Antibody-Drug Conjugates Comprising Cyclic Dinucleotide Derivatives

Abstract

It is desired to develop an antibody-drug conjugate that can be administered systemically and specifically deliver a STING agonist to target cells and organs (e.g., tumor sites), as well as a therapeutic agent and / or treatment method for diseases related to the STING agonist activity, such as diseases that can be treated based on immune activation (e.g., cancer). The present invention provides a novel antibody-CDN derivative conjugate that can be administered systemically and exhibits an anti-tumor effect in tumors expressing an antigen.

Description

Technical Field

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

[0002] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein that is locally present in the endoplasmic reticulum (Non-Patent Document 1). STING functions as a central molecule for activating innate immunity in mammals and is responsible for the front line against the entry of pathogens such as bacteria and viruses. It is known that the activation of STING is caused by signals when multiple cytoplasmic DNA sensors sense exogenous and endogenous DNA. Among the cytoplasmic DNA sensors, cGAS (Cyclic GMP-AMP Synthase) is considered an important DNA sensor. When cGAS senses DNA, a cyclic dinucleotide (2',3'-cGAMP) is produced, and this 2',3'-cGAMP directly binds to STING to activate STING (Non-Patent Document 2). The activated STING moves to the Golgi apparatus, where it promotes the autophosphorylation of TBK1 (Tank-binding kinase 1). The autophosphorylated and activated TBK1 activates both the IRF3 (Interferon regulatory factor 3) transcriptional pathway (Non-Patent Document 3) and the NFκB transcriptional pathway (Non-Patent Document 4), increasing the production of inflammatory proteins called interferons or cytokines (type I IFN (Interferon), IL-6 (Interleukin-6), TNF-α (Tumor Necrosis Factor-α)). These proteins initiate the acquired immune system, including T cells that destroy pathogens or cancer cells through a complex cascade.

[0003] According to recent studies, STING not only promotes host defense against microorganisms but also promotes anti-tumor immunity. For example, in the case of transplanting immunogenic tumors into STING-deficient mice, the tumors proliferate rapidly compared to wild-type mice or TRIF (Toll / Interleukin-1 (IL-1) receptor domain containing adaptor-inducing interferon-β) -deficient mice. In addition, unlike TLR (Toll-like receptor), MyD88 (Myeloid differentiation primary response 88), and MAVS (Mitochondrial antiviral-signaling protein) -deficient mice, the spontaneous CD8 + T cell priming in STING-deficient mice is also abolished. This indicates that the STING pathway activated by cytoplasmic DNA sensing is involved in the control of tumor proliferation (Non-Patent Document 5). Other studies have also shown that STING is necessary for the anti-tumor effects of radiotherapy (Non-Patent Document 6) or anti-CD47 antibody therapy (Non-Patent Document 7). After treatment with radiation or anti-CD47 antibody, DNA from dying tumor cells migrates into the cytoplasm of dendritic cells and activates the cGAS-STING pathway, inducing IFN production and acting as an intermediary between innate and acquired immunity. This study suggests that cross priming mediated by dendritic cells activated through the STING pathway is important for eliciting acquired immunity against tumors.

[0004] The flavonoid-based small molecule compound DMXAA, known as a vascular disrupting agent, shows strong anti-tumor activity in mouse tumor models because it induces type I IFN in macrophages (Non-Patent Document 8). Due to its excellent anti-tumor effects in preclinical studies, DMXAA was expected to be an immunotherapy drug for non-small cell lung cancer, but it failed in human clinical trials (Non-Patent Document 9). According to recent studies, it has been clearly shown that DMXAA is a specific agonist for mouse STING and cannot bind to human STING due to lack of species cross-reactivity (Non-Patent Document 10). As a result, DMXAA is ineffective in humans, but studies based on mouse models have shown that small molecule drugs intervene in STING and effectively prime CD8 + T cells, enhancing anti-tumor immunity.

[0005] As other low-molecular-weight compounds, cyclic dinucleotides (CDNs) have shown that when administered to tumor-bearing mice, anti-tumor immune responses are enhanced due to STING intervention, tumor proliferation is significantly inhibited, and the survival rate of mice can be improved (Non-Patent Document 11). CDNs are classified into CDNs with standard two 3'-5' phosphodiester bonds from bacteria (cyclic-di-GMP, cyclic-di-AMP, 3',3'-cGAMP) and hybrid-bound CDNs with non-standard 2'-5' phosphodiester bonds produced by mammalian cGAS (2',3'-cGAMP). According to recent studies, hybrid-bound CDNs can more generally activate diverse STINGs compared to standard CDNs (Non-Patent Document 12).

[0006] Natural CDNs cannot be administered in their original form because they are rapidly degraded by nucleases in the blood like most nucleic acid molecules. Therefore, synthetic low-molecular-weight compounds with STING agonist activity in vivo have been developed (for example, Patent Documents 1 to 26).

[0007] Currently, MIW-815, a STING agonist being advanced in clinical trials as an anti-tumor agent (sometimes also referred to as ADU-S100, ML RR-S2 CDA, or ML-RR-CDA·2Na + ) is directly administered into tumors. The method of directly administering a STING agonist into tumors can only administer the agent to a limited area within the tumor. In addition, it is difficult to directly administer to all multiple distant metastatic tumors, so there is a problem of limited treatable tumors. It is recorded in Non-Patent Document 13 that anti-tumor effects were shown by administering ML RR-S2CDA, but it was only for intratumoral administration, and anti-tumor effects produced by systemic administration (for example, intravenous administration) were not shown. It is recorded in Non-Patent Document 14 that anti-tumor effects were shown when the STING agonist SB11285 was intravenously administered to a mouse tumor model, but it is not clear what specific structure the compound SB11285 has. Patent Document 14 records a conjugate containing an immune-stimulating compound, an antibody construct, and a linker, but no specific examples of conjugates using a STING agonist as the immune-stimulating compound are recorded. Patent Document 26 records a conjugate in which a CDN with a specific structure is bound to an antibody via a linker, but no examples of in vivo administration of the conjugate are recorded, and the anti-tumor effect of the conjugate has not been confirmed.

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: WO 2014 / 099824 A1

[0011] Patent Document 2: WO 2014 / 179335 A1

[0012] Patent Document 3: WO 2014 / 189805 A1

[0013] Patent Document 4: WO 2014 / 189806 A1

[0014] Patent Document 5: WO 2015 / 074145 A1

[0015] Patent Document 6: WO 2015 / 185565 A1

[0016] Patent Document 7: WO 2016 / 096714 A1

[0017] Patent Document 8: WO 2016 / 012305 A1

[0018] Patent Document 9: WO 2016 / 145102 A1

[0019] Patent Document 10: WO 2017 / 027646 A1

[0020] Patent Document 11: WO 2017 / 027645 A1

[0021] Patent Document 12: WO 2017 / 075477 A1

[0022] Patent Document 13: WO 2017 / 093933 A1

[0023] Patent Document 14: WO 2017 / 100305 A1

[0024] Patent Document 15: WO 2017 / 123669 A1

[0025] Patent Document 16: WO 2017 / 161349 A1

[0026] Patent Document 17: WO 2017 / 175147 A1

[0027] Patent Document 18: WO 2017 / 175156 A1

[0028] Patent Document 19: WO 2018 / 009466 A1

[0029] Patent Document 20: WO 2018 / 045204 A1

[0030] Patent Document 21: WO 2018 / 060323

[0031] Patent Document 22: WO 2018 / 067423

[0032] Patent Document 23: WO 2018 / 065360

[0033] Patent Document 24: WO 2014 / 093936

[0034] Patent Document 25: WO 2018 / 009648

[0035] Patent Document 26: WO 2018 / 100558

[0036] Non-Patent Literature

[0037] Non-Patent Literature 1: Nature 2008, 455, 674 - 678

[0038] Non-Patent Literature 2: Mol.Cell, 2013, 51, 226 - 235

[0039] Non-Patent Literature 3: Science 2015a, 347, aaa2630

[0040] Non-Patent Literature 4: J.Virol.2014, 88, 5328 - 5341

[0041] Non-Patent Literature 5: Immunity 2014, 41, 830 - 842

[0042] Non-Patent Literature 6: Immunity 2014, 41, 843 - 852

[0043] Non-Patent Literature 7: Nat.Med.2015, 21, 1209 - 1215

[0044] Non-Patent Literature 8: J.Immunol.1994, 153, 4684 - 4693

[0045] Non-Patent Literature 9: J.Clin.Oncol.2011, 29, 2965 - 2971

[0046] Non-Patent Literature 10: J.Immunol.2013, 190, 5216 - 5225

[0047] Non-Patent Literature 11: Sci.Rep.2016, 6, 19049

[0048] Non-Patent Document 12: Mol. Cell, 2015, 59, 891-903

[0049] Non-Patent Document 13: Cell Rep. 2015, 11, 1018-1030

[0050] Non-Patent Document 14: AACR Tumor Immunology and Immunotherapy, 2017, Poster #A25 Summary of the Invention

[0051] Problems to be Solved by the Invention

[0052] It is desired to develop an antibody-drug conjugate that can be administered systemically and can specifically deliver a STING agonist to target cells and organs (e.g., tumor sites), as well as a therapeutic agent and / or treatment method for diseases related to the STING agonist activity, such as diseases that can be treated based on immune activation (e.g., cancer).

[0053] Solutions to the Problems

[0054] In order to solve the above technical problems, the present inventors found an antibody-drug conjugate in which a CDN derivative having a fused tricyclic substituent is bound to a specific antibody via a linker, and found that when this antibody-drug conjugate is administered systemically, it shows an antitumor effect in tumors expressing the antigen, thereby completing the present invention.

[0055] That is, the invention of the present application relates to the following, but is not limited thereto.

[0056] [1] An antibody-drug conjugate represented by the following formula (II):

[0057]

[0058] In the formula, m 1 is in the range of 1 to 10, Ab represents an antibody or a functional fragment of the antibody, and the sugar chain of the antibody can be optionally reconstructed. Here, the antibody represents 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 that connects Ab and D. Ab can optionally bind directly to L from its amino acid residues, or bind to L from the sugar chain or the reconstructed sugar chain of Ab. D represents a compound represented by the following formula (I):

[0059]

[0060] Here, L binds to any -NH2 or hydroxyl group contained in L 1 and L 1represents any one of the following three structural formulas:

[0061]

[0062] Herein, the wavy line represents the substitution position, Q and Q' each independently represent a hydroxyl group or a thiol group, and R 21 and R 22 each independently represent a hydroxyl group or a fluorine atom, and W represents -NH- or a sulfur atom.

[0063] [2] The antibody-drug conjugate according to [1], wherein D is represented by any one of the following two structural formulas:

[0064]

[0065] (Herein, L 1 , Q, Q' and W are as defined above).

[0066] [3] The antibody-drug conjugate according to [1] or [2], wherein D is represented by any one of the following four structural formulas:

[0067]

[0068] (Herein, the asterisk represents binding to L, and Q, Q' and W are as defined above).

[0069] [4] The antibody-drug conjugate according to any one of [1] to [3], wherein D is represented by any one of the following three structural formulas:

[0070]

[0071] (Herein, the asterisk represents binding to L, and W is as defined above).

[0072] [5] The antibody-drug conjugate according to any one of [1] to [4], wherein D is represented by any one of the following three structural formulas:

[0073]

[0074] (Herein, the asterisk represents binding to L).

[0075] [6] The antibody-drug conjugate according to any one of [1] to [4], wherein D is represented by any one of the following four structural formulas:

[0076]

[0077] (Herein, the asterisk represents binding to L).

[0078] [7] The antibody-drug conjugate according to any one of [1] to [4] or [6], wherein D is represented by the following formula:

[0079]

[0080] (Here, the asterisk indicates binding to L).

[0081] [8] The antibody-drug conjugate according to any one of [1] to [3], wherein D is represented by any one of the following two structural formulas:

[0082]

[0083] (Here, the asterisk indicates binding to L, and W is as defined above).

[0084] [9] The antibody-drug conjugate according to any one of [1] to [3] or [8], wherein D is represented by any one of the following four structural formulas:

[0085]

[0086] (Here, the asterisk indicates binding to L).

[0087]

[10] The antibody-drug conjugate according to any one of [1] to [9], wherein the linker L is represented by -Lb-La-Lp-Lc-*, where the asterisk indicates binding to the drug D, Lp represents a linker formed by an amino acid sequence that can be cleaved in the target cell or is absent, and 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 -O-C(=O)-, and, -(CH2)n 9 -C(=O)-, where n 2 represents an integer from 1 to 3, n 3 represents an integer from 1 to 5, n 4Represents integers 0 to 2, n 9 Represents integers 2 to 7, Lb represents a spacer that binds the sugar chain of La and Ab or a reconstructed sugar chain, or a spacer that binds La and the cysteine residue of Ab, and Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)-, or -NH-heteroaryl-CH2-O(C=O)-, or is absent.

[0088]

[11] The antibody-drug conjugate according to

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

[0089]

[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-, or -GGPP-.

[0090]

[13] The antibody-drug conjugate according to

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

[0091]

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

[10] to

[13] , wherein La represents any one selected from the group consisting of: -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)-.

[0092]

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

[10] to

[14] , wherein Lb is represented by any one of the following structural formulas:

[0093]

[0094] (In the structural formulas of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a reconstructed sugar chain).

[0095]

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

[10] to

[14] , wherein Lb is -(succinimidyl-3-yl-N)-, and herein, -(succinimidyl-3-yl-N)- represents the following structural formula:

[0096]

[0097] Here, an asterisk indicates binding to La, and a wavy line indicates binding by forming a thioether with the side chain of a cysteine residue of the antibody.

[0098]

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

[10] to

[15] , wherein the linker L is represented by -Lb-La-Lp-Lc-*, where the asterisk indicates binding to the drug D, Lp is -GGFG- or -GGPI-, La represents -C(=O)-CH2CH2-C(=O)-, and Lb represents the following formula:

[0099]

[0100] (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 the reconstructed sugar chain), and Lc represents -NH-CH2-.

[0101]

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

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

[0102]

[19] The antibody-drug conjugate according to

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

[0103]

[20] The antibody-drug conjugate according to

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

[0104]

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

[20] , wherein the antibody binds to L from the sugar chain (N297 sugar chain) that binds to Asn297 of the antibody.

[0105]

[22] The antibody-drug conjugate according to

[21] , wherein the N297 sugar chain is a reconstructed sugar chain.

[0106]

[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 shown by the following formula:

[0107]

[0108] In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5-CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 is an integer from 2 to 5.

[0109]

[0110] In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 is an integer from 2 to 5.

[0111]

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

[21] to

[23] is represented by the following formula:

[0112]

[0113] In the formula, m 2 represents the integer 1 or 2, L is a linker connecting the N297 sugar chain of Ab 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, and the N297 sugar chain of Ab is represented as N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula.

[0114]

[0115] In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents the integer 2 to 5.

[0116]

[0117] In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents an integer from 2 to 5, and D is represented by any one of the following four structural formulas:

[0118]

[0119] Here, in the formula, the asterisk indicates binding to L.

[0120]

[25] The antibody-drug conjugate according to

[24] is selected from the following formula:

[0121]

[0122] In each of the structural formulas shown above, m 2 represents an integer 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, and the N297 sugar chain of Ab represents either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula.

[0123]

[0124] In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents an integer from 2 to 5.

[0125]

[0126] In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5-CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of the Lb of the linker L, and, n 5 represents an integer from 2 to 5.

[0127]

[26] The antibody-drug conjugate according to

[25] , which is selected from the following formula:

[0128]

[0129] In each of the structural formulas shown above, m 2 represents an integer 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, and the N297 sugar chain of Ab represents any one of N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula.

[0130]

[0131] In the formula, the wavy line represents binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man branch of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of the Lb of the linker L, and, n 5 represents an integer from 2 to 5.

[0132]

[0133] In the formula, the wavy line represents binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain. The asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of the Lb of the linker L, and, n 5 represents an integer from 2 to 5.

[0134]

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

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

[0135]

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

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

[0136]

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

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

[0137]

[30] The antibody-drug conjugate according to

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

[0138]

[31] The antibody-drug conjugate according to

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

[0139]

[32] The antibody-drug conjugate according to

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

[0140]

[33] The antibody-drug conjugate according to

[27] , wherein the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2, or the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 4.

[0141]

[34] The antibody-drug conjugate according to

[28] , wherein the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 6, or the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 8.

[0142]

[35] The antibody-drug conjugate according to

[29] , wherein the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10, or the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO: 12.

[0143]

[36] The antibody-drug conjugate according to

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

[0144]

[37] The antibody-drug conjugate according to

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

[0145]

[38] The antibody-drug conjugate according to

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

[0146]

[39] An antibody-drug conjugate represented by the following formula:

[0147]

[0148] In the formula, Ab represents any one selected from the group consisting of: an antibody comprising a light chain formed of the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain formed of the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain formed of the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 6; an antibody comprising a light chain formed of the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 8; an antibody comprising a light chain formed of the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 10; an antibody comprising a light chain formed of the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 12. The N297 sugar chain of Ab is represented by the following formula:

[0149]

[0150] In the formula, the wavy line represents binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing ends 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 represents binding to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb of the linker L, and n 5 is 3, and, m 2 is 2.

[0151]

[40] An antibody-drug conjugate, which is represented by the following formula:

[0152]

[0153] In the formula, Ab represents any one selected from the group consisting of: an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 4; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 6; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 8; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO: 12; the N297 sugar chain of Ab is represented by the following formula:

[0154]

[0155] In the formula, the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends 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, and the asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, n 5 is 3, and, m 2 is 2.

[0156]

[41] An antibody-drug conjugate, which is represented by the following formula:

[0157]

[0158] In the formula, Ab represents any one selected from the group consisting of: an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 49, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 55, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 58; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 61, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 64;An antibody comprising the following light chain and heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 67, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 70; the N297 glycan of Ab is represented by the following formula:;

[0159]

[0160] In the formula, the wavy line represents binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) is amide-bonded to the 2-position carboxyl group of sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 glycan, and the asterisk represents binding to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb of the linker L, n 5 is 3, and, m 2 is 2.

[0161]

[42] An antibody-drug conjugate represented by the following formula:

[0162]

[0163] In the formula, Ab represents any one selected from the group consisting of: an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 6; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 8; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 10; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 12; the N297 glycan of Ab is represented by the following formula:

[0164]

[0165] In the formula, the wavy line represents binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5-CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain, and the asterisk indicates binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and n 5 is 3, and, m 2 is 1.

[0166]

[43] An antibody-drug conjugate, which is represented by the following formula:

[0167]

[0168] In the formula, Ab represents any one selected from the following group: an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2; an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 4; an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 6; an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 8; an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10; an antibody comprising a light chain and a heavy chain, the light chain comprising a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprising a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO: 12; the N297 sugar chain of Ab is represented by the following formula:

[0169]

[0170] In the formula, the wavy line represents binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5-CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain. The asterisk indicates binding to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of the Lb of the linker L, and n 5 is 3, and, m 2 is 1.

[0171]

[44] An antibody-drug conjugate represented by the following formula:

[0172]

[0173] In the formula, Ab represents any one selected from the group consisting of: an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 49, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 55, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 58; an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 61, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 64;An antibody comprising the following light chain and heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 67, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 70; the N297 glycan of Ab is represented by the following formula:;

[0174]

[0175] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 glycan, the asterisk indicates binding to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb of the linker L, n 5 is 3, and, m 2 is 1.

[0176]

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

[44] .

[0177]

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

[44] .

[0178]

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

[44] .

[0179]

[48] The antitumor agent according to

[47] , wherein the tumor is lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, testicular cancer, cervical cancer, choriocarcinoma of placenta, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, auditory organ cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

[0180]

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

[44] , the STING agonist described in

[45] , the pharmaceutical composition described in

[46] , and the anti-tumor agents described in

[47] or

[48] .

[0181]

[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, endometrial cancer, testicular cancer, cervical cancer, choriocarcinoma of placental villi, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, cancer of the auditory organ, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

[0182]

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

[27] ,

[30] ,

[33] or

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

[0183]

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

[42] to

[44] exhibits a stronger anti-tumor effect than the antibody contained in the antibody-drug conjugate in the animals described in the following (i) or (ii): (i) BALB / c mice subcutaneously transplanted with mouse colorectal cancer cell line CT26.WT (CRL2638), and the mouse colorectal cancer cell line CT26.WT (CRL2638) is transfected with a gene of a human-mouse chimeric antigen in which the epitope site on the antigen bound by the antibody contained in the antibody-drug conjugate is replaced with a human type; (ii) BALB / c-nu mice subcutaneously transplanted with human renal cancer cell line A-498 (HTB-44) cells.

[0184] Advantages of the Invention

[0185] According to the present invention, there is provided an antibody-CDN derivative conjugate capable of systemic administration and showing an anti-tumor effect in tumors expressing an antigen. Brief Description of the Drawings

[0186] Figure 1 Schematically showing the antibody-drug conjugate ((II) molecule) of the present invention, the antibody-drug conjugate obtained by reconstructing an antibody with an SG-type sugar chain ( Figure 1 molecule (II) of A) and the antibody-drug conjugate obtained by reconstructing an antibody with an MSG-type sugar chain ( Figure 1The molecule of (II) of B. (a) represents the drug D, (b) represents the linker L, (c) represents the PEG linker (L(PEG)), (d) represents the N297 sugar chain (here, the white circle represents NeuAc (Sia), the white hexagon represents Man, the black hexagon represents GlcNAc, the white rhombus represents Gal, and the white inverted triangle represents Fuc). The white pentagon represents the triazole ring formed by the reaction of the alkyne from the linker L and the azide from the PEG linker. The Y shape represents the antibody Ab. The PEG linker is connected to the 2-position carboxyl group of the sialic acid at the non-reducing end via an amide bond. Unless otherwise specified, such a representation method is applicable throughout this specification.

[0187] Figure 2 represents the (Fucα1,6)GlcNAc - antibody ([ Figure 2 the molecule of (III) of A) of the present invention as an intermediate for the production of the antibody-drug conjugate, the SG-type sugar chain reconstructed antibody ([ Figure 2 the molecule of (IV) of B) of, and the MSG-type sugar chain reconstructed antibody ([ Figure 2 the molecule of (IV) of C) of the present invention. In all the figures, the Y shape represents the antibody Ab in the same way as Figure 1 . In Figure 2 A of, (e) represents the N297 sugar chain containing the disaccharide in which the 1-position of Fuc and the 6-position of GlcNAc are α-glycosidically bonded. In Figure 2 B and C of, (d) represents the same N297 sugar chain as Figure 1 , (f) is the PEG linker having an azide group, and the azide group provided for the binding to the linker L is represented at the end. The binding mode of the PEG linker having an azide group is the same as that of the PEG linker in Figure 1 .

[0188] Figure 3 is a schematic diagram of the process for producing the SG-type sugar chain reconstructed antibody and the MSG-type sugar chain reconstructed antibody from the antibody produced in animal cells. The molecules (III) and (IV) in the figure are the same as Figure 2 , and represent the (Fucα1,6)GlcNAc - antibody and the SG-type sugar chain reconstructed antibody or the MSG-type sugar chain reconstructed antibody respectively. The molecule of (V) is the antibody produced in animal cells and is a mixture of molecules with heterogeneous N297 sugar chains. Figure 3 A of represents the process of producing the homogeneous (Fucα1,6)GlcNAc - antibody (III) by treating the heterogeneous N297 sugar chain of (V) with a hydrolase such as EndoS. Figure 3B represents the process of producing the SG-type glycan-reconstructed antibody of (IV) by transferring the glycan of a SG-type glycan donor molecule using a glycosyltransferase such as EndoSD233Q / Q303L variant to the GlcNAc of the N297 glycan of the antibody (III). Figure 3 C of Figure 3 Similar to B of Figure 2 C represents the process of producing the MSG-type glycan-reconstructed antibody of (IV) by transferring the glycan of a MSG-type glycan donor molecule to the antibody (III). The SG-type glycan donor molecule and the MSG-type glycan donor molecule used herein are each modified at the non-reducing end sialic acid with a PEG linker having an azide group. In the produced SG-type N297 glycan-reconstructed antibody and MSG-type N297 glycan-reconstructed antibody, as

[0189] Figure 4 represents 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 (in this specification, "anti-CD70 antibody 1" is also referred to as "engineered anti-CD70 antibody 1").

[0190] Figure 5 represents 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 (in this specification, "anti-CD70 antibody 2" is also referred to as "engineered anti-CD70 antibody 2").

[0191] Figure 6 represents the amino acid sequence of the light chain (SEQ ID NO: 5) and the amino acid sequence of the heavy chain (SEQ ID NO: 6) of anti-TROP2 antibody 1.

[0192] Figure 7 represents 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 (in this specification, "anti-TROP2 antibody 2" is also referred to as "engineered anti-TROP2 antibody").

[0193] Figure 8 represents 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 (in this specification, "anti-EGFR antibody 1" is also referred to as "engineered anti-EGFR antibody 1").

[0194] Figure 9 represents 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 (in this specification, "anti-EGFR antibody 2" is also referred to as "engineered anti-EGFR antibody 2").

[0195] Figure 10 Represent the amino acid sequences of (a) human STING wild type (SEQ ID NO: 13), (b) human STING REF variant (R232H) (SEQ ID NO: 15), and (c) human STING HAQ variant (R71H, G230A, R293Q) (SEQ ID NO: 17).

[0196] Figure 11 Represent the STING agonist activities 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.

[0197] Figure 12 Represent the activities 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 the co-culture analysis system of CT26.WT and CT26.WT-hCD70 cell lines and dendritic cells from mouse bone marrow.

[0198] Figure 13 Represent the antitumor effects of intravenous administration based on anti-TROP2 antibody 1 and anti-TROP2 antibody 1-CDN conjugate (1). The black square line in the figure represents the vehicle group, the white inverted triangle line represents the administration group of anti-TROP2 antibody 1-CDN conjugate (1) formed by conjugating the compound 6b of Example 1 with anti-TROP2 antibody 1 prepared in Reference Example 5, and the white circular line represents the anti-TROP2 antibody 1 administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.

[0199] Figure 14 Represent the antitumor effects of intravenous administration based on anti-TROP2 antibody 2 and anti-TROP2 antibody 2-CDN conjugate (1). The black square line in the figure represents the vehicle group, the white inverted triangle line represents the administration group of anti-TROP2 antibody 2-CDN conjugate (1) formed by conjugating the compound 34a of Example 2 with anti-TROP2 antibody 2 prepared in Reference Example 6, and the white circular line represents the anti-TROP2 antibody 2 administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.

[0200] Figure 15Indicates the anti-tumor effects of intravenous administration of anti-EGFR antibody 1, anti-EGFR antibody 2, and anti-EGFR antibody-CDN conjugate. Indicates the anti-tumor effects of intravenous administration of anti-EGFR antibody 1-CDN conjugate (1) formed by conjugating compound 34a of Example 2 with anti-EGFR antibody 1 prepared in Reference Example 7 and anti-EGFR antibody 2-CDN conjugate (1) formed by conjugating compound 34a of Example 2 with anti-EGFR antibody 2 prepared in Reference Example 8. The black square lines in the figure represent the vehicle group, the white triangle lines represent the anti-EGFR antibody 1 administration group, the black triangle lines represent the anti-EGFR antibody 1-CDN conjugate (1) administration group, the white circular lines represent the anti-EGFR antibody 2 administration group, and the black circular lines represent the anti-EGFR antibody 2-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.

