizing the coupling precursor represented by general formula (II) of the present invention, the "-LD" part and the "-L" part are different. 1 -D 1 When a part contains amino acid residues with an asymmetric center that may produce optical isomerism, any optical isomers of the D and L types are included in this invention.

[0634] The effects of the invention

[0635] It has been confirmed that the antibody-drug conjugate obtained by reacting the conjugate precursor (I) or its salt with an antibody can be selectively delivered to target tumor cells after administration into a living organism and release an antitumor drug within those cells, thereby becoming a cancer therapeutic agent with excellent antitumor efficacy and safety. Furthermore, it has been confirmed that the conjugate precursor synthesis intermediate (II) or its salt is useful for the synthesis of the conjugate precursor (I). Attached Figure Description

[0636] Figure 1 This is a graph showing the tumor volume inhibition rate of the compound (1 mg / kg) from ADC manufacturing example 2 in test example 4.

[0637] Figure 2This is a graph showing the tumor volume inhibition rate of the compound (1 mg / kg) from ADC manufacturing example 3 in test example 4.

[0638] Figure 3 This is a graph showing the tumor volume inhibition rate of the compound (1 mg / kg) from ADC manufacturing example 15 in test example 4.

[0639] Figure 4 This is a graph showing the tumor volume inhibition rate of the compound (1 mg / kg) from ADC manufacturing example 19 in test example 4.

[0640] Figure 5 This is a chromatogram representing the SEC analysis HPLC of ADC manufacturing example 2 in test example 8. Detailed Implementation

[0641] The following are definitions of terms used in this specification, with specific examples provided, but the invention is not limited thereto. Furthermore, the various compounds (raw materials, intermediates), drug molecules, and substituents / protecting groups defined or illustrated below can be arbitrarily selected, and by combining them, the conjugate precursor (I) or its salt, and the conjugate precursor synthesis intermediate (II) or its salt of the present invention can be manufactured.

[0642] Unless otherwise stated, the following terms and phrases used in this specification shall have the meanings described herein.

[0643] The term "tumor" as used in this instruction manual refers to "malignant tumor," including "carcinoma," "leukemia," "cancer," "sarcoma," and "brain tumor," among others.

[0644] The term "antibody" as used in this specification refers to complete monoclonal antibodies, polyclonal antibodies, single-specific antibodies, multi-specific antibodies (e.g., bispecific antibodies), heavy chain antibodies, and antibody fragments. A "monoclonal antibody" is an antibody derived from a substantially homogeneous population of antibodies, the individual antibodies in which the population is identical except for the very rare possibility of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. A "polyclonal antibody" refers to a heterogeneous population of antibody molecules derived from the serum of an immunized animal. A "monospecific antibody" is an antibody that exhibits binding specificity against a single antigen. A "multispecific antibody" is an antibody that exhibits binding specificity against two or more different antigens (two in the case of bispecific antibodies). Bispecific antibodies were prepared using methods described in Journal of Hematology & Oncology 8, 130 (2015), Frontiers in Immunology 8, 38 (2017), BioDrugs 24(2), 89-98 (2010), MAbs 1(6), 539-547 (2009), and Journal of Immunology 155(1), 219-25 (1995). The heavy chain antibody refers to an antibody consisting of only two heavy chains and lacking a light chain.

[0645] The "antibody fragment" includes an antigen-binding region or a variable region, comprising a portion of a complete antibody. For use in this invention, the antibody fragment needs to have functional groups (e.g., thiol, amino, etc.) capable of binding to a drug moiety via a linker, or disulfide bonds that can generate thiol groups through reduction. The "antigen" mentioned above refers to a substance that the antibody specifically binds to.

[0646] The "antibodies" used in this invention can be obtained by known methods, such as by immunizing rats, mice, and rabbits with various antigens and then screening and purifying the antibodies produced in vivo using various methods. Human monoclonal antibodies can be produced by known methods (e.g., Immunology Today, Vol. 472-79 (1983)). In cases involving species other than humans, chimerism or humanization using known techniques is preferred.

[0647] The "antibody" used in this invention can also be obtained by purchasing it as a commercially available reagent, extracting / purifying it from antibody pharmaceutical preparations, or preparing it using known methods. The nucleotide sequence of the antibody required for its preparation can be obtained, for example, from databases such as GenBank or from literature. Alternatively, it can be obtained by cloning and sequencing using known methods.

[0648] The "antibody" used in this invention is an immunoglobulin, a molecule containing an antigen-binding site that specifically binds to an antigen. This antibody can be any of IgG, IgE, IgM, IgD, IgA, and IgY, with IgG being preferred. Furthermore, as a subclass, it can be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG1, IgG2, and IgG4 being preferred. These antibodies can be polyclonal antibodies or monoclonal antibodies, with monoclonal antibodies being preferred.

[0649] The antibodies used in this invention can be any antibodies capable of targeting tumor cells. In the antibody-drug conjugates of this invention, the antibody binds to an antitumor active drug via a linker. Therefore, the antibody preferably possesses one or more of the following properties: the ability to recognize tumor cells, the ability to specifically bind to tumor cells, the ability to be absorbed into and internalized within tumor cells, and the ability to damage tumor cells. "Specific binding" refers to the antibody or antibody derivative binding to the corresponding target antigen in a highly selective manner, without binding to other antigens present in large quantities. This binding can be confirmed using flow cytometry. "Antibody internalization" refers to the process by which an antibody binds to an antigen on the cell membrane surface and is absorbed into the cell through endocytosis. This absorption can be confirmed by the following methods: using a secondary antibody (fluorescently labeled) bound to the antibody to visualize the antibody absorbed into the cell using a fluorescence microscope (Cell Death and Differentiation 15, 751-761 (2008)), or measuring its fluorescence intensity (Molecular Biology of the Cell 15, 5268-5282 (2004)), or using an immunotoxin bound to the antibody to inhibit cell proliferation by releasing the toxin upon absorption into the cell, as described by the Mab-ZAP method (Bio Techniques 28, 162-165 (2000)).

[0650] "Damaging tumor cells" refers to the killing of cells by antibodies, and their cell-damaging activity can be confirmed by measuring the inhibitory activity of cell proliferation in vitro.

[0651] The term "antibody" in this specification also includes modified versions of antibodies. Modified versions of antibodies refer to substances that have undergone chemical or biological modifications to the antibody. Chemical modifications include chemical modifications to the amino acid backbone of the antibody, as well as chemical modifications to N-binding or O-binding glycans. Biological modifications include post-translational modifications (e.g., addition to oxygen or nitrogen atoms in glycans, N-terminal or C-terminal treatment, deamidation, isomerization of aspartic acid, oxidation of methionine), and addition to N-terminal methionine residues expressed through prokaryotic host cells.

[0652] The aforementioned chemical or biological modifiers also include substances from which auxiliary linkers extend from functional groups (hydroxyl, thiol, amino, carboxyl, etc.) present in the original antibody, with "thiol, hydroxyl, amino, maleimide (formula (i) above), α-halomethylcarbonyl, ethynylphosphonic amide ester (formula (ii) above), carboxyl, active ester of carboxyl (e.g., formula (iii) above), azide (-N3), alkynyl, cycloalkynyl, azahexacyclic alkynyl, etc." It should be noted that in this specification, the "antibody" and the "modified antibody" defined above are sometimes collectively referred to as "antibody". The aforementioned "auxiliary linker" refers to the linkers "-L-" and "-L" in the above general formulas (I) and (II). 1 Part of "-".

[0653] The following are specific examples of antibodies used in the reaction with the conjugate precursor (I) of the present invention, but the present invention is not limited to these.

[0654] Anti-HER2 antibody, anti-EGFR antibody, anti-lymphocyte antigen 6E (Ly-6E) antibody, anti-hepatocyte growth factor receptor (HGFR) antibody, anti-CD22 antibody, anti-folate receptor α (FRα) antibody, anti-PSMA (FOLH1) antibody, anti-CD30 (TNFRSF8) antibody, anti-TROP-2 antibody, anti-CD19 antibody, anti-5T4 antibody, anti-mesothelin antibody, anti-CD20 antibody, anti-CD33 antibody, anti-CDB7-H3 antibody, anti-CD269 (BCMA) antibody, anti-CD142 antibody, anti-CD70 antibody, anti-CD123 antibody, anti-TAG-72 antibody, anti-TFRC antibody, anti-EPHA2 antibody, anti-EPHA3 antibody, anti-c-KIT antibody, anti-P D-L1 antibody, anti-epithelial cell adhesion molecule (EPCAM) antibody, anti-AXL antibody, anti-interleukin-1 receptor helper antibody, anti-mucin-16 (CA125) antibody, anti-MUC1 antibody, anti-GPC3 antibody, anti-CLEC12A (CLL1) antibody, anti-LRRC15 antibody, anti-ADAM9 antibody, anti-sodium-dependent phosphate transporter 2B (SLC34A2) antibody, anti-CD25 antibody, anti-ROR1 antibody, anti-TweakR antibody, anti-CD74 antibody, anti-CEACAM5 antibody, anti-CD105 antibody, anti-CD79b antibody, anti-CD147 antibody, anti-Sialyl-Tn antibody, anti-GCC antibody, anti-HER3 antibody, anti-integrin antibody, anti-EGFR Variant III antibody, anti-tight junction protein 6 antibody, anti-leucine-rich G protein-coupled receptor 5 antibody, anti-PTK7 antibody, anti-LYPD3 antibody, anti-ASCT2 antibody, anti-ASPH antibody, anti-GPC1 antibody, anti-CD174 antibody, anti-CD38 antibody, anti-ANGPT2 antibody, anti-CD44 antibody, anti-DDL3 antibody, anti-FGFR2 antibody, anti-KAAG1 antibody, anti-CD73 antibody, anti-CLDN18.2 antibody, anti-PRLR antibody, anti-SEZ6 antibody, anti-neprilysin antibody, anti-neuronal cell adhesion molecule 1 antibody, anti-CD166(ALCAM) antibody, anti-CD24 antibody, anti-CD27 antibody, anti-CDH3 antibody, anti-EPHA4 antibody, anti-FGFR3 antibody, anti-GFRAL antibody, anti-GloboH antibody, anti-IL-13Ra2 antibody, anti-STEAP-1 antibody, anti-TMCC3 antibody, anti-CD209 antibody, anti-CD37, anti-CD48 antibody, anti-CDCP1 antibody, anti-CD46 antibody, anti-CD200 antibody, anti-CTLA4 antibody, anti-CXCR5 antibody, anti-FLT3 antibody, anti-SDC1(CD138) antibody, anti-CLDN6 / 9 antibody, anti-CEACAM6 antibody, anti-CA9 antibody, anti-EDNRB antibody, anti-GD3 ganglioside antibody, anti-MICA / B antibody, anti-JAG1 / 2 antibody, anti-TM4SF1 antibody, anti-uPAR antibody, anti-carbonic anhydrase IX antibody, anti-ALK1 antibody, anti-IGF -1R antibody, anti-KDR antibody, anti-CEACAM8 antibody, anti-IL5R antibody, anti-CD205 (Ly75) antibody, anti-MSR1 antibody, anti-KREMEN2 antibody, anti-SSEA-4 antibody, anti-CD228 antibody, anti-CD5 antibody, anti-DLL4 antibody, anti-FAP antibody, anti-Notch3 antibody, anti-AG7 antibody, anti-guanylate cyclase antibody, anti-adhesion-4 antibody, anti-CD226 (DNAM-1) antibody, anti-FcRL5 antibody, anti-HLA-DR antibody, anti-ITGB3 antibody, anti-DLK1 antibody, anti-CD157 (BST1) antibody, anti-CD56 (NCAM1) antibody, anti-MICA (MHC) antibody Class I peptide-related sequence A) antibodies, anti-SSEA-1 antibody, anti-TRAIL-R2 (DR5) antibody, anti-GPNMB antibody, anti-CCR5 antibody, anti-LAMP1 antibody, anti-LGALS3BP antibody, anti-ROR2 antibody, anti-DLL3 antibody, anti-ETBR antibody, anti-LIV-1 antibody, anti-Integrinαvβ6 antibody, anti-TIM-1 antibody, anti-AGS-16 (ENPP3) antibody, anti-SLITRK6 antibody, anti-GD2 antibody, anti-CD52 antibody, anti-CCR4 antibody, anti-VEGFR2 antibody, anti-PDGFR antibody, anti-FGFR antibody, anti-SLAMF7 antibody, anti-GD2 ganglioside antibody, anti-EPHA3 antibody, anti- Integrin αvβ3 antibody, anti-AGS-5 antibody, anti-CA19-9 antibody, anti-PSA antibody, anti-MAGE3 antibody, anti-transferrin receptor 1 (CD71) antibody, anti-CEACAM1 antibody, anti-SLC3A2 (CD98) antibody, anti-ACVR1 antibody, anti-AG-7 antibody, anti-AMHR2 antibody, anti-ABCB1 antibody, anti-C16orf54 antibody, anti-cathepsin D antibody, anti-CCR7 antibody, anti-SLC44A4 antibody, anti-CD300LF antibody, anti-DPEP3 antibody, anti-FucGM1 antibody, anti-GPR20 antibody, anti-ITGB6 antibody, anti-Lewis-A carbohydrate antibody, anti-prolactin receptor antibody, anti-SialylTn antibody, anti-SLAMF6 antibody, anti-SLAMF7 antibody, anti-TRA-1-60 antibody, anti-protein lyase antibody, anti-B7-H4 antibody, anti-Cripto antibody, anti-CD99 antibody, anti-CanAg antibody, anti-A33 antibody, anti-α10β1 integrin antibody, anti-ALPP antibody, anti-CD248 antibody, anti-GPRC5D antibody.

[0655] The term "antibody functional group" as used in this specification refers to the groups present in the "antibody" and "modified antibody" as defined above, which react with the reactive groups present in the conjugate precursor (I) of this invention to form an antibody-drug conjugate. Specifically, examples include thiol, hydroxyl, amino, maleimide (formula (i) above), α-halomethylcarbonyl (formula (ii) above), ethynylphosphonic acid amide ester (formula (iii) above), carboxyl, active ester of carboxyl, azide (-N3), alkynyl, and cycloalkynyl.

[0656] The term "reactive group" as used in this specification refers to groups present in the conjugate precursor (I) or conjugate precursor intermediate (II) of this invention, which react with the "antibody functional groups" defined above to form antibody-drug conjugates. Specifically, examples include maleimide (formula (i) above), α-halomethylcarbonyl (formula (ii) above), ethynylphosphonic acid amide ester (formula (iii) above), carboxyl group, active ester body of carboxyl group, thiol group, hydroxyl group, amino group, alkynyl group, cycloalkynyl group, or azide group (-N3 group), etc.

[0657] The term "conjugating group" as used in this specification refers to a group present at the end of the linker component of the coupling precursor synthesis intermediate (II) of the present invention, and used to conjugate the aforementioned "reactive group" or the "antitumor drug residue" described later to the linker component via etherification, thioetherification, carboxyl esterification, phosphoric acid (mono- or di) esterification, amidation, reductive amination, disulfide reaction, or click reaction. Specifically, examples include hydroxyl, nitro, cyano, amide, oxo, azide, amino, optional monoalkylamino with substituents, imino (including -N=, cyclic amine imino), carboxyl, phosphoryl, alkynyl, cycloalkynyl, or mercapto groups. It should be noted that, within this group, nitro can be a precursor of amino, cyano can be a precursor of both amino and amide, and amide can be a precursor of both amino and carboxyl; therefore, they are also treated as "conjugating groups" in this specification.

[0658] The term "antitumor drug molecule" as used in this specification refers to any drug molecule that has antitumor effects, regardless of its mechanism of action, as long as it has the ability to combine with the linker (L in the conjugate precursor (I) and L in the conjugate precursor synthesis intermediate (II)).1 There are no particular restrictions on the substituents or partial structures that can be bound. In tumor cells, some or all of the linkers of antitumor drug molecules are cleaved and released, thus exerting their antitumor effect.

[0659] It should be noted that the term "antitumor drug residue" as used in this specification refers to a residue formed by removing one hydrogen atom or one hydroxyl group from any position of the aforementioned "antitumor drug molecule or its analogue or its derivative".

[0660] Examples of such antitumor drug molecules include: camptothecin; MMAE; maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; cazithromycin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytidine; vincristine; vinblastine; doxorubicin; salicylate 10; superdox; ciprofloxacin; cardiol (CS-940); or analogues of these antitumor drugs; or the above-mentioned antitumor drug molecules or their analogues, derivatives, etc.

[0661] The term "analyte" in "antitumor drug molecule or its analogue or its derivative" as used in this specification refers to a compound having a chemical structure and activity similar to the respective antitumor drug molecule. For example, "camptothecin analogue" refers to a compound having a chemical structure similar to camptothecin and having the same type I topoisomerase inhibitory activity as camptothecin.

[0662] In this specification, the term "derivative" for "antitumor drug molecule or its analogues, or their derivatives" refers to derivatives containing functional groups such as hydroxyl, thiol (-SH), amino (-NH2, -NH- (including amide groups and intra-ring -NH- of heteroaryl or heterocyclic groups)), and carboxyl groups within the molecule of the antitumor drug, protected by a "protecting group," or glucosinolate derivatives formed by the reaction of these functional groups with sugars. Sometimes, the protecting groups of these derivatives are deprotected in vivo through various chemical, biochemical, or metabolic reactions (e.g., pH changes, hydrolysis, redox reactions, etc.) to transform them into the parent compound (the original antitumor drug molecule) and exhibit antitumor activity. However, sometimes they remain unprotected and exhibit antitumor activity in their original form.

[0663] The "protecting group" can be selected arbitrarily from the protecting groups described in, for example, Greene's Protective Groups in Organic Synthesis, 5th edition, by PGMWuts, John Wiley & Sons Inc. (2014).

[0664] For protecting groups that function as hydroxyl groups, examples include: alkoxyalkyl groups such as methoxymethyl, methoxyethoxymethyl, and ethoxy-2-ethyl; arylalkoxymethyl groups such as benzyloxymethyl; aryloxymethyl groups such as phenoxymethyl; alkathiomethyl groups such as methylthiomethyl; arylalkathiomethyl groups such as benzylthiomethyl; arylthiomethyl groups such as phenylthiomethyl; aminomethyl groups where the N atom is optionally protected by an alkyl group or a "protecting group of amino" as described below; arylmethyl groups such as benzyl, 4-methoxybenzyl, and triphenylmethyl; alkylcarbonyl groups such as methylcarbonyl and ethylcarbonyl; alkenylcarbonyl groups such as allylcarbonyl; alkynylcarbonyl groups such as propargylcarbonyl; arylcarbonyl groups such as phenylcarbonyl; heteroarylcarbonyl groups such as pyridine-2,3 or 4-ylcarbonyl; heterocyclic carbonyl groups such as tetrahydrofuran-2 or 3-ylcarbonyl and 5-oxotetrahydrofuran-2-ylcarbonyl; tert-butoxycarbonyl and methoxycarbonyl. Alkoxycarbonyl groups such as ethoxycarbonyl; allyloxycarbonyl; phenoxycarbonyl, 4-nitrophenoxycarbonyl, 4-methoxyphenoxycarbonyl, etc. aryloxycarbonyl groups; 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4 (or 2)-nitrobenzyloxycarbonyl, 4 (or 2)-aminobenzyloxycarbonyl or its N-alkylcarbonyl or N-arylcarbonyl, 4 (or 2)-hydroxybenzyloxycarbonyl or its O-alkylcarbonyl or O-arylcarbonyl, etc. arylmethoxycarbonyl groups; tert-butylaminocarbonyl, methylaminocarbonyl, ethylaminocarbonyl, etc. alkylaminocarbonyl groups; trimethylsilyl, trimethylsilylethoxymethyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, etc. silyl groups; tetrahydropyranyl, 2 (or 4)-methoxytetrahydropyranyl, etc. In addition, the hydroxyl group can be protected in the form of an ester body with formic acid, boric acid, phosphoric acid, phosphonic acid, hypophosphonic acid, sulfuric acid, sulfonic acid, sulfinic acid, hyposulfonic acid, amino acid, or peptide.

[0665] In one embodiment, the protecting group of the hydroxyl group is an alkoxyalkyl group; an arylalkoxymethyl group; an aminomethyl group whose N atom is optionally protected by an alkyl group or the "amino protecting group" described below; an alkyl carbonyl group; an alkenyl carbonyl group; an alkoxycarbonyl group; an allyloxycarbonyl group; an aryloxycarbonyl group; an arylmethoxycarbonyl group; an alkylaminocarbonyl group; a tetrahydropyranyl group; a tetrahydrofuranyl group; a silyl group; a lactone group; a lactone alkyl group; a lactone carbonyl group; a lactone alkyl carbonyl group; a phosphoryl group; a phosphoryl alkyl group; or a phosphoryl alkyl carbonyl group.

[0666] For protecting groups that are thiol groups, the protecting groups that are hydroxyl groups described above can be listed. In addition, disulfide bonds (-SS-) can also be protecting groups.

[0667] Examples of protecting groups for the carboxyl group, which is a functional group, include alkyl groups such as methyl, ethyl, and tert-butyl; allyl; benzyl and other arylmethyl groups; lactone groups; and silyl groups such as trimethylsilyl, trimethylsilylethoxymethyl, tert-butyldimethylsilyl, and tert-butyldiphenylsilyl. In addition, the carboxyl group is sometimes protected in the form of an amide (e.g., an amide with ammonia, alkylamine, dialkylamine, lactone amine, amino acid ester, amino acid amide, etc.), and the amino group of this amide moiety may also have a lactone group, lactone alkyl group, or phosphoryl alkyl group as a substituent.

[0668] In one embodiment, the protecting group of the carboxyl group is alkyl; allyl; arylmethyl; lactone; lactone alkyl; or phosphoryl alkyl.

[0669] For the sulfenic acid group, sulfinic acid group, sulfonic acid group, phosphonic acid group, phosphonic acid group and boric acid group, which are functional groups, the same protecting groups as the carboxyl groups mentioned above can also be used.

[0670] Protecting groups for amino groups that are functional groups include, for example, alkoxycarbonyl groups such as tert-butoxycarbonyl, methoxycarbonyl, and ethoxycarbonyl; allyloxycarbonyl; aryloxycarbonyl groups such as phenoxycarbonyl, 4-nitrophenoxycarbonyl, and 4-methoxyphenoxycarbonyl; aryloxycarbonyl groups such as 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4 (or 2)-nitrobenzyloxycarbonyl, 4 (or 2)-aminobenzyloxycarbonyl or its N-alkylcarbonyl body or N-arylcarbonyl body, 4 (or 2)-hydroxybenzyloxycarbonyl or its O-alkylcarbonyl body or O-arylcarbonyl body; arylmethyl groups such as benzyl and triphenylmethyl; alkoxyalkyl groups such as methoxymethyl, methoxyethoxymethyl, and ethoxy-2-ethyl; arylalkoxymethyl groups such as benzyloxymethyl; and aryloxy groups such as phenoxymethyl. Methyl; alkylthiomethyl and other methylthiomethyl; arylalkylthiomethyl and other arylalkylthiomethyl; phenoxymethyl and other arylthiomethyl; aminomethyl with the N atom optionally protected by an alkyl or amino protecting group; alkylcarbonyl and other methylcarbonyl; alkenylcarbonyl and other allylcarbonyl; alkynylcarbonyl and other alkynylcarbonyl; arylcarbonyl and other phenylcarbonyl; heteroarylcarbonyl and other pyridine-2,3 or 4-ylcarbonyl; heterocyclic carbonyl and other heterocyclic carbonyl such as tetrahydrofuran-2 or 3-ylcarbonyl and 5-oxotetrahydrofuran-2-ylcarbonyl; silyl such as trimethylsilyl, trimethylsilylethoxymethyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl; or arylsulfonyl such as 2,4-dinitrobenzenesulfonyl and 4-nitrobenzenesulfonyl. In addition, the amino group can be protected in the form of an amide of formic acid, phosphoric acid, phosphonic acid, hypophosphonic acid, sulfuric acid, sulfonic acid, sulfinic acid, hyposulfonic acid, amino acid, or peptide.

[0671] In one embodiment, the protecting group of the amino group is alkoxycarbonyl; allyloxycarbonyl; arylmethoxycarbonyl; alkylcarbonyl; alkenylcarbonyl; arylcarbonyl; silyl, lactone; lactone alkyl; lactone carbonyl; lactone alkyl carbonyl; phosphoryl; phosphoryl alkyl; or phosphonyl alkyl carbonyl.

[0672] The attachment and detachment of these protective groups are carried out according to the methods usually implemented (e.g., see Greene's Protective Groups in Organic Synthesis, 5th Edition, by PGMUTS, John Wiley & Sons Inc. (2014)).

[0673] The functional group can react with various sugars as described above and be protected in the form of a glucosinolate. Specifically, it can be protected by a glucosyl group. Examples of such a glucosyl group include D-glucosyl, D-galactosyl, D-mannosyl, and D-glucuronic acid groups, and the functional groups of these sugar moieties (-OH, -SH, -NH2, -NH-, -COOH, etc.) can also be protected as described above. These glucosinolates and their protecting bodies can be produced using conventional methods commonly used in the field of glycochemistry (e.g., *Comprehensive Glycoscience From Chemistry to Systems Biology*, by Hans Kamerling (2007)).

[0674] In addition, the hydroxyl, mercapto (-SH), and amino (-NH2, -NH- (including amide group -NH- and heterocyclic group -NH- within the ring) of the above functional groups can also be protected in the form of esters, thioesters, amides or imides with various sugar acids (gluconic acid, glucuronic acid, galacturonic acid, etc.).

[0675] The above describes the "protecting groups" of various functional groups in the "derivatives" of "antitumor drug molecules or their analogues or derivatives" as described in this specification. However, this also applies to the "linkers (L, L)" described in this specification. 1 The functional group in the "()," "joining group", "various protecting groups or substituents".

[0676] It should be noted that the selection of the above-mentioned protecting group and its attachment and detachment methods are also applicable to the manufacture of compounds of general formulas (I) and (II) described later (coupling precursors and intermediates for the synthesis of coupling precursors).

[0677] Specific examples of "antitumor drug molecules or their analogues or derivatives" described in this specification are shown in Example 1 below, but the present invention is not limited thereto.

[0678] Example 1. Examples of "antitumor drug molecules or their analogues, or their derivatives".

[0679]

[0680] Camptothecin

[0681]

[0682] Topotecan

[0683] Letopecan

[0684] Iciticon

[0685] SN-38

[0686]

[0687] DB-67

[0688]

[0689] BNP-1350

[0690]

[0691] ST-1481

[0692]

[0693] CKD-602

[0694]

[0695] Dxd(N-(2-hydroxyacetyl)ecetane)

[0696]

[0697] U-001

[0698]

[0699] U-002

[0700]

[0701] U-003

[0702]

[0703] U-004

[0704]

[0705] U-005

[0706]

[0707] U-006

[0708]

[0709] U-007

[0710]

[0711] MMAE (methylauratestatin E)

[0712]

[0713] MMAF (methylauratestatin F)

[0714]

[0715] MMAQ (Methylauratestatin Q)

[0716]

[0717] MMAD (Monomethylaplysia 10)

[0718]

[0719] Tail hare 10

[0720]

[0721] U-008

[0722]

[0723] U-009

[0724]

[0725] U-010

[0726]

[0727] U-011

[0728]

[0729] U-012

[0730]

[0731] U-013

[0732]

[0733] U-014

[0734]

[0735] U-015

[0736]

[0737] U-016

[0738]

[0739] U-017

[0740]

[0741] U-018

[0742]

[0743] U-019

[0744]

[0745] U-020

[0746]

[0747] U-021

[0748]

[0749] U-022

[0750]

[0751] U-023

[0752]

[0753] U-024

[0754]

[0755] U-025

[0756]

[0757] U-026

[0758]

[0759] U-027

[0760]

[0761] U-028

[0762]

[0763] U-029

[0764]

[0765] U-030

[0766]

[0767] U-031

[0768]

[0769] U-032

[0770]

[0771] U-033

[0772]

[0773] U-034

[0774]

[0775] U-035

[0776]

[0777] U-036

[0778]

[0779] U-037

[0780]

[0781] U-038

[0782]

[0783] U-039

[0784]

[0785] U-040

[0786]

[0787] U-041

[0788]

[0789] U-042

[0790]

[0791] U-043

[0792]

[0793] U-044

[0794]

[0795] U-045

[0796]

[0797] Maytansine

[0798] DM-1 (Maytenyl)

[0799]

[0800] DM-3

[0801]

[0802] DM-4

[0803]

[0804] PBD dimer

[0805] Eriblin

[0806]

[0807] 5-FU

[0808]

[0809] PD-318088

[0810]

[0811] AS-703026

[0812]

[0813] TAK-733

[0814]

[0815] LY-3023414

[0816]

[0817] Kazimidic acid gamma1

[0818]

[0819] Paclitaxel

[0820] Dorsey

[0821] Mitomycin C

[0822]

[0823] Bleomycin A2

[0824]

[0825] Cyclocytidine

[0826] Changchun New Alkali

[0827] Vincristine

[0828] Donomycin

[0829] Doxorubicin

[0830] superdox

[0831]

[0832] Ciprofloxacin

[0833] Cardiol (CS-940)

[0834]

[0835] U-046

[0836]

[0837] U-047

[0838]

[0839] U-048

[0840]

[0841] U-049

[0842]

[0843] U-050

[0844]

[0845] U-051

[0846]

[0847] U-052

[0848]

[0849] U-053

[0850]

[0851] U-054

[0852]

[0853] U-055

[0854]

[0855] U-056

[0856]

[0857] U-057

[0858]

[0859] U-058

[0860]

[0861] U-059

[0862]

[0863] U-060

[0864]

[0865] U-061

[0866]

[0867] U-062

[0868]

[0869] U-063

[0870]

[0871] U-064

[0872]

[0873] U-065

[0874]

[0875] U-066

[0876]

[0877] U-067

[0878]

[0879] U-068

[0880]

[0881] U-069

[0882]

[0883] U-070

[0884]

[0885] U-071

[0886]

[0887] U-072

[0888]

[0889] U-073

[0890]

[0891] U-074

[0892]

[0893] U-075

[0894]

[0895] U-076

[0896]

[0897] U-077

[0898]

[0899] U-078

[0900]

[0901] U-079

[0902]

[0903] U-080

[0904]

[0905] U-081

[0906]

[0907] U-082

[0908]

[0909] U-083

[0910]

[0911] U-084

[0912]

[0913] U-085

[0914]

[0915] U-086

[0916]

[0917] U-087

[0918]

[0919] U-088

[0920]

[0921] U-089

[0922]

[0923] U-090

[0924]

[0925] U-091

[0926]

[0927] U-092

[0928]

[0929] U-093

[0930]

[0931] U-094

[0932]

[0933] U-095

[0934]

[0935] U-096

[0936]

[0937] U-097

[0938]

[0939] U-098

[0940]

[0941] U-099

[0942]

[0943] U-100

[0944]

[0945] U-101

[0946]

[0947] U-102

[0948]

[0949] U-103

[0950]

[0951] U-104

[0952] The term "carboxyl-active ester body" as used in this specification refers to a substance that enhances reactivity by forming an ester body of an alcohol with high acidity by causing the carboxyl group to form an ester body. Examples include N-hydroxysuccinimide ester, sulfosuccinimide ester, N-hydroxyphthalimide ester, N-hydroxysulfonylphthalimide ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxyl-4-nitrophenyl ester, pentafluorophenyl ester, etc., but the present invention is not limited to these.

[0953] In this specification, "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched saturated hydrocarbon (i.e., an alkane) having 1 to 10 carbon atoms. It should be noted that, unless otherwise specified, the alkyl group also includes "optionally substituted alkyl groups".

