Antibody-drug conjugate including novel cyclic dinucleotide derivative

Abstract

To develop an antibody-drug conjugate capable of being systemically administered and delivering a STING agonist specifically to a target cell or organ (for example, a tumor lesion), and a therapeutic agent and / or therapeutic method using the antibody-drug conjugate for diseases related to STING agonist activity, for example, diseases (for example, cancers) to which immunostimulation therapy can be applied.SOLUTION: The present invention provides a novel antibody-CDN derivative conjugate which can be systemically administered and exhibits an antitumor effect against an antigen-expressing tumor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an antibody-drug conjugate in which a cyclic dinucleotide derivative having a novel structure and STING agonist activity is linked via a linker to an antibody against a target cell, and a pharmaceutical composition containing the antibody-drug conjugate, etc. [Background technology]

[0002] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein localized in the endoplasmic reticulum (Non-Patent Document 1). STING functions as a central molecule in innate immune activation in mammals and serves as the first line of defense against the invasion of pathogens such as bacteria and viruses. STING activation is known to be triggered by signals from multiple cytoplasmic DNA sensors that sense exogenous and endogenous DNA. Among these cytoplasmic DNA sensors, cGAS (cyclic GMP-AMP synthase) is considered to be an important DNA sensor. When cGAS senses DNA, it produces cyclic dinucleotide (2',3'-cGAMP), which directly binds to STING and activates it (Non-Patent Document 2). Activated STING translocates to the Golgi apparatus, where it promotes the autophosphorylation of TBK1 (Tank-binding kinase 1). Activated TBK1 through autophosphorylation activates both the IRF3 (Interferon regulatory factor 3) transcription pathway (Non-Patent Document 3) and the NFκB transcription pathway (Non-Patent Document 4), increasing the production of inflammatory proteins called interferons and cytokines (type I IFN (Interferon), IL-6 (Interleukin-6), and TNF-α (Tumor Necrosis Factor-α)). These proteins trigger the adaptive immune system, including T cells, which destroy pathogens and cancer cells through a complex cascade.

[0003] Recent studies have demonstrated that STING not only promotes host defense against microorganisms but also promotes antitumor immunity. For example, when immunogenic tumors are implanted in STING-deficient mice, the tumors grow more rapidly than in wild-type mice or in mice lacking Toll / Interleukin-1 (IL-1) receptor domain containing adaptor-inducing interferon-β (TRIF). Furthermore, STING-deficient mice exhibited a reduced spontaneous CD8+ response to tumors, unlike mice lacking TLR (Toll-like receptor), MyD88 (Myeloid differentiation primary response 88), or MAVS (Mitochondrial antiviral-signaling protein). + T cell priming was also abolished. This suggests that the STING pathway, triggered by cytoplasmic DNA sensing, is involved in the control of tumor growth (Non-Patent Document 5). Other studies have also shown that STING is required for the antitumor effects of radiation therapy (Non-Patent Document 6) or anti-CD47 antibody therapy (Non-Patent Document 7). After treatment with radiation or anti-CD47 antibody, DNA from dead tumor cells migrates into the cytoplasm of dendritic cells, activates the cGAS-STING pathway, and then induces IFN production, bridging the gap between innate and adaptive immunity. This study suggests that cross-priming mediated by dendritic cells activated by the STING pathway is important for triggering adaptive immunity against tumors.

[0004] DMXAA, a flavonoid-based small molecule compound known as a vascular disrupting agent, has been shown to have potent antitumor activity in mouse tumor models due to its ability to induce type I IFN in macrophages (Non-Patent Document 8). Due to its excellent preclinical antitumor activity, DMXAA was expected to be a promising immunotherapeutic agent for non-small cell lung cancer, but human clinical trials failed (Non-Patent Document 9). Recent studies have revealed that DMXAA is a specific agonist for mouse STING and cannot bind to human STING due to its lack of species cross-reactivity (Non-Patent Document 10). Although DMXAA was ultimately ineffective in humans, studies in mouse models have demonstrated that small molecule drugs can effectively inhibit CD8 through STING. + It was suggested that this could prime T cells and enhance anti-tumor immunity.

[0005] Cyclic dinucleotides (CDNs), another small molecule compound, have been shown to enhance STING-mediated antitumor immune responses, significantly inhibit tumor growth, and improve survival in tumor-bearing mice (Non-Patent Document 11). CDNs are classified into bacterial CDNs with canonical two 3'-5' phosphate bonds (cyclic-di-GMP, cyclic-di-AMP, 3',3'-cGAMP) and mixed-linkage CDNs with non-canonical 2'-5' phosphate bonds (2',3'-cGAMP) produced by mammalian cGAS. Recent studies have shown that mixed-linkage CDNs are more versatile in activating diverse STINGs than canonical CDNs (Non-Patent Document 12).

[0006] Natural CDNs, like many nucleic acid molecules, are rapidly degraded by nucleases in the blood and cannot be administered as is. For this reason, synthetic small molecule compounds with in vivo STING agonist activity have been developed (e.g., Patent Documents 1 to 26).

[0007] MIW-815 (ADU-S100, ML-RR-S2 CDA or ML-RR-CDA 2Na), a STING agonist, is currently undergoing clinical trials as an antitumor agent. + (sometimes referred to as "STING agonist") is administered directly into the tumor. Direct administration of a STING agonist into a tumor limits the range of tumors that can be treated, and it is difficult to administer the drug directly to all distant metastatic tumors. This limits the number of tumors that can be treated. Non-Patent Document 13 describes that administration of ML RR-S2 CDA demonstrated antitumor effects, but only intratumor administration; systemic administration (e.g., intravenous administration) has not demonstrated antitumor effects. Non-Patent Document 14 describes that intravenous administration of the STING agonist SB11285 to a mouse tumor model demonstrated antitumor effects, but the specific structure of SB11285 is not disclosed. Patent Document 14 describes a conjugate containing an immunostimulatory compound, an antibody construct, and a linker, but does not describe specific examples of conjugates using a STING agonist as the immunostimulatory compound. Patent Document 26 describes a conjugate in which a CDN having a specific structure is linked to an antibody via a linker, but does not describe any examples of in vivo administration of the conjugate, and the antitumor effect of the conjugate has not been confirmed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2014 / 099824 [Patent Document 2] International Publication No. 2014 / 179335 [Patent Document 3] International Publication No. 2014 / 189805 [Patent Document 4] International Publication No. 2014 / 189806 [Patent Document 5] International Publication No. 2015 / 074145 [Patent Document 6] International Publication No. 2015 / 185565 [Patent Document 7] International Publication No. 2016 / 096714 [Patent Document 8] International Publication No. 2016 / 012305 [Patent Document 9] International Publication No. 2016 / 145102 [Patent Document 10] International Publication No. 2017 / 027646 [Patent Document 11] International Publication No. 2017 / 027645 [Patent Document 12] International Publication No. 2017 / 075477 [Patent Document 13] International Publication No. 2017 / 093933 [Patent Document 14] International Publication No. 2017 / 100305 [Patent Document 15] International Publication No. 2017 / 123669 [Patent Document 16] International Publication No. 2017 / 161349 [Patent Document 17] International Publication No. 2017 / 175147 [Patent Document 18] International Publication No. 2017 / 175156 [Patent Document 19] International Publication No. 2018 / 009466 [Patent Document 20] International Publication No. 2018 / 045204 [Patent Document 21] International Publication No. 2018 / 060323 [Patent Document 22] International Publication No. 2018 / 067423 [Patent Document 23] International Publication No. 2018 / 065360 [Patent Document 24] International Publication No. 2014 / 093936 [Patent Document 25] International Publication No. 2018 / 009648

Patent document 26

Non-licensed literature

[0009] [Non-licensed document 1] Nature 2008,455,674-678 [Non-licensed document 2] Mol.Cell,2013,51,226-235 [Non-licensed document 3] Science 2015a,347,aaa2630

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0010] There is a need for the development of antibody-drug conjugates that can be administered systemically and that can deliver STING agonists specifically to target cells or organs (e.g., tumor sites), as well as therapeutic agents and / or methods using such antibody-drug conjugates for diseases associated with STING agonist activity, such as diseases that can be treated by immunostimulation (e.g., cancer). [Means for solving the problem]

[0011] In order to solve the above problems, the present inventors discovered an antibody-drug conjugate in which a novel CDN derivative characterized by having a fused tricyclic substituent is linked to a specific antibody via a linker, and found that systemic administration of the antibody-drug conjugate exhibits an antitumor effect on tumors expressing the antigen, thereby completing the present invention.

[0012] That is, the present invention relates to, but is not limited to, the following: [1] Formula (II): [ka] (In the formula, m 1 is in the range of 1 to 10, Ab refers to an antibody or a functional fragment of the antibody, and the sugar chain of the antibody may be remodeled. (Here, the antibody refers to any antibody selected from the group consisting of an anti-CD70 antibody, an anti-TROP2 antibody, and an anti-EGFR antibody.) L represents a linker connecting Ab and D, Ab may be bound to L directly through its amino acid residue, or may be bound to L through a glycan or a remodeled glycan of Ab; D is a group represented by the following formula (I): [ka] (where, L is L 1 and binds to any -NH2 or hydroxy group contained in L 1 is the following three equations: [ka] (Here, the wavy lines indicate the substitution positions.) and Q and Q ’ each independently represents a hydroxy group or a thiol group, R 21 and R 22 each independently represents a hydroxy group or a fluorine atom, W represents -NH- or a sulfur atom. (This indicates a compound represented by the formula: an antibody-drug conjugate represented by: [2]D is the following two equations: [ka] (where L 1 , Q, Q' and W are as previously defined). The antibody-drug conjugate according to [1], wherein [3]D is the following four equations: [ka] where the asterisk indicates the bond to L, and Q, Q', and W are as previously defined. The antibody-drug conjugate according to [1] or [2], wherein [4]D is the following three equations: [ka] (where the asterisk indicates a bond to L and W is as previously defined) The antibody-drug conjugate according to any one of [1] to [3], wherein [5]D is the following three equations: [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of [1] to [4], wherein [6]D is the following four equations: [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of [1] to [4], wherein [7] D is of the formula: [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of [1] to [4] or [6], wherein [8]D is the following two equations: [ka] (where the asterisk indicates a bond to L and W is as previously defined) The antibody-drug conjugate according to any one of [1] to [3], wherein [9]D is the following four equations: [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of [1] to [3] or [8], wherein

[10] The linker L is represented by -Lb-La-Lp-Lc-*; In the formula, the asterisk indicates binding to drug D, Lp has or does not have a linker consisting of an amino acid sequence that is cleavable in the target cell, La represents any one selected from the following group: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -(CH2)n 4 -OC(=O)-, and -(CH2)n 9 -C(=O)- (where n 2 represents an integer from 1 to 3, and n 3 represents an integer from 1 to 5, and n 4 represents an integer from 0 to 2, and n 9 represents an integer from 2 to 7), Lb represents a spacer that connects the sugar chains or remodeled sugar chains of La and Ab, or a spacer that connects the cysteine ​​residues of La and Ab; and Lc is -NH-CH2-, -NH-phenyl group -CH2-O(C=O)- or -NH-heteroaryl group -CH2-O(C=O)-, or is absent; The antibody-drug conjugate according to any one of [1] to [9];

[11] The antibody-drug conjugate according to

[10] , wherein Lc is -NH-CH2-;

[12] The antibody-drug conjugate according to

[10] or

[11] , wherein Lp is any one of -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, and -GGPP-;

[13] The antibody-drug conjugate according to

[12] , wherein Lp is -GGFG- or -GGPI-;

[14] La is as follows: -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, and -(CH2)5-C(=O)- The antibody-drug conjugate according to any one of

[10] to

[13] , which is any one selected from the group consisting of:

[15] Lb is a compound of the formula: [ka] or [ka] (in the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain);

[16] Lb is -(succinimide-3-yl-N)-; wherein -(succinimide-3-yl-N)- has the following structural formula: [ka] indicates, wherein the asterisk indicates binding to La, and the wavy line indicates binding via thioether formation with the side chain of a cysteine ​​residue of the antibody; the antibody-drug conjugate according to any one of

[10] to

[14] ;

[17] The linker L is represented by -Lb-La-Lp-Lc-*; In the formula, the asterisk indicates binding to drug D, Lp is -GGFG- or -GGPI-; La represents -C(=O)-CHCH-C(=O)-; Lb is expressed by the following formula: [ka] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain), The antibody-drug conjugate according to any one of

[10] to

[15] , wherein Lc represents -NH-CH2-;

[18] The antibody-drug conjugate according to any one of [1] to

[17] , wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is in the range of 1 to 10;

[19] The antibody-drug conjugate according to

[18] , wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is in the range of 1 to 5;

[20] The antibody-drug conjugate according to

[19] , wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is in the range of 3 to 5;

[21] The antibody-drug conjugate according to any one of [1] to

[20] , wherein the antibody is bound to L via a sugar chain (N297 sugar chain) bound to Asn297 of the antibody;

[22] The antibody-drug conjugate according to

[21] , wherein the N297 sugar chain is a remodeled sugar chain;

[23] The antibody-drug conjugate according to

[21] or

[22] , wherein the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula: [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 is an integer between 2 and 5; [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 is an integer between 2 and 5;

[24] The following formula: [ka] (In the formula, m 2 represents an integer of 1 or 2, L is a linker connecting the N297 carbohydrate chain of the Ab and D, as defined above; Ab represents an anti-CD70 antibody, an anti-TROP2 antibody, an anti-EGFR antibody, or a functional fragment of these antibodies; The N297 sugar chain of Ab is N297-(Fuc)MSG1 or N297-(Fuc)SG, which has the structure shown in the following formula: [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5-CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 represents an integer from 2 to 5; [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 represents an integer from 2 to 5, D is expressed by one of the following four formulas: [ka] wherein in the formula, an asterisk indicates binding to L);

[25] The following formula: [ka] Selected from In each of the structural formulas shown above, m 2 indicates an integer of 1 or 2, Ab represents an anti-CD70 antibody, an anti-TROP2 antibody, an anti-EGFR antibody, or a functional fragment of these antibodies; The N297 sugar chain of Ab is either N297-(Fuc)MSG1 or N297-(Fuc)SG, which has the structure shown in the following formula: [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 represents an integer from 2 to 5; [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 represents an integer of 2 to 5; the antibody-drug conjugate according to

[24] ;

[26] The following formula: [ka] Selected from In each of the structural formulas shown above, m 2 indicates an integer of 1 or 2, Ab represents an anti-CD70 antibody, an anti-TROP2 antibody, an anti-EGFR antibody, or a functional fragment of these antibodies; The N297 sugar chain of Ab is either N297-(Fuc)MSG1 or N297-(Fuc)SG, which has the structure shown in the following formula: [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 represents an integer from 2 to 5; [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, and n 5 represents an integer of 2 to 5; the antibody-drug conjugate according to

[25] ;

[27] The antibody-drug conjugate according to any one of [1] to

[26] , wherein the antibody is an anti-CD70 antibody;

[28] The antibody-drug conjugate according to any one of [1] to

[26] , wherein the antibody is an anti-TROP2 antibody;

[29] The antibody-drug conjugate according to any one of [1] to

[26] , wherein the antibody is an anti-EGFR antibody;

[30] The antibody-drug conjugate according to

[27] , wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4;

[31] The antibody-drug conjugate according to

[28] , wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8;

[32] The antibody-drug conjugate according to

[29] , wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12;

[33] The antibody-drug conjugate according to

[27] , wherein the antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4;

[34] the antibody-drug conjugate according to

[28] , wherein the antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO: 6, or an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO: 8;

[35] the antibody-drug conjugate according to

[29] , wherein the antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO: 10, or an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO: 12;

[36] the antibody-drug conjugate according to

[27] , wherein the antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; or an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46;

[37] the antibody-drug conjugate according to

[28] , wherein the antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; or an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58;

[38] the antibody-drug conjugate according to

[29] , wherein the antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 64; or an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 70;

[39] The following formula: [ka] wherein Ab represents any one selected from the following group: an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12; The N297 glycan of Ab has the following formula: [ka] (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, n 5 is 3, and m 2 an antibody-drug conjugate represented by the formula (I) wherein

[40] The following formula: [ka] wherein Ab represents any one selected from the following group: an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO: 6; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO: 8; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO: 10; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO: 12; The N297 glycan of Ab has the following formula: [ka] (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, n 5 is 3, and m 2 an antibody-drug conjugate represented by the formula (I) wherein

[41] The following formula: [ka] wherein Ab represents any one selected from the following group: an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 64; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 70; The N297 glycan of Ab has the following formula: [ka] (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, n 5 is 3, and m 2an antibody-drug conjugate represented by the formula (I) wherein

[42] The following formula: [ka] wherein Ab represents any one selected from the following group: an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12; The N297 glycan of Ab has the following formula: [ka] (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, n 5 is 3, and m 2 an antibody-drug conjugate represented by the formula (I) wherein

[43] The following formula: [ka] wherein Ab represents any one selected from the following group: an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO: 6; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO: 8; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO: 10; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO: 12; The N297 glycan of Ab has the following formula: [ka] (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH2-CH2-O)n 5-CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, n 5 is 3, and m 2 an antibody-drug conjugate represented by the formula (I) wherein

[44] The following formula: [ka] wherein Ab represents any one selected from the following group: an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 64; an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 70; The N297 glycan of Ab has the following formula: [ka] (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, n 5 is 3, and m 2an antibody-drug conjugate represented by the formula (I) wherein

[45] A STING agonist comprising the antibody-drug conjugate according to any one of [1] to

[44] ;

[46] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[44] ;

[47] An antitumor agent comprising the antibody-drug conjugate according to any one of [1] to

[44] ;

[48] ​​The antitumor agent according to

[47] , wherein the tumor is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, ear cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma;

[49] A method for treating cancer, comprising administering any one selected from the group consisting of the antibody-drug conjugate according to any one of [1] to

[44] , the STING agonist according to

[45] , the pharmaceutical composition according to

[46] , and the antitumor agent according to

[47] or

[48] ;

[50] The method according to

[49] , wherein the cancer is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, ear cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma;

[51] The antibody-drug conjugate according to any one of

[27] ,

[30] ,

[33] or

[36] , which exhibits an antibody target-dependent antitumor effect in BALB / c-nu mice subcutaneously implanted with human renal cancer cell line Caki-1 cells;

[52] The antibody-drug conjugate according to any one of

[42] to

[44] , which exhibits a stronger antitumor effect than an antibody contained in the antibody-drug conjugate in an animal described in the following (i) or (ii): (i) a BALB / c mouse subcutaneously implanted with the mouse colon cancer cell line CT26.WT (CRL2638), which has been introduced with a gene for a human-mouse chimeric antigen in which the epitope site on the antigen to which the antibody contained in the antibody-drug conjugate binds has been replaced with a human type; (ii) BALB / c-nu mice subcutaneously implanted with human renal carcinoma cell line A-498 (HTB-44) cells; Regarding. [Effects of the Invention]

[0013] The present invention provides a novel antibody-CDN derivative conjugate that can be administered systemically and exhibits antitumor effects on tumors expressing the antigen. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows schematic representations of antibody-drug conjugates (molecule (II) in Figure 1A) obtained from an SG-type glycosylation remodeling antibody and antibody-drug conjugates (molecule (II) in Figure 1B) obtained from an MSG-type glycosylation remodeling antibody, which are antibody-drug conjugates (molecule (II)) of the present invention. (a) represents drug D, (b) represents linker L, (c) represents PEG linker (L(PEG)), and (d) represents N297 glycan (wherein the white circle represents NeuAc (Sia), the white hexagon represents Man, the black hexagon represents GlcNAc, the white diamond represents Gal, and the white inverted triangle represents Fuc). The white pentagon represents a triazole ring generated by the reaction of the alkyne derived from linker L with the azide group derived from the PEG linker. The Y-shape represents the antibody Ab. The PEG linker is linked to the carboxyl group at the 2-position of the sialic acid located at the non-reducing end via an amide bond. This notation method is applied throughout this specification unless otherwise stated. [Figure 2]2A and 2C show schematic diagrams illustrating the structures of (Fucα1,6)GlcNAc-antibody (molecule (III) in FIG. 2A), SG-glycan remodeling antibody (molecule (IV) in FIG. 2B), and MSG-glycan remodeling antibody (molecule (IV) in FIG. 2C), which are production intermediates of the antibody-drug conjugates of the present invention. In all figures, the Y-shape represents the antibody Ab, as in FIG. 1. In FIG. 2A, (e) represents the N297 glycan, which consists of a disaccharide α-glycosidically linked between position 1 of Fuc and position 6 of GlcNAc. In FIGS. 2B and 2C, (d) represents the same N297 glycan as in FIG. 1, and (f) represents a PEG linker having an azide group, which is used for binding to linker L at its terminal. The binding mode of the PEG linker having an azide group is the same as that of the PEG linker in FIG. 1. [Figure 3] This is a schematic diagram of the process for producing SG-type glycosylation-remodeled antibodies and MSG-type glycosylation-remodeled antibodies from antibodies produced in animal cells. Molecules (III) and (IV) in the diagram represent (Fucα1,6)GlcNAc-antibodies and SG-type glycosylation-remodeled antibodies or MSG-type glycosylation-remodeled antibodies, respectively, as in Figure 2. Molecule (V) is an antibody produced in animal cells and is a mixture of molecules with heterogeneous N297 glycans. Figure 3A shows the process for producing homogeneous (Fucα1,6)GlcNAc-antibody (III) by treating the heterogeneous N297 glycan of (V) with a hydrolase such as EndoS. Figure 3B shows the process for producing SG-type glycosylation-remodeled antibodies (IV) by glycosylation-transferring an SG-type glycosylation-remodeled antibody to the GlcNAc of the N297 glycan of antibody (III) using a glycosyltransferase such as the EndoS D233Q / Q303L mutant. Figure 3C, like Figure 3B, shows the process for preparing an MSG-type glycan remodeled antibody (IV) by glycosylation of an MSG-type glycan donor molecule to antibody (III). The SG-type glycan donor molecule and MSG-type glycan donor molecule used here have the sialic acid at their non-reducing ends modified with a PEG linker having an azide group, and the SG-type N297 glycan remodeled antibody and MSG-type N297 glycan remodeled antibody to be prepared also have the sialic acid at their non-reducing ends similarly modified, as shown in Figures 2B and C. [Figure 4] The amino acid sequence of the light chain (SEQ ID NO: 1) and the amino acid sequence of the heavy chain (SEQ ID NO: 2) of anti-CD70 antibody 1 are shown (herein, "anti-CD70 antibody 1" is also referred to as "modified anti-CD70 antibody 1"). [Figure 5] The amino acid sequence of the light chain (SEQ ID NO: 3) and the amino acid sequence of the heavy chain (SEQ ID NO: 4) of anti-CD70 antibody 2 are shown (herein, "anti-CD70 antibody 2" is also referred to as "modified anti-CD70 antibody 2"). [Figure 6] The amino acid sequence of the light chain (SEQ ID NO: 5) and the heavy chain (SEQ ID NO: 6) of anti-TROP2 antibody 1 are shown. [Figure 7] The amino acid sequence of the light chain (SEQ ID NO: 7) and the amino acid sequence of the heavy chain (SEQ ID NO: 8) of anti-TROP2 antibody 2 are shown (herein, "anti-TROP2 antibody 2" is also referred to as "modified anti-TROP2 antibody"). [Figure 8] The amino acid sequence of the light chain (SEQ ID NO: 9) and the amino acid sequence of the heavy chain (SEQ ID NO: 10) of anti-EGFR antibody 1 are shown (herein, "anti-EGFR antibody 1" is also referred to as "modified anti-EGFR antibody 1"). [Figure 9] The amino acid sequence of the light chain (SEQ ID NO: 11) and the amino acid sequence of the heavy chain (SEQ ID NO: 12) of anti-EGFR antibody 2 are shown (herein, "anti-EGFR antibody 2" is also referred to as "modified anti-EGFR antibody 2"). [Figure 10] (a) The amino acid sequence of human STING wild-type (SEQ ID NO: 13), (b) the amino acid sequence of human STING REF mutant (R232H) (SEQ ID NO: 15), and (c) the amino acid sequence of human STING HAQ mutant (R71H, G230A, R293Q) (SEQ ID NO: 17). [Figure 11] Figure 1 shows the STING agonist activity of anti-TROP2 antibody 2, anti-TROP2 antibody 2-CDN conjugate (1), anti-TROP2 antibody 2-CDN conjugate (2), and anti-TROP2 antibody 2-CDN conjugate (3) in TROP2-expressing cells. [Figure 12]This shows the activity of compound 34a, anti-CD70 antibody 1, anti-CD70 antibody 2, anti-CD70 antibody 1-CDN conjugate (1), and anti-CD70 antibody 2-CDN conjugate (1) in a co-culture assay system between CT26.WT and CT26.WT-hCD70 cell lines and mouse bone marrow-derived dendritic cells. [Figure 13] This figure shows the antitumor effect of intravenous administration of anti-TROP2 antibody 1 and anti-TROP2 antibody 1-CDN conjugate (1). In the figure, the black squares represent the vehicle group, the white inverted triangles represent the group administered with anti-TROP2 antibody 1-CDN conjugate (1) prepared in Reference Example 5 and conjugated with compound 6b of Example 1, and the white circles represent the group administered with anti-TROP2 antibody 1. The vertical axis represents tumor volume (mm), and the horizontal axis represents the number of days after tumor implantation. [Figure 14] This figure shows the antitumor effect of intravenous administration of anti-TROP2 antibody 2 and anti-TROP2 antibody 2-CDN conjugate (1). In the figure, the black square line represents the vehicle group, the white inverted triangle line represents the group administered with anti-TROP2 antibody 2-CDN conjugate (1) prepared in Reference Example 6 and conjugated with compound 34a of Example 2, and the white circle line represents the group administered with anti-TROP2 antibody 2. The vertical axis represents tumor volume (mm), and the horizontal axis represents the number of days after tumor implantation. [Figure 15] The figure shows the antitumor effects of intravenous administration of anti-EGFR antibody 1, anti-EGFR antibody 2, and anti-EGFR antibody-CDN conjugate. The figure also shows the antitumor effects of intravenous administration of anti-EGFR antibody 1-CDN conjugate (1), which was prepared in Reference Example 7 by conjugating compound 34a of Example 2 to anti-EGFR antibody 1, and anti-EGFR antibody 2-CDN conjugate (1), which was prepared in Reference Example 8 by conjugating compound 34a of Example 2 to anti-EGFR antibody 2. In the figure, the black squares represent the vehicle group, the white triangles represent the anti-EGFR antibody 1-administered group, the black triangles represent the anti-EGFR antibody 1-CDN conjugate (1)-administered group, the white circles represent the anti-EGFR antibody 2-administered group, and the black circles represent the anti-EGFR antibody 2-CDN conjugate (1)-administered group. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 16]The amino acid sequence of CDRL1 (sequence number 35), CDRL2 (sequence number 36), CDRL3 (sequence number 37), CDRH1 (sequence number 38), CDRH2 (sequence number 39), and CDRH3 (sequence number 40) of anti-CD70 antibody 1 are shown. [Figure 17] The amino acid sequence of CDRL1 (sequence number 41), CDRL2 (sequence number 42), CDRL3 (sequence number 43), CDRH1 (sequence number 44), CDRH2 (sequence number 45), and CDRH3 (sequence number 46) of anti-CD70 antibody 2 are shown. [Figure 18] The amino acid sequence of CDRL1 (sequence number 47), CDRL2 (sequence number 48), CDRL3 (sequence number 49), CDRH1 (sequence number 50), CDRH2 (sequence number 51), and CDRH3 (sequence number 52) of anti-TROP2 antibody 1 are shown. [Figure 19] The amino acid sequence of CDRL1 (sequence number 53), CDRL2 (sequence number 54), CDRL3 (sequence number 55), CDRH1 (sequence number 56), CDRH2 (sequence number 57), and CDRH3 (sequence number 58) of anti-TROP2 antibody 2 are shown. [Figure 20] The amino acid sequence of CDRL1 (sequence number 59), CDRL2 (sequence number 60), CDRL3 (sequence number 61), CDRH1 (sequence number 62), CDRH2 (sequence number 63), and CDRH3 (sequence number 64) of anti-EGFR antibody 1 are shown. [Figure 21] The amino acid sequence of CDRL1 (sequence number 65), CDRL2 (sequence number 66), CDRL3 (sequence number 67), CDRH1 (sequence number 68), CDRH2 (sequence number 69), and CDRH3 (sequence number 70) of anti-EGFR antibody 2 are shown. [Figure 22]Figure 1 shows the antitumor effects of intravenous administration of anti-CD70 antibody 1, anti-CD70 antibody 1-CDN conjugate (1), anti-CD70 antibody 2, and anti-CD70 antibody 2-CDN conjugate (1). In the figure, the black squares represent the vehicle group, the white triangles represent the anti-CD70 antibody 1-administered group, the white inverted triangles represent the anti-CD70 antibody 2-administered group, the white diamonds represent the anti-CD70 antibody 1-CDN conjugate (1)-administered group, and the white circles represent the anti-CD70 antibody 2-CDN conjugate (1)-administered group. The vertical axis represents tumor volume (mm), and the horizontal axis represents the number of days after tumor implantation. [Figure 23] This figure shows the antitumor effect of intravenous administration of anti-CD70 antibody 2 and anti-CD70 antibody 2-CDN conjugate (2). In the figure, the black squares represent the vehicle group, the white triangles represent the anti-CD70 antibody 2-administered group, and the inverted white triangles represent the anti-CD70 antibody 2-CDN conjugate (2)-administered group. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 24] This figure shows the antitumor effect of intravenous administration of anti-EGFR antibody 1, compound 34a, and anti-EGFR antibody 1-CDN conjugate (2). In the figure, the black squares represent the vehicle group, the white triangles represent the anti-EGFR antibody 1 administration group, the white diamonds represent the compound 34a administration group, and the white squares represent the anti-EGFR antibody 1-CDN conjugate (2) administration group. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to antibody-drug conjugates containing novel CDN derivatives with STING agonist activity and uses thereof. The novel CDN derivatives have STING agonist activity and activate immune cells to induce the production of interferons and cytokines. Furthermore, the novel CDN derivatives exert antitumor effects by activating the immune cells. The antibody-drug conjugates of the present invention are prepared by linking the CDN derivatives to antibodies capable of recognizing and binding to target cells (e.g., tumor cells or immune cells) via any linker, and can be administered systemically. Specific examples of systemic administration include intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.