[0201] Figure 16 Indicates the amino acid sequence of CDRL1 (SEQ ID NO: 35), CDRL2 (SEQ ID NO: 36), CDRL3 (SEQ ID NO: 37), CDRH1 (SEQ ID NO: 38), CDRH2 (SEQ ID NO: 39), and CDRH3 (SEQ ID NO: 40) of anti-CD70 antibody 1.

[0202] Figure 17 Indicates the amino acid sequence of CDRL1 (SEQ ID NO: 41), CDRL2 (SEQ ID NO: 42), CDRL3 (SEQ ID NO: 43), CDRH1 (SEQ ID NO: 44), CDRH2 (SEQ ID NO: 45), and CDRH3 (SEQ ID NO: 46) of anti-CD70 antibody 2.

[0203] Figure 18 Indicates the amino acid sequence of CDRL1 (SEQ ID NO: 47), CDRL2 (SEQ ID NO: 48), CDRL3 (SEQ ID NO: 49), CDRH1 (SEQ ID NO: 50), CDRH2 (SEQ ID NO: 51), and CDRH3 (SEQ ID NO: 52) of anti-TROP2 antibody 1.

[0204] Figure 19 Indicates the amino acid sequence of CDRL1 (SEQ ID NO: 53), CDRL2 (SEQ ID NO: 54), CDRL3 (SEQ ID NO: 55), CDRH1 (SEQ ID NO: 56), CDRH2 (SEQ ID NO: 57), and CDRH3 (SEQ ID NO: 58) of anti-TROP2 antibody 2.

[0205] Figure 20 The amino acid sequences of CDRL1 (SEQ ID NO: 59), CDRL2 (SEQ ID NO: 60), CDRL3 (SEQ ID NO: 61), CDRH1 (SEQ ID NO: 62), CDRH2 (SEQ ID NO: 63), and CDRH3 (SEQ ID NO: 64) of anti-EGFR antibody 1 are shown.

[0206] Figure 21 The amino acid sequences of CDRL1 (SEQ ID NO: 65), CDRL2 (SEQ ID NO: 66), CDRL3 (SEQ ID NO: 67), CDRH1 (SEQ ID NO: 68), CDRH2 (SEQ ID NO: 69), and CDRH3 (SEQ ID NO: 70) of anti-EGFR antibody 2 are shown.

[0207] Figure 22 The anti-tumor 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) are shown. The black square line in the figure represents the vehicle group, the white triangle line represents the anti-CD70 antibody 1 administration group, the white inverted triangle line represents the anti-CD70 antibody 2 administration group, the white diamond line represents the anti-CD70 antibody 1-CDN conjugate (1) administration group, and the white circle line represents the anti-CD70 antibody 2-CDN conjugate (1) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.

[0208] Figure 23 The anti-tumor effects of intravenous administration of anti-CD70 antibody 2 and anti-CD70 antibody 2-CDN conjugate (2) are shown. The black square line in the figure represents the vehicle group, the white triangle line represents the anti-CD70 antibody 2 administration group, and the white inverted triangle line represents the anti-CD70 antibody 2-CDN conjugate (2) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation.

[0209] Figure 24 The anti-tumor effects of intravenous administration of anti-EGFR antibody 1, compound 34a, and anti-EGFR antibody 1-CDN conjugate (2) are shown. The black square line in the figure represents the vehicle group, the white triangle line represents the anti-EGFR antibody 1 administration group, the white diamond line represents the compound 34a administration group, and the black square line represents the anti-EGFR antibody 1-CDN conjugate (2) administration group. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after tumor transplantation. Detailed implementation manners

[0210] The present invention relates to an antibody-drug conjugate comprising a CDN derivative having STING agonist activity and its use. The CDN derivative has STING agonist activity, activates immune cells and induces the production of interferons and cytokines. In addition, the CDN derivative exerts an anti-tumor effect through the activation of the immune cells. The antibody-drug conjugate of the present invention can be prepared by linking the CDN derivative to an antibody capable of recognizing and binding to target cells (e.g., tumor cells or immune cells) via an arbitrary linker, and can be administered systemically. Specific examples of systemic administration include intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.

[0211] STING (Stimulator of Interferon Genes) refers to a transmembrane adaptor protein that is locally present in the endoplasmic reticulum. It is known that there are congenital polymorphisms in STING at a high frequency (PLoS One, 2013 Oct 21, 8(10), e77846). Regarding STING variants, for example, the R232H variant in which the 232nd amino acid is mutated from arginine (R) to histidine (H), the 71st arginine (R) is mutated to histidine (H), the 230th glycine (G) is mutated to alanine (A), and the HAQ variant in which the 293rd arginine (R) is mutated to glutamine (Q) are known. It is known that such polymorphisms of STING differ in the response intensity such as the amount of cytokines produced by stimulation with a STING agonist (Genes and Immunity, 2011, 12, 263-269). Therefore, in order for a STING agonist to act stably on humans, it is preferably active against each type of STING.

[0212] In this specification, "cancer", "carcinoma" and "tumor" have the same meaning.

[0213] In the present invention, "immune activation" means the activation of immune cells involved in anti-tumor immunity, such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, neutrophils, etc., in any form, for example, the production of cytokines and chemokines, an increase in the expression of immune activation markers, a decrease in the expression of immunosuppressive markers, changes such as phosphorylation of intracellular signal transduction systems, and all structural and functional changes of immune cells such as changes in gene expression. In addition, it also includes changes that induce anti-tumor immunity in tumor cells, for example, activation of immune cells or induction of the production of free cytokines and chemokines, and induction of enhanced sensitivity to immune cells.

[0214] In the present invention, "anti-tumor effect" means directly or indirectly affecting tumor cells by a drug, thereby inducing a reduction or shrinkage of the tumor. For example, directly damaging tumor cells by a drug, activating anti-tumor immunity due to the stimulation of the drug on the tumor cells, releasing the drug delivered to the tumor cells extracellularly, etc., and activating anti-tumor immunity around the tumor cells, etc., resulting in a reduction in the number of tumor cells and damage, or shrinkage of the tumor, and these are referred to as anti-tumor effects.

[0215] In the present invention, "cytotoxicity" means causing pathological changes to cells in any form, not only referring to direct trauma, but also referring to all structural and functional damages of cells such as DNA cleavage, formation of base dimers, chromosome cleavage, damage to the cell division apparatus, and reduction of various enzyme activities.

[0216] In the present invention, "cell" also includes cells in an animal individual and cultured cells.

[0217] <1. CDN derivative>

[0218] The CDN derivative has a structure represented by the following formula (I):

[0219]

[0220] L 1 is a group represented by any one of the following three structural formulas.

[0221]

[0222] Q and Q' each independently represent a hydroxyl group or a thiol group. Preferably, both Q and Q' are thiol groups.

[0223] R 21 and R 22 each independently represent a hydroxyl group or a fluorine atom. R 21 is preferably a hydroxyl group. R 22 is preferably a fluorine atom.

[0224] W is -NH- or a sulfur atom. W is preferably -NH-.

[0225] The manufacturing method of this CDN derivative is described in <3. Manufacturing method> described later.

[0226] <2. Antibody-drug conjugate>

[0227] The antibody-drug conjugate of the present invention can be systemically administered as an antibody-drug conjugate formed by connecting the above CDN derivative and an antibody capable of recognizing and binding to a target cell (for example, a tumor cell or an immune cell) via an arbitrary linker.

[0228] The antibody-drug conjugate of the present invention is represented by the following formula (II):

[0229]

[0230] m 1 is in the range of 1 to 10 and represents the number of drug bindings per molecule of antibody in the antibody-drug conjugate. Ab represents an antibody or a functional fragment of the antibody, L represents a linker that connects Ab and D, and D represents the above-mentioned CDN derivative (in this specification, when the CDN derivative is used as a part of the antibody-drug conjugate, it is also simply referred to as "drug").

[0231] The drug D is a compound having the activity of activating immune cells, specifically having STING agonist activity. When a part or all of the linker is cleaved within a target cell (e.g., a tumor cell or an immune cell), the drug D is released in its original structure and exerts an immune activation effect. The target function is exerted by enhancing the sensitivity of the target cell to immune cells or activating immune cells via the target cell. As the target function, there is no particular limitation as long as it is related to STING agonist activity, and antitumor activity is preferred. That is, the drug D linked to an antibody targeting a tumor (e.g., anti-CD70 antibody, anti-TROP2 antibody, anti-EGFR antibody) via an arbitrary linker is delivered to the target cell or tissue, a part or all of the linker is cleaved, and an antitumor effect is exerted by enhancing the sensitivity of the target cell to immune cells or activating immune cells via the target cell (e.g., production of interferon, cytokine).

[0232] The drug D bound to the antibody-drug conjugate of the present invention is represented by the following formula (I):

[0233]

[0234] Here, L 1 , Q, Q', R 21 , R 22 and W are defined as in the above <1. CDN derivative>.

[0235] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two structural formulas:

[0236]

[0237] Here, L 1 , Q, Q' and W are defined as in the above <1. CDN derivative>.

[0238] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following four structural formulas:

[0239]

[0240] Here, the asterisk indicates binding to L, and Q, Q', and W are as defined in <1.CDN derivatives> above.

[0241] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following three structural formulas:

[0242]

[0243] Here, the asterisk indicates binding to L, and W is as defined in <1.CDN derivatives> above.

[0244] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following three structural formulas:

[0245]

[0246] Here, the asterisk indicates binding to L.

[0247] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following four structural formulas:

[0248]

[0249] Here, the asterisk indicates binding to L.

[0250] In addition, the drug D used in the antibody-drug conjugate of the present invention is more preferably represented by the following formula.

[0251]

[0252] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula.

[0253]

[0254] Here, the asterisk indicates binding to L, and W is as defined in <1.CDN derivatives> above.

[0255] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two structural formulas:

[0256]

[0257] Here, the asterisk indicates binding to L.

[0258] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula:

[0259]

[0260] Here, the asterisk indicates binding to L, and W is as defined in the above <1.CDN derivative>.

[0261] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two structural formulas:

[0262]

[0263] Here, the asterisk indicates binding to L.

[0264] <2.1. Linker structure>

[0265] The linker structure that binds the drug to the antibody in the antibody-drug conjugate of the present invention will be described. The linker used in the antibody-drug conjugate of the present invention is not particularly limited as long as it is a linker that is understood by those skilled in the art as a linker for linking an antibody and a drug. As the linker used in the antibody-drug conjugate of the present invention, for example, the linkers described in Protein Cell, 2018, 9(1): 33-46, Pharm Res, 2015, 32: 3526-3540, or Int. J. Mol. Sci., 2016, 17, 561 can be cited, but are not limited thereto. The linker can be a linker that is cleaved in vivo or a linker that is not cleaved in vivo, but a linker that is cleaved in vivo is preferred.

[0266] As the linker used in the antibody-drug conjugate of the present invention, for example, a linker that binds the drug to the sugar chain of the Fc portion of the antibody or a reconstructed sugar chain (sometimes referred to as "sugar chain conjugation" in this specification) (for example, described in WO2018 / 003983), or a linker that binds the drug to an arbitrary amino acid residue of the antibody (for example, a cysteine residue or a lysine residue) (for example, described in WO2014 / 057687) can be cited, but are not limited thereto. As the linker that binds the drug to an arbitrary amino acid residue of the antibody, preferably, the case of forming a thioether bond with the sulfhydryl group (SH group) of cysteine of Ab (sometimes referred to as "cysteine conjugation" in this specification) or the case of forming an amide bond with the amino group (NH2 group) of lysine of Ab (sometimes referred to as "lysine conjugation") can be cited, and cysteine conjugation is preferred.

[0267] The preferred linker L used in the antibody-drug conjugate of the present invention is represented by the following formula.

[0268] -Lb-La-Lp-Lc-*

[0269] Here, the asterisk indicates binding to drug D.

[0270] First, Lp will be described. Lp represents a linker formed by an amino acid sequence that can be cleaved in vivo or in a target cell (hereinafter, also referred to as a peptide linker in this specification) or is absent.

[0271] Lp is cleaved by the action of enzymes such as peptidases and esterases. Lp is a peptide composed of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond with the carbonyl group at the right end of La described below at its N-terminus and an amide bond with the amino group (-NH-) of Lc at its C-terminus. The amide bond on the C-terminal side of Lp is cleaved by the enzyme such as the peptidase.

[0272] The amino acids constituting Lp are not particularly limited. For example, they are L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, they can also be amino acids with structures such as β-alanine, ε-aminohexanoic acid, and γ-aminobutyric acid. Furthermore, they can be non-natural amino acids such as N-methylated amino acids. The amino acid sequence of Lp is not particularly limited. As the constituent amino acids, 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) can be mentioned. Among them, 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 can be repeated and have an amino acid sequence containing arbitrarily selected amino acids. In addition, the mode of drug release can be controlled according to the type of amino acid.

[0273] As specific examples of Lp, for example, -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, -GGPP- can be mentioned. The linker Lp is preferably -GGFG- or -GGPI-, more preferably -GGFG-.

[0274] Next, La will be described. La represents any one selected from the group consisting of: -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 -O-C(=O)- and -(CH2)n 9 -C(=O)-.

[0275] Here, in the formula, n 2 represents an integer from 1 to 3 (preferably 1 or 2), n 3 represents an integer from 1 to 5 (preferably an integer from 2 to 5, more preferably 3 or 4), n 4 represents an integer from 0 to 2 (preferably 0 or 1), n 9 represents an integer from 2 to 7 (preferably an integer from 2 to 5, more preferably 2, 3 or 5).

[0276] La preferably represents any one selected from the group consisting of: -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)-.

[0277] 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)-.

[0278] La is further preferably -C(=O)-CH2CH2-C(=O)-.

[0279] Next, Lb will be described. Lb represents a spacer used in a linker for sugar chain conjugation (also referred to as "the spacer of the linker for sugar chain conjugation" in this specification), or a spacer used in a linker for cysteine conjugation (also referred to as "the spacer of the linker for cysteine conjugation" in this specification).

[0280] <When Lb is "the spacer of the linker for sugar chain conjugation">

[0281] When Lb is "the spacer of the linker for sugar chain conjugation", Lb is not particularly limited, and examples thereof include the spacers represented by the following formulas.

[0282]

[0283] In each of the above - shown structural formulas, the asterisk (*) represents binding to - (C = O)-, - CH2 - or - OC(=O)- at the left end of La, and the wavy line represents binding to the sugar chain of Ab or the reconstructed sugar chain.

[0284] When Lb selects Lb - 1 or Lb - 3, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of the two structures or a mixture thereof. The antibody - drug conjugate of the present invention can bind multiple drugs to one molecule of antibody. When multiple drugs are bound to one molecule of antibody, there are also multiple Lbs (for example, refer to the schematic diagram of the antibody - drug conjugate shown in Method E of <3. Manufacturing method> (1e) described later). When Lb is selected from Lb - 1 or Lb - 3 and there are multiple such Lbs relative to one molecule of antibody (for example, when m 2 is 1 or 2 as described later), in each Lb, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of the two structures or a mixture thereof.

[0285] <When Lb is "the spacer of the linker for cysteine conjugation">

[0286] When Lb is "the spacer of the linker for cysteine conjugation", Lb is not particularly limited, and examples thereof include -(succinimidyl - 3 - yl - N)-. In the present invention, "-(succinimidyl - 3 - yl - N)-" has the structure represented by the following formula.

[0287]

[0288] In the above - shown structural formula, the asterisk represents binding to La, and the wavy line represents binding by forming a thioether with the side chain of the cysteine residue of the antibody.

[0289] Next, Lc will be described. Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)-, or -NH-heteroaryl-CH2-O(C=O)-, or it does not exist. Here, as the phenyl group, 1,4-phenyl is preferred, and as the heteroaryl group, 2,5-pyridyl, 3,6-pyridyl, 2,5-pyrimidinyl, or 2,5-thienyl is preferred. Lc is preferably -NH-CH2- or does not exist.

[0290] Regarding the more preferred linker L used in the antibody-drug conjugate of the present invention, when the binding mode of the drug to the antibody is "glycan conjugation", the linker L is -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-, -Z L1 -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 represents the following structural formula of the above Lb:

[0291]

[0292] Alternatively, when the binding mode between the drug and the antibody is "cysteine conjugation", the linker L is -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 represents -(succinimidyl-3-yl-N)- shown by the following structural formula of Lb above.

[0293]

[0294] Regarding the further preferred linker L used in the antibody-drug conjugate of the present invention, when the binding mode between the drug and the antibody is "glycan conjugation", the linker L is -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, or -Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-CH2-, where Z L1The following structural formula represents Lb described above:

[0295]

[0296] For the more preferred linker L used in the antibody-drug conjugate of the present invention, the binding mode of the drug to the antibody is "glycan conjugation", and the linker L is -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, where Z L1 The following structural formula represents Lb described above:

[0297]

[0298] The right end of the above-mentioned "preferred linker L", "more preferred linker L", "further preferred linker L" and "even more preferred linker L" binds to the drug D.

[0299] The "linker L-drug D" used in the antibody-drug conjugate of the present invention is preferably represented by the following two structural formulas:

[0300]

[0301] Here, the wavy line represents binding to the glycan or the reconstructed glycan of Ab.

[0302] The "linker L-drug D" used in the antibody-drug conjugate of the present invention is more preferably represented by the following two structural formulas:

[0303]

[0304] Here, the wavy line represents binding to the glycan or the reconstructed glycan of Ab.

[0305] The "linker L-drug D" used in the antibody-drug conjugate of the present invention is further preferably represented by the following formula:

[0306]

[0307] Here, the wavy line represents binding to the glycan or the reconstructed glycan of Ab.

[0308] <2.2. Antibody and Its Glycan Modification>

[0309] <2.2.1 Antibody>

[0310] In this specification, "gene" refers to a nucleotide or nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or its complementary strand. For example, 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, are also included in the meaning of "gene".

[0311] In this specification, "nucleotide", "polynucleotide" or "nucleotide sequence" have the same meaning as "nucleic acid". For example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence".

[0312] In this specification, "polypeptide", "peptide" and "protein" are used interchangeably.

[0313] In this specification, "functional fragment of an antibody" is also referred to as "antigen-binding fragment of an antibody", and refers to a partial fragment of an antibody having binding activity to an antigen, including Fab, F(ab’)2, Fv, scFv, diabody (bispecific antibody), linear antibody, and multispecific antibody formed by antibody fragments, etc. In addition, the monovalent fragment Fab’ of the variable region of an antibody obtained by treating F(ab’)2 under reducing conditions is also included in the antigen-binding fragments of the antibody. However, as long as it has the binding ability to an antigen, it is not limited to these molecules. In addition, these antigen-binding fragments include not only molecules obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using a genetically engineered antibody gene.

[0314] The functional fragment of the antibody used in the antibody-drug conjugate of the present invention includes asparagine (Asn297) which maintains the modification caused by the N-linked glycans well conserved in the Fc region of the IgG heavy chain and the surrounding amino acids, and has a functional fragment with binding ability to an antigen.

[0315] The antibody used in the antibody-drug conjugate of the present invention refers to an immunoglobulin, which is a molecule containing an antigen-binding site that binds immunologically specifically to an antigen. As the antibody used in the antibody-drug conjugate of the present invention, it can be any one of IgG, IgE, IgM, IgD, IgA and IgY, preferably IgG. In addition, as a subclass, it can be any one of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1, IgG2 or IgG4 (including antibodies having mutations affecting ADCC and ADCP activities in the Fc region of the IgG heavy chain).

[0316] When IgG1 is used as the isotype of the antibody used in the antibody-drug conjugate of the present invention, a part of the amino acid residues in the constant region can be substituted to adjust the effector function (see WO88 / 07089, WO94 / 28027, WO94 / 29351). As variants of IgG1, for example, IgG1 LALA variants (IgG1-L234A, L235A) can be mentioned. The L234A and L235A indicate that the leucines at positions 234 and 235 determined 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) are substituted with alanines.

[0317] It is known that there are multiple allotypes in the constant region of antibodies. For example, regarding the IgG1 heavy chain, G1m17, G1m3, G1m1, and G1m2 can be mentioned. The constant region of the antibody used in the present invention is not particularly limited, and G1m17 or G1m3 is preferably used.

[0318] It is known that the heavy chain and the light chain of an antibody molecule each have three complementarity determining regions (CDR: Complementarity determining region). CDR is also called the hypervariable region. In the variable regions of the heavy chain and the light chain of an antibody, it is a region where the variability of the primary structure is particularly high, and it is separated into three parts on the primary structure of the polypeptide chains of the heavy chain and the light chain, respectively. In this specification, for the CDR of an antibody, the CDR of the heavy chain is denoted as CDRH1, CDRH2, and CDRH3 starting from the amino-terminal side of the heavy chain amino acid sequence, and the CDR of the light chain is denoted as CDRL1, CDRL2, and CDRL3 starting from the amino-terminal side 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 be bound.

[0319] The antibody can be from any species, and preferably, humans, rats, mice, and rabbits can be exemplified. In the case of a species other than humans, it is preferable to use known techniques for chimerization or humanization. The antibody of the present invention can be a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody is preferred. Monoclonal antibodies include monoclonal antibodies from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, human antibodies, their functional fragments, or their modified forms.

[0320] The antibody is preferably an antibody targeting tumor cells or immune cells, but is not limited thereto. More preferably, the antibody is an antibody targeting tumor cells.

[0321] In the case of using an antibody targeting tumor cells, as the antibody, it preferably has one or more of the properties of being able to recognize tumor cells, being able to bind to tumor cells, being internalized by being engulfed into tumor cells, and killing tumor cells. The drug used in the antibody-drug conjugate of the present invention has STING agonist activity. This drug activates the signal of interferon regulatory factor-3 (IRF3) to induce interferon. Therefore, in the case of using an antibody targeting tumor cells in the antibody-drug conjugate of the present invention, after the antibody-drug conjugate is administered in vivo, it reaches the tumor site, and after being engulfed into tumor cells, the linker part is cleaved by a peptidase or the like, and the drug part is released. It is considered that the released drug part enhances the sensitivity of tumor cells to immune cells and activates anti-tumor immunity through STING agonist activity, thereby exerting an anti-tumor effect. Alternatively, it can also be considered that even if the antibody-drug conjugate aggregated on tumor cells is not internalized, the tumor cells and / or the antibody-drug conjugate are engulfed into immune cells by phagocytosis, and anti-tumor immunity is activated through STING agonist activity, thereby exerting an anti-tumor effect.

[0322] The binding property of the antibody to tumor cells can be confirmed using a flow cytometer. The engulfment of the antibody into tumor cells can be confirmed by the following methods: (1) an analysis of visualizing the antibody engulfed into cells by a fluorescence microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an analysis of measuring the fluorescence amount engulfed into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP analysis using an immunotoxin that binds to the therapeutic antibody and releases the toxin to inhibit cell proliferation when engulfed into cells (Bio Techniques 28: 162-165, January 2000). As the immunotoxin, a recombinant complex protein of the catalytic region of diphtheria toxin and protein G can also be used.

[0323] When the antibody-drug conjugate of the present invention uses an antibody targeting tumor cells, it is preferred that the antibody itself has an anti-tumor effect, but this is not necessary.

[0324] The anti-tumor activities of a drug and an antibody-drug conjugate refer to the cytotoxicity to tumor cells, the anti-cell effect, and the shrinkage of tumor volume. The anti-tumor activities can be confirmed using a well-known in vitro or in vivo evaluation system.

[0325] The effects and immune activation activities of a drug and an antibody-drug conjugate refer to the enhanced sensitivity of tumor cells to immune cells or the activation of immune cells by tumor cells. The effects and immune activation activities of a drug and an antibody-drug conjugate can be confirmed using a well-known in vitro or in vivo evaluation system.

[0326] Examples of the in vitro or in vivo evaluation systems that can be used in the present invention include: the co-culture analysis system of CT26.WT and CT26.WT-hCD70 cell lines and dendritic cells derived from mouse bone marrow described in Test Example 4; the system of BALB / c mice subcutaneously transplanted with CT26.WT-hTROP2 cells obtained by introducing the human TROP2 gene into the mouse colon cancer cell line CT26.WT described in Test Example 5; the system of BALB / c mice subcutaneously transplanted with CT26.WT-hEGFR cells obtained by introducing the human EGFR gene into the mouse colon cancer cell line CT26.WT described in Test Example 6; the system of BALB / c-nu mice subcutaneously transplanted with the human renal cancer cell line Caki-1 cells described in Test Example 7; the system of BALB / c-nu mice subcutaneously transplanted with the human renal cancer cell line A-498 (HTB-44) cells described in Test Example 8; the system of BALB / c mice subcutaneously transplanted with the mouse colon cancer cell line CT26.WT (CRL2638) into which a human-mouse chimeric antigen gene in which the epitope site on the antigen bound by the antibody contained in the antibody-drug conjugate has been replaced with a human type has been introduced described in Test Example 9, etc., but are not limited thereto.

[0327] Examples of the antibody used in the present invention include an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody.

[0328] The antibody used in the present invention can be obtained by immunizing an animal with a polypeptide as an antigen using a method commonly practiced in the art and collecting and purifying the antibody produced in vivo. The source of the antigen is not limited to humans, and an animal other than humans such as a mouse or a rat can also be immunized with an antigen. In this case, an antibody that can be used for human diseases can be selected by testing the cross-reactivity of the antibody that binds to the obtained heterologous antigen with a human antigen.

[0329] In addition, monoclonal antibodies can be obtained by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to known methods (for example, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennett, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)).

[0330] It should be noted that an antigen can be obtained by genetically engineering a host cell to produce a gene encoding an antigen protein.

[0331] 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. U.S.A., 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525, WO90 / 07861).

[0332] 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 methods.

[0333] The anti-CD70 antibody used in the present invention is not particularly limited, and preferably an antibody having the following characteristics, for example.

[0334] (1) An anti-CD70 antibody that specifically binds to CD70.

[0335] (2) The antibody as described in (1) above, which binds to the extracellular domain of human CD70.

[0336] (3) The antibody as described in (1) or (2) above, which is a monoclonal antibody.

[0337] (4) The antibody as described in any one of (1) to (3) above, which has antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0338] (5) The antibody as described in any one of (1) to (4) above, which is a murine monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody.

[0339] (6) The antibody as described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains mutations that reduce ADCC and ADCP activities.

[0340] (7) The antibody as described in (5) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 shown by EU INDEX are replaced with alanines.

[0341] (8) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain formed of the amino acid sequence set forth in SEQ ID NO: 1.

[0342] (9) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a heavy chain formed of the amino acid sequence set forth in SEQ ID NO: 4 and a light chain formed of the amino acid sequence set forth in SEQ ID NO: 3.

[0343] (10) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed of the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 formed of the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 formed of the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain comprises CDRH1 formed of the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 formed of the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 formed of the amino acid sequence set forth in SEQ ID NO: 40.

[0344] (11) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed of the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 formed of the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 formed of the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain comprises CDRH1 formed of the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 formed of the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 formed of the amino acid sequence set forth in SEQ ID NO: 46.

[0345] (12) The antibody as described in any one of (1) to (11) above has one or two amino acids deleted at the carboxyl terminus of the heavy chain.

[0346] (13) An antibody obtained by a method for producing the antibody comprising the following steps: a step of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody as described in any one of (1) to (12) above; and a step of collecting the target antibody from the culture obtained by this step.