[0954] As a substituent for the alkyl group that may be substituted, one or more substituents may be selected independently from group A below.

[0955] Group A

[0956] Halogen atoms (fluorine, chlorine, bromine, or iodine atoms);

[0957] Alkoxy;

[0958] hydroxyl group;

[0959] thiol;

[0960] amino;

[0961] Alkylamino;

[0962] Dialkylamino (or two alkyl groups together to form a heterocyclic group containing a nitrogen atom);

[0963] Trialkylammonium group;

[0964] carboxyl;

[0965] Cyano;

[0966] Nitro;

[0967] Alkyl carbonyl;

[0968] alkenyl carbonyl;

[0969] alkynyl carbonyl;

[0970] aryl carbonyl;

[0971] heteroaryl carbonyl;

[0972] Heterocyclic carbonyl group;

[0973] lactone carbonyl;

[0974] Phosphorylalkylcarbonyl;

[0975] alkoxycarbonyl;

[0976] aryloxycarbonyl;

[0977] heteroaryloxycarbonyl;

[0978] amino carbonyl group;

[0979] The amino group is the amino carbonyl group of any amino acid or peptide.

[0980] N-alkylaminocarbonyl;

[0981] N,N-dialkylaminocarbonyl (or two alkyl groups together to form a heterocyclic group containing a nitrogen atom);

[0982] aminocarbonyloxy group;

[0983] N-alkylaminocarbonyloxy;

[0984] N,N-dialkylaminocarbonyloxy group (or two alkyl groups together to form a heterocyclic group containing an N atom);

[0985] alkoxycarbonyloxy;

[0986] aryloxycarbonyloxy;

[0987] heteroaryloxycarbonyloxy;

[0988] Urea group;

[0989] N-alkylurea;

[0990] N,N-dialkylurea (or two alkyl groups together to form a heterocyclic group containing a nitrogen atom);

[0991] N,N,N'-trialkylurea (or two alkyl groups on the same N atom can be combined to form a heterocyclic group containing the N atom);

[0992] Guanidino;

[0993] Amidoyl;

[0994] hydrazine group;

[0995] Alkylhydrazine;

[0996] N,N-dialkylhydrazide;

[0997] N,N'-dialkylhydrazine;

[0998] Alkyl thio;

[0999] alkylsulfinyl;

[1000] alkylsulfonyl;

[1001] Arylthio;

[1002] arylsulfinyl;

[1003] arylsulfonyl;

[1004] heteroarylthio;

[1005] heteroarylsulfinyl;

[1006] heteroarylsulfonyl;

[1007] sulfenic acid group;

[1008] sulfinic acid group;

[1009] Sulfonic acid group;

[1010] sulfate group;

[1011] sulfinamide group;

[1012] sulfonamide group;

[1013] Phosphine oxide;

[1014] Phosphino acid group;

[1015] Phosphoryl group (phosphonic acid group -P(=O)(OH)2);

[1016] Diphosphorylmethyl (-C(H, OH, NH2, Cl, or optionally substituted alkyl group)(P(=O)(OH)2)2);

[1017] Phosphoric acid group (phosphate ester group -OP(=O)(OH)2);

[1018] Boronic acid group;

[1019] alkenyl;

[1020] alkynyl group;

[1021] cycloalkyl;

[1022] Cycloalkenyl;

[1023] Aryl;

[1024] Mixed aromatics;

[1025] Heterocyclic groups;

[1026] lactone group;

[1027] D-glucose-(1, 2, 3, 4, or 6)-O-yl, and

[1028] -(OCH2CH2) x -Y base

[1029] (x is an integer from 1 to 30,

[1030] Y is the reason

[1031] hydrogen atom;

[1032] alkyl;

[1033] Halogen atoms (fluorine, chlorine, bromine, or iodine atoms);

[1034] Alkoxy;

[1035] Alkyl thio;

[1036] hydroxyl group;

[1037] thiol;

[1038] amino;

[1039] Alkylamino;

[1040] Dialkylamino (or two alkyl groups together to form a heterocyclic group containing a nitrogen atom);

[1041] Trialkylammonium group;

[1042] carboxyl;

[1043] Cyano;

[1044] Nitro;

[1045] Alkyl carbonyl;

[1046] alkenyl carbonyl;

[1047] alkynyl carbonyl;

[1048] aryl carbonyl;

[1049] heteroaryl carbonyl;

[1050] Heterocyclic carbonyl group;

[1051] lactone carbonyl;

[1052] Phosphorylalkylcarbonyl;

[1053] alkoxycarbonyl;

[1054] aryloxycarbonyl;

[1055] Heteroaryloxycarbonyl;

[1056] amino carbonyl group;

[1057] The amino group is the amino carbonyl group of any amino acid or peptide.

[1058] N-alkylaminocarbonyl;

[1059] N,N-dialkylaminocarbonyl (or two alkyl groups together to form a heterocyclic group containing a nitrogen atom);

[1060] aminocarbonyloxy group;

[1061] N-alkylaminocarbonyloxy;

[1062] N,N-dialkylaminocarbonyloxy group (or two alkyl groups together to form a heterocyclic group containing an N atom);

[1063] alkoxycarbonyloxy;

[1064] aryloxycarbonyloxy;

[1065] heteroaryloxycarbonyloxy;

[1066] Urea group;

[1067] N-alkylurea;

[1068] N,N-dialkylurea (or two alkyl groups together to form a heterocyclic group containing a nitrogen atom);

[1069] N,N,N'-trialkylurea (or two alkyl groups on the same N atom can be combined to form a heterocyclic group containing the N atom);

[1070] Guanidino;

[1071] Amidoyl;

[1072] hydrazine group;

[1073] Alkylhydrazine;

[1074] N,N-dialkylhydrazide;

[1075] N,N'-dialkylhydrazine;

[1076] Alkyl thio;

[1077] alkylsulfinyl;

[1078] alkylsulfonyl;

[1079] Arylthio;

[1080] arylsulfinyl;

[1081] arylsulfonyl;

[1082] heteroarylthio;

[1083] heteroarylsulfinyl;

[1084] heteroarylsulfonyl;

[1085] sulfenic acid group;

[1086] sulfinic acid group;

[1087] Sulfonic acid group;

[1088] sulfate group;

[1089] sulfinamide group;

[1090] sulfonamide group;

[1091] Phosphine oxide;

[1092] Phosphino acid group;

[1093] Phosphoryl group (phosphonic acid group -P(=O)(OH)2);

[1094] Diphosphorylmethyl (-C(H, OH, NH2, Cl, or optionally substituted alkyl group)(P(=O)(OH)2)2);

[1095] Phosphoric acid group (phosphate ester group -OP(=O)(OH)2);

[1096] Boronic acid group;

[1097] alkyl;

[1098] alkenyl;

[1099] alkynyl group;

[1100] cycloalkyl;

[1101] Cycloalkenyl;

[1102] Aryl;

[1103] Mixed aromatics;

[1104] Heterocyclic groups;

[1105] lactone group); or

[1106] D-glucose-(1, 2, 3, 4, or 6)-O- group; a group consisting of...

[1107] Specifically, examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[1108] It should be noted that, when the term "alkane" is used in this specification, the alkane refers to a "saturated hydrocarbon with 1 to 10 carbon atoms, either straight-chain or branched, consisting of an alkyl group plus one hydrogen atom as defined above."

[1109] The term "alkenyl" as used in this specification refers to a straight-chain or branched unsaturated hydrocarbon group containing one or more double bonds (-C=C-) in the chain of the alkyl group defined above.

[1110] As a substituent for the substituted alkenyl group, one or more substituents selected independently from group A may be listed.

[1111] Specifically, examples of this alkenyl group include methylene (=CH2 group), vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-methyl-2-propenyl, 1,1-dimethyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 3-methyl-1-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 3-methyl-1-pentenyl, 2-heptenyl, 4-octenyl, 1-nonenyl, 2-decenyl, etc.

[1112] The term "alkynyl" as used in this specification refers to a straight-chain or branched unsaturated hydrocarbon group containing one or more triple bonds (C triple bond C) in the chain of an alkyl group (wherein having 2 to 10 carbon atoms) as defined above.

[1113] As a substituent for the alkynyl group that may be substituted, one or more substituents may be selected independently from group A.

[1114] Specifically, examples of this alkynyl group include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1,1-dimethyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-2-butynyl, 3-methyl-1-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 3-methyl-1-pentynyl, 2-heptyynyl, 4-octyynyl, 1-nonynyl, 2-decynyl, etc.

[1115] The term "alkoxy" as used in this specification refers to a monovalent group (alkyl-O-yl) formed by the bonding of an alkyl group having 1 to 10 carbon atoms with an oxygen group, as defined above. Examples of such alkoxy groups include, for instance, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexoxy, n-heptoxy, n-octoxy, n-nonoxy, and n-decoxy.

[1116] The term "alkathioyl" as used in this specification refers to a monovalent group (alkyl-S-yl) formed by the bonding of an alkyl group having 1 to 10 carbon atoms with a thioyl group, as defined above. Examples of such alkathioyl groups include methylthioyl, ethylthioyl, n-propylthioyl, isopropylthioyl, n-butylthioyl, isobutylthioyl, sec-butylthioyl, tert-butylthioyl, n-pentylthioyl, n-hexylthioyl, n-heptylthioyl, n-octylthioyl, n-nonylthioyl, and n-decylthioyl.

[1117] As optional substituents for alkoxy and alkylthio groups, one or more substituents can be independently selected from group A.

[1118] The term "cycloalkyl" as used in this specification refers to a group obtained by removing one hydrogen atom from a monocyclic cyclic hydrocarbon (i.e., cycloalkanes) having 3 to 10 carbon atoms, and a group obtained by removing one hydrogen atom from a bicyclic cyclic hydrocarbon (i.e., bicyclic cycloalkanes) having 4 to 10 carbon atoms.

[1119] Substituents for the cycloalkyl group that can be optionally substituted include oxo (=O); thio (=S); imino (=N(R)); and others. 8 )); Oxime base (=N-OR 9 ); Hydrazine group (=NN(R) 10 (R) 11 )); alkyl; and group A (hereinafter, the group consisting of oxo; thio; imino; oxime; hydrazine; alkyl; and group A will be referred to as group B) independently selected one or more substituents (in the above, R 8 R 9 R 10 and R 11 Each can be independently a hydrogen atom, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic.

[1120] Specifically, examples of such cycloalkyl groups include: monocyclic cycloalkyl groups with 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl; and dicyclic cycloalkyl groups with 4 to 10 carbon atoms, such as bicyclic [1.1.0]butyl, bicyclic [2.1.0]pentyl, bicyclic [3.1.0]hexyl, bicyclic [3.2.0]heptyl, bicyclic [2.2.1]heptyl, bicyclic [3.3.0]octyl, bicyclic [3.2.1]octyl, bicyclic [2.2.2]octyl, bicyclic [6.1.0]nonyl, and bicyclic [4.4.0]decyl.

[1121] It should be noted that when the term "cycloalkanes" is used in this specification, "cycloalkanes" refers to "monocyclic cyclic hydrocarbons with 3 to 10 carbon atoms and bicyclic cyclic hydrocarbons with 4 to 10 carbon atoms" which are formed by adding one hydrogen atom to the "cycloalkyl" group as defined above.

[1122] The term "cycloalkenyl" as used in this specification refers to a monocyclic or bicyclic cyclic unsaturated hydrocarbon group containing one or more double bonds (-C=C-) within the ring of a cycloalkyl group as defined above.

[1123] As a substituent for the cyclic alkenyl group that may be substituted, one or more substituents may be selected independently from group B.

[1124] Specifically, examples of monocyclic cycloalkenyl groups with 3 to 10 carbon atoms include 1-cyclopropenyl, 1-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1-cycloheptenyl, 2-cyclooctenyl, 3-cyclooctenyl, 4-cyclooctenyl, 1-cyclononenyl, and 1-cyclodecenyl; bicyclic [1.1.0]but-1-enyl, ... Cyclo[2.1.0]pent-2-enyl, bicyclo[3.1.0]hex-2-di-enyl, bicyclo[3.2.0]hept-3-enyl, bicyclo[2.2.1]hept-2-enyl, bicyclo[3.3.0]oct-2-enyl, bicyclo[3.2.1]oct-2-enyl, bicyclo[2.2.2]oct-2-enyl, bicyclo[6.1.0]non-4-enyl, bicyclo[4.4.0]dec-3-enyl, and other bicyclic cycloalkenyl groups with 4 to 10 carbon atoms.

[1125] The term "aryl" as used in this specification refers to a group obtained by removing one hydrogen atom from a monocyclic or bicyclic aromatic hydrocarbon (i.e., aromatic hydrocarbon) having 6 to 11 carbon atoms.

[1126] As a substituent for the aryl group that may be substituted, one or more substituents may be independently selected from alkyl groups and group A (hereinafter, the group consisting of alkyl groups and group A will be referred to as group C).

[1127] Specifically, examples of this aryl group include monocyclic aryl groups such as phenyl; and cyclic carbon groups with 9 to 11 (C9 to C11) carbon atoms, such as naphthyl, tetrahydronaphthyl, indenyl, and indenyl. 11 (e.g., bicyclic aryl groups)

[1128] It should be noted that when the term "aromatic hydrocarbon" is used in this specification, the aromatic hydrocarbon refers to "a monocyclic or bicyclic aromatic hydrocarbon with 6 to 11 cyclic carbon atoms, which is formed by adding one hydrogen atom to the "aryl" group as defined above".

[1129] The term "heteroaryl" as used in this specification refers to a group obtained by removing one hydrogen atom from a 5- to 14-membered monocyclic, bicyclic, or tricyclic aromatic heterocycle (i.e., heteroaromatic hydrocarbon) containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to carbon atoms.

[1130] As a substituent for the optionally substituted heteroaryl group, one or more substituents selected independently from group C may be listed.

[1131] Specifically, examples of such heteroaryl groups include pyrroleyl, furanyl, thiopheneyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl, which are 5- to 6-membered monocyclic heteroaryl groups containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to a carbon atom; indoleyl, isindoleyl, indazoleyl, tetrahydroindazoleyl, benzofuranyl, dihydrobenzofuranyl, dihydroisobenzofuranyl, benzothiopheneyl, dihydrobenzothiopheneyl, and dihydroisobenzothiopheneyl. Benzoxazolyl, dihydrobenzooxazolyl, benzothiazolyl, dihydrobenzothiazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, naphridinyl, tetrahydronaphridinyl, quinoxalinyl, tetrahydroquinoxalinyl, and quinazolinyl, etc., contain 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to carbon atoms, forming 8-11 member bicyclic heteroaryl groups; dibenzofuran, dibenzopyrrole, dibenzothiophene, dibenzopyridine, etc., contain 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to carbon atoms, forming 11-14 member tricyclic heteroaryl groups, etc.

[1132] It should be noted that when the term "heteroaromatic hydrocarbon" is used in this specification, it refers to a 5- to 14-membered monocyclic, bicyclic, or tricyclic aromatic heterocycle that contains 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to a hydrogen atom added to a "heteroaryl" as defined above.

[1133] The term "heterocyclic group" as used in this specification refers to a monovalent group obtained by removing one hydrogen atom from a 3- to 12-membered monocyclic non-aromatic heterocycle (i.e., heterocyclic hydrocarbon) containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to carbon atoms.

[1134] Examples of substituents that can be substituted for heterocyclic groups include oxo groups (=O); thio groups (=S); and imino groups (=NR). 9 ;R 9 (Same meaning as above); Hydrazine (=NN(R) 10 (R) 11 ); R 10 and R 11(Same meaning as above); alkyl; and one or more substituents independently selected from group A (hereinafter, the group consisting of oxo; thio; imino; hydrazine; alkyl; and group A will be referred to as group D).

[1135] Specifically, examples of such heterocyclic groups include, for instance, azirrobutyl, oxacyclobutyl, thiohexacyclobutyl, pyrrolithyl, piperidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, piperazinyl, morpholinyl, perhydroazepinyl, azirrobutyl, azirrobutyl-3-enyl, azirrobutyl-4-enyl, azirrobutyl-nonyl, azirrobutyl-decyl, and 6- to 12-membered azirrobicycloalkyl groups (e.g., azirrobicyclohexyl, azirrobicyclohepyl, azirrobicyclohexyl, azirrobicyclohexyl, etc.). Octyl, azibicyclononyl, azibicyclodecyl, azibicycloundecyl or azibicyclododecyl), 6- to 12-membered azibicycloalkenyl (e.g., azibicyclohexenyl, azibicycloheptenyl, azibicyclooctenyl, azibicyclononenyl, azibicyclodecenyl, azibicycloundecenyl or azibicyclododecenyl), 6- to 12-membered azispiryl (e.g., azispirohexyl, azispirohepyl, azispiroctyl, azispirnonyl, azispirodel, azispirundecyl or azispirdodecyl), etc.

[1136] It should be noted that, when the term "heterocycle" is used in this specification, the heterocycle refers to a 4- to 12-membered monocyclic non-aromatic heterocycle that contains 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen atoms in addition to a carbon atom, by adding one hydrogen atom to a "heterocyclic group" as defined above.

[1137] The aforementioned cyclic compounds, including cycloalkanes, aromatics, heteroaromatics, and heterocycles, can be fused with other arbitrary cyclic compounds to form 2- to 4-cyclic compounds, and can generate monovalent or divalent groups obtained by removing one or two hydrogen atoms from these 2- to 4-cyclic compounds.

[1138] The term "lactone group" as used in this specification refers to one of the aforementioned heterocyclic groups. It is a heterocyclic group in which an oxygen atom is used as a heteroatom within the ring, and the carbon atom adjacent to the oxygen atom is replaced by an oxo group. Examples of such lactone groups include α-ethinolone, β-propinolone, γ-butinolone, δ-pentanolactone, ε-hexanolactone, γ-nonanolactone, γ-decanolactone, γ-undecaprolone, gluconic acid-δ-lactone, or pantothenic acid lactone (a monovalent residue of pantothenic acid lactone). These groups can be substituted in the same way as the aforementioned heterocyclic groups. In addition, they can be fused with any other cyclic compound to form 2 to 4-cyclic cyclic compounds.

[1139] It should be noted that, in the coupling precursor or coupling precursor synthesis intermediate of the present invention shown in general formula (I) or (II), the lactone group can become a component of L, L 1 D or D 1 Substituents bonded to carbon atoms or heteroatoms (N, O, S, Ser, P, etc.) present in the matrix.

[1140] As a lactone group in L, L 1 D or D 1 Examples of substitution on heteroatoms present in the sample are not limited to these, such as α-ethanolide-3-yl, β-propiolide-3-yl, β-propiolide-4-yl, γ-butyrolactone-3-yl, γ-butyrolactone-4-yl, γ-butyrolactone-5-yl, δ-valerolide-3-yl, δ-valerolide-4-yl, δ-valerolide-5-yl, δ-valerolide-6-yl, ε-caprolactone-3-yl, ε-caprolactone-4-yl, ε-caprolactone-5-yl, ε-caprolactone-6-yl, ε-caprolactone-7-yl, (3 (3,4-Dihydroxy-γ-butyrolactone-5-yl)methyl, 4-hydroxy-γ-butyrolactone, (3,4-dihydroxy-γ-butyrolactone-5-yl)methyl, gluconic acid-γ-lactone-3-yl, 2-(3,4-dihydroxy-γ-butyrolactone-5-yl)-1-hydroxyethyl, 4,4-dimethyl-β-propiolactone-3-yl, 4,5-dihydroxy-6-hydroxymethyl-methylvalerolactone-3-yl, 4,5-dihydroxy-hydroxyvalerolactone-3-yl, and (3,4,5-trihydroxy-hydroxyvalerolactone-3-yl)methyl, etc.

[1141] Both the (S) configuration and the (R) configuration are included in this invention as the lactone group.

[1142] In this specification, "alkylene" refers to a divalent group obtained by removing one arbitrary hydrogen atom from an "alkyl" group as defined above. Furthermore, if the alkyl group has a substituent, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from a carbon atom of an alkyl chain that is not substituent.

[1143] It should be noted that in general formulas (I) and (II) of the present invention, L and L 1 Each is an independently substituted alkylene group, wherein one or more methylene groups in the chain of the alkylene group are optionally replaced by one or more divalent groups selected independently from the group consisting of the following groups.

[1144] -C(R 1 (R) 2 )-;-O-;-N(R 3 )-;-N(R 3 )-N(R 3 )-;-S-;-Se-;-Si(R4 (R) 5 )-;-SS-;-Se-Se-;-SOm-;-SeOn-;-C(=C(R 6 (R) 7 ))-;-C(=O)-;-C(=S)-;-C(=N(R 8 ))-;-C(=N-OR 9 )-;-C(=NN(R 10 (R) 11 ))-;-P(=O)(R 12 )-;-P(=O)(OR 13 )-;-OP(=O)(R 12 -O-;-OP(=O)(OR 13 )-O-;-C(R 14 =; =C(R) 14 )-;-C(R 14 )=C(R 14 )-;-N=;=N-;-C≡C-;-(OC(R 1 (R) 2 )-C(R 1 (R) 2 )) 1-30 -;-(C(R 1 (R) 2 )-C(R 1 (R) 2 )-0) 1-30 -(R in the above) 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 and R 14 Independently, R is a group selected from hydrogen atom, alkyl, alkenyl, ynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic groups. 3 When it is an alkyl group, the alkyl group may optionally form a cyclic structure together with the alkyl group on the adjacent methylene group (m and n are independently integers from 0 to 2); optionally substituted alkenyl group, optionally substituted alynyl group, optionally substituted cycloalkyl group; optionally substituted cycloalkenyl group; optionally substituted aryl group; optionally substituted heteroaryl group; and optionally substituted heterocyclic group.

[1145] As optional substituents for alkylene groups, one or more substituents selected independently from group A may be listed.

[1146] Specifically, examples of such alkylene groups include methylene, ethylene, methylmethylene, trimethylene, ethylmethylene, dimethylmethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, n-heptylene, n-octylene, n-nonylene, n-decylene, n-undecylene, n-dodecylene, n-tridecylene, n-tetradecylene, n-pentadecanylene, n-hexadecylene, n-heptadecylene, n-octadecylene, n-nonadecanylene, n-eicosylene, n-triacylene, n-eicosylene, n-tetradecylene, and n-pentadecanylene.

[1147] The term "alkenyl" as used in this specification refers to L and Li in general formulas (I) and (II) above, respectively. 1 The linker chain shown refers to a divalent group obtained by removing one arbitrary hydrogen atom from the "alkenyl" group as defined above. Furthermore, when the alkenyl group has a substituent, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from a carbon atom constituting the alkenyl chain that is not a substituent.

[1148] As a substituent for the optional substituted alkenyl group, one or more substituents selected independently from group A may be listed.

[1149] Specifically, examples of this alkenyl group include vinylidene, 1-propenyne, 2-propenyne, 1-butenyne, 2-butenyne, 3-butenyne, 1,3-butadienyne, 1-methyl-2-propenyne, 1,1-dimethyl-2-propenyne, 1-pentenyne, 2-pentenyne, 3-pentenyne, 4-pentenyne, 1-methyl-2-butenyne, 3-methyl-1-butenyne, 1-hexenyne, 2-hexeneyl. Alkenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 3-methyl-1-pentenyl, 2-heptenyl, 4-octenyl, 1-nonenyl, 2-decenyl, 1-undecenyl, 3-dodecenyl, 2-tridecenyl, 4-tetradecenyl, 1-pentadecenyl, 2-hexadecenyl, 3-heptadecenyl, 2-octadecenyl, 1-nonadecenyl, 1-eicoseneyl, etc.

[1150] The term "acetylenic" as used in this specification refers to L and Li as defined in formulas (I) and (II) above, respectively. 1 The linker chain shown refers to a divalent group obtained by removing one arbitrary hydrogen atom from the "alkynyl" group as defined above. Furthermore, if the alkynyl group has a substituent, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from the carbon atom constituting the alkynyl chain, which is not a substituent.

[1151] As a substituent for the substituted alynyne group, one or more substituents selected independently from group A may be listed.

[1152] Specifically, examples of this ynyl group include, for instance, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 3-butynylene, 1-methyl-2-propynylene, 1,1-dimethyl-2-propynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 4-pentynylene, 1-methyl-2-butynylene, 3-methyl-1-butynylene, 1-hexynylene, 2-hexynylene, 3- Hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 3-methyl-1-pentynyl, 2-heptenynyl, 4-octyynyl, 1-nonynyl, 2-decynyl, 1-undecanynyl, 3-dodecynyl, 2-tridecynyl, 4-tetradecynyl, 1-pentadecanynyl, 2-hexadecynyl, 3-heptadecynyl, 2-octadecynyl, 1-nonadecanynyl, 1-eicosynyl, etc.

[1153] The term "cycloalkylene" as used in this specification refers to L and L that constitute the general formulas (I) and (II) above, respectively. 1 The linker chain shown refers to a divalent group obtained by removing one arbitrary hydrogen atom from a "cycloalkyl" group as defined above. Furthermore, when the cycloalkyl group has substituents, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from the cycloalkyl ring carbon atom that is not a substituent.

[1154] As a substituent for the optionally substituted cycloalkyl group, one or more substituents selected independently from group B may be listed.

[1155] Specifically, examples of such cycloalkyl groups include monocyclic cycloalkyl groups with 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl; and bicyclic cycloalkyl groups with 4 to 10 carbon atoms, such as bicyclic[1.1.0]butylene, bicyclic[2.1.0]pentylene, bicyclic[3.1.0]hexylene, bicyclic[3.2.0]heptylene, bicyclic[2.2.1]heptylene, bicyclic[3.3.0]octylene, bicyclic[3.2.1]octylene, bicyclic[2.2.2]octylene, bicyclic[6.1.0]nonylene, and bicyclic[4.4.0]decylene.

[1156] The term "cycloene group" as used in this specification refers to L and L in general formulas (I) and (II) above, respectively. 1The linker chain shown refers to a divalent group obtained by removing one arbitrary hydrogen atom from the "cycloalkenyl" group as defined above. Furthermore, if the cycloalkenyl group has a substituent, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from the cycloalkenyl carbon atom that is not a substituent.

[1157] As a substituent for the optionally substituted cycloene group, one or more substituents selected independently from group B may be listed.

[1158] Specifically, examples of this cyclohexene group include monocyclic cyclohexene groups with 3 to 10 carbon atoms, such as 1-cyclopropenyl, 1-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1-cycloheptenyl, 2-cyclooctenyl, 3-cyclooctenyl, 4-cyclooctenyl, 1-cyclononenyl, and 1-cyclodecenyl; bicyclic [1.1.0] Butyl-1-enyl, bicyclo[2.1.0]pentyl-2-enyl, bicyclo[3.1.0]hexyl-2-enyl, bicyclo[3.2.0]heptyl-3-enyl, bicyclo[2.2.1]heptyl-2-enyl, bicyclo[3.3.0]octyl-2-enyl, bicyclo[3.2.1]octyl-2-enyl, bicyclo[2.2.2]octyl-2-enyl, bicyclo[6.1.0]nonyl-4-enyl, bicyclo[4.4.0]decyl-3-enyl, and other bicyclic cycloalkenyl groups with 4 to 10 carbon atoms.

[1159] The term "triaryl" as used in this specification refers to L and La in general formulas (I) and (II) above, respectively. 1 The linker group shown refers to a divalent group obtained by removing one arbitrary hydrogen atom from the "aryl" group as defined above. Furthermore, if the aryl group has a substituent, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from the cyclic carbon atom of the aryl group (which is not a substituent).

[1160] As a substituent for the optionally substituted aryl group, one or more substituents may be independently selected from group C.

[1161] Specifically, examples of such arylene groups include monocyclic arylene groups with 6 to 11 cyclic carbon atoms, such as phenylene; and bicyclic arylene groups with 9 to 11 cyclic carbon atoms, such as naphthylene, tetrahydronaphthylene, indenylene, and indenylene.

[1162] The term "heteroaryl" as used in this specification refers to L and L that constitute the components of general formulas (I) and (II) above, respectively. 1The linker group shown refers to a divalent group obtained by removing one arbitrary hydrogen atom from a "heteroaryl" group as defined above. Furthermore, if the heteroaryl group has a substituent, it refers to a divalent group obtained by further removing another arbitrary hydrogen atom from a non-substituent heteroaryl cyclizing atom.

[1163] As a substituent for the optionally substituted heteroaryl group, one or more substituents selected independently from group C may be listed.

[1164] Specifically, examples of such heteroaryl groups include pyridine, furanyl, thiophene, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridinyl, pyridazinyl, and triazinyl, which are 5- to 6-membered monocyclic heteroaryl groups containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to a carbon atom; indoleyl, isindoleyl, indazoleyl, tetrahydroindazoleyl, benzofuranyl, etc. Dihydrobenzofuranyl, dihydroisobenzofuranyl, benzothiopheneyl, dihydrobenzothiopheneyl, dihydroisobenzothiopheneyl, benzoxazolyl, dihydrobenzoxazolyl, benzothiazolyl, dihydrobenzothiazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, naphthidyl, tetrahydronaphthidyl, quinoxalinyl, tetrahydroquinoxalinyl, and quinoxalinyl are 8- to 11-membered bicyclic heteroaryl groups that contain 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen atoms in addition to a carbon atom.

[1165] The term "heterocyclic group" as used in this specification refers to the group that forms L1 and L2 in the above general formulas (I) and (II), respectively. 1 The linker group shown is a divalent group obtained by removing one arbitrary hydrogen atom from the "heterocyclic group" defined above.

[1166] As a substituent for the optionally substituted heterocyclic group, one or more substituents can be independently selected from group D.

[1167] Specifically, examples of such heterocyclic groups include azapyrocyclobutane, oxocyclobutane, thionylcyclobutane, pyrrolithane, piperidinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, piperazineyl, morpholinyl, perhydroazapyroxyl, azapyrocyclooctyl, azapyrocyclooct-3-yl, azapyrocyclooct-4-yl, azapyrocyclononyl, azapyrocyclodecyl, and 6- to 12-membered azapyrobicycloalkyl groups (e.g., azapyrobicyclohexyl, azapyrobicycloheptyl, azapyrobicyclooctyl, azapyrobicyclononyl). 6- to 12-membered azidocyclodecyl, azidocycloundecyl, or azidocyclododecyl, etc.

[1168] The heterocyclic group can fuse with one or more of the above-mentioned aryl rings and / or the above-mentioned heteroaryl rings to form a 2- to 4-cyclic heterocyclic group.

[1169] It should be noted that when the above-mentioned alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and heterocyclic groups have hydroxyl, mercapto, amino (-NH2, -NH- (including -NH- within the ring of heteroaryl and heterocyclic groups)), carboxyl, sulfinic acid, sulfinic acid, sulfonic acid, phosphonic acid, phosphonic acid, or borate groups as substituents, these groups can be protected by the protecting groups described in "antitumor drug molecules or their analogues or derivatives thereof".

[1170] The term "salt" as used in this specification includes, but is not limited to, alkali metal salts such as lithium, sodium, and potassium; Group II metal salts such as magnesium and calcium; salts with aluminum or zinc; salts with amines such as ammonia, choline, diethanolamine, lysine, ethylenediamine, tert-butylamine, tert-octylamine, tris(hydroxymethyl)aminomethane, N-methylglucosamine, triethanolamine, and dehydrorosinamine; salts with inorganic acids such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, and benzenesulfonic acid; or salts with acidic amino acids such as aspartic acid and glutamic acid. Furthermore, "salt" also includes intramolecular salts.

[1171] It should be noted that the coupling precursor (I) and coupling precursor synthesis intermediate (II) of the present invention, as well as their salts, are sometimes obtained in the form of hydrates or solvates, and the present invention also includes any of them.

[1172] The specific examples of the conjugate precursor (I) of the present invention are shown in Example 2 below, and the specific examples of the conjugate precursor synthesis intermediate (II) of the present invention are shown in Example 3, but the present invention is not limited thereto.