[0016] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein localized in the endoplasmic reticulum. Congenital STING polymorphisms are known to occur frequently (PLoS One, 2013 Oct, 21, 8(10), e77846). Examples of STING mutants include the R232H mutation, in which the 232nd amino acid is mutated from arginine (R) to histidine (H), and the HAQ mutation, in which the 71st amino acid is mutated to histidine (H), the 230th amino acid is glycine (G) to alanine (A), and the 293rd amino acid is mutated to glutamine (Q). These STING polymorphisms are known to affect the strength of responses, such as cytokine production, induced by STING agonist stimulation (Genes and Immunity, 2011, 12, 263-269). Therefore, for a STING agonist to act stably in humans, it is desirable that it has activity against each form of STING.

[0017] In this specification, the terms "cancer," "carcinoma," and "tumor" are used interchangeably.

[0018] In the present invention, "immunostimulation" refers to inducing in some way the activation of immune cells involved in anti-tumor immunity, such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, and neutrophils, and refers to causing any structural or functional changes in immune cells, such as the production of cytokines and chemokines, increased expression of immune activation markers, decreased expression of immunosuppressive markers, changes such as phosphorylation in intracellular signaling pathways, and changes in gene expression. It also includes causing changes in tumor cells that induce anti-tumor immunity, such as the production of cytokines and chemokines that activate or induce migration of immune cells, and increased sensitivity to immune cells.

[0019] In the present invention, the term "antitumor effect" refers to the direct or indirect effect of a drug on tumor cells, thereby inducing a reduction or regression of tumors. For example, an antitumor effect can be caused by a drug directly damaging tumor cells, by tumor cells stimulating with the drug to activate antitumor immunity, or by a drug delivered to tumor cells being released extracellularly to activate antitumor immunity around the tumor cells, thereby reducing the number of tumor cells, damaging them, or causing tumor regression.

[0020] In the present invention, "cytotoxic activity" refers to causing pathological changes in cells in some form, including not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes.

[0021] In the present invention, the term "cells" includes cells in an animal body and cultured cells.

[0022] <1. New CDN derivatives> The novel CDN derivatives have the following formula (I):

[0023] [ka] It has the structure shown below.

[0024] L 1 is a group represented by one of the following three formulas:

[0025] [ka] is. Q and Q ’ each independently represents a hydroxy group or a thiol group. Preferably, Q and Q' are both thiol groups.

[0026] R 21 and R 22 R each independently represents a hydroxy group or a fluorine atom. 21is preferably a hydroxy group. 22 is preferably a fluorine atom.

[0027] W is —NH— or a sulfur atom, and preferably —NH—.

[0028] The method for producing the novel CDN derivative will be described later in <3. Production Method>.

[0029] 2. Antibody-drug conjugates The antibody-drug conjugate of the present invention can be administered systemically as an antibody-drug conjugate in which the above-mentioned novel CDN derivative is linked to an antibody capable of recognizing and binding to a target cell (e.g., a tumor cell or an immune cell) via an optional linker.

[0030] The antibody-drug conjugate of the present invention has the following formula (II):

[0031] [ka] It is shown by m 1 is in the range of 1 to 10 and indicates the number of drugs bound per antibody molecule in the antibody-drug conjugate. Ab represents an antibody or a functional fragment of the antibody, L represents a linker connecting Ab and D, and D represents the novel CDN derivative described above (herein, when the novel CDN derivative is used as a part of the antibody-drug conjugate, it is also simply referred to as a "drug").

[0032] Drug D is a compound that has immune cell activation activity, specifically, STING agonist activity. When part or all of the linker is cleaved in target cells (e.g., tumor cells or immune cells), Drug D is released in its original structure, thereby exerting its immunostimulatory effect. The desired function is exerted by enhancing the sensitivity of target cells to immune cells or by target cell-mediated activation of immune cells. The desired function is not particularly limited as long as it is related to STING agonist activity, but is preferably antitumor activity. Specifically, Drug D, linked to a tumor-targeting antibody (e.g., an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody) via an optional linker, is delivered to target cells or tissues, where part or all of the linker is cleaved, thereby exerting an antitumor effect through enhancing the sensitivity of target cells to immune cells or target cell-mediated activation of immune cells (e.g., production of interferon or cytokines).

[0033] The drug D attached to the antibody-drug conjugate of the present invention has the following formula (I):

[0034] [ka] (where L 1 , Q, Q', R 21 , R 22 and W is as defined in <1. Novel CDN derivatives> above). Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulae:

[0035] [ka] (where L 1 , Q, Q', and W are as defined above in <1. Novel CDN derivatives>).

[0036] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following four formulas:

[0037] [ka] (wherein the asterisk indicates binding to L, and Q, Q', and W are as defined above in <1. Novel CDN derivatives>).

[0038] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula:

[0039] [ka] (wherein the asterisk indicates binding to L, and W is as defined in <1. Novel CDN derivatives> above).

[0040] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula:

[0041] [ka] (where the asterisk indicates binding to L).

[0042] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following four formulas:

[0043] [ka] (where the asterisk indicates binding to L).

[0044] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is more preferably represented by the following formula:

[0045] [ka] It is shown as follows.

[0046] Furthermore, the drug D used in the antibody-drug conjugate of the present invention preferably has the following formula:

[0047] [ka] (wherein the asterisk indicates binding to L, and W is as defined in <1. Novel CDN derivatives> above).

[0048] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulae:

[0049] [ka] (where the asterisk indicates binding to L).

[0050] Furthermore, the drug D used in the antibody-drug conjugate of the present invention preferably has the following formula:

[0051] [ka] (wherein the asterisk indicates binding to L, and W is as defined in <1. Novel CDN derivatives> above). Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulae:

[0052] [ka] (where the asterisk indicates binding to L).

[0053] <2.1. Linker structure> The linker structure that binds a drug to an antibody in the antibody-drug conjugate of the present invention is described below. The linker used in the antibody-drug conjugate of the present invention is not particularly limited as long as it is understood by those skilled in the art as a linker that connects an antibody and a drug. Examples of linkers used in the antibody-drug conjugate of the present invention include, but are not limited to, those described in Protein Cell, 2018, 9(1):33-46; Pharm Res, 2015, 32:3526-3540; or Int. J. Mol. Sci., 2016, 17, 561. The linker may be either a linker that is cleaved in vivo or a linker that is not cleaved in vivo, but is preferably a linker that is cleaved in vivo.

[0054] Linkers used in the antibody-drug conjugates of the present invention include, but are not limited to, linkers that bind a drug to a sugar chain or remodeled sugar chain of the Fc portion of an antibody (sometimes referred to herein as "sugar chain conjugation") (e.g., as described in WO2018 / 003983), or linkers that bind a drug to any amino acid residue of an antibody (e.g., a cysteine ​​residue or a lysine residue) (e.g., as described in WO2014 / 057687). Linkers that bind a drug to any amino acid residue of an antibody preferably include those that bind via a thioether bond with a sulfhydryl group (SH group) of a cysteine ​​of an Ab (sometimes referred to herein as "cysteine ​​conjugation") or a amide bond with an amino group (NH group) of a lysine of an Ab (sometimes referred to herein as "lysine conjugation"), with cysteine ​​conjugation being preferred.

[0055] A preferred linker L for use in the antibody drug conjugates of the invention is represented by the following formula: -Lb-La-Lp-Lc-* (where the asterisk indicates binding to drug D).

[0056] First, Lp will be explained. Lp either exhibits a linker (hereinafter also referred to as a peptide linker in this specification) consisting of an amino acid sequence that is cleavable in vivo or in target cells, or is absent.

[0057] Lp is cleaved by the action of an enzyme such as peptidase or esterase. Lp is a peptide composed of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond at its N-terminus with the carbonyl group at the right end of La (described below), and forms an amide bond at its C-terminus with the amino group (—NH—) of Lc. The amide bond at the C-terminus of Lp is cleaved by the enzyme such as peptidase.

[0058] The amino acids constituting Lp are not particularly limited, but may be, for example, L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, amino acids with structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may also be used, and non-natural amino acids such as N-methylated amino acids may also be used. The amino acid sequence of Lp is not particularly limited, but examples of the constituting amino acids include glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), methionine (Met; M), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D). Among these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), citrulline (Cit), isoleucine (Ile; I), and proline (Pro; P) are preferred. These amino acids may overlap, and the amino acid sequence may include any selected amino acid. Furthermore, the drug release pattern can be controlled by the type of amino acid.

[0059] Specific examples of Lp include -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, and -GGPP-. The linker Lp is preferably -GGFG- or -GGPI-, and more preferably -GGFG-.

[0060] Next, we will explain La. La is as follows: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -(CH2)n 4 -OC(=O)-, and -(CH2)n 9 -C(=O)- (wherein, n 2 represents an integer of 1 to 3 (preferably 1 or 2), and n 3 represents an integer of 1 to 5 (preferably an integer of 2 to 5, more preferably 3 or 4), and n 4 represents an integer of 0 to 2 (preferably 0 or 1), and n 9 represents any one selected from the group consisting of integers of 2 to 7 (preferably an integer of 2 to 5, more preferably 2, 3, or 5).

[0061] La is preferably one of the following: -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, and -(CH2)5-C(=O)- represents any one selected from the group consisting of.

[0062] La is more preferably -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, or -(CH2)5-C(=O)- is.

[0063] La is even more preferably -C(=O)-CH2CH2-C(=O)-.

[0064] Next, Lb will be described. Lb represents a spacer used for a linker of sugar chain conjugation (also referred to as "spacer of linker of sugar chain conjugation" in this specification), or a spacer used for a linker of cysteine conjugation (also referred to as "spacer of linker of cysteine conjugation" in this specification).

[0065] <When Lb is "spacer of linker of sugar chain conjugation"> When Lb is the "spacer of the linker for sugar chain conjugation", Lb is not particularly limited, and examples thereof include spacers represented by the following formulae.

[0066] [Chemical formula] Or

[0067] [Chemical formula]

[0068] In each of the structural formulae shown above, the asterisk (*) indicates that it is bonded to the -(C=O)-, -CH2- or -OC(=O)- at the left end of La, and the wavy line indicates that it is bonded to the sugar chain of Ab or the remodeled sugar chain.

[0069] When Lb-1 or Lb-3 is selected for Lb, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of the two types of structures or a mixture thereof. The antibody-drug conjugate of the present invention can bind a plurality of drugs to one molecule of an antibody. When a plurality of drugs are bound to one molecule of an antibody, a plurality of Lbs will also be present (for example, refer to the schematic diagram (1e) of the antibody-drug conjugate shown in Method E of <3. Production method> described later). When Lb is selected from Lb-1 or Lb-3 and a plurality of such Lbs are present with respect to one molecule of an antibody (for example, when m 2 is 1 or 2), in each Lb, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of the two types of structures or a mixture thereof.

[0070] <When Lb is the "spacer of the linker for cysteine conjugation"> When Lb is the "spacer of the linker for cysteine conjugation", Lb is not particularly limited, and examples thereof include -(succinimid-3-yl-N)-. In the present invention, "-(succinimid-3-yl-N)-" has the following formula:

[0071] [ka] In the structural formula shown above, the asterisk indicates a bond to La, and the wavy line indicates a bond via a thioether with the side chain of a cysteine ​​residue in the antibody.

[0072] Next, Lc will be explained. Lc represents -NH-CH2-, -NH-phenyl group -CH2-O(C=O)-, or -NH-heteroaryl group -CH2-O(C=O)-, or is absent. Here, the phenyl group is preferably a 1,4-phenyl group, and the heteroaryl group is preferably a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group. Lc is preferably -NH-CH2- or is absent.

[0073] A more preferred linker L used in the antibody-drug conjugate of the present invention is, when the binding mode between the drug and the antibody is "glycoconjugation", -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGVA-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGVCit-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFCit-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGICit-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFM-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGLM-, -ZL1 -C(=O)-CH2CH2-C(=O)-FG-, -Z L1 -C(=O)-CH2CH2-C(=O)-VA-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-CH2-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-CH2-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFCit-NH-CH2-, -Z L1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, or -Z L1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)- (where Z L1 is the structural formula of Lb shown below:

[0074] [ka] or when the binding mode between the drug and the antibody is "cysteine ​​conjugation", -Z L2 -(CH2)5-C(=O)-GGFG-, -Z L2 -(CH2)5-C(=O)-GGVA-, -Z L2 -(CH2)5-C(=O)-GGVCit-, -Z L2 -(CH2)5-C(=O)-GGFCit-, -Z L2 -(CH2)5-C(=O)-GGICit-, -Z L2 -(CH2)5-C(=O)-GGFM-, -Z L2-(CH2)5-C(=O)-GGPI-, -Z L2 -(CH2)5-C(=O)-GGLM-, -Z L2 -(CH2)5-C(=O)-FG-, -Z L2 -(CH2)5-C(=O)-VA-, -Z L2 -(CH2)5-C(=O)-GGFG-NH-CH2-, -Z L2 -(CH2)5-C(=O)-GGVA-NH-CH2-, -Z L2 -(CH2)5-C(=O)-GGVCit-NH-CH2-, -Z L2 -(CH2)5-C(=O)-GGFCit-NH-CH2-, -Z L2 -(CH2)5-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, or -Z L2 -(CH2)5-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, (where Z L2 is the structural formula of Lb shown below:

[0075] [ka] It represents -(succinimide-3-yl-N)-. is.

[0076] A more preferred linker L used in the antibody-drug conjugate of the present invention is one in which the binding mode between the drug and the antibody is "glycoconjugation", -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, or -Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-CH2-, (where Z L1 is the structural formula of Lb shown below:

[0077] [ka] (Indicates

[0078] An even more preferred linker L used in the antibody-drug conjugate of the present invention is one in which the binding mode between the drug and the antibody is "glycoconjugation", -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, (where Z L1 is the structural formula of Lb shown below:

[0079] [ka] (Indicates

[0080] The right ends of the above-mentioned "preferred linker L", "more preferred linker L", "even more preferred linker L" and "even more preferred linker L" are bonded to the drug D.

[0081] The "linker L-drug D" used in the antibody drug conjugates of the present invention preferably has the following two formulas:

[0082] [ka] (Here, the wavy line indicates binding to the Ab glycan or remodeled glycan).

[0083] The "linker L-drug D" used in the antibody drug conjugates of the present invention more preferably has the following two formulas:

[0084] [ka] (where the wavy line indicates binding to the Ab glycan or remodeled glycan).

[0085] The "linker L-drug D" used in the antibody drug conjugates of the present invention is more preferably represented by the following formula:

[0086] [ka] (Here, the wavy line indicates binding to the Ab glycan or remodeled glycan).

[0087] 2.2. Antibodies and their glycosylation <2.2.1 Antibodies> As used herein, the term "gene" refers to a nucleotide or nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof. For example, the term "gene" includes polynucleotides, oligonucleotides, DNA, mRNA, cDNA, RNA, etc., which are nucleotide sequences containing a nucleotide sequence encoding the amino acids of a protein, or their complementary strands.

[0088] As used herein, "nucleotide," "polynucleotide," or "nucleotide sequence" is synonymous with "nucleic acid," and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence."

[0089] In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably.

[0090] As used herein, the term "functional antibody fragment," also referred to as "antigen-binding fragment of antibody," refers to a partial fragment of an antibody that retains antigen-binding activity, and includes Fab, F(ab'), Fv, scFv, diabody, linear antibody, and multispecific antibody fragments formed from antibody fragments. Also included in the antigen-binding fragment of an antibody is Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab') under reducing conditions. However, the antigen-binding fragment is not limited to these molecules, as long as it retains antigen-binding ability. Furthermore, these antigen-binding fragments include not only those obtained by treating the full-length antibody protein molecule with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0091] The functional fragments of antibodies used in the antibody-drug conjugates of the present invention include functional fragments that retain the well-conserved asparagine (Asn297) and its surrounding amino acids that are modified by N-linked sugar chains in the Fc region of IgG heavy chains, and that have antigen-binding ability.

[0092] The antibody used in the antibody-drug conjugates of the present invention refers to an immunoglobulin, and is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody used in the antibody-drug conjugates of the present invention may be of any class, including IgG, IgE, IgM, IgD, IgA, and IgY, with IgG being preferred. The subclass may also be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG1, IgG2, or IgG4 being preferred (including antibodies with mutations in the Fc region of the IgG heavy chain that affect ADCC and ADCP activity).

[0093] When the antibody used in the antibody-drug conjugates of the present invention is of the IgG1 isotype, effector function can be adjusted by substituting some of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, and WO94 / 29351). Examples of IgG1 mutants include IgG1 LALA mutations (IgG1-L234A, L235A). L234A and L235A represent substitutions of leucine with alanine at positions 234 and 235, as defined by the EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No. 1 (May 15, 1969), pp. 78-85).

[0094] It is known that there are multiple allotypes of antibody constant regions. For example, for IgG1 heavy chains, allotypes include G1m17, G1m3, G1m1, and G1m2. The antibody constant region used in the present invention is not particularly limited, but it is preferable to use G1m17 or G1m3.

[0095] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). CDRs, also known as hypervariable regions, are located in the variable regions of the heavy and light chains of an antibody and are particularly highly variable in their primary structure. They are separated into three regions in the primary structure of the heavy and light chain polypeptide chains. Herein, with regard to antibody CDRs, the heavy chain CDRs are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and the light chain CDRs are referred to as CDRL1, CDRL2, and CDRL3 from the amino-terminal end of the light chain amino acid sequence. These regions are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0096] The antibody may be derived from any species, preferably human, rat, mouse, or rabbit. When derived from a species other than human, it is preferable to chimerize or humanize the antibody using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, and modified versions thereof.

[0097] The antibody is preferably, but not limited to, an antibody that targets tumor cells or immune cells, and more preferably, an antibody that targets tumor cells.

[0098] When an antibody targeting tumor cells is used, the antibody preferably has one or more of the following properties: the ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be taken up and internalized into tumor cells, and the ability to damage tumor cells. The drug used in the antibody-drug conjugate of the present invention has STING agonist activity. The drug activates the signal of interferon regulatory factor-3 (IRF3) to induce interferon. Therefore, when an antibody targeting tumor cells is used in the antibody-drug conjugate of the present invention, the antibody-drug conjugate is administered into the body, delivered to the tumor site, and taken up into tumor cells. After that, the linker moiety is cleaved by peptidase or the like, releasing the drug moiety. The released drug moiety is thought to enhance the sensitivity of tumor cells to immune cells and activate anti-tumor immunity through its STING agonist activity, thereby exerting an anti-tumor effect. Alternatively, even if the antibody-drug conjugates that accumulate in tumor cells are not internalized, the tumor cells and / or the antibody-drug conjugates may be taken up by immune cells by phagocytosis, activating anti-tumor immunity through STING agonist activity and exerting an anti-tumor effect.

[0099] Antibody binding to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to visualize the antibody uptake by the cell under a fluorescent microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to measure the amount of fluorescence uptake by the cell (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released upon intracellular uptake, suppressing cell proliferation (BioTechniques 28:162-165, January 2000). A recombinant complex protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin.

[0100] When an antibody that targets tumor cells is used in the antibody-drug conjugate of the present invention, it is preferable, but not essential, that the antibody itself have an anti-tumor effect.

[0101] The antitumor activity of a drug or antibody-drug conjugate refers to cytotoxic activity against tumor cells, anticellular effect, and tumor volume regression. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems.

[0102] The effects and immunostimulatory activity of drugs and antibody-drug conjugates refer to the enhancement of tumor cell sensitivity to immune cells or tumor cell-mediated activation of immune cells. The effects and immunostimulatory activity of drugs and antibody-drug conjugates can be confirmed using known in vitro or in vivo evaluation systems.

[0103] Examples of in vitro or in vivo evaluation systems that can be used in the present invention include a co-culture assay system of CT26.WT and CT26.WT-hCD70 cell lines with mouse bone marrow-derived dendritic cells as described in Test Example 4, a BALB / c mouse system in which CT26.WT-hTROP2 cells, which are mouse colon cancer cell line CT26.WT introduced with the human TROP2 gene, are subcutaneously implanted as described in Test Example 5, a BALB / c mouse system in which CT26.WT-hEGFR cells, which are mouse colon cancer cell line CT26.WT introduced with the human EGFR gene, are subcutaneously implanted as described in Test Example 6, and a human kidney-derived dendritic cell system in which human kidney-derived dendritic cells are subcutaneously implanted as described in Test Example 7. Examples of the antibody-drug conjugate include, but are not limited to, a BALB / c-nu mouse strain subcutaneously implanted with the human renal cancer cell line Caki-1 cells, a BALB / c-nu mouse strain subcutaneously implanted with the human renal cancer cell line A-498 (HTB-44) cells as described in Test Example 8, and a BALB / c mouse strain subcutaneously implanted with the mouse colon cancer cell line CT26.WT (CRL2638) introduced with a gene for a human-mouse chimeric antigen in which the epitope site on the antigen to which the antibody contained in the antibody-drug conjugate binds has been replaced with a human type as described in Test Example 9.

[0104] Antibodies used in the present invention include anti-CD70 antibodies, anti-TROP2 antibodies, and anti-EGFR antibodies.

[0105] The antibodies used in the present invention can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies that are applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0106] Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennett, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)), and a monoclonal antibody can be obtained.

[0107] The antigen can be obtained by genetically manipulating a gene encoding the antigen protein in a host cell to produce it.

[0108] The antibodies used in the antibody-drug conjugates of the present invention can be obtained according to known methods (for example, Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525, WO90 / 07861).

[0109] For example, anti-CD70 antibodies (WO2004 / 073656, WO2007 / 038637, etc.), anti-TROP2 antibodies (WO2015 / 098099, etc.), and anti-EGFR antibodies (WO1998 / 050433, WO2002 / 092771, etc.) can be obtained by known means.

[0110] The anti-CD70 antibody used in the present invention is not particularly limited, but preferably has the following properties: (1) An anti-CD70 antibody that specifically binds to CD70. (2) The antibody according to (1) above, which binds to the extracellular domain of human CD70. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that reduces ADCC and ADCP activity. (7) The antibody according to (5) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (8) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1. (9) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3. (10) The antibody described in (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 40. (11) The antibody described in (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 46. (12) The antibody according to any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (13) An antibody obtained by a method for producing the antibody, comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above, and collecting the antibody of interest from the culture obtained in the step.

[0111] Examples of anti-CD70 antibodies include vorsetuzumab, MDX-1115, and cusatuzumab, and preferred examples include vorsetuzumab and MDX-1115.

[0112] The anti-TROP2 antibody used in the present invention is not particularly limited, but preferably has the following properties: (1) An anti-TROP2 antibody that specifically binds to TROP2. (2) The antibody described in (1) above, which binds to the extracellular domain of human TROP2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that reduces ADCC and ADCP activity. (7) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 5. (8) The antibody according to (5) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 7. (10) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 52. (11) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 58. (12) The antibody according to any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (13) An antibody obtained by a method for producing the antibody, comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above, and collecting the antibody of interest from the culture obtained in the step.

[0113] The anti-EGFR antibody used in the present invention is not particularly limited, but preferably has the following properties, for example: (1) Anti-EGFR antibodies that specifically bind to EGFR. (2) The antibody according to (1) above, which binds to the extracellular domain of human EGFR. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that reduces ADCC and ADCP activity. (7) The antibody according to (5) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (8) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9. (9) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11. (10) The antibody described in (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 61, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 64. (11) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 70. (12) The antibody according to any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (13) An antibody obtained by a method for producing the antibody, comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above, and collecting the antibody of interest from the culture obtained in the step.

[0114] Examples of anti-EGFR antibodies include panitumumab, nimotuzumab, cetuximab, ametuximab (SY-101), SYN-004, SCT-200, tomuzotuximab, GC-1118, GR-1401, and depatuxizumab (ABT-806), with panitumumab and ABT806 being preferred.

[0115] The antibody used in the present invention may have 80% to 99% amino acid identity with the heavy and / or light chains of the above-mentioned antibodies. Here, the term "identity" has the general definition used in the art. The percent identity refers to the percentage of identical amino acids per total number of amino acids (including gaps) when two amino acid sequences are aligned to maximize amino acid identity. Such identity is generally 80% or higher, preferably 90, 91, 92, 93, or 94% or higher, more preferably 95, 96, 97, or 98% or higher, and even more preferably 99% or higher. Furthermore, antibodies having various functions equivalent to those of the above-mentioned antibodies can be selected by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, and / or added to the amino acid sequences of the heavy and / or light chains. The number of amino acid residues to be substituted, deleted, and / or added is generally 10 amino acid residues or less, preferably 5 to 6 amino acid residues or less, more preferably 2 to 3 amino acid residues or less, and even more preferably 1 amino acid residue.