[0347] As anti-CD70 antibodies, vorsetuzumab, MDX-1115, and Cusatuzumab (anti-CD70 monoclonal antibody) can be mentioned, and vorsetuzumab and MDX-1115 are preferably mentioned.

[0348] The anti-TROP2 antibody used in the present invention is not particularly limited, and an antibody having the following characteristics is preferably used.

[0349] (1) An anti-TROP2 antibody that specifically binds to TROP2.

[0350] (2) The antibody according to (1) above, which binds to the extracellular domain of human TROP2.

[0351] (3) The antibody according to (1) or (2) above, which is a monoclonal antibody.

[0352] (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cell cytotoxicity (CDC).

[0353] (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.

[0354] (6) The antibody according to (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a mutation that reduces ADCC and ADCP activities.

[0355] (7) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 6 and a light chain formed by the amino acid sequence set forth in SEQ ID NO: 5.

[0356] (8) The antibody according to (5) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and leucines at positions 234 and 235 indicated by EU INDEX are replaced with alanines.

[0357] (9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 8 and a light chain formed by the amino acid sequence set forth in SEQ ID NO: 7.

[0358] (10) The antibody as described in (8) above, which is a humanized monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 49, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 52.

[0359] (11) The antibody as described in (8) above, which is a humanized monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 55, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 58.

[0360] (12) The antibody as described in any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

[0361] (13) An antibody obtained by a method for manufacturing the antibody comprising the following steps: a step of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody as described in any one of (1) to (12) above; and a step of collecting the target antibody from the culture obtained by this step.

[0362] The anti-EGFR antibody used in the present invention is not particularly limited, and an antibody having the following characteristics is preferably used, for example.

[0363] (1) An anti-EGFR antibody that specifically binds to EGFR.

[0364] (2) The antibody as described in (1) above, which binds to the extracellular domain of human EGFR.

[0365] (3) The antibody as described in (1) or (2) above, which is a monoclonal antibody.

[0366] (4) The antibody as described in any one of (1) to (3) above, which has antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0367] (5) The antibody as described in 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.

[0368] (6) The antibody as described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a variation that causes a decrease in ADCC and ADCP activities.

[0369] (7) The antibody as described in (5) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 indicated by EU INDEX are replaced by alanines.

[0370] (8) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 10 and a light chain formed by the amino acid sequence set forth in SEQ ID NO: 9.

[0371] (9) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 12 and a light chain formed by the amino acid sequence set forth in SEQ ID NO: 11.

[0372] (10) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 61, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 64.

[0373] (11) The antibody as described in (7) above, which is a humanized monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 67, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 70.

[0374] (12) The antibody as described in any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

[0375] (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 the above (1) to (12); and collecting the target antibody from the culture obtained by this step.

[0376] Examples of the anti-EGFR antibody include panitumumab, nimotuzumab, cetuximab, ametumumab (SY-101), SYN-004, SCT-200, tomuzotuximab, GC-1118, GR-1401, and depatuxizumab (ABT-806). Preferred examples include panitumumab and ABT806.

[0377] The antibody used in the present invention may be an antibody having 80% to 99% amino acid identity compared to the heavy chain and / or light chain of the above antibody. Here, the term "identity" has the ordinary definition used in the art. The % of identity refers to the percentage of the number of identical amino acids relative to the total number of amino acids (including gaps) when two amino acid sequences are aligned in such a way that the amino acid identity is maximized. Such identity is usually 80% or more, preferably 90%, 91%, 92%, 93% or 94% or more, more preferably 95%, 96%, 97% or 98% or more, and further preferably 99% or more. In addition, by combining amino acid sequences in which 1 to several amino acid residues are substituted, deleted and / or added in the amino acid sequence of the heavy chain and / or light chain, an antibody having various actions equivalent to those of the above antibodies can also be selected. The number of amino acid residues substituted, deleted and / or added is usually 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 further preferably 1 amino acid residue.

[0378] <2.2.2 Glycan remodeling of the antibody>

[0379] In recent years, methods for reconstructing heterogeneous glycans of antibodies and uniformly introducing glycans with functional groups through enzymatic reactions have been reported (ACS Chem. Biol. 2012, 7, 110 - 122, ACS Med. Chem. Lett. 2016, 7, 1005 - 1008). The glycan reconstruction technology has been attempted to site - specifically introduce drugs and synthesize uniform ADCs (Bioconjugate Chem. 2015, 26, 2233 - 2242, Angew. Chem. Int. Ed. 2016, 55, 2361 - 2367, US2016361436).

[0380] For glycan reconstruction, first, a hydrolase is used to excise the heterogeneous glycans added to a protein (such as an antibody), leaving only the terminal GlcNAc, and a uniform protein moiety added with GlcNAc (hereinafter referred to as "receptor") is prepared. Then, any separately prepared glycan (hereinafter referred to as "donor") is prepared, and the receptor and the donor are linked using a glycosyltransferase. Thus, a uniform glycoprotein with an arbitrary glycan structure can be synthesized.

[0381] In the present invention, a "glycan" refers to a structural unit formed by the binding of two or more monosaccharides through glycosidic bonds. Specific monosaccharides and glycans are sometimes represented in abbreviated forms, such as "GlcNAc -", "SG -". When recorded in these abbreviated forms in a structural formula, unless otherwise specifically defined, the oxygen atom or nitrogen atom attributed to the glycosidic bond with other structural units at the reducing end is not included in the abbreviation representing the glycan.

[0382] In the present invention, unless otherwise specified, for the convenience of description, regarding the monosaccharide that is the basic unit of the glycan, the carbon atom bonded to the oxygen atom forming the ring and directly bonded to a hydroxyl group (or the oxygen atom attributed to the glycosidic bond) in its ring structure is denoted as the 1 - position (2 - position only in sialic acid). The names of the example compounds are named based on the overall chemical structure and do not necessarily follow this rule.

[0383] In the present invention, when a glycan is recorded using symbols (such as SG, MSG, GlcNAc, etc.), unless otherwise defined, the carbon at the reducing end is included in the symbol, but the N or O attributed to the N - or O - glycosidic bond is not included in the symbol.

[0384] The antibody - drug conjugate of the present invention is represented by the following formula:

[0385]

[0386] The antibody Ab or its functional fragment binds directly to L from the side chains of its amino acid residues (e.g., cysteine, lysine, etc.), or binds to L from the sugar chain of Ab or the reconstructed sugar chain.

[0387] The sugar chain of the Ab of the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

[0388] The N-linked sugar chain binds to the amino acid side chain of the antibody through an N-glycosidic bond, and the O-linked sugar chain binds to the amino acid side chain of the antibody through an O-glycosidic bond.

[0389] The Ab of the present invention is IgG, preferably IgG1, IgG2 or IgG4.

[0390] It is known that IgG has a highly conserved N-linked sugar chain (hereinafter referred to as "Asn297 sugar chain or N297 sugar chain") at the asparagine residue at position 297 (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which contributes to the activity, dynamics, etc. of the antibody molecule (Eon-Duval, A. et al, Biotechnol. Prog. 2012, 28, 608-622, Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0391] The amino acid sequence in the constant region of IgG is highly conserved. In the report of Edelman et al (Proc. Natl. Acad. Sci. U.S.A., 63, 78-85, (1969)), each amino acid is determined by the EU numbering (EU INDEX). For example, Asn297 to which the N-linked sugar chain is added in the Fc region corresponds to the 297th position in the EU numbering. Even when the actual amino acid position changes due to fragmentation or regional deletion of the molecule, the amino acid can be uniquely determined by representing it with the EU numbering.

[0392] The following figure shows the binding of the antibody-drug conjugate of the present invention to L from the N297 sugar chain of the antibody or its functional fragment.

[0393]

[0394] It should be noted that the antibody having the reconstructed sugar chain is called a sugar chain-reconstructed antibody.

[0395] SGP (α2,6-SGP) is an abbreviation for Sialylglycopeptide and is a representative example of N-linked glycopeptides. SGP can be isolated and purified from the egg yolk according to the method described in, for example, WO2011 / 027868. In addition, purified products of SGP are sold by Tokyo Chemical Industry and Fushimi Pharmaceutical. In this specification, the sugar chain part of SGP is denoted as SG, and the sugar chain lacking the GlcNAc at the reducing end of one SG is denoted as SG(10). SG(10) can be prepared, for example, by enzymatic hydrolysis of SGP with reference to the report by Umemura et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209). In addition, SG(10) can also be purchased from Tokyo Chemical Industry and Fushimi Pharmaceutical.

[0396] In this specification, the sugar chain structure in which the non-reducing terminal sialic acid is missing from either one of the branches of β-Man in SG(10) is denoted as MSG(9), the sugar chain having sialic acid only in the 1-3 sugar chain of the branch is denoted as MSG1, and the sugar chain having sialic acid only in the 1-6 sugar chain of the branch is denoted as MSG2.

[0397] The reconstructed sugar chains used in the antibody-drug conjugate 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. N297-(Fuc)SG is represented by the following structural formula or sequence formula.

[0398]

[0399]

[0400] In the above formula, the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) forms an amide bond with the 2-position carboxyl group of the non-reducing terminal sialic acid on both the 1-3 chain side and the 1-6 chain side of the branch of β-Man of the N297 sugar chain, and the asterisk represents binding to the linker L, particularly to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb of the linker L, where n 5 is an integer from 2 to 10, preferably an integer from 2 to 5.

[0401] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula.

[0402]

[0403] In the above formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the linker L, particularly to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb of the linker L, where n 5 is an integer from 2 to 10, preferably an integer from 2 to 5.

[0404] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula.

[0405]

[0406] In the above formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of this L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing end of the 1-6 chain side of the branch of β-Man of the N297 sugar chain. The asterisk indicates binding to the linker L, particularly to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb of the linker L, where n 5 is an integer from 2 to 10, preferably an integer from 2 to 5.

[0407] 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, so the antibody-drug conjugate is a molecule that binds 4 linkers L and 4 drugs D (the above m 2 =2).

[0408] 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, so the antibody-drug conjugate is a molecule that binds 2 linkers L and 2 drugs D (the above m 2= 1) (Refer to Figure N297. The 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.)

[0409] When the N297 sugar chain of the antibody of the antibody-drug conjugate of the present invention is N297-(Fuc)SG or N297-(Fuc)MSG1 or N297-(Fuc)MSG2, an ADC with high uniformity can be obtained.

[0410] <3. Manufacturing Method>

[0411] A representative manufacturing method of the antibody-drug conjugate containing the CDN derivative of the present invention or its manufacturing intermediate will be described. It should be noted that in the following, in order to represent compounds, the numbers of the compounds shown in each reaction formula are used. That is, it is called "the compound of formula (1)", "compound (1)", etc. In addition, the compounds with other numbers are also described in the same way.

[0412] In the following Method A to Method E, the substituent L 1 has the same meaning as described above. The substituent L 2 represents a group selected from the following (i) or (ii): (i) When combined with L, L 2 represents -NHR', hydroxy C1-C6 alkyl or amino C1-C6 alkyl, where R' represents a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl, and the C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl may be substituted with 1 to 6 halogen atoms; or (ii) When not combined with L, L 2 represents a hydrogen atom or a halogen atom, and the substituent W 1 represents -NH- or a sulfur atom. The substituent W 2 represents -CH=. The substituent Z 1 ~Z 3 together represent -CH2-CH2-CH2-. The substituent R 1 ~R 3 each independently represents a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N3, -NHR', -NR'R" or -NHC(=O)R' (where R' is as defined above, and R" represents C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C6 cycloalkyl). The substituent R 4 represents a hydrogen atom. Regarding the substituent R 5 , when W 1 is a nitrogen atom, R 5 represents a hydrogen atom. When W1 When it is an oxygen atom, R 5 does not exist. The substituent R a , R c , R e and R g represent the side chains of natural α-amino acids. For example, they are methyl, isopropyl, sec-butyl, isobutyl, benzyl, etc. PRO 1 represents a protecting group for primary alcohols. Preferably, it is 4,4'-dimethoxytrityl, 4-methoxytrityl, etc. PRO 2 , PRO 3 , PRO 7 , PRO 8 represent protecting groups for secondary alcohols. Preferably, they are tert-butyldimethylsilyl, triisopropylsilyloxymethyl, benzoyl, 2-nitrobenzyl, 4-methoxytetrahydropyran-4-yl, etc. PRO 6 represents a protecting group for carboxylic acids. Preferably, it is tert-butyl, benzyl, etc. PRO 5 , PRO 9 represent protecting groups for amines. PRO 5 Preferably, they are tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, etc., PRO 9 Preferably, it is 9-fluorenylmethyloxycarbonyl or 2-(trimethylsilyl)ethoxycarbonyl. PRO 4 represents a protecting group for alcohols or amines. In the case of alcohols, preferably tert-butyldimethylsilyl, benzoyl, etc., and in the case of amines, preferably 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, tert-butoxycarbonyl, etc. Q a represents an oxygen atom or a sulfur atom, Q b represents a hydroxyl group or a thiol group. Q a’ and Q b’ each independently represent a negatively charged oxygen atom (O － ) or sulfur atom (S － ). R x and R y each independently represent a halogen atom or -O-PRO 2 . n represents an integer from 1 to 3.

[0413] Method A

[0414] The CDN derivative shown in (1) used in the antibody-drug conjugate of the present invention can be produced according to Method A described below.

[0415]

[0416] This manufacturing method is a method for manufacturing the compound represented by the general formula (1). The A-1 to A-5 steps of this manufacturing method can be carried out by one pot synthesis, and this manufacturing method can be implemented with reference to the report by Gaffney et al. (Org. Lett. 2010, 12, 3269-3271).

[0417]

[0418] (Step A-1)

[0419] This step is a step for manufacturing the compound of formula (2a) by continuously performing a hydrolysis reaction and removing a cyanoethyl group on the compound of formula (1a) using a known organic chemical method. In a solvent (acetonitrile, tetrahydrofuran, N,N-dimethylformamide, or a mixed solvent thereof), at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C, the hydrolysis reaction is carried out by treating the compound (1a) with water and an acid (pyridine trifluoroacetate, 4,5-dicyanoimidazole, 1H-tetrazole, etc.). Relative to 1 mole of the compound (1a), 2 moles to an excess mole of water is used, preferably 2 moles to 10 moles, and 1 mole to an excess mole of acid is used, preferably 1 mole to 5 moles. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Then, a base (tert-butylamine, etc.) is added to the reaction solution to remove the cyanoethyl group. Relative to 1 mole of the compound (1a), an excess mole of the base is used, preferably 30 moles to 50 moles. The reaction time is 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction solution is concentrated under reduced pressure to obtain a crude product of the compound (2a). The crude product of the compound (2a) can be carried on to the next step without purification.

[0420] (Step A-2)

[0421] This step is a step for manufacturing the compound of formula (3a) by removing the protecting group of the hydroxyl group from the compound of formula (2a) using a known organic chemical method. Before starting the reaction of this step, the crude product of the compound (2a) is dried by azeotroping with acetonitrile 1 to 3 times as needed. In PRO 1In the case of 4,4'-dimethoxytrityl, the compound (2a) is treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C to remove 4,4'-dimethoxytrityl. Relative to 1 mole of the compound (2a), an excess molar amount of water is used, preferably 10 moles to 20 moles, and the acid is diluted to 1% to 50% (v / v), preferably 5% to 10% (v / v) with the solvent used in the reaction. An excess molar amount of this diluted solution is used, preferably 5 moles to 15 moles. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Pyridine is added to the reaction solution to stop the reaction. The amount of pyridine used is sufficient to neutralize the acid used, preferably 2 moles to 10 moles of pyridine relative to 1 mole of the acid. The reaction solution is concentrated under reduced pressure to obtain a crude product of the compound (3a). The crude product of the compound (3a) is azeotroped with dehydrated acetonitrile 3 to 5 times. Acetonitrile remains at the last azeotropy to obtain a 0.01M to 1M acetonitrile solution of the compound (3a). The obtained acetonitrile solution is directly fed into the next step.

[0422] (Step A-3)

[0423] This step is a step of manufacturing the compound of formula (5a) by successively performing a coupling reaction of the compound of formula (3a) with the compound of formula (4a) and a sulfidation reaction of the obtained coupling product using a known organic chemical method. Before starting the reaction of this step, the compound (4a) is azeotroped with dehydrated acetonitrile 3 to 5 times. Acetonitrile remains at the last azeotropy to prepare a 0.01M to 1M acetonitrile solution of the compound (4a). A desiccant (powdered or pellet-shaped molecular sieve 3A or molecular sieve 4A) is added to this solution and stored under a nitrogen or argon atmosphere before using the solution. The coupling reaction is carried out by adding the acetonitrile solution of the azeotropically dried compound (4a) to the acetonitrile solution of the compound (3a) at 5°C to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Then, a sulfiding agent (N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine, 3H-1,2-benzodithiol-3-one, etc.) is added to the reaction solution to carry out the sulfidation reaction. Relative to 1 mole of the compound (3a), 1 mole to 5 moles of the sulfiding agent is used, preferably 1 mole to 2 moles. The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product of the compound (5a). The obtained crude product of the compound (5a) is directly fed into the next step.

[0424] (Step A-4)

[0425] This step is a step of removing the protecting group of the hydroxyl group from the compound of formula (5a) using a known organic chemical method to produce the compound of formula (6a). In the case of PRO 1 When it is 4,4'-dimethoxytrityl, the compound (5a) is treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C, to remove 4,4'-dimethoxytrityl. With respect to 1 mole of the compound (5a), an excess molar amount of water is used, preferably 10 to 20 moles, and the acid is diluted to 1% to 50% (v / v), preferably 5% to 10% (v / v) with the solvent used in the reaction, and an excess molar amount of this diluted solution is used, preferably 5 moles to 15 moles. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Pyridine is added to the reaction solution to stop the reaction. The amount of pyridine used is such that it can sufficiently neutralize the acid used, preferably 10 moles to 200 moles of pyridine are used with respect to 1 mole of the acid. The reaction solution is concentrated under reduced pressure to obtain a crude product of the compound (6a). The obtained crude product of the compound (6a) is directly fed into the next step.

[0426] (Step A-5)

[0427] This step is a step of continuously performing a cyclization reaction and a sulfidation reaction on the compound of formula (6a) using a known organic chemical method to produce the compound of formula (7a). After dissolving the compound (6a) in pyridine, it is concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. By adding a dehydrating condensing agent (2-chloro-5,5-dimethyl-1,3,2λ 5-2-oxaphosphorinane-2-one, etc.) to carry out the cyclization reaction. Per 1 mole of compound (6a), 1 mole to an excess mole of the dehydrating condensing agent is used, and preferably 3 moles to 5 moles are used. The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Then, water and a sulfurizing agent (3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine, etc.) are added to the reaction solution to carry out the sulfurization reaction. Per 1 mole of compound (6a), an excess mole of water is used, preferably 30 moles to 50 moles, and 1 mole to 5 moles of the sulfurizing agent is used, preferably 1 mole to 2 moles. The reaction time is 5 minutes to 12 hours, preferably 30 minutes to 3 hours. After adding the reaction solution to an aqueous sodium bicarbonate solution (0.1M to 1M), stirring is carried out for 15 minutes to 24 hours to stop the reaction. The reaction solution is extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixed solvent thereof), and the extracted solutions are combined and dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant is filtered off and the filtrate is concentrated under reduced pressure. The obtained residue is 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) in the form of a mixture of two or more diastereoisomers or two or more pure diastereoisomers. In this step, in most cases, two diastereoisomers can be obtained, but depending on the starting materials (1a) and (4a), sometimes one or two more diastereoisomers can be further obtained. Even if the obtained compound (7a) is a mixture of multiple diastereoisomers, it can enter the next step without further purification.

[0428] (Step A-6)

[0429] This step is a step of simultaneously removing the cyanoethyl group and all acyl group protecting groups from the compound of formula (7a) using a known organic chemical method to produce the compound of formula (8a). This step is carried out in an autoclave or a sealed tube as required. In PRO 4When it is a benzoyl group, the cyanoethyl and benzoyl groups are removed by treating compound (7a) with 28% (v / v) aqueous ammonia in a solvent (methanol, ethanol, tetrahydrofuran or a mixed solvent thereof) at 5 °C to the boiling point of the solvent used in the reaction. With respect to 1 mole of compound (7a), an excess molar amount of ammonia is used, preferably 300 to 3000 moles. The reaction time is 30 minutes to 96 hours, preferably 2 to 48 hours. If necessary, the reaction solution is concentrated, and the residue is 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 obtained compound (8a) is a mixture of diastereoisomers, it can be directly carried on to the next step without further purification. In addition, in this step, it is also possible to directly carry on to the next step without purification.

[0430] (Step A-7)

[0431] This step is a step of simultaneously removing all silyl-based protecting groups from the compound of formula (8a) using a known organic chemical method to produce the compound of formula (9a). In PRO 2 and PRO 3 When it is tert-butyldimethylsilyl, the tert-butyldimethylsilyl group is removed by directly treating compound (8a) with triethylamine trihydrofluoride at 5 °C to 100 °C, preferably at 35 °C to 60 °C. With respect to 1 mole of compound (8a), an excess molar amount of triethylamine trihydrofluoride is used, preferably 100 to 200 moles. The reaction time is 30 minutes to 24 hours, preferably 2 to 12 hours. After cooling the reaction solution to room temperature, an ice-cold 1M aqueous solution of triethylammonium bicarbonate and a 3:1 to 10:1 (v / v) mixed solution of triethylamine are gradually injected into the reaction solution to stop the reaction. It is also possible to inject the reaction solution into an ice-cold mixed solution of 1M aqueous solution of triethylammonium bicarbonate and triethylamine as needed. In this case, the reaction vessel is washed with acetonitrile and water. Triethylamine is used in an amount sufficient to make the liquid property of the reaction solution weakly basic, preferably about 2 moles of triethylamine with respect to 1 mole of triethylamine trihydrofluoride. After distilling off the organic solvent component of the reaction solution under reduced pressure, the remaining aqueous solution is 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) in the form of a single diastereoisomer.

[0432] (Step A-8)

[0433] This step is a step of producing the compound of formula (1) by subjecting the compound of formula (9a) to ion exchange using a known organic chemical method. A cation exchange resin (BT AG (registered trademark) 50W-X2 resin, 100-200 mesh, hydrogen form) is suspended in pure water and packed into an empty column cartridge. The cation exchange resin is used in an amount of 10 to 50 times the amount of the compound (9a) by weight. After allowing an excessive amount of pure water to flow down naturally, a 1M aqueous sodium hydroxide solution is allowed to flow down naturally for 3 column volumes, and then 6 column volumes of pure water are allowed to flow down naturally. The compound (9a) is dissolved in about 3 column volumes of pure water and loaded into the column. In the case where the compound is not easily soluble in pure water, a mixed solution with a small amount of an organic solvent (acetonitrile, methanol, etc.) can be used. After collecting the solution that has flowed down naturally, it is further eluted with 6 column volumes of pure water or the like, and the components are collected. The components containing the target substance are combined and freeze-dried to obtain the compound (1) in the form of a single diastereoisomer.

[0434] Method A'

[0435] 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.

[0436]

[0437] This production method is a method for producing the compound represented by the general formula (1') by changing a part of Method A. Specifically, the compound of the general formula (1') can be produced by changing Step A-5 of Method A to Step A'-5 shown below. In addition, when substituents R x and R y are both halogen atoms, Step A-7 can be omitted.

[0438]

[0439] (Step A'-5)

[0440] This step is a step of producing the compound of formula (7a') by continuously performing a cyclization reaction and an oxidation reaction on the compound of formula (6a') using a known organic chemical method. The compound (6a') is dissolved in pyridine and concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. In this pyridine solution, a dehydrating condensing agent (2-chloro-5,5-dimethyl-1,3,2λ 5-2-oxaphosphorinane-2-one, etc.) to carry out a cyclization reaction. Relative to 1 mole of compound (6a'), 1 mole to an excess mole of a dehydrating condensing agent is used, and preferably 3 moles to 5 moles are used. The reaction time is 1 minute to 6 hours, and preferably 5 minutes to 1 hour. Then, water and an oxidizing agent (such as iodine) are added to the reaction solution to carry out an oxidation reaction. Relative to 1 mole of compound (6a'), 0 mole to an excess mole of water is used, and preferably 30 moles to 50 moles are used, and 2 moles to 10 moles of an oxidizing agent are used, and preferably 3 moles to 5 moles are used. The reaction time is 5 minutes to 12 hours, and preferably 30 minutes to 3 hours. The reaction solution is added to an aqueous sodium bicarbonate solution (0.1 M to 1 M), and stirred for 15 minutes to 24 hours to carry out a reaction stopping treatment. The reaction solution is extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixed solvent thereof), and the extracted solutions are combined and dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent is filtered off, and the filtrate is concentrated under reduced pressure. The obtained residue is 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').

[0441] Method A”

[0442] 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.

[0443]

[0444] This production method is a method for producing a compound represented by the general formula (1”) by changing a part of Method A. Specifically, the compound of the general formula (1”) can be produced by changing Step A-3 of Method A to Step A”-3 shown below. In addition, when substituents R x and R y are both halogen atoms, Step A-7 can be omitted.

[0445]

[0446] (Step A”-3)

[0447] This step is a step of producing the compound of formula (5a”) by continuously performing a coupling reaction of the compound of formula (3a”) with the compound of formula (4a”) and an oxidation reaction of the obtained coupling product using a known organic chemical method. Before starting the reaction of this step, the compound (4a”) is subjected to azeotropic distillation 3 to 5 times with dehydrated acetonitrile. Acetonitrile remains after the last azeotropic distillation, and a 0.01 M to 1 M acetonitrile solution of the compound (4a”) is prepared. A desiccant (powdered or lumped molecular sieve 3A or molecular sieve 4A) is added to this solution and stored under a nitrogen or argon atmosphere before using the solution. The coupling reaction is carried out by adding the acetonitrile solution of the compound (4a”) dried by azeotropic distillation to the acetonitrile solution of the compound (3a”) at 5°C to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Then, an oxidation reaction is carried out by adding an oxidizing agent (such as tert-butyl hydroperoxide) to the reaction solution. The amount of the oxidizing agent is 1 mole to 5 moles, preferably 2 moles to 3 moles, relative to 1 mole of the compound (3a”). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. An aqueous saturated sodium thiosulfate solution is added to the reaction solution, and the reaction is stopped by stirring for 10 minutes to 12 hours. The reaction solution is extracted 1 to 5 times with an organic solvent (such as a mixed solvent of dichloromethane and methanol), and the extracted solutions are combined and dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant is filtered off, and the filtrate is concentrated under reduced pressure to obtain a crude product of the compound (5a”). The obtained crude product of the compound (5a”) directly enters the next step.

[0448] Method A”’

[0449] 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.

[0450]

[0451] This production method is a method for producing the compound represented by the general formula (1”’) by changing a part of Method A. Specifically, the compound of the general formula (1”’) can be produced by changing Step A-3 of Method A to Step A”-3 and Step A-5 of Method A to Step A’-5. In addition, when both substituents R x and R y are halogen atoms, Step A-7 can be omitted.

[0452]

[0453] Method B: Coupling precursor (glycan coupling)

[0454] The coupling precursor represented by (2) used in the antibody-drug conjugate of the present invention can be produced according to Method B described below.

[0455]

[0456] This manufacturing method is a method for manufacturing a coupling precursor (2) in which -NH2 is substituted at an arbitrary position in L 1 The case of.