[1173] Example 2. Example of "Conjugate Precursor (I)"

[1174] Example 2-1

[1175]

[1176] Example 2-2

[1177]

[1178] Example 2-3

[1179]

[1180] Example 2-4

[1181]

[1182] Example 2-5

[1183]

[1184] Example 2-6

[1185]

[1186] Example 2-7

[1187]

[1188] Example 2-8

[1189]

[1190] Example 2-9

[1191]

[1192] Example 2-10

[1193]

[1194] Example 2-11

[1195]

[1196] Example 2-12

[1197]

[1198] Example 2-13

[1199]

[1200] Example 2-14

[1201]

[1202] Example 2-15

[1203]

[1204] Example 2-16

[1205]

[1206] Example 2-17

[1207]

[1208] Example 2-18

[1209]

[1210] Example 2-19

[1211]

[1212] Example 2-20

[1213]

[1214] Example 2-21

[1215]

[1216] Example 2-22

[1217]

[1218] Example 2-23

[1219]

[1220] Example 2-24

[1221]

[1222] Example 2-25

[1223]

[1224] Example 2-26

[1225]

[1226] Example 2-27

[1227]

[1228] Example 2-28

[1229]

[1230] Example 2-29

[1231]

[1232] Example 2-30

[1233]

[1234] Example 2-31

[1235]

[1236] Example 2-32

[1237]

[1238] Example 2-33

[1239]

[1240] Example 2-34

[1241]

[1242] Example 2-35

[1243]

[1244] Example 2-36

[1245]

[1246] Example 2-37

[1247]

[1248] Example 2-38

[1249]

[1250] Example 2-39

[1251]

[1252] Example 2-40

[1253]

[1254] Example 2-41

[1255]

[1256] Example 2-42

[1257]

[1258] Example 2-43

[1259]

[1260] Example 2-44

[1261]

[1262] Example 2-45

[1263]

[1264] Example 2-46

[1265]

[1266] Example 2-47

[1267]

[1268] Example 2-48

[1269]

[1270] Example 2-49

[1271]

[1272] Example 2-50

[1273]

[1274] Example 2-51

[1275]

[1276] Example 2-52

[1277]

[1278] Example 2-53

[1279]

[1280] Example 2-54

[1281]

[1282] Example 2-55

[1283]

[1284] Example 2-56

[1285]

[1286] Example 2-57

[1287]

[1288] Example 2-58

[1289]

[1290] Example 2-59

[1291]

[1292] Example 2-60

[1293]

[1294] Example 2-61

[1295]

[1296] Example 2-62

[1297]

[1298] Example 2-63

[1299]

[1300] Example 2-64

[1301]

[1302] Example 2-65

[1303]

[1304] Example 2-66

[1305]

[1306] Example 2-67

[1307]

[1308] Example 2-68

[1309]

[1310] Example 2-69

[1311]

[1312] Example 3. “Conjunctive precursor synthesis intermediate body (II)” Example 3-1-1

[1313]

[1314] Example 3-1-2

[1315]

[1316] Example 3-1-3

[1317]

[1318] Example 3-2-1

[1319]

[1320] Example 3-3-1

[1321]

[1322] Example 3-3-3

[1323]

[1324] Example 3-4-1

[1325]

[1326] Example 3-5-1

[1327]

[1328] Example 3-6-1

[1329]

[1330] Example 3-6-2

[1331]

[1332] Example 3-6-3

[1333]

[1334] Example 3-7-3

[1335]

[1336] Example 3-9-1

[1337]

[1338] Example 3-10-1

[1339]

[1340] Example 3-11-1

[1341]

[1342] Example 3-12-1

[1343]

[1344] Example 3-13-1

[1345]

[1346] Example 3-13-2

[1347]

[1348] Example 3-13-3

[1349]

[1350] Example 3-14-1

[1351]

[1352] Example 3-15-3

[1353]

[1354] Example 3-16-1

[1355]

[1356] Example 3-16-2

[1357]

[1358] Example 3-16-3

[1359]

[1360] Example 3-17-3

[1361]

[1362] Example 3-18-1

[1363]

[1364] Example 3-18-2

[1365]

[1366] Example 3-18-3

[1367]

[1368] Example 3-19-1

[1369]

[1370] Example 3-19-2

[1371]

[1372] Example 3-19-3

[1373]

[1374] Example 3-20-1

[1375]

[1376] Example 3-21-3

[1377]

[1378] Example 3-22-3

[1379]

[1380] Example 3-23-1

[1381]

[1382] Example 3-24-1

[1383]

[1384] Example 3-25-1

[1385]

[1386] Example 3-26-1

[1387]

[1388] Example 3-27-3

[1389]

[1390] Example 3-28-3

[1391]

[1392] Example 3-29-3

[1393]

[1394] Example 3-30-1

[1395]

[1396] Example 3-31-1

[1397]

[1398] Example 3-32-1

[1399]

[1400] Example 3-32-2

[1401]

[1402] Example 3-32-3

[1403]

[1404] Example 3-33-1

[1405]

[1406] Example 3-33-2

[1407]

[1408] Example 3-33-3

[1409]

[1410] Example 3-34-1

[1411]

[1412] Example 3-34-2

[1413]

[1414] Example 3-34-3

[1415]

[1416] Example 3-35-1

[1417]

[1418] Example 3-35-2

[1419]

[1420] Example 3-35-3

[1421]

[1422] Example 3-36-1

[1423]

[1424] Example 3-36-2

[1425]

[1426] Example 3-36-3

[1427]

[1428] Example 3-37-1

[1429]

[1430] Example 3-37-2

[1431]

[1432] Example 3-37-3

[1433]

[1434] Example 3-38-1

[1435]

[1436] Example 3-38-2

[1437]

[1438] Example 3-38-3

[1439]

[1440] Example 3-39-1

[1441]

[1442] Example 3-39-2

[1443]

[1444] Example 3-39-3

[1445]

[1446] Example 3-40-1

[1447]

[1448] Example 3-40-2

[1449]

[1450] Example 3-40-3

[1451]

[1452] Example 3-41-1

[1453]

[1454] Example 3-41-2

[1455]

[1456] Example 3-41-3

[1457]

[1458] Example 3-42-1

[1459]

[1460] Example 3-42-2

[1461]

[1462] Example 3-42-3

[1463]

[1464] Example 3-43-1

[1465]

[1466] Example 3-43-2

[1467]

[1468] Example 3-43-3

[1469]

[1470] Example 3-44-1

[1471]

[1472] Example 3-44-2

[1473]

[1474] Example 3-44-3

[1475]

[1476] Example 3-45-1

[1477]

[1478] Example 3-45-2

[1479]

[1480] Example 3-45-3

[1481]

[1482] Example 3-46-1

[1483]

[1484] Example 3-46-2

[1485]

[1486] Example 3-46-3

[1487]

[1488] Example 3-47-1

[1489]

[1490] Example 3-47-2

[1491]

[1492] Example 3-47-3

[1493]

[1494] Example 3-48-1

[1495]

[1496] Example 3-48-2

[1497]

[1498] Example 3-48-3

[1499]

[1500] Example 3-49-1

[1501]

[1502] Example 3-49-2

[1503]

[1504] Example 3-49-3

[1505]

[1506] Example 3-50-1

[1507]

[1508] Example 3-50-2

[1509]

[1510] Example 3-50-3

[1511]

[1512] Example 3-51-1

[1513]

[1514] Example 3-51-2

[1515]

[1516] Example 3-51-3

[1517]

[1518] Example 3-52-1

[1519]

[1520] Example 3-52-2

[1521]

[1522] Example 3-52-3

[1523]

[1524] Example 3-53-3

[1525]

[1526] Example 3-54-3

[1527]

[1528] Example 3-55-3

[1529]

[1530] Example 3-56-3

[1531]

[1532] Example 3-57-1

[1533]

[1534] Example 3-57-2

[1535]

[1536] Example 3-57-3

[1537]

[1538] Example 3-58-1

[1539]

[1540] Example 3-58-2

[1541]

[1542] Example 3-58-3

[1543]

[1544] Example 3-59-1

[1545]

[1546] Example 3-59-2

[1547]

[1548] Example 3-59-3

[1549]

[1550] Example 3-60-1

[1551]

[1552] Example 3-60-2

[1553]

[1554] Example 3-60-3

[1555]

[1556] Example 3-61-1

[1557]

[1558] Example 3-61-2

[1559]

[1560] Example 3-61-3

[1561]

[1562] Example 3-62-1

[1563]

[1564] Example 3-62-2

[1565]

[1566] Example 3-62-3

[1567]

[1568] Example 3-63-1

[1569]

[1570] Example 3-63-2

[1571]

[1572] Example 3-63-3

[1573]

[1574] Example 3-64-1

[1575]

[1576] Example 3-64-2

[1577]

[1578] Example 3-64-3

[1579]

[1580] Example 3-65-1

[1581]

[1582] Example 3-65-2

[1583]

[1584] Example 3-65-3

[1585]

[1586] Example 3-66-1

[1587]

[1588] Example 3-66-2

[1589]

[1590] Example 3-66-3

[1591]

[1592] Example 3-67-1

[1593]

[1594] Example 3-67-2

[1595]

[1596] Example 3-67-3

[1597]

[1598] Example 3-68-1

[1599]

[1600] Example 3-68-2

[1601]

[1602] Example 3-68-3

[1603]

[1604] Example 3-69-3

[1605]

[1606] Actual example

[1607] Hereinafter, examples of the synthesis of "antitumor drug molecules or their analogues or derivatives" used in this invention are described as "manufacturing examples", examples of the synthesis of the conjugate precursor (I) and the conjugate precursor synthesis intermediate (II) of this invention are described as "examples", and further, examples of the manufacture of antibody-drug conjugates (ADCs) based on the reaction of the conjugate precursor with an antibody and examples of evaluation tests of the ADC are described as "reference example 1" and "reference example 2", respectively, but this invention is not limited to these.

[1608] It should be noted that in the synthesis examples (i.e., examples) of the conjugate precursor (I) and the intermediate for the synthesis of the conjugate precursor (II), the amino acid residues of the linker portion are all of the L-type stereoisomerism, but in this invention they can each be of the D-type independently, and the reaction proceeds in the same way in the case of the D-type.

[1609] In addition, the reagents, solvents and starting materials not specifically described in the manufacturing examples, embodiments and reference examples can be readily obtained from commercially available sources.

[1610] It should be noted that the abbreviations and structures used in the following description are as follows.

[1611]

[1612] Fmoc-(L)-Val-(L)―Cit-PAB-PNP

[1613]

[1614] (L)-Val-(L)―Cit-PAB

[1615]

[1616] 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[1617] (Hereinafter referred to as HATU)

[1618]

[1619] MC-Val-Cit-PAB-MMAE

[1620]

[1621] Mal-PEG2-Val-Cit-PAB-Eribulin

[1622] [Manufacturing Example] Synthesis of antitumor drug molecules or their analogues, or their derivatives.

[1623] Manufacturing Example 1.U-001

[1624]

[1625] In a 30 mL cylindrical flask, a solution of eczemab mesylate (284.1 mg, 0.534 mmol) and triethylamine (0.70 mL, 508.2 mg, 5.02 mmol) in 5 mL of dichloromethane was added. While stirring under an argon atmosphere, 0.14 mL of acetic anhydride (151.2 mg, 1.481 mmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After the reaction was complete, the precipitate was filtered and washed with dichloromethane to obtain U-001 (239.9 mg, 94.0% yield) as a light brown solid.

[1626] MS(ESI)m / z 478(M+H) +

[1627] Manufacturing Example 2.U-002

[1628]

[1629] In a 10 mL cylindrical flask, a solution of 20.4 mg (0.038 mmol) of eczemanetide in N,N-dimethylformamide (0.6 mL) was added. While stirring under an argon atmosphere, 0.015 mL (4.86 mg (0.054 mmol) of 3-hydroxypropionic acid, 15.6 mg (0.115 mmol) of 1-hydroxybenzotriazole, 21.6 mg (0.113 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.040 mL (29.04 mg (0.287 mmol) of triethylamine were added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, a saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was subjected to medium-pressure preparation at a mountain (silica, M (16g), ethyl acetate / methanol = 97 / 3 (V / V) → 60 / 40 (V / V)). The component containing the target substance (Rf value = 0.44 (ethyl acetate / methanol = 85 / 15 (V / V))) was concentrated under reduced pressure to obtain U-002 (9.8mg, yield 50.31%) as a pale yellow solid.

[1630] MS(ESI)m / z 508(M+H) +

[1631] Manufacturing Example 3.U-003

[1632] 3-1.U-003-1

[1633]

[1634] In a 10 mL cylindrical flask, a solution of 22 mg (0.041 mmol) of eczemanetide in N,N-dimethylformamide (0.8 mL) was added. While stirring under an argon atmosphere, N-(tert-butoxycarbonyl)-N-methylglycine (11 mg (0.058 mmol), 1-hydroxybenzotriazole (13 mg (0.096 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (13 mg (0.068 mmol), and 0.032 mL (23.23 mg (0.230 mmol)) of triethylamine were added (11 mg (0.058 mmol)). The mixture was stirred at room temperature for 2 hours and then allowed to stand overnight. After the reaction was complete, saturated sodium bicarbonate aqueous solution (5 mL × 2) was added to the reaction solution, and the mixture was extracted with 15 mL of dichloromethane. The organic layer was washed with 5 mL of saturated saline solution, dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain U-003-1 (29 mg, yield quantification).

[1635] MS(ESI)m / z 607(M+H) +

[1636] 3-2.U-003

[1637]

[1638] A solution of U-003-1 (25 mg, 0.041 mmol) in 0.6 mL of dichloromethane was added to a 20 mL cylindrical flask. Trifluoroacetic acid (74 mg, 0.649 mmol) was added while stirring under an argon atmosphere at room temperature. After stirring for 1.5 hours at room temperature, the solution was concentrated under reduced pressure. Methanol was added to the residue, and the solution was concentrated under reduced pressure. Ethyl acetate was then added, and the resulting solid was filtered off. The solid was dried under reduced pressure to obtain a brown solid, trifluoroacetate of U-003 (13 mg, yield 50.83%).

[1639] MS(ESI)m / z 507(M+H) +

[1640] Manufacturing Example 4.U-004

[1641] 4-1.U-004-1

[1642]

[1643] In a 10 mL cylindrical flask, a solution of 20.6 mg eczematrix (0.039 mmol) and 0.020 mL N,N-diisopropylethylamine (14.8 mg 0.115 mmol) in 1 mL dichloromethane was added. While stirring, 9.6 mg benzoyl chloride (0.052 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was concentrated under reduced pressure to give U-004-1 (22.6 mg, yield 99.76%).

[1644] 4-2.U-004

[1645]

[1646] A mixture of 1 mL ethanol / 1 mL dichloromethane solution containing U-004-1 (22.6 mg, 0.039 mmol) and 10% palladium on carbon NX type (20.6 mg, 0.0968 mmol) was added to a 30 mL piezoelectric flask and stirred at room temperature for 2 hours under a hydrogen atmosphere. After the reaction was complete, the catalyst was filtered off, and the solvent was concentrated under reduced pressure. The residue was subjected to medium-pressure preparation (silica, M (16 g), dichloromethane / methanol = 100 / 0 (V / V) → 94 / 6 (V / V)). The component containing the target analyte was concentrated under reduced pressure to obtain U-004 (9.4 mg, yield 43.84%) as a pale yellow solid.

[1647] MS(DUIS)m / z 555(M+H) +

[1648] Manufacturing Example 5.U-005

[1649] 5-1.U-005-1

[1650]

[1651] In a 10 mL cylindrical flask, a solution of 21.2 mg (0.040 mmol) of eczematrix and 0.020 mL (14.8 mg, 0.115 mmol) of N,N-diisopropylethylamine in 1 mL of dichloromethane was added. While stirring, 10.6 mg (0.057 mmol) of 4-nitrobenzoyl chloride was added at room temperature, and the mixture was stirred for 1 hour. Then, 10.6 mg (0.057 mmol) of 4-nitrobenzoyl chloride was added again at room temperature, and the mixture was stirred for another 1 hour. After the reaction was complete, water and tert-butyl methyl ether were added to the reaction solution, and the mixture was stirred. The resulting solid was filtered and washed sequentially with water and tert-butyl methyl ether to obtain U-005-1 (22.1 mg, yield 94.79%).

[1652] 5-2.U-005

[1653]

[1654] A mixture of 1 mL ethanol / 1 mL dichloromethane solution containing U-005-1 (22.6 mg, 0.039 mmol) and 10% palladium on carbon NX type (15.4 mg, 0.0724 mmol) was added to a 30 mL piezoelectric flask and stirred at room temperature for 2 hours under a hydrogen atmosphere. After the reaction was complete, the catalyst was filtered off, and the solvent was concentrated under reduced pressure. The resulting residue was subjected to a medium-pressure preparation (silica, M (16 g), dichloroethane / methanol = 100 / 0 (V / V) → 94 / 6 (V / V)) to concentrate the component containing the target analyte under reduced pressure, thereby obtaining crude U-005. The crude product was subjected to preparative HPLC chromatography under the following conditions: the component containing the target analyte was neutralized with a saturated sodium bicarbonate aqueous solution, concentrated under reduced pressure, water was added to the precipitated solid, the insoluble matter was filtered off, and the solid was washed with water to obtain U-005 (7.1 mg, yield 33.11%) as a pale yellow solid.

[1655] MS(ESI)m / z 555(M+H) +

[1656] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1657] Flow rate: 17 mL / min.

[1658] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1659] Gradient (solution B): 30% (0.00 min.) → 95% (10.00 min.)

[1660] Manufacturing Example 6.U-006

[1661] 6-1.U-006-1

[1662]

[1663] In a 10 mL cylindrical flask, a solution of eczemab methanesulfonate (23.1 mg, 0.043 mmol), 2-(3-nitrophenyl)acetic acid (15.7 mg, 0.087 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (16.7 mg, 0.087 mmol), and 1-hydroxybenzotriazole (13.3 mg, 0.087 mmol) in N,N-dimethylformamide was added. While stirring under an argon atmosphere, 0.012 mL of triethylamine (8.71 mg, 0.086 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature. After the reaction was complete, a saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was concentrated under reduced pressure to obtain the crude product U-006-1 (38.4 mg).

[1664] MS(ESI)m / z 599(M+H) +

[1665] 6-2.U-006

[1666]

[1667] A mixture of 1 mL ethanol / 1 mL dichloromethane solution containing U-006-1 (38.4 mg, 0.044 mmol) and 10% palladium on carbon NX type (21.3 mg, 0.200 mmol) was added to a 30 mL piezoelectric flask and stirred at room temperature for 2 hours under a hydrogen atmosphere. After the reaction was complete, the catalyst was filtered off, and the solvent was concentrated under reduced pressure. The resulting residue was subjected to a medium-pressure preparation (silica, M (16 g), dichloroethane / methanol = 100 / 0 (V / V) → 94 / 6 (V / V)) to concentrate the component containing the target analyte under reduced pressure. Ethanol was added to the residue, and the precipitated solid was filtered and washed with ethanol to obtain U-006 (16.6 mg, total yield of 6-1 and 6-2 66.92%) as a colorless solid.

[1668] MS(DUIS)m / z 569(M+H) +

[1669] Manufacturing Example 7.U-007

[1670] 7-1.U-007-1

[1671]

[1672] In a 10 mL cylindrical flask, a solution of 0.61 mL of N,N-dimethylformamide containing eczemab (32.5 mg, 0.061 mmol), 2-(4-nitrophenyl)acetic acid (22.1 mg, 0.122 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (23.4 mg, 0.122 mmol), and 1-hydroxybenzotriazole (16.7 mg, 0.109 mmol) was added. While stirring, 0.017 mL of triethylamine (12.34 mg, 0.122 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature. After the reaction was complete, a saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was concentrated under reduced pressure to obtain U-007-1 (26.8 mg, yield 73.23%).

[1673] MS(ESI)m / z 598(M+H) +

[1674] 7-2.U-007

[1675]

[1676] A mixture of 1 mL ethanol / 1 mL dichloromethane solution of U-007-1 (26.8 mg, 0.045 mmol) and 10% palladium on carbon NX type (20.3 mg, 0.0954 mmol) was added to a 30 mL piezoelectric flask and stirred at room temperature under a hydrogen atmosphere for 2 hours. After the reaction was complete, the catalyst was filtered off, and the solvent was concentrated under reduced pressure. Ethanol was added to the residue, the precipitated solid was filtered off, and the solid was washed with ethanol to obtain U-007 (24.2 mg, yield 95.06%) as a light brown solid.

[1677] MS(DUIS)m / z 569(M+H) +

[1678] Manufacturing Example 8.U-008

[1679] 8-1.U-008-1

[1680]

[1681] In a 30 mL cylindrical flask, a solution of MMAE (0.36 g, 0.501 mmol) and 2-fluoro-5-nitrobenzaldehyde (0.25 g, 1.478 mmol) in 5 mL of dichloromethane was added. While stirring under an argon atmosphere, sodium triacetoxyborohydride (0.32 g, 1.510 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Then, 0.25 g, 1.478 mmol, and sodium triacetoxyborohydride (0.32 g, 1.510 mmol) were added again, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was subjected to medium-pressure preparation (silica, M (16g), ethyl acetate:methanol = 100 / 0 (V / V) → 90 / 10 (V / V)) and the component containing the target substance was concentrated under reduced pressure to obtain U-008-1 (406.6mg, yield 93.09%), which was a pale yellow bubbly substance.

[1682] MS(DUIS)m / z 873(M+H) +

[1683] 8-2.U-008

[1684]

[1685] A 0.2 mL solution of U-008-1 (45.1 mg, 0.052 mmol) and zinc (89.3 mg, 1.366 mmol) in acetic acid was added to a 30 mL cylindrical flask and stirred at room temperature under an argon atmosphere for 2 hours. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was subjected to a medium-pressure preparation (silica, M (16 g), ethyl acetate / methanol = 100 / 0 (V / V) → 90 / 10 (V / V)) to concentrate the component containing the target analyte under reduced pressure. The resulting residue was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target analyte was concentrated under reduced pressure and distilled off the acetonitrile, followed by freeze-drying to obtain the formate of U-008 as a colorless bubbly substance (13.3 mg, yield 28.96%).

[1686] MS(DUIS)m / z 841(M+H) +

[1687] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1688] Flow rate: 17 mL / min.

[1689] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1690] Gradient (solution B): 30% (0.00 min) → 95% (10.00 min)

[1691] Manufacturing Example 9.U-009

[1692]

[1693] In a 10 mL cylindrical flask, a solution of (tert-butoxycarbonyl)glycylglycine (8.4 mg, 0.036 mmol), 1-hydroxybenzotriazole (6.2 mg, 0.046 mmol), and MMAE (22.3 mg, 0.031 mmol) in N,N-dimethylformamide was added. While stirring under an argon atmosphere, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (8.8 mg, 0.046 mmol) was added at room temperature, and the mixture was stirred at 60 °C for 1 hour. The resulting reaction solution was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target analyte was concentrated under reduced pressure to obtain a colorless oil.

[1694] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1695] Flow rate: 17 mL / min.

[1696] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1697] Gradient (solution B): 30% (0.00 min.) → 95% (10.00 min.)

[1698] The obtained compound was dissolved in 1 mL of dichloromethane and 1 mL of trifluoroacetic acid, stirred at room temperature for 1 hour, and then concentrated under reduced pressure. The resulting reaction solution was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target compound was concentrated under reduced pressure. Next, it was subjected to DNH silica gel column chromatography, eluted with dichloromethane / methanol = 9 / 1 (V / V), and the fraction containing the target compound was concentrated under reduced pressure. Then, it was dissolved in a water / acetonitrile mixed solvent and freeze-dried to obtain Preparation Example 9 (U-009) (2.2 mg, yield 8.51%) as a colorless solid.

[1699] MS(ESI)m / z 833(M+H) +

[1700] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1701] Flow rate: 17 mL / min.

[1702] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1703] Gradient (solution B): 30% (0.00 min.) → 95% (10.00 min.)

[1704] Manufacturing Example 10.U-010

[1705]

[1706] In a 30 mL cylindrical flask, 0.37 g (0.515 mmol) of MMAE and 0.37 mL (0.4 g, 4.97 mmol) of 37% formaldehyde aqueous solution were added to a 5 mL methanol solution. While stirring under an argon atmosphere, sodium triacetoxyborohydride (0.32 g, 1.510 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was subjected to a medium-pressure preparation (silica, M (16 g), dichloromethane / methanol = 100 / 0 (V / V) → 90 / 10 (V / V)) to concentrate the component containing the target analyte under reduced pressure, yielding a colorless bubbly substance, U-010 (0.18 g, yield 47.72%).

[1707] MS(DUIS)m / z 732(M+H) +

[1708] Manufacturing Example 11.U-011

[1709] 11-1.U-011-1

[1710]

[1711] Add 5 mL of water to a 5 mL tetrahydrofuran solution of MMAE (0.4996 g, 0.696 mmol) and sodium carbonate (0.1503 g, 1.418 mmol) in a 30 mL cylindrical flask. While stirring, add 0.185 mL (0.19 g, 0.848 mmol) of di-tert-butyl dicarbonate at room temperature and stir for 18 hours. Then, stir at 50°C for 1 hour. Add 0.015 mL (0.02 g, 0.069 mmol) of di-tert-butyl dicarbonate and stir at 50°C for 1 hour. Then, add another 0.015 mL (0.02 g, 0.069 mmol) of di-tert-butyl dicarbonate and stir at 50°C for 2 hours. After the reaction is complete, add ethyl acetate and saturated saline solution to the reaction mixture and stir briefly at room temperature. The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain U-011-1 (570.5 mg, yield quantification), which was a white bubbly substance.

[1712] MS(ESI)m / z 818(M+H) +

[1713] 11-2.U-011-2

[1714]

[1715] A solution of U-011-1 (51.0 mg, 0.062 mmol) and bis(4-nitrophenyl) carbonate (38.8 mg, 0.128 mmol) in dichloromethane was added to 0.2-0.5 mL of a microwave-safe container. 0.035 mL of N,N-diisopropylethylamine (25.97 mg, 0.201 mmol) was then added, and the container was sealed with nitrogen and stirred at room temperature for 3 hours. Subsequently, the mixture was placed in a microwave reactor (Biotage) and reacted at 80°C for 1 hour. Then, bis(4-nitrophenyl) carbonate (100.0 mg, 0.329 mmol) and 0.5 mL of dichloromethane were added, and the mixture was stirred at room temperature for 66 hours. The reaction solution was prepared under medium pressure in a general-purpose high-quality silica gel, S (7g) (2-linked), and eluted with hexane / ethyl acetate = 33 / 67 (V / V) → 12 / 88 (V / V). The component containing the target substance was concentrated under reduced pressure to obtain U-011-2 (46.7mg, yield 76.19%), which was a slightly yellow bubbly substance.

[1716] MS(ESI)m / z 983(M+H) +

[1717] 11-3.U-011-3

[1718]

[1719] A solution of U-011-2 (46.7 mg, 0.047 mmol) and 3,6,9,12,15,18,21,24-octaoxococtidecane-1-amine (34.3 mg, 0.089 mmol) in dichloromethane was added to a 20 mL flask. 0.030 mL of N,N-diisopropylethylamine (22.26 mg, 0.172 mmol) was added, and the mixture was stirred at room temperature under an argon atmosphere for 4 hours, followed by standing at room temperature for 16.75 hours. The reaction solution was diluted with dichloromethane and filled into a Fuji SILYSIA Chromatorex Q-Pack DIOL 60 Size 20 container. Elution was performed with hexane / ethyl acetate = 0→100. The fraction containing the target compound was concentrated under reduced pressure to obtain U-011-3 (49.2 mg, yield 84.38%) as a colorless oil.

[1720] MS(ESI)m / z 1227(M+H) +

[1721] 11-4.U-011

[1722]

[1723] A 2 mL solution of U-011-3 (47 mg, 0.038 mmol) in dichloromethane was added to a 30 mL round-bottom flask. 0.50 mL of trifluoroacetic acid (744.5 mg, 6.53 mmol) was added at room temperature, and the mixture was stirred for 0.67 hours at room temperature. The reaction solution was then concentrated under reduced pressure. 0.1% formic acid-acetonitrile / water = 1 / 9 (V / V) was added to the concentrated residue, and the mixture was freeze-dried to obtain a white solid of trifluoroacetate of U-011 (47.3 mg, yield 99.51%).

[1724] MS(ESI)m / z 1127(M+H) +

[1725] Manufacturing Example 12.U-012

[1726] 12-1.U-012-1

[1727]

[1728] To a 0.3 mL solution of pyridine containing U-011-1 (12.95 mg, 0.016 mmol) and 4-dimethylaminopyridine (1.46 mg, 0.012 mmol) in a 0.2 mL-0.5 mL microwave reaction vessel, add 0.010 mL (9.17 mg, 0.076 mmol) of 2,4,6-trimethylpyridine and 0.050 mL (59.6 mg, 0.43 mmol) of 2-methoxyethyl 2-chloroformate. Seal the container with nitrogen and place it in a microwave reaction apparatus (Biotage) at 100 °C for 3 hours. Dilute the reaction solution with ethyl acetate, wash once with water, three times with 5% potassium bisulfate solution, once with saturated sodium bicarbonate solution, and once with saturated brine. Dry with anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure. The concentrated residue was dissolved in hexane / ethyl acetate = 4 / 6 (V / V), and the solution was packed into a Fuji SILYSIA Chromatorex Q-Pack DIOL60 Size 10. Elution was performed with hexane / ethyl acetate = 39 / 61 (V / V) → 18 / 82 (V / V). The fraction containing the target compound was concentrated under reduced pressure to obtain U-012-1 (9.4 mg, yield 64.53%), a colorless oil.

[1729] MS(ESI)m / z 920(M+H) +

[1730] 12-2.U-012

[1731]

[1732] A 1 mL solution of U-012-1 (9.4 mg, 0.01022 mmol) in dichloromethane was added to a 20 mL pear-shaped flask. 0.050 mL of trifluoroacetic acid (74.45 mg, 0.653 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature. Subsequently, 0.450 mL of trifluoroacetic acid (670.05 mg, 5.88 mmol) was added at room temperature, and the mixture was stirred for 0.5 hours at room temperature. The reaction solution was then concentrated under reduced pressure. The concentrated residue was prepared by HPLC (column: Waters SunFire Prep C18 5 μm ODB 19*150 mm, 0.1% formic acid-acetonitrile / water (V / V) = 30 / 70 → 95 / 5), and the fraction containing the target analyte was concentrated under reduced pressure. Add 0.1% formic acid aqueous solution to the concentrated residue and freeze-dry to obtain formate of U-012 as a white solid (3.8 mg, yield 42.95%).

[1733] MS(ESI)m / z 820(M+H) +

[1734] Manufacturing Example 13.U-013

[1735] 13-1.U-013-1

[1736]

[1737] In a 200 mL flask, a solution of tert-butyl hydroxy(methyl)carbamate (7.40 g, 50.3 mmol) and potassium carbonate (13.84 g, 100 mmol) in acetonitrile was added. While stirring under an argon atmosphere, ((2-bromoethoxy)methyl)benzene (11.36 g, 52.8 mmol) was added at room temperature, and the mixture was stirred at 80 °C for 5 hours. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure. The residue was extracted twice with diisopropyl ether by adding a saturated aqueous solution of sodium bicarbonate. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was subjected to medium-pressure preparation in a shale gas atmosphere (silica, L (40 g), hexane / ethyl acetate = 95 / 5 (V / V) → 80 / 20 (V / V)), and the component containing the target substance was concentrated under reduced pressure to obtain U-013-1 (15.11 g, yield quantitative) as a colorless oil.