[0116] 2.2.2 Antibody Glycosylation Remodeling Recently, a method for remodeling heterogeneous antibody glycans by enzymatic reactions to uniformly introduce functionalized glycans has been reported (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Using this glycan remodeling technique, attempts have also been made to site-specifically introduce drugs and synthesize homogeneous ADCs (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0117] Glycan remodeling involves first using a hydrolase to remove heterogeneous glycans attached to a protein (e.g., an antibody) leaving only the terminal GlcNAc, thereby preparing a homogeneous protein portion with GlcNAc attached (hereafter referred to as the "acceptor"). Next, a separately prepared glycan of your choice (hereafter referred to as the "donor") is prepared, and this acceptor and donor are linked using a glycosyltransferase. This allows the synthesis of a homogeneous glycoprotein with a desired glycan structure.

[0118] In the present invention, the term "glycan" refers to a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and glycans may be represented by abbreviations such as "GlcNAc-" or "SG-." When these abbreviations are used in structural formulae, the oxygen or nitrogen atom at the reducing end that forms a glycosidic bond with another structural unit is not included in the abbreviation representing the glycan, unless otherwise defined.

[0119] In the present invention, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described by assuming that the carbon atom bonded to the oxygen atom constituting the ring and directly bonded to a hydroxy group (or an oxygen atom belonging to a glycosidic bond) in the ring structure is at position 1 (position 2 only in sialic acid). The names of the example compounds are given based on the entire chemical structure, and this rule does not necessarily apply.

[0120] In the present invention, when a sugar chain is described as a symbol (e.g., SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon atom at the reducing end, and does not include the N or O attributable to the N- or O-glycosidic bond.

[0121] The antibody drug conjugate of the present invention has the following formula:

[0122] [ka] The antibody Ab or a functional fragment thereof is bound to L either directly from the side chain of its amino acid residue (e.g., cysteine, lysine, etc.) or via a glycan or remodeled glycan of the Ab.

[0123] The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

[0124] N-linked glycans are bound to amino acid side chains of antibodies via N-glycosidic bonds, and O-linked glycans are bound to amino acid side chains of antibodies via O-glycosidic bonds.

[0125] The Ab in the present invention is an IgG, preferably an IgG1, IgG2, or IgG4.

[0126] IgG has a well-conserved N-linked glycan (hereinafter referred to as "Asn297 glycan or N297 glycan") at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which is known to contribute to the activity and kinetics of antibody molecules (Eon-Duval, A. et al., Biotechnol. Prog. 2012, 28, 608-622; Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0127] The amino acid sequence in the constant region of IgG is well conserved, and in a report by Edelman et al. (Proc. Natl. Acad. Sci. USA, 63, 78-85, (1969)), each amino acid is identified by an EU number (EU INDEX). For example, Asn297, to which N-linked glycosylation is added in the Fc region, corresponds to position 297 in the EU numbering. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by indicating it by EU numbering.

[0128] The diagram below shows the case where the antibody-drug conjugate of the present invention is bound to L from the N297 sugar chain of the antibody or functional fragment thereof.

[0129] [ka] An antibody having such a remodeled sugar chain is called a sugar chain remodeling antibody.

[0130] SGP (α2,6-SGP) is an abbreviation for sialyglycopeptide and is a representative N-linked glycopeptide. SGP can be isolated and purified from chicken egg yolk, for example, according to the method described in WO2011 / 027868. Purified SGP products are also commercially available from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd. In this specification, the glycan portion of SGP is referred to as SG, and a glycan lacking one GlcNAc at the reducing end of SG is referred to as SG(10). SG(10) can be prepared by enzymatic hydrolysis of SGP, for example, according to the report by Umekawa et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209). SG(10) can also be purchased from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd.

[0131] In this specification, the glycan structure in which sialic acid at the non-reducing end of only one of the branched β-Man chains of SG(10) is deleted is referred to as MSG(9), the one with sialic acid only on the 1-3 glycan of the branched chain is referred to as MSG1, and the one with sialic acid only on the 1-6 glycan of the branched chain is referred to as MSG2.

[0132] The remodeled sugar chains used in the antibody-drug conjugates of the present invention are N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, preferably N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1.

[0133] N297-(Fuc)SG is represented by the following structural formula or sequence formula:

[0134] [ka]

[0135] [ka]

[0136] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0137] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula:

[0138] [ka]

[0139] [ka]

[0140] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0141] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula:

[0142] [ka]

[0143] [ka]

[0144] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5-CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0145] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which four linkers L and four drugs D are bound (the above m 2 =2).

[0146] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which two linkers L and two drugs D are bound (the above m 2 =1) (see Figure 1).

[0147] The N297 sugar chain is preferably N297-(Fuc)SG or N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1, and even more preferably N297-(Fuc)SG.

[0148] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, a highly homogeneous ADC can be obtained.

[0149] <3. Manufacturing method> Representative methods for producing antibody-drug conjugates containing the novel CDN derivatives of the present invention or their production intermediates are described below. In the following, the compound numbers shown in each reaction scheme are used to indicate the compounds. That is, they are referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers are also referred to in the same manner.

[0150] In the following Methods A to E, the substituent L 1 has the same meaning as defined above. 2 is either (i) or (ii) below: (i) When combined with L, L 2 represents -NHR', a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group, where R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group is optionally substituted with 1 to 6 halogen atoms; or (ii) When not bonded to L, L 2 represents a hydrogen atom or a halogen atom, The substituent W represents a group selected from 1 represents -NH- or a sulfur atom. 2 represents -CH=. Substituent Z 1 ~Z 3 together represent -CH2-CH2-CH2-. 1 ~R 3 are each independently a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N3, -NHR', -NR'R'', or -NHC(=O)R' (wherein R' is as defined above and R'' represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group). 4 represents a hydrogen atom. 5 is W 1 When is a nitrogen atom, R 5 indicates a hydrogen atom, and W 1 When is an oxygen atom, R 5 does not exist. Substituent Ra , R c , R e and R g represents the side chain of a natural α-amino acid, such as a methyl group, an isopropyl group, a sec-butyl group, an isobutyl group, or a benzyl group. 1 represents a protecting group for a primary alcohol. Preferred examples include a 4,4'-dimethoxytrityl group and a 4-methoxytrityl group. PRO 2 , PRO 3 , PRO 7 , PRO 8 represents a protecting group for a secondary alcohol. Preferred examples include a tert-butyldimethylsilyl group, a triisopropylsilyloxymethyl group, a benzoyl group, a 2-nitrobenzyl group, and a 4-methoxytetrahydropyran-4-yl group. PRO 6 represents a protecting group for carboxylic acid. Preferred is a tert-butyl group, a benzyl group, etc. 5 , PRO 9 indicates an amine protecting group. 5 is preferably a tert-butyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, a benzyloxycarbonyl group, or the like, and PRO 9 is preferably a 9-fluorenylmethyloxycarbonyl group or a 2-(trimethylsilyl)ethoxycarbonyl group. 4 represents a protecting group for an alcohol or amine. In the case of an alcohol, preferred protecting groups include a tert-butyldimethylsilyl group, a benzoyl group, etc., and in the case of an amine, preferred protecting groups include a 2-(trimethylsilyl)ethoxycarbonyl group, an allyloxycarbonyl group, a tert-butyloxycarbonyl group, etc. a represents an oxygen atom or a sulfur atom, and Q b indicates a hydroxyl group or a thiol group. a’ and Q b’ are each independently a negatively charged oxygen atom (O - ) or sulfur atom (S - ) indicates R x and R y each independently represents a halogen atom or -O-PRO2 n is an integer of 1 to 3.

[0151] A method The CDN derivative represented by (1) used in the antibody-drug conjugate of the present invention can be produced according to Method A described below.

[0152] [ka]

[0153] This production method is a method for producing a compound represented by general formula (1). Steps A-1 to A-5 of this production method can be performed as a one-pot synthesis, which can be carried out by referring to the report by Gaffney et al. (Org. Lett. 2010, 12, 3269-3271).

[0154] [ka]

[0155] (A-1 process) This step involves sequentially hydrolyzing compound (1a) and removing the cyanoethyl group using known organic chemistry techniques to produce compound (2a). Compound (1a) was hydrolyzed in a solvent (acetonitrile, tetrahydrofuran, N,N-dimethylformamide, or a mixture thereof) at temperatures ranging from -10°C to the boiling point of the solvent, preferably 15°C to 35°C, by treatment with water and an acid (pyridine trifluoroacetate, 4,5-dicyanoimidazole, 1H-tetrazole, etc.). Water was used in an amount ranging from 2 moles to an excess, preferably 2 to 10 moles, per mole of compound (1a), and acid was used in an amount ranging from 1 mole to an excess, preferably 1 to 5 moles. The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. A base (tert-butylamine, etc.) was then added to the reaction mixture to remove the cyanoethyl group. The base was used in excess molar amount, preferably 30 to 50 moles, relative to 1 mole of compound (1a). The reaction time was 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction solution was concentrated under reduced pressure to obtain crude compound (2a). The crude compound (2a) can be used in the next step without purification.

[0156] (A-2 process) In this step, the protecting group of the hydroxy group is removed from the compound of formula (2a) using a known organic chemistry method to produce a compound of formula (3a). Before starting the reaction of this step, the crude product of formula (2a) was dried by azeotropy with acetonitrile one to three times, if necessary. PRO 1When 2a is a 4,4'-dimethoxytrityl group, the 4,4'-dimethoxytrityl group was removed by treating compound (2a) with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at temperatures ranging from -10°C to the boiling point of the reaction solvent, preferably 15°C to 35°C. Water was used in excess, preferably 10 to 20 moles, per mole of compound (2a). The acid was diluted with the reaction solvent to 1% to 50% (v / v), preferably 5% to 10% (v / v), and the diluted solution was used in excess, preferably 5 to 15 moles. The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. The reaction was quenched by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to neutralize the acid, preferably 2 to 10 moles per mole of acid. The reaction mixture was concentrated under reduced pressure to obtain crude compound (3a). The crude compound (3a) was azeotroped three to five times with dehydrated acetonitrile. The acetonitrile was left over from the final azeotrope to obtain a 0.01M to 1M acetonitrile solution of compound (3a). The resulting acetonitrile solution was used directly in the next step.

[0157] (A-3 process) This step involves the preparation of a compound of formula (5a) by sequentially coupling a compound of formula (3a) with a compound of formula (4a) and sulfurizing the resulting coupling product using known organic chemistry techniques. Prior to the start of this step, compound (4a) was azeotropically distilled three to five times with dehydrated acetonitrile. A 0.01 M to 1 M acetonitrile solution of compound (4a) was prepared by leaving acetonitrile after the final azeotropic distillation. A desiccant (powdered or pelleted molecular sieves 3A or molecular sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use. The coupling reaction was carried out by adding the azeotropically dried acetonitrile solution of compound (4a) to the acetonitrile solution of compound (3a) at 5°C to 35°C. The reaction time was 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, a sulfurizing agent (N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, 3H-1,2-benzodithiol-3-one, etc.) was added to the reaction mixture to carry out a sulfurization reaction. The sulfurizing agent was used in an amount of 1 to 5 moles, preferably 1 to 2 moles, per mole of compound (3a). The reaction time was 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction mixture was concentrated under reduced pressure to obtain crude compound (5a). The crude compound (5a) obtained was used directly in the next step.

[0158] (A-4 process) In this step, the protecting group of the hydroxy group is removed from the compound of formula (5a) using a known organic chemistry method to produce a compound of formula (6a). 1When the 4,4'-dimethoxytrityl group is present, compound (5a) is treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at temperatures ranging from -10°C to the boiling point of the reaction solvent, preferably 15°C to 35°C, to remove the 4,4'-dimethoxytrityl group. Water is used in excess, preferably 10 to 20 moles, per mole of compound (5a). The acid is diluted with the solvent to 1% to 50% (v / v), preferably 5% to 10% (v / v), and the diluted solution is used in excess, preferably 5 to 15 moles. The reaction time is 1 minute to 3 hours, preferably 5 to 30 minutes. The reaction was quenched by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to neutralize the acid, preferably 10 to 200 moles per mole of acid. The reaction mixture was concentrated under reduced pressure to give crude compound (6a), which was used directly in the next step.

[0159] (A-5 process) In this step, a compound of formula (6a) is subjected to a cyclization reaction and a sulfurization reaction in succession using known organic chemistry techniques to produce a compound of formula (7a). Compound (6a) is dissolved in pyridine and then concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. This pyridine solution is added with a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) at 5°C to 35°C. 5The cyclization reaction was carried out by adding a sulfurizing agent (e.g., 3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, etc.) to the reaction mixture. The dehydration condensation agent was used in an amount of 1 mole to an excess of 1 mole, preferably 3 to 5 moles, per mole of compound (6a). The reaction time was 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and a sulfurizing agent (e.g., 3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, etc.) were added to the reaction mixture to carry out a sulfurization reaction. The amount of water used was in excess of 1 mole, preferably 30 to 50 moles, per mole of compound (6a), and the amount of sulfurizing agent used was 1 mole to 5 moles, preferably 1 to 2 moles. The reaction time was 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction mixture was added to an aqueous sodium bicarbonate solution (0.1 M to 1 M), and the mixture was stirred for 15 minutes to 24 hours to terminate the reaction. The reaction mixture was extracted one to five times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixture thereof), and the combined extracts were dried over anhydrous sodium sulfate or magnesium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol, ethyl acetate / methanol, hexane / ethyl acetate, etc.), C18 silica gel column chromatography (buffer / acetonitrile), or a combination thereof to obtain compound (7a) as a mixture of two or more diastereomers or as two or more pure diastereomers. This step generally yields two diastereomers, but depending on the starting materials (1a) and (4a), one or two additional diastereomers may also be obtained. Even if the resulting compound (7a) is a mixture of multiple diastereomers, it can be carried on to the next step without further purification.

[0160] (A-6 process) In this step, the cyanoethyl group and all acyl protecting groups are simultaneously removed from the compound of formula (7a) using a known organic chemistry method to produce the compound of formula (8a). This step was carried out in an autoclave or sealed tube as necessary. PRO 4When is a benzoyl group, compound (7a) was treated with 28% (v / v) aqueous ammonia in a solvent (methanol, ethanol, tetrahydrofuran, or a mixture thereof) at temperatures between 5°C and the boiling point of the solvent used in the reaction to remove the cyanoethyl and benzoyl groups. Ammonia was used in excess molar amount, preferably 300 to 3000 moles, per mole of compound (7a). The reaction time was 30 minutes to 96 hours, preferably 2 to 48 hours. If necessary, the reaction mixture was concentrated, and the residue was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof to obtain compound (8a). Even if the resulting compound (8a) is a diastereomeric mixture, it can be advanced to the next step without further purification. Alternatively, it can be advanced to the next step without further purification in this step.

[0161] (A-7 process) In this step, all silyl protecting groups are simultaneously removed from the compound of formula (8a) using a known organic chemistry method to produce the compound of formula (9a). 2 and PRO 3When is a tert-butyldimethylsilyl group, compound (8a) was directly treated with triethylamine trihydrofluoride at 5°C to 100°C, preferably 35°C to 60°C, to remove the tert-butyldimethylsilyl group. Triethylamine trihydrofluoride was used in excess, preferably 100 to 200 moles, per mole of compound (8a). The reaction time was 30 minutes to 24 hours, preferably 2 to 12 hours. After cooling the reaction solution to room temperature, the reaction was quenched by gradually pouring into an ice-cold mixture of 1 M aqueous triethylammonium bicarbonate and triethylamine (3:1 to 10:1 (v / v)). If necessary, the reaction solution may be poured into an ice-cold mixture of 1 M aqueous triethylammonium bicarbonate and triethylamine. In this case, the reaction vessel was washed with acetonitrile and water. Triethylamine is used in an amount sufficient to change the pH of the reaction mixture to weakly basic, preferably about 2 moles of triethylamine per mole of triethylamine trihydrofluoride. After the organic solvent component of the reaction mixture was distilled off under reduced pressure, the remaining aqueous solution was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof to obtain compound (9a) as a single diastereomer.

[0162] (A-8 process) This step involves ion-exchanging the compound of formula (9a) using a known organic chemistry technique to produce the compound of formula (1). A cation exchange resin (BT AG® 50W-X2 resin, 100-200 mesh, hydrogen type) was suspended in purified water and packed into an empty column cartridge. The cation exchange resin was used in an amount 10 to 50 times the weight of the compound (9a). After allowing excess purified water to flow by gravity, 3 column volumes of 1 M aqueous sodium hydroxide were added by gravity, followed by 6 column volumes of purified water. Compound (9a) was dissolved in approximately 3 column volumes of purified water and charged onto the column. If the compound is poorly soluble in purified water, a mixture with a small amount of organic solvent (e.g., acetonitrile, methanol) may be used. The solution that flowed by gravity was collected and then eluted with 6 column volumes of purified water, etc., to obtain the desired fractions. The fractions containing the target product were combined and lyophilized to obtain compound (1) as a single diastereomer.

[0163] A' method The CDN derivative represented by (1') used in the antibody-drug conjugate of the present invention can be produced according to Method A' described below.

[0164] [ka]

[0165] This production method is a method for producing a compound represented by general formula (1') by partially modifying Method A. Specifically, the compound of general formula (1') can be produced by modifying step A-5 of Method A to step A'-5 shown below. x and R y When both are halogen atoms, step A-7 can be omitted.

[0166] [ka]

[0167] (A'-5 process) In this step, a compound of formula (6a') is subjected to a cyclization reaction and an oxidation reaction in succession using known organic chemistry techniques to produce a compound of formula (7a'). Compound (6a') is dissolved in pyridine and then concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. This pyridine solution is added with a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) at 5°C to 35°C. 5 A cyclization reaction was carried out by adding an oxidizing agent (e.g., -dioxaphosphinan-2-one) to the reaction mixture. The dehydration condensation agent was used in an amount of 1 mole to an excess mole, preferably 3 to 5 moles, relative to 1 mole of compound (6a'). The reaction time was 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and an oxidizing agent (e.g., iodine) were added to the reaction mixture to carry out an oxidation reaction. Water was used in an amount of 0 mole to an excess mole, preferably 30 to 50 moles, relative to 1 mole of compound (6a'), and the oxidizing agent was used in an amount of 2 moles to 10 moles, preferably 3 to 5 moles. The reaction time was 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction mixture was added to an aqueous sodium bicarbonate solution (0.1 M to 1 M) and stirred for 15 minutes to 24 hours to quench the reaction. The reaction mixture was extracted once to five times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixture thereof), and the combined extracts were dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol, ethyl acetate / methanol, hexane / ethyl acetate, etc.), C18 silica gel column chromatography (buffer / acetonitrile), or a combination thereof to obtain compound (7a').

[0168] A'' method The CDN derivative represented by (1″) used in the antibody-drug conjugate of the present invention can be produced according to Method A″ described below.

[0169] [ka]

[0170] This production method is a method for producing a compound represented by general formula (1'') by partially modifying Method A. Specifically, the compound of general formula (1'') can be produced by modifying Step A-3 of Method A to Step A''-3 shown below. In addition, when the substituent R x and R y When both are halogen atoms, step A-7 can be omitted.

[0171] [ka]

[0172] (A''-3 process) This step is a step of producing a compound of formula (5a'') by sequentially carrying out a coupling reaction of a compound of formula (3a'') with a compound of formula (4a'') using known organic chemistry techniques and an oxidation reaction of the resulting coupling product. Before starting the reaction of this step, compound (4a'') was azeotropically distilled three to five times with dehydrated acetonitrile. After the final azeotropic distillation, acetonitrile was left over to prepare a 0.01 M to 1 M acetonitrile solution of compound (4a''). A desiccant (powdered or pelleted molecular sieves 3A or molecular sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use. The coupling reaction was carried out by adding the acetonitrile solution of compound (4a'') dried by azeotropy to the acetonitrile solution of compound (3a'') at 5 to 35°C. The reaction time was 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, an oxidizing agent (such as tert-butyl hydroperoxide) was added to the reaction mixture to carry out an oxidation reaction. The oxidizing agent was used in an amount of 1 to 5 moles, preferably 2 to 3 moles, per mole of compound (3a''). The reaction time was 5 minutes to 24 hours, preferably 30 minutes to 6 hours. A saturated aqueous solution of sodium thiosulfate was added to the reaction mixture, and the mixture was stirred for 10 minutes to 12 hours to terminate the reaction. The reaction mixture was extracted once to five times with an organic solvent (such as a mixed solvent of dichloromethane and methanol), and the combined extracts were dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain crude compound (5a''). The crude compound (5a'') obtained was directly used in the next step.

[0173] A''' method The CDN derivative represented by (1''') used in the antibody-drug conjugate of the present invention can be produced according to Method A''' described below.

[0174] [ka]

[0175] This production method is a method for producing a compound represented by general formula (1''') by partially modifying Method A. Specifically, the compound of general formula (1''') can be produced by changing Step A-3 of Method A to Step A''-3 and Step A-5 to Step A'-5. In addition, the substituent R x and R y When both are halogen atoms, step A-7 can be omitted.

[0176] [ka]

[0177] Method B: Conjugation precursor (glycan conjugation) The conjugation precursor represented by (2) used in the antibody-drug conjugate of the present invention can be produced according to Method B described below.

[0178] [ka]

[0179] This manufacturing method is L 1 This is a method for producing a conjugation precursor (2) in which -NH2 is substituted at any position of the above.

[0180] [ka]

[0181] (B-1 process) In this step, the protecting group of the compound of formula (1b) is removed using a known organic chemistry method to produce the compound of formula (2b). 5When is a tert-butyloxycarbonyl group, the protecting group was removed by treating compound (1b) with trifluoroacetic acid in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixture thereof) at temperatures ranging from −10°C to the boiling point of the solvent used in the reaction, preferably from 15°C to 35°C. Trifluoroacetic acid was used in excess, preferably 20 to 50 moles, per mole of compound (1b). The reaction time ranged from 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure, suspended in toluene, and then concentrated again under reduced pressure. This procedure was repeated two to five times. A solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate, or a mixture thereof) was added to form a slurry, and the solid was collected by filtration to obtain crude compound (2b). The crude compound (2b) was carried on to the next step without further purification.

[0182] (B-2 process) In this step, compound (2b) is amidated with compound (3b) using known organic chemistry techniques to produce compound (4b). Compound (2b) was amidated by reacting it with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (3b) in a solvent (N,N-dimethylformamide, N-methylpyrrolidone, N,N-dimethylacetamide, acetonitrile, etc.) at 5°C to 35°C. Relative to 1 mole of compound (2b), 1 mole to 5 moles of base and 0.5 moles to 1.5 moles of compound (3b) were used. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction mixture was poured into a bilayer consisting of an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol, or a mixture thereof) and water or an acidic aqueous solution (0.1 to 1 M hydrochloric acid, citric acid, etc.), followed by extraction with the organic solvent one to five times. The combined extracts were washed with saturated saline and then dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. Alternatively, the above-mentioned separation procedure can be omitted, and the reaction mixture can be directly concentrated under reduced pressure and subjected to subsequent silica gel column purification. The resulting residue was purified by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, etc.] to obtain compound (4b). If necessary, the purity can be increased by dissolving the obtained compound (4b) in a good solvent (ethyl acetate, acetonitrile, dichloromethane, methanol, or a mixture thereof) and then adding a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) to cause reprecipitation, and then filtering the solid.

[0183] (B-3 process) This step involves esterifying compound (4b) using a known organic chemistry method to produce compound (5b). Compound (4b) was esterified by reacting it with N-hydroxysuccinimide and a condensing agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, etc.) in a solvent (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at 5°C to 35°C. N-hydroxysuccinimide and the condensing agent were used in amounts of 1 to 3 moles per mole of compound (4b). The reaction time was 30 minutes to 72 hours, preferably 2 to 24 hours. The reaction solution was diluted with an organic solvent (e.g., dichloromethane, chloroform, ethyl acetate, or a mixture thereof) and then washed with ice water three to five times. The organic layer was dried over anhydrous salts (e.g., anhydrous sodium sulfate or anhydrous magnesium sulfate). After filtering off the drying agent, the filtrate was concentrated under reduced pressure to obtain crude compound (5b). If necessary, the resulting compound (5b) may be purified by C18 silica gel column chromatography (acetonitrile only). Alternatively, the purity can be increased by dissolving the resulting compound (5b) in a good solvent (ethyl acetate, acetonitrile, dichloromethane, or a mixture thereof) and then reprecipitating it with a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.). The solid can then be filtered off.

[0184] (B-4 process) This step involves the production of a compound of formula (2) by condensing a compound of formula (5b) with a compound of formula (6b) using known organic chemistry techniques. The condensation reaction was carried out by reacting compound (6b) with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (5b) in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at temperatures ranging from -10°C to 100°C, preferably from 15°C to 35°C. Two to five moles of base and one to two moles of compound (5b) were used per mole of compound (6b). The reaction time was 5 minutes to 24 hours, preferably 1 to 6 hours. The reaction was quenched by adding benzylamine to the reaction mixture. Four to 10 moles of benzylamine were used per mole of compound (6b). If necessary, the reaction mixture was partially concentrated under reduced pressure, and the residual solution was purified by preparative HPLC [buffer / acetonitrile, buffer / methanol, etc.], C18 silica gel column chromatography [buffer / acetonitrile, buffer / methanol, etc.], or a combination thereof to obtain compound (2).

[0185] Method B': Conjugation precursor (cysteine ​​conjugation) The conjugation precursor represented by (2') used in the antibody-drug conjugate of the present invention can be produced according to Method B' described below.

[0186] [ka]

[0187] This manufacturing method is L 1 is a method for producing a conjugation precursor (2') in which -NH2 is substituted at any position of the above.

[0188] [ka]

[0189] (B-5 process) In this step, a compound of formula (2b') is amidated with a compound of formula (7b) using a known organic chemistry method to produce a compound of formula (8b). Compound (8b) was obtained according to the method described in Step B-2 of Method B, except that a base was not used.

[0190] (B-6 process) In this step, the compound of formula (9b) is produced by removing the protecting group from the compound of formula (8b) using a known organic chemistry method. 6 When is a tert-butyl group, compound (9b) was obtained according to the procedure described in Step B-1 of Method B, except that silica gel column chromatography [dichloromethane / methanol] was used for purification.

[0191] (B-7 process) In this step, the compound of formula (9b) is esterified using a known organic chemistry method to produce the compound of formula (10b). Compound (10b) was obtained according to the method described in Step B-3 of Method B.

[0192] (B-8 process) In this step, a compound of formula (6b) is condensed with a compound of formula (10b) using a known organic chemistry method to produce a compound of formula (2'). Compound (2') was obtained according to the method described in Step B-4 of Method B.

[0193] C method The conjugation precursor represented by (3) used in the antibody-drug conjugate of the present invention can be produced according to Method C described below.

[0194] [ka]

[0195] This manufacturing method is L 1The present invention relates to a method for producing a conjugation precursor (3) in which a hydroxy group is substituted at any position of the above.

[0196] [ka]

[0197] (C-1 process) In this step, a compound of formula (1c) is amidated with a compound of formula (2c) using a known organic chemistry method to produce a compound of formula (3c). Compound (3c) was obtained according to the method described in Step B-2 of Method B.