[0457]

[0458] (Step B-1)

[0459] This step is a step of manufacturing a compound of formula (2b) by removing a protecting group from the compound of formula (1b) using a known organic chemical method. In the case where PRO 5 is tert-butoxycarbonyl, the protecting group is removed by treating the compound (1b) with trifluoroacetic acid in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixed solvent thereof) at -10 °C to the boiling point of the solvent used in the reaction, preferably at 15 °C to 35 °C. An excess molar amount of trifluoroacetic acid is used relative to 1 mole of the compound (1b), preferably 20 moles to 50 moles. The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. After concentrating the reaction solution under reduced pressure, it is suspended in toluene and then concentrated under reduced pressure. This operation is repeated 2 to 5 times. After adding a solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate, or a mixed solvent thereof) to form a slurry, the solid is filtered to obtain a crude product of the compound (2b). The crude product of the compound (2b) is carried on to the next step without further purification.

[0460] (Step B-2)

[0461] This step is a step of producing the compound of formula (4b) by amidating the compound of formula (2b) with the compound of formula (3b) using a known organic chemical method. The amidation is carried out by reacting the compound (2b) with a base (triethylamine, N,N-diisopropylethylamine, etc.) and the compound (3b) in a solvent (N,N-dimethylformamide, N-methylpyrrolidone, N,N-dimethylacetamide, acetonitrile, etc.) at 5°C to 35°C. With respect to 1 mole of the compound (2b), 1 mole to 5 moles of the base and 0.5 mole to 1.5 moles of the compound (3b) are used. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction solution is poured into two layers of an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol or a mixed solvent thereof) and water or an acidic aqueous solution (0.1 to 1 M hydrochloric acid, citric acid aqueous solution, etc.), and extracted with the organic solvent 1 to 5 times. The extraction liquids are combined, washed with saturated brine, and then dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant is filtered off, and the filtrate is concentrated under reduced pressure. It should be noted that the above liquid separation operation can also be omitted, and the reaction solution can be directly concentrated under reduced pressure for the next silica gel column purification. The obtained residue is purified by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, etc.] to obtain the compound (4b). If necessary, the obtained compound (4b) can be dissolved in a good solvent (ethyl acetate, acetonitrile, dichloromethane, methanol or a mixed solvent thereof), and then a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) is added for reprecipitation, and the solid is filtered to improve the purity.

[0462] (Step B-3)

[0463] This step is a step of producing the compound of formula (5b) by esterifying the compound of formula (4b) using a known organic chemical method. Esterification is carried out by reacting compound (4b) with N-hydroxysuccinimide and a condensing agent (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at 5°C to 35°C. 1 to 3 moles of N-hydroxysuccinimide and the condensing agent are used respectively per mole of compound (4b). The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. The reaction solution is diluted with an organic solvent (dichloromethane, chloroform, ethyl acetate or a mixed solvent thereof), and then washed 3 to 5 times with ice water. The organic layer is dried using an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). After filtering off the desiccant, the filtrate is concentrated under reduced pressure to obtain the crude product of compound (5b). The obtained compound (5b) can be purified by C18 silica gel column chromatography [using only acetonitrile] as needed. In addition, the obtained compound (5b) can be dissolved in a good solvent (ethyl acetate, acetonitrile, dichloromethane or a mixed solvent thereof), and then a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) is added for reprecipitation, and the solid is filtered to improve the purity.

[0464] (Step B-4)

[0465] This step is a step of producing the compound of formula (2) by carrying out a condensation reaction of the compound of formula (5b) with the compound of formula (6b) using a known organic chemical method. The condensation reaction is 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 -10°C to 100°C, preferably at 15°C to 35°C. 2 to 5 moles of the base and 1 to 2 moles of compound (5b) are used per mole of compound (6b). The reaction time is 5 minutes to 24 hours, preferably 1 hour to 6 hours. Benzylamine is added to the reaction solution to terminate the reaction. 4 to 10 moles of benzylamine are used per mole of compound (6b). If necessary, the reaction solution is partially concentrated under reduced pressure, and the remaining solution is 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).

[0466] Method B': Coupling precursor (cysteine coupling)

[0467] The coupling precursor shown in (2') used in the antibody-drug conjugate of the present invention can be produced according to Method B' described below.

[0468]

[0469] This manufacturing method is for manufacturing a coupling precursor (2') in which -NH2 is substituted at any position of L 1

[0470]

[0471] (Step B-5)

[0472] This step is to manufacture the compound of formula (8b) by amidating the compound of formula (2b') with the compound of formula (7b) using a known organic chemical method. Except for not using a base, the compound (8b) is obtained according to the method described in Step B-2 of Method B.

[0473] (Step B-6)

[0474] This step is to manufacture the compound of formula (9b) by removing the protecting group from the compound of formula (8b) using a known organic chemical method. When PRO 6 is tert-butyl, except for using silica gel column chromatography [dichloromethane / methanol] in the purification operation, the compound (9b) is obtained according to the method described in Step B-1 of Method B.

[0475] (Step B-7)

[0476] This step is to manufacture the compound of formula (10b) by esterifying the compound of formula (9b) using a known organic chemical method. The compound (10b) is obtained according to the method described in Step B-3 of Method B.

[0477] (Step B-8)

[0478] This step is to manufacture the compound of formula (2') by carrying out a condensation reaction of the compound of formula (6b) with the compound of formula (10b) using a known organic chemical method. The compound (2') is obtained according to the method described in Step B-4 of Method B.

[0479] Method C

[0480] The coupling precursor shown in (3) used in the antibody-drug conjugate of the present invention can be manufactured according to Method C described below.

[0481]

[0482] This manufacturing method is for manufacturing a coupling precursor (3) in which a hydroxyl group is substituted at any position of L 1

[0483] ​​

[0484] (Step C-1)

[0485] This step is to produce the compound of formula (3c) by amidating the compound of formula (1c) with the compound of formula (2c) using a well-known organic chemistry method. The compound (3c) is obtained according to the method described in Step B-2 of Method B.

[0486] (Step C-2)

[0487] This step is to produce the compound of formula (4c) by esterifying the compound of formula (3c) using a well-known organic chemistry method. The compound (4c) is obtained according to the method described in Step B-3 of Method B.

[0488] (Step C-3)

[0489] This step is to produce the compound of formula (7c) by successively performing a coupling reaction (aminomethylenation) of the compound of formula (5c) with the compound of formula (6c) and deprotecting the resulting conjugate using a well-known organic chemistry method. In the case where PRO 9 is 9-fluorenylmethyloxycarbonyl, aminomethylenation is carried out by reacting the compound (5c) with the compound (6c) and an acid (such as p-toluenesulfonic acid) in tetrahydrofuran at 5 °C to 35 °C. With respect to 1 mole of the compound (5c), 1 mole to 20 moles of the compound (6c) are used, preferably 2 moles to 10 moles, and the acid is used in an amount of 0.05 mole to an excess amount, preferably 0.1 mole to 3 moles. The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. Then, a base (such as 1,8-diazabicyclo[5.4.0]-7-undecene) is added to the reaction solution to carry out deprotection. When the reaction solution is in suspension, a solvent (such as N,N-dimethylformamide) can be added as needed and dissolved before the reaction. With respect to 1 mole of the compound (5c), the base is used in an excess amount, preferably 5 moles to 20 moles. The reaction time is 10 minutes to 24 hours, preferably 2 hours to 12 hours. Water is added to the reaction solution, and purification is directly carried out by C18 silica gel column chromatography [buffer / acetonitrile, etc.] to obtain the compound (7c).

[0490] (Step C-4)

[0491] This step is to produce the compound of formula (8c) by removing the protecting group from the compound of formula (7c) using a well-known organic chemistry method. In the case where PRO 7 and PRO 8 are tert-butyldimethylsilyl, the compound (8c) is obtained according to the method described in Step A-7 of Method A.

[0492] (Step C-5)

[0493] This step is to produce the compound of formula (3) by subjecting the compound of formula (8c) to a condensation reaction with the compound of formula (4c) using a known organic chemical method. The compound (3) is obtained according to the method described in Step B-4 of Method B.

[0494] Method C’

[0495] The conjugate precursor shown in (3’) used in the antibody-drug conjugate of the present invention can be produced according to Method C’ described below.

[0496]

[0497] This production method is for producing the conjugate precursor (3’) substituted with a hydroxyl group at any position of L 1

[0498]

[0499] (Step C’-1)

[0500] This step is to produce the compound of formula (2c’) by subjecting the compound of formula (1c’) to a hydrolysis reaction and removal of the cyanoethyl group continuously using a known organic chemical method. The compound (2c’) is obtained according to the method described in Step A-1 of Method A.

[0501] (Step C’-2)

[0502] This step is to produce the compound of formula (3c’) by removing the protecting group of the hydroxyl group from the compound of formula (2c’) using a known organic chemical method. The compound (3c’) is obtained according to the method described in Step A-2 of Method A.

[0503] (Step C’-3)

[0504] This step is to produce the compound of formula (5c’) by subjecting the compound of formula (3c’) to a coupling reaction with the compound of formula (4c’) continuously and then subjecting the resulting conjugate to a sulfurization reaction or an oxidation reaction using a known organic chemical method. The compound (5c’) is obtained according to the method described in Step A-3 of Method A or Step A”-3 of Method A”.

[0505] (Step C’-4)

[0506] This step is to produce the compound of formula (6c’) by removing the protecting group of the hydroxyl group from the compound of formula (5c’) using a known organic chemical method. The compound (6c’) is obtained according to the method described in Step A-4 of Method A.

[0507] (Step C’-5)

[0508] This step is a step of manufacturing the compound of formula (7c’) by continuously performing a cyclization reaction and a sulfidation reaction or an oxidation reaction on the compound of formula (6c’) using a known organic chemical method. The compound (7c’) is obtained according to the method described in Step A-5 of Method A or Step A’-5 of Method A’.

[0509] (Step C’-6)

[0510] This step is a step of manufacturing the compound of formula (8c’) by simultaneously removing the cyanoethyl group and all acyl group protecting groups from the compound of formula (7c’) using a known organic chemical method. The compound (8c’) is obtained according to the method described in Step A-6 of Method A.

[0511] (Step C’-7)

[0512] This step is a step of manufacturing the compound of formula (9c’) by simultaneously removing all silyl group protecting groups from the compound of formula (8c’) using a known organic chemical method. In the case where PRO 9 is 2-(trimethylsilyl)ethoxycarbonyl, the compound (8c’) is treated with a solution of tetrabutylammonium fluoride in tetrahydrofuran at 5°C to 100°C, preferably at 35°C to 60°C, to remove 2-(trimethylsilyl)ethoxycarbonyl. An excess molar amount of tetrabutylammonium fluoride is used relative to 1 mole of the compound (8c’), and preferably 10 to 30 moles are used. The reaction time is 1 hour to 48 hours, preferably 4 hours to 24 hours. After adding a buffer solution to dilute the reaction solution, the organic solvent component is distilled off under reduced pressure as needed. The residue is 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 the compound (9c’).

[0513] (Step C’-8)

[0514] This step is a step of manufacturing the compound of formula (3’) by performing a condensation reaction of the compound of formula (9c’) with the compound of formula (4c) using a known organic chemical method. The compound (3’) is obtained according to the method described in Step B-4 of Method B.

[0515] Method D: Manufacture of Glycan-Reduced Antibodies

[0516] Glycan-reduced antibodies can be manufactured by the method shown in the following formula according to the method described in, for example, WO2018 / 003983, etc.

[0517]

[0518] (Step D-1)

[0519] In this step, a known enzymatic reaction is used on the target antibody, and the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing-terminal chitobiose structure of the N-linked glycan (N297-linked glycan) that binds to asparagine at the 297th position of the amino acid sequence of the antibody is cleaved by hydrolysis to produce a glycan-cleaved antibody. In a buffer (such as phosphate buffer), at 0°C to 40°C, a hydrolytic enzyme such as wild-type EndoS enzyme is used to perform a hydrolysis reaction on the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing-terminal chitobiose structure of the target antibody (1d) (10 mg / mL). The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. For 100 mg of antibody (1d), 0.1 mg to 10 mg of wild-type EndoS enzyme is used, preferably 0.1 mg to 3 mg. After the reaction is completed, purification is carried out using affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE HEALTH CARE)) and / or a hydroxyapatite column (Bio-Scale Mini CHT Type I tube (5 ml) (manufactured by BIO-RAD)) to obtain the (Fucα1,6)GlcNAc antibody (2d).

[0520] (Step D-2)

[0521] In this step, a known enzymatic reaction is used to bind an SG-type or MSG (MSG1, MSG2)-type glycan oxazoline body having a PEG linker containing an azide group (hereinafter referred to as "azide glycan oxazoline body") to the (Fucα1,6)GlcNAc antibody (2d) obtained in Step D-1 to produce a glycan-reconstituted antibody (3d).

[0522] In a buffer (such as phosphate buffer), at 0°C to 40°C, in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L), the antibody (2d) is reacted with the azide glycan oxazoline body to perform a glycan transfer reaction. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. For 100 mg of antibody, 1 mg to 10 mg of EndoS enzyme (D233Q / Q303L) is used, preferably 1 mg to 3 mg, and 2 equivalents to an excess equivalent of the azide glycan oxazoline body is used, preferably 4 equivalents to 20 equivalents. After the reaction is completed, purification is carried out using affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE HEALTH CARE)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I tube (5 ml) (manufactured by BIO-RAD)) to obtain the glycan-reconstituted antibody (3d).

[0523] In the preparation of the sugar chain - reconstructed antibody described above, concentration, concentration measurement, and buffer exchange of the antibody aqueous solution can be carried out according to the common operations A to C described below.

[0524] It should be noted that the azide - sugar chain oxazoline of the SG type is 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 formula.

[0525]

[0526] The azide - sugar chain oxazoline of the MSG type is 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 formula.

[0527]

[0528] Method E: Conjugation of antibody and drug (sugar chain conjugation 1)

[0529]

[0530] (Here, the two asterisks (*) on the left side of the antibody - drug conjugate (1e) represent the drug linker part indicated by the asterisk on the right side.)

[0531] This manufacturing method is a method for producing an antibody - drug conjugate (1e) by binding the sugar chain - reconstructed antibody (3d) obtained in the D - 2 step of Method D and the precursor conjugate (2) obtained in the B - 4 step of Method B through SPAAC (strain - promoted azide - alkyne cycloaddition: J. Am. Chem. Soc. 2004, 126, 15046 - 15047) reaction.

[0532] (Step E - 1)

[0533] The SPAAC reaction is carried out by mixing a buffer solution (phosphate buffer, acetate buffer, borate buffer, etc.) of the glycan - reconstructed antibody (3d) with a solution obtained by dissolving the coupling precursor (2) in a suitable solvent (dimethyl sulfoxide, N,N - dimethylformamide, N,N - dimethylacetamide, N - methylpyrrolidone, propylene glycol, or a mixed solvent thereof). With respect to 1 mole of the glycan - reconstructed antibody (3d), the coupling precursor (2) is 2 moles to an excess mole, preferably 4 moles to 30 moles. With respect to the buffer solution of the antibody, the ratio of the organic solvent is preferably 1% to 200% (v / v). The reaction temperature is 0°C to 37°C, preferably 15°C to 25°C, the reaction time is 1 hour to 150 hours, preferably 6 hours to 72 hours. The pH of the reaction solution is preferably 5 to 9. The reaction solution is purified according to the method described in the common operation D described below to obtain the antibody - drug conjugate (1e).

[0534] Method E': Conjugation of antibody and drug (cysteine conjugation)

[0535] The antibody - drug conjugate of the present invention having cysteine conjugation can be produced by using the target antibody prepared according to Reference Example 3, etc. and the coupling precursor (2') having a maleimide group obtained in Step B - 8 of Method B' according to the method described in WO2014 / 057687, etc.

[0536] Method E'': Conjugation of antibody and drug (glycan conjugation 2)

[0537] In Method E, the coupling precursor (2) is changed to the coupling precursor (3') obtained in Step C' - 8 of Method C' to obtain the antibody - drug conjugate (1e'') shown by the following formula.

[0538]

[0539] (Here, the two asterisks (* 1 ) on the left side of the antibody - drug conjugate (1e'') represent the drug linker part indicated by the asterisk on the right side.)

[0540] For the antibody - drug conjugate, buffer exchange, purification, determination of the antibody concentration, and determination of the average number of drug bindings per antibody molecule can be carried out through the common operations D - G described below to identify the antibody - drug conjugate.

[0541] Common operation A: Concentration of the antibody aqueous solution

[0542] An antibody or antibody-drug conjugate solution was added to an Amicon (registered trademark) Ultra centrifugal filter device (50000 NMWL, Merck Millipore Ltd.), and the antibody and antibody-drug conjugate solution was concentrated by centrifugation (centrifugation at 2000 G to 4000 G for 5 minutes to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

[0543] Common operation B: Determination of antibody concentration

[0544] Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific, Inc.), the antibody concentration was determined according to the method specified by the manufacturer. At this time, different extinction coefficients at 280 nm (1.3 mL / mg － 1 cm －1 ~1.8 mL / mg －1 cm －1 ) were used for each antibody.

[0545] Common operation C: Buffer exchange of antibody

[0546] A buffer solution (phosphate-buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to the aqueous antibody solution, and concentration was carried out according to the method described in Common operation A. After performing this operation several times, the antibody concentration was determined according to the method described in Common operation B. A buffer solution (phosphate-buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was appropriately added to this antibody buffer solution to prepare an antibody buffer solution with a target concentration (for example, about 10 mg / mL).

[0547] Common operation D: Purification of antibody-drug conjugate (gel filtration chromatography)

[0548] An NAP column (NAP-5, NAP-10, NAP-25 (manufactured by GE HEALTH CARE)) was equilibrated with an acetate buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5; referred to as ABS in this specification) or other appropriate buffer solution. The antibody-drug conjugate reaction solution was loaded into this NAP column, and the buffer solution specified by the manufacturer was allowed to flow down naturally, and the antibody component was fractionated. This component was loaded into the NAP column again, and the buffer solution specified by the manufacturer was allowed to flow down naturally, and the antibody component was fractionated. This operation was repeated a total of 2 to 3 times, whereby an antibody-drug conjugate from which unbound drug linker, dimethyl sulfoxide, and propylene glycol were removed was obtained. The concentration of the antibody-drug conjugate solution was adjusted by Common operation A and C as needed.

[0549] Common operation E: Determination of the antibody concentration of an antibody-drug conjugate and the average number of drug molecules bound per antibody molecule (UV method)

[0550] The conjugated drug concentration of the antibody-drug conjugate can be calculated by measuring the absorbances of the antibody-drug conjugate aqueous solution at two wavelengths, 280 nm and 250 nm, using an absorptiometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.), and then performing the following calculations. The total absorbance at a certain wavelength is equal to the sum of the absorbances of all the absorbing chemical species present in the system (additivity of absorbance). Therefore, if it is assumed that the molar extinction coefficients of the antibody and the drug do not change before and after the conjugation of the antibody and the drug, the antibody concentration and the drug concentration of the antibody-drug conjugate are as shown in the following relational expressions.

[0551] A 280 =A D , 280 +A A , 280 =ε D , 280 C D +ε A , 280 C A Equation (I)

[0552] A 250 =A D , 250 +A A , 250 =ε D , 250 C D +ε A , 250 C A Equation (II)

[0553] Here, A 280 represents the absorbance of the antibody-drug conjugate aqueous solution at 280 nm, A 250 represents the absorbance of the antibody-drug conjugate aqueous solution at 250 nm, A A , 280 represents the absorbance of the antibody at 280 nm, A A , 250 represents the absorbance of the antibody at 250 nm, A D , 280 represents the absorbance of the conjugate precursor at 280 nm, A D , 250 represents the absorbance of the conjugate precursor at 250 nm, ε A , 280Represents the molar extinction coefficient of the antibody at 280 nm, ε A , 250 Represents the molar extinction coefficient of the antibody at 250 nm, ε D , 280 represents the molar extinction coefficient of the conjugate precursor at 280 nm, ε D , 250 Represents the molar extinction coefficient of the conjugate precursor at 250 nm, C A Represents the antibody concentration in the antibody-drug conjugate, C D Represents the drug concentration in the antibody-drug conjugate. Here, ε A , 280 , ε A , 250 , ε D , 280 , ε D , 250 Pre-prepared values (calculated estimated values or measured values) can be used. For example, ε A , 280 Can be estimated from the amino acid sequence of the antibody by known calculation methods (Protein Science, 1995, vol.4, 2411-2423). ε A , 250 Use the value calculated from the measured value obtained by UV measurement of the antibody and the estimated value of ε A , 280 In the examples, the molar extinction coefficient of anti-TROP2 antibody 1 uses ε A , 280 = 223400 and ε A , 250 = 63482. The molar extinction coefficient of anti-TROP2 antibody 2 uses ε A , 280 = 223400 and ε A , 250 = 69027 or 71411. The molar extinction coefficient of anti-CD70 antibody 1 uses ε A , 280 = 226380 and ε A , 250 = 73432. The molar extinction coefficient of anti-CD70 antibody 2 uses ε A , 280 = 212400 and ε A , 250 = 72355. The molar extinction coefficient of anti-EGFR antibody 1 uses ε A , 280 = 203460 and ε A , 250= 62692. The molar extinction coefficient of anti-EGFR antibody 2 was determined using ε A , 280 = 217440 and ε A , 250 = 75731. ε D , 280 and ε D , 250 can be obtained by measuring the absorbance of a solution prepared by dissolving the conjugate precursor used into a certain molar concentration, according to the Lambert-Beer 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 each time. The A 280 and A 250 of the antibody-drug conjugate aqueous solution can be measured, and these values are substituted into equations (I) and (II) to solve the simultaneous equations to obtain C A and C D . Furthermore, the average number of drug molecules bound per antibody molecule can be obtained by dividing C D by C A .

[0554] Common operation F: Determination of the antibody concentration and the average number of drug molecules bound per antibody molecule in the antibody-drug conjugate (Reverse-phase high performance liquid chromatography: RP-HPLC)

[0555] The antibody concentration and the average number of drug molecules bound per antibody molecule in the antibody-drug conjugate can be obtained by high performance liquid chromatography analysis using the following method in addition to the above common operation E.

[0556] [F-1. Preparation of the sample for HPLC analysis (Reduction of the antibody-drug conjugate)]

[0557] Mix the antibody-drug conjugate solution (about 1 mg / mL, 60 μL) with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). Incubate the mixture at 37 °C for 30 minutes to cleave the disulfide bond between the L chain and the H chain of the antibody-drug conjugate. Use the reaction solution directly for HPLC analysis.

[0558] [F-2. HPLC analysis]

[0559] Typical analysis conditions are as follows.

[0560] HPLC system: Agilent 1290 HPLC system (Agilent Technologies)

[0561] Detector: UV spectrophotometer (measurement wavelength: 280 nm)

[0562] Column: Acquity BEH Phenyl (2.1×50 mm, 1.7 μm, manufactured by Waters)

[0563] Column temperature: 75 °C

[0564] Flow rate: 0.8 mL / min

[0565] Sample injection volume: 10 μL

[0566] Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropanol aqueous solution

[0567] Mobile phase B: 0.075% TFA, 15% isopropanol acetonitrile solution

[0568] Gradient program (mobile phase B): 14% - 36% (0 min - 15 min), 36% - 80% (15 - 17 min), 80% - 14% (17 min - 17.1 min), 14% - 14% (17.1 min - 23 min)

[0569] [F - 3. Data analysis]

[0570] [F - 3 - 1] In the case of sugar chain coupling under SPAAC reaction, for the L chain (L0) and H chain (H0) of the antibody without bound drug, the H chain with bound drug (H chain with one bound drug: H1, H chain with two bound drugs: H2) increases in hydrophobicity proportionally to the number of bound drugs and has a longer retention time. Therefore, in principle, they are eluted in the order of L0, H0, H1, H2. By comparing the retention times with L0 and H0, the detected peaks can be assigned to any one of L0, H0, H1, H2. The same is true for the case of cysteine coupling. The L chain with bound drug (L chain with one bound drug: L1) and the H chain with bound drug (H chain with one bound drug: H1, H chain with two bound drugs: H2, H chain with three bound drugs: H3) increase in hydrophobicity proportionally to the number of bound drugs and have a longer retention time. Therefore, in principle, they are eluted in the order of L0, L1, H0, H1, H2, H3. By comparing the retention times with L0 and H0, the detected peaks can be assigned to any one of L0, L1, H0, H1, H2, H3.

[0571] 〔F-3-2〕Since the drug linker has UV absorption, in the case of sugar chain coupling under SPAAC reaction, the peak area is corrected according to the following formula using the molar extinction coefficients of the H chain and the drug linker based on the number of bindings of the drug linker. In the case of cysteine coupling where the drug is also bound to the L chain, the peak area is corrected for the L chain in the same manner.

[0572]

[0573] Here, the molar extinction coefficients (280 nm) of the L chain and H chain of each antibody are the estimated values calculated using the known calculation methods described in the common operation E. In the case of anti-TROP2 antibody 1 and anti-TROP2 antibody 2, the molar extinction coefficient of the L chain is 27702 and that of the H chain is 83998. In the case of anti-CD70 antibody 1, the molar extinction coefficient of the L chain is 30222 and that of the H chain is 82968. In the case of anti-CD70 antibody 2, the molar extinction coefficient of the L chain is 30222 and that of the H chain is 75978. In the case of anti-EGFR antibody 1, the molar extinction coefficient of the L chain is 23232 and that of the H chain is 78498. In the case of anti-EGFR antibody 2, the molar extinction coefficient of the L chain is 30222 and that of the H chain is 78498. The molar extinction coefficient (280 nm) of the drug linker in the case of sugar chain coupling under SPAAC reaction uses the measured value of the coupling precursor, and in the case of cysteine coupling, it uses the measured value of the compound obtained by reacting the coupling precursor in mercaptoethanol or N-acetylcysteine to convert the maleimide group to succinimide thioether.

[0574] 〔F-3-3〕Calculate the peak area ratio (%) of each chain relative to the total peak area correction value according to the following formula.

[0575]

[0576] 〔F-3-4〕Calculate the average number of drug bindings per molecule of antibody (DAR) of the antibody-drug conjugate according to the following formula.

[0577]

[0578] 〔F-3-5〕Calculate the antibody concentration in the antibody-drug conjugate according to the following formula.

[0579]

[0580] Here, the absorbance (280 nm) of the antibody-drug conjugate is the measured value of the aqueous solution of the antibody-drug conjugate. The dilution factor indicates how many times the aqueous solution of the antibody-drug conjugate is diluted when measuring the absorbance, usually diluted 4 times. The molar extinction coefficient of the antibody (280 nm) uses the estimated value calculated by the known calculation method described in the common operation E. The average number of drug bindings uses the value obtained in [F-3-4]. The molar extinction coefficient of the drug linker (280 nm) in the case of sugar chain conjugation under SPAAC reaction uses the measured value of the coupling precursor, and in the case of cysteine conjugation, uses the measured value of the compound obtained by reacting the coupling precursor in mercaptoethanol or N-acetylcysteine to convert the maleimide group to succinimide thioether.

[0581] Common operation G: Determination of the antibody concentration in the antibody-drug conjugate and the average number of drug bindings per molecule of antibody (hydrophobic interaction - high performance liquid chromatography: HI-HPLC)

[0582] The antibody concentration in the antibody-drug conjugate and the average number of drug bindings per molecule of antibody can be determined by high performance liquid chromatography analysis using the following methods in addition to the above common operations E and F.