[1738] 13-2.U-013-2

[1739]

[1740] A 50 mL ethanol solution of U-013-1 (2.8 g, 9.95 mmol) and 10% palladium on carbon (1.4 g, 0.658 mmol) was added to a 200 mL pear-shaped flask and stirred at room temperature under a hydrogen atmosphere for 4 hours. After the reaction was complete, the catalyst was filtered off, and the reaction solution was concentrated under reduced pressure to obtain U-013-2 (1.90 g, 99.84% yield), which was a pale yellow oil.

[1741] 13-3.U-013-3

[1742]

[1743] A 5 mL solution of U-013-2 (0.19 g, 0.994 mmol) in dichloromethane was added to a 30 mL cylindrical flask. While stirring, Dysmartin oxidant (0.51 g, 1.202 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to the reaction solution, followed by a sodium thiosulfate aqueous solution. After stirring for 20 minutes at room temperature, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain U-013-3 (162.7 mg, yield 86.54%), a pale yellow oil.

[1744] 13-4.U-013-4

[1745]

[1746] A 5 mL solution of MMAE (0.30 g, 0.418 mmol) and U-013-3 (0.16 g, 0.846 mmol) in dichloromethane was added to a 30 mL cylindrical flask. Sodium triacetoxyborohydride (0.18 g, 0.849 mmol) was added while stirring at room temperature under an argon atmosphere. The mixture was stirred for 2 hours at room temperature. After the reaction was complete, a saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product U-013-4 as a yellow solid. The crude product was subjected to medium-pressure preparation at a pressure of 10 ...

[1747] MS(DUIS)m / z 893(M+H) +

[1748] 13-5.U-013

[1749]

[1750] A 0.2 mL solution of U-013-4 (45.3 mg, 0.051 mmol) in dichloromethane was added to a 30 mL cylindrical flask. While stirring under an argon atmosphere, 1 mL (4 mmol) of a 1,4-dioxane solution of 4M hydrogen chloride was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After the reaction was complete, the reaction solution was concentrated under reduced pressure, dissolved in a mixture of water and acetonitrile, and then freeze-dried to obtain U-013 hydrochloride (44.3 mg, yield quantification), which was a pale yellow bubbly substance.

[1751] MS(DUIS)m / z 791(M+H) +

[1752] Manufacturing Example 14.U-014

[1753] 14-1.U-014-1

[1754]

[1755] A solution of U-011-1 (102.1 mg, 0.125 mmol) and triacetic acid (2R,3R,4S,5R,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triester (463.2 mg, 1.17 mmol) in dichloromethane was added to a 10 mL cylindrical flask. Then, 0.255 mL of 2,4,6-trimethylpyridine (233.8 mg, 1.93 mmol) was added at room temperature. Silver trifluoromethanesulfonate (387.2 mg, 1.93 mmol) was added under an argon atmosphere, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was diluted with ethyl acetate, filtered through a Celite 545 filter, and washed with ethyl acetate. The filtrate and washings were washed sequentially with 5% potassium bisulfate aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The mixture was dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a concentrated residue. Purification was performed under the conditions described below to obtain U-014-1 (128 mg, yield 90.4%) as a white bubbly substance.

[1756] MS(DUIS)m / z 1134(M+H) +

[1757] Recirculation Separation Unit (YMC): LC Forte / R

[1758] Column (YMC Company): Connect T-30000 (21.2mmφx600mm, 50nm), T-4000 (21.2mmφx600mm, 10nm), and T-2000 (21.2mmφx600mm, 5nm) for use.

[1759] Elution buffer: ethyl acetate

[1760] Temperature: Room temperature

[1761] 14-2.U-014

[1762]

[1763] In a 100 mL round-bottom flask, a solution of U-014-1 (166 mg, 0.146 mmol) and 2,6-dimethylpyridine (0.090 mL, 83.27 mg, 0.777 mmol) in 5 mL of dichloromethane was added. While stirring under an argon atmosphere, 0.135 mL of trimethylsilyl trifluoromethanesulfonate (166.05 mg, 0.747 mmol) was added under ice-cooling. The mixture was stirred for 1 hour under ice-cooling. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to the reaction mixture under ice-cooling, and the layers were separated. The aqueous layer was extracted with dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate to obtain a concentrated residue. Purification was performed under the conditions shown below, and the fraction containing the target compound was concentrated under reduced pressure to obtain a colorless oil. After adding 2 mL of acetonitrile and 5 mL of distilled water, the mixture was freeze-dried to obtain U-014 (43.3 mg, yield 28.61%) as a white powder.

[1764] MS(DUIS)m / z 1034(M+H) +

[1765] Recirculation Separation Unit (YMC): LC Forte / R

[1766] Column (YMC Company): Connect T-30000 (21.2mmφx600mm, 50nm), T-4000 (21.2mmφx600mm, 10nm), and T-2000 (21.2mmφx600mm, 5nm) for use.

[1767] Elution buffer: ethyl acetate

[1768] Temperature: Room temperature

[1769] Manufacturing Example 15.U-015

[1770] 15-1.U-015-1

[1771]

[1772] A solution of U-011-2 (101.3 mg, 0.103 mmol) and 2,5,8,11,14,17,20,23,26,29,32,35-dodecoxaheptadecane-37-amine (98.1 mg, 0.175 mmol) in dichloromethane was added to a 30 mL cylindrical flask. Then, 0.060 mL of N,N-diisopropylethylamine (44.52 mg, 0.344 mmol) was added, and the mixture was stirred at room temperature under an argon atmosphere for 7 hours. After the reaction was complete, the solution was concentrated under reduced pressure. Separation and purification were performed under the following conditions: 150 mL of the obtained ethyl acetate solution was washed twice with 50 mL of water and once with 50 mL of saturated brine. The solution was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain U-015-1 (91.8 mg, yield 63.47%) as a colorless oil.

[1773] MS(DUIS)m / z 702(M+2H) 2+

[1774] Recirculation Separation Unit (YMC): LC Forte / R

[1775] Column (YMC Company): Used to connect T-30000 (21.2mmφx600mm, 50nm), T-4000 (21.2mmφx600mm, 10nm), and T-2000 (21.2mmφx600mm, 5nm).

[1776] Elution buffer: ethyl acetate

[1777] Temperature: Room temperature

[1778] 15-2.U-015

[1779]

[1780] A 5 mL solution of U-015-1 (88.2 mg, 0.063 mmol) in dichloromethane was added to a 50 mL pear-shaped flask. 0.48 mL of trifluoroacetic acid (714.72 mg, 6.27 mmol) was added under ice-cooling, and the mixture was stirred for 1.25 hours under ice-cooling. Subsequently, the mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. 5 mL solutions of 0.1% formic acid in acetonitrile / water (1 / 9, V / V) and 0.1% formic acid in acetonitrile / water (5 / 5, V / V) were added to the concentrated residue, and the mixture was freeze-dried to obtain a colorless oily trifluoroacetate of U-015 (96.1 mg, yield quantitative).

[1781] MS(ESI)m / z 1304(M+H)+

[1782] Manufacturing Example 16.U-016

[1783] 16-1.U-016-1

[1784]

[1785] In a 50 mL flask, a solution of tert-butyl (3-fluoro-4-(2-oxoethyl)phenyl)carbamate (57.0 mg, 0.225 mmol) and MMAE (106.3 mg, 0.148 mmol) in dichloromethane (2.5 mL) was added. Then, 0.015 mL of acetic acid (15.83 mg, 0.264 mmol) was added, and sodium triacetoxyborohydride (65.0 mg, 0.307 mmol) was added while stirring under an argon atmosphere and ice-cooled conditions. The mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction solution was diluted with 5 mL of ethyl acetate, and 3 mL of saturated sodium bicarbonate aqueous solution was added. After stirring briefly at room temperature, the mixture was separated. The organic layer was washed with saturated brine, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was dissolved in hexane / ethyl acetate = 50 / 50 (V / V) and fed into a Fuji SILYSIA Chromatorex Q-Pack DIOL60 Size 10. It was eluted with hexane / ethyl acetate = 50 / 50 (V / V) → 19 / 81 (V / V). The fraction containing the target analyte was concentrated under reduced pressure to obtain U-016-1 (140.2 mg, yield 99.13%) as a white foamy substance.

[1786] MS(ESI)m / z 955(M+H) +

[1787] 16-2.U-016

[1788]

[1789] A solution of U-016-1 (136 mg, 0.142 mmol) in 3.6 mL of dichloromethane was added to a 50 mL pear-shaped flask. While stirring under an argon atmosphere, 0.545 mL of trifluoroacetic acid (811.51 mg, 7.12 mmol) was added under ice-cooling. The mixture was stirred for 0.5 hours under ice-cooling. Then, the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure. 5 mL of dichloromethane and 5 mL of saturated sodium bicarbonate solution were added, and the mixture was separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a concentrated residue. The residue was then purified under the following conditions and concentrated under reduced pressure to obtain U-016 (93.9 mg, yield 77.13%) as a white bubbly substance.

[1790] MS(ESI)m / z 855(M+H) +

[1791] Recirculation Separation Unit (YMC): LC Forte / R

[1792] Column (YMC Company): Used to connect T-30000 (21.2mmφx600mm, 50nm), T-4000 (21.2mmφx600mm, 10nm), and T-2000 (21.2mmφx600mm, 5nm).

[1793] Eluent: Acetonitrile

[1794] Temperature: Room temperature

[1795] Manufacturing Example 17.U-017

[1796] 17-1.U-017-1

[1797]

[1798] A solution of MMAE (71.8 mg, 0.100 mmol) and methyl 4-oxobutyrate (34.8 mg, 0.300 mmol) in 1 mL of dichloromethane was added to a 30 mL cylindrical flask. Sodium triacetoxyborohydride (63.6 mg, 0.300 mmol) was added while stirring at room temperature under an argon atmosphere. The mixture was stirred for 1 hour at room temperature. After the reaction was complete, a saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product U-017-1 as a yellow solid. The obtained solid was subjected to medium-pressure preparation (silica, M (16g), dichloroethane / methanol = 100 / 0 (V / V) → 90 / 10 (V / V)), and the component containing the target substance was concentrated under reduced pressure to obtain U-017-1 (20.2mg, yield 24.69%), which was a colorless bubble.

[1799] 17-2.U-017

[1800]

[1801] A 0.4 mL solution of U-017-1 (20.2 mg, 0.025 mmol) in ethanol was added to a 10 mL cylindrical flask. While stirring under an argon atmosphere, 0.050 mL of 2 equivalents of sodium hydroxide aqueous solution was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After neutralization with 0.2 mL of 2 equivalents of hydrochloric acid, the solvent was removed by vacuum distillation. 0.4 mL of N,N-dimethylformamide was added to the residue, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.5 mg, 0.039 mmol), 1-hydroxybenzotriazole (6.5 mg, 0.042 mmol), eczemab mesylate (13.1 mg, 0.025 mmol), and 0.01 mL of triethylamine (7.26 mg, 0.072 mmol). The mixture was stirred for 14 hours at room temperature. After the reaction was complete, water was added to the reaction solution, followed by separation with ethyl acetate. The insoluble residue was filtered off, and the organic layer was subjected to a preparative HPLC system (silica, S (7g), dichloroethane / methanol = 90 / 10 (V / V) → 80 / 20 (V / V)). The fraction containing the target compound (Rf value = 0.4 (dichloroethane / methanol = 90 / 10 (V / V))) was concentrated under reduced pressure. The residue was then subjected to preparative HPLC under the following conditions: the fraction containing the target compound was neutralized with saturated sodium bicarbonate aqueous solution, concentrated under reduced pressure, and water was added to the residue. The residue was then extracted twice with ethyl acetate. The organic layer was dried with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, dissolved in water / acetonitrile, and freeze-dried to obtain a colorless solid, U-017 (3.8 mg, yield 12.6%).

[1802] MS(DUIS)m / z 1222(M+H) +

[1803] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1804] Flow rate: 17 mL / min.

[1805] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1806] Gradient (solution B): 30% (0.00 min.) → 50% (10.00 min.)

[1807] Manufacturing Example 18.U-018

[1808] 18-1.U-018-1

[1809]

[1810] In a 20 mL cylindrical flask, a solution of U-011-2 (61.5 mg, 0.063 mmol) in 0.4 mL of dichloromethane was added. While stirring under an argon atmosphere, 0.008 mL of propyne-2-yn-1-amine (6.88 mg, 0.125 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The resulting residue was subjected to a medium-pressure preparation at a pressure of 0.25 (hexane / ethyl acetate = 20 / 80 (V / V)) (silica, M (16 g)) to concentrate the component containing the target analyte under reduced pressure, yielding U-018-1 (57.6 mg, yield quantification) as a white solid.

[1811] MS(ESI)m / z 900(M+H) +

[1812] 18-2.U-018-2

[1813]

[1814] A 0.2 mL solution of U-018-1 (67.6 mg, 0.075 mmol) in dichloromethane was added to a 20 mL cylindrical flask. Trifluoroacetic acid (95.36 mg, 0.836 mmol) was added while stirring under an argon atmosphere at room temperature, and the mixture was stirred for 3 hours at room temperature. After the reaction was complete, the solution was concentrated under reduced pressure. The residue was then subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target analyte was concentrated under reduced pressure to obtain U-018-2 (17.2 mg, yield 28.63%) as a white solid.

[1815] MS(ESI)m / z 799(M+H) +

[1816] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1817] Flow rate: 17 mL / min.

[1818] Elution solvent: 0.1% formic acid aqueous solution (solution A) → 0.1% formic acid acetonitrile solution (solution B)

[1819] Gradient (solution B): 20% (0.00 min.) → 95% (10.00 min.)

[1820] 18-3.U-018

[1821]

[1822] In a 20 mL cylindrical flask, a solution of U-018-2 (8.8 mg, 0.011 mmol) in 0.1 mL of water was added. While stirring under an argon atmosphere, (2R,3R,4S,5S,6R)-2-azido-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (3.8 mg, 3.09 μmol), copper(II) sulfate pentahydrate (3.5 mg, 0.022 mmol), and sodium ascorbate (7.1 mg, 0.033 mmol) were added at room temperature. The mixture was stirred for 5 minutes at room temperature. The resulting residue was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target analyte was lyophilized to obtain U-018 (3.1 mg, yield 28.03%) as a white solid.

[1823] MS(DUIS)m / z 1046(M+HCOO) -

[1824] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1825] Flow rate: 17 mL / min.

[1826] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile solution (solution B)

[1827] Gradient (solution B): 20% (0.00 min.) → 61% (6.00 min.)

[1828] Manufacturing Example 19.U-019

[1829]

[1830] In a 10 mL cylindrical flask, 0.2 mL of a water solution containing Preparation Example 18-1 (U-018-1) (8.2 mg, 10.26 μmol) was added to (2R,3R,4S,5S,6R)-6-azido-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2.8 mg, 0.013 mmol), copper(II) sulfate pentahydrate (2.0 mg, 0.013 mmol), and sodium ascorbate (3.5 mg, 0.016 mmol) were added while stirring at room temperature under an argon flow. The mixture was stirred for 5 minutes at room temperature. The resulting residue was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target analyte was freeze-dried to obtain U-019 (5.4 mg, yield 51.68%) as a white solid.

[1831] MS(ESI)m / z 1016(MH) -

[1832] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1833] Flow rate: 17 mL / min.

[1834] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile solution (solution B)

[1835] Gradient (solution B): 20% (0.00 min.) → 61% (7.00 min.)

[1836] Manufacturing Example 20.U-020

[1837] 20-1.U-020-1

[1838]

[1839] To a 20 mL cylindrical flask containing a 0.2 mL solution of U-011-2 (50.0 mg, 0.051 mmol) in dichloromethane, 0.00587 mL (5.71 mg, 0.102 mmol) of propargyl-1-ol was added while stirring under an argon atmosphere at room temperature. The mixture was stirred for 45 minutes at room temperature. Next, 0.8 mg (6.55 μmol) of 4-dimethylaminopyridine was added while stirring at room temperature, and the mixture was stirred for 2.5 hours at room temperature. Finally, 0.00587 mL (5.71 mg, 0.102 mmol) of propargyl-1-ol was added, and the mixture was stirred for 14 hours. The residue obtained was subjected to medium-pressure preparation in a shale gas atmosphere (Rf value = 0.3 (hexane / ethyl acetate = 20 / 80 (V / V) (silica, M (16g))), and the component containing the target substance was concentrated under reduced pressure to obtain U-020-1 (37.8 mg, yield 82.57%) as a white solid.

[1840] MS(ESI)m / z 900(M+H) +

[1841] 20-2.U-020-2

[1842]

[1843] A 0.2 mL solution of U-020-1 (36.8 mg, 0.041 mmol) in dichloromethane was added to a 20 mL cylindrical flask. Trifluoroacetic acid (0.069 mL, 102.81 mg, 0.902 mmol) was added while stirring under an argon atmosphere at room temperature, and the mixture was stirred for 2.5 hours at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was then subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain U-020-2 (18.2 mg, yield 55.65%) as a white solid.

[1844] MS(ESI)m / z 800(M+H) +

[1845] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1846] Flow rate: 17 mL / min.

[1847] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile solution (solution B)

[1848] Gradient (solution B): 20% (0.00 min.) → 75% (6.50 min.)

[1849] 20-3.U-020

[1850]

[1851] In a 20 mL cylindrical flask, a solution of U-020-2 (5.8 mg, 7.25 μmol) in 0.3 mL of water was added. While stirring under an argon atmosphere, (2R,3R,4S,5S,6R)-6-azido-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2.3 mg, 10.49 μmol), copper(II) sulfate pentahydrate (0.6 mg, 2.403 μmol), and sodium ascorbate (0.8 mg, 3.77 μmol) were added. The mixture was stirred overnight at room temperature. The resulting residue was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target analyte was freeze-dried to obtain U-020 as a white solid (6.3 mg, yield 85.26%).

[1852] MS(ESI)m / z 1020(M+H) +

[1853] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1854] Flow rate: 17 mL / min.

[1855] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile solution (solution B)

[1856] Gradient (solution B): 20% (0.00 min.) → 61% (6.00 min.)

[1857] Manufacturing Example 21.U-021

[1858] 21-1.U-021-1

[1859]

[1860] In a 2 mL dichloromethane solution containing 25 mg (0.025 mmol) of U-011-2, added to a 20 mL cylindrical flask, 7.0 mg (0.052 mmol) of (1H-1,2,3-triazol-4-yl)methylamine hydrochloride and 0.00354 mL (2.57 mg (0.025 mmol) of triethylamine were added under an argon atmosphere and stirred at room temperature for 1 hour. Then, 1 mL of N,N-dimethylformamide was added, and the mixture was stirred for another hour. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product U-021-1. No further purification was performed, and the product proceeded to the next step.

[1861] MS(ESI)m / z 942(M+H) +

[1862] 21-2.U-021

[1863]

[1864] In a 20 mL cylindrical flask, a solution of crude U-021-1 (284 mg, 8.5% purity) in dichloromethane (0.5 mL) was added. Trifluoroacetic acid (0.10 mL, 149 mg, 1.307 mmol) was added while stirring under an argon atmosphere at room temperature, and the mixture was stirred for 1.5 hours at room temperature. After the reaction was complete, the solution was concentrated under reduced pressure. The residue was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a white solid, 8.8 mg, of trifluoroacetic acid salt of U-021.

[1865] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1866] Flow rate: 17 mL / min.

[1867] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile solution (solution B)

[1868] Gradient (solution B): 5% (0.00 min.) → 95% (10.50 min.)

[1869] The obtained solid was dissolved in dichloromethane, washed with sodium bicarbonate aqueous solution and saturated brine, and dried with anhydrous sodium sulfate. The residue was concentrated under reduced pressure to obtain U-021 as a white solid (7.0 mg, total yield of 21-1 and 21-2 32.63%).

[1870] MS(ESI)m / z 840(MH) -

[1871] Manufacturing Example 22.U-022

[1872] 22-1.U-022-1

[1873]

[1874] To a 6 mL toluene solution of U-011-1 (400 mg, 0.489 mmol) added to a 50 mL flask, 0.11 mL (173.8 mg, 1.461 mmol) of 3-bromoprop-1-yne, 33.2 mg (0.103 mmol) of tetrabutylammonium bromide, and 0.061 mL of 8 equivalents of sodium hydroxide were added at room temperature under an argon atmosphere with stirring. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, a saturated aqueous solution of ammonium chloride was added to the reaction solution, and the mixture was extracted with dichloromethane. The residue was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target analyte was concentrated under reduced pressure to obtain U-022-1 (142.1 mg, yield 33.95%) as a white solid.

[1875] MS(ESI)m / z 857(M+H) +

[1876] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1877] Flow rate: 17 mL / min.

[1878] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1879] Gradient (solution B): 50% (0.00 min.) → 100% (10.00 min.)

[1880] 22-2.U-022-2

[1881]

[1882] A solution of U-022-1 (142.1 mg, 0.166 mmol) in 0.4 mL of dichloromethane was added to a 20 mL cylindrical flask. Trifluoroacetic acid (378 mg, 3.32 mmol) was added while stirring under an argon atmosphere at room temperature, and the mixture was stirred for 20 hours at room temperature. After the reaction was complete, the mixture was neutralized with triethylamine and concentrated under reduced pressure. The residue was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target analyte was concentrated under reduced pressure, acetonitrile was distilled off, and the fraction was lyophilized to obtain U-022-2 (69.6 mg, yield 55.47%) as a white solid.

[1883] MS(DUIS)m / z 757(M+H) +

[1884] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1885] Flow rate: 17 mL / min.

[1886] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1887] Gradient (solution B): 0% (0.00 min.) → 50% (10.00 min.)

[1888] 22-3.U-022

[1889]

[1890] A 1 mL water solution of U-022-2 (30 mg, 0.040 mmol) was added to a 30 mL cylindrical flask. While stirring under an argon atmosphere, (2S,3S,4S,5R,6R)-6-azido-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (11.2 mg, 0.051 mmol), copper sulfate pentahydrate (3.1 mg, 0.012 mmol), and sodium ascorbate (12.7 mg, 0.060 mmol) were added. The mixture was stirred at room temperature for 15 hours. The resulting residue was subjected to vacuum concentration of the component containing the target analyte under the following conditions to obtain a crude product of U-022 as a yellow solid.

[1891] Column: Waters XSelect HSS C18 SB OBD 5μm ODB 19*150mm

[1892] Flow rate: 17 mL / min.

[1893] Elution solvent:

[1894] 0.1% formic acid aqueous solution / acetonitrile with 0.1% formic acid added = 90 / 10 (solution A)

[1895] 0.1% formic acid aqueous solution / acetonitrile with 0.1% formic acid added = 10 / 90 (solution B)

[1896] Gradient (Solution B): 0% (0.00 min.) → 0% (5.00 min.) → 50% (5.10 min.) → 50% (20.00 min.)

[1897] The crude product of U-022 was purified under the conditions shown below, and the residue was freeze-dried to give U-022 as a white solid (18.8 mg, yield 37.72%).

[1898] MS(ESI)m / z 976(M+H) +

[1899] Columns: Asahipak GS-510-20G, GS-310-20G, GS-310-20G (triple connector) (Shodex Corporation) 20*500mm, 13μm

[1900] Flow rate: 7.5 mL / min.

[1901] Elution solvent: 0.1% formic acid / 0.1% formic acid in acetonitrile = 90 / 10

[1902] Detection wavelength: 254nm

[1903] Manufacturing Example 23.U-023

[1904] 23-1.U-023-1

[1905]

[1906] In a 50 mL flask, a solution of triacetic acid (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-tolyl ester (0.7982 g, 1.318 mmol) in dichloromethane was added sequentially to the solution. The mixture was stirred at room temperature under an argon atmosphere for 72 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The residue was diluted with 30 mL of ethyl acetate, washed five times with 10 mL of saturated sodium bicarbonate aqueous solution, then washed with 20 mL of saturated brine, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was used for medium-pressure preparation of silicon dioxide (40 g, hexane / ethyl acetate = 10 / 90 (V / V), Rf = 0.51 (hexane / ethyl acetate = 10 / 90 (V / V)))). The component containing the target analyte was concentrated under reduced pressure to obtain U-023-1 (677.8 mg, yield 87.21%), which was a white bubbly substance.

[1907] MS(ESI) m / z 635(M+HCOOH) +

[1908] 23-2.U-023-2

[1909]

[1910] In a 20 mL cylindrical flask, a 1.5 mL solution of U-023-1 (100.3 mg, 0.170 mmol) in dichloromethane was added. While stirring under an argon atmosphere, paraformaldehyde (9.30 mg, 0.310 mmol) and trimethylchlorosilane (33 μL, 28.25 mg, 0.260 mmol) were added sequentially at room temperature. The mixture was stirred for 5 hours at room temperature. The reaction solution was filtered, washed with dichloromethane, and the filtrate was concentrated under reduced pressure at room temperature to obtain U-023-2 (107.1 mg, yield 98.67%), a white bubbly substance.

[1911] MS(ESI)m / z 651(M+H2O) +

[1912] 23-3.U-023-3

[1913]

[1914] A 0.5 mL solution of dichloromethane containing U-011-1 (100.8 mg, 0.123 mmol) was added to a 20 mL cylindrical flask. While stirring under an argon atmosphere, 150 μL of N,N-diisopropylethylamine (111.3 mg, 0.861 mmol) and 1 mL of dichloromethane containing U-023-2 (108.55 mg, 0.170 mmol) were added sequentially at room temperature. The mixture was stirred at room temperature for 22.5 hours. Subsequently, 110 μL of 2,4,6-trimethylpyridine (101.2 mg, 0.835 mmol) was added at room temperature, followed by 2 mL of dichloromethane containing U-023-2 (171.3 mg, 0.268 mmol). After stirring at room temperature for 21 hours, the solvent was distilled off by purging the reaction mixture with nitrogen. After adding 0.75 mL of dichloromethane, 250 μL of N,N-diisopropylethylamine (185.5 mg, 1.435 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Subsequently, the mixture was ice-cooled, and 3 mL of a dichloromethane solution containing U-023-2 (446.7 mg, 0.700 mmol) was added dropwise. After the addition was complete, the mixture was brought back to room temperature and stirred for 18 hours. The reaction solution was concentrated, diluted with ethyl acetate, washed successively with 5% potassium bisulfate aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was subjected to medium-pressure preparation at a Fujifilm (Silysia Chromatorex Q-Pack DIOL60 Size 60, hexane / ethyl acetate = 30 / 70 (V / V) (Rf = 0.30 (hexane / ethyl acetate = 30 / 70 (V / V))))) to concentrate the component containing the target substance under reduced pressure, thereby obtaining the crude product U-023-3, which is a white bubbly substance.

[1915] The obtained white bubbly substance was purified under the conditions shown below to obtain U-023-3 (46.0 mg, yield 26.3%) as a white solid.

[1916] MS(ESI)m / z 1420(M+H) +

[1917] Columns: YMC-GPC T-30000, T-4000, T-2000 (three-connector) 21.2*600mm, 10μm

[1918] Flow rate: 15 mL / min.

[1919] Elution solvent: ethyl acetate

[1920] Temperature: Room temperature

[1921] Detection wavelengths: 254 nm, 285 nm

[1922] 23-4.U-023-4

[1923]

[1924] In a 100 mL flask, a solution of U-023-3 (43.2 mg, 0.030 mmol) and 160 μL (143 mg, 1.381 mmol) of 2,6-dimethylpyridine in 20 mL of dichloromethane was added. Under an argon atmosphere, with stirring, 240 μL (295.2 mg, 1.328 mmol) of trimethylsilyl trifluoromethanesulfonate was added under ice-cooling. The mixture was stirred for 20 hours under ice-cooling. Subsequently, 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction mixture under ice-cooling. After stirring briefly at room temperature, dichloromethane was added for extraction. The aqueous layer was extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated. Purification was performed under the following conditions: the fraction containing the target compound was concentrated under reduced pressure to obtain a white vesicular residue. After adding 2 mL of acetonitrile and 4 mL of distilled water, the mixture was freeze-dried to obtain U-023-4 (18.3 mg, yield 45.58%) as a white powder.

[1925] MS(ESI)m / z 1320(M+H) +

[1926] Posts: Asahipak GS-510-20G, GS-310-20G, GS-310-20G (Triple Connector) (Shodex) 20*500mm, 13μm

[1927] Flow rate: 17 mL / min.

[1928] Elution solvent: acetonitrile

[1929] Detection wavelength: 220nm

[1930] 23-5.U-023

[1931]

[1932] A 4 mL methanol solution of U-023-4 (17.1 mg, 0.013 mmol) was added to a 20 mL flask. Under air atmosphere and ice cooling, 52 μL of one equivalent of lithium hydroxide was added while stirring at room temperature. The mixture was then allowed to return to room temperature and stirred for 9.5 hours. Subsequently, 4 mL of a 50 mM ammonium formate aqueous solution (12.61 mg, 0.200 mmol) was added. Purification was carried out under the following conditions: the fraction containing the target compound was concentrated under reduced pressure, and 15 mL of acetonitrile was added to the concentrated residue, followed by freeze-drying to obtain U-023 as a white solid (13.10 mg, yield 85.71%).

[1933] MS(ESI)m / z 1180(M+H) +

[1934] Column: Xselect CSH, Prep Fluoro-Phenyl (Waters Corporation)

[1935] 5μm 19*150mm

[1936] Flow rate: 17 mL / min.

[1937] Elution solvent: 10% methanol aqueous solution (solution A) - 90% methanol aqueous solution (solution B)

[1938] Gradient (solution B): 50% (0.00 min.) → 50% (3.00 min.)

[1939] →62% (9.50 min.)

[1940] Detection wavelength: 220nm

[1941] Manufacturing Example 24.U-024

[1942]

[1943] In a 30 mL cylindrical flask, 58 μL of methanesulfonic acid (0.09 g, 0.893 mmol) was added to a 1 mL solution of MMAF (0.22 g, 0.301 mmol) in dichloromethane. The mixture was stirred under an argon atmosphere at room temperature and stirred at 60 °C for 4 hours. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to a medium-pressure preparation (silica, M (16 g), ethyl acetate / methanol = 100 / 0 (V / V) → 60 / 40 (V / V)) to concentrate the component containing the target analyte under reduced pressure, yielding a colorless bubbly substance, U-024 (183.9 mg, yield 74.61%).

[1944] MS(ESI)m / z 821(M+H) +

[1945] Manufacturing Example 25.U-025

[1946] 25-1.U-025-1

[1947]

[1948] A 1 mL solution of MMAE (43.5 mg, 0.061 mmol) in dichloromethane was added to a 5 mL sample vial. While stirring at room temperature, 10.3 μL (11.01 mg, 0.067 mmol) of 2-(benzyloxy)acetaldehyde, 5.2 μL (5.49 mg, 0.091 mmol) of acetic acid, and 25.1 mg (25.118 mmol) of sodium triacetoxyborohydride were added sequentially. The mixture was stirred for 1.5 hours at room temperature. After the reaction was complete, 1.5 mL of saturated sodium bicarbonate aqueous solution was added dropwise while stirring at room temperature for 30 minutes. The organic layer was separated, the aqueous layer was extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and subjected to medium-pressure preparation (silica, S (7g), dichloromethane / 2-propanol = 95 / 5 (V / V)) (Rf = 0.47 (dichloromethane / 2-propanol = 95 / 5 (V / V))). The component containing the target analyte was concentrated under reduced pressure to obtain U-025-1 (43.8mg, yield 84.84%), which was a white bubbly substance.