[0198] (C-2 process) In this step, the compound of formula (3c) is esterified using a known organic chemistry method to produce the compound of formula (4c). Compound (4c) was obtained according to the method described in Step B-3 of Method B.

[0199] (C-3 process) In this step, a compound of formula (7c) is produced by sequentially carrying out a coupling reaction (aminomethylenation) of a compound of formula (5c) with a compound of formula (6c) using a known organic chemistry technique and deprotecting the resulting coupling product. 9When R is a 9-fluorenylmethyloxycarbonyl group, aminomethylenation was carried out by reacting compound (5c) with compound (6c) and an acid (e.g., p-toluenesulfonic acid) in tetrahydrofuran at 5°C to 35°C. Compound (6c) was used in an amount of 1 to 20 moles, preferably 2 to 10 moles, per mole of compound (5c), and acid was used in an amount of 0.05 moles to an excess of molar, preferably 0.1 to 3 moles. The reaction time was 30 minutes to 72 hours, preferably 2 to 24 hours. Next, a base (e.g., 1,8-diazabicyclo[5.4.0]-7-undecene) was added to the reaction mixture to carry out deprotection. If the reaction mixture was suspended, a solvent (e.g., N,N-dimethylformamide) could be added to dissolve the mixture before reaction, as necessary. The base was used in an excess of molar amounts, preferably 5 to 20 moles, per mole of compound (5c). The reaction time is 10 minutes to 24 hours, preferably 2 to 12 hours. Water is added to the reaction mixture, which is then purified by C18 silica gel column chromatography (buffer solution / acetonitrile, etc.) to obtain compound (7c).

[0200] (C-4 process) In this step, the protecting group is removed from the compound of formula (7c) using a known organic chemistry method to produce a compound of formula (8c). 7 and PRO 8 When is a tert-butyldimethylsilyl group, compound (8c) was obtained according to the procedure described in Step A-7 of Method A.

[0201] (C-5 process) In this step, a compound of formula (8c) is condensed with a compound of formula (4c) using a known organic chemistry method to produce a compound of formula (3). Compound (3) was obtained according to the method described in Step B-4 of Method B.

[0202] C' method The conjugation precursor represented by (3') used in the antibody-drug conjugate of the present invention can be produced according to Method C' described below.

[0203] [ka]

[0204] This manufacturing method is L 1 The present invention relates to a method for producing a conjugation precursor (3') in which a hydroxy group is substituted at any position of the above.

[0205] [ka]

[0206] (C'-1 process) In this step, the compound of formula (1c') is subjected to hydrolysis and removal of the cyanoethyl group in succession using known organic chemistry techniques to produce the compound of formula (2c'). Compound (2c') was obtained according to the method described in Method A, Step A-1.

[0207] (C'-2 process) In this step, the protecting group of the hydroxy group of the compound of formula (2c') is removed using a known organic chemistry method to produce a compound of formula (3c'). Compound (3c') was obtained according to the method described in Step A-2 of Method A.

[0208] (C'-3 process) In this step, compound (3c') is subjected to a coupling reaction with compound (4c') using known organic chemistry techniques, followed by a sulfurization or oxidation reaction of the resulting coupling product, to produce compound (5c'). Compound (5c') was obtained according to the method described in Step A-3 of Method A or Step A''-3 of Method A''.

[0209] (C'-4 process) In this step, the protecting group of the hydroxy group of compound (5c') is removed using a known organic chemistry method to produce compound (6c'). Compound (6c') was obtained according to the method described in Step A-4 of Method A.

[0210] (C'-5 process) In this step, compound (6c') is subjected to a cyclization reaction and a sulfurization or oxidation reaction in succession using known organic chemistry techniques to produce compound (7c'). Compound (7c') was obtained according to the method described in Step A-5 of Method A or Step A'-5 of Method A'.

[0211] (C'-6 process) In this step, the cyanoethyl group and all acyl protecting groups are simultaneously removed from compound (7c') using a known organic chemistry method to produce compound (8c'). Compound (8c') was obtained according to the method described in Step A-6 of Method A.

[0212] (C'-7 process) In this step, all silyl protecting groups are simultaneously removed from the compound of formula (8c') using a known organic chemistry method to produce the compound of formula (9c'). 9 When is a 2-(trimethylsilyl)ethoxycarbonyl group, compound (8c') was treated with a tetrahydrofuran solution of tetrabutylammonium fluoride at 5°C to 100°C, preferably 35°C to 60°C, to remove the 2-(trimethylsilyl)ethoxycarbonyl group. Tetrabutylammonium fluoride was used in excess, preferably 10 to 30 moles, per mole of compound (8c'). The reaction time was 1 to 48 hours, preferably 4 to 24 hours. After diluting the reaction mixture with a buffer solution, the organic solvent was removed under reduced pressure, if necessary. The residue was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof, to obtain compound (9c').

[0213] (C'-8 process) In this step, a compound of formula (9c') is condensed with a compound of formula (4c) using a known organic chemistry method to produce a compound of formula (3'). Compound (3') was obtained according to the method described in Step B-4 of Method B.

[0214] Method D: Production of glycosylation remodeling antibodies The glycan remodeling antibody can be produced by the method shown in the following scheme, for example, in accordance with the method described in WO2018 / 003983.

[0215] [ka]

[0216] (D-1 process) This step involves hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing-end chitobiose structure of the N-linked glycan (N297-linked glycan) attached to asparagine at position 297 of the antibody's amino acid sequence, using a known enzymatic reaction, to produce a glycosylated antibody. The target antibody (1d) (10 mg / mL) was placed in a buffer (e.g., phosphate buffer) at 0°C to 40°C, and the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing-end chitobiose structure was hydrolyzed using a hydrolase such as wild-type EndoS enzyme. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. The wild-type EndoS enzyme was used in an amount of 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, per 100 mg of antibody (1d). After the reaction was completed, the antibody was purified using affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and / or a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (BIO-RAD)) to obtain (Fucα1,6)GlcNAc antibody (2d).

[0217] (D-2 process) This step involves conjugating an SG-type or MSG (MSG1, MSG2)-type glycan oxazoline having an azide group-containing PEG linker (hereinafter referred to as "azidoglycan oxazoline") to the (Fucα1,6)GlcNAc antibody (2d) obtained in step D-1 using a known enzymatic reaction to produce a glycan remodeling antibody (3d).

[0218] Antibody (2d) was reacted with an azido-glycosylated oxazoline derivative in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L) in a buffer solution (e.g., phosphate buffer) at temperatures ranging from 0°C to 40°C to carry out the glycosylation reaction. The reaction time ranged from 10 minutes to 72 hours, preferably 1 to 6 hours. EndoS enzyme (D233Q / Q303L) was used in an amount of 1 mg to 10 mg, preferably 1 to 3 mg, per 100 mg of antibody. The azido-glycosylated oxazoline derivative was used in an excess amount, preferably 4 to 20 equivalents. After the reaction, the antibody was purified using affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (Bio-Rad)) to obtain glycosylated remodeling antibody (3d).

[0219] In the preparation of the above-mentioned glycosylation-remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the common procedures A to C described below.

[0220] The SG-type azide sugar chain oxazoline compound was synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]2-SG(10)-Ox (compound 1-10 described in WO2018 / 003983) is shown in the following scheme.

[0221] [ka]

[0222] The MSG-type azidoglycan oxazoline derivative was also synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]-MSG1(9)-Ox (compound 1-11 described in WO2018 / 003983) is shown in the following scheme.

[0223] [ka] Method E: Antibody-drug conjugation (glycoconjugation 1)

[0224] [ka] (Here, the two asterisks (*) on the left side of antibody drug conjugate (1e) indicate the drug linker moiety indicated by the asterisk on the right side.) This production method involves the conjugation of the glycosylated remodeling antibody (3d) obtained in step D-2 of Method D with the conjugation precursor (2) obtained in step B-4 of Method B via the SPAAC (strain-promoted azide-alkyne cycloaddition: J. Am. Chem. Soc. 2004, 126, 15046-15047) reaction to produce the antibody-drug conjugate (1e).

[0225] (E-1 process) The SPAAC reaction was carried out by mixing a buffer solution of the glycosylation-remodeling antibody (3d) (e.g., phosphate buffer, acetate buffer, borate buffer, etc.) with a solution of the conjugation precursor (2) dissolved in an appropriate solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixture thereof). The conjugation precursor (2) was used in an amount of 2 moles to a molar excess, preferably 4 to 30 moles, per mole of glycosylation-remodeling antibody (3d). The organic solvent ratio relative to the antibody buffer solution was preferably 1% to 200% (v / v). The reaction temperature was 0°C to 37°C, preferably 15°C to 25°C, and the reaction time was 1 hour to 150 hours, preferably 6 hours to 72 hours. The pH of the reaction solution was preferably 5 to 9. The reaction solution was purified according to the method described in Common Procedure D below to obtain the antibody-drug conjugate (1e).

[0226] Method E': Antibody-drug conjugation (cysteine ​​conjugation) The antibody-drug conjugate of the present invention having a cysteine ​​conjugation can be produced in accordance with the method described in WO2014 / 057687, etc., using the antibody of interest prepared according to Reference Example 3, etc., and the conjugation precursor (2') having a maleimide group obtained in Step B-8 of Method B'.

[0227] E'' Method: Antibody-Drug Conjugation (Sugar Conjugation 2) In Method E, the conjugation precursor (2) was converted to the conjugation precursor (3') obtained in Step C'-8 of Method C' to obtain the antibody-drug conjugate (1e'') shown in the following formula.

[0228] [ka] (where the two asterisks (*) on the left of the antibody-drug conjugate (1e'') 1 ) shows the drug linker moiety indicated by an asterisk on the right.

[0229] The antibody-drug conjugate can be identified by buffer exchange, purification, measurement of antibody concentration, and measurement of the average number of drugs bound per antibody molecule according to the common procedures D to G described below.

[0230] Common Procedure A: Concentration of antibody aqueous solution The antibody or antibody-drug conjugate solution was placed in an Amicon® Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and the antibody or antibody-drug conjugate solution was concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000 to 4000 G for 5 to 20 minutes.

[0231] Common Procedure B: Antibody concentration measurement Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The 280 nm extinction coefficient (1.3 mL mg) varied depending on the antibody. -1 cm -1 to 1.8 mL mg -1 cm -1 ) was used.

[0232] Common Procedure C: Antibody Buffer Exchange A buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to the aqueous antibody solution, and the solution was concentrated according to the method described in Common Procedure A. This procedure was repeated several times, and then the antibody concentration was measured according to the method described in Common Procedure B. An appropriate buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to this antibody buffer solution to prepare an antibody buffer solution of the desired concentration (for example, approximately 10 mg / mL).

[0233] Common Procedure D: Purification of Antibody-Drug Conjugates (Gel Filtration Chromatography) A NAP column (NAP-5, NAP-10, NAP-25 (GE Healthcare)) was equilibrated with acetate buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5; herein referred to as ABS) or another appropriate buffer. The antibody-drug conjugate reaction solution was charged onto this NAP column, and the manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This fraction was again charged onto the NAP column, and the manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This procedure was repeated two to three times to obtain an antibody-drug conjugate from which unbound drug linker, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-drug conjugate solution was adjusted using common procedures A and C.

[0234] Common Procedure E: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (UV method) The bound drug concentration in an antibody-drug conjugate can be calculated by measuring the absorbance of an aqueous antibody-drug conjugate solution at two wavelengths, 280 nm and 250 nm, using an absorptiometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.), and then performing the following calculation: Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (additivity of absorbance), assuming that there is no change in the molar extinction coefficients of the antibody and drug before and after conjugation of the antibody and drug, the antibody concentration and drug concentration in the antibody-drug conjugate are expressed by the following relationship: A 280 =A D , 280 +A A , 280 =ε D , 280 C D +ε A , 280 C A Formula (I) A 250 =A D , 250 +A A , 250 =εD , 250 C D +ε A , 250 C A Formula (II) where A 280 indicates the absorbance of the antibody-drug conjugate aqueous solution at 280 nm, and A 250 indicates the absorbance of the antibody-drug conjugate aqueous solution at 250 nm, and A A , 280 indicates the absorbance of the antibody at 280 nm, and A A , 250 indicates the absorbance of the antibody at 250 nm, and A D , 280 denotes the absorbance of the conjugate precursor at 280 nm, and A D , 250 denotes the absorbance of the conjugate precursor at 250 nm, and ε A , 280 denotes the molar extinction coefficient of the antibody at 280 nm, and ε A , 250 denotes the molar extinction coefficient of the antibody at 250 nm, and ε D , 280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D , 250 is the molar extinction coefficient of the conjugate precursor at 250 nm, and C A indicates the antibody concentration in the antibody-drug conjugate, and C D denotes the drug concentration in the antibody-drug conjugate, where ε A , 280 , ε A , 250 , ε D , 280 , ε D , 250 is a value prepared in advance (calculated estimated value or actual measured value). For example, ε A , 280 can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A , 250 is the difference between the measured value obtained from UV measurement of the antibody and εA , 280 In the examples, the molar extinction coefficient of anti-TROP2 antibody 1 was calculated as ε A , 280 = 223400 and ε A , 250 The molar extinction coefficient of anti-TROP2 antibody 2 was ε = 63482. A , 280 = 223400 and ε A , 250 The molar extinction coefficient of anti-CD70 antibody 1 was ε = 69027 or 71411. A , 280 = 226380 and ε A , 250 The molar extinction coefficient of anti-CD70 antibody 2 was ε = 73432. A , 280 = 212400 and ε A , 250 = 72355. The molar extinction coefficient of anti-EGFR antibody 1 was ε A , 280 = 203460 and ε A , 250 The molar extinction coefficient of anti-EGFR antibody 2 was ε = 62692. A , 280 = 217440 and ε A , 250 =75731 was used. D , 280 and ε D , 250 The A of the antibody-drug conjugate solution was measured using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length) by dissolving the conjugate precursor to be used at a certain molar concentration. The A of the antibody-drug conjugate solution was measured using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). The molar extinction coefficient of the conjugate precursor in the examples was obtained by UV measurement. 280 and A 250 By measuring these values ​​and substituting them into equations (I) and (II) and solving the simultaneous equations, C A and C D Furthermore, C D C A By dividing by this, the average number of drugs bound per antibody molecule can be calculated.

[0235] Common Procedure F: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (reversed-phase high-performance liquid chromatography: RP-HPLC) The antibody concentration and the average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography analysis using the following method in addition to the above-mentioned common procedure E.

[0236] [F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes to cleave the disulfide bond between the L chain and H chain of the antibody-drug conjugate. This reaction solution was used directly for HPLC analysis.

[0237] [F-2.HPLC analysis] Typical analytical conditions are as follows: HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: Acquity BEH Phenyl (2.1 x 50 mm, 1.7 μm, manufactured by Waters) Column temperature: 75℃ Flow rate: 0.8mL / min Sample injection volume: 10 μL Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropyl alcohol aqueous solution Mobile phase B: 0.075% TFA, 15% isopropyl alcohol in acetonitrile Gradient program (mobile phase B): 14%-36% (0-15 min), 36%-80% (15-17 min), 80%-14% (17-17.1 min), 14%-14% (17.1-23 min)

[0238] [F-3. Data Analysis] [F-3-1] In the case of glycan conjugation using the SPAAC reaction, the drug-bound H chains (H chains with one drug bound: H1, H chains with two drugs bound: H2) are eluted in the order of L0, H0, H1, and H2 in principle, compared to the drug-free antibody L chain (L0) and H chain (H0). Because the hydrophobicity of these H chains increases in proportion to the number of drugs bound, the retention time increases. By comparing the retention times of L0 and H0, the detected peak can be assigned to either L0, H0, H1, or H2. Similarly, in the case of cysteine ​​conjugation, drug-bound L chains (L chains with one drug bound: L1) and drug-bound H chains (H chains with one drug bound: H1, H chains with two drugs bound: H2, H chains with three drugs bound: H3) become more hydrophobic and have longer retention times in proportion to the number of drugs bound, so they are eluted in the following order: L0, L1, H0, H1, H2, H3. By comparing the retention times of L0 and H0, the detected peak can be assigned to one of L0, L1, H0, H1, H2, or H3.

[0239] [F-3-2] Because the drug linker has UV absorption, in the case of sugar chain conjugation by SPAAC reaction, the peak area was corrected according to the number of drug linkers bound using the molar extinction coefficients of the heavy chain and drug linker according to the following formula: In the case of cysteine ​​conjugation in which the drug is also bound to the light chain, the peak area was corrected in the same way for the light chain.

[0240]

number

[0241] Here, the molar extinction coefficients (280 nm) of the L chain and H chain of each antibody were estimated values ​​calculated by the known calculation method described in Common Procedure E. For anti-TROP2 antibody 1 and anti-TROP2 antibody 2, the molar extinction coefficients of the L chain and H chain were 27702 and 83998, respectively. For anti-CD70 antibody 1, the molar extinction coefficients of the L chain and H chain were 30222 and 82968, respectively. For anti-CD70 antibody 2, the molar extinction coefficients of the L chain and H chain were 30222 and 75978, respectively. For anti-EGFR antibody 1, the molar extinction coefficients of the L chain and H chain were 23232 and 78498, respectively. For anti-EGFR antibody 2, the molar extinction coefficients of the L chain and H chain were 30222 and 78498, respectively. For the molar extinction coefficient (280 nm) of the drug linker, the actual measured value of the conjugation precursor was used in the case of sugar chain conjugation by SPAAC reaction, and for cysteine ​​conjugation, the actual measured value of the compound in which the conjugation precursor was reacted with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether was used.

[0242] [F-3-3] The ratio (%) of each chain peak area to the total corrected peak area was calculated according to the following formula.

[0243]

number

[0244] [F-3-4] The average number of drugs bound per antibody molecule (DAR) in the antibody-drug conjugate was calculated according to the following formula.

[0245]

number

[0246] [F-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the following formula.

[0247]

number

[0248] Here, the absorbance (280 nm) of the antibody-drug conjugate was measured using the actual value of the antibody-drug conjugate aqueous solution. The dilution factor indicates how many times the antibody-drug conjugate aqueous solution was diluted when measuring absorbance, and is usually 4-fold. The molar extinction coefficient (280 nm) of the antibody was an estimated value calculated using the known calculation method described in Common Procedure E. The average number of drugs bound was the value obtained in [F-3-4]. For the molar extinction coefficient (280 nm) of the drug linker, the actual measured value of the conjugation precursor was used in the case of sugar chain conjugation via SPAAC reaction. For cysteine ​​conjugation, the actual measured value of the compound obtained by reacting the conjugation precursor with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether was used.

[0249] Common Procedure G: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (hydrophobic interaction-high performance liquid chromatography: HI-HPLC) The antibody concentration and the average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography analysis using the following method in addition to the above-mentioned common procedures E and F.

[0250] [G-1. Preparation of samples for HPLC analysis] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was used directly for HPLC analysis.

[0251] [G-2.HPLC analysis] The typical analytical conditions are as follows: HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: TSK-gel Butyl-NPR (4.6 x 100 mm, 2.5 μm, manufactured by TOSOH) Column temperature: constant temperature around 25°C Mobile phase A: 25 mM phosphate buffer (pH 7.0) containing 1.5 M ammonium sulfate Mobile phase B: 25 mM phosphate buffer (pH = 7.0) / isopropyl alcohol mixture (3:1) Flow rate: 0.8mL / min Sample injection volume: 15 μL Gradient program (mobile phase B): 10%-15% (0 min-5 min), 15%-65% (5 min-20 min) or HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: PolyPROPYL A (4.6 x 100 mm, 3 μm, 1500 Å, manufactured by PolyLC) Column temperature: constant temperature around 40°C Mobile phase A: 20 mM phosphate buffer (pH 7.4) containing 1.5 M ammonium sulfate Mobile phase B: 20mM phosphate buffer (pH=7.4) Flow rate: 0.8mL / min Sample injection volume: 15 μL Gradient program (mobile phase B): 40%-80% (0 min-20 min)

[0252] [G-3. Data Analysis] [G-3-1] Because hydrophobicity increases in proportion to the number of drugs bound to the antibody and retention time increases, in the case of glycan conjugation by SPAAC reaction, elution generally occurs in the order of DAR=0, DAR=2, and DAR=4. By comparing the retention time with DAR=0, the detected peak can be assigned to either DAR=2 or DAR=4. Depending on the type of antibody and drug linker, peaks of DAR=1 and DAR=3 may also be detected. The DAR of the detected peak may also be estimated by measuring the mass spectrum after fractionating the peak by HI-HPLC.

[0253] [G-3-2] Because the drug linker has UV absorption, the peak area value was corrected according to the molar absorption coefficients of the antibody and drug linker according to the following formula, depending on the number of drug linkers bound.

[0254]

number

[0255] Here, the molar extinction coefficient (280 nm) of the antibody was an estimated value calculated by the known calculation method described in Common Procedure E. The molar extinction coefficient (280 nm) of the drug linker was the actually measured value of the conjugation precursor.

[0256] [G-3-3] The antibody peak area ratio (%) to the total corrected peak area was calculated according to the following formula.

[0257]

number

[0258] [G-3-4] The average number of drugs bound per antibody molecule in the antibody-drug conjugate was calculated according to the following formula.

[0259]

number

[0260] [G-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the formula described in [F-3-5]. The average number of drugs bound was calculated using the value obtained in [G-3-4].

[0261] The antibody-drug conjugates of the present invention or their production intermediates may exist as stereoisomers, optical isomers derived from asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers, and all of these isomers, optical isomers, and mixtures thereof are included in the present invention.

[0262] In the antibody-drug conjugates of the present invention, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-drug conjugates are produced by specifying reaction conditions, such as the amounts of raw materials and reagents used, so that a certain number of drugs are bound. However, unlike chemical reactions of small molecules, a mixture of drugs bound to one antibody molecule is usually obtained, with different numbers of drugs bound. The number of drugs bound to one antibody molecule can be specified as an average value, i.e., the drug binding average (DAR). The number of cyclic dinucleotide derivatives bound to an antibody molecule can be controlled, and the average number of drugs bound to one antibody can range from 1 to 10, preferably 1 to 8, and more preferably 1 to 5.

[0263] In the antibody-drug conjugate of the present invention, when the antibody Ab is bound to L via a remodeled sugar chain of the antibody Ab, the number of drugs bound per antibody molecule in the antibody-drug conjugate, m 2 is an integer of 1 or 2. When the sugar chain is an N297 sugar chain, and the sugar chain is N297-(Fuc)SG, m 2 is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.2). When the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1, and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

[0264] Furthermore, a person skilled in the art would be able to design a reaction for binding the required number of drugs to an antibody based on the description of the examples of the present application, and obtain an antibody in which the number of cyclic dinucleotide derivatives bound is controlled.

[0265] The antibody-drug conjugate of the present invention or a production intermediate thereof may absorb moisture, become adsorbed with water, or become a hydrate when left in the air or when recrystallized, and such water-containing compounds and salts are also encompassed by the present invention.

[0266] When the antibody-drug conjugate of the present invention or a production intermediate thereof has a basic group such as an amino group, it can be converted into a pharmaceutically acceptable salt, if desired. Examples of such salts include hydrohalides such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0267] The antibody-drug conjugates of the present invention generally can form base addition salts because they contain a phosphate group and / or a thiophosphate group in their structure. Furthermore, when their production intermediates contain an acidic group such as a carboxy group, they can generally form base addition salts. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

[0268] The antibody-drug conjugates of the present invention and their production intermediates may exist as hydrates due to, for example, absorption of moisture from the air. The solvates of the present invention are not particularly limited as long as they are pharmaceutically acceptable, but specifically, hydrates, ethanol solvates, 2-propanol solvates, etc. are preferred. Furthermore, when nitrogen atoms are present in the antibody-drug conjugates of the present invention and their production intermediates, they may be in the form of N-oxides, and these solvates and N-oxides are also within the scope of the present invention. Furthermore, when sulfur atoms are present in the antibody-drug conjugates of the present invention and their production intermediates, they may be in the form of sulfoxides, and these solvates and sulfoxides are also within the scope of the present invention.

[0269] The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugates of the present invention and their production intermediates may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium (H), tritium (H), iodine-125 (I), or carbon-14 (C). The compounds of the present invention may also be radiolabeled with radioactive isotopes such as tritium (H), iodine-125 (I), or carbon-14 (C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo diagnostic imaging agents. All isotopic variants of the antibody-drug conjugates of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0270] <4. Pharmaceuticals> The antibody-drug conjugate of the present invention exhibits antitumor immune activity or cytotoxic activity against cancer cells, and therefore can be used as a pharmaceutical, particularly as a therapeutic and / or preventive agent for cancer, or an antitumor agent.

[0271] The types of cancer to which the antibody-drug conjugates of the present invention can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer (seminoma, nonseminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, and gastrointestinal stromal tumor. Examples of cancer cells to be treated include cancers such as gastrointestinal stromal tumors (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, pharyngeal cancer, tongue cancer, ear cancer, thymus cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, and sarcoma; however, the antibody-drug conjugate is not limited to these, as long as the cancer cells to be treated express a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0272] The antibody-drug conjugate of the present invention can be suitably administered to mammals, and the mammal is more preferably a human.

[0273] Substances used in pharmaceutical compositions containing the antibody-drug conjugates of the present invention can be appropriately selected and applied in terms of dosage and administration concentration from pharmaceutical additives and other substances commonly used in this field.

[0274] The antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation corresponds to the mode of administration.

[0275] Various delivery systems are known and can be used to administer the antibody-drug conjugate of the present invention.Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.Administration can be, for example, by infusion or bolus injection.In certain preferred embodiments, the antibody-drug conjugate is administered by infusion.Parenteral administration is the preferred administration route.

[0276] In a representative embodiment, a pharmaceutical composition comprising the antibody-drug conjugate is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the medicament may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the pharmaceutical composition is to be administered by infusion, it can be dispensed, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration. The pharmaceutical composition may also be provided as a solution.