[0583] [G-1. Preparation of HPLC analysis sample]

[0584] The antibody-drug conjugate solution (about 1 mg / mL, 60 μL) is directly used for HPLC analysis.

[0585] [G-2. HPLC analysis]

[0586] Typical analysis conditions are as follows.

[0587] HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation)

[0588] Detector: Ultraviolet spectrophotometer (measurement wavelength: 280 nm)

[0589] Column: TSK-gel Butyl-NPR (4.6×100 mm, 2.5 μm, manufactured by TOSOH)

[0590] Column temperature: Fixed temperature around 25°C

[0591] Mobile phase A: 25 mM phosphate buffer (pH = 7.0) containing 1.5 M ammonium sulfate

[0592] Mobile phase B: 25 mM phosphate buffer (pH = 7.0) / isopropanol mixture (3:1)

[0593] Flow rate: 0.8 mL / min

[0594] Sample injection volume: 15 μL

[0595] Gradient program (mobile phase B): 10% - 15% (0 min - 5 min), 15% - 65% (5 min - 20 min)

[0596] Or, HPLC system: SHIMADZU CBM - 20A (Shimadzu Corporation)

[0597] Detector: UV spectrophotometer (measurement wavelength: 280 nm)

[0598] Column: PolyPROPYL A (4.6 × 100 mm, 3 μm, manufactured by PolyLC)

[0599] Column temperature: Fixed temperature near 40°C

[0600] Mobile phase A: 20 mM phosphate buffer containing 1.5 M ammonium sulfate (pH = 7.4)

[0601] Mobile phase B: 20 mM phosphate buffer (pH = 7.4)

[0602] Flow rate: 0.8 mL / min

[0603] Sample injection volume: 15 μL

[0604] Gradient program (mobile phase B): 40% - 80% (0 min - 20 min)

[0605] [G - 3. Data analysis]

[0606] [G - 3 - 1] The hydrophobicity increases proportionally to the number of drugs bound to the antibody, and the retention time becomes longer. Therefore, in the case of sugar chain conjugation under SPAAC reaction, in principle, it is eluted in the order of DAR = 0, DAR = 2, and DAR = 4. By comparing with the retention time of DAR = 0, the detected peak can be assigned to either DAR = 2 or DAR = 4. Sometimes, depending on the types 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 separating the peak by HI - HPLC.

[0607] [G - 3 - 2] Since the drug linker has UV absorption, the peak area value is corrected according to the following formula using the molar extinction coefficients of the antibody and the drug linker based on the number of bindings of the drug linker.

[0608]

[0609] Here, the molar extinction coefficient (280 nm) of the antibody is the estimated value calculated using the known calculation method described in Common Operation E. The molar extinction coefficient (280 nm) of the drug linker is the measured value of the coupling precursor.

[0610] 〔G-3-3〕Calculate the antibody peak area ratio (%) relative to the total corrected peak area according to the following formula.

[0611]

[0612] 〔G-3-4〕Calculate the average number of drugs bound per molecule of antibody of the antibody-drug conjugate according to the following formula.

[0613]

[0614] 〔G-3-5〕The antibody concentration in the antibody-drug conjugate is calculated according to the formula described in 〔F-3-5〕. At this time, the average number of drug bindings is the value obtained in 〔G-3-4〕.

[0615] The antibody-drug conjugate of the present invention or its production intermediate may sometimes have stereoisomers or optical isomers, geometric isomers, tautomers or optical isomers such as d-form, l-form, atropisomers, etc. from asymmetric carbon atoms. These isomers, optical isomers and their mixtures are all included in the present invention.

[0616] In the antibody-drug conjugate of the present invention, the number of drugs bound to one molecule of antibody is an important factor affecting its efficacy and safety. The production of the antibody-drug conjugate is carried out by specifying reaction conditions such as the usage amounts of raw materials / reagents of the reaction so that the number of drug bindings becomes a fixed number. However, different from the chemical reaction of low-molecular compounds, a mixture in which different amounts of drugs are bound is usually obtained. The number of drugs bound to one molecule of antibody can be determined by the average value, that is, the average drug binding number (DAR). The number of bindings of the cyclic dinucleotide derivative to the antibody molecule can be controlled. As the average number of drugs per antibody, cyclic dinucleotide derivatives in the range of 1 to 10 can be bound, preferably 1 to 8, more preferably 1 to 5.

[0617] In the antibody-drug conjugate of the present invention, when the antibody Ab binds to L through the reconstructed sugar chain of the antibody Ab, the number of drug bindings m per molecule of antibody of the antibody-drug conjugate 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 2is 2, and the 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 the 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).

[0618] It should be noted that those skilled in the art can design a reaction to bind an antibody to a desired number of drugs according to the description of the embodiments of the present application, and can obtain an antibody with the number of bound cyclic dinucleotide derivatives controlled.

[0619] It should be noted that the antibody-drug conjugate of the present invention or its manufacturing intermediate may absorb moisture, sometimes attach adsorbed water or form a hydrate by being placed in the atmosphere or recrystallized. Such hydrated compounds and salts are also included in the present invention.

[0620] When the antibody-drug conjugate of the present invention or its manufacturing intermediate has a basic group such as an amino group, it can be made into a pharmaceutically acceptable salt as needed. Examples of such salts include hydrohalic acid salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; aryl sulfonates 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 salt, glutamate salt, and aspartate salt.

[0621] Regarding the antibody-drug conjugate of the present invention, since its structure contains a phosphate group and / or a phosphorothioate group, it can usually form a base addition salt. In addition, when its manufacturing intermediate has an acidic group such as a carboxyl group, it can usually also form a base addition salt. 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; organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucosamine 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, etc.

[0622] The antibody-drug conjugates of the present invention and their manufacturing intermediates sometimes exist in the form of hydrates by absorbing moisture in the air or the like. As the solvates of the present invention, as long as they are pharmaceutically acceptable, there is no particular limitation. Specifically, hydrates, ethanolates, 2-propanolates, etc. are preferred. In addition, when a nitrogen atom is present in the antibody-drug conjugates of the present invention and their manufacturing intermediates, N-oxides can be formed, and these solvates and N-oxides are also included within the scope of the present invention. In addition, when a sulfur atom is present in the antibody-drug conjugates of the present invention and their manufacturing intermediates, sulfoxides can also be formed, and these solvates and sulfoxides are also included within the scope of the present invention.

[0623] In addition, the present invention also includes compounds labeled with various radioactive or non-radioactive isotopes. One or more of the atoms constituting the antibody-drug conjugates of the present invention and their manufacturing intermediates may also contain an unnatural proportion of atomic isotopes. Examples of atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), or carbon-14 (14C), etc. In addition, the compounds of the present invention may also be radioactively labeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or carbon-14 (14C). The radioactively labeled compounds are useful as therapeutic or prophylactic agents, research reagents (such as analytical reagents), and diagnostic agents (such as in vivo diagnostic imaging agents). All isotope variants of the antibody-drug conjugates of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0624] <4. Medicine>

[0625] The antibody-drug conjugates of the present invention exhibit anti-tumor immune activity or cytotoxicity against cancer cells, and thus can be used as medicines, particularly as therapeutic and / or prophylactic agents for cancer, or anti-tumor agents.

[0626] Examples of cancers for which the antibody-drug conjugate of the present invention is applicable include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumors, stromal tumors, germ cell tumors, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumors, head and neck cancers, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, pharyngeal cancer, tongue cancer, auditory organ cancer, thymic cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, etc. Regarding the antibody-drug conjugate, as long as it is a cancer cell expressing a protein that can be recognized by the antibody in the antibody-drug conjugate as a treatment target, it is not limited thereto.

[0627] The antibody-drug conjugate of the present invention can be suitably administered to mammals, and mammals are more preferably humans.

[0628] As a substance used in the pharmaceutical composition containing the antibody-drug conjugate of the present invention, in terms of the dosage and administration concentration, it can be appropriately selected and used from the formulation additives commonly used in the art.

[0629] The antibody-drug conjugate of the present invention can be administered in the form of a pharmaceutical composition containing one or more pharmaceutically compatible components. For example, the above-mentioned pharmaceutical composition typically contains one or more pharmaceutical carriers (e.g., sterilized liquids (e.g., containing water and oils (derived from petroleum, animals, plants, or synthetic oils (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)))). In the case of intravenous administration of the above-mentioned pharmaceutical composition, water is a more representative carrier. Saline solution, as well as glucose aqueous solution and glycerol aqueous solution, can also be used as liquid carriers, and can be particularly used for injection solutions. Appropriate pharmaceutical excipients are well-known in the art. If necessary, the above composition may also contain trace amounts of wetting agents or emulsifiers, or pH buffering agents. Examples of appropriate pharmaceutical carriers are described in "Remington’s Pharmaceutical Sciences" by E.W. Martin. Its formulation corresponds to the administration method.

[0630] A variety of delivery systems are known and can be used for the administration of the antibody-drug conjugates of the present invention. As the introduction methods, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes can be mentioned, but are not limited thereto. The administration can be carried out, for example, by injection or bolus injection. In a specific preferred embodiment, the administration of the above antibody-drug conjugate is carried out by injection. Parenteral administration is the preferred route of administration.

[0631] In a representative embodiment, the pharmaceutical composition comprising the above antibody-drug conjugate is formulated according to conventional procedures to form a pharmaceutical composition suitable for intravenous administration to humans. The composition typically used for intravenous administration is a solution in a sterile, isotonic aqueous buffer. If necessary, the above medicine may also contain a solubilizer and a local anesthetic (such as lidocaine) for alleviating the pain at the injection site. Generally, the above components are separately supplied, for example, in the form of a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or a sachet showing the amount of the active agent, or are supplied together in a unit dosage form. In the case where the above pharmaceutical composition is administered by injection, it can be administered, for example, through an injection bottle containing sterile pharmaceutical-grade water or saline. In the case of administering the above medicine by injection, an ampoule of sterile water for injection or saline can be provided, for example, in such a manner that the above components can be mixed before administration. The above pharmaceutical composition is sometimes provided in the form of a solution.

[0632] 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 other cancer therapeutic agents. The antibody-drug conjugate of the present invention can be administered together with or in combination with other cancer therapeutic agents, thereby enhancing the anti-tumor effect. Other cancer therapeutic agents used for such a purpose can be administered to an individual simultaneously with, separately from, or continuously with the antibody-drug conjugate, or the administration intervals of each can be changed for administration. As such cancer therapeutic agents,Examples include: chemotherapeutic agents such as metabolic antagonists, alkylating agents, and microtubule inhibitors (albumin-bound paclitaxel (abraxane), carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, vinblastine, or the agents described in WO2003 / 038043 pamphlet, etc.), hormone regulators (leuprorelin, etc. as LH-RH analogs, goserelin, estramustine, tamoxifen, raloxifene, etc. as estrogen antagonists), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, molecular target drugs (anti-EGFR antibodies, anti-VEGF antibodies, anti-VEGFR antibodies, etc.), immune checkpoint inhibitors (nivolumab, pembrolizumab, etc. as anti-PD-1 antibodies, atezolizumab, avelumab, durvalumab, etc. as anti-PD-L1 antibodies, anti-PD-L2 antibodies, ipilimumab, etc. as anti-CTLA4 antibodies, 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.), immune modulators (anti-CD47 antibodies, anti-SIRPα antibodies, inhibitory myeloid regulators, etc.), antibody drugs with ADCC (Antibody Dependent Cellular Cytotoxicity) activity, ADCP (Antibody Dependent Cellular Phagocytosis) activity, or complement activity, BiTE (Bi-specific T-cell engagers), Antibody-Drug-Conjugate (ADC) (for example,Drug conjugates containing Deruxtecan, DM1, Pyrrolobenzodiazepine, MMAF, etc. (anti-HER2-ADC, anti-TROP2-ADC, anti-HER3-ADC, etc.), ADCs combined with photodynamic therapy, etc., as well as anti-tumor vaccines, anti-tumor cell therapies (CAR-T, TCR-T, dendritic cells, NK cells, etc.), anti-tumor bacterial therapies, anti-tumor viral therapies, etc. are not limited as long as they are agents with anti-tumor activity. Furthermore, the antibody-drug conjugate of the present invention can also be administered together with other antibody-drug conjugates other than the present invention, thereby enhancing the anti-tumor effect. In addition, the antibody-drug conjugate of the present invention can enhance the anti-tumor effect not only by combining with drugs, but also by combining with treatments that bring anti-tumor effects, such as radiation, heavy particle radiation, surgery, bone marrow transplantation, etc., and there is no limitation as long as it is a treatment with anti-tumor effects.

[0633] Such a pharmaceutical composition can be formulated into a freeze-dried preparation or a liquid preparation as a preparation with the selected composition and required purity. When formulating into a freeze-dried preparation, it can be a preparation containing appropriate formulation additives used in the art. In addition, similarly, the liquid preparation can be formulated into a liquid preparation containing various formulation additives used in the art.

[0634] The composition and concentration of the pharmaceutical composition also vary depending on the administration method. Regarding the antibody-drug conjugate contained in the pharmaceutical composition of the present invention, in terms of the affinity of the antibody-drug conjugate for the antigen, that is, the dissociation constant (Kd value) for the antigen, the higher the affinity (the lower the Kd value), the more effective can be exerted even with a small dosage. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can also be set based on the affinity status of the antibody-drug conjugate for the antigen. When the antibody-drug conjugate of the present invention is administered to humans, for example, it can be administered once at about 0.001 to 100 mg / kg or administered multiple times at intervals of 1 to 180 days.

[0635] The present invention will be described below by way of examples, but the present invention is not limited thereto.

[0636] Examples

[0637] In the following examples, room temperature means 15°C to 35°C. Dehydrated acetonitrile used was acetonitrile (dehydrated)-Super- sold by Kanto Chemical or acetonitrile (ultra-dehydrated) sold by Wako Pure Chemical Industries, Ltd. Pyridine used was pyridine (dehydrated)-Super- sold by Kanto Chemical. Silica gel chromatography was carried out using Biotage SNAP Ultra (manufactured by Biotage), Chromatorex Q-PackSI (manufactured by Fuji Silysia), or Purif-Pack-Ex SI (manufactured by Shoko Science). DIOL silica column chromatography was carried out using Chromatorex Q-pack DIOL (manufactured by Fuji Silysia). C18 silica column chromatography was carried out using BiotageSNAP Ultra C18 (manufactured by Biotage). Elution of column chromatography was carried out under observation based on thin layer chromatography (TLC). 0.1% triethylamine for the elution solvent means that 0.1% of triethylamine is contained in the total volume of the elution solvent. Preparative HPLC was carried out using a SHIMADZU SPD-M10A HPLC system (manufactured by Shimadzu Corporation), etc. The preparative column used was Kinetex (5μm, C18, 250×30.0mm, manufactured by Phenomenex) or Kinetex (5μm, C18, 250×21.2mm, manufactured by Phenomenex).

[0638] The measurement of various spectral data was carried out using the following equipment. 1 1H-NMR spectra were measured using JEOL ECS-400 (400 MHz), Varian 400-MR (400 MHz), or Varian Unity Inova 500 (500 MHz). 31 31P-NMR spectra were measured using JEOL ECS-400 (160 MHz). Mass spectrometry was carried out using an Agilent 6130 Quadrupole LC / MS system (Agilent Technologies). The LC / MS measurement was carried out under the following conditions [column: Develosil Combi-RP, 5μm, 50×2.0mm (manufactured by Nomura Chemical), mobile phase: 0.1% formic acid acetonitrile solution / 0.1% formic acid aqueous solution, 0.1% formic acid acetonitrile solution: 2% - 100% (0 minutes - 5 minutes or 0 minutes - 10 minutes)].

[0639] Example 1: Synthesis of CDN6

[0640] (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-epoxy-1,6-dihydro-9H-purin-9-yl]-2,10-bis(mercapto)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenz[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]pentaoxadiphosphetacyclotetradecine-2,10-dione

[0641]

[0642] [Synthetic Route]

[0643]

[0644]

[0645] (Step 1)

[0646] 7-{2-O-[tert-Butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylene)-β-D-ribofuranosyl}-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine

[0647] To a solution of 5-iodotubercidin (1.0 g) in N,N-dimethylformamide (10 mL), which is known in the literature (Tetrahedron 2007, 63, 9850 - 9861), di-tert-butylsilyl bis(trifluoromethanesulfonate) (1.24 mL) was slowly added dropwise at 0 °C, and then the mixture was stirred at the same temperature for 30 minutes. Imidazole (868 mg) was added at 0 °C, and the temperature was then raised to room temperature and stirred for 30 minutes. tert-Butyldimethylchlorosilane was added at room temperature, and the mixture was stirred overnight at the same temperature. Saturated aqueous sodium bicarbonate was added to the reaction solution to stop the reaction, and then extraction was carried out with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was filtered off, 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).

[0648] MS(ESI) m / z: 647(M + H) + .

[0649] 11H-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, 4.8 Hz), 4.17 (1H, m), 4.00 (1H, t, J = 9.7 Hz), 1.09 (9H, s), 1.04 (9H, s), 0.91 (9H, s), 0.13 (3H, s), 0.11 (3H, s).

[0650] (Step 2)

[0651] 7-{2-O-[tert-Butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylene)-β-D-ribofuranosyl}-5-(3,3-diethoxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine

[0652] To a mixed solution of N,N-dimethylformamide (3.0 mL) - tetrahydrofuran (9.0 mL) of the compound (910 mg) obtained in the above Step 1, sequentially add propargylaldehyde dimethyl acetal (1.01 mL), triethylamine (0.392 mL), tetrakis(triphenylphosphine)palladium(0) (163 mg), and copper(I) iodide (53.6 mg), and stir at 40 °C for 18 hours. Add saturated aqueous sodium bicarbonate solution and ethyl acetate to the reaction solution, and extract with ethyl acetate. Wash the organic layer with saturated brine, and dry over anhydrous sodium sulfate. Filter off the desiccant, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (878 mg).

[0653] MS(ESI) m / z: 647 (M + H) + .

[0654] 1 1H-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, 4.9 Hz), 4.42 (1H, d, J = 4.9 Hz), 4.25 (1H, dd, J = 9.4, 4.6 Hz), 4.17 (1H, m), 4.00 (1H, t, J = 9.7 Hz), 3.85 - 3.77 (2H, m), 3.66 (2H, m), 1.28 (6H, t, J = 7.3 Hz), 1.08 (9H, s), 1.04 (9H, s), 0.91 (9H, s), 0.13 (3H, s), 0.11 (3H, s).

[0655] (Step 3)

[0656] 2-{2-O-[tert-Butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylene)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0657] To a solution of the compound (878 mg) obtained in the above Step 2 in ethanol (8.8 mL) was added 10% palladium on carbon (M) wet (500 mg), and the mixture was stirred under a hydrogen atmosphere at room temperature for 9 hours. After filtering off the catalyst, the residue was washed with dichloromethane and the filtrate was concentrated under reduced pressure. To a solution of the residue in acetic acid (8.8 mL) was added 10% palladium on carbon (M) wet (500 mg), and the mixture was stirred under a hydrogen atmosphere at 40 °C for 2 days. After filtering off the catalyst, the residue 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).

[0658] MS(ESI) m / z: 561 (M+H) + .

[0659] 1 1H-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, 4.8 Hz), 4.15 (1H, m), 3.99 (1H, t, J = 9.7 Hz), 3.55 (2H, m), 2.89 (2H, t, J = 5.4 Hz), 2.04 (2H, m), 1.09 (9H, s), 1.04 (9H, s), 0.90 (9H, s), 0.10 (3H, s), 0.10 (3H, s).

[0660] (Step 4)

[0661] 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylene)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0662] To a solution of the compound (2.17 g) obtained in the above step 3 in dichloromethane (21.7 mL), pyridine (1.56 mL), N,N-dimethylaminopyridine (94.5 mg), and benzoyl chloride (0.898 mL) were successively added at room temperature, and the mixture was stirred at 50 °C for 15 hours. Saturated aqueous sodium bicarbonate was added to the reaction solution to stop the reaction. After extraction with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The desiccant was filtered off, 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).

[0663] MS(ESI) m / z: 665 (M+H) + .

[0664] 1 1H-NMR (CDCl3) δ: 8.08 (1H, s), 7.37 - 7.33 (3H, m), 7.23 (2H, t, J = 7.6 Hz), 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 = 10.0 Hz), 3.03 (2H, t, J = 6.3 Hz), 2.29 - 2.17 (2H, m), 1.10 (9H, s), 1.05 (9H, s), 0.90 (9H, s), 0.10 (6H, s).

[0665] (Step 5)

[0666] 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

[0667] To a solution of the compound (1.91 g) obtained in the above step 4 in dichloromethane (15 mL), a mixture of the prepared hydrogen fluoride - pyridine (0.30 mL) and pyridine (1.88 mL) was added at 0 °C, and the mixture was stirred at 0 °C for 2 hours. Saturated aqueous sodium bicarbonate was added to the reaction solution to stop the reaction. The reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in pyridine (15 mL), 4,4'-dimethoxytriphenylmethyl chloride (1.17 g) was added, and the mixture was stirred at 0 °C for 12 hours. Methanol was added and stirred for 30 minutes, and then saturated aqueous sodium bicarbonate was added to stop the reaction. The reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was filtered off, 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 (1.98 g).

[0668] MS(ESI) m / z: 827 (M + H) + .

[0669] 1 1H-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 = 5.5 Hz), 4.75 (1H, t, J = 5.2 Hz), 4.38 - 4.20 (4H, m), 3.80 (6H, s), 3.53 (1H, dd, J = 10.7, 2.8 Hz), 3.40 (1H, dd, J = 11.0, 3.1 Hz), 2.83 (1H, d, J = 3.7 Hz), 2.78 (2H, t, J = 6.4 Hz), 2.17 (2H, m), 0.81 (9H, s), -0.03 (3H, s), -0.21 (3H, s).

[0670] (Step 6)

[0671] 6-Benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyanoethoxy)[bis(propane-2-yl)amino]phosphino}-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0672] To a solution of the compound (1.98 g) obtained in the above step 5 in dichloromethane (23.9 mL) were added N,N-diisopropylethylamine (1.02 mL) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.07 mL), and the mixture was stirred at room temperature for 15 hours. The reaction was stopped by adding saturated aqueous sodium bicarbonate to the reaction mixture. The reaction mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was filtered off, 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 (2.06 g) as a mixture of diastereomers on the phosphorus atom (diastereomer ratio = 7:3).

[0673] MS(ESI) m / z: 1027 (M+H) + .

[0674] 1 1H-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 = 6.7 Hz), 6.31 (0.3H, d, J = 6.1 Hz), 4.84 (0.7H, dd, J = 7.0, 4.6 Hz), 4.78 (0.3H, t, J = 5.8 Hz), 4.43 - 4.17 (4H, m), 4.04 - 3.85 (1.3H, m), 3.80 - 3.76 (6H, m), 3.69 - 3.43 (3H, m), 3.50 (0.7H, dd, J = 10.6, 3.3 Hz), 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 = 6.7 Hz), 2.23 - 2.11 (2H, m), 1.21 - 1.13 (7.8H, m), 1.04 (4.2H, d, J = 6.7 Hz), 0.73 (2.7H, s), 0.72 (6.3H, s), -0.03 (0.9H, s), -0.06 (2.1H, s), -0.24 (3H, s).

[0675] (Step 7)

[0676] 6-benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[hydroxy(bridging oxy)-λ 5 -phosphino]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0677] To a solution of the compound (935 mg) obtained in the above step 6 in acetonitrile (4.55 mL) were added water (33 μL) and pyridinium trifluoroacetate (229 mg), and the mixture was stirred at room temperature for 15 minutes. To the reaction solution was added tert-butylamine (4.55 mL), and the mixture was stirred at room temperature for 15 minutes. After concentrating the reaction solution under reduced pressure, the residue was azeotroped twice with acetonitrile (5 mL). To a solution of the residue in dichloromethane (11.4 mL) was added water (0.164 mL), and then a solution of dichloroacetic acid (0.651 mL) in dichloromethane (11.4 mL) was added, and the mixture was stirred at room temperature for 15 minutes. After adding pyridine (1.25 mL) to stop the reaction, the reaction solution was concentrated under reduced pressure. The residue was azeotroped three times with dehydrated acetonitrile (10 mL), and about 5 mL of acetonitrile remained in the last azeotropy. The obtained acetonitrile solution of the title compound was directly used in the following step 12.

[0678] (Step 8)

[0679] 2’,3’,5’-Tri-O-acetyl-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine

[0680] To a suspension of commercially available (Ark Pharm) 2’,3’,5’-tri-O-acetylinosine (10.0 g) in tetrahydrofuran (100 mL) were added 2-{[tert-butyl(dimethyl)silyl]oxy}ethan-1-ol (5.37 g) and triphenylphosphine (7.69 g), and then diisopropyl (E)-diazene-1,2-dicarboxylate (6.10 mL) was added, and the mixture was stirred at room temperature for 6 hours. After concentrating the reaction solution under reduced pressure, the residue was purified by silica gel column chromatography [hexane / ethyl acetate / dichloromethane] to obtain the title compound in the form of a mixture with triphenylphosphine oxide (10.6 g).

[0681] MS(ESI) m / z: 553 (M+H) + .

[0682] 1 1H-NMR (CDCl3) δ: 8.05 (1H, s), 7.92 (1H, s), 6.12 (1H, d, J = 5.4 Hz), 5.86 (1H, t, J = 5.4 Hz), 5.59 (1H, dd, J = 5.4, 4.2 Hz), 4.47 - 4.41 (2H, m), 4.38 - 4.31 (1H, m), 4.22 - 4.17 (2H, m), 3.89 (2H, t, J = 4.8 Hz), 2.15 (3H, s), 2.14 (3H, s), 2.08 (3H, s), 0.83 (9H, s), -0.06 (3H, s), -0.06 (3H, s).

[0683] (Step 9)

[0684] 5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine

[0685] Potassium carbonate (150 mg) was added to a mixed solution of tetrahydrofuran (30 mL) and methanol (30 mL) of the compound (10.6 g) obtained in the above Step 8, and the mixture was stirred at room temperature for 3 hours. Acetic acid (125 μL) was added to the reaction solution, and after concentration under reduced pressure, the residue was azeotroped with pyridine. To a pyridine (60 mL) solution of the residue, 4,4'-dimethoxytrityl chloride (6.50 g) was added at 0 °C, and after stirring for 30 minutes, it was stored in a refrigerator overnight. Methanol (2 mL) was added to the reaction solution, and after stirring for 30 minutes, it 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 in the form of a mixture with triphenylphosphine oxide (7.21 g).

[0686] MS(ESI) m / z: 729 (M+H) + .

[0687] 1 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 = 5.4 Hz), 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 = 4.5 Hz), 3.77 (6H, s), 3.42 (1H, dd, J = 10.3, 3.6 Hz), 3.34 (1H, dd, J = 10.3, 3.6 Hz), 3.10 (1H, brs), 0.83 (9H, s), -0.06 (3H, s), -0.07 (3H, s).

[0688] (Step 10)

[0689] 5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine

[0690] To a solution of the compound (7.21 g) obtained in the above step 9 in dichloromethane (36 mL) were added imidazole (1.41 g) and tert-butyl(chloro)dimethylsilane (1.49 g), and the mixture was stirred at room temperature for 16 hours. Saturated aqueous sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, 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 (2.17 g) and the positional isomer of the title compound, i.e., 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).