[1949] MS(ESI)m / z 853(M+H) +

[1950] Synthesis of 25-2.U-025

[1951]

[1952] In a 50 mL flask, a 2 mL solution of U-025-1 (43.8 mg, 0.051 mmol) in ethanol was added. Under a nitrogen atmosphere, 10.94 mg (0.273 mg, 2.57 μmol) of 5% palladium on carbon (ASCA-2 type, 50% aqueous) was added. After degassing under reduced pressure, the mixture was stirred at room temperature for 1 hour under a hydrogen atmosphere. Then, 0.2 mL of acetic acid was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, the mixture was heated and stirred at a bath temperature of 60 °C for 4.5 hours. After the reaction was complete, the mixture was cooled, filtered using Celite 545 (trade name), washed with ethanol, and the filtrate was concentrated under reduced pressure. The concentrated residue was diluted with dichloromethane, washed with a saturated sodium bicarbonate aqueous solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Add 2 mL of acetonitrile and 3 mL of distilled water to the concentrated residue, and freeze-dry to obtain U-025 (34.6 mg, yield 88.34%) as a white solid.

[1953] MS(ESI)m / z 763(M+H) +

[1954] Manufacturing Example 26.U-026

[1955]

[1956] To a 5 mL sample vial containing 1.5 mL of dichloromethane solution containing 63.2 mg MMAE (0.088 mmol), the following solutions were added sequentially with stirring at room temperature: 8.00 μL (4.49 mg 0.045 mmol) of 5.6 M glutaraldehyde aqueous solution, 8.00 μL (8.44 mg 0.141 mmol) of acetic acid, and 39.6 mg 0.187 mmol of sodium triacetoxyborohydride. The mixture was stirred for 2.5 hours at room temperature. After the reaction was complete, 2 mL of saturated sodium bicarbonate aqueous solution was added dropwise with stirring at room temperature. After the addition was complete, the mixture was stirred for 30 minutes at room temperature. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified under the following conditions: the component containing the target substance was concentrated under reduced pressure, dissolved in dichloromethane, added with diethyl ether, subjected to ultrasonic treatment, the solid generated by filtration was washed with diethyl ether and dried under reduced pressure, thereby obtaining U-026 (36.32 mg, yield 53.9%) as a white solid.

[1957] MS(ESI)m / z 753(M+2H) 2+

[1958] Columns: YMC-GPC T-30000, T-4000, T-2000 (three-connector) 21.2*600mm, 10μm

[1959] Flow rate: 15 mL / min.

[1960] Elution solvent: ethyl acetate

[1961] Temperature: Room temperature

[1962] Detection wavelength: 260nm

[1963] Manufacturing Example 27.U-027

[1964] 27-1.U-027-1

[1965]

[1966] In a 30 mL flask, a 1.3 mL solution of U-011-1 (102.1 mg, 0.125 mmol) in toluene was added. While stirring in air, 16 μL of tetrabutylammonium bisulfate (2.16 mg, 6.36 μmol) and 16 μL of 3-bromoprop-1-ene (22.37 mg, 0.185 mmol) were added at room temperature. Subsequently, 230 μL of a 50% (345 mg, 4.31 mmol) aqueous solution of sodium hydroxide was added at room temperature, and the mixture was stirred for 5 hours. After the reaction was complete, 2 mL of toluene and 3 mL of water were added to the reaction solution, and the mixture was stirred and separated. The organic layer was washed with 2 mL of water. The organic layer was diluted with ethyl acetate and washed successively with 5% potassium bisulfate aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine. The solution was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a white bubbly substance. Dichloromethane was added to the residue to dissolve it, and the solution was prepared under medium pressure using Chromatorex_COOH_MB100-40 / 75 (11.0 g), dichloromethane / 2-propanol = 100 / 0 (V / V) → 90 / 10 (V / V), (Rf = 0.90 (dichloromethane / 2-propanol = 80 / 20 (V / V))). The component containing the target analyte was concentrated under reduced pressure, resulting in a white bubbly substance. 2 mL of acetonitrile and 2 mL of ultrapure water were added to this substance, and the mixture was freeze-dried to obtain U-027-1 (58.3 mg, yield 54.44%) as a white solid.

[1967] MS(ESI)m / z 859(M+H) +

[1968] 27-2.U-027-2

[1969]

[1970] A 1 mL solution of U-027-1 (54.6 mg, 0.064 mmol) in tetrahydrofuran was added to a 20 mL pear-shaped flask. While stirring under an argon atmosphere, 640 μL of a 0.5 M tetrahydrofuran solution of 9-BBN (39.05 mg, 0.320 mmol) was added at room temperature, and the mixture was stirred for 2.33 hours at room temperature. Subsequently, 360 μL of a 0.5 M tetrahydrofuran solution of 9-BBN (21.96 mg, 0.180 mmol) was added, and the mixture was stirred for another 2.33 hours at room temperature. The mixture was then ice-cooled, 1 mL of methanol was added, and the mixture was allowed to return to room temperature. After stirring for 1 hour at room temperature, the mixture was concentrated under reduced pressure. 1 mL of tetrahydrofuran, 1 mL of tetrahydrofuran, and 1.0 mL of sodium hydroxide (1 equivalent) were added sequentially to the concentrated residue. The mixture was then ice-cooled, and 110 μL of a 30 wt% hydrogen peroxide aqueous solution (122.1 mg, 1.077 mmol) was added. The mixture was stirred under ice-cooling conditions for 1 hour at room temperature. Subsequently, 5 mL of water and 5 mL of ethyl acetate were added, and the mixture was separated. The organic layer was washed sequentially with a 5 wt% potassium bisulfate aqueous solution, a saturated sodium bicarbonate aqueous solution, and a saturated brine solution. The solution was dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a colorless oily substance. Dichloromethane was added to the residue to dissolve it, and the solution was prepared under medium pressure using Chromatorex_COOH(Lot No. HU20999)_MB100-40 / 75 (11.0 g), dichloromethane / 2-propanol = 100 / 0 (V / V) → 90 / 10 (V / V) to concentrate the component containing the target analyte under reduced pressure, thereby obtaining a colorless oil. 1 mL of acetonitrile and 1 mL of ultrapure water were added to the oil, and the mixture was freeze-dried to obtain U-027-2 (39.2 mg, yield 70.32%) as a white solid.

[1971] MS(ESI)m / z 877(M+H) +

[1972] 27-2.U-027

[1973]

[1974] In a 30 mL flask containing a solution of U-027-2 (35.6 mg, 0.041 mmol) in dichloromethane, 99 μL (91.6 mg, 0.856 mmol) of 2,6-dimethylpyridine and 139 μL (171 mg, 0.771 mmol) of trimethylsilyl trifluoromethanesulfonate were added under ice-cooling and stirring under an argon atmosphere. The mixture was stirred for 6 hours under ice-cooling. Then, 4 mL of dichloromethane and 8 mL of saturated sodium bicarbonate aqueous solution were added to the reaction mixture under ice-cooling, and the mixture was separated. The aqueous layer was extracted with dichloromethane, the dichloromethane layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow oil. Purification was carried out under the following conditions: the component containing the target substance was concentrated under reduced pressure, acetonitrile and water were added to the concentrated residue, and then freeze-dried to obtain U-027 (21.69 mg, yield 68.79%) as a white solid.

[1975] MS(ESI)m / z 777(M+H) +

[1976] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1977] Flow rate: 17 mL / min.

[1978] Elution solvent: acetonitrile

[1979] Detection wavelength: 220nm

[1980] Manufacturing Example 28.U-028

[1981]

[1982] In a 20 mL cylindrical flask, U-022-2 (26.4 mg, 0.035 mmol), 0.095 mL of 11–15% toluene solution of 4-methylbenzenesulfonyl azide (85.5 mg, 0.048 mmol), and 0.3 mL of acetonitrile solution of 2-aminophenol (4.0 mg, 0.037 mmol) were added. Copper(II) acetate (3.2 mg, 0.018 mmol) was added simultaneously with stirring under an argon atmosphere at room temperature. After stirring for 5 hours at room temperature, the mixture was allowed to stand for 15 hours. After the reaction was complete, acetonitrile was removed by nitrogen purging, and 1 mL of methanol was added. The mixture was then heated under reflux for 2 hours. The residue was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target analyte was concentrated under reduced pressure to remove the acetonitrile, and then freeze-dried to obtain crude U-028 as a white solid.

[1983] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1984] Flow rate: 17 mL / min.

[1985] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[1986] Gradient (solution B): 20% (0.00 min.) → 100% (10.00 min.)

[1987] After purification under the conditions shown below, the product was freeze-dried to obtain U-028 as a white solid (2.1 mg, yield 7.53%).

[1988] MS(DUIS)m / z 799(M+H) +

[1989] Device: LC Forte / R (YMC Corporation)

[1990] Column: CHIRALPAK IB (20x 250mm, 5um) (Dairalpak Co., Ltd.)

[1991] Elution buffer: MeOH

[1992] Flow rate: 15 mL / min

[1993] Wavelengths: 220, 254, 285 nm

[1994] Manufacturing Example 29.U-029

[1995]

[1996] MMAF (49.7 mg, 0.068 mmol) and (S)-(-)-α-amino-γ-butyrolactone hydrochloride (i.e., L-homoserine lactone hydrochloride) (14.1 mg, 0.102 mmol) added to a 30 mL cylindrical tube were dissolved in 1.0 mL of N,N-dimethylformamide, and 38 μL of triethylamine (27.59 mg, 0.273 mmol) was added. Then, HATU (28.6 mg, 0.075 mmol) was added at room temperature, and the mixture was stirred for 12 hours at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target analyte was concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain formate of U-029 as a colorless bubbly substance (19.6 mg, yield 33.52%).

[1997] MS(ESI)m / z 816(M+H) +

[1998] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[1999] Flow rate: 17 mL / min.

[2000] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2001] Gradient (solution B): 25% (0.00 min.) → 38% (6.00 min.)

[2002] Manufacturing Example 30.U-030

[2003] 30-1.U-030-1

[2004]

[2005] In a 50 mL pear-shaped flask, a solution of MMAE (0.7545 g, 1.051 mmol) in 15 mL of dichloromethane was added. While stirring under an argon atmosphere and ice-cooling, 0.73 mL (0.53 g, 5.24 mmol) of triethylamine and 0.73 mL (1.1 g, 5.25 mmol) of 2,2,2-trifluoroacetic anhydride were added sequentially, and the mixture was stirred for 1 hour under ice-cooling. Subsequently, while ice-cooling, 3.8 mL (3.01 g, 94 mmol) of methanol and 0.38 mL (0.28 g, 2.73 mmol) of triethylamine were added sequentially, and the mixture was stirred for 1 hour under ice-cooling. The mixture was then concentrated under reduced pressure. 30 mL of ethyl acetate was added, and the solution was washed sequentially with 30 mL of 5% potassium bisulfate aqueous solution, 30 mL of saturated sodium bicarbonate aqueous solution, and 30 mL of saturated brine. The solution was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Add 10 mL of acetonitrile and 8 mL of ultrapure water to the residue, freeze, and freeze-dry to obtain U-030-1 (0.8320 g, yield 97.27%) as a white solid.

[2006] MS(ESI)m / z 815(M+H) +

[2007] 30-2.U-030-2

[2008]

[2009] In a 50 mL flask containing a solution of U-030-1 (0.8011 g, 0.984 mmol) and 1H-tetrazole (0.4840 g, 6.91 mmol), 1.28 mL (1.33 g, 3.85 mmol) of dibenzyl diisopropyl phosphonoamidate was added to a 30 mL dichloromethane solution. The mixture was stirred at room temperature under an argon atmosphere for 2 hours. Subsequently, 0.60 mL (0.67 g, 5.87 mmol) of 30% hydrogen peroxide solution was added under ice-cooling conditions, and the mixture was stirred for 1.5 hours under ice-cooling conditions.

[2010] Subsequently, under ice cooling, saturated sodium bicarbonate aqueous solution was added to 12.75 g of sodium thiosulfate to make a volume of 100 mL, and then 20 mL of this solution was added. After separating the organic layer, the solution was washed with saturated brine, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure.

[2011] The obtained residue was subjected to medium-pressure preparation at a pressure of 99 / 1 (V / V) to 95 / 5 (V / V) of dichloromethane / isopropanol. The component containing the target analyte was subjected to reduced pressure to obtain the crude product U-030-2.

[2012] The crude product was purified under the following conditions: the fraction containing the target compound was concentrated under reduced pressure, 10 mL of acetonitrile and 8 mL of ultrapure water were added to the residue, the mixture was frozen, and then freeze-dried to obtain U-030-2 (0.6915 g, yield 65.41%) as a white solid.

[2013] MS(ESI) m / z 1075(M+H) +

[2014] Device: LC Forte / R (YMC Corporation)

[2015] Columns: YMC-GPC T-30000, T-4000, T-2000 (three-connector) 21.2*600mm, 10μm

[2016] Temperature: Room temperature

[2017] Eluent: Acetonitrile

[2018] Flow rate: 25 mL / min

[2019] Wavelength: 220nm

[2020] 30-3.U-030-3

[2021]

[2022] Add 5 mL of a methanol solution of U-030-2 (331 mg, 0.308 mmol) to a 50 mL pear-shaped flask. While stirring under an argon atmosphere and under ice-cooling, add sodium borohydride (34.6 mg, 0.915 mmol) and stir for 1.25 hours. Then, add sodium borohydride (107.4 mg, 2.838 mmol) in three portions, stirring for 3 hours each time while under ice-cooling.

[2023] After the reaction was completed, 20 mL of dichloromethane and 20 mL of saturated sodium bicarbonate aqueous solution were added under ice cooling to separate the organic layer. The resulting aqueous layer was extracted three times with 20 mL of dichloromethane. The organic layers were combined and dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain U-030-3 (325 mg, yield quantification), a colorless oil.

[2024] MS(ESI)m / z 979(M+H) +

[2025] 30-4.U-030

[2026]

[2027] Add 30 mL of ethanol solution of U-030-3 (301 mg, 0.308 mmol) to a 100 mL flask, and while stirring under a nitrogen atmosphere, add 5 wt% palladium on carbon STD type (50 wt% water, 82.3 mg, 0.019 mmol), and stir at room temperature under a hydrogen atmosphere for 2 hours.

[2028] After the reaction was complete, the mixture was filtered through Celite 545 (trade name), washed with ethanol, and concentrated under reduced pressure. Acetonitrile was added to the residue, the resulting solid was filtered off, washed with ice-cooled acetonitrile, and dried under reduced pressure to obtain U-030 (220.3 mg, yield 89.72%) as a white solid.

[2029] MS(ESI)m / z 799(M+H) +

[2030] Manufacturing Example 31.U-031

[2031]

[2032] In a 20 mL cylindrical flask, sodium triacetoxyborohydride (77.8 mg, 0.367 mmol) was added to a 1 mL methanol solution containing 1 mL of Preparation Example 30 (100.4 mg, 0.126 mmol) and 0.093 mL of 37% formaldehyde aqueous solution (101.37 mg, 1.249 mmol), and stirred at room temperature under an argon atmosphere for 1 hour.

[2033] After the reaction was completed, the solution was concentrated under reduced pressure, and water was added. The resulting aqueous solution was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target analyte was concentrated under reduced pressure to obtain 41.0 mg of crude purified U-031 as a white solid.

[2034] Column: Waters XBrige Prep C18 5μm ODB 19*150mm

[2035] Flow rate: 17 mL / min.

[2036] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2037] Gradient (Solution B): 10% (0.00 min.) → 30% (4.00 min.) → 30% (9.00 min.)

[2038] Under the following conditions, 41.0 mg of the crude purified product of U-031 was subjected to preparative HPLC chromatography, and the fraction containing the target analyte was freeze-dried to obtain U-031 (29.5 mg, yield 28.87%) as a white solid.

[2039] MS(ESI)m / z 810(MH) -

[2040] Column: Waters XBrige Prep C18 5μm ODB 19*150mm

[2041] Flow rate: 17 mL / min.

[2042] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2043] Gradient (Solution B): 10% (0.00 min.) → 30% (5.00 min.) → 30% (10.00 min.)

[2044] Manufacturing Example 32.U-032

[2045]

[2046] In a 10 mL cylindrical flask, a solution of eczemab mesylate (13.6 mg, 0.026 mmol) in 1 mL of dichloromethane was added. While stirring under an argon atmosphere, 35 μL of triethylamine (25.41 mg, 0.251 mmol) was added, followed by ice cooling. Then, under ice cooling, 10 μL of 2,2-difluoroacetic anhydride (16 mg, 0.092 mmol) was added, and the mixture was stirred at 0 °C for 2 hours.

[2047] Next, 17 μL of triethylamine (12.34 mg, 0.122 mmol) and 10 μL of 2,2-difluoroacetic anhydride (16 mg, 0.092 mmol) were added at room temperature with stirring, and the mixture was stirred at room temperature for 1 hour.

[2048] Next, 100 μL of methanol (79.2 mg, 2.472 mmol) was added at room temperature with stirring, and the mixture was stirred at room temperature for 0.5 hours.

[2049] Next, 60 μL of triethylamine (43.56 mg, 0.430 mmol) was added at room temperature with stirring, and the mixture was stirred at room temperature for 0.5 hours.

[2050] After the reaction was completed, 2 mL of diethyl ether was added, the mixture was stirred for 5 minutes, filtered through a membrane filter, and the resulting solid was washed with ethyl acetate and water and dried under reduced pressure to obtain U-032 (7.9 mg, yield 60.13%), which was a pale yellow solid.

[2051] MS(ESI)m / z 514(M+H) +

[2052] Manufacturing Example 33.U-033

[2053] 33-1.U-033-1

[2054]

[2055] In a 100 mL cylindrical flask containing a 12 mL solution of U-032 (419 mg, 0.816 mmol) in dichloromethane, diallyl N,N-diisopropylphosphonamide (820 μL, 760.96 mg, 3.10 mmol) and 1H-tetrazole (373 mg, 5.32 mmol) were added sequentially at room temperature while stirring under an argon atmosphere. The mixture was stirred at room temperature for 5 hours.

[2056] Subsequently, 480 μL (532.8 mg, 4.70 mmol) of 30% hydrogen peroxide water was added under ice cooling, and the mixture was stirred for 1 hour under ice cooling.

[2057] Subsequently, 6 mL of a solution prepared by adding 100 mL of water to 20.0 g (80.6 mmol) of sodium thiosulfate decahydrate and 7.0 g (83.3 mmol) of sodium bicarbonate, along with 6 mL of dichloromethane, were added, and the mixture was separated. The aqueous layer was extracted with 6 mL of dichloromethane and combined with the organic layer. The mixture was dried with 0.80 g of anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a concentrated residue of 967 mg.

[2058] Add 5% acetonitrile aqueous solution to the obtained residue, perform ultrasonic treatment, filter under reduced pressure, and dry the obtained solid to obtain U-033-1 (250 mg, yield 45.48%), which is a brown solid.

[2059] MS(ESI) m / z 1348(2M+H) +

[2060] 33-2.U-033

[2061]

[2062] Add 5 mL of tetrahydrofuran solution of U-033-1 (243 mg, 0.361 mmol) to a 50 mL flask, and while stirring under an argon atmosphere, add 84 μL of N-methylaniline (83.16 mg, 0.776 mmol) and tetra(triphenylphosphine)palladium (43 mg, 0.037 mmol) sequentially at room temperature, and stir at room temperature for 1 hour.

[2063] After the reaction was completed, 15 mL of tert-butyl methyl ether was added, and the mixture was stirred for 10 minutes. The precipitated solid was filtered, washed with tert-butyl methyl ether, and dried under reduced pressure to obtain 256 mg of crude product U-033.

[2064] The obtained solid was dissolved in 15 mL of a 30% acetonitrile aqueous solution containing 2% ammonium acetate, and subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was freeze-dried to obtain U-033 (81 mg, yield 37.83%) as a pale yellow solid.

[2065] MS(ESI)m / z 592(MH) -

[2066] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2067] Flow rate: 17 mL / min.

[2068] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2069] Gradient (Solution B): 30% (0.00 min.) → 60% (6.00 min.) → 90% (6.50 min.) → 90% (9.00 min.)

[2070] Manufacturing Example 34.U-034

[2071]

[2072] In a 10 mL cylindrical flask containing 1 mL of N,N-dimethylformamide, along with 53.2 mg (0.100 mmol) of eczemamate, 18.3 mg (0.120 mmol) of 2-(3-hydroxyphenyl)acetic acid, 38.2 mg (0.199 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 15.3 mg (0.100 mmol) of 1-hydroxybenzotriazole, 28 μL (20.33 mg, 0.201 mmol) of triethylamine was added while stirring at room temperature under an argon atmosphere. The mixture was stirred at room temperature for 2 hours.

[2073] After the reaction was completed, the reaction solution was subjected to preparative HPLC chromatography under the following conditions. The component containing the target compound was concentrated under reduced pressure, the precipitated solid was filtered off, and washed with water to obtain U-033 (25.4 mg, yield 44.56%) as a colorless solid.

[2074] MS(ESI)m / z 568(MH) -

[2075] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2076] Flow rate: 17 mL / min.

[2077] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2078] Gradient (solution B): 50% (0.00 min.) → 95% (5.00 min.)

[2079] Manufacturing Example 35.U-035

[2080] 35-1.U-035-1

[2081]

[2082] In a 50 mL flask containing 4 mL of N,N-dimethylformamide solution containing DXd (162.6 mg, 0.329 mmol), 29.0 μL of trifluoromethanesulfonic acid (49.3 mg, 0.329 mmol) was added while stirring at room temperature under an argon atmosphere. The mixture was stirred for 30 minutes at room temperature. Subsequently, 350 μL of diallylN,N-diisopropylphosphonamide (324.8 mg, 1.324 mmol) and 1H-tetrazole (163.7 mg, 2.337 mmol) were added at room temperature, and the mixture was stirred for 2 hours at room temperature.

[2083] Subsequently, 270 μL (253.8 mg, 1.971 mmol) of 70% tert-butyl peroxide aqueous solution was added at room temperature, and the mixture was stirred at room temperature for 1.5 hours.

[2084] Subsequently, 40 mL of diethyl ether was added to the reaction solution, and the mixture was stirred for a short time under ice cooling. The resulting solid was filtered, washed with diethyl ether, and dried under reduced pressure to obtain a light brown solid of U-035-1 trifluoromethanesulfonate (249.6 mg, yield 94.26%).

[2085] MS(ESI)m / z 654(M+H) +

[2086] 35-2.U-035

[2087]

[2088] In a 20 mL flask, 22 μL of N-methylaniline (21.78 mg, 0.203 mmol) and 10.4 mg of tetra(triphenylphosphine)palladium (9.00 μmol) were added sequentially to a 5 mL solution of trifluoromethanesulfonate (51.4 mg, 0.064 mmol) in tetrahydrofuran under an argon atmosphere and stirred at room temperature. The mixture was stirred for 1.25 hours at room temperature. Subsequently, tetra(triphenylphosphine)palladium (27.9 mg, 0.024 mmol) was added, followed by 0.5 mL of N,N-dimethylformamide, and the mixture was stirred for 0.75 hours at room temperature.

[2089] The reaction solution was concentrated under reduced pressure. Diethyl ether was added to the concentrated residue, and the mixture was subjected to ultrasonic treatment. The resulting solid was filtered off, washed with diethyl ether, and dried under reduced pressure to obtain a grayish-brown solid (55.2 mg).

[2090] The resulting grayish-brown solid was recycled as shown below, and the component containing the target compound was freeze-dried to obtain U-035 (6.4 mg, yield 17.45%) as a pale yellow solid.

[2091] MS(ESI)m / z 574(M+H) +

[2092] Device: LC Forte / R (YMC Corporation)

[2093] Column: Xbridege C18, 30x150mm, 5um

[2094] Eluent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile (solution B)

[2095] Solution composition (solution B): 30%

[2096] Flow rate: 25 mL / min, 21 MPa

[2097] Wavelength: 254nm

[2098] Manufacturing Example 36.U-036

[2099]

[2100] In a 20 mL cylindrical flask, a solution of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-ethylglycine (15.06 mg, 0.046 mmol) in N,N-dimethylformamide (0.6 mL) was added. While stirring under an argon atmosphere at room temperature, HATU (17.60 mg, 0.046 mmol) and N,N-diisopropylethylamine (0.022 mL, 16.32 mg, 0.126 mmol) were added, and the mixture was stirred for 15 minutes at room temperature.

[2101] Next, eczema mesylate (21.7 mg, 0.042 mmol) was added at room temperature with stirring, and the mixture was stirred at room temperature for 1 hour.

[2102] Next, piperidine 4,16 μL (3.58 mg, 0.042 mmol) was added at room temperature with stirring, and the mixture was stirred at room temperature for 3 hours.

[2103] The obtained residue was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target analyte (around 7.5 min.) was freeze-dried to obtain formate of U-036 as a white solid (6.9 mg, yield 31.5%).

[2104] MS(ESI)m / z 521(M+H) +

[2105] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2106] Flow rate: 17 mL / min.

[2107] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2108] Gradient (solution B): 15% (0.00 min.) → 55% (10.00 min.)

[2109] [Example] Synthesis of conjugate precursor (I) and conjugate precursor synthesis intermediate (II)

[2110] Synthesis of Example 1

[2111]

[2112] Example 1-1

[2113]

[2114] (S)-(-)-α-amino-γ-butyrolactone hydrochloride (i.e., L-homoserine lactone hydrochloride) (1.75 g, 12.72 mmol) was dissolved in 60 mL of N,N-dimethylformamide in a 200 mL orb-shaped flask. Then, Fmoc-(L)-Asp-tert-butyl (5.23 g, 12.71 mmol) and triethylamine (3.65 mL, 2.65 g, 26.2 mmol) were added. Next, HATU (4.83 g, 12.70 mmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layer was concentrated under reduced pressure to give the labeled compound as a colorless oil (6.16 g, 97.9% yield).

[2115] Examples 1-2

[2116]

[2117] The compound from Example 1-1 (6.16 g, 12.46 mmol) added to a 500 mL pear-shaped flask was dissolved in 60 mL of dichloromethane, followed by the addition of 60 mL of trifluoroacetic acid (1.421 g, 12.46 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was removed by vacuum distillation. The resulting residue was dissolved in 50 mL of acetonitrile, followed by the addition of 50 mL of ethyl acetate. The precipitated solid was filtered off and washed with ethyl acetate to give the labeled compound as a colorless solid (3.86 g, yield 70.68%).

[2118] MS(ESI)m / z 439(M+H) +

[2119] Examples 1-3

[2120]

[2121] In a 30 mL cylindrical flask, 4 mL of N,N-dimethylformamide solution (298 mg, 0.680 mmol) from Examples 1-2 was added, and piperidine 0.270 mL (232.2 mg, 2.73 mmol) was added while stirring under an argon flow at room temperature. The mixture was stirred at room temperature for 1 hour.

[2122] After the reaction was complete, xylene was added, and the solvent was removed under reduced pressure.

[2123] Add 4 mL of diethyl ether and 8 mL of ethyl acetate to the residue, sonicate, and filter to collect the solid. Add 20% acetonitrile aqueous solution to the filtered solid and concentrate under reduced pressure.

[2124] The residue was added to ethyl acetate and filtered. Diethyl ether was added to the solid, filtered, and dried under reduced pressure to give the labeled compound as a white solid (143 mg, yield 97.32%).

[2125] MS(ESI)m / z 215(MH) -

[2126] Examples 1-4

[2127]

[2128] A solution of Fmoc-(L)-Val-(L)―Cit-PAB-PNP (Angene) (292.7 mg, 0.382 mmol), MMAE (MedChemExpress) (259.7 mg, 0.362 mmol), and 1-hydroxy-7-azabenzotriazole (56.3 mg, 0.414 mmol) in N,N-dimethylformamide was added to a 30 mL cylindrical flask with stirring under a nitrogen atmosphere. The mixture was stirred for 19.5 hours at room temperature. After the reaction was complete, the solvent was removed by vacuum distillation. 20 mL of diethyl ether was added to the residue, and the precipitated solid was filtered off, washed with water, and then washed with diethyl ether to give the labeled compound as a beige solid (415.8 mg, yield 85.43%).

[2129] MS(DUIS)m / z 673(M+2H) 2+

[2130] Examples 1-5

[2131]

[2132] In a 30 mL flask containing 5 mL of N,N-dimethylformamide solution (414.7 mg, 0.308 mmol) from Examples 1-4, 0.244 mL of piperidine (210 mg, 2.465 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and ethyl acetate, acetonitrile, and dichloromethane were added to the residue to dissolve it. Diethyl ether was then added, and the mixture was sonicated. The resulting solid was filtered off, washed with diethyl ether, and dried under reduced pressure to obtain a pale yellow solid labeled compound (280.3 mg, yield 80.96%).

[2133] MS(ESI)m / z 1124(M+H) +

[2134] Examples 1-6

[2135]

[2136] In a 30 mL cylindrical flask, 5 mL of N,N-dimethylformamide solution (130.1 mg, 0.297 mmol) from Examples 1-2 and 1-5 (309 mg, 0.275 mmol) was added. Then, 41 μL of triethylamine (29.77 mg, 0.294 mmol) was added at room temperature, followed by the addition of HATU (112.9 mg, 0.297 mmol). The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure by adding dichloromethane and 2-propanol. Then, 10 mL of acetonitrile and 10 mL of water were added, and the mixture was sonicated. The resulting solid was filtered off, washed with 10 mL of acetonitrile / water (1 / 1 (V / V)), then washed with 20 mL of diethyl ether, and dried under reduced pressure to obtain the labeled compound as a white solid (342.3 mg, yield 80.61%).

[2137] MS(ESI)m / z 773(M+2H) 2+

[2138] Examples 1-7

[2139]

[2140] In a 10 mL flask containing 450 μL of N,N-dimethylformamide solution (49.7 mg, 0.032 mmol) from Examples 1-6, 10 μL of piperidine (8.62 mg, 0.101 mmol) was added to the solution under an argon stream while stirring at room temperature. After stirring for 50 minutes at room temperature, the solvent was removed under reduced pressure, and the solution was washed with 10 mL of diethyl ether. The resulting solid was dried under reduced pressure to obtain the labeled compound as a white solid (36.4 mg, yield 85.56%).

[2141] MS(ESI)m / z 1323(M+H) +

[2142] Example 1

[2143]

[2144] In a 5 mL cylindrical flask, a 0.4 mL solution of N,N-dimethylformamide (10.0 mg, 7.57 μmol) from Examples 1-7 was added. While stirring under an argon atmosphere, a 400 μL solution of acetonitrile containing 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester (8.2 mg, 0.027 mmol) was added, and the mixture was stirred at room temperature for 1 hour.

[2145] The reaction solution obtained under the following conditions was subjected to preparative HPLC chromatography, and the component containing the target analyte was freeze-dried to obtain Example 1 (3.2 mg, yield 24.59%) as a white solid.

[2146] MS(ESI)m / z 861(M+2H) 2 +

[2147] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2148] Flow rate: 17 mL / min.

[2149] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2150] Gradient (solution B): 40% (0.00 min.) → 90% (10.00 min.)

[2151] Synthesis of Example 2

[2152]

[2153] Example 2-1

[2154]

[2155] (L)-Val-(L)-Cit-PAB (manufactured by Angene) (1.90 g, 5.01 mmol) and those from Examples 1-2 (2.24 g, 5.11 mmol) were suspended in 50 mL of N,N-dimethylformamide in a 200 mL pear-shaped flask. 0.71 mL of triethylamine (0.52 g, 5.09 mmol) was added, followed by HATU (1.94 g, 5.10 mmol). The mixture was stirred at room temperature for 1 hour. 30 mL of water and 20 mL of acetone were added to the resulting reaction mixture, and the mixture was stirred for 30 minutes. The mixture was then filtered, washed with water, and then washed with acetone to give the labeled compound as a pale yellow solid (3.37 g, yield 84.13%).