[0277] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition containing the antibody-drug conjugate of the present invention and another cancer therapeutic agent. The antibody-drug conjugate of the present invention can also be administered together with or in combination with other cancer therapeutic agents, thereby enhancing the anti-tumor effect. The other cancer therapeutic agents used for such purposes may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or consecutively, or at different administration intervals. Examples of such cancer therapeutic agents include chemotherapeutic agents such as antimetabolites, alkylating agents, and microtubule inhibitors (e.g., abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, vinblastine, or drugs described in International Publication No. WO 2003 / 038043), hormone modulating agents (e.g., LH-RH analogs such as leuprorelin, goserelin, and estramustine, and estrogen antagonists such as tamoxifen and raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, and exemestane), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, molecular targeted drugs (e.g., anti-EGFR antibodies, anti- Anti-VEGF antibodies, anti-VEGFR antibodies, etc.), immune checkpoint inhibitors (anti-PD-1 antibodies such as nivolumab and pembrolizumab, anti-PD-L1 antibodies such as atezolizumab, avelumab, and durvalumab, anti-PD-L2 antibodies, anti-CTLA4 antibodies such as ipilimumab, anti-A2aR antibodies, A2a receptor antagonists, anti-LAG3 antibodies, anti-TIM3 antibodies, etc.), anti-regulatory T cell drugs (anti-CTLA4 antibodies, anti-CD25 antibodies, anti-GITR antibodies, anti-GARP antibodies, anti-TIGIT antibodies, anti-CCR8 antibodies, etc.), immune activators (anti-4-1BB antibodies, anti-OX40 antibodies, anti-CD40 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, IL-2 analogs, cytokines, TLR agonists, etc.), immunomodulators (anti-CD47 antibodies, anti-SIRPα antibodies, inhibitory myeloid modulators, etc.), ADCC (Antibody Dependent Cellular Cytotoxicity) activity, ADCP (Antibody Dependent CellularExamples of the antitumor conjugates include antibody drugs with phagocytosis activity or complement activity, BiTEs (Bi-specific T-cell engagers), antibody-drug conjugates (ADCs) (e.g., drug conjugates containing deruxtecan, DM1, pyrrolobenzodiazepine, MMAF, etc. (anti-HER2-ADC, anti-TROP2-ADC, anti-HER3-ADC, etc.)), ADCs combined with photodynamic therapy, as well as antitumor vaccines, antitumor cell therapies (CAR-T, TCR-T, dendritic cells, NK cells, etc.), antitumor bacterial therapies, antitumor viral therapies, etc., but are not limited thereto as long as they have antitumor activity. Furthermore, the antibody-drug conjugates of the present invention can be administered together with other antibody-drug conjugates of the present invention, thereby enhancing the antitumor effect. Furthermore, the antibody-drug conjugates of the present invention can enhance the antitumor effect when used in combination with not only drugs but also treatments that produce antitumor effects, such as radiation, heavy ion beams, surgery, bone marrow transplantation, etc., but are not limited thereto as long as they have antitumor effects.

[0278] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0279] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its therapeutic effect even at a smaller dose, as the affinity of the antibody-drug conjugate to the antigen, i.e., the dissociation constant (Kd value) for the antigen, of the antibody-drug conjugate increases (the lower the Kd value). Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0280] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Example]

[0281] In the following examples, room temperature refers to 15°C to 35°C. Anhydrous acetonitrile was used as Acetonitrile (Anhydrous)-Super- available from Kanto Chemical or Acetonitrile (Ultra Anhydrous) available from Wako Pure Chemical Industries, Ltd. Pyridine was used as Pyridine (Anhydrous)-Super- available from Kanto Chemical. Silica gel chromatography was performed using Biotage SNAP Ultra (Biotage), Chromatorex Q-Pack SI (Fuji Silysia), or Purif-Pack-Ex SI (Shoko Science). DIOL silica gel column chromatography was performed using Chromatorex Q-pack DIOL (Fuji Silysia). C18 silica gel column chromatography was performed using Biotage SNAP Ultra C18 (Biotage). Column chromatographic elution was performed under thin-layer chromatography (TLC) observation. The 0.1% triethylamine used as the elution solvent means that the total volume of the elution solvent contained 0.1% triethylamine. Preparative HPLC was performed using a SHIMADZU SPD-M10A HPLC system (Shimadzu Corporation) etc. The preparative columns used were Kinetex (5 μm, C18, 100 Å, 250 × 30.0 mm, manufactured by Phenomenex) or Kinetex (5 μm, C18, 100 Å, 250 × 21.2 mm, manufactured by Phenomenex). The following instruments were used to measure various spectral data. 1 H-NMR spectra were measured using a JEOL ECS-400 (400 MHz), a Varian 400-MR (400 MHz), or a Varian Unity Inova 500 (500 MHz). 31P-NMR spectra were measured using a JEOL ECS-400 (160 MHz). Mass spectra were measured using an Agilent 6130 Quadrupole LC / MS system (Agilent Technologies). LC / MS measurements were performed under the following conditions: column: Develosil Combi-RP, 5 μm, 50 × 2.0 mm (Nomura Chemical Co., Ltd.); mobile phase: 0.1% formic acid acetonitrile solution / 0.1% formic acid aqueous solution; 0.1% formic acid acetonitrile solution: 2%-100% (0-5 min or 0-10 min).

[0282] Example 1: Synthesis of CDN6 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0283] [ka]

[0284] [Synthetic scheme]

[0285] [ka] TIFF2025023046000103.tif187170

[0286] (Process 1) 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine To a solution of 5-iodotubercidin (1.0 g) in N,N-dimethylformamide (10 mL) as previously described (Tetrahedron 2007, 63, 9850-9861), di-tert-butylsilylbis(trifluoromethanesulfonate) (1.24 mL) was slowly added dropwise at 0°C, followed by stirring at the same temperature for 30 minutes. Imidazole (868 mg) was added at 0°C, and the mixture was warmed to room temperature and stirred for 30 minutes. tert-Butyldimethylchlorosilane was added at room temperature, followed by stirring overnight at the same temperature. The reaction mixture was quenched with saturated aqueous sodium bicarbonate and then extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (910 mg). MS(ESI)m / z:647(M+H) + . 1 H-NMR(CDCl3)δ:8.25(1H,s),7.03(1H,s),6.10(1H,s),5.63(2H,brs),4.49-4.44(2H,m),4.26(1H,dd,J=9.7 ,4.8Hz),4.17(1H,m),4.00(1H,t,J=9.7Hz),1.09(9H,s),1.04(9H,s),0.91(9H,s),0.13(3H,s),0.11(3H,s).

[0287] (Process 2) 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-(3,3-diethoxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine To a solution of the compound obtained in Step 1 (910 mg) in N,N-dimethylformamide (3.0 mL) and tetrahydrofuran (9.0 mL), propargylaldehyde dimethyl acetal (1.01 mL), triethylamine (0.392 mL), tetrakis(triphenylphosphine)palladium(0) (163 mg), and copper(I) iodide (53.6 mg) were added in that order, and the mixture was stirred at 40°C for 18 hours. A saturated aqueous solution of sodium bicarbonate and ethyl acetate were added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to yield the title compound (878 mg). MS(ESI)m / z:647(M+H) + . 1 H-NMR(CDCl3)δ:8.27(1H,s),7.17(1H,s),6.09(1H,s),5.56(2H,brs),5.50(1H ,s),4.48(1H,dd,J=9.1,4.9Hz),4.42(1H,d,J=4.9Hz),4.25(1H,dd,J=9.4,4.6 Hz),4.17(1H,m),4.00(1H,t,J=9.7Hz),3.85-3.77(2H,m),3.66(2H,m),1.28(6 H,t,J=7.3Hz),1.08(9H,s),1.04(9H,s),0.91(9H,s),0.13(3H,s),0.11(3H,s).

[0288] (Step 3) 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 2 (878 mg) in ethanol (8.8 mL), 10% palladium on carbon (M) wet (500 mg) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 9 hours. The catalyst was removed by filtration, and the mixture was washed with dichloromethane, and the filtrate was concentrated under reduced pressure. To a solution of the residue in acetic acid (8.8 mL), 10% palladium on carbon (M) wet (500 mg) was added, and the mixture was stirred under a hydrogen atmosphere at 40°C for 2 days. The catalyst was removed by filtration, and the mixture was washed with dichloromethane, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (603 mg). MS(ESI)m / z:561(M+H) + . 1 H-NMR(CDCl3)δ:8.47(1H,brs),8.07(1H,s),6.70(1H,s),6.14(1H,s),4.47-4.43(2H,m),4.29(1H,dd,J=9.1,4.8Hz),4.15(1H,m),3. 99(1H,t,J=9.7Hz),3.55(2H,m),2.89(2H,t,J=5.4Hz),2.04(2H,m),1.09(9H,s),1.04(9H,s),0.90(9H,s),0.10(3H,s),0.10(3H,s).

[0289] (Step 4) 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0290] To a solution of the compound obtained in Step 3 (2.17 g) in dichloromethane (21.7 mL) was added pyridine (1.56 mL), N,N-dimethylaminopyridine (94.5 mg), and benzoyl chloride (0.898 mL) in that order at room temperature, and the mixture was stirred at 50°C for 15 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution to the reaction mixture. After extraction with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (1.91 g). MS(ESI)m / z:665(M+H) + . 1 H-NMR(CDCl3)δ:8.08(1H,s),7.37-7.33(3H,m),7.23(2H,t,J=7.6Hz),6.97(1H,s),6.21(1H,s),4.50-4.46(2H,m),4.37-4.30(2H,m), 4.28-4.09(2H,m),4.02(1H,t,J=10.0Hz),3.03(2H,t,J=6.3Hz),2.29-2.17(2H,m),1.10(9H,s),1.05(9H,s),0.90(9H,s),0.10(6H,s).

[0291] (Step 5) 6-Benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene A mixture of hydrogen fluoride-pyridine (0.30 mL) and pyridine (1.88 mL) prepared at 0°C was added to a dichloromethane (15 mL) solution of the compound obtained in Step 4 (1.91 g) above, and the mixture was stirred at 0°C for 2 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution. The reaction was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in pyridine (15 mL), and 4,4'-dimethoxytrityl chloride (1.17 g) was added. The mixture was stirred at 0°C for 12 hours. Methanol was added and the mixture was stirred for 30 minutes. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution. The reaction was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (1.98 g). MS(ESI)m / z:827(M+H) + . 1 H-NMR(CDCl3)δ:8.07(1H,s),7.47(2H,m),7.37-7.19(13H,m),6.84(4H,m),6 .37(1H,d,J=5.5Hz),4.75(1H,t,J=5.2Hz),4.38-4.20(4H,m),3.80(6H,s),3. 53(1H,dd,J=10.7,2.8Hz),3.40(1H,dd,J=11.0,3.1Hz),2.83(1H,d,J=3.7Hz) ,2.78(2H,t,J=6.4Hz),2.17(2H,m),0.81(9H,s),-0.03(3H,s),-0.21(3H,s).

[0292] (Step 6) 6-Benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 5 (1.98 g) in dichloromethane (23.9 mL), N,N-diisopropylethylamine (1.02 mL) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.07 mL) were added, and the mixture was stirred at room temperature for 15 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution to the reaction mixture. The reaction mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (2.06 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 7:3). MS(ESI)m / z:1027(M+H) + . 1 H-NMR(CDCl3)δ:8.06(0.3H,s),8.04(0.7H,s),7.50-7.16(15H,m),6.85-6.79(4H,m),6.35(0.7H,d,J=6.7Hz),6.31(0.3H,d,J=6.1 Hz),4.84(0.7H,dd,J=7.0,4.6Hz),4.78(0.3H,t,J=5.8Hz),4.43-4.17(4H,m),4.04-3.85(1.3H,m),3.80-3.76(6H,m),3.69-3.43(3 H,m),3.50(0.7H,dd,J=10.6,3.3Hz),3.33-3.26(1H,m),2.87-2.76(2H,m),2.74-2.60(1.4H,m),2.31(0.6H,t,J=6.7Hz),2.23-2.11 (2H,m),1.21-1.13(7.8H,m),1.04(4.2H,d,J=6.7Hz),0.73(2.7H,s),0.72(6.3H,s),-0.03(0.9H,s),-0.06(2.1H,s),-0.24(3H,s).

[0293] (Step 7) 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound (935 mg) obtained in Step 6 above in acetonitrile (4.55 mL), water (33 μL) and pyridine trifluoroacetate (229 mg) were added and stirred at room temperature for 15 minutes. tert-Butylamine (4.55 mL) was added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was azeotroped twice with acetonitrile (5 mL). Water (0.164 mL) was added to a solution of the residue in dichloromethane (11.4 mL), followed by a solution of dichloroacetic acid (0.651 mL) in dichloromethane (11.4 mL) and stirring at room temperature for 15 minutes. Pyridine (1.25 mL) was added to quench the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was azeotroped three times with dehydrated acetonitrile (10 mL), leaving approximately 5 mL of acetonitrile in the final distillation. The resulting acetonitrile solution of the title compound was used directly in Step 12 below.

[0294] (Step 8) 2',3',5'-Tri-O-acetyl-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine To a suspension of commercially available 2',3',5'-tri-O-acetylinosine (10.0 g) in tetrahydrofuran (100 mL), 2-{[tert-butyl(dimethyl)silyl]oxy}ethan-1-ol (5.37 g) and triphenylphosphine (7.69 g) were added, followed by dipropan-2-yl (E)-diazene-1,2-dicarboxylate (6.10 mL) and stirring at room temperature for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane / ethyl acetate / dichloromethane] to give the title compound as a mixture with triphenylphosphine oxide (10.6 g). MS(ESI)m / z:553(M+H) + . 1H-NMR(CDCl3)δ:8.05(1H,s),7.92(1H,s),6.12(1H,d,J=5.4Hz),5.86(1H,t,J=5.4Hz),5.59(1H,dd,J=5.4,4.2Hz),4.47-4.41(2H,m),4. 38-4.31(1H,m),4.22-4.17(2H,m),3.89(2H,t,J=4.8Hz),2.15(3H,s ),2.14(3H,s),2.08(3H,s),0.83(9H,s),-0.06(3H,s),-0.06(3H,s).

[0295] (Step 9) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine Potassium carbonate (150 mg) was added to a solution of the compound obtained in Step 8 (10.6 g) in tetrahydrofuran (30 mL) and methanol (30 mL) and stirred at room temperature for 3 hours. Acetic acid (125 μL) was added to the reaction mixture, which was then concentrated under reduced pressure. The residue was azeotroped with pyridine. 4,4'-Dimethoxytrityl chloride (6.50 g) was added to a solution of the residue in pyridine (60 mL) at 0°C, stirred for 30 minutes, and then stored in a refrigerator overnight. Methanol (2 mL) was added to the reaction mixture, which was stirred for 30 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol / 0.1% triethylamine] to obtain the title compound as a mixture with triphenylphosphine oxide (7.21 g). MS(ESI)m / z:729(M+H) + . 1H-NMR(CDCl3)δ:8.01(1H,s),7.97(1H,s),7.35-7.30(2H,m),7.25-7.17(7H,m),6.81-6.76 (4H,m),5.95(1H,d,J=5.4Hz),5.13(1H,brs),4.68-4.61(1H,m),4.43-4.36(2H,m),4.31-4 .23(1H,m),4.15-4.08(1H,m),3.89(2H,t,J=4.5Hz),3.77(6H,s),3.42(1H,dd,J=10.3,3.6 Hz),3.34(1H,dd,J=10.3,3.6Hz),3.10(1H,brs),0.83(9H,s),-0.06(3H,s),-0.07(3H,s).

[0296] (Step 10) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine To a solution of the compound obtained in Step 9 (7.21 g) in dichloromethane (36 mL), imidazole (1.41 g) and tert-butyl(chloro)dimethylsilane (1.49 g) were added, and the mixture was stirred at room temperature for 16 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (2.17 g) and its regioisomer, 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine (2.55 g). MS(ESI)m / z:843(M+H) + . 1H-NMR(CDCl3)δ:7.99(1H,s),7.97(1H,s),7.43-7.39(2H,m),7.33-7.19(7H,m),6.83-6.77(4 H,m),5.96(1H,d,J=4.2Hz),4.56-4.50(2H,m),4.33-4.25(1H,m),4.19-4.02(2H,m),3.89(2H, t,J=4.8Hz),3.78(6H,s),3.45(1H,dd,J=10.9,4.2Hz),3.27(1H,dd,J=10.9,4.2Hz),3.03(1H, d,J=6.0Hz),0.88(9H,s),0.82(9H,s),0.07(3H,s),-0.01(3H,s),-0.07(3H,s),-0.07(3H,s). Positional isomer (2'-O-TBS form) MS(ESI)m / z:843(M+H) + . 1 H-NMR(CDCl3)δ:7.98(1H,s),7.94(1H,s),7.46-7.42(2H,m),7.35-7.20(7H,m),6.85-6.79(4H,m),5.9 9(1H,d,J=5.4Hz),4.83(1H,t,J=5.1Hz),4.33-4.29(1H,m),4.27-4.24(1H,m),4.24-4.12(2H,m),3.90 (2H,t,J=4.5Hz),3.79(3H,s),3.78(3H,s),3.48(1H,dd,J=10.3,3.0Hz),3.40(1H,dd,J=10.3,3.0Hz), 2.71(1H,d,J=3.6Hz),0.86(9H,s),0.83(9H,s),0.01(3H,s),-0.07(3H,s),-0.07(3H,s),-0.11(3H,s).

[0297] (Step 11) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}inosine To a solution of the compound obtained in Step 10 (2.17 g) in dichloromethane (25.7 mL), 4,5-dicyanoimidazole (334 mg) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.980 mL) were added and stirred at room temperature for 16 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution to the reaction mixture. The reaction mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by DIOL silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (2.65 g) as a diastereomeric mixture at the phosphorus atom. MS(ESI)m / z:1043(M+H) + . 1 H-NMR(CDCl3)δ:8.03(0.53H,s),8.01(0.47H,s),7.97(0.53H,s),7.93( 0.47H,s),7.45-7.41(2H,m),7.35-7.19(7H,m),6.83-6.78(4H,m),6.17 (0.53H,d,J=4.2Hz),6.05(0.47H,d,J=4.2Hz),4.87-4.80(0.47H,m),4. 64-4.58(0.53H,m),4.46-4.40(1H,m),4.30-4.05(3H,m),3.92-3.87(2H ,m),3.78(6H,s),3.86-3.40(5H,m),3.33-3.24(1H,m),2.54(0.94H,t,J =6.0Hz),2.43(1.06H,t,J=6.7Hz),1.16-1.09(9H,m),1.01-0.97(3H,m) ,0.83(4.23H,s),0.83(4.77H,s),0.82(9H,s),0.07(1.41H,s),0.04(1. 59H,s),-0.02(3H,s),-0.07(1.41H,s),-0.08(1.59H,s),-0.08(3H,s).

[0298] (Step 12) The compound (950 mg) obtained in step 11 above was azeotropically distilled three times with dehydrated acetonitrile (5 mL). For the final distillation, approximately 3 mL of acetonitrile was left, and molecular sieves 3A, 1 / 16 (five pellets) were added. This acetonitrile solution was added to the acetonitrile solution prepared in step 7 above, and the mixture was stirred at room temperature for 20 minutes under a nitrogen atmosphere. N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide (206 mg) was added to the reaction mixture, which was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure. Water (0.164 mL) was added to a solution of the residue in dichloromethane (13.0 mL), and a solution of dichloroacetic acid (0.822 mL) in dichloromethane (13.0 mL) was then added and the mixture was stirred at room temperature for 15 minutes. Pyridine (9.01 mL) was added to the reaction mixture to quench the reaction, and the mixture was then concentrated under reduced pressure. The resulting crude product was used as is in the next reaction.

[0299] (Step 13) 3-({(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-yl}oxy)propanenitrile

[0300] The pyridine (27.1 mL) solution of the crude product obtained in step 12 was concentrated to about 20 mL, and then 2-chloro-5,5-dimethyl-1,3,2λ 53H-Dioxaphosphinan-2-one (622 mg) was added and stirred at room temperature for 30 minutes. Water (0.57 mL) and 3H-1,2-benzodithiol-3-one (230 mg) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction mixture was poured into an aqueous solution (130 mL) of sodium bicarbonate (3.60 g) and stirred at room temperature for 30 minutes, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol] to obtain the title compound (494 mg) as a diastereomeric mixture at the phosphorus atom. MS(ESI)m / z:1274(M+H) + .

[0301] (Step 14) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (494 mg) obtained in Step 13 above in methanol (5 mL) was added 28% aqueous ammonia (5 mL), and the mixture was stirred at room temperature for 15 hours. The reaction mixture was concentrated, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to give diastereomer 1 (88.5 mg, containing impurities) and diastereomer 2 (70.7 mg, containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI) m / z: 1003 (M-C6H 15 Si+2H)+ . Diastereomer 2 (high polarity) MS(ESI) m / z: 1003 (M-C6H 15 Si+2H) + .

[0302] (Step 15-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1)

[0303] Triethylamine trihydrofluoride (2.0 mL) was added to the compound (diastereomer 1) (88.5 mg, containing impurities) obtained in Step 14 above, and the mixture was stirred at 45°C for 3 hours. An ice-cooled mixture of 1M aqueous triethylammonium bicarbonate (10 mL) and triethylamine (2 mL) was added to the reaction mixture at room temperature. The reaction mixture was concentrated under reduced pressure and then purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)]. The resulting compound (triethylamine salt) was converted to the sodium salt using the following method.

[0304] [Conversion to sodium salt] BT AG® 50W-X2 Resin (biotechnology grade, 100-200 mesh, hydrogen form) (500 mg) was suspended in pure water and packed into an empty column. After allowing excess pure water to flow by gravity, 1 M aqueous sodium hydroxide solution (5 mL) and pure water (10 mL) were added sequentially by gravity flow. The compound obtained above was dissolved in pure water (5 mL) and charged onto the column. The solution that flowed by gravity was collected and then eluted with pure water (10 mL). The fractions containing the target compound were combined and lyophilized to obtain the title compound (25.7 mg). MS(ESI)m / z:775(M+H) + . 1 H-NMR(CD3OD)δ:8.63(1H,s),8.22(1H,s),8.02(1H,s),7.11(1H,s),6.30 -6.24(2H,m),5.46-5.37(1H,m),5.23-5.15(1H,m),4.83-4.79(1H,m),4.7 8-4.74(1H,m),4.53-4.42(2H,m),4.35-4.16(3H,m),4.16-3.97(3H,m),3 .83-3.78(2H,m),3.52-3.47(2H,m),2.88-2.81(2H,m),2.03-1.95(2H,m). 31 P-NMR(CD3OD)δ:57.8(s),54.4(s).

[0305] (Step 15-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2)

[0306] The compound (diastereomer 2) (70.7 mg: containing impurities) obtained in the above step 14 was reacted in the same manner as in the above step 15-1, and then purified under the following [purification conditions] to obtain the title compound as a triethylamine salt. Purification conditions: C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 15%-70% (0 min-40 min)].

[0307] The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 above to obtain the title compound (17.8 mg). MS(ESI)m / z:775(M+H) + . 1 H-NMR(CD3OD)δ:8.72(1H,s),8.23(1H,s),8.02(1H,s),7.11(1H,s),6.30 (2H,dd,J=13.6,7.6Hz),5.48-5.39(2H,m),4.78(1H,dd,J=6.7,4.2Hz),4. 51-4.28(5H,m),4.26-4.13(2H,m),4.06-4.00(1H,m),3.93-3.86(1H,m),3 .85-3.80(2H,m),3.52-3.47(2H,m),2.94-2.88(2H,m),2.05-1.97(2H,m). 31 P-NMR(CD3OD)δ:62.9(s),60.0(s).

[0308] Example 2: Synthesis of CDN34 (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0309] [ka]

[0310] [Synthetic scheme]

[0311] [ka] TIFF2025023046000106.tif188170

[0312] (Process 1) 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine To a solution of commercially available inosine (10.0 g) (Tokyo Chemical Industry Co., Ltd.) in pyridine (50 mL) and N,N-dimethylacetamide (50 mL), 4,4'-dimethoxytrityl chloride (15.2 g) was added at 0°C and the mixture was stirred at 4°C for 64 hours. Methanol (2 mL) was added to the reaction mixture, which was stirred for 10 minutes and then concentrated to approximately 50 mL. 2-Bromoethyl benzoate (7.02 mL) and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (13.9 mL) were added to the residue and stirred at room temperature for 1 day. Saturated aqueous sodium bicarbonate and water were added to the reaction mixture, which was then extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol / 0.1% triethylamine] to obtain the title compound (15.2 g). MS(ESI)m / z:719(M+H) + . 1 H-NMR(CDCl3)δ:8.00(1H,s),7.98(1H,s),7.98-7.94(2H,m),7.62-7.15(12H,m),6.80-6.75(4H,m),5.95(1H,d,J=5.4Hz),4. 82-4.79(1H,m),4.72-4.64(3H,m),4.55-4.34(5H,m),3.77(6H,s),3.43(1H,dd,J=10.6,3.9Hz),3.34(1H,dd,J=10.6,3.6Hz).

[0313] (Process 2) 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]inosine Using the compound (3.01 g) obtained in Step 1 above, synthesis was carried out in the same manner as in Step 10 of Example 1 to obtain the title compound (1.20 g) and its positional isomer, 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]inosine (1.22 g). MS(ESI) m / z: 833 (M + H) + . 1 H-NMR(CDCl3) δ: 8.03(1H, s), 7.98 - 7.96(1H, m), 7.96(1H, s), 7.96 - 7.94(1H, m), 7.59 - 7.52(1H, m), 7.44 - 7.38(4H, m), 7.32 - 7.15(7H, m), 6.83 - 6.77(4H, m), 5.94(1H, d, J = 4.8Hz), 4.69 - 4.63(2H, m), 4.59 - 4.35(4H, m), 4.16(1H, dd, J = 3.8, 1.9Hz), 3.77(6H, d, J = 1.8Hz), 3.47(1H, dd, J = 10.9, 3.0Hz), 3.27(1H, dd, J = 10.9, 4.2Hz), 3.00(1H, d, J = 6.7Hz), 0.87(9H, s), 0.06(3H, s), -0.01(3H, s). (2’-O-TBS form) MS(ESI) m / z: 833 (M + H) + . 1 H-NMR(CDCl3) δ: 8.01(1H, s), 7.97 - 7.93(2H, m), 7.91(1H, s), 7.59 - 7.53(1H, m), 7.45 - 7.38(4H, m), 7.35 - 7.17(7H, m), 6.83 - 6.77(4H, m), 5.97(1H, d, J = 6.0Hz), 4.84(1H, t, J = 5.4Hz), 4.71 - 4.60(2H, m), 4.52 - 4.37(2H, m), 4.33 - 4.28(1H, m), 4.28 - 4.24(1H, m), 3.78(3H, s), 3.77(3H, s), 3.47(1H, dd, J = 10.9, 3.0Hz), 3.38(1H, dd, J = 10.9, 3.6Hz), 2.71(1H, d, J = 3.0Hz), 0.80(9H, s), -0.03(3H, s), -0.19(3H, s).

[0314] (Step 3) 1-[2-(Benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}inosine

[0315] The compound (1.20 g) obtained in Step 2 above was used to carry out synthesis in the same manner as in Step 11 of Example 1, to obtain the title compound (1.41 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 0.55:0.45). MS(ESI)m / z:1033(M+H) + . 1 H-NMR(CDCl3)δ:8.05(0.45H,s),8.04(0.55H,s),7.99-7.95(2H,m),7.95(0.55H,s),7.92(0.45H,s),7.59-7.53(1H,m),7.45-7.3 9(4H,m),7.35-7.10(7H,m),6.83-6.78(4H,m),6.15(0.55H,d,J=5.4Hz),6.08(0.45H,d,J=6.0Hz),4.86-4.49(3H,m),4.49-4.35( 3H,m),4.25-4.10(1H,m),3.78(6H,s),3.72-3.41(5H,m),3.35-3.25(1H,m),2.47(1H,t,J=6.7Hz),2.32(1H,t,J=6.3Hz),1.33-1. 24(6H,m),1.13-1.03(6H,m),0.84(4.05H,s),0.84(4.95H,s),0.08(1.35H,s),0.05(1.65H,s),0.00(1.35H,s),-0.01(1.65H,s).