[0691] MS(ESI) m / z: 843 (M+H) + .

[0692] 1 H-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(4H, m), 5.96(1H, d, J = 4.2 Hz), 4.56−4.50(2H, m), 4.33−4.25(1H, m), 4.19−4.02(2H, m), 3.89(2H, t, J = 4.8 Hz), 3.78(6H, s), 3.45(1H, dd, J = 10.9, 4.2 Hz), 3.27(1H, dd, J = 10.9, 4.2 Hz), 3.03(1H, d, J = 6.0 Hz), 0.88(9H, s), 0.82(9H, s), 0.07(3H, s), −0.01(3H, s), −0.07(3H, s), −0.07(3H, s).

[0693] Positional isomer (2’-O-TBS form)

[0694] MS(ESI) m / z: 843 (M+H) + .

[0695] 11H-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), 5.99 (1H, d, J = 5.4 Hz), 4.83 (1H, t, J = 5.1 Hz), 4.33 - 4.29 (1H, m), 4.27 - 4.24 (1H, m), 4.24 - 4.12 (2H, m), 3.90 (2H, t, J = 4.5 Hz), 3.79 (3H, s), 3.78 (3H, s), 3.48 (1H, dd, J = 10.3, 3.0 Hz), 3.40 (1H, dd, J = 10.3, 3.0 Hz), 2.71 (1H, d, J = 3.6 Hz), 0.86 (9H, s), 0.83 (9H, s), 0.01 (3H, s), -0.07 (3H, s), -0.07 (3H, s), -0.11 (3H, s).

[0696] (Step 11)

[0697] 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)[bis(propane-2-yl)amino]phosphino}inosine

[0698] To a solution of the compound (2.17 g) obtained in the above step 10 in dichloromethane (25.7 mL) were added 4,5-dicyanoimidazole (334 mg) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.980 mL), and the mixture was stirred at room temperature for 16 hours. The reaction was stopped 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 filtered off, 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) in the form of a mixture of diastereomers on the phosphorus atom.

[0699] MS (ESI) m / z: 1043 (M + H) + .

[0700] 11H-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 = 4.2 Hz), 6.05 (0.47H, d, J = 4.2 Hz), 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 = 6.0 Hz), 2.43 (1.06H, t, J = 6.7 Hz), 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).

[0701] (Step 12)

[0702] The compound (950 mg) obtained in the above Step 11 was azeotroped three times with dehydrated acetonitrile (5 mL), and about 3 mL of acetonitrile remained after the last time. Molecular sieve 3A, 1 / 16 (5 lumps) was added. This acetonitrile solution was added to the acetonitrile solution prepared in the above Step 7, and the mixture was stirred for 20 minutes under a nitrogen atmosphere at room temperature. N,N-Dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine (206 mg) was added to the reaction solution, and after stirring at room temperature for 30 minutes, the reaction solution was concentrated under reduced pressure. After adding water (0.164 mL) to a dichloromethane (13.0 mL) solution of the residue, a dichloromethane (13.0 mL) solution of dichloroacetic acid (0.822 mL) was added, and the mixture was stirred at room temperature for 15 minutes. After adding pyridine (9.01 mL) to the reaction solution to stop the reaction, it was concentrated under reduced pressure. The obtained crude product was directly used in the next reaction.

[0703] (Step 13)

[0704] 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-bridged oxy-1,6-dihydro-9H-purin-9-yl]-2-bridged oxy-2-mercapto-10-thione octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradec-10-yl}oxy)propanenitrile

[0705] After concentrating the pyridine (27.1 mL) solution of the crude product obtained in the above step 12 to about 20 mL, 2-chloro-5,5-dimethyl-1,3,2λ 5 -dioxaphosphinane-2-one (622 mg) was added, and the mixture was 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 solution, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was poured into an aqueous solution (130 mL) of sodium bicarbonate (3.60 g), stirred at room temperature for 30 minutes, and then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, 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) in the form of a mixture of diastereoisomers on the phosphorus atom.

[0706] MS(ESI) m / z: 1274 (M + H) + .

[0707] (Step 14)

[0708] (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-bridged oxy-1,6-dihydro-9H-purin-9-yl]-2,10-dibridged oxy-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]pentaoxadiphosphacyclotetradec-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[0709] 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. After concentrating the reaction mixture, the residue was purified by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] to obtain diastereomer 1 (88.5 mg: containing impurities) and diastereomer 2 (70.7 mg: containing impurities) of the title compound.

[0710] Diastereomer 1 (low polarity)

[0711] MS (ESI) m / z: 1003 (M−C6H 15 Si+2H) + .

[0712] Diastereomer 2 (high polarity)

[0713] MS (ESI) m / z: 1003 (M−C6H 15 Si+2H) + .

[0714] (Step 15-1)

[0715] (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-Dihydroxy-7-[1-(2-hydroxyethyl)-6-epoxy-1,6-dihydro-9H-purin-9-yl]-2,10-epoxy-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]pentaoxadiphosphecyclotetradecyne-2,10-bis(thiol) disodium

[0716] (Diastereomer 1)

[0717] To the compound (diastereomer 1) (88.5 mg: containing impurities) obtained in Step 14 above was added triethylamine trihydrofluoride (2.0 mL), and the mixture was stirred at 45 °C for 3 hours. To the reaction mixture at room temperature was added a mixture of ice-cold 1 M triethylammonium bicarbonate aqueous solution (10 mL) and triethylamine (2 mL). After concentrating the reaction mixture under reduced pressure, it was purified by 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-40 min)]. The resulting compound (triethylamine salt) was converted to the sodium salt by the following method.

[0718] [Conversion to sodium salt]

[0719] Suspend 500 mg of BT AG (registered trademark) 50W-X2 resin (biotechnology grade, 100-200 mesh, hydrogen form) in pure water and fill it into an empty column. After allowing the excess pure water to flow down naturally, sequentially allow 5 mL of 1 M sodium hydroxide aqueous solution and 10 mL of pure water to flow down naturally. Dissolve the above-obtained compound in 5 mL of pure water and load it into the column. After collecting the solution that flows down naturally, further elute with 10 mL of pure water. Combine the fractions containing the target compound and perform lyophilization to obtain the title compound (25.7 mg).

[0720] MS (ESI) m / z: 775 (M+H) + .

[0721] 1 1H-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), 4.78 - 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).

[0722] 31 31P-NMR (CD3OD) δ: 57.8 (s), 54.4 (s).

[0723] (Step 15-2)

[0724] (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-epoxy-1,6-dihydro-9H-purin-9-yl]-2,10-diepoxy-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]pentaoxadiphosphetacyclotetradecine-2,10-bis(thiol) disodium

[0725] (Diastereomer 2)

[0726] The compound (diastereomer 2) (70.7 mg: containing impurities) obtained by the above-mentioned step 14 was reacted in the same manner as in the above-mentioned step 15-1, and then purified according to the following [Purification Conditions] to obtain the title compound in the form of a triethylamine salt.

[0727] [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 minutes - 40 minutes)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 15% - 70% (0 minutes - 40 minutes)].

[0728] The obtained triethylamine salt was subjected to salt exchange in the same manner as [Conversion to Sodium Salt] described in the above-mentioned step 15-1 to obtain the title compound (17.8 mg).

[0729] MS (ESI) m / z: 775 (M + H) + .

[0730] 1 1H-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, 7.6 Hz), 5.48 - 5.39 (2H, m), 4.78 (1H, dd, J = 6.7, 4.2 Hz), 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).

[0731] 31 31P-NMR (CD3OD) δ: 62.9 (s), 60.0 (s).

[0732] Example 2: Synthesis of CDN34

[0733] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-15-Fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-epoxy-1,6-dihydro-9H-purin-9-yl]-2,10-bis(mercapto)-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]pentaoxadiphosphetacyclotetradecine-2,10-dione

[0734]

[0735] [Synthetic Route]

[0736]

[0737]

[0738] (Step 1)

[0739] 1-[2-(Benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine

[0740] To a solution of commercially available (Tokyo Chemical Industry) inosine (10.0 g) in pyridine (50 mL) and N,N-dimethylacetamide (50 mL) at 0 °C was added 4,4'-dimethoxytriphenylmethyl chloride (15.2 g), and the mixture was stirred at 4 °C for 64 hours. Methanol (2 mL) was added to the reaction solution, and after stirring for 10 minutes, the mixture was concentrated to about 50 mL. To the residue were added 2-bromoethyl benzoate (7.02 mL) and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (13.9 mL), and the mixture was stirred at room temperature for 1 day. Saturated aqueous sodium bicarbonate solution and water were added to the reaction solution, and extraction was carried out with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was filtered off, 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).

[0741] MS(ESI) m / z: 719 (M+H) + .

[0742] 1H-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 = 5.4 Hz), 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, 3.9 Hz), 3.34 (1H, dd, J = 10.6, 3.6 Hz).

[0743] (Step 2)

[0744] 1-[2-(Benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]inosine

[0745] Using the compound (3.01 g) obtained in the above Step 1, it was synthesized in the same manner as in Step 10 of Example 1 to obtain the title compound (1.20 g) and the positional isomer of the title compound, i.e., 1-[2-(benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]inosine (1.22 g).

[0746] MS (ESI) m / z: 833 (M + H) + .

[0747] 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 = 4.8 Hz), 4.69 - 4.63 (2H, m), 4.59 - 4.35 (4H, m), 4.16 (1H, dd, J = 3.8, 1.9 Hz), 3.77 (6H, d, J = 1.8 Hz), 3.47 (1H, dd, J = 10.9, 3.0 Hz), 3.27 (1H, dd, J = 10.9, 4.2 Hz), 3.00 (1H, d, J = 6.7 Hz), 0.87 (9H, s), 0.06 (3H, s), -0.01 (3H, s).

[0748] (2'-O-TBS form)

[0749] MS(ESI) m / z: 833 (M+H) + .

[0750] 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 = 6.0 Hz), 4.84 (1H, t, J = 5.4 Hz), 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, 3.0 Hz), 3.38 (1H, dd, J = 10.9, 3.6 Hz), 2.71 (1H, d, J = 3.0 Hz), 0.80 (9H, s), -0.03 (3H, s), -0.19 (3H, s).

[0751] (Step 3)

[0752] 1-[2-(Benzoyloxy)ethyl]-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[bis(propane-2-yl)amino]phosphino}inosine

[0753] Using the compound obtained in the above Step 2 (1.20 g), the title compound (1.41 g) was synthesized in the same manner as in Step 11 of Example 1 to obtain a mixture of diastereoisomers on the phosphorus atom (diastereoisomer ratio = 0.55:0.45).

[0754] MS(ESI) m / z: 1033 (M+H) + .

[0755] 11H-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 - 7.39 (4H, m), 7.35 - 7.10 (7H, m), 6.83 - 6.78 (4H, m), 6.15 (0.55H, d, J = 5.4 Hz), 6.08 (0.45H, d, J = 6.0 Hz), 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 = 6.7 Hz), 2.32 (1H, t, J = 6.3 Hz), 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).

[0756] (Step 4)

[0757] 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

[0758] To a mixed solution of dichloromethane (322 mL) - pyridine (35 mL) containing the compound obtained in Step 4 of Example 1 (35.80 g), a solution of hydrogen fluoride - pyridine (6.33 g) in dichloromethane (36 mL) was added dropwise over 5 minutes under ice-cooling, and the mixture was stirred at the same temperature for 3 hours. Saturated aqueous sodium bicarbonate solution (268 mL) and saturated brine (143 mL) were successively added to the reaction mixture to stop the reaction, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure. Hexane / ethyl acetate (1:1) (108 mL) was added to the residue to make a slurry, and the mixture was stirred at 50 °C for 30 minutes. Hexane (161 mL) was added and the mixture was further stirred for 2 hours. The precipitated solid was filtered off and washed with hexane / ethyl acetate (4:1) (143 mL) to obtain the title compound (26.81 g).

[0759] MS (ESI) m / z: 525 (M + H) + .

[0760] 11H-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 = 6.5 Hz), 5.15 (1H, t, J = 5.6 Hz), 5.00 (1H, d, J = 4.8 Hz), 4.48 (1H, t, J = 5.6 Hz), 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).

[0761] (Step 5)

[0762] 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-bis-O-(tetrahydropyran-2-yl)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0763] To a solution of the compound obtained in the above 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. Under ice-cooling, saturated aqueous sodium hydrogen carbonate was added to the reaction mixture to stop the reaction, 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 filtered off, 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).

[0764] 1 1H-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), 4.90 - 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).

[0765] (Step 6)

[0766] 6-Benzoyl-2-[3,5-bis-O-(tetrahydropyran-2-yl)-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0767] To a solution of the compound obtained in the above step 5 (24.73 g) and acetic acid (3.1 mL) in tetrahydrofuran (250 mL), a solution of tetrabutylammonium fluoride in tetrahydrofuran (ca. 1 M, 55 mL) was added under ice-cooling, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, 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 (18.74 g).

[0768] 1 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).

[0769] (Step 7)

[0770] 6-Benzoyl-2-[3,5-bis-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0771] To a solution of the compound obtained in the above 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. Saturated brine was added to the reaction solution to stop the reaction, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate. The desiccant was filtered off, 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 solution was concentrated under reduced pressure, ethyl acetate was added to the residue, and the mixture was washed successively with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, 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).

[0772] 11H-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).

[0773] (Step 8)

[0774] 6-Benzoyl-2-[2-deoxy-2-fluoro-3,5-bis-O-(tetrahydropyran-2-yl)-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0775] To a solution of the compound obtained in the above 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 brine was added to the reaction mixture to stop the reaction, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (200 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (ca. 1 M, 150 mL) was added under ice-cooling, and the mixture was stirred at the same temperature for 3 hours. Aqueous saturated ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, 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 (7.65 g).

[0776] 1 1H-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).

[0777] (Step 9)

[0778] 6-Benzoyl-2-(2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene

[0779] To a solution of the compound (7.65 g) obtained in the above step 8 in ethanol (150 mL) was added pyridinium p-toluenesulfonate (6.62 g), and the mixture was stirred at 50 °C for 3 hours. The reaction solution was concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was washed successively with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, 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 (3.55 g).

[0780] 1 H-NMR (CDCl3) δ: 8.05 (1H, s), 7.41 - 7.35 (3H, m), 7.30 - 7.24 (2H, m), 7.06 (1H, s), 6.07 - 6.00 (2H, m), 5.85 (1H, ddd, J = 52.8, 6.7, 4.7 Hz), 4.66 (1H, d, J = 3.9 Hz), 4.42 - 4.31 (2H, m), 4.20 (1H, m), 3.93 (1H, dd, J = 12.9, 1.6 Hz), 3.74 (1H, td, J = 12.3, 1.6 Hz), 3.12 - 2.96 (2H, m), 2.51 (1H, s), 2.33 - 2.15 (2H, m).

[0781] (Step 10)

[0782] 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

[0783] To a solution of the compound (3.55 g) obtained in the above step 9 in dehydrated pyridine (50 mL) was added 4,4'-dimethoxytrityl chloride (4.43 g), and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. Methanol (1 mL) was added to the reaction solution, and after stirring for about 10 minutes, the mixture 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 (5.77 g).

[0784] 11H-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, 2.3 Hz), 5.40 (1H, ddd, J = 53.2, 4.8, 2.3 Hz), 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, 2.7 Hz), 3.45 (1H, dd, J = 11.0, 3.5 Hz), 2.79 (2H, t, J = 6.3 Hz), 2.45 (1H, s), 2.24 - 2.11 (2H, m).

[0785] (Step 11)

[0786] 6-Benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[bis(2-methylpropyl)amino]phosphino}-2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene Using the compound (5.77 g) obtained in the above Step 10, the reaction was carried out in the same manner as in Step 6 of Example 1 to obtain the title compound (5.95 g) in the form of a mixture of diastereoisomers on the phosphorus atom (diastereoisomer ratio = 1:1).

[0787] 1 1H-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, 1.6 Hz), 2.40 (1H, td, J = 6.4, 2.1 Hz), 2.21 - 2.12 (2H, m), 1.21 - 1.13 (9H, m), 1.03 (3H, d, J = 6.7 Hz).

[0788] (Step 12)

[0789] The compound (1.02 g) obtained by the above-mentioned step 11 was reacted in the same manner as in step 7 of Example 1 to obtain an acetonitrile solution of 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(bridging oxy)-λ 5 -phosphino]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene. Using the obtained acetonitrile solution and the compound (1.15 g) obtained in the above-mentioned step 3, the reaction was carried out in the same manner as in step 12 of Example 1, and the obtained crude product was directly used for the next reaction.

[0790] (Step 13)

[0791] 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-bridging oxy-2-mercapto-10-thione octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradec-7-yl]-6-bridging oxy-6,9-dihydro-1H-purin-1-yl}ethyl benzoate

[0792] Using the crude product obtained in the above-mentioned step 12, the reaction was carried out in the same manner as in step 13 of Example 1 to obtain the title compound (818 mg: containing impurities) in the form of a mixture of diastereoisomers on the phosphorus atom.

[0793] MS(ESI) m / z: 1152 (M+H) + .

[0794] (Step 14)

[0795] (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-7-[1-(2-hydroxyethyl)-6-bridging oxy-1,6-dihydro-9H-purin-9-yl]-2,10-dibridging oxy-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]pentaoxadiphosphecyclotetradecine-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[0796] Using the compound (818 mg) obtained in the above step 13, the reaction was carried out in the same manner as in step 14 of Example 1 to obtain diastereomer 1 (107 mg: containing impurities) and diastereomer 2 (101 mg: containing impurities) of the title compound.

[0797] Diastereomer 1 (low polarity)

[0798] MS(ESI) m / z: 891 (M+H) + .

[0799] Diastereomer 2 (high polarity)

[0800] MS(ESI) m / z: 891 (M+H) + .

[0801] (Step 15-1)

[0802] (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-Fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-epoxy-1,6-dihydro-9H-purin-9-yl]-2,10-diepoxy-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]pentaoxadiphosphecyclotetradecine-2,10-bis(thiol) disodium

[0803] (Diastereomer 1)

[0804] Using the compound (diastereomer 1) (107 mg: containing impurities) obtained in the above step 14, the reaction was carried out in the same manner as in step 15-1 of Example 1, and then purified according to the following [Purification conditions] to obtain the title compound in the form of triethylamine salt.

[0805] [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)].

[0806] The obtained triethylamine salt was subjected to salt exchange in the same manner as described in Step 15-1 of Example 1 [converted to sodium salt] to obtain the title compound (29.1 mg).

[0807] MS(ESI) m / z: 777 (M+H) + .

[0808] 1 1H-NMR (CD3OD) δ: 8.58 (1H, m), 8.11 (1H, m), 8.03 (1H, s), 7.11 (1H, s), 6.47 (1H, d, J = 17.5 Hz), 6.26 (1H, d, J = 8.5 Hz), 5.53 - 5.36 (2H, m), 5.29 - 5.17 (1H, m), 4.77 (1H, d, J = 4.2 Hz), 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 (1H, 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).

[0809] 31 31P-NMR (CD3OD) δ: 57.5 (s), 53.0 (s).

[0810] (Step 15-2)

[0811] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-15-Fluoro-16-hydroxy-7-[1-(2-hydroxyethyl)-6-epoxy-1,6-dihydro-9H-purin-9-yl]-2,10-epoxy-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]pentaoxadiphosphetacyclotetradecyne-2,10-bis(thiol) disodium

[0812] (Diastereomer 2)

[0813] The compound (diastereomer 2) (101 mg: containing impurities) obtained by the above-mentioned process 14 was reacted in the same manner as in Step 15-1 of Example 1, and then purified according to the following [Purification Conditions] to obtain the title compound in the form of triethylamine salt.

[0814] [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)].

[0815] The obtained triethylamine salt was subjected to salt exchange in the same manner as [Conversion to Sodium Salt] described in Step 15-1 of Example 1 to obtain the title compound (11.2 mg).

[0816] MS (ESI) m / z: 777 (M + H) + .

[0817] 1 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, 2.1 Hz), 6.28 (1H, d, J = 8.5 Hz), 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 (2H, t, J = 5.1 Hz), 3.48 (2H, dd, J = 6.7, 3.6 Hz), 2.90 - 2.72 (2H, m), 2.00 - 1.90 (2H, m).

[0818] 31 31P-NMR (CD3OD) δ: 59.5 (s), 57.7 (s).

[0819] Example 3: Synthesis of CDN49

[0820] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-7-[1-(2-aminoethyl)-6-bridged oxy-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(mercapto)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadispiro[4.2.48.211]tetradecane-2,10-dione

[0821]

[0822] [Synthetic Route]

[0823]

[0824]

[0825] (Step 1)

[0826] 5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine

[0827] To a suspension of commercially available (Aamdis Chemical) 5’-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (13.0 g) in N,N-dimethylacetamide (60 mL) were added N-(2-bromoethyl)phthalimide (7.02 g) and 1,8-diazabicyclo[5.4.0]-7-undecene (4.1 mL), and the mixture was 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 stirring was continued for 1 day. The reaction was stopped by adding water to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [ethyl acetate / methanol] to obtain the title compound (12.4 g).

[0828] 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), 7.25 - 7.11 (7H, m), 6.74 - 6.70 (4H, m), 5.93 (1H, d, J = 5.1 Hz), 5.68 (1H, d, J = 3.9 Hz), 4.71 (1H, q, J = 4.8 Hz), 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, 3.9 Hz), 3.32 (1H, dd, J = 11.0, 5.5 Hz).

[0829] (Step 2)

[0830] 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

[0831] The compound obtained in the above-mentioned step 1 (12.4 g) was reacted in the same manner as in step 10 of Example 1 to obtain the title compound (4.18 g) and the positional isomer of the title compound, namely 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).

[0832] 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 = 5.1 Hz), 4.53 - 4.41 (3H, m), 4.32 - 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, 3.1 Hz), 3.24 (1H, dd, J = 10.8, 4.1 Hz), 2.98 (1H, d, J = 6.7 Hz), 0.85 (9H, s), 0.04 (3H, s), -0.03 (3H, s).

[0833] Positional isomer (2'-O-TBS form)

[0834] 11H-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 = 7.8 Hz), 5.91 (1H, d, J = 5.9 Hz), 4.82 (1H, t, J = 5.5 Hz), 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, 2.7 Hz), 3.36 (1H, dd, J = 10.6, 3.5 Hz), 2.70 (1H, d, J = 3.1 Hz), 0.83 (9H, s), -0.04 (3H, s), -0.19 (3H, s).

[0835] (Step 3)

[0836] 5'-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[bis(propane-2-yl)amino]phosphino}-1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]inosine

[0837] Using the compound obtained in Step 2 above (8.89 g), the reaction was carried out in the same manner as in Step 6 of Example 1 to obtain the title compound (9.45 g) in the form of a mixture of diastereoisomers on the phosphorus atom (diastereoisomer ratio = 1:1).

[0838] 11H-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 = 7.8 Hz), 7.33 - 7.18 (7H, m), 6.81 (4H, d, J = 8.6 Hz), 6.10 (0.5H, d, J = 5.5 Hz), 6.04 (0.5H, d, J = 5.1 Hz), 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 = 6.3 Hz), 2.34 (1H, t, J = 6.7 Hz), 1.12 - 1.08 (9H, m), 0.91 (3H, d, J = 7.0 Hz), 0.82 (9H, s), 0.06 (1.5H, s), 0.03 (1.5H, s), -0.03 (3H, s).

[0839] (Step 4)

[0840] 4-Chloro-5-iodo-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

[0841] 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) was added sodium hydride (containing 45% mineral oil) (13.3 g) under ice-cooling, and the mixture was stirred for 40 minutes while warming to room temperature. After re-cooling in ice, [2-(chloromethoxy)ethyl](trimethyl)silane (51.0 mL) was added over 10 minutes, and the mixture was stirred at the same temperature for 30 minutes. Water (260 mL) was added portionwise to the mostly solidified reaction mixture to stop the reaction. The solid was collected by filtration, washed with water (1500 mL) and hexane (600 mL), and dried under reduced pressure at 40 °C to give the title compound (97.63 g).

[0842] MS (ESI) m / z: 410 (M + H) + .

[0843] 11H-NMR (CDCl3) δ: 8.64 (1H, s), 7.54 (1H, s), 5.61 (2H, s), 3.52 (2H, t, J = 8.3 Hz), 0.92 (2H, t, J = 8.3 Hz), -0.04 (9H, s).

[0844] (Step 5)

[0845] 4-(Benzyloxy)-5-iodo-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

[0846] To a solution of sodium hydride (45% in mineral oil) (12 g) in N,N-dimethylformamide (170 mL) at ice-cooling was added benzyl alcohol (27 mL), and the mixture was stirred for 40 minutes while warming to room temperature. After re-cooling in ice, a suspension of the compound obtained in the above Step 4 (97.63 g) in N,N-dimethylformamide (360 mL) was added over 40 minutes, and the mixture was stirred at the same temperature for 35 minutes. Ice chips and saturated aqueous ammonium chloride solution were added to the reaction mixture to stop the reaction. The reaction mixture was poured into a two-layer mixture of saturated aqueous ammonium chloride solution and ethyl acetate, and extracted with ethyl acetate:toluene (9:1). The organic layer was washed twice with water and twice with saturated brine, and then dried over anhydrous sodium sulfate. The desiccant was filtered off, 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).

[0847] MS (ESI) m / z: 482 (M+H) + .

[0848] 1 1H-NMR (CDCl3) δ: 8.47 (1H, s), 7.61 (2H, d, J = 7.3 Hz), 7.41 (2H, t, J = 7.6 Hz), 7.36 - 7.30 (1H, m), 7.30 (1H, s), 5.65 (2H, s), 5.57 (2H, s), 3.52 (2H, t, J = 8.3 Hz), 0.91 (2H, t, J = 8.3 Hz), -0.05 (9H, s).

[0849] (Step 6)

[0850] 4-(Benzyloxy)-5-(3,3-diethoxyprop-1-yn-1-yl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

[0851] To a mixed solution of the compound (113.4 g) obtained in the above step 5 in acetonitrile (1000 mL) - triethylamine (98 mL), under a nitrogen atmosphere and at room temperature, copper(I) iodide (4.49 g), tetrakis(triphenylphosphine)palladium(0) (8.17 g), and 3,3 - diethoxyprop - 1 - yne (104 mL) were added, and the mixture was stirred at the same temperature for 4.5 hours. After concentrating the reaction mixture under reduced pressure, ethyl acetate and hexane were added to the residue, and the precipitated solid was filtered off. The solid was washed with a mixed solution of ethyl acetate:hexane (1:1), and then 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).

[0852] MS(ESI) m / z: 482 (M + H) + .

[0853] (Step 7)

[0854] 5-(3,3 - Diethoxypropyl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H - pyrrolo[2,3 - d]pyrimidin - 4 - ol

[0855] To a solution of the compound (145.5 g) obtained in the above step 6 in ethanol (900 mL), 10% palladium on carbon catalyst (M) wet (50.2 g) was added, and the mixture was stirred under a hydrogen atmosphere and at room temperature for 5 hours. Dichloromethane (500 mL) was added to the reaction mixture, and the catalyst was filtered off through diatomaceous earth. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] twice to obtain the title compound (59.6 g).

[0856] MS(ESI) m / z: 418 (M + Na) + , 394 [M - H] － .