[2156] MS(ESI)m / z 800(M+H) +

[2157] Example 2-2

[2158]

[2159] In a 100 mL flask containing 12 mL of N,N-dimethylformamide solution (1.027 g, 1.284 mmol) from Example 2-1, bis(4-nitrophenyl) carbonate (760 mg, 2.498 mmol) and N,N-diisopropylethylamine (440 μL, 0.33 g, 2.52 mmol) were added at room temperature with stirring under an argon atmosphere. The mixture was stirred for 3 hours at room temperature. Next, 8 mL of N,N-dimethylformamide was added at room temperature with stirring. After stirring for 0.5 hours at room temperature, the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. Diethyl ether and hexane were added to the residue, and the resulting solid was filtered, washed four times with water, then washed with diethyl ether, and dried under reduced pressure to obtain a labeled compound (760 mg, yield 61.34%) as a pale yellow solid.

[2160] MS(ESI)m / z 965(M+H) +

[2161] Example 2-3

[2162]

[2163] In a 10 mL flask containing 1 mL of N,N-dimethylformamide, along with 76.2 mg (0.079 mmol) of Example 2-2, U-030 (55.1 mg, 0.069 mmol), and 1-hydroxy-7-azabenzotriazole (13.0 mg, 0.096 mmol), 40 μL of N,N-diisopropylethylamine (29.68 mg, 0.230 mmol) was added, and the mixture was stirred at room temperature for 18 hours. Subsequently, 22.3 mg (0.023 mmol) of Example 2-2 was added, and the mixture was stirred at room temperature for 6.5 hours.

[2164] After the reaction was completed, the mixture was concentrated under reduced pressure. Diethyl ether was added to the residue, and the resulting solid was filtered off. 10 mL of acetonitrile / water (1 / 1 (V / V)) was added to the solid, and after removing insoluble matter by filtration, the mixture was freeze-dried to obtain the labeled compound as a white solid (57.4 mg, yield 51.19%).

[2165] MS(ESI)m / z 812(M+2H) 2+

[2166] Examples 2-4

[2167]

[2168] In a 100 mL flask containing 2 mL of N,N-dimethylformamide (57.4 mg, 0.035 mmol) from Examples 2-3, 11 μL of piperidine (9.48 mg, 0.111 mmol) was added to the solution while stirring under an argon stream at room temperature. After stirring for 1 hour at room temperature, the solvent was removed under reduced pressure, and the mixture was washed with 10 mL of diethyl ether. The resulting solid was dried under reduced pressure to obtain the labeled compound (51.8 mg, yield 93.23%) as a pale yellow solid.

[2169] MS(ESI)m / z 1402(M+H) +

[2170] Example 2

[2171]

[2172] Using 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester (11 mg, 0.021 mmol), and Examples 2-4 (18 mg, 13 μmol) instead of Examples 1-7, the reaction was carried out in the same manner as in Example 1 to give a labeled compound as a white solid (5.2 mg, yield 22.49%).

[2173] MS(ESI)m / z 901(M+2H) 2+

[2174] Synthesis of Example 3

[2175]

[2176] Example 3-1

[2177]

[2178] Add 10 mL of acetonitrile solution of di-tert-butyl phosphite (1.94 g, 9.99 mmol) to a 100 mL flask containing a gai flask, and while stirring under a nitrogen stream, add 1.68 mL of benzyl acrylate (1.78 g, 10.98 mmol) at room temperature, and stir at 80 °C for 6 hours.

[2179] After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was then used for medium-pressure preparation (silica, L (40 g), hexane / ethyl acetate = 70 / 30 (V / V) → 50 / 50 (V / V)). The component containing the target compound was concentrated under reduced pressure to obtain a colorless oily labeled compound (2.23 g, yield 62.64%).

[2180] MS(ESI)m / z 357(M+H) +

[2181] Example 3-2

[2182]

[2183] In a 22 mL ethanol solution of Example 3-1 (2.23 g, 6.26 mmol) added to a 200 mL flask, 10 wt% palladium on carbon NX type (50 wt% water, 1.30 g, 0.611 mmol) was added while stirring under a nitrogen atmosphere, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours.

[2184] After the reaction was complete, the mixture was filtered through Celite 545 (trade name), washed with ethanol, and concentrated under reduced pressure to obtain the labeled compound (1.68 g, yield quantification) as a colorless oil.

[2185] MS(ESI)m / z 267(M+H) +

[2186] Example 3-3

[2187]

[2188] In a 100 mL flask containing a 4 mL solution of (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid (i.e., (L)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid (1.21 g, 4.26 mmol / L) (as described in WO2019 / 195665), 21.3 mL (3.11 g, 85 mmol / L) of 1,4-dioxane solution of 4M hydrogen chloride was added to the solution while stirring under an argon atmosphere at room temperature. The mixture was stirred at room temperature for 1 hour.

[2189] After the reaction was complete, the solvent was removed under reduced pressure, and ethyl acetate was added to the residue. The resulting solid was then filtered. The solid was dissolved in methanol and concentrated under reduced pressure to obtain the labeled compound (1.17 g, yield quantitative) as a pale yellow bubbly substance.

[2190] Examples 3-4

[2191]

[2192] To a 3 mL solution of N,N-dimethylformamide (0.26 g, 0.976 mmol) from Example 3-2, which was added to a 30 mL cylindrical flask, 0.13 mL (0.09 g, 0.933 mmol) of triethylamine and HATU (0.37 g, 0.973 mmol) were added at room temperature while stirring under an argon atmosphere. The mixture was stirred at room temperature for 1 hour. This solution was then used as solution A.

[2193] In a 20 mL cylindrical flask containing 3 mL of N,N-dimethylformamide solution (0.21 g, 0.952 mmol), 0.065 mL of triethylamine (0.045 g, 0.466 mmol) was added while stirring under an argon stream at room temperature, and the mixture was stirred for 10 minutes at room temperature.

[2194] Add solution A to the 20 mL cylindrical flask at room temperature and stir for 6 hours at room temperature.

[2195] After the reaction was completed, water was added to the reaction solution, and the solution was filtered through a membrane filter. The resulting solution was then subjected to preparative HPLC chromatography under the following conditions. The component containing the target compound was freeze-dried to obtain a colorless solid labeled compound (143.9 mg, yield 34.96%).

[2196] MS(ESI)m / z 433(M+H) +

[2197] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2198] Flow rate: 17 mL / min.

[2199] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2200] Gradient (solution B): 30% (0.00 min.) → 36% (5.00 min.)

[2201] Examples 3-5

[2202]

[2203] In a 30 mL flask containing 3 mL of N,N-dimethylformamide (143.8 mg, 0.333 mmol) from Examples 3-4, 56 μL of triethylamine (40.66 mg, 0.402 mmol) and HATU (152.5 mg, 0.401 mmol) were added at room temperature while stirring under an argon atmosphere. The mixture was stirred for 2 minutes at room temperature. Next, a 0.5 mL solution of N,N-dimethylformamide containing 1-amino-3,6,9,12-tetraoxapentadecano-15-oate tert-butyl ester (112.5 mg, 0.350 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature.

[2204] After the reaction was completed, the reaction solution was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a colorless solid labeled compound (143.5 mg, yield 58.64%).

[2205] MS(ESI)m / z 734(MH) -

[2206] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2207] Flow rate: 17 mL / min.

[2208] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2209] Gradient (solution B): 20% (0.00 min.) → 60% (8.00 min.)

[2210] Examples 3-6

[2211]

[2212] In a 30 mL flask containing 1.5 mL of dichloromethane solution (63.3 mg, 0.086 mmol) from Examples 3-5, 150 μL of trifluoroacetic acid (223.35 mg, 1.959 mmol) was added while stirring under a nitrogen stream at room temperature. The mixture was stirred for 2.5 hours at room temperature. Then, 0.5 mL of trifluoroacetic acid (744.5 mg, 6.530 mmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature.

[2213] After the reaction was completed, the reaction solution was concentrated under reduced pressure, and 2 mL of acetonitrile / water (1 / 1 (V / V)) was added to the residue. The mixture was then freeze-dried to obtain a labeled compound as a white solid (56.6 mg, yield quantification).

[2214] MS(ESI)m / z 568(M+H) +

[2215] Examples 3-7

[2216]

[2217] In a 100 mL flask containing 6 mL of N,N-dimethylformamide solution (340.9 mg, 0.426 mmol) from Example 2-1, 255 μL (242.25 mg, 0.804 mmol) and 1H-tetrazole (56.6 mg, 0.808 mmol) were added to 2,N',N',-tetraisopropylphosphonic diamine at room temperature while stirring under an argon atmosphere. The mixture was stirred for 15 minutes at room temperature. Subsequently, 215 μL (245.1 mg, 0.800 mmol) of triisopropylsilyl trifluoromethanesulfonate was added at room temperature, and the mixture was stirred for 30 minutes at room temperature.

[2218] Subsequently, U-010 (146 mg, 0.199 mmol) and 5-(ethylthio)-1H-tetrazole (109.4 mg, 0.840 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Then, 330 μL of tert-butyl hydroperoxide (310.2 mg, 2.409 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 2 hours.

[2219] Subsequently, 600 μL (612 mg, 4.02 mmol) of diazabicycloundecene was added under ice cooling, and the mixture was stirred at room temperature for 1 hour.

[2220] After the reaction was complete, 50 mL of diethyl ether was added, and the supernatant was removed. Then, 25 mL of diethyl ether was added, and the supernatant was removed. The mixture was then dried under reduced pressure. The residue was purified under the recycling conditions shown below, and the fraction containing the target compound was subjected to reduced pressure to obtain the labeled compound as a colorless oil (185.6 mg, yield 67.8%).

[2221] MS(ESI)m / z 1372(M+H) +

[2222] Device: LC Forte / R (YMC Corporation)

[2223] Column: Xserect HSS C18 19*150mm, 5μm

[2224] Flow rate: 17 mL / min.

[2225] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile (solution B)

[2226] Solution composition (solution B): 50%

[2227] Example 3

[2228]

[2229] In a 10 mL cylindrical flask, 600 μL of N,N-dimethylformamide solution (11.9 mg, 0.021 mmol) from Examples 3-6 was added. While stirring under an argon atmosphere, 9 μL of triethylamine (6.53 mg, 0.065 mmol) was added at room temperature, followed by HATU (8.7 mg, 0.023 mmol). The mixture was stirred for 5 minutes at room temperature. Next, 400 μL of N,N-dimethylformamide solution (12.5 mg, 9.11 μmol) from Examples 3-7 was added at room temperature, and the mixture was stirred for 3 hours at room temperature.

[2230] After the reaction was completed, the reaction solution was concentrated under reduced pressure. The residue obtained was subjected to preparative HPLC chromatography under the following conditions, and the fraction containing the target compound was freeze-dried to obtain the labeled compound as a white solid (1.29 mg, yield 7.37%).

[2231] MS(ESI)m / z 961(M+2H) 2+

[2232] Column: Waters XSELECT HSS T3 Prep 5μm ODB 19*150mm

[2233] Flow rate: 17 mL / min.

[2234] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2235] Gradient (Solution B): 20% (0.00 min.) → 35% (6.00 min.) → 80% (6.50 min.) → 80% (9.00 min.)

[2236] Synthesis of Example 4

[2237]

[2238] Example 4-1

[2239]

[2240] Using Fmoc-(L)-Val-(L)-Cit-PAB-PNP (manufactured by Angene) (263.5 mg, 0.344 mmol), and the formate of the free form of U-003 in place of MMAE (167.8 mg, 0.304 mmol), the compound was reacted in the same manner as in Examples 1-4, thereby yielding a crude product of the labeled compound as a light brown solid (390 mg, yield quantification).

[2241] MS(ESI)m / z 1134(M+H) +

[2242] Example 4-2

[2243]

[2244] In a 2 mL solution of N,N-dimethylformamide (103.3 mg, 0.091 mmol) from Example 4-1, which was added to a 30 mL cylindrical flask, 87 μL of piperidine (74.99 mg, 0.881 mmol) was added at room temperature while stirring under a nitrogen stream. After stirring at room temperature for 0.5 hours, the solvent was removed under reduced pressure.

[2245] Add the residue obtained from Examples 1-2 (115.5 mg, 0.263 mmol), 72 μL of triethylamine (53.42 mg, 0.528 mmol), 2 mL of N,N-dimethylformamide, and HATU (106.4 mg, 0.280 mmol) to the mixture at room temperature, and stir for 10 minutes at room temperature. Then, add the residue from Examples 1-2 (115.5 mg, 0.263 mmol), 72 μL of triethylamine (53.42 mg, 0.528 mmol), and HATU (106.4 mg, 0.280 mmol) to the mixture at room temperature, and stir for 2 minutes at room temperature.

[2246] The reaction solution was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a labeled compound as a white solid (57.1 mg, yield 47.05%).

[2247] MS(ESI)m / z 1333(M+H) +

[2248] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2249] Flow rate: 17 mL / min.

[2250] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2251] Gradient (solution B): 30% (0.00 min.) → 80% (10.00 min.)

[2252] Example 4

[2253]

[2254] In a 5 mL sample tube, 4 μL of piperidine (3.44 mg, 0.040 mmol) was added to a 0.4 mL solution of N,N-dimethylformamide (10.0 mg, 7.51 μmol) from Example 4-2 at room temperature while stirring under a nitrogen stream. After stirring at room temperature for 1 hour, the solvent was removed under reduced pressure.

[2255] Add 0.4 mL of N,N-dimethylformamide to the residue, and while stirring, add 0.4 mL of acetonitrile solution containing 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester (15.4 mg, 0.030 mmol), and stir for 1 hour. Then, add 4 μL of triethylamine (2.9 mg, 0.029 mmol), and stir for 1 hour.

[2256] After the reaction was completed, 3 mL of 50% acetonitrile aqueous solution was added to the reaction solution and subjected to preparative HPLC chromatography under the following conditions. The component containing the target compound was freeze-dried to obtain a colorless solid labeled compound (5.3 mg, yield 46.81%).

[2257] MS(ESI)m / z 755(M+2H) 2+

[2258] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2259] Flow rate: 17 mL / min.

[2260] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2261] Gradient (solution B): 30% (0.00 min.) → 60% (10.00 min.)

[2262] Synthesis of Example 5

[2263]

[2264] Example 5-1

[2265]

[2266] Using Example 2-2 (343.0 mg, 0.356 mmol / L) and U-029 (290 mg, 0.356 mmol / L) instead of U-30, the reaction was carried out in the same manner as in Example 2-3 to obtain the labeled compound (49.7 mg, yield 8.51%) as a colorless oil.

[2267] MS(DUIS)m / z 821(M+2H) 2+

[2268] Example 5

[2269]

[2270] Using 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester (47 mg, 0.092 mmol), and Example 5-1 (25 mg, 0.015 mmol) instead of Examples 4-2, the reaction was carried out in the same manner as in Example 4 to give a labeled compound as a white solid (21.0 mg, yield 75.86%).

[2271] MS(DUIS)m / z 1815(MH) -

[2272] Synthesis of Example 6

[2273]

[2274] Example 6-1

[2275]

[2276] In a 20 mL cylindrical tube, 5 mL of N,N-dimethylformamide solution (0.49 g, 0.991 mmol) from Example 1-1 was added. While stirring under a nitrogen stream, 75 μL (0.08 g, 0.498 mmol) of diazabicycloundecene was added at room temperature, and the mixture was stirred for 1 hour at room temperature. This solution was used as reaction solution A.

[2277] In addition, in a 20 mL cylindrical flask, 3 mL of N,N-dimethylformamide solution (0.44 g, 1.004 mmol) from Examples 1-2 was added. While stirring under a nitrogen stream, the above reaction solution A was added at room temperature, followed by 0.28 mL of triethylamine (0.2 g, 2.009 mmol) and HATU (0.46 g, 1.210 mmol). The mixture was stirred at room temperature for 1 hour.

[2278] After the reaction was complete, 8 mL of tert-butyl methyl ether, 2.5 mL of 1 equivalent hydrochloric acid, and then 8 mL of water were added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes. The precipitate was filtered, washed with water, and then washed with tert-butyl methyl ether to obtain a colorless solid labeled compound (0.53 g, yield 77.22%).

[2279] MS(ESI)m / z 693(M+H) +

[2280] Example 6-2

[2281]

[2282] Dissolve Example 6-1 (0.21 g, 0.303 mmol) in 2 mL of dichloromethane in a 30 mL cylindrical tube, then add 2 mL of trifluoroacetic acid and stir at room temperature for 1 hour.

[2283] After the reaction was completed, the solvent was removed by vacuum distillation. 3 mL of acetonitrile was added to the residue, the precipitated solid was filtered off, and washed with acetonitrile to obtain the labeled compound as a colorless solid (0.21 g, yield quantification).

[2284] MS(ESI)m / z 637(M+H) +

[2285] Example 6-3

[2286]

[2287] To a 2 mL solution of N,N-dimethylformamide (0.21 g, 0.303 mmol) from Example 6-1, which was added to a 20 mL cylindrical tube, 23 μL (0.02 g, 0.153 mmol) of diazabicycloundecene was added while stirring under a nitrogen stream, and the mixture was stirred at room temperature for 1 hour. This solution was used as reaction solution A.

[2288] In a separate 20 mL cylindrical tube, a 2 mL solution of N,N-dimethylformamide (0.22 g, 0.306 mmol) from Example 6-2 was added. While stirring under a nitrogen stream, reaction solution A, 85 μL of triethylamine (0.06 g, 0.610 mmol), and HATU (0.14 g, 0.368 mmol) were added at room temperature, and the mixture was stirred at room temperature for 1 hour.

[2289] After the reaction was complete, 10 mL of tert-butyl methyl ether, 0.76 mL of 1 equivalent hydrochloric acid, and 10 mL of water were added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes. The precipitated solid was filtered, washed with water and tert-butyl methyl ether, and dried to obtain a colorless solid labeled compound (0.31 g, yield 93.89%).

[2290] MS(ESI) m / z 1089(M+H) +

[2291] Example 6-4

[2292]

[2293] In Example 6-3 (10.0 mg, 9.18 μmol) added to a 30 mL cylindrical tube, 0.1 mL of trifluoroacetic acid was added while stirring under a nitrogen stream at room temperature, and the mixture was stirred at room temperature for 1 hour.

[2294] After the reaction was completed, the solvent was concentrated under reduced pressure, and 0.3 mL of acetonitrile was added to the residue. The precipitated solid was filtered, washed with acetonitrile, and dried to obtain the labeled compound as a colorless solid (8.4 mg, yield 88.56%).

[2295] MS(ESI)m / z 1033(M+H) +

[2296] Example 6-5

[2297]

[2298] Instead of Examples 1-5, Examples 1-7 (6.9 mg, 5.22 μmol) were used, and instead of Examples 1-2, Examples 6-4 (6.6 mg, 6.39 μmol) were used, and the reaction was carried out in the same manner as in Examples 1-6 to obtain a labeled compound as a colorless bubbly substance (6.76 mg, yield 55.41%).

[2299] MS(ESI) m / z 1169(M+2H) 2+

[2300] Example 6

[2301]

[2302] Using 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester (7.2 mg, 0.014 mmol), and Examples 6-5 (5.3 mg, 2.268 μmol) instead of Examples 4-2, the reaction was carried out in the same manner as in Example 4 to give a labeled compound as a colorless solid (2.62 mg, yield 45.97%).

[2303] MS(ESI) m / z 1257(M+2H) 2+

[2304] Synthesis of Example 7

[2305]

[2306] Example 7-1

[2307]

[2308] In a 100 mL flask containing a solution of (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid (i.e., (L)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid (1.20 g, 4.22 mmol / L) (as described in WO2019 / 195665) in 50 mL of dichloromethane, 3.24 mL of trifluoroacetic acid was added while stirring at room temperature under a nitrogen atmosphere, and the mixture was stirred at room temperature for 4 hours.

[2309] After the reaction was completed, the reaction solution was concentrated under reduced pressure, 20 mL of diethyl ether was added, the precipitated solid was filtered, washed with diethyl ether, and dried to obtain the labeled compound as a beige solid (0.9734 g, yield 77.33%).

[2310] MS(ESI)m / z 185(M+H)+

[2311] Example 7-2

[2312]

[2313] In a 10 mL tetrahydrofuran solution containing 591 mg, 1.982 mmol of Example 7-1 and methyl chloroformate (9H-fluorene-9-yl) (536 mg, 2.072 mmol) added to a 100 mL flask, 5 mL of an aqueous solution containing sodium bicarbonate (973 mg, 11.58 mmol) was added while stirring in air at 0 °C, and the mixture was stirred at room temperature for 1 hour.

[2314] After the reaction was complete, a 10% (w / w) aqueous solution of citric acid was added to the reaction solution, and the mixture was extracted with ethyl acetate. The residue was subjected to a medium-pressure preparation (silica, L (40 g), ethyl acetate / methanol = 100 / 0 (V / V) → 60 / 40 (V / V) (Rf = 0.13 (ethyl acetate / methanol = 95 / 5 (V / V))) under reduced pressure to concentrate the component containing the target compound, thereby obtaining the labeled compound as a white bubbly substance (761 mg, yield 94.48%).

[2315] MS(ESI)m / z 407(M+H) +

[2316] Example 7-3

[2317]

[2318] Using tert-butyl 1-amino-3,6,9,12-tetraoxapentadecano-15-oate (477 mg, 1.484 mmol) and Example 7-2 (692 mg, 1.703 mmol), which replaced Examples 3-4, the reaction was carried out in the same manner as in Examples 3-5 to give the labeled compound (918 mg, yield 87.15%) as an orange oil.

[2319] MS(ESI)m / z 710(M+H) +

[2320] Example 7-4

[2321]

[2322] To a 12 mL solution of dichloromethane (780 mg, 1.099 mmol) from Example 7-3, which was added to a 30 mL cylindrical flask, 75 μL (75.75 mg, 0.498 mmol) of diazabicycloundecene was added at room temperature while stirring under an argon atmosphere. The mixture was stirred for 40 minutes at room temperature. This solution was used as reaction solution A.

[2323] In a separate 100 mL flask containing a solution of (S)-5-oxotetrahydrofuran-2-carboxylic acid (211 mg, 1.622 mmol) and 1-hydroxybenzotriazole (223 mg, 1.650 mmol), in 12 mL of dichloromethane, the above reaction solution A and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (320 mg, 1.669 mmol) were added to the solution while stirring under an argon atmosphere at room temperature. The mixture was stirred at room temperature for 1 hour.

[2324] After the reaction was complete, 10 mL of 5% citric acid aqueous solution was added to the reaction solution, and the mixture was extracted with dichloromethane. The solution was then washed with 5 mL of saturated saline, dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure.

[2325] The residue was subjected to medium-pressure preparation at a pressure of COOH, M(16g)2, dichloroethane / methanol = 100 / 0 (V / V) → 92 / 8 (V / V) to concentrate the component containing the target compound under reduced pressure, thereby obtaining a labeled compound (187mg, yield 28.38%) that is a light brown oil.

[2326] MS(ESI)m / z 598(MH) -

[2327] Example 7-5

[2328]

[2329] Instead of Examples 3-5, Examples 7-4 (21 mg, 0.035 mmol) were used, and the reaction was carried out in the same manner as in Examples 3-6 to obtain the labeled compound (20 mg, yield quantification) as a pale yellow oil.

[2330] MS(ESI)m / z 544(M+H) +

[2331] Example 7

[2332]

[2333] Instead of Examples 3-7, Examples 1-7 (18 mg, 0.014 mmol / L) were used, and instead of Examples 3-6, Examples 7-5 (20 mg, 0.037 mmol / L) were used, and the reaction was carried out in the same manner as in Example 3 to obtain a labeled compound as a white solid (14.2 mg, yield 56.44%).

[2334] MS(ESI) m / z 924(M+2H) 2+

[2335] Synthesis of Example 8

[2336]

[2337] Using Examples 3-6 (6.10 mg, 10.75 μmol) and Examples 1-7 (14.33 mg, 10.84 μmol) instead of Examples 3-7, the reaction was carried out in the same manner as in Example 3 to give a labeled compound as a white solid (5.91 mg, yield 29.38%).

[2338] MS(ESI) m / z 936(M+2H) 2+

[2339] Synthesis of Example 9

[2340]

[2341] Example 9-1

[2342]

[2343] Using the reagents from Examples 2-2 (198.0 mg, 0.205 mmol / L) and ethyl glycine (25.9 mg, 0.251 mmol / L) instead of U-030, the reaction was carried out in the same manner as in Examples 2-3 to obtain a labeled compound as a pale yellow solid (80.5 mg, yield 42.23%).

[2344] MS(ESI)m / z 929(M+H) +

[2345] Example 9-2

[2346]

[2347] In a 10 mL cylindrical flask, a solution of 0.2 mL of N,N-dimethylformamide containing eczemab mesylate (65.3 mg, 0.123 mmol), Example 9-1 (125 mg, 0.135 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (46.9 mg, 0.245 mmol), and 1-hydroxybenzotriazole (18.7 mg, 0.122 mmol) was added. While stirring under an argon atmosphere, 0.034 mL of triethylamine (24.68 mg, 0.244 mmol) was added at room temperature, and the mixture was stirred for 12 hours at room temperature.

[2348] The resulting reaction solution was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a colorless solid labeled compound (80.3 mg, yield 48.55%).

[2349] MS(DUIS)m / z 673(M+2H) 2+

[2350] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2351] Flow rate: 17 mL / min.

[2352] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2353] Gradient (solution B): 50% (0.00 min.) → 62% (6.00 min.)

[2354] Example 9

[2355]

[2356] To a 0.8 mL solution of N,N-dimethylformamide (12.1 mg, 8.99 μmol) from Example 9-2, which was added to a 5 mL sample tube, 1.74 μL of piperidine (1.5 mg, 0.018 mmol) was added at room temperature while stirring under an air stream. After stirring at room temperature for 1 hour, the solvent was removed under reduced pressure.

[2357] Add 0.8 mL of N,N-dimethylformamide to the residue, and while stirring, add 11 μL of N,N-diisopropylethylamine (8.14 mg, 0.063 mmol) from Examples 3-6 (10.1 mg, 0.018 mmol), HATU (8.2 mg, 0.022 mmol), and N,N-diisopropylethylamine (8.14 mg, 0.063 mmol) and stir at room temperature for 30 minutes.

[2358] After the reaction was completed, 5 mL of 50% acetonitrile aqueous solution was added to the reaction solution and subjected to preparative HPLC chromatography under the following conditions. The component containing the target compound was freeze-dried to obtain a white solid labeled compound (4.7 mg, yield 31.25%).

[2359] MS(ESI)m / z 837(M+2H) 2+

[2360] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2361] Flow rate: 17 mL / min.

[2362] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2363] Gradient (Solution B): 20% (0.00 min.) → 20% (2.00 min.) → 70% (8.00 min.)

[2364] Synthesis of Example 10

[2365]

[2366] Example 10-1

[2367]

[2368] In a 200 mL flask containing 50 mL of N,N-dimethylformamide solution (4.7 g, 5.88 mmol) from Example 2-1, 1.45 mL (1.25 g, 14.64 mmol) was added to piperidine at room temperature while stirring under an argon atmosphere. The mixture was stirred for 1 hour at room temperature. Then, 0.29 mL (0.25 g, 2.93 mmol) of piperidine was added at room temperature while stirring. After stirring for 2 hours at room temperature, the solvent was removed under reduced pressure.

[2369] After the reaction was completed, ethyl acetate and diethyl ether were added to the residue, the resulting solid was filtered off, and dried under reduced pressure at 50°C for 4 hours to obtain the labeled compound as a white solid (4.98 g, yield quantification).

[2370] MS(ESI)m / z 578(M+H) +

[2371] Example 10-2

[2372]

[2373] In a 300 mL flask containing N,N-dimethylformamide (4.24 g, 7.34 mmol) and anhydrous potassium carbonate (1.52 g, 11.00 mmol), 0.3 mL of water was added to 60 mL of the N,N-dimethylformamide solution from Example 10-1. Triphenylchloromethane (1.03 g, 3.69 mmol) was added while stirring at room temperature under air, and the mixture was stirred for 0.5 hours. Next, triphenylchloromethane (1.03 g, 3.69 mmol) was added while stirring at room temperature, and the mixture was stirred for 0.5 hours. Next, triphenylchloromethane (1.01 g, 3.62 mmol) was added while stirring at room temperature, and the mixture was stirred for 0.5 hours. Finally, 0.3 mL of water and triphenylchloromethane (0.496 g, 1.779 mmol) were added while stirring at room temperature, and the mixture was stirred for 15 minutes.

[2374] Add 100 mL of ethyl acetate, 50 mL of diethyl ether, and 25 mL of tetrahydrofuran to the reaction solution, followed by 75 mL of water and 25 mL of saturated brine. Stir and separate the layers. Concentrate the aqueous layer and extract again with ethyl acetate. Combine the organic layers, wash with saturated brine, dry with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure.

[2375] The residue was treated with tetrahydrofuran, ethyl acetate, diethyl ether, and hexane, and filtered. The solid was washed with tert-butyl methyl ether to obtain the labeled compound as a white solid (2.69 g, yield 44.69%).

[2376] MS(ESI)m / z 820(M+H) +

[2377] Example 10-3

[2378]

[2379] In a 300 mL flask, a mixed solution of 90 mL acetonitrile and 30 mL water containing eczemanetta-methyl sulfonate (1.60 g, 3.01 mmol) was added. Sodium bicarbonate (1.26 g, 15.00 mmol) and (9H-fluorene-9-yl)methyl chloroformate (0.93 g, 3.59 mmol) were added while stirring at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 2.5 hours.

[2380] After the reaction was complete, 150 mL of water was added to the reaction solution, and the mixture was stirred for 10 minutes under ice cooling. The precipitated solid was separated and dried under reduced pressure to obtain the labeled compound (1.91 g, yield 96.48%), which was a pale yellow solid.

[2381] MS(ESI)m / z 658(M+H)+

[2382] Example 10-4

[2383]

[2384] In a 500 mL flask containing 200 mL of dichloromethane solution (6.44 g, 0.79 mmol) from Example 10-3, 4-nitrophenyl chloroformate (5.92 g, 29.4 mmol), triethylamine (5.45 mL, 3.96 g, 39.1 mmol), and 4-dimethylaminopyridine (1.79 g, 14.65 mmol) were added at room temperature while stirring under a nitrogen atmosphere. The mixture was stirred at room temperature for 4 hours.

[2385] After the reaction was complete, 120 mL of 0.2 equivalent hydrochloric acid, 150 mL of dichloromethane, and 100 mL of water were added to the reaction solution, and the organic layer was separated by Celite filtration.

[2386] The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was subjected to medium-pressure preparation (silica, L (40 g), hexane / ethyl acetate = 50 / 50 (V / V) → 0 / 100 (V / V), and the component containing the target compound was concentrated under reduced pressure to obtain a labeled compound as a brown solid (5.06 g, yield 62.81%).

[2387] MS(ESI)m / z 823(M+H) +

[2388] Examples 10-5

[2389]

[2390] In a 500 mL flask containing 108 mL of dichloromethane solution from Example 10-4 (3.56 g, 4.33 mmol), while stirring under an argon atmosphere, 1.22 mL of triethylamine (0.89 g, 8.75 mmol) and 4-dimethylaminopyridine (1.02 g, 8.35 mmol) were added to the solution from Example 10-4 (3.25 g, 3.96 mmol), and stirred at room temperature for 7.5 hours.

[2391] After the reaction was completed, saturated saline solution was added to the reaction solution, the mixture was extracted with dichloromethane, and the organic layer was concentrated under reduced pressure.

[2392] The residue obtained was subjected to medium-pressure preparation at a pressure of 3L (135g) of silica and ethyl acetate / methanol = 100 / 0 (V / V) → 70 / 30 (V / V) (Rf = 0.22 (ethyl acetate / methanol = 90 / 10 (V / V)). The component containing the target compound was concentrated under reduced pressure to obtain a labeled compound (1.86g, yield 31.21%) as a pale yellow solid.