[0316] (Step 4) 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 4 of Example 1 (35.80 g) in dichloromethane (322 mL) and pyridine (35 mL), a solution of hydrogen fluoride-pyridine (6.33 g) in dichloromethane (36 mL) was added over 5 minutes under ice-cooling, and the mixture was stirred at the same temperature for 3 hours. Saturated aqueous sodium bicarbonate (268 mL) and saturated brine (143 mL) were added sequentially to the reaction mixture to quench the reaction, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. Hexane / ethyl acetate (1:1) (108 mL) was added to the residue to form a slurry, which was stirred at 50°C for 30 minutes. Hexane (161 mL) was added and the mixture was stirred for an additional 2 hours. The precipitated solid was collected by filtration and washed with hexane / ethyl acetate (4:1) (143 mL) to obtain the title compound (26.81 g). MS(ESI)m / z:525(M+H) + . 1 H-NMR(DMSO-d6)δ:7.98(1H,s),7.65(1H,s),7.39(1H,m),7.26-7.20(4H,m) ,6.19(1H,d,J=6.5Hz),5.15(1H,t,J=5.6Hz),5.00(1H,d,J=4.8Hz)4.48(1H ,t,J=5.6Hz),4.27(1H,m),4.11-4.02(2H,m),3.97(1H,m),3.67-3.57(2H,m ),2.99(2H,m),2.23-2.07(2H,m),0.68(9H,s),-0.11(3H,s),-0.26(3H,s).

[0317] (Step 5) 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-bis-O-(oxan-2-yl)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 4 (19.93 g) and 3,4-dihydro-2H-pyran (35 mL) in N,N-dimethylformamide (200 mL), p-toluenesulfonic acid monohydrate (7.25 g) was added under ice-cooling, and the mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate to the reaction mixture under ice-cooling, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and saturated brine, and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (24.73 g). 1 H-NMR(CDCl3)δ:8.10-8.07(1H,m),7.59-7.35(1H,m),7.35-7.27(3H,m),7.25-7.17(2H,m),6.44-6.36(1H,m),4.9 0-3.36(13H,m),3.06-2.96(2H,m),2.31-2.15(2H,m),2.01-1.43(12H,m),0.84-0.73(9H,m),0.04-(-0.35)(6H,m).

[0318] (Step 6) 6-Benzoyl-2-[3,5-bis-O-(oxan-2-yl)-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0319] To a solution of the compound obtained in Step 5 above (24.73 g) and acetic acid (3.1 mL) in tetrahydrofuran (250 mL), a solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 55 mL) was added under ice cooling, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue, which was then washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate, and the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to give the title compound (18.74 g). 1H-NMR(CDCl3)δ:8.12-8.09(1H,m),7.49-7.30(4H,m),7.28-7.20(2H,m),6.41-6.30(1H,m),4 .83-4.18(7H,m),4.12-3.50(7H,m),3.06-2.97(2H,m),2.31-2.17(2H,m),1.96-1.47(12H,m).

[0320] (Step 7) 6-Benzoyl-2-[3,5-bis-O-(oxan-2-yl)-β-D-arabinofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0321] To a solution of the compound obtained in Step 6 (18.74 g) and pyridine (13.1 mL) in dichloromethane (300 mL), trifluoromethanesulfonic anhydride (11 mL) was added dropwise under ice-cooling, and the mixture was stirred for 10 minutes. The reaction mixture was quenched by adding saturated saline solution, extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (300 mL), and a solution of tetrabutylammonium nitrite (28.34 g) in tetrahydrofuran (150 mL) was added dropwise under ice-cooling, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue, which was then washed successively with water and saturated saline. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (10.46 g). 1 H-NMR(CDCl3)δ:8.13-8.06(1H,m),7.63-7.30(4H,m),7.29-7.18(2H,m),6.79-6.55( 1H,m),4.93-3.45(14H,m),3.11-2.95(2H,m),2.32-2.14(2H,m),1.98-1.44(12H,m).

[0322] (Step 8) 6-Benzoyl-2-[2-deoxy-2-fluoro-3,5-bis-O-(oxan-2-yl)-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0323] To a solution of the compound obtained in Step 7 (10.46 g) and pyridine (7.3 mL) in dichloromethane (200 mL), trifluoromethanesulfonic anhydride (6.1 mL) was added dropwise under ice-cooling, and the mixture was stirred for 10 minutes. Saturated saline was added to the reaction mixture to terminate the reaction, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (200 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 150 mL) was added under ice-cooling, and the mixture was stirred at the same temperature for 3 hours. Saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (7.65 g). 1 H-NMR(CDCl3)δ:8.13-8.08(1H,m),7.53-7.31(4H,m),7.26-7.22(2H,m),6.68-6.53(1H,m),5.42-5.08(1H,m),4 .93-4.18(6H,m),4.10-3.76(3H,m),3.71-3.47(3H,m),3.06-2.96(2H,m),2.29-2.18(2H,m),1.96-1.47(12H,m).

[0324] (Step 9) 6-Benzoyl-2-(2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene Pyridinium p-toluenesulfonate (6.62 g) was added to a solution of the compound (7.65 g) obtained in Step 8 above in ethanol (150 mL), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was concentrated under reduced pressure, and ethyl acetate was added to the residue, which was then washed successively with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, and the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to give the title compound (3.55 g). 1 H-NMR(CDCl3)δ:8.05(1H,s),7.41-7.35(3H,m),7.30-7.24(2H,m),7.06(1 H,s),6.07-6.00(2H,m),5.85(1H,ddd,J=52.8,6.7,4.7Hz),4.66(1H,d,J= 3.9Hz),4.42-4.31(2H,m),4.20(1H,m),3.93(1H,dd,J=12.9,1.6Hz),3.74 (1H,td,J=12.3,1.6Hz),3.12-2.96(2H,m),2.51(1H,s),2.33-2.15(2H,m).

[0325] (Step 10) 6-Benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound (3.55 g) obtained in Step 9 above in dehydrated pyridine (50 mL) was added 4,4'-dimethoxytrityl chloride (4.43 g) and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. Methanol (1 mL) was added to the reaction mixture, which was stirred for approximately 10 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (5.77 g). 1H-NMR(CDCl3)δ:8.09(1H,s),7.45-7.41(2H,m),7.36-7.17(13H,m),6.85-6.79(4H,m) ,6.53(1H,dd,J=17.2,2.3Hz),5.40(1H,ddd,J=53.2,4.8,2.3Hz),4.83-4.72(1H,m),4. 32-4.21(2H,m),4.19-4.14(1H,m),3.79(3H,s),3.79(3H,s),3.59(1H,dd,J=11.0,2.7H z),3.45(1H,dd,J=11.0,3.5Hz),2.79(2H,t,J=6.3Hz),2.45(1H,s),2.24-2.11(2H,m).

[0326] (Step 11) 6-Benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene The compound (5.77 g) obtained in Step 10 above was used in a reaction in the same manner as in Step 6 of Example 1 to obtain the title compound (5.95 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 1:1). 1 H-NMR(CDCl3)δ:8.10(0.5H,s),8.09(0.5H,s),7.45-7.12(15H,m),6.84-6.75(4H,m),6. 57-6.46(1H,m),5.61-5.33(1H,m),5.07-4.83(1H,m),4.34-4.18(3H,m),3.93-3.72(7H, m),3.69-3.49(4H,m),3.38-3.27(1H,m),2.87-2.68(2H,m),2.61(1H,td,J=6.3,1.6Hz), 2.40(1H,td,J=6.4,2.1Hz),2.21-2.12(2H,m),1.21-1.13(9H,m),1.03(3H,d,J=6.7Hz).

[0327] (Step 12) The compound (1.02 g) obtained in Step 11 above was reacted in the same manner as in Step 7 of Example 1 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 An acetonitrile solution of {-phosphanyl}-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene was obtained. Using the obtained acetonitrile solution and the compound obtained in Step 3 above (1.15 g), a reaction was carried out in the same manner as in Step 12 of Example 1, and the obtained crude product was used directly in the next reaction.

[0328] (Step 13) 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate The crude product obtained in Step 12 above was used in a reaction in the same manner as in Step 13 of Example 1 to give the title compound (818 mg: containing impurities) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1152(M+H) + .

[0329] (Step 14) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (818 mg) obtained in Step 13 above was reacted in the same manner as in Step 14 of Example 1 to give Diastereomer 1 (107 mg: containing impurities) and Diastereomer 2 (101 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:891(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:891(M+H) + .

[0330] (Step 15-1) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1)

[0331] The compound (diastereomer 1) (107 mg: containing impurities) obtained in Step 14 above was reacted in the same manner as in Step 15-1 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-30% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 of Example 1 to obtain the title compound (29.1 mg). MS(ESI)m / z:777(M+H) + . 1 H-NMR(CD3OD)δ:8.58(1H,m),8.11(1H,m),8.03(1H,s),7.11(1H,s),6.47(1H,d,J=17.5Hz),6.26(1H ,d,J=8.5Hz),5.53-5.36(2H,m),5.29-5.17(1H,m),4.77(1H,d,J=4.2Hz),4.54-4.46(1H,m),4.44-4 .38(1H,m),4.35-4.32(1H,m),4.30-4.25(2H,m),4.25-4.16(1H,m),4.06-3.99(1H,m),3.96-3.85(1 H,m),3.82-3.71(2H,m),3.54-3.42(2H,m),2.77-2.68(1H,m),2.66-2.55(1H,m),2.02-1.81(2H,m). 31 P-NMR(CD3OD)δ:57.5(s),53.0(s).

[0332] (Step 15-2) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2)

[0333] The compound (diastereomer 2) (101 mg: containing impurities) obtained in Step 14 above was reacted in the same manner as in Step 15-1 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-20% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 of Example 1 to obtain the title compound (11.2 mg). MS(ESI)m / z:777(M+H) + . 1H-NMR(CD3OD)δ:8.61(1H,m),8.16(1H,m),8.02(1H,m),7.36(1H,s),6.49(1H,dd ,J=16.0,2.1Hz),6.28(1H,d,J=8.5Hz),5.56-5.33(3H,m),4.58-4.49(2H,m),4. 45-4.37(2H,m),4.31-4.27(1H,m),4.25-4.16(1H,m),4.10-3.98(3H,m),3.80(2 H,t,J=5.1Hz),3.48(2H,dd,J=6.7,3.6Hz),2.90-2.72(2H,m),2.00-1.90(2H,m). 31 P-NMR(CD3OD)δ:59.5(s),57.7(s).

[0334] Example 3: Synthesis of CDN49 (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0335] [ka]

[0336] [Synthetic scheme]

[0337] [ka] TIFF2025023046000109.tif234170

[0338] (Process 1) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine To a suspension of commercially available 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (13.0 g) in N,N-dimethylacetamide (60 mL), N-(2-bromoethyl)phthalimide (7.02 g) and 1,8-diazabicyclo[5.4.0]-7-undecene (4.1 mL) were added and stirred overnight at room temperature. N-(2-bromoethyl)phthalimide (1.75 g) and 1,8-diazabicyclo[5.4.0]-7-undecene (1.1 mL) were added and stirred for an additional day. The reaction mixture was quenched with water and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate / methanol] to give the title compound (12.4 g). 1 H-NMR(CDCl3)δ:7.83(1H,s),7.76-7.67(4H,m),7.64(1H,s),7.35-7.33(2H,m),7.2 5-7.11(7H,m),6.74-6.70(4H,m),5.93(1H,d,J=5.1Hz),5.68(1H,d,J=3.9Hz),4.71 (1H,q,J=4.8Hz),4.43(1H,m),4.37-4.18(3H,m),4.10-4.06(2H,m),3.730(3H,s),3 .728(3H,s),3.51(1H,m),3.36(1H,dd,J=10.6,3.9Hz),3.32(1H,dd,J=11.0,5.5Hz).

[0339] (Process 2) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine The compound (12.4 g) obtained in Step 1 above was used to carry out a reaction in the same manner as in Step 10 of Example 1, to give the title compound (4.18 g) and the positional isomer of the title compound, 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine (6.31 g). 1 H-NMR(CDCl3)δ:8.00(1H,s),7.82-7.77(2H,m),7.74(1H,s),7.72-7.67(2H,m),7.41-7.39 (2H,m),7.32-7.19(7H,m),6.83-6.78(4H,m),5.90(1H,d,J=5.1Hz),4.53-4.41(3H,m),4.3 2-4.25(1H,m),4.19-4.11(3H,m),3.79(3H,s),3.78(3H,s),3.46(1H,dd,J=10.6,3.1Hz),3 .24(1H,dd,J=10.8,4.1Hz),2.98(1H,d,J=6.7Hz),0.85(9H,s),0.04(3H,s),-0.03(3H,s). Positional isomer (2'-O-TBS form) 1 H-NMR(CDCl3)δ:7.97(1H,s),7.82-7.78(2H,m),7.73-7.69(2H,m),7.66(1H,s),7.44-7.41( 2H,m),7.33-7.18(7H,m),6.81(4H,d,J=7.8Hz),5.91(1H,d,J=5.9Hz),4.82(1H,t,J=5.5Hz), 4.43(1H,m),4.34-4.23(3H,m),4.18-4.08(2H,m),3.79(6H,s),3.46(1H,dd,J=10.6,2.7Hz), 3.36(1H,dd,J=10.6,3.5Hz),2.70(1H,d,J=3.1Hz),0.83(9H,s),-0.04(3H,s),-0.19(3H,s).

[0340] (Step 3) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine The compound (8.89 g) obtained in Step 2 above was used in a reaction in the same manner as in Step 6 of Example 1 to obtain the title compound (9.45 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 1:1). 1 H-NMR(CDCl3)δ:8.01(0.5H,s),8.00(0.5H,s),7.82-7.77(2H,m),7.74(0.5H,s),7.72-7.67(2.5H,m),7.42(2H,d,J=7.8Hz),7.3 3-7.18(7H,m),6.81(4H,d,J=8.6Hz),6.10(0.5H,d,J=5.5Hz),6.04(0.5H,d,J=5.1Hz),4.75(0.5H,m),4.60(0.5H,m),4.49-4.41( 1H,m),4.38-4.23(2H,m),4.22-4.05(3H,m),3.79(6H,s),3.78-3.65(1H,m),3.62-3.39(4H,m),3.33-3.23(1H,m),2.49(1H,t,J= 6.3Hz),2.34(1H,t,J=6.7Hz),1.12-1.08(9H,m),0.91(3H,d,J=7.0Hz),0.82(9H,s),0.06(1.5H,s),0.03(1.5H,s),-0.03(3H,s).

[0341] (Step 4) 4-chloro-5-iodo-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine To a solution of commercially available (PharmaBlock) 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (73.8 g) in N,N-dimethylformamide (10 mL), sodium hydride (containing 45% mineral oil) (13.3 g) was added under ice cooling, and the mixture was stirred for 40 minutes while warming to room temperature. The mixture was again ice-cooled, and [2-(chloromethoxy)ethyl](trimethyl)silane (51.0 mL) was added over 10 minutes, followed by stirring at the same temperature for 30 minutes. The reaction mixture, which had mostly solidified, was quenched by adding water (260 mL) in small portions. The solid was collected by filtration, washed with water (1500 mL) and hexane (600 mL), and dried under reduced pressure at 40 °C to obtain the title compound (97.63 g). MS(ESI)m / z:410(M+H) + . 1 H-NMR(CDCl3)δ:8.64(1H,s),7.54(1H,s),5.61(2H,s),3.52(2H,t,J=8.3Hz),0.92(2H,t,J=8.3Hz),-0.04(9H,s).

[0342] (Step 5) 4-(Benzyloxy)-5-iodo-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine To a solution of benzyl alcohol (27 mL) in N,N-dimethylformamide (170 mL), sodium hydride (containing 45% mineral oil) (12 g) was added under ice cooling, and the mixture was stirred for 40 minutes while warming to room temperature. The mixture was again ice-cooled, and a suspension of the compound obtained in Step 4 (97.63 g) in N,N-dimethylformamide (360 mL) was added over 40 minutes, followed by stirring at the same temperature for 35 minutes. The reaction was quenched by adding ice chips and saturated aqueous ammonium chloride solution. The reaction mixture was poured into a bilayer of saturated aqueous ammonium chloride and ethyl acetate, and extracted with ethyl acetate:toluene (9:1). The organic layer was washed twice with water and saturated brine, then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (107.7 g). MS(ESI)m / z:482(M+H)+ . 1 H-NMR(CDCl3)δ:8.47(1H,s),7.61(2H,d,J=7.3Hz),7.41(2H,t,J=7.6Hz),7.36-7.30(1H,m),7. 30(1H,s),5.65(2H,s),5.57(2H,s),3.52(2H,t,J=8.3Hz),0.91(2H,t,J=8.3Hz),-0.05(9H,s).

[0343] (Step 6) 4-(Benzyloxy)-5-(3,3-diethoxyprop-1-yn-1-yl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

[0344] To a solution of the compound (113.4 g) obtained in Step 5 above in acetonitrile (1000 mL) and triethylamine (98 mL) was added copper iodide (4.49 g), tetrakistriphenylphosphinepalladium(0) (8.17 g), and 3,3-diethoxyprop-1-yne (104 mL) at room temperature under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 4.5 hours. The reaction solution was concentrated under reduced pressure, and then ethyl acetate and hexane were added to the residue, and the precipitated solid was removed by filtration. The solid was washed with a mixture of ethyl acetate and hexane (1:1), and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (145.5 g, containing impurities). MS(ESI)m / z:482(M+H) + .

[0345] (Step 7) 5-(3,3-diethoxypropyl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-ol To a solution of the compound obtained in Step 6 (145.5 g) in ethanol (900 mL), 10% palladium-carbon catalyst (M) wet (50.2 g) was added and stirred at room temperature under a hydrogen atmosphere for 5 hours. Dichloromethane (500 mL) was added to the reaction mixture, the catalyst was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified twice by silica gel column chromatography [hexane / ethyl acetate] to give the title compound (59.6 g). MS(ESI)m / z:418(M+Na) + ,394[MH] - . 1 H-NMR(CDCl3)δ:11.23(1H,brs),7.85(1H,s),6.79(1H,s),5.47(2H,s),4.58(1H,t,J=5.9Hz),3.69(2H,m),3. 57-3.49(4H,m),2.90(2H,t,J=7.8Hz),2.07(2H,m),1.23(6H,t,J=7.1Hz),0.91(2H,t,J=8.1Hz),-0.04(9H,s).

[0346] (Step 8) 5-(3,3-diethoxypropyl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine-4-thiol To a solution of the compound (59.6 g) obtained in Step 7 above in dehydrated dichloromethane (300 mL) was added 2,6-lutidine (42 mL) under a nitrogen atmosphere. Trifluoromethanesulfonic anhydride (31 mL) was added dropwise over 20 minutes at -20°C, followed by stirring at the same temperature for 20 minutes. N,N-Dimethylformamide (500 mL) and sodium hydrogen monosulfide n-hydrate (33.5 g) were added under ice-cooling, and the mixture was warmed to room temperature and stirred for 2.5 hours. The reaction mixture was concentrated under reduced pressure, and low-boiling components were distilled off. The residue was poured into a bilayer of ethyl acetate and ice-cooled saturated aqueous ammonium chloride solution and extracted with a mixture of ethyl acetate:toluene (9:1). The organic layer was washed once with saturated aqueous ammonium chloride and twice with saturated brine, then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to give a mixture of the title compound and 2,6-lutidine. The resulting mixture was poured into a two-layer solution of ethyl acetate and 1N hydrochloric acid and extracted twice with ethyl acetate. The organic layer was washed three times with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (57.6 g). MS(ESI)m / z:410[MH] - . 1 H-NMR(CDCl3)δ:11.69(1H,brs),7.90(1H,s),6.96(1H,s),5.49(2H,s),4.61(1H,t,J=5.9Hz),3.71(2H,m),3.55(2H,m ),3.49(2H,t,J=8.1Hz),3.14(2H,t,J=7.8Hz),2.08(2H,m),1.23(6H,t,J=7.1Hz),0.90(2H,t,J=8.3Hz),-0.04(9H,s).

[0347] (Step 9) 3-(4-Sulfanyl-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propan-1-ol The compound obtained in Step 8 above (31.62 g) was dissolved in 80% aqueous acetic acid (300 mL) and stirred at room temperature for 30 minutes. After confirming the disappearance of the raw materials, the mixture was cooled on ice, and sodium borohydride (1.45 g) was carefully added in small portions and stirred at the same temperature for 30 minutes. Sodium triacetoxyborohydride (24.4 g) was then added over 15 minutes and stirred at the same temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure to approximately one-fifth of its original volume. The residue was neutralized to some extent by careful addition of sodium bicarbonate (solid) and then extracted with ethyl acetate. The organic layer was washed successively with saturated sodium bicarbonate and saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (17.93 g). MS(ESI)m / z:340[M+H] + . 1 H-NMR(CDCl3)δ:11.92(1H,brs),7.95(1H,s),7.01(1H,s),5.51(2H,s),3.70(2H,t,J=5.9Hz),3.50(2 H,t,J=8.1Hz),3.23(2H,t,J=7.3Hz),2.33(1H,brs),1.99(2H,m),0.91(2H,t,J=8.3Hz),-0.04(9H,s).

[0348] (Step 10) 2-{[2-(trimethylsilyl)ethoxy]methyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

[0349] To a solution of the compound (31.31 g) obtained in Step 9 above in dehydrated tetrahydrofuran (600 mL), triphenylphosphine (25.4 g) and diisopropyl azodicarboxylate (21.8 g) were added at 0°C under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane / ethyl acetate] and silica gel column chromatography [hexane / ethyl acetate] in turn to obtain the title compound (35.93 g, containing impurities). MS(ESI)m / z:322[M+H] + . 1 H-NMR(CDCl3)δ:8.57(1H,s),7.08(1H,s),5.58(2H,s),3.52(2H,t,J=8.3Hz),3.1 7(2H,m),3.06(2H,t,J=5.6Hz),2.36(2H,m),0.92(2H,t,J=8.3Hz),-0.05(9H,s).

[0350] (Step 11) (8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)methanol To a solution of the compound (35.93 g) obtained in step 10 above in dichloromethane (150 mL) was added trifluoroacetic acid (150 mL) at room temperature, and the mixture was stirred at the same temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and then azeotroped four times with toluene. A mixture of dichloromethane and hexane (1:2) was added to the residue, and the precipitated solid was collected by filtration (Solid 1). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane / ethyl acetate → ethyl acetate / methanol] to obtain Solid 2. Solid 1 and Solid 2 were combined to obtain the title compound (20.13 g). MS(ESI)m / z:222[M+H] + . 1 H-NMR (CDCl) δ: 8.60 (1H, s), 7.19 (1H, s), 5.71 (2H, s), 3.21 (2H, m), 3.07 (2H, m), 2.38 (2H, m). (Only observable peaks are shown.)

[0351] (Step 12) 2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene To a suspension of the compound (20.13 g) obtained in step 11 above in methanol (250 mL), 28% aqueous ammonia (150 mL) was added and stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure to approximately half its volume. The precipitated solid was collected by filtration and washed with ethanol to obtain solid 1. The filtrate was concentrated under reduced pressure, and solid 2 was obtained by the same procedure. The filtrate was applied to silica gel and purified by silica gel column chromatography [dichloromethane / methanol]. The fraction containing the target product was concentrated under reduced pressure, and after slurry washing with ethanol, the solid was collected by filtration (solid 3). Solid 1, solid 2, and solid 3 were combined to obtain the title compound (12.36 g). MS(ESI)m / z:192[M+H] + . 1 H-NMR(CDCl3)δ:10.53(1H,brs),8.57(1H,s),7.10(1H,s),3.18(2H,m),3.08(2H,t,J=5.6Hz),2.37(2H,m).

[0352] (Step 13) 2-(2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene To a suspension of the compound obtained in Step 12 (13.47 g) in dehydrated acetonitrile (350 mL) was added powdered potassium hydroxide (10.3 g) and tris[2-(2-methoxyethoxy)ethyl]amine (1.13 mL) under a nitrogen atmosphere and stirred at room temperature for 1.5 hours. Under ice cooling, a solution of 2,3,5-tri-O-benzyl-α-D-arabinofuranosyl chloride (40.2 g) in acetonitrile (100 mL) was added in small portions, as previously described (J. Med. Chem. 1976, 19, 6, 814-816). The mixture was then warmed to room temperature and stirred for 4 hours. Insoluble material was removed by filtration and washed with acetonitrile. The filtrate was concentrated under reduced pressure, and the residue was purified twice by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (26.19 g). MS(ESI)m / z:594[M+H] + . 1H-NMR(CDCl3)δ:8.51(1H,s),7.37-7.17(14H,m),6.86(2H,m),6.82(1H,d,J=4.9Hz) ,4.68(1H,d,J=11.7Hz),4.59(1H,d,J=11.7Hz),4.54(1H,d,J=13.2Hz),4.52(1H,d,J =11.7Hz),4.36-4.33(2H,m),4.22(1H,d,J=11.7Hz),4.14-4.08(2H,m),3.77(1H,dd, J=10.7,3.9Hz),3.72(1H,dd,J=10.5,4.1Hz),3.13(2H,m),2.81(2H,m),2.27(2H,m).

[0353] (Step 14) 2-β-D-arabinofuranosyl-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene To a solution of the compound (26.19 g) obtained in Step 13 above in dehydrated dichloromethane (300 mL), a solution of boron trichloride in dichloromethane (1 M, 200 mL) was added at -78°C under a nitrogen atmosphere. The mixture was stirred at the same temperature for 2 hours, then warmed to 0°C and stirred for an additional 4 hours. The reaction mixture was cooled again to -78°C, and a solution of methanol (80 mL) in dichloromethane (160 mL) was added. The mixture was stirred for 30 minutes while warming to room temperature. The reaction mixture was concentrated under reduced pressure and azeotroped twice with ethanol. The residue was slurried with ethanol (200 mL) and diethyl ether (100 mL), and the solid was collected by filtration (Solid 1). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol). The fraction containing the target product was concentrated under reduced pressure, slurried with ethanol, and the solid was collected by filtration (Solid 2). Solid 1 and Solid 2 were combined to give the title compound (13.2 g). MS(ESI)m / z:324[M+H] + . 1H-NMR(CD3OD)δ:8.73(1H,s),7.96(1H,s),6.70(1H,d,J=4.9Hz),4.32(1H,t,J=4.6Hz),4.25(1H,t,J=4.6Hz), 3.97(1H,m),3.90(1H,dd,J=12.0,3.2Hz),3.85(1H,dd,J=12.0,4.6Hz),3.53(2H,m),3.17(2H,m),2.43(2H,m).

[0354] (Step 15) 2-[3,5-bis-O-(oxan-2-yl)-β-D-arabinofuranosyl]-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene To a solution of the compound obtained in Step 14 (15.35 g) in anhydrous dimethyl sulfoxide (160 mL) were added 3,4-dihydro-2H-pyran (17.2 mL) and p-toluenesulfonic acid monohydrate (9.02 g) at 0°C and stirred at room temperature for 3 hours. 3,4-Dihydro-2H-pyran (8.6 mL) was added, and the mixture was stirred for 45 minutes. The reaction was then quenched by the immediate addition of triethylamine (13 mL). The reaction mixture was poured into a bilayer of ethyl acetate and saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was washed once with water and twice with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (10.81 g) as a mixture of four diastereomers. MS(ESI)m / z:492[M+H] + . 1H-NMR(CDCl3)δ:8.548(0.2H,s),8.546(0.3H,s),8.54(0.3H,s),8.53(0.2H,s),7.54(0. 2H,s),7.53(0.3H,s),7.51(0.2H,s),7.44(0.3H,s),6.75(0.2H,d,J=5.4Hz),6.71(0.2H, d,J=5.9Hz),6.57(0.3H,d,J=5.9Hz),6.56(0.3H,d,J=5.9Hz),4.87-4.69(2H,m),4.55-3 .54(10H,m),3.18-3.12(2H,m),3.10-2.96(2H,m),2.40-2.30(2H,m),1.92-1.51(12H,m).