[0857] 1 1H - NMR(CDCl3) δ: 11.23(1H, brs), 7.85(1H, s), 6.79(1H, s), 5.47(2H, s), 4.58(1H, t, J = 5.9 Hz), 3.69(2H, m), 3.57 - 3.49(4H, m), 2.90(2H, t, J = 7.8 Hz), 2.07(2H, m), 1.23(6H, t, J = 7.1 Hz), 0.91(2H, t, J = 8.1 Hz), -0.04(9H, s).

[0858] (Step 8)

[0859] 5-(3,3-Diethoxypropyl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine-4-thiol

[0860] To a solution of the compound (59.6 g) obtained in Step 7 above in dehydrated dichloromethane (300 mL), 2,6-dimethylpyridine (42 mL) was added under a nitrogen atmosphere. Trifluoromethanesulfonic anhydride (31 mL) was added dropwise over 20 minutes at -20 °C, and the mixture was stirred at the same temperature for 20 minutes. N,N-Dimethylformamide (500 mL) and sodium hydrosulfide 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 two-layer mixture of ethyl acetate and ice-cooled saturated ammonium chloride aqueous solution, and extracted with a mixture of ethyl acetate:toluene (9:1). The organic layer was washed once with saturated ammonium chloride aqueous solution, twice with saturated brine, and then dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain a mixture of the title compound and 2,6-dimethylpyridine. The obtained mixture was poured into a two-layer mixture 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 desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain the title compound (57.6 g).

[0861] MS(ESI) m / z: 410 [M-H] － .

[0862] 1 1H-NMR (CDCl3) δ: 11.69 (1H, brs), 7.90 (1H, s), 6.96 (1H, s), 5.49 (2H, s), 4.61 (1H, t, J = 5.9 Hz), 3.71 (2H, m), 3.55 (2H, m), 3.49 (2H, t, J = 8.1 Hz), 3.14 (2H, t, J = 7.8 Hz), 2.08 (2H, m), 1.23 (6H, t, J = 7.1 Hz), 0.90 (2H, t, J = 8.3 Hz), -0.04 (9H, s).

[0863] (Step 9)

[0864] 3-(4-Mercapto-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propan-1-ol

[0865] The compound obtained in the above step 8 (31.62 g) was dissolved in 80% aqueous acetic acid solution (300 mL), and stirred at room temperature for 30 minutes. After confirming the disappearance of the raw material, it was cooled with ice, and sodium borohydride (1.45 g) was carefully added portionwise, and stirred at the same temperature for 30 minutes. Then, sodium triacetoxyborohydride (24.4 g) was added over 15 minutes, and stirred at the same temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure to about one-fifth of the original volume. After carefully adding solid sodium bicarbonate to the residue to neutralize it to a certain extent, it was 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 filtered off, 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).

[0866] MS(ESI) m / z: 340 [M+H] + .

[0867] 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 = 5.9 Hz), 3.50(2H, t, J = 8.1 Hz), 3.23(2H, t, J = 7.3 Hz), 2.33(1H, brs), 1.99(2H, m), 0.91(2H, t, J = 8.3 Hz), -0.04(9H, s).

[0868] (Step 10)

[0869] 2-{[2-(Trimethylsilyl)ethoxy]methyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenz[cd]azulene

[0870] To a solution of the compound obtained in the above step 9 (31.31 g) in dehydrated tetrahydrofuran (600 mL) was added triphenylphosphine (25.4 g) and diisopropyl azodicarboxylate (21.8 g) under a nitrogen atmosphere at 0 °C, and stirred at the same temperature for 1 hour. After concentrating the reaction solution under reduced pressure, the residue was purified successively by silica gel column chromatography [dichloromethane / ethyl acetate] and silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (35.93 g: containing impurities).

[0871] MS(ESI) m / z: 322 [M+H] + .

[0872] 11H-NMR (CDCl3) δ: 8.57 (1H, s), 7.08 (1H, s), 5.58 (2H, s), 3.52 (2H, t, J = 8.3 Hz), 3.17 (2H, m), 3.06 (2H, t, J = 5.6 Hz), 2.36 (2H, m), 0.92 (2H, t, J = 8.3 Hz), -0.05 (9H, s).

[0873] (Step 11)

[0874] (8,9-Dihydro-6-thia-2,3,5-triazabenz[cd]azulen-2(7H)-yl)methanol

[0875] To a solution of the compound obtained in the above step 10 (35.93 g) 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 h. After concentrating the reaction mixture under reduced pressure, it was azeotroped with toluene 4 times. To the residue was added a mixture of dichloromethane:hexane (1:2), and the precipitated solid (Solid 1) was collected by filtration. After concentrating the filtrate under reduced pressure, 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).

[0876] MS (ESI) m / z: 222 [M+H] + .

[0877] 1 1H-NMR (CDCl3) 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 the observable peaks are recorded)

[0878] (Step 12)

[0879] 2,7,8,9-Tetrahydro-6-thia-2,3,5-triazabenz[cd]azulene

[0880] To a suspension of the compound obtained in the above step 11 (20.13 g) in methanol (250 mL) was added 28% aqueous ammonia solution (150 mL), and the mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated to about half of its original volume under reduced pressure. 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 in the same manner. The filtrate was applied to silica gel, and then purified by silica gel column chromatography [dichloromethane / methanol]. The fraction containing the target compound was concentrated under reduced pressure, slurried and washed with ethanol, and then the solid (Solid 3) was collected by filtration. Solid 1, Solid 2, and Solid 3 were combined to obtain the title compound (12.36 g).

[0881] MS(ESI) m / z: 192 [M+H] + .

[0882] 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).

[0883] (Step 13)

[0884] 2-(2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

[0885] To a suspension of the compound (13.47 g) obtained in the above Step 12 in dehydrated acetonitrile (350 mL), powdered potassium hydroxide (10.3 g) and tris[2-(2-methoxyethoxy)ethyl]amine (1.13 mL) were added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1.5 hours. After adding dropwise a solution of 2,3,5-tri-O-benzyl-α-D-arabinofuranosyl chloride (40.2 g), which is known in the literature (J. Med. Chem. 1976, 19, 6, 814 - 816), in acetonitrile (100 mL) under ice-cooling, the temperature was raised to room temperature and the mixture was stirred for 4 hours. The insoluble matter 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).

[0886] MS(ESI) m / z: 594 [M+H] + .

[0887] 1 H-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).

[0888] (Step 14)

[0889] 2-β-D-Arabinofuranosyl-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

[0890] To a solution of the compound (26.19 g) obtained in the above Step 13 in dehydrated dichloromethane (300 mL), a solution of boron trichloride in dichloromethane (1 M, 200 mL) was added under a nitrogen atmosphere at -78 °C. After stirring at the same temperature for 2 hours, the temperature was raised to 0 °C and stirring was continued for 4 hours. The reaction solution was cooled again to -78 °C, and a solution of methanol (80 mL) in dichloromethane (160 mL) was added, and the mixture was stirred for 30 minutes while warming to room temperature. The reaction solution was concentrated under reduced pressure and azeotroped with ethanol twice. Ethanol (200 mL) and diethyl ether (100 mL) were added to the residue to form a slurry, and the solid (Solid 1) was collected by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [dichloromethane / methanol]. After concentrating the fraction containing the target compound under reduced pressure, ethanol was added to form a slurry, and the solid (Solid 2) was collected by filtration. Solid 1 and Solid 2 were combined to obtain the title compound (13.2 g).

[0891] MS(ESI) m / z: 324 [M+H] + .

[0892] 1 1H-NMR(CD3OD) δ: 8.73(1H, s), 7.96(1H, s), 6.70(1H, d, J = 4.9 Hz), 4.32(1H, t, J = 4.6 Hz), 4.25(1H, t, J = 4.6 Hz), 3.97(1H, m), 3.90(1H, dd, J = 12.0, 3.2 Hz), 3.85(1H, dd, J = 12.0, 4.6 Hz), 3.53(2H, m), 3.17(2H, m), 2.43(2H, m).

[0893] (Step 15)

[0894] 2-[3,5-Bis-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl]-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulene

[0895] To a solution of the compound (15.35 g) obtained in the above step 14 in dehydrated dimethyl sulfoxide (160 mL), 3,4-dihydro-2H-pyran (17.2 mL) and p-toluenesulfonic acid monohydrate (9.02 g) were added at 0 °C, and the mixture was stirred at room temperature for 3 hours. 3,4-Dihydro-2H-pyran (8.6 mL) was added additionally, and after stirring for 45 minutes, triethylamine (13 mL) was immediately added to stop the reaction. The reaction solution was poured into two layers of ethyl acetate and saturated aqueous sodium bicarbonate, and extracted with ethyl acetate. The organic layer was washed once with water, twice with saturated brine, and then dried over anhydrous sodium sulfate. The desiccant was filtered off, 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 (10.81 g) in the form of a mixture of 4 diastereoisomers.

[0896] MS(ESI) m / z: 492 [M+H] + .

[0897] 1 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 = 5.4 Hz), 6.71 (0.2H, d, J = 5.9 Hz), 6.57 (0.3H, d, J = 5.9 Hz), 6.56 (0.3H, d, J = 5.9 Hz), 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).

[0898] (Step 16)

[0899] 2-[2-Deoxy-2-fluoro-3,5-bis-O-(tetrahydropyran-2-yl)-β-D-ribofuranosyl]-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenz[cd]azulene

[0900] To a solution of the compound (10.81 g) obtained in Step 15 in dehydrated dichloromethane (150 mL) was added pyridine (5.3 mL) and trifluoromethanesulfonic anhydride (5.6 mL) under a nitrogen atmosphere at 0 °C, and the mixture was stirred at the same temperature for 1 hour. After adding crushed ice to the reaction mixture to stop the reaction, the reaction mixture was poured into a two-layer system 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 filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product of the amorphous trifluoromethanesulfonate. The obtained crude product of the trifluoromethanesulfonate was dissolved in dehydrated tetrahydrofuran (150 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (ca. 1 M, 154 mL) was added portionwise under ice-cooling, and the mixture was stirred at the same temperature overnight. The reaction was stopped by adding saturated aqueous ammonium chloride to the reaction mixture. The reaction mixture was concentrated under reduced pressure to about half of its original volume. The residue was poured into a two-layer system of ethyl acetate and saturated aqueous ammonium chloride, and extracted 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 organic layers were combined and dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain a crude product of the title compound (40.37 g).

[0901] MS(ESI) m / z: 494 [M+H] + .

[0902] (Step 17)

[0903] 2-(2-Deoxy-2-fluoro-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenz[cd]azulene

[0904] To a solution of the compound (40.37 g) obtained in Step 16 in methanol (400 mL) was added p-toluenesulfonic acid monohydrate (2.09 g), and the mixture was stirred at 60 °C for 4 hours. The reaction was stopped by adding triethylamine (16 mL) to the reaction mixture. 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 fractions containing the target compound were concentrated under reduced pressure until a slurry was formed, 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).

[0905] MS(ESI) m / z: 326 [M+H] + .

[0906] 11H-NMR (CDCl3) δ: 8.51 (1H, s), 7.01 (1H, s), 6.00 (1H, dd, J = 13.7, 6.3 Hz), 5.95 (1H, dd, J = 11.7, 2.0 Hz), 5.87 (1H, ddd, J = 52.7, 6.3, 4.9 Hz), 4.69 (1H, m), 4.32 (1H, brs), 3.96 (1H, d, J = 12.7 Hz), 3.77 (1H, m), 3.17 (2H, m), 3.04 (2H, m), 2.41 - 2.31 (3H, m).

[0907] (Step 18)

[0908] 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-triazabenz[cd]azulene

[0909] Using the compound (5.32 g) obtained in the above Step 17, the reaction was carried out in the same manner as in Step 10 of Example 2 to obtain the title compound (10.1 g).

[0910] MS (ESI) m / z: 628 [M+H] + .

[0911] 1 1H-NMR (CDCl3) δ: 8.54 (1H, s), 7.42 (2H, d, J = 7.3 Hz), 7.32 - 7.21 (8H, m), 6.81 (4H, m), 6.52 (1H, dd, J = 17.3, 2.2 Hz), 5.37 (1H, ddd, J = 53.3, 4.4, 2.4 Hz), 4.76 (1H, m), 4.16 (1H, m), 3.789 (3H, s), 3.786 (3H, s), 3.59 (1H, dd, J = 10.7, 2.4 Hz), 3.44 (1H, dd, J = 10.7, 3.4 Hz), 3.12 (2H, m), 2.76 (2H, t, J = 5.6 Hz), 2.27 (2H, m), 2.18 (1H, dd, J = 7.8, 2.9 Hz).

[0912] (Step 19)

[0913] 2-(5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[bis(propane-2-yl)amino]phosphino}-2-deoxy-2-fluoro-β-D-ribofuranosyl)-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenz[cd]azulene

[0914] The compound (10.1 g) obtained by the above-mentioned step 18 was reacted in the same manner as in step 6 of Example 1 to obtain the title compound (12.6 g) in the form of a mixture of diastereoisomers on the phosphorus atom (diastereoisomer ratio = 1:1).

[0915] 1 H-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, 2.2 Hz), 6.48 (0.5H, dd, J = 17.6, 1.5 Hz), 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, 2.4 Hz), 2.39 (1H, m), 2.28 - 2.21 (2H, m), 1.19 - 1.15 (9H, m), 1.03 (3H, d, J = 6.8 Hz).

[0916] (Step 20)

[0917] The compound (1.80 g) obtained by the above-mentioned step 19 was reacted in the same manner as in step 7 of Example 1 to obtain an acetonitrile solution of 2-{2-deoxy-2-fluoro-3-O-[hydroxy(bridged oxy)-λ 5 -phosphino]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6-thia-2,3,5-triazabenz[cd]azulene. Using the obtained acetonitrile solution and the compound (2.30 g) obtained in the above-mentioned step 3, the reaction was carried out in the same manner as in step 12 of Example 1, and the obtained crude product was directly used in the subsequent reaction.

[0918] (Step 21)

[0919] 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-thioxooctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadispiro[tetradecane-10,2'-furan]-10-yl]oxy}propanenitrile

[0920] Using the crude product obtained in Step 20 above, the reaction was carried out in the same manner as in Step 13 of Example 1 to obtain the title compound (1.22 g) in the form of a mixture of diastereoisomers on the phosphorus atom.

[0921] MS(ESI) m / z: 1090 (M+H) + .

[0922] (Step 22)

[0923] (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]pentaoxadispiro[tetradecane-2,10'-furan]-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[0924] Hydrazine monohydrate (0.544 mL) was added to a mixed solution of ethanol (10 mL) - tetrahydrofuran (10 mL) of the compound (1.22 g) obtained in Step 21 above, and the mixture was stirred at room temperature for 15 hours. After concentrating the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] to obtain diastereoisomer 1 (108 mg: containing impurities) and diastereoisomer 2 (111 mg: containing impurities) of the title compound.

[0925] Diastereoisomer 1 (low polarity)

[0926] MS(ESI) m / z: 907 (M+H) + .

[0927] Diastereomer 2 (high polarity)

[0928] MS(ESI) m / z: 907 (M+H) + .

[0929] (Step 23-1)

[0930] (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-bridged oxy-1,6-dihydro-9H-purin-9-yl]-14-(8,9-dihydro-6-thia-2,3,5-triazabenz[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dibridged oxyoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphetacyclotetradecyne-2,10-bis(thiol) disodium

[0931] (Diastereomer 1)

[0932] Using the compound (diastereomer 1) (108 mg: containing impurities) obtained in the above Step 22, reacting it in the same manner as in Step 15-1 of Example 1, and then purifying it according to the following [Purification Conditions], the title compound in the form of triethylamine salt was obtained.

[0933] [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 minutes - 40 minutes)].

[0934] The obtained triethylamine salt was subjected to salt exchange in the same manner as [Conversion to Sodium Salt] described in Step 15-1 of Example 1 to obtain the title compound (44.4 mg).

[0935] MS(ESI) m / z: 793 (M+H) + .

[0936] 11H-NMR (CD3OD) δ: 8.50 (1H, s), 8.42 (1H, s), 7.92 (1H, s), 7.56 (1H, s), 6.56 (1H, d, J = 16.3 Hz), 6.21 (1H, d, J = 6.0 Hz), 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).

[0937] 31 31P-NMR (CD3OD) δ: 57.5 (s), 52.9 (s).

[0938] (Step 23-2)

[0939] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-7-[1-(2-aminoethyl)-6-bridged oxy-1,6-dihydro-9H-purin-9-yl]-14-(8,9-dihydro-6-thia-2,3,5-triazabenz[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dibridged oxyoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphetacyclotetradecyne-2,10-bis(thiol) disodium

[0940] (Diastereomer 2)

[0941] Using the compound (diastereomer 2) (111 mg: containing impurities) obtained in the above Step 22, after reacting in the same manner as in Step 15-1 of Example 1, it was purified according to the following [Purification Conditions] to obtain the title compound in the form of a triethylamine salt.

[0942] [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 minutes - 40 minutes)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 20% - 60% (0 minutes - 40 minutes)].

[0943] The obtained triethylamine salt was subjected to salt exchange in the same manner as described in Step 15-1 of Example 1 [converted to sodium salt] to obtain the title compound (40.6 mg).

[0944] MS(ESI) m / z: 793 (M+H) + .

[0945] 1 1H-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, 1.8 Hz), 6.26 (1H, d, J = 8.5 Hz), 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).

[0946] 31 31P-NMR (CD3OD) δ: 58.7 (s), 57.8 (s).

[0947] Example 4: Synthesis of CDN50

[0948] N-(2-{9-[(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenz[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxido-2,10-bis(mercapto)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxido-6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[0949]

[0950] [Synthetic route]

[0951]

[0952] (Step 1-1)

[0953] (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-bridged oxy-1,6-dihydro-9H-purin-9-yl}-2,10-dibridged oxyoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphetacyclotetradecyne-2,10-bis(thiol) disodium

[0954] (Diastereomer 1)

[0955] To a solution of the compound (20.0 mg) obtained in Step 23-1 of Example 3 in N,N-dimethylformamide (0.5 mL) were added triethylamine (17 μL) and 1-[(hydroxyacetyl)oxy]pyrrolidine-2,5-dione (10.3 mg), and the mixture was stirred at room temperature for 3 hours. The reaction solution was diluted with a 10 mM aqueous solution of triethylammonium acetate and purified by C18 silica gel column chromatography [10 mM aqueous solution of triethylammonium acetate / acetonitrile] and preparative HPLC [10 mM aqueous solution of triethylammonium acetate / acetonitrile, acetonitrile: 10%-30% (0 minutes - 40 minutes)]. The resulting compound (triethylamine salt) was subjected to salt exchange in the same manner as described in Step 15-1 of Example 1 [converted to sodium salt] to obtain the title compound (15.6 mg).

[0956] MS(ESI) m / z: 851 (M+H) + .

[0957] 1H-NMR (CD3OD) δ: 8.43 (1H, s), 8.40 (1H, brs), 7.66 (1H, brs), 7.58 (1H, s), 6.53 (1H, d, J = 16.3 Hz), 6.14 (1H, d, J = 8.5 Hz), 5.73 - 5.64 (1H, m), 5.59 - 5.42 (1H, m), 5.42 - 5.29 (1H, m), 4.80 - 4.74 (1H, m), 4.53 - 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).

[0958] 31 P-NMR (CD3OD) δ: 57.0 (s), 52.6 (s).

[0959] (Step 1-2)

[0960] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenz[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-7-{1-[2-(2-hydroxyacetamido)ethyl]-6-epoxy-1,6-dihydro-9H-purin-9-yl}-2,10-diepoxyoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphetacyclotetradecyne-2,10-bis(thiol) disodium

[0961] (Diastereomer 2)

[0962] Using the compound (10.0 mg) obtained in Step 23-2 of Example 3, after reacting in the same manner as in Step 1-1 above, it was purified by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 7% - 25% (0 minutes - 40 minutes)]. The resulting compound (triethylamine salt) was subjected to salt exchange in the same manner as described in Step 15-1 of Example 1 [converted to sodium salt] to obtain the title compound (6.6 mg).

[0963] MS (ESI) m / z: 851 (M + H)+ .

[0964] 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 = 15.1 Hz), 6.20 (1H, d, J = 7.9 Hz), 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).

[0965] 31 P-NMR (CD3OD) δ: 58.2 (s), 57.6 (s).

[0966] Example 5: Synthesis of Drug Linker 2

[0967] [Synthesis Route]

[0968]

[0969] (Step 1)

[0970] Carry out the same reaction as in Step 7 of Example 1 on the following scale (starting material: 1.40 g). Use the acetonitrile solution of the resulting compound and the compound obtained in Step 3 of Example 2 (1.41 g), and carry out the reaction in the same manner as in Step 12 of Example 1. Use the resulting crude product directly in the next reaction.

[0971] (Step 2)

[0972] 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-bridged oxy-2-mercapto-10-thione octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadispiro[tetradecane-7,1'-purine]-6'-yl}ethyl benzoate

[0973] Using the crude product obtained in Step 1 above, the reaction was carried out in the same manner as in Step 13 of Example 1 to obtain the title compound (778 mg) in the form of a mixture of diastereoisomers on the phosphorus atom.

[0974] MS(ESI) m / z: 1264 (M+H) + .

[0975] (Step 3)

[0976] (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-hydroxyethyl)-6-oxido-1,6-dihydro-9H-purin-9-yl]-2,10-dioxido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methanospiro[2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadispiro[tetradecane-2,10-bis(thiol)]bis(N,N-diethylethylammonium)

[0977] Using the compound (778 mg) obtained in Step 2 above, the reaction was carried out in the same manner as in Step 14 of Example 1 to obtain diastereoisomer 1 (255 mg) and diastereoisomer 2 (containing impurities) of the title compound. Diastereoisomer 2 was purified again by preparative HPLC [water / acetonitrile containing 0.2% triethylamine, acetonitrile containing 0.2% triethylamine: 5%-50% (0 minutes - 40 minutes)] to obtain diastereoisomer 2 (94.6 mg) of the title compound.

[0978] Diastereoisomer 1 (low polarity)

[0979] MS(ESI) m / z: 1003 (M+H) + .

[0980] 11H-NMR (CD3OD) δ: 8.66 (1H, s), 8.21 (1H, s), 8.04 (1H, s), 7.33 (1H, s), 6.27 (1H, d, J = 5.1 Hz), 6.25 (1H, d, J = 3.6 Hz), 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 = 7.3 Hz), 2.98 - 2.75 (2H, m), 2.05 - 1.88 (2H, m), 1.28 (18H, t, J = 7.3 Hz), 0.98 (9H, s), 0.85 (9H, s), 0.31 (3H, s), 0.27 (3H, s), 0.25 (3H, s), 0.09 (3H, s).

[0981] Diastereomer 2 (high polarity)

[0982] MS (ESI) m / z: 1003 (M + H) + .

[0983] 1 1H-NMR (CD3OD) δ: 8.50 (1H, s), 8.22 (1H, s), 8.07 (1H, s), 7.20 (1H, s), 6.33 (1H, d, J = 7.3 Hz), 6.26 (1H, d, J = 9.1 Hz), 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 = 7.3 Hz), 2.96 - 2.89 (2H, m), 2.07 - 1.98 (2H, m), 1.30 (18H, t, J = 7.3 Hz), 0.99 (9H, s), 0.74 (9H, s), 0.27 (3H, s), 0.27 (3H, s), 0.20 (3H, s), -0.05 (3H, s).

[0984] (Process 4 - 1)

[0985] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-bridgedoxy-1,6-dihydro-9H-purin-9-yl)-2,10-dibridgedoxy-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]pentaoxadiphosphetecyclotetradecyne-2,10-bis(thiol)bis(N,N-diethylethylammonium)

[0986] (diastereomer 1)

[0987] To a solution of the compound (diastereomer 1) (30 mg) obtained in Step 3 above in tetrahydrofuran (0.5 mL) were added [(N-{[(9H-fluoren-9-yl)methoxy]carbonyl}glycyl)amino]methyl acetate (91.7 mg) and p-toluenesulfonic acid monohydrate (11.8 mg), and the mixture was 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 Triethylammonium acetate aqueous solution was added to the reaction solution, and purification was carried out by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] to obtain the title compound (25.6 mg) containing the starting material as an impurity.

[0988] MS(ESI) m / z: 1089 (M+H) + .

[0989] (Step 4-2)

[0990] (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-bridgedoxy-1,6-dihydro-9H-purin-9-yl)-2,10-dibridgedoxy-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]pentaoxadiphosphetecyclotetradecyne-2,10-bis(thiol)bis(N,N-diethylethylammonium)

[0991] (Diastereomer 2)

[0992] The compound (diastereomer 2) (84.6 mg) obtained by the above-mentioned step 3 was reacted in the same manner as in the above-mentioned step 4-1 to obtain the title compound (70.9 mg) containing the starting material as an impurity.

[0993] MS (ESI) m / z: 1089 (M+H) + .

[0994] (Step 5-1)

[0995] (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-epoxy-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-diepoxy-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]pentaoxadiphosphetacyclotetradecyne-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[0996] Diastereomer 1

[0997] Triethylamine trihydrofluoride (2 mL) was added to the compound (25.6 mg) obtained in the above-mentioned step 4-1, and the mixture was stirred at 45 °C for 3 hours. At room temperature, a mixture of ice-cold 1 M triethylammonium bicarbonate solution (10 mL) and triethylamine (2 mL) was added to the reaction solution. After the reaction solution was concentrated under reduced pressure, it was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate aqueous solution / acetonitrile) to obtain the title compound (16.6 mg: containing impurities from the starting material of step 7-1).

[0998] MS (ESI) m / z: 861 (M+H) + .

[0999] (Step 5-2)

[1000] (5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R)-7-(1-{2-[(glycylamino)methoxy]ethyl}-6-epoxy-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-diepoxy-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]pentaoxadiphosphetecyclotetradecyne-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[1001] (Diastereomer 2)

[1002] The compound obtained in the above Step 4-2 (70.9 mg) was reacted in the same manner as in the above Step 5-1 to obtain the title compound (51.7 mg: containing impurities from the raw material of Step 4-2).

[1003] MS(ESI) m / z: 861 (M+H) + .

[1004] (Step 6-1)

[1005] N-[4-(11,12-Didehydrodibenz[b,f]azocine-5(6H)-yl)-4-epoxybutyryl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-diepoxy-2,10-dithio-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]pentaoxadiphosphetecyclotetradecyne-7-yl]-6-epoxy-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide bis(N,N-diethylethylammonium)

[1006] (Drug linker 2a: Diastereomer 1)

[1007] To a solution of the compound obtained in the above step 5-1 (16.6 mg) in N,N-dimethylformamide (0.5 mL) were added triethylamine (6 μL) and the compound obtained in step 8 described below (15.5 mg), and the mixture was 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 triethylammonium acetate aqueous solution and methanol were added, and purification was carried out by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 10% - 45% (0 min - 30 min)] to obtain the title compound (5.1 mg).

[1008] MS(ESI) m / z: 1409 (M+H) + .

[1009] 1 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 (1H, 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 = 7.3 Hz). (Only the observable peaks are recorded)

[1010] (Step 6-2)

[1011] N-[4-(11,12-Didehydrodibenz[b,f]azocine-5(6H)-yl)-4-bridged oxybutyryl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dibridged oxy-2,10-dithio-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]pentaoxadispiro[4.1.4.2]tetradec-7-yl]-6-bridged oxy-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide bis(N,N-diethylethylammonium)

[1012] (Drug linker 2b: Diastereomer 2)

[1013] The compound (51.7 mg) obtained by the above-mentioned step 5-2 was reacted in the same manner as in the above-mentioned step 6-1, and then purified according to the following [Purification conditions] to obtain the title compound (33.7 mg).