[2393] MS(ESI)m / z 1262(M+H-Trt) +

[2394] Examples 10-6

[2395]

[2396] In a 1 mL solution of N,N-dimethylformamide (15 mg, 9.98 μmol) from Example 10-5, which was added to a 10 mL cylindrical flask, 10 μL of piperidine (8.6 mg, 0.101 mmol) was added while stirring at room temperature under an air atmosphere, and the mixture was stirred at room temperature for 1 hour.

[2397] The reaction solution was concentrated under reduced pressure, and 1 mL of dichloromethane was added to the residue. Then, 4 μL of triethylamine (2.9 mg, 0.029 mmol) was added at room temperature with stirring, followed by 3.0 μL of acetic anhydride (3.24 mg, 0.032 mmol). The mixture was stirred at room temperature for 36 hours.

[2398] The solvent was concentrated under reduced pressure, and 1 mL of N,N-dimethylformamide was added to the residue. Then, 0.50 mL of formic acid (600 mg, 13.04 mmol) was added at room temperature with stirring, and the mixture was stirred at room temperature for 1 hour.

[2399] The obtained residue was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a colorless solid labeled compound (4.9 mg, yield 43.58%).

[2400] MS(ESI)m / z 1081(M+H) +

[2401] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2402] Flow rate: 17 mL / min.

[2403] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2404] Gradient (solution B): 20% (0.00 min.) → 60% (10.00 min.)

[2405] Example 10

[2406]

[2407] Using Examples 3-6 (16.1 mg, 0.028 mmol / L) and Examples 10-6 (4.9 mg, 4.35 μmol / L) instead of Examples 3-7, the reaction was carried out in the same manner as in Example 3 to give a labeled compound as a white solid (4.1 mg, yield 57.84%).

[2408] MS(ESI)m / z 816(M+2H) 2+

[2409] Synthesis of Example 11

[2410]

[2411] Example 11-1

[2412]

[2413] Add 2 mL of N,N-dimethylformamide solution (66.8 mg, 0.044 mmol) from Example 10-5 to a 30 mL flask, and while stirring in air, add 22 μL of piperidine (18.92 mg, 0.222 mmol) at room temperature, and stir for 1 hour at room temperature.

[2414] The reaction solution was concentrated under reduced pressure. 2 mL of N,N-dimethylformamide was added to the residue. 19 μL of triethylamine (13.79 mg, 0.136 mmol) was added at room temperature with stirring, followed by 2-hydroxyacetic acid (10.1 mg, 0.133 mmol) and HATU (50.7 mg, 0.133 mmol). The mixture was stirred at room temperature for 3 hours.

[2415] Next, 1 mL of formic acid (1200 mg, 26.1 mmol) was added at room temperature and stirred for 1 hour at room temperature.

[2416] The obtained residue was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a labeled compound as a light brown solid (30.6 mg, yield 60.25%).

[2417] MS(ESI) m / z 1097(M+H) +

[2418] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2419] Flow rate: 17 mL / min.

[2420] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2421] Gradient (solution B): 20% (0.00 min.) → 60% (10.00 min.)

[2422] Example 11

[2423]

[2424] Using Examples 3-6 (16.5 mg, 0.029 mmol / L) and Examples 11-1 (6.6 mg, 5.77 μmol / L) instead of Examples 3-7, the reaction was carried out in the same manner as in Example 3 to give a labeled compound as a white solid (3.6 mg, yield 37.87%).

[2425] MS(ESI)m / z 824(M+2H) 2+

[2426] Synthesis of Example 12

[2427]

[2428] Example 12-1

[2429]

[2430] In a 50 mL flask containing 3 mL of N,N-dimethylformamide solution (162.2 mg, 0.203 mmol) from Example 2-1, 130 μL (123.5 mg, 0.410 mmol) of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine and 1H-tetrazole (29.8 mg, 0.425 mmol) were added at room temperature while stirring under an argon atmosphere. The mixture was stirred for 15 minutes at room temperature. Subsequently, 110 μL (125.4 mg, 0.409 mmol) of triisopropylsilyl trifluoromethanesulfonate was added at room temperature, and the mixture was stirred for 30 minutes at room temperature.

[2431] Subsequently, DXd (100.7 mg, 0.204 mmol) and 5-(ethylthio)-1H-tetrazole (53.8 mg, 0.413 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Next, 170 μL of 70% tert-butyl hydrogen peroxide aqueous solution (159.8 mg, 1.241 mmol) was added at room temperature, and the mixture was stirred at room temperature for 1.6 hours.

[2432] Subsequently, 300 μL (306 mg, 2.01 mmol) of diazabicycloundecene was added under ice cooling, and the mixture was stirred at room temperature for 1.5 hours.

[2433] After the reaction was complete, 30 mL of diethyl ether was added, and the supernatant was removed. Then, 15 mL of diethyl ether was added, and the supernatant was removed. Next, 15 mL of diethyl ether was added, and the supernatant was removed. Then, 20 mL of acetonitrile was added, and the supernatant was removed. Next, 10 mL of acetonitrile was added, and the supernatant was removed. Then, 20 mL of diethyl ether was added, and the supernatant was removed. The mixture was dried under reduced pressure to obtain the crude product of the labeled compound. This crude product was purified under the recycling conditions shown below, and the fraction containing the target compound was subjected to reduced pressure to obtain the labeled compound as a beige solid (39.8 mg, yield 17.32%).

[2434] MS(ESI)m / z 1133(M+H) +

[2435] Device: LC Forte / R (YMC Corporation)

[2436] Column: XBridge Prep C18 5μm ODB 19*150mm

[2437] Flow rate: 25 mL / min.

[2438] Elution solvent: 0.1% formic acid aqueous solution (solution A) - 0.1% formic acid acetonitrile (solution B)

[2439] Solution composition (solution B): 40%

[2440] Example 12

[2441]

[2442] Using Examples 3-6 (13.2 mg, 0.023 mmol / L) and Example 12-1 (11.5 mg, 10.15 μmol / L) instead of Examples 3-7, the reaction was carried out in the same manner as in Example 3 to give a labeled compound as a white solid (5.78 mg, yield 33.85%).

[2443] MS(ESI)m / z 842(M+2H) 2+

[2444] Synthesis of Example 13

[2445]

[2446] Example 13-1

[2447]

[2448] In a 0.3 mL solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycine amide (compound described in JP6186045) (102.8 mg, 0.040 mmol, 41%), 9 μL (9.09 mg, 0.06 mmol) of diazabicycloundecene was added at room temperature while stirring under an argon atmosphere, and the mixture was stirred at room temperature for 12 hours. Add 30 μL of 2 equivalents of hydrochloric acid. Use this as reaction solution A.

[2449] Separately from the above, in a 0.2 mL solution of N,N-dimethylformamide (26.1 mg, 0.060 mmol) from Examples 1-2, added to a 10 mL cylindrical flask, 8.5 μL of triethylamine (6.17 mg, 0.061 mmol) and HATU (22.8 mg, 0.060 mmol) were added at room temperature while stirring. The mixture was stirred at room temperature for 30 minutes and then added to the above reaction solution A. The mixture was stirred at room temperature for 1 hour.

[2450] The resulting reaction solution was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a colorless and amorphous labeled compound (17.9 mg, yield 35.8%).

[2451] MS(ESI)m / z 1262(M+H) +

[2452] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2453] Flow rate: 17 mL / min.

[2454] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2455] Gradient (solution B): 40% (0.00 min.) → 60% (8.00 min.)

[2456] Example 13

[2457]

[2458] Instead of 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester, 6-maleimidehexanoic acid N-succinimide ester (8.75 mg, 0.028 mmol) was used, and instead of Examples 4-2, Example 13-1 (17.9 mg, 0.014 mmol) was used. The reaction was carried out in the same manner as in Example 4 to give a labeled compound as a colorless solid (3.6 mg, yield 20.59%).

[2459] MS(ESI)m / z 1233(M+H) +

[2460] Synthesis of Example 14

[2461]

[2462] Example 14-1

[2463]

[2464] Using Example 2-2 (23.1 mg, 0.024 mmol / L) and eribulin mesylate (16.4 mg, 0.020 mmol / L) instead of U-030, the reaction was carried out in the same manner as in Example 2-3 to give a colorless and amorphous labeled compound (21.0 mg, yield 67.98%).

[2465] MS(DUIS)m / z 1555(M+H) +

[2466] Example 14

[2467]

[2468] Using Examples 3-6 (11.7 mg, 0.021 mmol / L) and Example 14-1 (8.3 mg, 5.33 μmol / L) instead of Examples 9-2, the reaction was carried out in the same manner as in Example 9 to give a labeled compound as a pale yellow solid (6.9 mg, yield 68.69%).

[2469] MS(ESI) m / z 942(M+2H) 2+

[2470] Synthesis of Example 15

[2471]

[2472] Example 15-1

[2473]

[2474] In a 100 mL cylindrical flask, a solution of bis(2,5-dioxopyrrolidone-1-yl)4,7,10,13,16-pentaenodecanoic acid ester (1.08 g, 2.028 mmol) and 1-hydroxypyrrolidone-2,5-dione (46.6 mg, 0.405 mmol) in N,N-dimethylformamide was added dropwise at room temperature while stirring under an argon atmosphere. The mixture was stirred at room temperature for 20 minutes. Next, add 4 mL of water, and then add dropwise a mixture of alendronic acid (0.50 g, 2.007 mmol) and triethylamine (0.84 mL, 0.61 g, 6.03 mmol) in 4 mL of water / 2 mL of N,N-dimethylformamide at room temperature while stirring. Stir at room temperature for 20 minutes, and then add 3 mL of 2 equivalents of hydrochloric acid.

[2475] The obtained residue was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a colorless oily labeled compound (0.31 g, yield 23.17%).

[2476] MS(ESI)m / z 667(M+H) +

[2477] Column: Waters XSelect HSS T3 OBD 5um (19*150mm)

[2478] Flow rate: 17 mL / min.

[2479] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2480] Gradient (solution B): 5% (0.00 min.) → 30% (10.00 min.)

[2481] Example 15-2

[2482]

[2483] In a 20 mL cylindrical flask, 9 mL of N,N-dimethylformamide solution (0.31 g, 0.465 mmol) from Example 15-1 was added. While stirring under an argon atmosphere, 0.21 g, 0.952 mmol of Example 3-3 and 0.65 mL of triethylamine (0.47 g, 4.66 mmol) were added. The mixture was stirred at room temperature for 1 hour, and then 2.3 mL of 2 equivalents of hydrochloric acid was added.

[2484] The residue was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the labeled compound as a colorless oil (272.7 mg, yield 79.71%).

[2485] MS(ESI)m / z 736(M+H) +

[2486] Column: Waters XSelect HSS T3 OBD 5um (19*150mm)

[2487] Flow rate: 17 mL / min.

[2488] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2489] Gradient (solution B): 5% (0.00 min.) → 20% (6.00 min.)

[2490] Example 15-3

[2491]

[2492] Using tert-butyl 1-amino-3,6,9,12-tetraoxapentadecano-15-oate (17.5 mg, 0.054 mmol) and Example 15-2 (40.0 mg, 0.054 mmol), which replaced Examples 3-4, the reaction was carried out in the same manner as in Examples 3-5 to give the labeled compound as a colorless oil (20.4 mg, yield 36.11%).

[2493] MS(ESI) m / z 1040(M+H) +

[2494] Example 15-4

[2495]

[2496] Instead of Examples 3-5, Examples 15-3 (20.3 mg, 0.020 mmol) were used, and the reaction was carried out in the same manner as in Examples 3-6 to obtain the labeled compound as a colorless oil (9.0 mg, yield 46.87%).

[2497] MS(ESI)m / z 983(M+H) +

[2498] Example 15

[2499]

[2500] Using Examples 3-7 (8.4 mg, 6.12 μmol), and Examples 15-4 (9.0 mg, 9.16 μmol) which replaced Examples 3-6, the reaction was carried out in the same manner as in Example 3 to give a labeled compound as a colorless solid (9.97 mg, yield 69.68%).

[2501] MS(ESI) m / z 1169(M+2H) 2+

[2502] Synthesis of Example 16

[2503]

[2504] Example 16-1

[2505]

[2506] In a 30 mL cylindrical flask, a 5 mL methanol / 5 mL dichloromethane mixture of (0.28 g, 0.823 mmol) fluorene-9-yl)methoxy)carbonyl)amino)ethyl)glycine was added. While stirring, 0.28 mL (0.31 g, 3.76 mmol) of 37% formaldehyde aqueous solution, 0.26 mL (0.31 g, 6.78 mmol) of formic acid, and 0.26 g, 1.227 mmol of sodium triacetoxyborohydride were added. The mixture was stirred at room temperature for 1 hour. Then, 0.26 g, 1.227 mmol of sodium triacetoxyborohydride was added.

[2507] After the reaction was completed, the solvent was concentrated under reduced pressure, acetonitrile and water were added, and the mixture was filtered through a membrane filter.

[2508] The obtained solution was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a colorless solid labeled compound (0.15 g, yield 51.45%).

[2509] MS(ESI)m / z 355(M+H) +

[2510] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2511] Flow rate: 17 mL / min.

[2512] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2513] Gradient (solution B): 30% (0.00 min.) → 45% (6.00 min.)

[2514] Example 16-2

[2515]

[2516] To a 0.5 mL solution of N,N-dimethylformamide (7.1 mg, 4.56 μmol) from Example 14-1, which was added to a 5 mL sample tube, 5 μL of piperidine (4.3 mg, 0.050 mmol) was added at room temperature while stirring under air. After stirring at room temperature for 1 hour, the solution was concentrated under reduced pressure. This was recorded as residue A.

[2517] In another 5 mL sample tube, 0.5 mL of N,N-dimethylformamide solution (3.4 mg, 9.59 μmol) from Example 16-1 was added. While stirring under an argon atmosphere, 1.5 μL of dimethylbenzylamine (1.35 mg, 9.98 μmol) was added at room temperature, followed by HATU (3.4 mg, 8.94 μmol). The mixture was stirred at room temperature for 1 hour.

[2518] The above reaction solution was added to residue A and stirred at room temperature for 1 hour. Then, HATU (1 mg, 2.63 μmol) was added.

[2519] The resulting reaction solution was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a colorless solid labeled compound (4.5 mg, yield 59.05%).

[2520] MS(ESI) m / z 836(M+2H) 2+

[2521] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2522] Flow rate: 17 mL / min.

[2523] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2524] Gradient (solution B): 30% (0.00 min) → 80% (8.00 min)

[2525] Example 16

[2526]

[2527] Using Examples 3-6 (6.0 mg, 10.57 μmol) and Examples 16-2 (4.5 mg, 2.69 μmol) instead of Examples 9-2, the reaction was carried out in the same manner as in Example 9 to obtain a labeled compound as a colorless solid (2.40 mg, yield 44.59%).

[2528] MS(ESI)m / z 1000(M+2H) 2+

[2529] Synthesis of Example 17

[2530]

[2531] Example 17-1

[2532]

[2533] In a 30 mL cylindrical flask, 5 mL of N,N-dimethylformamide solution containing 0.54 g (1.470 mmol) of Example 3-2 and 0.41 mL (0.3 g, 2.94 mmol) of triethylamine was added. HATU (0.59 g, 1.552 mmol) was added while stirring under an argon atmosphere at room temperature, and the mixture was stirred for 0.5 hours at room temperature. This solution was designated as solution A.

[2534] In a 20 mL cylindrical flask, 5 mL of N,N-dimethylformamide solution (0.32 g, 1.451 mmol) from Example 3-3 was added, and solution A was added while stirring under an argon flow at room temperature. The mixture was stirred at room temperature for 6 hours.

[2535] After the reaction was completed, water was added to the reaction solution, and the solution was filtered through a membrane filter. The resulting solution was then subjected to preparative HPLC chromatography under the following conditions. The component containing the target analyte was freeze-dried to obtain Example 3-4 (62.5 mg) as a colorless oil.

[2536] MS(ESI)m / z 433(M+H) +

[2537] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2538] Flow rate: 17 mL / min.

[2539] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2540] Gradient (solution B): 30% (0.00 min.) → 36% (5.00 min.)

[2541] The obtained Examples 3-4 were placed at room temperature for one month, thereby obtaining the labeled compounds as colorless solids.

[2542] MS(ESI)m / z 321(M+H) +

[2543] Example 17-2

[2544]

[2545] In a 10 mL flask containing a solution of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propionate (0.21 g, 0.896 mmol) in N,N-dimethylformamide (5 mL), 0.32 mL (0.3 g, 1.009 mmol) of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine and 1H-tetrazole (70.0 mg, 0.999 mmol) were added sequentially, and the mixture was stirred at room temperature for 15 minutes.

[2546] Subsequently, (9H-fluorene-9-yl)methyl(2-hydroxyethyl)carbamate (0.31 g, 1.094 mmol) was added, followed by 5-(ethylthio)-1H-tetrazole (0.18 g, 1.383 mmol), and the mixture was stirred at room temperature for 20 minutes.

[2547] Subsequently, 0.75 mL (0.71 g, 5.48 mmol) of 70% tert-butyl hydrogen peroxide aqueous solution was added at room temperature, and the mixture was stirred for 0.5 hours at room temperature. Next, 1.09 mL (1.11 g, 7.30 mmol) of diazabicycloundecene was added and stirred for 10 minutes. Then, 2 mL of 50% acetonitrile aqueous solution was added, followed by 1.2 mL of 6 equivalents of hydrochloric acid.

[2548] The obtained residue was subjected to preparative HPLC chromatography under the following conditions to concentrate the component containing the target compound under reduced pressure, thereby obtaining a colorless solid labeled compound (0.12 g, yield 37.47%).

[2549] MS(ESI)m / z 358(M+H) +

[2550] Column: Waters XSelect HSS T3 OBD 5um (19*150mm)

[2551] Flow rate: 17 mL / min.

[2552] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2553] Gradient (solution B): 10% (0.00 min.) → 50% (8.00 min.)

[2554] Example 17-3

[2555]

[2556] Instead of Examples 3-4, Example 17-1 (10.4 mg, 0.032 mmol) was used, and instead of tert-butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate, Example 17-2 (15.6 mg, 0.044 mmol) was used, and the reaction was carried out in the same manner as in Examples 3-5 to obtain the labeled compound as a colorless oil (10.3 mg, yield 48.08%).

[2557] MS(ESI)m / z 660(M+H) +

[2558] Example 17-4

[2559]

[2560] Instead of Examples 3-5, Examples 17-3 (10.3 mg, 0.016 mmol) were used, and the reaction was carried out in the same manner as in Examples 3-6 to obtain the labeled compound as a colorless oil (8.0 mg, yield 84.89%).

[2561] MS(ESI)m / z 604(M+H) +

[2562] Example 17

[2563]

[2564] Instead of Examples 9-2, Examples 14-1 (4.4 mg, 2.83 μmol) were used, and instead of Examples 3-6, Examples 17-4 (8.5 mg, 0.014 mmol) were used, and the reaction was carried out in the same manner as in Example 9 to obtain a labeled compound as a colorless solid (4.05 mg, yield 74.63%).

[2565] MS(ESI) m / z 960(M+2H) 2+

[2566] Synthesis of Example 18

[2567]

[2568] Example 18-1

[2569]

[2570] Using Example 14-1 (9.5 mg, 6.11 μmol), and instead of Example 16-1, N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6,N6-dimethyl-L-lysine hydrochloride (5.3 mg, 0.012 mmol), the reaction was carried out in the same manner as in Example 16-2 to give a labeled compound as a colorless solid (7.5 mg, yield 71.74%).

[2571] MS(ESI)m / z 857(M+2H) 2+

[2572] Example 18

[2573]

[2574] Using Examples 3-6 (5.0 mg, 8.81 μmol) and Example 18-1 (7.5 mg, 4.38 μmol) instead of Examples 9-2, the reaction was carried out in the same manner as in Example 9 to obtain a labeled compound as a colorless solid (3.8 mg, yield 42.54%).

[2575] MS(ESI) m / z 1021(M+2H) 2+

[2576] Synthesis of Example 19

[2577]

[2578] Example 19-1

[2579]

[2580] In a 500 mL flask, 10.78 g (36.0 mmol) of 2-((tert-butyldiphenylsilyl)oxy)ethane-1-amine was added to a 100 mL dichloromethane solution of 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecane-12-acid (11.28 g, 37.9 mmol) under water cooling and argon atmosphere. Then, 7.28 g (38.0 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole (1.11 g, 7.25 mmol) were added sequentially at room temperature, and the mixture was stirred at room temperature for 2 hours.

[2581] Add 75 mL of water and 75 mL of saturated saline solution to the reaction solution, separate the layers, extract the aqueous layer twice with 30 mL of dichloromethane, combine the organic phases, dry with 10 g of anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 23.19 g of orange slurry residue.

[2582] Add 100 mL of hexane / ethyl acetate (57 / 43 (V / V)) to the residue and dissolve it uniformly. Then, prepare the sample under medium pressure (silica, 5 L (3000 g), hexane / ethyl acetate = 40 / 60 (V / V) (Rf = 0.40 (hexane / ethyl acetate = 40 / 60 (V / V))). Concentrate the component containing the target compound under reduced pressure to obtain the labeled compound (20.04 g, yield 96.19%), which is a colorless oil.

[2583] MS(ESI)m / z 579(M+H) +

[2584] Example 19-2

[2585]

[2586] In a 150 mL ethanol solution of Example 19-1 (21.18 g, 36.6 mmol) added to a 500 mL flask, a Pearlman catalyst (20% Pd, 50% water content, manufactured by Tokyo Chemical Industry Co., Ltd.) (1.28 g, 0.911 mmol) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 5 hours under a hydrogen atmosphere.

[2587] After the reaction solution was placed under a nitrogen atmosphere, it was filtered using Celite 545 (trade name), washed with ethanol, and the filtrate / washing liquid was concentrated under reduced pressure to obtain the labeled compound (16.15 g, yield 99.25%) as a colorless oil.

[2588] MS(ESI)m / z 445(M+H) +

[2589] Example 19-3

[2590]

[2591] In a 100 mL flask containing 20 mL of dichloromethane solution from Examples 1-2 (2.27 g, 5.11 mmol), Example 19-2 (2.31 g, 5.27 mmol) was added under an argon atmosphere. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.08 g, 5.63 mmol) and 1-hydroxybenzotriazole (0.1595 g, 1.042 mmol) were added sequentially at room temperature, and the mixture was stirred at room temperature for 2 hours.

[2592] Add 10 mL of water and 10 mL of saturated saline solution to the reaction solution, separate the layers, extract the aqueous layer twice with 10 mL of dichloromethane, combine the organic phases, dry with anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a colorless solid residue of 6.09 g.

[2593] Add 50 mL of diethyl ether to the residue and sonicate. Add 50 mL of diethyl ether to the resulting solid and sonicate again. Filter the solid, wash it with diethyl ether, and dry it under reduced pressure to obtain the labeled compound as a white solid (4.26 g, yield 96.46%).

[2594] MS(ESI)m / z 865(M+H) +

[2595] Example 19-4

[2596]

[2597] Add 1.46 mL of piperidine (1.26 g, 14.78 mmol) to a 20 mL solution of N,N-dimethylformamide (4.26 g, 4.92 mmol) in Example 19-3 to a 100 mL flask, while stirring at room temperature under an argon atmosphere, and stir at room temperature for 0.5 hours.

[2598] The reaction solution was concentrated under reduced pressure to obtain a white solid residue of 7.72 g. 50 mL of diethyl ether was added to the residue, followed by sonication and standing at -20°C for 12 hours. The supernatant was then removed. Next, 25 mL of diethyl ether was added to the residue, followed by sonication and standing at -20°C. The supernatant was then removed. This process was repeated, followed by drying under reduced pressure to obtain the labeled compound (3.05 g, yield 96.35%), which was a pale yellow oil.

[2599] MS(ESI)m / z 643(M+H) +

[2600] Example 19-5

[2601]

[2602] In a 100 mL flask containing 20 mL of N,N-dimethylformamide solution (3.05 g, 4.74 mmol) from Example 19-4, potassium carbonate (1.0075 g, 7.29 mmol) and triphenylchloromethane (1.70 g, 6.10 mmol) were added sequentially at room temperature while stirring under an argon atmosphere. The mixture was then stirred at room temperature for 2 hours.

[2603] After the reaction was completed, the reaction solution was concentrated, tetrahydrofuran was added to the concentrated residue, and the mixture was subjected to ultrasonic treatment. The insoluble matter was filtered out, and the filter was washed with tetrahydrofuran. The resulting filtrate and washings were combined and concentrated under reduced pressure to obtain 5.61 g of a concentrated residue that was a pale yellow oil.

[2604] The residue was dissolved in dichloromethane, and 27.0 g of Fuji SILYSIA Chromatrex Q-Pack Diol 60 Size 60 was added. The mixture was then dried under reduced pressure. The solution was prepared under medium pressure in a Yamazaki process (Fuji SILYSIA Chromatrex Q-Pack Diol 60 (85.0 g), hexane / ethyl acetate = 7 / 93 (V / V) → 0 / 100 (V / V), (Rf = 0.50 (ethyl acetate)). The component containing the target compound was concentrated under reduced pressure to obtain a colorless oily labeled compound (2.82 g, yield 67.15%).

[2605] MS(ESI)m / z 885(M+H) +

[2606] Example 19-6

[2607]

[2608] Add 50 mL of tetrahydrofuran solution (2.82 g, 3.19 mmol) from Example 19-5 to a 300 mL flask, and while stirring under an argon atmosphere, add 3.5 mL (0.96 g, 3.50 mmol) of 1 M tetrabutylammonium fluoride tetrahydrofuran solution at room temperature, and stir for 2 hours at room temperature.

[2609] The reaction solution was concentrated under reduced pressure. 50 mL of dichloromethane was added to the 4.42 g residue for dissolution, followed by washing with 50 mL of saturated ammonium chloride aqueous solution. The aqueous layer was extracted twice with 25 mL of dichloromethane, the organic layers were combined, dried with 10 g of anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 4.28 g of a colorless oily residue.

[2610] Dissolve the residue in 45 mL of acetonitrile and prepare it under medium pressure in solution A (silica, L (40 g), A / B = 100 / 0 (V / V) → 50 / 50 (V / V), followed by ethyl acetate / methanol = 100 / 0 (V / V) → 40 / 60 (V / V) (Rf = 0.47 (ethyl acetate)); acetonitrile / water / triethylamine = 950 / 50 / 1 (V / V / V) in solution B; acetonitrile / water / triethylamine = 850 / 150 / 5 (V / V / V). Concentrate the component containing the target compound under reduced pressure to obtain 1.76 g of crude product of the labeled compound as a white bubbly substance.

[2611] The preparation was then carried out under the conditions shown below, and the component containing the target compound was concentrated under reduced pressure to obtain a white bubbly labeled compound (1.58 g, yield 71.62%).

[2612] MS(ESI)m / z 647(M+H) +

[2613] • Device: LC Forte / R

[2614] • Column: GPC system

[2615] • Eluent: dichloromethane → acetonitrile

[2616] • Flow rate: 15 mL / min.

[2617] • Pressure: 12 MPa (dichloromethane), 8.4 MPa (acetonitrile)

[2618] Wavelength: 220nm

[2619] Example 19-7

[2620]

[2621] In a 200 mL flask containing a solution of 30 mL dichloromethane (1.58 g, 2.282 mmol) from Examples 19-6, diallyl N,N-diisopropylphosphonamide (2.58 mL, 2.39 g, 9.76 mmol) and 1H-tetrazole (1.20 g, 17.13 mmol) were added sequentially to the solution under an argon atmosphere and stirred at room temperature for 2 hours.

[2622] Subsequently, the mixture was ice-cooled, and 0.650 mL (0.72 g, 6.36 mmol) of 30% hydrogen peroxide water was added. The mixture was then stirred for 1 hour while still ice-cooled.

[2623] Subsequently, 30 mL of a solution prepared by adding 100 mL of water to 20.0 g (80.6 mmol) of sodium thiosulfate decahydrate and 7.0 g (83.3 mmol) of sodium bicarbonate was added, and the mixture was separated. The aqueous layer was extracted twice with 15 mL of dichloromethane, and the two layers were combined. The solution was dried with 10.0 g of anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3.49 g of concentrated residue as a pale yellow oil.

[2624] Dissolve the compound in 30 mL of dichloromethane, then add 22.0 g of Fuji SILYSIA Chromatrex Q-PackDiol60 Size 60 and dry under reduced pressure. Prepare the compound under medium pressure (Fuji SILYSIA Chromatrex Q-PackDiol60 (88.0 g), hexane / ethyl acetate = 0 / 100 (V / V), (Rf = 0.33 (ethyl acetate)). Concentrate the component containing the target compound under reduced pressure to obtain the labeled compound as a white bubbly substance (1.55 g, yield 84.19%).

[2625] MS(ESI)m / z 807(M+H) +

[2626] Examples 19-8

[2627]

[2628] In a 33 mL solution of tetrahydrofuran (1.55 g, 1.921 mmol) from Examples 19-7, added to a 300 mL flask, 0.440 mL (0.44 g, 4.07 mmol) of N-methylaniline and 0.2263 g (0.196 mmol) of tetra(triphenylphosphine)palladium were added sequentially at room temperature while stirring under an argon atmosphere. The mixture was stirred at room temperature for 1 hour.

[2629] After the reaction solution was concentrated under reduced pressure, 50 mL of diethyl ether was added to 2.38 g of the concentrated residue of the pale yellow bubbly substance. The mixture was subjected to ultrasonic treatment, and the resulting solid was filtered off, washed with diethyl ether, and dried under reduced pressure. Thus, the N-allyl-N-methylaniline salt of the labeled compound (1.89 g, yield 96.34%), which was a pale yellow solid, was obtained.

[2630] MS(ESI)m / z 727(M+H) +

[2631] Examples 19-9

[2632]

[2633] In a 50 mL flask containing 2.5 mL of N,N-dimethylformamide solution (0.3958 g, 0.388 mmol) from Examples 19-8, carbonyl diimidazole (0.1575 g, 0.971 mmol) was added while stirring at room temperature under an argon atmosphere, and the mixture was stirred at room temperature for 0.75 hours.

[2634] Subsequently, U-031 (0.3096 g, 0.381 mmol) was added, followed by ice cooling, and then zinc chloride (0.4098 g, 3.01 mmol) was added. The mixture was then brought back to room temperature and stirred at room temperature for 6 hours.

[2635] Add 25 mL of diethyl ether to the reaction solution, sonicate, and remove the supernatant. Add 10 mL of diethyl ether to the residue and remove the supernatant. Repeat this operation again, and dry the residue under reduced pressure to obtain 1.64 g of a pale yellow slurry.

[2636] 20 mL of acetonitrile / water / triethylamine = 850 / 150 / 5 (V / V / V) was added to dissolve the compound and filled into a Yamazaki injection column (silica gel) of size L for Yamazaki medium-pressure preparation (silica, L (40 g), acetonitrile / water / triethylamine = 850 / 150 / 5 (V / V / V)). The component containing the target compound was concentrated under reduced pressure, and 30 mL of acetonitrile / water (1 / 1 (V / V)) was added to the residue to dissolve it. The residue was then freeze-dried to obtain a white solid labeled compound (517 mg, yield 83.6%).

[2637] MS(ESI)m / z 761(M+2H) 2+

[2638] Examples 19-10

[2639]

[2640] In a 50 mL flask containing 3 mL of N,N-dimethylformamide solution (512.8 mg, 0.316 mmol) from Examples 19-9, 3 mL of formic acid (3660 mg, 80 mmol) was added while stirring under an argon atmosphere and ice cooling, and then stirred at room temperature for 45 minutes.