[0355] (Step 16) 2-[2-Deoxy-2-fluoro-3,5-bis-O-(oxan-2-yl)-β-D-ribofuranosyl]-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene To a solution of the compound (10.81 g) obtained in Step 15 above in anhydrous dichloromethane (150 mL), pyridine (5.3 mL) and trifluoromethanesulfonic anhydride (5.6 mL) were added at 0°C under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 1 hour. After adding ice chips to the reaction mixture to quench the reaction, the reaction mixture was poured into a two-layer mixture of ethyl acetate and saturated aqueous sodium bicarbonate, and extracted with ethyl acetate. The organic layer was washed twice with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the crude triflate as an amorphous substance. The resulting crude triflate was dissolved in anhydrous tetrahydrofuran (150 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 154 mL) was added portionwise under ice cooling, followed by stirring overnight at the same temperature. The reaction was quenched by adding saturated aqueous ammonium chloride to the reaction mixture. The reaction mixture was concentrated under reduced pressure to approximately half its volume. The residue was poured into a mixture of two layers of ethyl acetate and saturated aqueous ammonium chloride, followed by extraction with ethyl acetate. The organic layer was washed once with saturated aqueous ammonium chloride and twice with saturated brine. The aqueous layer was extracted again with ethyl acetate, and the extract was washed with saturated brine. The combined organic layers were dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the title compound (40.37 g) in crude form. MS(ESI)m / z:494[M+H] + .

[0356] (Step 17) 2-(2-Deoxy-2-fluoro-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene To a solution of the compound (40.37 g) obtained in Step 16 above in methanol (400 mL) was added p-toluenesulfonic acid monohydrate (2.09 g), and the mixture was stirred at 60°C for 4 hours. Triethylamine (16 mL) was added to the reaction mixture to quench the reaction. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane / ethyl acetate → ethyl acetate / methanol]. The fraction containing the target product was concentrated under reduced pressure to a slurry, and the solid was collected by filtration. The obtained solid was washed with hexane / ethyl acetate (1:1) to obtain Solid 1. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane / methanol] to obtain Solid 2. Solid 1 and Solid 2 were combined to obtain the title compound (5.32 g). MS(ESI)m / z:326[M+H] + . 1 H-NMR(CDCl3)δ:8.51(1H,s),7.01(1H,s),6.00(1H,dd,J=13.7,6.3Hz),5.95(1H,dd,J=11.7,2.0Hz),5.87(1H,ddd,J=52.7 ,6.3,4.9Hz),4.69(1H,m),4.32(1H,brs),3.96(1H,d,J=12.7Hz),3.77(1H,m),3.17(2H,m),3.04(2H,m),2.41-2.31(3H,m).

[0357] (Step 18) 2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene The compound (5.32 g) obtained in Step 17 above was reacted in the same manner as in Step 10 of Example 2 to give the title compound (10.1 g). MS(ESI)m / z:628[M+H] + . 1H-NMR(CDCl3)δ:8.54(1H,s),7.42(2H,d,J=7.3Hz),7.32-7.21(8H,m),6.81(4H,m), 6.52(1H,dd,J=17.3,2.2Hz),5.37(1H,ddd,J=53.3,4.4,2.4Hz),4.76(1H,m),4.16(1 H,m),3.789(3H,s),3.786(3H,s),3.59(1H,dd,J=10.7,2.4Hz),3.44(1H,dd,J=10.7, 3.4Hz),3.12(2H,m),2.76(2H,t,J=5.6Hz),2.27(2H,m),2.18(1H,dd,J=7.8,2.9Hz).

[0358] (Step 19) 2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-2-deoxy-2-fluoro-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene The compound (10.1 g) obtained in Step 18 above was used in a reaction in the same manner as in Step 6 of Example 1 to obtain the title compound (12.6 g) as a diastereomeric mixture on the phosphorus atom (diastereomeric ratio = 1:1). 1H-NMR(CDCl3)δ:8.53(1H,s),7.40(2H,m),7.34-7.17(8H,m),6.84-6.74(4H,m),6.53(0.5H,dd,J=17.3,2.2Hz),6.48( 0.5H,dd,J=17.6,1.5Hz),5.50-5.31(1H,m),4.99(0.5H,m),4.85(0.5H,m),4.31-4.26(1H,m),3.93-3.76(1H,m),3.792 (1.5H,s),3.789(1.5H,s),3.779(1.5H,s),3.776(1.5H,s),3.67-3.51(4H,m),3.34-3.30(1H,m),3.13-3.10(2H,m),2 .76-2.69(2H,m),2.61(1H,td,J=6.3,2.4Hz),2.39(1H,m),2.28-2.21(2H,m),1.19-1.15(9H,m),1.03(3H,d,J=6.8Hz).

[0359] (Step 20) The compound (1.80 g) obtained in Step 19 above was reacted in the same manner as in Step 7 of Example 1 to obtain 2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 An acetonitrile solution of {-phosphanyl}-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene was obtained. Using the obtained acetonitrile solution and the compound obtained in Step 3 above (2.30 g), a reaction was carried out in the same manner as in Step 12 of Example 1, and the obtained crude product was used directly in the next reaction.

[0360] (Step 21) 3-{[(5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-7-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-15-fluoro-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-yl]oxy}propanenitrile

[0361] The crude product obtained in Step 20 above was used in a reaction in the same manner as in Step 13 of Example 1 to give the title compound (1.22 g) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1090(M+H) + .

[0362] (Step 22) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-16-{[tert-butyl(dimethyl)silyl]oxy}-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

[0363] To a solution of the compound (1.22 g) obtained in Step 21 above in ethanol (10 mL) and tetrahydrofuran (10 mL) was added hydrazine monohydrate (0.544 mL) and the mixture was stirred at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to obtain diastereomer 1 (108 mg, containing impurities) and diastereomer 2 (111 mg, containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:907(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:907(M+H) + .

[0364] (Step 23-1) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1)

[0365] The compound (diastereomer 1) (108 mg: containing impurities) obtained in Step 22 above was reacted in the same manner as in Step 15-1 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10%-50% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 of Example 1 to obtain the title compound (44.4 mg). MS(ESI)m / z:793(M+H) + . 1 H-NMR(CD3OD)δ:8.50(1H,s),8.42(1H,s),7.92(1H,s),7.56(1H,s),6.56(1H,d,J=16 .3Hz),6.21(1H,d,J=6.0Hz),5.57-5.40(2H,m),5.35-5.22(1H,m),4.73-4.67(1H,m) ,4.58-4.49(1H,m),4.45-4.26(4H,m),4.24-4.15(1H,m),4.05-3.96(1H,m),3.78-3. 51(1H,m),3.26-3.06(4H,m),2.93-2.82(1H,m),2.70-2.51(1H,m),2.29-2.07(2H,m). 31 P-NMR(CD3OD)δ:57.5(s),52.9(s).

[0366] (Step 23-2) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2)

[0367] The compound (diastereomer 2) (111 mg: containing impurities) obtained in Step 22 above was reacted in the same manner as in Step 15-1 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. Purification conditions: C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 20%-60% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 of Example 1 to obtain the title compound (40.6 mg). MS(ESI)m / z:793(M+H) + . 1 H-NMR(CD3OD)δ:8.57(1H,s),8.41(1H,s),8.13(1H,s),7.72(1H,s),6.59(1H ,dd,J=15.7,1.8Hz),6.26(1H,d,J=8.5Hz),5.61-5.34(3H,m),4.57-4.48(2H, m),4.48-4.38(2H,m),4.38-4.28(2H,m),4.08-3.98(3H,m),3.29-3.21(2H,m ),3.20-3.12(2H,m),3.02-2.92(1H,m),2.92-2.81(1H,m),2.29-2.15(2H,m). 31 P-NMR(CD3OD)δ:58.7(s),57.8(s).

[0368] Example 4: Synthesis of CDN50 N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[0369] [ka]

[0370] [Synthetic scheme]

[0371] [ka]

[0372] (Process 1-1) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-7-{1-[2-(2-hydroxyacetamido)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1)

[0373] To a solution of the compound (20.0 mg) obtained in Step 23-1 of Example 3 in N,N-dimethylformamide (0.5 mL), triethylamine (17 μL) and 1-[(hydroxyacetyl)oxy]pyrrolidine-2,5-dione (10.3 mg) were added and stirred at room temperature for 3 hours. The reaction solution was diluted with 10 mM aqueous triethylammonium acetate solution and purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 10%-30% (0 min-40 min)]. The resulting compound (triethylamine salt) was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 of Example 1 to obtain the title compound (15.6 mg). MS(ESI)m / z:851(M+H) + . 1 H-NMR(CD3OD)δ:8.43(1H,s),8.40(1H,brs),7.66(1H,brs),7.58(1H,s),6.53(1H,d,J=16.3Hz),6. 14(1H,d,J=8.5Hz),5.73-5.64(1H,m),5.59-5.42(1H,m),5.42-5.29(1H,m),4.80-4.74(1H,m),4.5 3-4.26(5H,m),4.21-4.12(1H,m),3.99-3.92(1H,m),3.83(2H,s),3.66-3.56(1H,m),3.43-3.26(2H ,m),3.23-3.06(2H,m),2.89-2.79(1H,m),2.49-2.33(1H,m),2.27-2.15(1H,m),2.15-2.02(1H,m). 31 P-NMR(CD3OD)δ:57.0(s),52.6(s).

[0374] (Step 1-2) Disodium (5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-7-{1-[2-(2-hydroxyacetamido)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2)

[0375] The compound (10.0 mg) obtained in Step 23-2 of Example 3 was reacted in the same manner as in Step 1-1 above, and then purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate / acetonitrile, acetonitrile: 7%-25% (0 min-40 min)]. The obtained compound (triethylamine salt) was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 15-1 of Example 1 to give the title compound (6.6 mg). MS(ESI)m / z:851(M+H) + . 1 H-NMR(CD3OD)δ:8.46(1H,s),8.42(1H,s),7.84(1H,s),7.78(1H,s),6.59(1H,d,J=1 5.1Hz),6.20(1H,d,J=7.9Hz),5.69-5.38(3H,m),4.60-4.50(2H,m),4.48-4.38(2H, m),4.31-4.20(2H,m),4.10-3.93(2H,m),3.87(2H,s),3.73-3.57(2H,m),3.52-3.41 (1H,m),3.25-3.10(2H,m),3.01-2.90(1H,m),2.83-2.71(1H,m),2.30-2.11(2H,m). 31P-NMR(CD3OD)δ:58.2(s),57.6(s).

[0376] Example 5: Synthesis of Drug Linker 2 [Synthetic scheme]

[0377] [ka]

[0378] (Process 1) The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 1.40 g). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 3 of Example 2 (1.41 g), a reaction was carried out in the same manner as in Step 12 of Example 1. The obtained crude product was used as is in the next reaction.

[0379] (Process 2) 2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl benzoate The crude product obtained in Step 1 above was used in a reaction in the same manner as in Step 13 of Example 1 to give the title compound (778 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1264(M+H) + .

[0380] (Step 3) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

[0381] Using the compound (778 mg) obtained in Step 2 above, a reaction was carried out in the same manner as in Step 14 of Example 1 to give Diastereomer 1 (255 mg) of the title compound and Diastereomer 2 (containing impurities). Diastereomer 2 was purified again by preparative HPLC [water / acetonitrile containing 0.2% triethylamine, acetonitrile containing 0.2% triethylamine: 5%-50% (0 min-40 min)] to give Diastereomer 2 (94.6 mg) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1003(M+H) + . 1H-NMR(CD3OD)δ:8.66(1H,s),8.21(1H,s),8.04(1H,s),7.33(1H,s),6.27(1H,d,J=5.1Hz),6.25(1H,d,J=3.6Hz),5.39-5 .29(1H,m),5.18-5.11(1H,m),4.85-4.81(1H,m),4.79-4.74(1H,m),4.71-4.66(1H,m),4.50-4.42(1H,m),4.36-4.21(2H, m),4.09-3.98(2H,m),3.85-3.78(2H,m),3.78-3.69(2H,m),3.55-3.46(2H,m),3.17(12H,q,J=7.3Hz),2.98-2.75(2H,m) ,2.05-1.88(2H,m),1.28(18H,t,J=7.3Hz),0.98(9H,s),0.85(9H,s),0.31(3H,s),0.27(3H,s),0.25(3H,s),0.09(3H,s). ジアステレオマー2 (high polarity) MS (ESI) m / z: 1003 (M+H) + . 1 H-NMR(CD3OD)δ:8.50(1H,s),8.22(1H,s),8.07(1H,s),7.20(1H,s),6.33(1H,d,J=7.3Hz),6.26(1H,d,J=9.1Hz ),5.59-5.44(1H,m),5.38-5.32(1H,m),5.21-5.11(1H,m),4.99-4.89(2H,m),4.68-4.54(2H,m),4.25-4.12(3H, m),4.09-4.03(1H,m),3.90-3.80(3H,m),3.59-3.51(2H,m),3.20(12H,q,J=7.3Hz),2.96-2.89(2H,m),2.07-1.9 8(2H,m),1.30(18H,t,J=7.3Hz),0.99(9H,s),0.74(9H,s),0.27(3H,s),0.27(3H,s),0.20(3H,s),-0.05(3H,s).

[0382] (Project 4-1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1)

[0383] To a solution of the compound (diastereomer 1) (30 mg) obtained in Step 3 above in tetrahydrofuran (0.5 mL), [(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycyl)amino]methyl acetate (91.7 mg) and p-toluenesulfonic acid monohydrate (11.8 mg) were added and stirred at room temperature for 6 hours. N,N-Dimethylformamide (0.5 mL) and 1,8-diazabicyclo[5.4.0]-7-undecene (56 μL) were added to the reaction solution, and the mixture was stirred at room temperature for 3 hours. 10 mM aqueous triethylammonium acetate solution was added to the reaction solution, and the mixture was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to obtain the title compound (25.6 mg) containing the starting material as an impurity. MS(ESI)m / z:1089(M+H) + .

[0384] (Step 4-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2)

[0385] The compound (diastereomer 2) (84.6 mg) obtained in the above step 3 was reacted in the same manner as in the above step 4-1 to give the title compound (70.9 mg) containing the starting material as an impurity. MS(ESI)m / z:1089(M+H) + .

[0386] (Step 5-1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) diastereomer 1

[0387] Triethylamine trihydrofluoride (2 mL) was added to the compound (25.6 mg) obtained in step 4-1 above, and the mixture was stirred at 45°C for 3 hours. To the reaction mixture was added an ice-cooled mixture of 1 M triethylammonium bicarbonate solution (10 mL) and triethylamine (2 mL) at room temperature. The reaction mixture was concentrated under reduced pressure and then purified by C18 silica gel column chromatography (10 mM aqueous triethylammonium acetate solution / acetonitrile) to obtain the title compound (16.6 mg: containing impurities derived from the raw materials of step 7-1). MS(ESI)m / z:861(M+H) + .

[0388] (Step 5-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (70.9 mg) obtained in the above step 4-2 was reacted in the same manner as in the above step 5-1 to obtain the title compound (51.7 mg: containing impurities derived from the raw materials in step 4-2). MS(ESI)m / z:861(M+H) + .

[0389] (Step 6-1) Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (Drug Linker 2a: Diastereomer 1)

[0390] To a solution of the compound obtained in Step 5-1 (16.6 mg) in N,N-dimethylformamide (0.5 mL), triethylamine (6 μL) and the compound obtained in Step 8 (15.5 mg) described below were added and stirred at room temperature for 3 hours. Benzylamine (3 μL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. 10 mM aqueous triethylammonium acetate solution and methanol were added, and the mixture was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 10%-45% (0 min-30 min)] to obtain the title compound (5.1 mg). MS(ESI)m / z:1409(M+H) + . 1H-NMR(CD3OD)δ:8.66-8.60(1H,m),8.17(1H,s),8.02(1H,s),7.65-7.48(2H,m),7.43-7.36(3H,m),7.31-7.13(8H,m),7 .11(1H,s),6.30-6.21(2H,m),5.46-5.37(1H,m),5.23-5.16(1H,m),5.08-4.99(1H,m),4.86-4.81(1H,m),4.80-4.75(1 H,m),4.70-4.40(7H,m),4.40-4.20(3H,m),4.10-3.97(3H,m),3.86-3.58(8H,m),3.51-3.43(3H,m),3.18(12H,q,J=7.3 Hz),3.01-2.93(1H,m),2.85-2.72(3H,m),2.37-2.15(2H,m),2.01-1.93(2H,m),1.29(18H,t,J=7.3Hz).(Only observable peaks are listed)

[0391] (Step 6-2) Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (Drug Linker 2b: Diastereomer 2) The compound (51.7 mg) obtained in the above step 5-2 was reacted in the same manner as in the above step 6-1, and then purified under the following [purification conditions] to obtain the title compound (33.7 mg). [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 10%-50% (0 min-30 min)]. MS(ESI)m / z:1409(M+H) + . 1 H-NMR(CD3OD)δ:8.73(1H,d,J=6.7Hz),8.19(1H,d,J=3.0Hz),8.02(1H,s),7.66-7.50(2H,m),7.43-7.37(3H,m ),7.33-7.13(8H,m),7.11(1H,s),6.33-6.23(2H,m),5.51-5.38(2H,m),5.04(1H,t,J=13.6Hz),4.83-4.77(1H, m),4.64-4.55(2H,m),4.52-4.26(6H,m),4.25-3.97(2H,m),3.93-3.45(13H,m),3.19(12H,q,J=7.3Hz),3.17-3 .11(1H,m),3.02-2.92(1H,m),2.91-2.73(3H,m),2.40-2.24(2H,m),2.07-1.95(3H,m),1.30(18H,t,J=7.3Hz).

[0392] (Step 7) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanine A solution of commercially available (BACHEM) (2S)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-phenylpropanoic acid (2.86 g) in N,N-dimethylformamide (51.2 mL) was added with triethylamine (2.56 mL) and commercially available (Click Chemistry Tools) 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (3.69 g) and stirred at room temperature for 24 hours. A solution of citric acid monohydrate (24.0 g) in water (500 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a mixed solution of ethyl acetate / acetonitrile, and then precipitated with diisopropyl ether and collected by filtration to obtain the title compound (4.30 g). 1 H-NMR(DMSO-d6)δ:12.8(1H,brs),8.15-7.95(3H,m),7.68-7.17(13H,m),5.01(1H,d,J=14.2Hz),4.41-4.37(1H,m),3.74-3.57(5 H,m),3.05-3.01(1H,m),2.87(1H,dd,J=14.2,9.3Hz),2.68-2.59(1H,m),2.32-2.25(1H,m),2.09-2.03(1H,m),1.82-1.76(1H,m).

[0393] (Step 8) 2,5-Dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalaninate To a solution of the compound obtained in Step 7 (2.10 g) in N,N-dimethylformamide (75.9 mL), N-hydroxysuccinimide (961 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.60 g) were added, and the mixture was stirred at room temperature for 21 hours under a nitrogen atmosphere. The reaction mixture was diluted with dichloromethane, washed three times with ice water, and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. Toluene was added to the residue, and the mixture was concentrated under reduced pressure again. The residue was dissolved in acetonitrile and purified by C18 silica gel column chromatography [acetonitrile: 100%]. The fraction containing the target product was concentrated under reduced pressure, and diisopropyl ether was added to the residue to form a slurry. The resulting solid was collected by filtration to give the title compound (2.59 g). 1 H-NMR(DMSO-d6)δ:8.58-8.51(1H,m),8.17-8.00(2H,m),7.66-7.20(13H,m),5.02-4.98(1H,m),4.90-4.85(1H,m),3.78-3.57(5H,m) ),3.24-3.19(1H,m),3.06-3.00(1H,m),2.82(4H,brs),2.67-2.58(1H,m),2.32-2.23(1H,m),2.09-2.02(1H,m),1.82-1.75(1H,m).

[0394] Example 6: Synthesis of Drug Linker 17 [Synthetic scheme]

[0395] [ka]

[0396] (Process 1) [(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methyl acetate To a mixture of commercially available N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycylglycine (9.32 g) (SUNDIA) in tetrahydrofuran (100 mL) and toluene (33.3 mL), pyridine (3.26 mL) and lead tetraacetate (17.9 g) were added at room temperature, and the mixture was stirred at 65°C for 3 hours. Insoluble matter was removed by filtration, washed with tetrahydrofuran, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to give the title compound (8.24 g). 1 H-NMR(CDCl3)δ:7.14(1H,brs),5.27(2H,d,J=7.3Hz),5.20(1H,brs),4.22-4. 16(2H,m),3.88(2H,d,J=6.0Hz),2.08(3H,s),1.04-0.97(2H,m),0.05(9H,s).

[0397] (Process 2) 2',3',5'-Tris-O-[tert-butyl(dimethyl)silyl]-1-(2-hydroxyethyl)inosine A literature-described (Chem. Pharm. Bull. 1987, 35(1), 72-79) solution of 2',3',5'-tris-O-[tert-butyl(dimethyl)silyl]inosine (31.3 g) in tetrahydrofuran (75 mL) and N,N-dimethylacetamide (75 mL) was added with 2-bromoethanol (4.82 mL) and 1,8-diazabicyclo[5.4.0]-7-undecene (7.65 mL) and stirred at room temperature for 23 hours. Water and ethyl acetate were added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (29.4 g). MS(ESI)m / z:655(M+H) + . 1H-NMR(CDCl3)δ:8.16(1H,s),7.99(1H,d,J=2.4Hz),5.97(1H,d,J=4.2Hz), 4.40-4.25(3H,m),4.18-4.06(3H,m),4.03-3.92(2H,m),3.79(1H,dd,J=11. 5,2.4Hz),3.08-2.83(1H,brm),0.96(9H,s),0.92(9H,s),0.82(9H,s),0.15 (3H,s),0.14(3H,s),0.09(3H,s),0.08(3H,s),-0.02(3H,s),-0.15(3H,s).

[0398] (Step 3) 2',3',5'-Tris-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine To a solution of the compound obtained in Step 2 (15.6 g) in toluene (46.8 mL), the compound obtained in Step 1 (10.4 g) and pyridine (9.63 mL) were added, and the mixture was stirred at 110°C for 12 hours. To the reaction solution, the compound obtained in Step 1 (3.46 g) was added, and the mixture was stirred at 110°C for 1 day. A saturated aqueous solution of sodium bicarbonate and dichloromethane were added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (20.6 g: containing impurities). MS(ESI)m / z:885(M+H) + .

[0399] (Step 4) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine To a solution of the compound obtained in Step 3 (20.6 g) in tetrahydrofuran (50 mL), triethylamine trihydrofluoride (10 mL) was added and stirred at room temperature for 17 hours. A mixture of 1 M triethylammonium bicarbonate solution (50 mL) and triethylamine (10 mL) was slowly added to the reaction mixture under ice cooling, and the reaction mixture was then concentrated under reduced pressure. The residue was crudely purified by C18 silica gel column chromatography (water / acetonitrile) and then lyophilized. The resulting crude product was azeotroped with pyridine. 4,4'-Dimethoxytrityl chloride (4.73 g) was added to a solution of the residue in pyridine (50 mL) at 0°C and stirred at 4°C for 17 hours. Methanol (2 mL) was added to the reaction mixture, which was stirred at room temperature for 15 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate / methanol / 0.1% triethylamine) to obtain the title compound (9.18 g, containing impurities). MS(ESI)m / z:845(M+H) + . 1 H-NMR(CDCl3)δ:7.94(1H,s),7.88(1H,s),7.65(1H,brs),7.41-7.36(2H,m),7.32-7.15 (7H,m),6.83-6.76(4H,m),5.96(1H,d,J=6.1Hz),5.73-5.65(2H,m),4.87-4.80(1H,m),4 .76-4.61(2H,m),4.44-4.39(1H,m),4.35-4.30(1H,m),4.22-4.05(4H,m),3.83-3.73(2 H,m),3.77(6H,s),3.72-3.67(2H,m),3.48-3.32(3H,m),0.99-0.91(2H,m),0.02(9H,s).

[0400] (Step 5) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine The compound (5.96 g) obtained in Step 4 above was used to carry out a reaction in the same manner as in Step 10 of Example 1, to give the title compound (2.33 g) and the positional isomer of the title compound, 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (2.45 g). MS(ESI)m / z:959(M+H) + . 1 H-NMR(CDCl3)δ:7.99(1H,s),7.93(1H,s),7.45-7.39(2H,m),7.35-7.18(7H,m),7.05(1H,brs),6.84-6.77 (4H,m),5.92(1H,d,J=5.4Hz),5.46(1H,brs),4.71-4.61(3H,m),4.54-4.51(1H,m),4.22-4.10(5H,m),3.81 -3.76(2H,m),3.78(3H,s),3.78(3H,s),3.74(2H,d,J=6.0Hz),3.48(1H,dd,J=10.9,4.2Hz),3.26(1H,dd,J= 10.9,4.2Hz),3.16(1H,d,J=6.7Hz),1.00-0.93(2H,m),0.89(9H,s),0.09(3H,s),0.02(9H,s),0.02(3H,s). Positional isomer (2'-O-TBS form) MS(ESI)m / z:959(M+H) + . 1H-NMR(CDCl3)δ:7.99(1H,s),7.91(1H,s),7.48-7.42(2H,m),7.37-7.18(8H,m),6.85-6.78(4H,m),5.96(1H, d,J=5.4Hz),5.63(1H,brs),4.88(1H,t,J=5.1Hz),4.66(2H,d,J=6.7Hz),4.36-4.32(1H,m),4.27-4.19(2H,m) ,4.18-4.10(3H,m),3.81-3.74(4H,m),3.78(3H,s),3.78(3H,s),3.50(1H,dd,J=10.9,3.6Hz),3.38(1H,dd,J =10.9,3.6Hz),2.73(1H,d,J=4.2Hz),0.97-0.90(2H,m),0.86(9H,s),0.02(3H,s),0.01(9H,s),-0.09(3H,s).

[0401] (Step 6) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine

[0402] The compound (2.33 g) obtained in Step 5 above was reacted in the same manner as in Step 11 of Example 1 to give the title compound (2.72 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 6:4). MS(ESI)m / z:1159(M+H) + . 1H-NMR(CDCl3)δ:8.03(0.4H,s),8.02(0.6H,s),7.95(0.6H,s),7.92(0.4H,s),7.46-7.40(2H,m),7.35-7.17(7H,m),6.88(1H,brs),6.84- 6.78(4H,m),6.15(0.6H,d,J=4.2Hz),6.10(0.4H,d,J=4.8Hz),5.34(1H,brs),4.86-4.61(3H,m),4.48-4.42(1H,m),4.29-4.09(5H,m),3.8 3-3.44(9H,m),3.79(3H,s),3.78(3H,s),3.32-3.23(1H,m),2.58-2. 49(1H,m),2.44-2.38(1H,m),1.15(3.6H,d,J=6.7Hz),1.11(6H,d,J=6 .7Hz),1.04-0.92(2H,m),0.97(2.4H,d,J=6.7Hz),0.85(3.6H,s),0.84(5.4H,s),0.09(1.2H,s),0.06(1.8H,s),0.03(9H,s),0.00(3H,s).