[2641] Subsequently, 30 mL of diethyl ether was added, and the mixture was ultrasonically treated and placed at -20°C for 3 hours. After removing the supernatant, 15 mL of diethyl ether was added to the residue and stirred for 5 minutes, then the supernatant was removed. Another 15 mL of diethyl ether was added to the residue and stirred for 5 minutes, then the supernatant was removed. The resulting residue was dried under reduced pressure, yielding 0.7368 g of a colorless slurry.

[2642] After dissolving the acetonitrile / water / triethylamine in 15 mL (850 / 150 / 5 (V / V / V)), the solution was packed into a Yamazaki injection column (silica gel) of size M for Yamazaki medium-pressure preparation (silica, M (16 g), acetonitrile / water / triethylamine = 850 / 150 / 5 (V / V / V)). The component containing the target compound was concentrated under reduced pressure, and 20 mL of acetonitrile / water (1 / 1 (V / V)) was added to the residue to dissolve it. The residue was then freeze-dried to obtain a white solid labeled compound (328.1 mg, yield 75.22%).

[2643] MS(ESI)m / z 640(M+2H) 2+

[2644] Example 19

[2645]

[2646] Using 1-maleimide-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic acid N-succinimide ester (10.0 mg, 0.019 mmol), and Examples 19-10 (9.5 mg, 6.89 μmol), which replaced Examples 1-7, the reaction was carried out in the same manner as in Example 1 to give a labeled compound as a white solid (2.5 mg, yield 21.65%).

[2647] MS(ESI) m / z 839(M+2H) 2+

[2648] Synthesis of Example 20

[2649]

[2650] Example 20-1

[2651]

[2652] In a 10 mL flask containing 2 mL of N,N-dimethylformamide in benzyl 2-(3-hydroxyphenyl)acetate (72.2 mg, 0.298 mmol), 95 μL of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (90.25 mg, 0.299 mmol) and 1H-tetrazole (21.7 mg, 0.310 mmol) were added sequentially, and the mixture was stirred at room temperature for 1 hour. Then, 80.0 mg (0.100 mmol) of the mixture from Example 2-1 was added, followed by 5-(ethylthio)-1H-tetrazole (26.7 mg, 0.205 mmol), and the mixture was stirred at room temperature for 0.5 hours.

[2653] Subsequently, 41 μL (38.54 mg, 0.299 mmol) of 70% tert-butyl hydrogen peroxide aqueous solution was added at room temperature, and the mixture was stirred for 0.5 hours at room temperature. Then, 149 μL (151.98 mg, 0.998 mmol) of diazabicycloundecene was added, and the mixture was stirred for 20 minutes at room temperature. Finally, 38 μL (45.6 mg, 0.991 mmol) of formic acid was added, and the mixture was left to stand overnight.

[2654] After adding 50% acetonitrile aqueous solution to the obtained reaction solution, the mixture was filtered. The filtrate was subjected to preparative HPLC chromatography under the following conditions. The fraction containing the target compound was concentrated under reduced pressure to remove the acetonitrile, and then freeze-dried to obtain the labeled compound as a colorless bubbly substance (16.9 mg, yield 19.16%).

[2655] MS(ESI)m / z 882(M+H) +

[2656] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2657] Flow rate: 17 mL / min.

[2658] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2659] Gradient (solution B): 25% (0.00 min.) → 46% (6.00 min.)

[2660] Example 20-2

[2661]

[2662] To a 10 mL flask containing 200 μL of N,N-dimethylformamide solution (11.7 mg, 0.013 mmol) from Example 20-1, 33 μL of a 2-equivalent sodium hydroxide aqueous solution (2.64 mg, 0.066 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Subsequently, 33 μL of a 2-equivalent hydrochloric acid was added.

[2663] Next, 4 μL of N,N-dimethylbenzylamine (3.64 mg, 0.027 mmol) and 9-fluorenylmethyl-succinimide carbonate (9.0 mg, 0.027 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Then, 4 μL of N,N-dimethylbenzylamine (3.64 mg, 0.027 mmol) was added again, and the mixture was stirred for 16 hours.

[2664] N,N-dimethylformamide and 50% acetonitrile aqueous solution were added to the obtained reaction solution and filtered. The filtrate was subjected to preparative HPLC chromatography under the following conditions. The component containing the target compound was concentrated under reduced pressure and then freeze-dried to obtain the labeled compound as a colorless bubbly substance (9.4 mg, yield 68.65%).

[2665] MS(ESI)m / z 1033(M+H) +

[2666] Column: Waters XBridge Prep C18 5μm ODB 19*150mm

[2667] Flow rate: 17 mL / min.

[2668] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2669] Gradient (solution B): 25% (0.00 min.) → 60% (8.00 min.)

[2670] Example 20-3

[2671]

[2672] In a 10 mL cylindrical flask, 500 μL of N,N-dimethylformamide solution containing eczemab (5.5 mg, 10.35 μmol), Example 20-2 (9.4 mg, 9.11 μmol), and 5.71 μL (5.2 mg, 0.038 mmol) of N,N-dimethylbenzylamine was added. HATU (19.0 mg, 0.050 μmol) was added while stirring at room temperature under an argon atmosphere, and the mixture was stirred at room temperature for 2 hours.

[2673] After the reaction was completed, the reaction solution was subjected to preparative HPLC chromatography under the following conditions, and the component containing the target compound was freeze-dried to obtain a colorless solid labeled compound (6.8 mg, yield 52.15%).

[2674] MS(ESI)m / z 1432(M+H) +

[2675] Column: Waters SunFire Prep C18 5μm ODB 19*150mm

[2676] Flow rate: 17 mL / min.

[2677] Elution solvent: 0.1% formic acid aqueous solution (solution A) - acetonitrile (solution B)

[2678] Gradient (solution B): 25% (0.00 min.) → 65% (8.00 min.)

[2679] Example 20

[2680]

[2681] Using Examples 3-6 (10.8 mg, 0.019 mmol / L) and Examples 20-3 (6.8 mg, 4.75 μmol / L) instead of Examples 9-2, the reaction was carried out in the same manner as in Example 9 to obtain a labeled compound as a colorless solid (5.16 mg, yield 61.76%).

[2682] MS(ESI)m / z 880(M+2H) 2+

[2683] Synthesis of Example 21

[2684]

[2685] Instead of Examples 9-2, Example 18-1 (8.0 mg, 4.67 μmol) was used, and instead of Examples 3-6, Example 17-4 (15.2 mg, 0.025 mmol) was used, reacting in the same manner as in Example 9 to obtain a colorless, amorphous labeled compound (4.3 mg, yield 44.34%).

[2686] MS(ESI) m / z 1036(M-2H) 2-

[2687] Synthesis of Example 22

[2688]

[2689] Instead of Examples 9-2, Examples 16-2 (6.8 mg, 4.07 μmol) were used, and instead of Examples 3-6, Examples 17-4 (6.2 mg, 10.27 μmol) were used, and the reaction was carried out in the same manner as in Example 9 to obtain a labeled compound as a colorless solid (2.1 mg, yield 25.37%).

[2690] MS(ESI) m / z 1016(M+2H) 2+

[2691] Synthesis of Example 23

[2692]

[2693] Example 23-1

[2694]

[2695] In a 500 mL flask, a solution of (L)-Val-(L)-Cit-PAB (10.0 g, 26.4 mmol) in 100 mL of tetrahydrofuran was added. 1H-imidazole (3.59 g, 52.7 mmol) was added while stirring under an argon atmosphere. The flask was then ice-cooled, and 7.45 mL of tert-butyldiphenylchlorosilane (7.97 g, 29.0 mmol) was added. The mixture was stirred at room temperature for 1.5 hours.

[2696] Next, 0.5 mL (0.54 g, 1.946 mmol) of tert-butyldiphenylchlorosilane was added under stirring and ice cooling, and the mixture was stirred at room temperature for 1 hour.

[2697] After the reaction was complete, 300 mL of ethyl acetate was added to the reaction solution, and the insoluble matter was filtered off. The solution was then washed with 15 mL of ethyl acetate. 100 mL of saturated sodium bicarbonate aqueous solution and 100 mL of saturated saline solution were added to the filtrate. The mixture was stirred at room temperature for 15 minutes, and then separated. The aqueous layer was extracted twice with 20 mL of ethyl acetate. The organic layers were combined, dried over 5 g of anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.

[2698] The obtained solid was added to 150 mL of tetrahydrofuran, followed by 150 mL of saturated ammonium chloride aqueous solution and 20 mL of water. The aqueous layer was extracted twice with 20 mL of ethyl acetate, washed with 50 mL of saturated brine, and the organic layers were combined. After drying with 5 g of sodium sulfate, the mixture was concentrated under reduced pressure.

[2699] In a 200 mL solution of the obtained residue in dichloromethane, 36.7 mL of triethylamine (26.64 g, 263 mmol) and 36.7 g of triphenylchloromethane (132 mmol) were added under ice-cooling, and the mixture was stirred at room temperature for 1 hour. Then, 36.7 g of triphenylchloromethane (132 mmol) was added under stirring at room temperature, and the mixture was stirred at room temperature for another 1 hour.

[2700] Next, 36.7 mL of triethylamine (26.64 g, 263 mmol) and 36.7 g of triphenylchloromethane (132 mmol) were added at room temperature with stirring, and the mixture was stirred at room temperature for 1 hour.

[2701] Next, triphenylchloromethane (15.0 g, 53.8 mmol) was added at room temperature with stirring, and the mixture was stirred for 30 minutes at room temperature.

[2702] After the reaction was complete, 40 mL of water was added for separation. The aqueous layer was extracted twice with 20 mL of dichloromethane. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The precipitated solid was filtered off to obtain a solution containing the target analyte.

[2703] The above solution was concentrated under reduced pressure, and the concentration was stopped when a white solid precipitated. The mixture was stirred for 30 minutes under ice cooling. The solid was filtered, washed with 50 mL of a mixed solvent of hexane / ethyl acetate (70 / 30, V / V), and dried under reduced pressure to obtain the labeled compound as a white solid (30.2459 g, yield quantification).

[2704] MS(ESI)m / z 1103(M+H) +

[2705] Example 23-2

[2706]

[2707] In a 270 mL solution of tetrahydrofuran (30.0 g, 27.2 mmol) from Example 23-1, which was added to a 500 mL flask, 77 mL of tetra-n-butylammonium fluoride (1 equivalent) was added at room temperature, and the mixture was stirred at room temperature for 5 hours.

[2708] After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in 30 mL of dichloromethane. The solution was then used for medium-pressure preparation of silica (3 L (135 g), dichloromethane / ethyl acetate = 92 / 8 (V / V) → 44 / 56 (V / V). The fraction containing the target compound (Rf = 0.45 (dichloromethane / ethyl acetate = 50 / 50 (V / V))) was collected. The precipitated solid was filtered, washed with dichloromethane, and dried under reduced pressure to obtain the labeled compound as a white solid (16.2604 g, yield 69.16%).

[2709] MS(ESI)m / z 864(M+H) +

[2710] Example 23-3

[2711]

[2712] In a 50 mL flask containing 15 mL of dichloromethane solution (434 mg, 0.502 mmol) from Example 23-2, 190 μL (171.57 mg, 0.595 mmol) and 1H-tetrazole (44 mg, 0.628 mmol) were added sequentially at room temperature while stirring under an argon atmosphere. The mixture was stirred at room temperature for 1 hour.

[2713] Next, U-032 (99 mg, 0.193 mmol) and 5-(ethylthio)-1H-tetrazole (80 mg, 0.615 mmol) were added, and the mixture was heated and stirred at 43°C for 3 hours.

[2714] Next, ice cooling was performed, and 145 μL (136.3 mg, 1.059 mmol) of 70% tert-butyl hydrogen peroxide aqueous solution was added under ice cooling, and the mixture was stirred for 1 hour under ice cooling.

[2715] After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted with 15 mL of dichloromethane. The organic layer was washed with water, dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow oil.

[2716] To a 15 mL solution of the above-mentioned pale yellow oily substance in tetrahydrofuran, which was added to a 100 mL flask, 57 μL of N-methylaniline (56.43 mg, 0.527 mmol) and 47 mg of tetra(triphenylphosphine)palladium (0.041 mmol) were added sequentially at room temperature while stirring under an argon atmosphere. After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure.

[2717] tert-butyl methyl ether was added to the residue, and the mixture was ultrasonically treated to remove the supernatant. Next, water was added to the residue, and the mixture was ultrasonically treated to remove the supernatant. The residue was then dried under reduced pressure to obtain 770 mg of residue.

[2718] In a 10 mL dichloromethane solution of the above residue in a 100 mL pear-shaped flask, 166 μL of trifluoroacetic acid (245.68 mg, 2.155 mmol) was added at room temperature, and the mixture was stirred for 3 hours at room temperature. Then, 83 μL of trifluoroacetic acid (122.84 mg, 1.077 mmol) was added at room temperature, and the mixture was stirred for 1 hour at room temperature.

[2719] After the reaction was completed, 20 mL of tert-butyl methyl ether was added, the supernatant was removed, and the residue was dissolved in 30% acetonitrile aqueous solution. The residue was then subjected to preparative HPLC chromatography unde...

Claims

1. An antibody-drug conjugate precursor or a salt thereof represented by the following general formula (I): Z—LD (I) In the formula, Z is a reactive group capable of reacting with a functional group present in an antibody or a modified antibody, D is an antitumor drug residue obtained by removing one hydrogen atom or one hydroxyl group from any position of an antitumor drug molecule or its analog or derivative, L is a linker connecting Z and D, At least one of D and L has one or more lactone groups as substituents or protecting groups at any positions.

2. The antibody-drug conjugate precursor or a salt thereof according to claim 1, wherein: Z is a maleimide group (the following formula (i)), an α-halomethylcarbonyl group (the following formula (ii)), an ethynylphosphonic acid amide ester group (the following formula (iii)), a carboxyl group, an active ester of a carboxyl group, a mercapto group, a hydroxyl group, an amino group, an alkynyl group, a cycloalkynyl group, an azacycloalkynyl group, or an azido group (-N3 group), Formula (i) Formula (ii) Hal-CH2-C(=O)* Formula (iii) In the above formula, * is the bonding point with L, Hal is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and R 16 It is a methyl group, an ethyl group, or a -CH2CH2OCH2CH2OH group.

3. The antibody-drug conjugate precursor or a salt thereof according to claim 1 or 2, wherein: Z is a maleimide group (the formula (i)), an α-halomethylcarbonyl group (the formula (ii)), an ethynylphosphonic acid amide ester group (the formula (iii)), a carboxyl group, an active ester of a carboxyl group, an alkynyl group, a cycloalkynyl group, an azacycloalkynyl group, or an azide group (-N3 group).

4. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 3, wherein Z is a maleimide group (the above formula (i)), an α-halogenated methylcarbonyl group (the above formula (ii), a carboxyl group, or an active ester of a carboxyl group.

5. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 4, wherein D is an antitumor drug residue formed by removing one hydrogen atom or one hydroxyl group from any position of: camptothecin; auristatin E (AE) or monomethyl auristatin E (MMAE); maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; calicheamicin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytidine; vincristine; vinblastine; daunomycin; doxorubicin; dorystia 10; superdox; ciprofloxacin; caderfloxacin (CS-940); or analogs of these antitumor drugs; or the antitumor drug molecules or their analogs and derivatives; 6. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 5, wherein D is an antitumor drug residue formed by removing one hydrogen atom or one hydroxyl group from any position of: camptothecin; auristatin E (AE) or monomethyl auristatin E (MMAE); maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; 5-FU; PD-318088; AS-703026; TAK-733; LY-3023414; calicheamicin; paclitaxel; docetaxel; mitomycin C; bleomycin; cyclocytidine; vincristine; vinblastine; daunomycin; doxorubicin; doxorubicin; caudastatin 10; superdox; or analogs of these antitumor drugs; or the antitumor drug molecules or their analogs and derivatives; 7. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 6, wherein D is an antitumor drug residue formed by removing one hydrogen atom or one hydroxyl group from any position of: camptothecin; auristatin E (AE) or monomethyl auristatin E (MMAE); maytansine; PBD (pyrrolobenzodiazepine) dimer; eribulin; or analogs of these antitumor drugs; or the antitumor drug molecules or their analogs and derivatives.

8. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 7, wherein D is an antitumor drug residue formed by removing one hydrogen atom or one hydroxyl group from any position of: an antitumor drug or an analogue of these antitumor drugs in which at least one hydroxyl group present in the molecule is phosphated; or the antitumor drug molecule or their analogues and derivatives; 9. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 8, wherein L is an optionally substituted alkylene group; One or more methylene groups in the chain of the alkylene group may be independently selected from -C(R 1 )(R 2 )-;-O-;-N(R 3 )-;-N(R 3 )-N(R 3 )-;-S-;-Se-;-Si(R 4 )(R 5 )-;-S-S-;-Se-Se-;-SOm-;-SeOn-;-C(=C(R 6 )(R 7 ))-; -C(=O)-;-C(=S)-;-C(=N(R 8 ))-;-C(=N-OR 9 )-;-C(=NN(R 10 )(R 11 ))-;-P(=O)(R 12 )-;-P(=O)(OR 13 )-;-OP(=O)(R 12 )-O-;-OP(=O)(OR 13 )-O-;-C(R 14 )=;=C(R 14 )-;-C(R 14 )=C(R 14 )-;-N=;=N-;-C≡C-;-(OC(R 1 )(R 2 )-C(R 1 )(R 2 )) 1-30 -;-(C(R 1 )(R 2 )-C(R 1 )(R 2 )-0) 1-30 -; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted cycloalkylene; optionally substituted cycloalkenylene; optionally substituted cycloalkynylene; optionally substituted azacycloalkynylene; optionally substituted arylene; optionally substituted heteroarylene; and optionally substituted heterocyclylene are replaced by one or more divalent groups in the group consisting of, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 are each independently a group selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted cycloalkynyl group, an optionally substituted azacycloalkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, and an optionally substituted heterocyclic group, and R 3 When it is an alkyl group, the alkyl group may form a ring structure together with the alkyl group in the adjacent methylene group. m and n are each independently an integer of 0-2.

10. The antibody-drug conjugate precursor or a salt thereof according to claim 9, wherein: L is an optionally substituted alkylene group; One or more methylene groups in the chain of the alkylene group are replaced with one or more divalent groups independently selected from the following chemical formula group: Chemical formula group: -OC(H or C1-C4 alkyl)(H or C1-C4 alkyl)-; -SC(H or C1-C4 alkyl)(H or C1-C4 alkyl)-; -N(H or C1-C4 alkyl)-; -N(H or C1-C4 alkyl)-OC(H or C1-C4 alkyl)(C(H or C1-C4 alkyl)-; -C(H or C1-C4 alkyl)-SO 0-2 -C(H or C1-C4 alkyl)-; -C(H or C1-C4 alkyl)-SSC(H or C1-C4 alkyl)-; -C(H or C1-C4 alkyl)=C(H or C1-C4 alkyl)-; -C(H or C1-C4 alkyl)=N-; -C(H or C1-C4 alkyl)=NO-; -C(H or C1-C4 alkyl)=NN(H or C1-C4 alkyl)-; -C(=C(H or C1-C4 alkyl)(H or C1-C4 alkyl))-; -C(=NO(H or C1-C4 alkyl))-; -C(=NN(H or C1-C4 alkyl)(H or C1-C4 alkyl))-; -O-Si(C1-C4 alkylCH3)(C1-C4 alkyl)-O-; -C(H or C1-C4 alkyl)-N(C1-C4 alkyl)-CH2-; -C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-C(=O)-C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-; -C(=O)-O-; -C(=O)-S-; -C(=O)-N(H or C1-C4 alkyl)-; -OC(=O)-O-; -N(H or C1-C4 alkyl)-C(=O)-N(H or C1-C4 alkyl)-; -C(=O)-N(H or C1-C4 alkyl)-C(H or C1-C4 alkyl)(H or C1-C4 alkyl)-; -C(=O)-(CH2) 1-10 -(O-CH2CH2) 1-20 -O-(CH2) 1-10 -C(=O)-; -C(=O)-(CH2) 1-10 -(O-CH2CH2) 1-20 -O-(CH2) 1-10 -CH2-NH-; -NH-CH2-(CH2) 1-10 (O-CH2CH2) 1-20 -O-(CH2) 1-10 -CH2-NH-; -C(=O)-N(C1-C4 alkyl)-CH2CH2-N(C1-C4 alkyl)-C(=O)-; In the formula, * is the bonding point with the adjacent group; -P(=O)(OH)-O-; -OP(=O)(OH)-O-; -OP(=S)(OH)-O-; -OP(=O)(OH)-S-; -OP(=O)(OH)-OP(=O)(OH)-O-; -OP(=O)(OH)-OP(=O)(OH)-OP(=O)(OH)-O-; -CH(CH2-NH2)-; -CH(CH2-NH(C1-C4 alkyl))-; -CH(CH2-N(C1-C4alkyl)2)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 0-20 -C1-C4 alkyl)-; -CH(CH2-NH-C(=O)-(CH2) 0-20 -lactone)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-lactone)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-NH-lactone)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-NH-C(=O)-lactone)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-lactone)-; -CH(CH2-NH-(CH2) 1-20 -(O) 0-1 -(P(=O)(OH)2)-; -CH(CH2-NH-C(=O)-(CH2) 1-20 -(P(=O)(OH)2)-; -CH(CH2-NH-C(=O)-(CH2) 1-20 -C(=O)-NH-(CH2) 1-10 -(P(=O)(OH)2)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -CH2-(P(=O)(OH)2)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -(P(=O)(OH)2)-; -CH(CH2-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-; -CH(CH2-NH-C(=O)-(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-; -CH(CH2-NH-C(=O)-(CH2CH2-O-) 1-20 -(CH2) 1-10 -C(=O)-NH-(CH2) 1-10 -C(H, OH, Cl, NH2, or C1-C4 alkyl)(P(=O)(OH)2)2)-; -(cis)-CH=CH-P(=O)(O-CH2CH3)-NH-(phenylene)-C(=O)-; -C(=O)-(cyclohexylene)-; -(succinimidyl)-; -Ser-; -Cys-; -Ama-: -Asp-; -Glu-; -Orn-; -Lys-; -ValLys-; -ValCit-; -ValAla-; -GlyGly-C(=O)-NH-(optionally substituted phenylene)-CH2-; -PheLys-C(=O)-NH-(optionally substituted phenylene)-CH2-; -ValLys-C(=O)-NH-(optionally substituted phenylene)-CH2-; -ValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-; -ValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-; -AspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-; -LysAspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-; -GluValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-; -LysValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-; -SerValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-; -AspValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-; -GluValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-; -LysValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-; -SerValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-; -GlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-; -AspGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-; -GluGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-; -LysGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-; -SerGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-; -AspAspAspAspAsp-C(=O)-NH-(optionally substituted phenylene)-CH2-; -GlyGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -PheLys-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -ValLys-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -ValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -ValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -AspValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -GluValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -LysValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -SerValCit-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -AspValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -GluValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -LysValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -SerValAla-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -GlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -AspGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -GluGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -LysGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -SerGlyGlyPheGly-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -AspAspAspAspAsp-C(=O)-NH-(optionally substituted phenylene)-CH2-OC(=O)-; -GlyGlyPheGly-C(=O)-NH-CH2-; -AspGlyGlyPheGly-C(=O)-NH-CH2-; and The 1,2,3-triazolylene group of the following formula, Among the amino acid residues, and the amino acid residues in the peptide, The carboxyl groups of the side chains of Ama, Asp, and Glu may form (alkyl)esters, (aminoalkyl)esters, ((N-alkylamino)alkyl)esters, ((N,N-dialkylamino)alkyl)esters, ((trialkylammonium)alkyl)esters, (lactonyl)esters, ((lactonyl)alkyl)esters, ((phosphoryl)alkyl)esters, or ((bisphosphoryl)alkyl)esters, or may form unsubstituted amides, alkylamides, dialkylamides, ((N-alkylamino)alkyl)amides, ((N,N-dialkylamino)alkyl)amides, ((trialkylammonium)alkyl)amides, (lactonyl)amides, ((lactonyl)alkyl)amides, (phosphoryl)amides, ((phosphoryl)alkyl)amides, or ((bisphosphoryl)alkyl)amides, The amino group of the side chain of Lys and Orn may have one or two alkyl groups, alkylcarbonyl groups, (aminoalkyl)carbonyl groups, ((N-alkylamino)alkyl)carbonyl groups, ((N,N-dialkylamino)alkyl)carbonyl groups, ((trialkylammonium)alkyl)carbonyl groups, lactone groups, (lactonyl)alkyl groups, (lactonyl)carbonyl groups, ((lactonyl)alkyl)carbonyl groups, phosphoryl groups, (phosphoryl)alkyl groups, ((phosphoryl)alkyl)carbonyl groups, (bisphosphoryl)alkyl groups, or ((bisphosphoryl)alkyl)carbonyl groups as substituents, The hydroxyl group of the side chain of Ser and the thiol group of the side chain of Cys may have an alkyl group, an alkylcarbonyl group, an aminoalkyl group, an (aminoalkyl)carbonyl group, an (N-alkylamino)alkyl group, a ((N-alkylamino)alkyl)carbonyl group, an (N,N-dialkylamino)alkyl group, a ((N,N-dialkylamino)alkyl)carbonyl group, a (trialkylammonium)alkyl group, a ((trialkylammonium)alkyl)carbonyl group, a lactone group, a (lacone group)alkyl group, a (lacone group)carbonyl group, a ((lacone group)alkyl)carbonyl group, a phosphoryl group, a (phosphoryl group)alkyl group, a ((phosphoryl group)alkyl)carbonyl group, a (bisphosphoryl)alkyl group, or a ((bisphosphoryl)alkyl)carbonyl group as a substituent, In the formula, * is the bonding point with the adjacent group.

11. The antibody-drug conjugate precursor or a salt thereof according to claim 9, wherein: L is any one divalent group selected from the following chemical formula group, Chemical formula group In the formula, * is the bonding point with the reactive group Z, and # is the bonding point with the anti-tumor drug residue D.

12. The antibody-drug conjugate precursor or a salt thereof according to any one of claims 1 to 11, wherein L has at any position one or more groups selected from lactone group, phosphoryl group (-P(=O)(OH)2); phosphorylalkylcarbonyl group (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl group (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino group; monoalkylamino group; dialkylamino group; a cyclic amino group in which the two alkyl groups of the dialkylamino group form a ring together with the nitrogen atom to which they are bonded; and trialkylammonium group as a substituent or protecting group, and when the total number of such groups is plural, they may be optionally the same or different.

13. An antibody-drug conjugate precursor or a salt thereof selected from the group consisting of the following chemical formula:

14. A synthetic intermediate (II) of a conjugate precursor (I) represented by the following general formula (II), or a salt thereof: Z 1 -L 1 -D 1 (II) In the formula, Z 1 A reactive group as defined in claim 1, or A connecting group for connecting the reactive group or the anti-tumor drug residue defined in claim 1 to a linker, wherein the connecting group is optionally protected by a protecting group, D 1 is an anti-tumor drug residue, or a conjugated group or a protected body thereof, Z 1 When D is a reactive group, 1 For the bonding base, L 1 To Z 1 and D 1 Connected linkers, D 1 When it is an antitumor drug residue, D 1 and L 1 At least one of the following has one or more lactone groups as substituents or protecting groups at any position, D 1 When it is a bonding base, L 1 At any position, it has one or more groups selected from lactone group; phosphoryl group (-P(=O)(OH)2); phosphorylalkylcarbonyl group (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl group (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino group; monoalkylamino group; dialkylamino group; a cyclic amino group in which the two alkyl groups of the dialkylamino group form a ring together with the nitrogen atom to which they are bonded; and trialkylammonium group, and when the total number of such groups is plural, they may be the same or different.

15. The synthetic intermediate (II) or a salt thereof according to claim 14, wherein The bonding group is a hydroxyl group, a nitro group, a cyano group, an amide group, an oxo group, an azido group, an amino group, a monoalkylamino group optionally having a substituent, an imino group (including -N=, the imino group of a cyclic amine), a carboxyl group, a phosphoryl group, an alkynyl group, a cycloalkynyl group, an azacycloalkynyl group, or a thiol group, or a protected body thereof.

16. The synthetic intermediate (II) or a salt thereof according to claim 14 or 15, wherein The bonding group is a hydroxyl group, a nitro group, a cyano group, an oxo group, an azido group, an amino group, a monoalkylamino group optionally having a substituent, an imino group (including -N=, the imino group of a cyclic amine), a carboxyl group, a phosphoryl group, an alkynyl group, a cycloalkynyl group, an azacycloalkynyl group, or a thiol group, or a protected form thereof.

17. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 16, wherein The bonding group is a hydroxyl group, a nitro group, an oxo group, an azido group, an amino group, a monoalkylamino group optionally having a substituent, an imino group (including -N=, the imino group of a cyclic amine), a carboxyl group, a phosphoryl group, an alkynyl group, a cycloalkynyl group, an azacycloalkynyl group, or a thiol group, or a protected form thereof.

18. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 17, wherein The bonding group is a hydroxyl group, a nitro group, an oxo group, an azido group, an amino group, a monoalkylamino group optionally having a substituent, an imino group (including -N=, the imino group of a cyclic amine), a carboxyl group, a phosphoryl group, an alkynyl group, a cycloalkynyl group, or an azacycloalkynyl group, or a protected form thereof.

19. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 18, wherein L 1 is an optionally substituted alkylene group, One or more methylene groups in the alkylene chain are replaced with one or more groups selected from the divalent group according to any one of claims 9 to 11.

20. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 19, wherein Z 1 is a group selected from the bonding groups according to any one of claims 15 to 18, and D1 is a group selected from the antitumor drug residue according to any one of claims 5 to 8.

21. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 19, wherein Z 1 and D 1 Each independently is a group selected from the bonding group according to any one of claims 15 to 18.

22. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 19, wherein Z 1 is a group selected from the reactive groups according to any one of claims 2 to 4, and D1 is a group selected from the bonding groups according to any one of claims 15 to 18.

23. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 20, wherein Z 1 is a group selected from the bonding groups according to any one of claims 15 to 18, D 1 is a group selected from the antitumor drug residues according to any one of claims 5 to 8, D 1 and L 1 At least one of the compounds has one or more lactone groups as substituents or protecting groups at any position, and, independently of this, D 1 and L 1 At least any one of them has at any position as a substituent or protecting group one or more groups selected from phosphoryl (-P(=O)(OH)2); phosphorylalkylcarbonyl (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino; monoalkylamino; dialkylamino; a cyclic amino group in which the two alkyl groups of the dialkylamino group form a ring together with the nitrogen atom to which they are bonded; and trialkylammonium groups, and when the total number of such groups is plural, they may be the same or different.

24. The synthetic intermediate (II) or a salt thereof according to any one of claims 14 to 19, 21 and 22, wherein Z 1 is a group selected from the reactive groups according to any one of claims 2 to 4, or a group selected from the bonding groups according to any one of claims 15 to 18, D 1 is a group selected from the bonding groups according to any one of claims 15 to 18, L 1 At any position, it has one or more groups selected from lactone group; phosphoryl group (-P(=O)(OH)2); phosphorylalkylcarbonyl group (-C(=O)-alkylene-P(=O)(OH)2); bisphosphorylmethyl group (-C(H, OH, NH2, Cl, or alkyl)(P(=O)(OH)2)2); amino group; monoalkylamino group; dialkylamino group; cyclic amino group in which the dialkyl groups together form a ring; and trialkylammonium group as substituents or protecting groups, and when the total number of such groups is plural, they may be optionally the same or different.

25. A synthetic intermediate (II) selected from the group consisting of the following chemical formula, or a salt thereof,

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