[0403] (Step 7) The compound (2.15 g) obtained in Step 11 of Example 2 was reacted in the same manner as in Step 7 of Example 1 to obtain 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 An acetonitrile solution of {-phosphanyl}-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene was obtained. Using the obtained acetonitrile solution and the compound obtained in Step 6 above (2.72 g), a reaction was carried out in the same manner as in Step 12 of Example 1, and the obtained crude product was used directly in the next reaction.

[0404] (Step 8) 2-(Trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-15-fluoro-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate The crude product obtained in Step 7 above was used in a reaction similar to that in Step 13 of Example 1 to give the title compound (1.47 g: containing impurities) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1278(M+H) + .

[0405] (Step 9) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (1.47 g) obtained in Step 8 above in a methanol (10 mL)-tetrahydrofuran (10 mL) mixture, 28% aqueous ammonia (10 mL) was added and the mixture was stirred for 6 hours at 50° C. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to give diastereomer 1 (204 mg, containing impurities) and diastereomer 2 (205 mg, containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1121(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:1121(M+H) + .

[0406] (Step 10-1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (diastereomer 1) (204 mg) obtained in Step 9 above in tetrahydrofuran (6 mL), a solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 3 mL) was added and stirred at room temperature for 33 hours. After storage at 4°C for 3 days, 10 mM aqueous triethylammonium acetate solution was added to the reaction mixture, and the mixture was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10%-50% (0 min-40 min)] to obtain the title compound (40.7 mg, containing impurities). MS(ESI)m / z:863(M+H) + .

[0407] (Step 10-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-oxo-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate)

[0408] The compound (diastereomer 2) (205 mg) obtained in the above step 9 was reacted in the same manner as in the above step 10-1, and then purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile-methanol solution (1:1), acetonitrile-methanol solution (1:1): 10%-50% (0 min-40 min)] to obtain the title compound (50.8 mg: containing impurities). MS(ESI)m / z:863(M+H) + .

[0409] (Step 11-1) Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (Drug Linker 17a: Diastereomer 1)

[0410] To a solution of the compound obtained in Step 10-1 (40.7 mg) in N,N-dimethylformamide (0.5 mL), triethylamine (11 μL) and the compound obtained in Step 8 of Example 5 (25.4 mg) were added, and the mixture was stirred at room temperature for 2 hours. Benzylamine (8 μL) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour. 10 mM aqueous triethylammonium acetate solution and methanol were added, and the mixture was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 20%-45% (0 min-40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate solution / methanol, methanol: 40%-90% (0 min-40 min)] to obtain the title compound (25.1 mg). MS(ESI)m / z:1411(M+H) + . 1H-NMR(CD3OD)δ:8.58(1H,s),8.09(1H,s),8.04(1H,s),7.57-7.49(2H,m),7.43-7.34(3H,m),7.32-7.08(9H,m),6. 47(1H,d,J=16.9Hz),6.23(1H,d,J=7.9Hz),5.56-5.37(2H,m),5.31-5.17(1H,m),5.03(1H,d,J=13.9Hz),4.79(1H,d ,J=4.2Hz),4.64-4.38(6H,m),4.36-4.21(4H,m),4.05-3.60(10H,m),3.53-3.42(3H,m),3.18(12H,q,J=7.3Hz),3. 01-2.92(1H,m),2.86-2.73(1H,m),2.70-2.54(2H,m),2.37-2.16(2H,m),2.06-1.77(3H,m),1.28(18H,t,J=7.3Hz).

[0411] (Step 11-2) Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (Drug Linker 17b: Diastereomer 2)

[0412] The compound obtained in Step 10-2 (50.8 mg) and the compound obtained in Step 8 of Example 5 (31.7 mg) were reacted in the same manner as in Step 11-1 above, and then purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 25%-45% (0 min-40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate solution / methanol, methanol: 45%-90% (0 min-40 min)] to obtain the title compound (23.4 mg). MS(ESI)m / z:1411(M+H) + . 1 H-NMR(CD3OD)δ:8.67(1H,s),8.14(1H,s),8.02(1H,s),7.67-7.50(2H,m),7.43-7.36(3H,m), 7.34-7.12(9H,m),6.48(1H,d,J=15.1Hz),6.26(1H,t,J=8.8Hz),5.60-5.31(3H,m),5.09-5.00 (1H,m),4.61-4.22(9H,m),4.11-3.59(13H,m),3.50-3.44(2H,m),3.18(12H,q,J=7.3Hz),3.04 -2.93(1H,m),2.87-2.74(3H,m),2.39-2.22(2H,m),2.06-1.85(3H,m),1.28(18H,t,J=7.3Hz).

[0413] Example 7: Synthesis of Drug Linker 20 [Synthetic scheme]

[0414] [ka]

[0415] (Process 1) N-(Azaniumylacetyl)glycyl-L-phenylalanylglycine trifluoroacetate Trifluoroacetic acid (15 mL) was added to a solution of commercially available N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (3.00 g) in dichloromethane (30 mL) at room temperature, and the mixture was stirred at the same temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, then suspended in toluene and concentrated under reduced pressure again. This concentration procedure was repeated two more times. The residue was slurried with diethyl ether (100 mL) and collected by filtration to obtain the title compound (3.27 g) in crude form. MS(ESI)m / z:337(M+H) + . 1 H-NMR(DMSO-d6)δ:12.60(1H,brs),8.48(1H,t,J=5.6Hz),8.44(1H,t,J=5.9Hz) ,8.31(1H,d,J=8.8Hz),7.97(3H,brs),7.28-7.16(5H,m),4.58(1H,m),3.87(1H ,dd,J=16.8,5.6Hz),3.78(2H,d,J=5.9Hz),3.67(1H,dd,J=17.1,5.4Hz),3.56( 2H,brd,J=4.4Hz),3.05(1H,dd,J=13.7,3.9Hz),2.74(1H,dd,J=13.7,10.3Hz).

[0416] (Process 2) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanylglycine To a solution of the compound (2.09 g) obtained in Step 1 above in N,N-dimethylformamide (46.4 mL), triethylamine (0.804 mL) and 1-{[4-(11,12-didehydrodibenzo[b,f]azocine-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (1.87 g) were added and stirred at room temperature for 21 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane / methanol]. Diethyl ether was added to a dichloromethane solution of the obtained compound to form a slurry, which was then filtered to obtain the title compound (2.10 g). MS(ESI)m / z:624(M+H)+ . 1 H-NMR (DMSO-d6) δ: 8.20-7.91 (4H, m), 7.68-7.13 (13H, m), 4.98 (1H, dd, J = 13.9, 3.2 Hz), 4.51-4.46 (1H, m), 3.73-3.47 (7H, m), 3.00 (1H, dd, J = 13.9, 4.1 Hz), 2.73 (1H, t, J = 11.7 Hz), 2.67-2.57 (1H, m), 2.29-2.22 (1H, m), 2.06-2.01 (1H, m), 1.80-1.73 (1H, m). (Only observable peaks are shown.)

[0417] (Step 3) 2,5-Dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanylglycinate To a solution of the compound (2.10 g) obtained in Step 2 above in N,N-dimethylformamide (33.7 mL), N-hydroxysuccinimide (426 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (710 mg) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction solution was diluted with dichloromethane, washed three times with ice water, and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the oily residue to precipitate a solid. The solvent was evaporated under reduced pressure, and diethyl ether was added to the resulting solid to form a slurry, which was then collected by filtration to give the title compound (2.18 g). 1H-NMR(DMSO-d6)δ:8.74-8.69(1H,m),8.16-8.08(2H,m),8.00-7.93(1H,m),7.71- 7.15(13H,m),5.00(1H,dd,J=13.9,3.0Hz),4.55-4.49(1H,m),4.27(2H,t,J=6.0Hz ),3.77-3.68(1H,m),3.64-3.50(4H,m),3.02(1H,dd,J=13.9,4.2Hz),2.82-2.73( 5H,m),2.69-2.58(1H,m),2.33-2.24(1H,m),2.10-2.02(1H,m),1.83-1.75(1H,m).

[0418] (Step 4) Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfide octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)glycinamide (Drug Linker 20)

[0419] To a solution of the compound obtained in Step 23-2 of Example 3 (25.0 mg) in N,N-dimethylformamide (0.5 mL), triethylamine (8 μL) and the compound obtained in Step 3 above (25.8 mg) were added and stirred at room temperature for 2 hours. Benzylamine (7 μL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. 10 mM aqueous triethylammonium acetate solution and methanol were added to the reaction solution, and the mixture was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 30%-50% (0 min-40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate solution / methanol, methanol: 50%-90% (0 min-40 min)] to obtain the title compound (33.1 mg). MS(ESI)m / z:1398(M+H) + . 1 H-NMR(CD3OD)δ:8.58(1H,brs),8.39(1H,d,J=8.5Hz),8.00(1H,brs),7.71(1H,brs),7.64-7.49(2H,m),7.44-7.3 7(3H,m),7.32-7.10(8H,m),6.59(1H,d,J=15.1Hz),6.23(1H,d,J=8.5Hz),5.65-5.36(3H,m),5.03(1H,dd,J=16.6, 14.2Hz),4.57-4.38(5H,m),4.30-4.17(2H,m),4.07-3.95(2H,m),3.94-3.50(9H,m),3.50-3.35(1H,m),,3.19(12 H,q,J=7.3Hz),3.18-3.07(3H,m),3.04-2.73(4H,m),2.40-2.11(4H,m),2.05-1.92(1H,m),1.29(18H,t,J=7.3Hz).

[0420] Example 8: Synthesis of Drug Linker 21 [Synthetic scheme]

[0421] [ka]

[0422] (Process 1) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-({2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethoxy}methyl)glycinamide 1,8-diazabicyclo[5.4.0]-7-undecene (0.74 mL) was added to a solution of {[(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycyl)amino]methoxy}acetic acid (955 mg) in N,N-dimethylformamide (8.0 mL) as described in the literature (WO2014 / 057687), and the mixture was stirred at room temperature for 1 hour (reaction solution A). To a solution of the compound obtained in Example 5, Step 7 (938 mg) in N,N-dimethylformamide (8.0 mL), N-hydroxysuccinimide (229 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (380 mg) were added and the mixture was stirred at room temperature for 50 minutes (reaction solution B). Reaction solution A was added to reaction solution B, and the mixture was stirred at room temperature for 1 hour. Dichloromethane (50 mL) and 10% aqueous citric acid (10 mL) were added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [chloroform / (chloroform / methanol / water = 7:3:1 lower layer)]. N-Hydroxysuccinimide (229 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (380 mg) were added to a solution of the obtained compound in N,N-dimethylformamide (8.0 mL), and the mixture was stirred at room temperature for 30 minutes. Dichloromethane (100 mL) and water (25 mL) were added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [chloroform / methanol]. The fraction containing the target product was concentrated under reduced pressure, and the residue was slurried with diethyl ether, and the resulting solid was collected by filtration to give the title compound (412 mg). 1H-NMR(DMSO-d6)δ:8.72(1H,m),8.32(1H,m),8.17-7.96(3H,m),7.71-7.15(13H,m),5.01(1H,d,J=13.9Hz),4.70-4.48(5H,m), 3.81-3.51(7H,m),3.05(1H,dd,J=14.2,3.9Hz),2.83(4H,s),2.80(1H,m),2.64(1H,m),2.28(1H,m),2.07(1H,m),1.79(1H,m).

[0423] (Process 2) Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-({2-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfidooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)amino]-2-oxoethoxy}methyl)glycinamide (Drug Linker 21)

[0424] The compound obtained in Step 23-2 of Example 3 (15.0 mg) and the compound obtained in Step 1 (17.4 mg) were reacted in the same manner as in Step 4 of Example 7, and then purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 25%-50% (0 min-40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate solution / methanol, methanol: 40%-90% (0 min-40 min)] to give the title compound (21.8 mg). MS(ESI)m / z:1485(M+H) + . 1 H-NMR(CD3OD)δ:8.61(1H,brs),8.41-8.34(1H,m),8.10-8.05(1H,m),7.72-7.37(6H,m ),7.32-7.12(8H,m),6.63-6.50(1H,m),6.27-6.22(1H,m),5.65-5.31(3H,m),5.07-4. 94(1H,m),4.68-4.20(10H,m),4.11-3.53(14H,m),3.26-3.08(2H,m),3.19(12H,q,J=7 .3Hz),3.06-2.67(4H,m),2.40-2.11(4H,m),2.05-1.88(1H,m),1.29(18H,t,J=7.3Hz).

[0425] Example 9: Synthesis of glycosylation remodeling antibody 1 Preparation of anti-Trop2 antibody 1-[SG-(N3)2]2

[0426] [Synthetic scheme] [ka]

[0427] (Process 1) Preparation of (Fucα1,6)GlcNAc-anti-Trop2 antibody 1 A solution of wild-type EndoS in phosphate-buffered saline (0.064 mL, 7.70 mg / mL, pH 6.0) was added to a solution of anti-Trop2 antibody 1 (6.0 mL, 16.55 mg / mL, pH 6.0) prepared according to Reference Example 5, and the mixture was shaken at 37°C for 2 hours. The progress of the reaction was monitored using an Experion electrophoresis station (BIO-RAD). After completion of the reaction, the product was purified by affinity chromatography and hydroxyapatite column chromatography according to the following methods. (1) Purification by affinity chromatography Purification device: AKTA avant 25 (GE Healthcare) Column: HiTrap rProtein A FF (5 mL) (GE Healthcare) Flow rate: 5 mL / min (1.25 mL / min when charging) For binding to the column, the reaction mixture was loaded directly onto the column, and 2 CV (column volume) of binding buffer (20 mM phosphate buffer, pH 6.0) was applied at 1.25 mL / min, followed by 5 CV at 5 mL / min. For intermediate washes, 15 CV of wash solution (20 mM phosphate buffer, pH 7.0, 0.5 M sodium chloride solution) was applied. For elution, 6 CV of elution buffer (ImmunoPure IgG Elution buffer, Pierce) was applied. The eluate was immediately neutralized with 1 M Tris buffer (pH 9.0). The fractions containing the target product were buffer-exchanged into 5 mM phosphate buffer, 50 mM 2-morpholinoethanesulfonic acid (MES) solution (pH 6.8) according to the method described in Common Procedure C. The antibody concentration in the resulting buffer was measured according to the method described in Common Procedure B, and a crudely purified titled antibody solution (12.38 mg / mL, 8 mL) was obtained. (2) Purification by hydroxyapatite chromatography Purification device: AKTA avant 25 (GE Healthcare) Column: Bio-Scale Mini CHT Type I Cartridge (5 mL) (BIO-RAD) Flow rate: 5 mL / min (1.25 mL / min when charging)

[0428] The solution obtained in (1) above was applied to the column, and 2 CV of solution A [5 mM phosphate buffer, 50 mM MES solution (pH 6.8)] was applied at 1.25 mL / min, followed by 3 CV at 5 mL / min. Elution was then performed using solutions A and B [5 mM phosphate buffer, 50 mM MES solution (pH 6.8), 2 M sodium chloride solution]. The elution conditions were solution A:solution B = 10...

Claims

1. The following formula (II): 【Chemistry 1】 (In the formula, m １ is in the range of 1 to 10, Ab refers to an antibody or a functional fragment of the antibody, and the sugar chain of the antibody may be remodeled. wherein the antibody is any antibody selected from the group consisting of an anti-CD70 antibody, an anti-TROP2 antibody, and an anti-EGFR antibody; The antibody is an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:2, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:3 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:4; an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:6, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:8, an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:10, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:11 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:8, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:9, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:10, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:11, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:70, L represents a linker connecting Ab and D; Ab may be linked to L directly through its amino acid residue, or through a glycan or remodeled glycan of Ab; D is represented by the following formula (I): 【Chemistry 2】 (where: L is L １ Any -NH included in ２ or bonded to a hydroxy group, L １ is expressed by the following three formulas: 【Chemistry 3】 (wherein the wavy lines indicate the substitution positions) represents any one of the groups Q and Q ’ each independently represents a hydroxyl group or a thiol group, R ２１ and R ２２ each independently represents a hydroxy group or a fluorine atom, W represents -NH- or a sulfur atom. (representing a compound represented by the formula: An antibody-drug conjugate represented by the formula:

2. D is represented by the following two formulas: 【Chemistry 4】 (Here, L １ , Q, Q′ and W are as previously defined. The antibody-drug conjugate of claim 1, wherein

3. D is a compound of the following formula: 【Chemistry 5】 where the asterisk indicates the bond to L, and Q, Q' and W are as previously defined. The antibody-drug conjugate according to claim 1 or 2, wherein

4. D is a compound represented by the following formula: 【Chemistry 6】 where the asterisk indicates a bond to L and W is as previously defined. The antibody-drug conjugate according to any one of claims 1 to 3, wherein

5. D is a compound represented by the following formula: 【Chemistry 7】 (where the asterisk indicates the bond to L) The antibody-drug conjugate according to any one of claims 1 to 4, wherein

6. D is a compound of the following formula: 【Chemistry 8】 (where the asterisk indicates the bond to L) The antibody-drug conjugate according to any one of claims 1 to 4, wherein

7. D is of the formula: 【Chemistry 9】 (where the asterisk indicates the bond to L) The antibody-drug conjugate according to any one of claims 1 to 4 or claim 6, wherein

8. D is represented by the following two formulas: 【Chemistry 10】 where the asterisk indicates a bond to L and W is as previously defined. The antibody-drug conjugate according to any one of claims 1 to 3, wherein

9. D is the following four formulas: 【Chemistry 11】 (where the asterisk indicates the bond to L) The antibody-drug conjugate according to any one of claims 1 to 3 or claim 8, wherein

10. The linker L is represented by -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates binding to drug D, Lp exhibits or is absent a linker consisting of an amino acid sequence cleavable in the target cell; La represents one selected from the following group: -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -CH ２ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-NH-(CH ２ CH ２ )n ３ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-NH-(CH ２ CH ２ )n ３ -CH ２ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-NH-(CH ２ CH ２ O)n ３ -CH ２ -C(=O)-、 - (CH ２ ) n ４ -O-C(=O)-, and -(CH ２ )n ９ -C(=O)- (where n ２ represents an integer of 1 to 3, n ３ represents an integer of 1 to 5, n ４ represents an integer of 0 to 2, n ９ represents an integer from 2 to 7), Lb represents a spacer that binds the glycan or remodeled glycan of La and Ab, or a spacer that binds the cysteine ​​residues of La and Ab; and Lc is -NH-CH ２ -, -NH-phenyl group -CH ２ -O(C=O)- or -NH-heteroaryl group -CH ２ -O(C=O)- or not present; The antibody-drug conjugate according to any one of claims 1 to 9.

11. Lc is -NH-CH ２ The antibody-drug conjugate of claim 10,

12. 12. The antibody drug conjugate of claim 10 or 11, wherein Lp is any one of -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ- or -GGPP-.

13. The antibody drug conjugate of claim 12, wherein Lp is -GGGFG- or -GGPI-.

14. La is the following: -C(=O)-CH ２ CH ２ -C(=O)-、 -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ３ -CH ２ -C(=O)-、 -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)-, and -(CH ２ ) ５ -C(=O)- The antibody-drug conjugate according to any one of claims 10 to 13, which exhibits any one selected from the group consisting of:

15. Lb is represented by the following formula: 【Chemistry 12】 or 【Chemistry 13】 (in the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain).

16. Lb is -(succinimid-3-yl-N)-; where -(succinimid-3-yl-N)- has the following structural formula: 【Chemistry 14】 indicates, Here, the asterisk indicates binding to La, and the wavy line indicates binding via thioether formation with a side chain of a cysteine ​​residue of the antibody. The antibody-drug conjugate according to any one of claims 10 to 14.

17. The linker L is represented by -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates binding to drug D, Lp is -GGGFG- or -GGPI-; La is -C(=O)-CH ２ CH ２ -C(=O)-, Lb is represented by the following formula: 【Chemistry 15】 (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain), Lc is -NH-CH ２ The antibody-drug conjugate of any one of claims 10 to 15, wherein

18. The antibody-drug conjugate according to any one of claims 1 to 17, wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is in the range of 1 to 10.

19. The antibody-drug conjugate of claim 18 , wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is in the range of 1 to 5.

20. 20. The antibody-drug conjugate of claim 19, wherein the average number of drugs bound per antibody molecule in the antibody-drug conjugate is in the range of 3 to 5.

21. The antibody-drug conjugate according to any one of claims 1 to 15 or 17 to 20, wherein the antibody is bound to L via a sugar chain that binds to Asn297 (N297 sugar chain) of the antibody.

22. The antibody-drug conjugate of claim 21 , wherein the N297 glycan is a remodeled glycan.

23. The antibody-drug conjugate of claim 21 or 22, wherein the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula: 【Chemistry 16】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates that the linker L or Lb in the linker L is bonded to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring; and n ５ is an integer from 2 to 5, or 【Chemistry 17】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates that the linker L or Lb in the linker L is bonded to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring; and n ５ is an integer from 2 to 5.

24. The following formula: 【Chemistry 18】 (In the formula, m ２ represents an integer of 1 or 2, L is a linker connecting the N297 glycan and D, as defined above; Ab represents an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody, or a functional fragment of these antibodies; The antibody is an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:2, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:3 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:4; an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:6, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:8, an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:10, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:11 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:8, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:9, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:10, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:11, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:70, The N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula, 【Chemistry 19】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates the bond to the linker L, and n ５ represents an integer from 2 to 5, or 【Chemistry 20】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates the bond to the linker L, and n ５ represents an integer from 2 to 5, D is represented by any one of the following four formulas: 【Chemistry 21】 wherein the asterisk indicates binding to L.

25. The following formula: 【Chemical 22】 【Chemistry 23】 Selected from In each of the structural formulas shown above, m ２ represents an integer of 1 or 2, Ab represents an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody, or a functional fragment of these antibodies; The antibody is an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:2, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:3 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:4; an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:6, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:8, an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:10, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:11 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:8, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:9, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:10, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:11, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:70, The N297 sugar chain is either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula, 【Chemistry 24】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates attachment to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring, and n ５ represents an integer from 2 to 5, or 【Chemistry 25】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates attachment to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring, and n ５ The antibody-drug conjugate according to any one of claims 1 to 4, wherein represents an integer of 2 to 5.

26. The following formula: 【Chemistry 26】 【Chemistry 27】 Selected from In each of the structural formulas shown above, m ２ represents an integer of 1 or 2, Ab represents an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody, or a functional fragment of these antibodies; The antibody is an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:2, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:3 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:4; an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:6, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:8, an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:10, or an antibody comprising a light chain having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:11 and a heavy chain having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:8, an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:9, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:10, or an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:11, and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:12, The antibody is an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 58; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64; or an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:70, The N297 sugar chain is either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula, 【Chemistry 28】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates attachment to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring, and n ５ represents an integer from 2 to 5, or 【Chemical 29】 In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates attachment to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring, and n ５ The antibody-drug conjugate of claim 25, wherein represents an integer from 2 to 5.

27. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:

4.

28. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:6, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:

8.

29. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:

12.

30. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO:

4.

31. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6, or an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:

8.

32. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO: 10, or an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:

12.

33. The antibody-drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; or an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:

46.

34. The antibody drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52, or an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:

58.

35. The antibody drug conjugate according to any one of claims 1 to 26, wherein the antibody is an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64, or an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:

70.

36. The following formula: 【Chemistry 30】 In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:6; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:8; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:10; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12; The N297 glycan has the following formula: 【Chemistry 31】 (wherein the wavy line indicates binding to Asn297 of the antibody; L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates that the bond is to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring. n ５ is 3, and m ２ (i) wherein R is an integer from 1 to 2;

37. The following formula: 【Chemistry 32】 In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:8; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:10; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO: 12; The N297 glycan has the following formula: 【Chemical 33】 (wherein the wavy line indicates binding to Asn297 of the antibody; L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates that the bond is to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring. n ５ is 3, and m ２ (i) wherein R is an integer from 1 to 2;

38. The antibody-drug conjugate of claim 37, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO:

2.

39. The antibody-drug conjugate of claim 37, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO:

4.

40. The antibody-drug conjugate of claim 37, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:

6.

41. The antibody-drug conjugate of claim 37, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:

8.

42. The antibody-drug conjugate of claim 37, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:

10.

43. The antibody-drug conjugate of claim 37, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:

12.

44. The following formula: 【Chemical 34】 In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:53, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:54, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:55, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:56, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:57, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:58; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:70; The N297 glycan has the following formula: 【Chemistry 35】 (wherein the wavy line indicates binding to Asn297 of the antibody; L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, The asterisk indicates that the bond is to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring. n ５ is 3, and m ２ (i) wherein R is an integer from 1 to 2;

45. The following formula: 【Chemical 36】 In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:5 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:6; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:7 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:8; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO:9 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:10; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12; The N297 glycan has the following formula: 【Chemical 37】 (wherein the wavy line indicates binding to Asn297 of the antibody; L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates that the bond is to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring. n ５ is 3, and m ２ (I) wherein R is 1;

46. The following formula: 【Chemical 38】 In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 4; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:6; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:8; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:10; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO: 12; The N297 glycan has the following formula: 【Chemical 39】 (wherein the wavy line indicates binding to Asn297 of the antibody; L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates that the bond is to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring. n ５ is 3, and m ２ (I) wherein R is 1;

47. The antibody-drug conjugate of claim 46, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO:

2.

48. The antibody-drug conjugate of claim 46, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO:

4.

49. The antibody-drug conjugate of claim 46, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO:5, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:

6.

50. The antibody-drug conjugate of claim 46, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 7, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 121 of SEQ ID NO:

8.

51. The antibody-drug conjugate of claim 46, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO:

10.

52. The antibody-drug conjugate of claim 46, wherein the Ab is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:

12.

53. The following formula: 【Chemistry 40】 In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 35, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 36, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 41, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 44, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 50, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 51, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:53, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:54, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:55, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:56, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:57, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:58; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:59, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:60, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:62, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:63, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:64; an antibody comprising a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:65, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:66, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:67, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:68, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:69, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:70; The N297 glycan has the following formula: 【Chemistry 41】 (wherein the wavy line indicates binding to Asn297 of the antibody; L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain, The asterisk indicates that the bond is to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring. n ５ is 3, and m ２ (I) wherein R is 1;

54. The antibody-drug conjugate according to any one of claims 1 to 29, 36 and 45, wherein 2 to 3 or less amino acid residues have been substituted, deleted and / or added in the amino acid sequence of the antibody heavy chain and / or light chain shown in SEQ ID NOs: 1 to 12.

55. The antibody drug conjugate of any one of claims 1 to 54, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

56. A method for producing an antibody-drug conjugate according to any one of claims 1 to 55, comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding an antibody contained in the antibody-drug conjugate, and collecting the antibody of interest from the culture obtained in the step.

57. A method for producing a functional fragment of an antibody contained in the antibody-drug conjugate according to any one of claims 1 to 55, comprising producing the functional fragment of the antibody in a suitable host cell using a gene modified by genetic engineering.

58. A STING agonist comprising an antibody-drug conjugate according to any one of claims 1 to 55.

59. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 55.

60. An antitumor agent comprising the antibody-drug conjugate according to any one of claims 1 to 55.

61. The antitumor agent according to claim 60, wherein the tumor is lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, auditory cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

62. The antitumor agent of claim 60, wherein the tumor is lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, cervical cancer, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, thymic cancer, or small intestine cancer.

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