[1014] [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 10%-50% (0 minutes - 30 minutes)].

[1015] MS (ESI) m / z: 1409 (M+H) + .

[1016] 1 1H-NMR (CD3OD) δ: 8.73 (1H, d, J = 6.7 Hz), 8.19 (1H, d, J = 3.0 Hz), 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 = 13.6 Hz), 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 = 7.3 Hz), 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 = 7.3 Hz).

[1017] (Step 7)

[1018] N-[4-(11,12-Didehydrodibenzo[b,f]azocine-5(6H)-yl)-4-bridged oxybutyryl]glycylglycyl-L-phenylalanine

[1019] To a solution of commercially available (2S)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-phenylpropanoic acid (2.86 g) in N,N-dimethylformamide (51.2 mL) was added triethylamine (2.56 mL) and commercially available 1-{[4-(11,12-didehydrodibenzo[b,f]azocine-5(6H)-yl)-4-bridged oxybutanoyl]oxy}pyrrolidine-2,5-dione (3.69 g, Click Chemistry Tools), and the mixture was stirred at room temperature for 24 hours. Aqueous solution of citric acid monohydrate (24.0 g) in water (500 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a mixed solution of ethyl acetate / acetonitrile, and precipitated with diisopropyl ether and filtered to obtain the title compound (4.30 g).

[1020] 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 = 14.2 Hz), 4.41 - 4.37 (1H, m), 3.74 - 3.57 (5H, m), 3.05 - 3.01 (1H, m), 2.87 (1H, dd, J = 14.2, 9.3 Hz), 2.68 - 2.59 (1H, m), 2.32 - 2.25 (1H, m), 2.09 - 2.03 (1H, m), 1.82 - 1.76 (1H, m).

[1021] (Step 8)

[1022] N-[4-(11,12-Didehydrodibenzo[b,f]azocine-5(6H)-yl)-4-bridged oxybutanoyl]glycylglycyl-L-phenylalanine 2,5-dibridged oxypyrrolidin-1-yl ester

[1023] To a solution of the compound (2.10 g) obtained in the above step 7 in N,N-dimethylformamide (75.9 mL) were added N-hydroxysuccinimide (961 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.60 g), and the mixture was stirred at room temperature under a nitrogen atmosphere for 21 hours. The reaction solution was diluted with dichloromethane, washed three times with ice water, and then dried over anhydrous sodium sulfate. The desiccant was filtered off, 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 compound was concentrated under reduced pressure, and diisopropyl ether was added to the residue to form a slurry. The resulting solid was filtered to obtain the title compound (2.59 g).

[1024] 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).

[1025] Example 6: Synthesis of Drug Linker 17

[1026] [Synthetic Route]

[1027]

[1028] (Step 1) Methyl [(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]acetate

[1029] To a mixture of commercially available (SUNDIA) N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycylglycine (9.32 g) in tetrahydrofuran (100 mL)-toluene (33.3 mL) were added pyridine (3.26 mL) and lead tetraacetate (17.9 g) at room temperature, and the mixture was stirred at 65 °C for 3 hours. The insoluble material was filtered off, washed with tetrahydrofuran, 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 (8.24 g).

[1030] 11H-NMR (CDCl3) δ: 7.14 (1H, brs), 5.27 (2H, d, J = 7.3 Hz), 5.20 (1H, brs), 4.22 - 4.16 (2H, m), 3.88 (2H, d, J = 6.0 Hz), 2.08 (3H, s), 1.04 - 0.97 (2H, m), 0.05 (9H, s).

[1031] (Step 2)

[1032] 2',3',5'-Tri-O-[tert-butyl(dimethyl)silyl]-1-(2-hydroxyethyl)inosine

[1033] To a mixed solution of 2',3',5'-tri-O-[tert-butyl(dimethyl)silyl]inosine (31.3 g) in tetrahydrofuran (75 mL) - N,N-dimethylacetamide (75 mL), which is known in the literature (Chem. Pharm. Bull. 1987, 35(1), 72 - 79), 2-bromoethanol (4.82 mL) and 1,8-diazabicyclo[5.4.0]-7-undecene (7.65 mL) were added, and the mixture was stirred at room temperature for 23 hours. Water and ethyl acetate were added to the reaction solution, and extraction was carried out with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, 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).

[1034] MS (ESI) m / z: 655 (M + H) + .

[1035] 1 1H-NMR (CDCl3) δ: 8.16 (1H, s), 7.99 (1H, d, J = 2.4 Hz), 5.97 (1H, d, J = 4.2 Hz), 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, 2.4 Hz), 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).

[1036] (Step 3)

[1037] 2’,3’,5’-Tri-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine

[1038] To a solution of the compound obtained in Step 2 (15.6 g) in toluene (46.8 mL) were added the compound obtained in Step 1 (10.4 g) and pyridine (9.63 mL), and the mixture was stirred at 110 °C for 12 hours. The compound obtained in Step 1 (3.46 g) was added to the reaction mixture, and the mixture was stirred at 110 °C for 1 day. Saturated aqueous sodium hydrogen carbonate and dichloromethane were added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, 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).

[1039] MS(ESI) m / z: 885 (M+H) + .

[1040] (Step 4)

[1041] 5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine

[1042] To a solution of the compound obtained in Step 3 (20.6 g) in tetrahydrofuran (50 mL) was added triethylamine trihydrofluoride (10 mL), and the mixture was stirred at room temperature for 17 hours. A mixture of 1M triethylammonium bicarbonate solution (50 mL) and triethylamine (10 mL) was slowly added to the reaction mixture under ice-cooling, and the reaction mixture was concentrated under reduced pressure. The residue was roughly purified by C18 silica gel column chromatography [water / acetonitrile] and then freeze-dried. The resulting crude product was azeotroped with pyridine, and 4,4’-dimethoxytrityl chloride (4.73 g) was added to a pyridine (50 mL) solution of the residue at 0 °C, and the mixture was stirred at 4 °C for 17 hours. Methanol (2 mL) was added to the reaction mixture, and the mixture 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).

[1043] MS(ESI) m / z: 845 (M+H) + .

[1044] 11H-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 = 6.1 Hz), 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 (2H, 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).

[1045] (Step 5)

[1046] 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

[1047] Using the compound (5.96 g) obtained in the above Step 4, the reaction was carried out in the same manner as in Step 10 of Example 1 to obtain 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).

[1048] MS(ESI) m / z: 959 (M+H) + .

[1049] 11H-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 = 5.4 Hz), 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 = 6.0 Hz), 3.48 (1H, dd, J = 10.9, 4.2 Hz), 3.26 (1H, dd, J = 10.9, 4.2 Hz), 3.16 (1H, d, J = 6.7 Hz), 1.00 - 0.93 (2H, m), 0.89 (9H, s), 0.09 (3H, s), 0.02 (9H, s), 0.02 (3H, s).

[1050] Position isomer (2'-O-TBS form)

[1051] MS (ESI) m / z: 959 (M+H) + .

[1052] 1 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 = 5.4 Hz), 5.63 (1H, brs), 4.88 (1H, t, J = 5.1 Hz), 4.66 (2H, d, J = 6.7 Hz), 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, 3.6 Hz), 3.38 (1H, dd, J = 10.9, 3.6 Hz), 2.73 (1H, d, J = 4.2 Hz), 0.97 - 0.90 (2H, m), 0.86 (9H, s), 0.02 (3H, s), 0.01 (9H, s), -0.09 (3H, s).

[1053] (Step 6)

[1054] 5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-[tert-butyl(dimethyl)silyl]-2’-O-{(2-cyanoethoxy)[bis(propane-2-yl)amino]phosphino}-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine

[1055] The compound (2.33 g) obtained in Step 5 above was reacted in the same manner as in Step 11 of Example 1 to obtain the title compound (2.72 g) in the form of a mixture of diastereoisomers on the phosphorus atom (diastereoisomer ratio = 6:4).

[1056] MS(ESI) m / z: 1159 (M+H) + .

[1057] 1 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 = 4.2 Hz), 6.10 (0.4H, d, J = 4.8 Hz), 5.34 (1H, brs), 4.86 - 4.61 (3H, m), 4.48 - 4.42 (1H, m), 4.29 - 4.09 (5H, m), 3.83 - 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 = 6.7 Hz), 1.11 (6H, d, J = 6.7 Hz), 1.04 - 0.92 (2H, m), 0.97 (2.4H, d, J = 6.7 Hz), 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).

[1058] (Step 7)

[1059] 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(bridging oxy)-λ 5Acetonitrile solution of {[(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-bridged oxy-2-mercapto-10-thione octahydro-2H,10H,12H-5,8-methano-2λ

[1060] (Step 8)

[1061] (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-bridged oxy-2-mercapto-10-thione octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospacyclotetradec-7

[1062] -yl]-6-bridged oxy-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2

[1063] -bridged oxyethyl)carbamic acid 2-(trimethylsilyl)ethyl ester

[1064] Using the crude product obtained in the above step 7, the reaction was carried out in the same manner as in step 13 of Example 1 to obtain the title compound (1.47 g: containing impurities) in the form of a mixture of diastereoisomers on the phosphorus atom.

[1065] MS(ESI) m / z: 1278 (M+H) + .

[1066] (Step 9)

[1067] (5R,7R,8R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dibridged oxy-7-[6-bridged oxy-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]pentaoxadispiro[tetradecane]-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[1068] To a mixed solution of methanol (10 mL) - tetrahydrofuran (10 mL) of the compound (1.47 g) obtained in the above step 8 was added 28% aqueous ammonia (10 mL), and the mixture was stirred at 50 °C for 6 hours. After concentrating the reaction solution under reduced pressure, the residue was purified by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] to obtain diastereomer 1 (204 mg: containing impurities) and diastereomer 2 (205 mg: containing impurities) of the title compound.

[1069] Diastereomer 1 (low polarity)

[1070] MS(ESI) m / z: 1121 (M+H) + .

[1071] Diastereomer 2 (high polarity)

[1072] MS(ESI) m / z: 1121 (M+H) + .

[1073] (Step 10-1)

[1074] (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-Fluoro-7-(1-{2

[1075] -[(glycylamino)methoxy]ethyl}-6-bridged oxy-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-dibridged oxy-14-(6,7,8,9-tetrahydro-2H-2,3,5,6

[1076] -tetraazabenzo[cd]azulene-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5

[1077] -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadispiro[tetradecane]-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[1078] To a solution of the compound (diastereomer 1) (204 mg) obtained in the above step 9 in tetrahydrofuran (6 mL) was added a solution of tetrabutylammonium fluoride in tetrahydrofuran (ca. 1 M, 3 mL), and the mixture was stirred at room temperature for 33 hours. After storing at 4 °C for 3 days, 10 mM triethylammonium acetate aqueous solution was added to the reaction mixture, and purification was carried out by 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)] to obtain the title compound (40.7 mg: containing impurities).

[1079] MS(ESI) m / z: 863 (M + H) + .

[1080] (Step 10-2)

[1081] (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-Fluoro-7-(1-{2

[1082] -[(glycylamino)methoxy]ethyl}-6-epoxy-1,6-dihydro-9H-purin-9-yl)-16-hydroxy-2,10-diepoxy-14-(6,7,8,9-tetrahydro-2H-2,3,5,6

[1083] -tetrazabenz[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5

[1084] -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphecyclotetradecyne-2,10-bis(thiol) bis(N,N-diethylethylammonium)

[1085] Using the compound (diastereomer 2) (205 mg) obtained in the above step 9, the reaction was carried out in the same manner as in the above step 10-1, and purification was carried out by 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)] to obtain the title compound (50.8 mg: containing impurities).

[1086] MS(ESI) m / z: 863 (M + H) + .

[1087] (Step 11-1)

[1088] N-[4-(11,12-Didehydrodibenz[b,f]azocine-5(6H)-yl)-4-bridged oxybutyryl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dibridged oxy-2,10-dithioether-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]pentaoxadiphosphetecyclotetradecyne-7-yl]-6-bridged oxy-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide bis(N,N-diethylethylammonium)

[1089] (Drug linker 17a: Diastereomer 1)

[1090] To a solution of the compound obtained in Step 10-1 above (40.7 mg) in N,N-dimethylformamide (0.5 mL) were added triethylamine (11 μL) and the compound obtained in Step 8 of Example 5 (25.4 mg), and the mixture was stirred at room temperature for 2 hours. Benzylamine (8 μL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. 10 mM triethylammonium acetate aqueous solution and methanol were added, and purification was carried out by C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 20%-45% (0 minutes - 40 minutes)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 40%-90% (0 minutes - 40 minutes)] to obtain the title compound (25.1 mg).

[1091] MS(ESI) m / z: 1411 (M+H) + .

[1092] 11H-NMR (CD3OD) δ: 8.58 (1H, s), 8.09 (1H, s), 8.04 (1H, s), 7.57 ...

Claims

1. An antibody-drug conjugate represented by the following formula (II): where m 1 ranges from 1 to 10 Ab represents an antibody or a functional fragment of the antibody, wherein the antibody represents any antibody selected from the group consisting of an anti-CD70 antibody, an anti-TROP2 antibody, and an anti-EGFR antibody, the antibody represents any one selected from the following group: an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 39, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 40; an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 44, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 45, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 46; an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 49, and the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 52; an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 55, and the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 58; an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 61, and the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 64; An antibody comprising the following light chain and heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 67, and the heavy chain comprises CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 70; L represents a linker connecting Ab and D, Ab binds directly to L from its amino acid residues or binds to L from the sugar chain of Ab, D represents a compound represented by the following formula (I): Here, L binds to any -NH2 or hydroxyl group contained in L 1 ​ L 1 represents any one of the following three structural groups: Here, the wavy line indicates the substitution position, Q and Q' each independently represent a hydroxyl group or a thiol group, R 21 and R 22 each independently represents a hydroxyl group or a fluorine atom, W represents -NH- or a sulfur atom, The linker L is represented by -Lb-La-Lp-Lc-*, In the formula, the asterisk represents binding to the drug D, Lp represents a linker formed by an amino acid sequence that can be cleaved in a target cell or is absent, 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 -O-C(=O)-, and, -(CH2)n 9 -C(=O)-, Here, n 2 represents an integer from 1 to 3, n 3 represents an integer from 1 to 5, n 4 represents an integer from 0 to 2, n 9 represents an integer from 2 to 7, Lb is represented by any one of the following structural formulas: In the structural formula of Lb shown above, the asterisk represents binding to La, the wavy line represents binding to the sugar chain of Ab, and, Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)- or -NH-heteroaryl-CH2-O(C=O)- or is absent.

2. An antibody-drug conjugate represented by the following formula (II): where m 1 ranges from 1 to 10 Ab represents an antibody or a functional fragment of the antibody, Here, the antibody represents any antibody selected from the group consisting of an anti-CD70 antibody, an anti-TROP2 antibody, and an anti-EGFR antibody, The antibody represents any one selected from the following group: An antibody comprising the following light chain and heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2; An antibody comprising the following light chain and heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 4; An antibody comprising the following light chain and heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 6; An antibody comprising the following light chain and heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 8; An antibody comprising the following light chain and heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10; An antibody comprising the following light chain and heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO: 12, L represents a linker connecting Ab and D, Ab binds directly to L from its amino acid residues or binds to L from the sugar chain of Ab, D represents a compound represented by the following formula (I): Here, L binds to any -NH2 or hydroxyl group contained in L 1 and L 1 represents any one of the following three structural groups: Herein, the wavy line represents the substitution position, Q and Q' each independently represent a hydroxyl group or a thiol group, R 21 and R 22 each independently represents a hydroxyl group or a fluorine atom, W represents -NH- or a sulfur atom, The linker L is represented by -Lb - La - Lp - Lc - *, In the formula, the asterisk represents binding to the drug D, Lp represents a linker formed by an amino acid sequence that can be cleaved in target cells or is absent, 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 -O-C(=O)-, and, -(CH2)n 9 -C(=O)-, Here, n 2 represents an integer from 1 to 3, n 3 represents an integer from 1 to 5, n 4 represents an integer from 0 to 2, n 9 represents an integer from 2 to 7, Lb is represented by any one of the following structural formulas: In the structural formula of Lb shown above, the asterisk represents binding to La, the wavy line represents binding to the sugar chain of Ab, and, Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)- or -NH-heteroaryl-CH2-O(C=O)- or is absent.

3. An antibody-drug conjugate represented by the following formula (II): where m 1 ranges from 1 to 10 Ab represents an antibody or a functional fragment of the antibody, Herein, the antibody represents any antibody selected from the group consisting of an anti-CD70 antibody, an anti-TROP2 antibody, and an anti-EGFR antibody, The antibody represents any one selected from the following group: An antibody comprising a light chain formed by the amino acid sequence of SEQ ID NO: 1 and a heavy chain formed by the amino acid sequence of SEQ ID NO: 2; An antibody comprising a light chain formed by the amino acid sequence of SEQ ID NO: 3 and a heavy chain formed by the amino acid sequence of SEQ ID NO: 4; An antibody comprising a light chain formed by the amino acid sequence of SEQ ID NO: 5 and a heavy chain formed by the amino acid sequence of SEQ ID NO: 6; An antibody comprising a light chain formed by the amino acid sequence of SEQ ID NO: 7 and a heavy chain formed by the amino acid sequence of SEQ ID NO: 8; An antibody comprising a light chain formed by the amino acid sequence of SEQ ID NO: 9 and a heavy chain formed by the amino acid sequence of SEQ ID NO: 10; An antibody comprising a light chain formed by the amino acid sequence of SEQ ID NO: 11 and a heavy chain formed by the amino acid sequence of SEQ ID NO: 12, L represents a linker connecting Ab and D, Ab binds directly to L from its amino acid residues or binds to L from the sugar chain of Ab, D represents a compound represented by the following formula (I): Here, L binds to any -NH2 or hydroxyl group contained in L 1 and L 1 represents any one of the following three structural groups: Herein, the wavy line represents the substitution position, Q and Q' each independently represent a hydroxyl group or a thiol group, R 21 and R 22 each independently represents a hydroxyl group or a fluorine atom, W represents -NH- or a sulfur atom, The linker L is represented by -Lb - La - Lp - Lc - *, In the formula, the asterisk represents binding to the drug D, Lp represents a linker formed by an amino acid sequence that can be cleaved in a target cell or is absent. 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 -O-C(=O)-, and, -(CH2)n 9 -C(=O)-, Here, n 2 represents an integer from 1 to 3, n 3 represents an integer from 1 to 5, n 4 represents an integer from 0 to 2, n 9 represents an integer from 2 to 7, Lb is represented by any one of the following structural formulas: In the structural formula of Lb shown above, the asterisk represents binding to La, the wavy line represents binding to the sugar chain of Ab, and Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)-, or -NH-heteroaryl-CH2-O(C=O)- or is absent.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following two structural formulas: Here, L 1 , Q, Q', and W are defined as described above.

5. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following four structural formulas: Here, the asterisk represents binding to L, and Q, Q', and W are as defined above.

6. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following three structural formulas: Here, the asterisk represents binding to L, and W is as defined above.

7. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following three structural formulas: Here, the asterisk represents binding to L.

8. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following four structural formulas: Here, the asterisk represents binding to L.

9. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by the following formula: Here, the asterisk represents binding to L.

10. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following two structural formulas: Here, the asterisk represents binding to L, and W is as defined above.

11. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any one of the following four structural formulas: Here, the asterisk represents binding to L.

12. The antibody-drug conjugate according to any one of claims 1 to 3, wherein Lc is -NH-CH2-.

13. The antibody-drug conjugate according to any one of claims 1 to 3, wherein Lp is any one of -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, or -GGPP-.

14. The antibody-drug conjugate according to claim 13, wherein Lp is -GGFG- or -GGPI-.

15. The antibody-drug conjugate according to any one of claims 1 to 3, wherein La represents any one selected from the group consisting of -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)-.

16. The antibody-drug conjugate according to any one of claims 1 to 3, wherein, The linker L is represented by -Lb-La-Lp-Lc-*, In the formula, the asterisk represents binding to the drug D, Lp is -GGFG- or -GGPI-, La represents -C(=O)-CH2CH2-C(=O)-, Lb represents the following formula: In the structural formula of Lb shown above, the asterisk represents binding to La, and the wavy line represents binding to the sugar chain of Ab, Lc represents -NH-CH2-.

17. The antibody-drug conjugate according to any one of claims 1 to 3, wherein, The average number of drug bindings per molecule of antibody in the antibody-drug conjugate is in the range of 1 to 10.

18. The antibody-drug conjugate according to claim 17, wherein, The average number of drug bindings per molecule of antibody in the antibody-drug conjugate is in the range of 1 to 5.

19. The antibody-drug conjugate according to claim 18, wherein, The average number of drug bindings per molecule of antibody in the antibody-drug conjugate is in the range of 3 to 5.

20. The antibody-drug conjugate according to any one of claims 1 to 3, wherein, The antibody binds to the sugar chain through Asn297 of the antibody and binds to L through the sugar chain, and the sugar chain is the N297 sugar chain.

21. The antibody-drug conjugate according to any one of claims 1 to 3, wherein, The sugar chain of the Ab is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula: In the formula, the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man of the N297 sugar chain. The asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 is an integer from 2 to 5; In the formula, the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain The asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 is an integer from 2 to 5.

22. The antibody-drug conjugate according to claim 20, which is represented by the following formula: where m 2 represents the integer 1 or 2, L is a linker connecting the N297 sugar chain of Ab and D, as defined above, Ab is as defined in any one of claims 1 to 3, and the N297 sugar chain of Ab is represented as N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula, In the formula, the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) undergoes amide bonding with the 2-position carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man of the N297 sugar chain The asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents integers from 2 to 5; In the formula, the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain. The asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents integers from 2 to 5, D is represented by any one of the following four structural formulas, Here, in the formula, the asterisk represents binding to L.

23. The antibody-drug conjugate according to claim 22, which is selected from the following formulas: In each of the above - shown structural formulas, m 2 represents an integer 1 or 2, Ab is as defined in any one of claims 1 to 3; The N297 glycan of Ab is represented by either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula, wherein the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the branch of β-Man of the N297 sugar chain the asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents integers from 2 to 5; wherein the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain the asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents integers from 2 to 5.

24. The antibody-drug conjugate according to claim 23, which is selected from the following formula: In each of the above - shown structural formulas, m 2 represents an integer 1 or 2, Ab is as defined in any one of claims 1 to 3; The N297 glycan of Ab is represented by either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown by the following formula, wherein the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man of the N297 sugar chain the asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents integers 2 to 5; wherein the wavy line represents binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain the asterisk represents binding to the 1-position or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb of the linker L, and, n 5 represents integers from 2 to 5.

25. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the antibody or an antigen-binding fragment of the antibody is an anti-CD70 antibody or an antigen-binding fragment of the antibody.

26. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the antibody or an antigen-binding fragment of the antibody is an anti-TROP2 antibody or an antigen-binding fragment of the antibody.

27. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the antibody or an antigen-binding fragment of the antibody is an anti-EGFR antibody or an antigen-binding fragment of the antibody.

28. The antibody-drug conjugate according to claim 25, wherein the antibody is an antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 2, or an antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO:

4.

29. The antibody-drug conjugate according to claim 26, wherein the antibody is an antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 6, or an antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO:

8.

30. The antibody-drug conjugate according to claim 27, wherein the antibody is an antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 10, or an antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO:

12.

31. The antibody-drug conjugate according to claim 25, wherein The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO: 2, or, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 118 of SEQ ID NO:

4.

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

8.

33. The antibody-drug conjugate according to claim 27, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 119 of SEQ ID NO: 10, or, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence set forth in amino acid numbers 1 to 116 of SEQ ID NO:

12.

34. The antibody-drug conjugate according to claim 25, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence of SEQ ID NO: 35, CDRL2 formed by the amino acid sequence of SEQ ID NO: 36, and CDRL3 formed by the amino acid sequence of SEQ ID NO: 37, and the heavy chain comprises CDRH1 formed by the amino acid sequence of SEQ ID NO: 38, CDRH2 formed by the amino acid sequence of SEQ ID NO: 39, and CDRH3 formed by the amino acid sequence of SEQ ID NO: 40, or, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises CDRL1 formed by the amino acid sequence of SEQ ID NO: 41, CDRL2 formed by the amino acid sequence of SEQ ID NO: 42, and CDRL3 formed by the amino acid sequence of SEQ ID NO: 43, and the heavy chain comprises CDRH1 formed by the amino acid sequence of SEQ ID NO: 44, CDRH2 formed by the amino acid sequence of SEQ ID NO: 45, and CDRH3 formed by the amino acid sequence of SEQ ID NO:

46.

35. The antibody-drug conjugate according to claim 26, wherein, the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 49, the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 50, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 51, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 52, or, the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 55, the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 56, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 57, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO:

58.

36. The antibody-drug conjugate according to claim 27, wherein, the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 59, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 60, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 61, the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 62, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 63, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO: 64, or, the antibody is an antibody comprising a light chain and a heavy chain, the light chain comprising CDRL1 formed by the amino acid sequence set forth in SEQ ID NO: 65, CDRL2 formed by the amino acid sequence set forth in SEQ ID NO: 66, and CDRL3 formed by the amino acid sequence set forth in SEQ ID NO: 67, the heavy chain comprising CDRH1 formed by the amino acid sequence set forth in SEQ ID NO: 68, CDRH2 formed by the amino acid sequence set forth in SEQ ID NO: 69, and CDRH3 formed by the amino acid sequence set forth in SEQ ID NO:

70.

37. The antibody-drug conjugate according to any one of claims 25, 28, 31 or 34, which exhibits an antibody target-dependent anti-tumor effect in BALB / c-nu mice subcutaneously transplanted with human renal cancer cell line Caki-1 cells.

38. An antibody-drug conjugate represented by the following formula: wherein Ab represents any one selected from the group consisting of: an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 2; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 4; an antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 6; An antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 8; An antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 10; An antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 12; The N297 glycan of Ab is represented by the following formula: In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain The asterisk indicates binding to the nitrogen atom at the 1-position or 3-position of the 1,2,3-triazole ring in the antibody-drug conjugate structure of the above formula, and n 5 is 3, and, m 2 is 2.

39. An antibody-drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the group consisting of: An antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2; An antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 4; An antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 6; An antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 8; An antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10; An antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO: 12; The N297 glycan of Ab is represented by the following formula: In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-position carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain The asterisk indicates binding to the nitrogen atom at the 1-position or 3-position of the 1,2,3-triazole ring in the antibody-drug conjugate structure of the above formula, and n 5 is 3, and, m 2 is 2.

40. The antibody-drug conjugate according to claim 39, wherein the antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO:

2.

41. The antibody-drug conjugate according to claim 39, wherein The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO:

4.

42. The antibody-drug conjugate according to claim 39, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO:

6.

43. The antibody-drug conjugate according to claim 39, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO:

8.

44. The antibody-drug conjugate according to claim 39, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:

10.

45. The antibody-drug conjugate according to claim 39, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO:

12.

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

47. An antibody-drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following group: An antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 2; An antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 3 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 4; An antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 5 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 6; An antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 7 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 8; An antibody comprising a light chain formed by the amino acid sequence set forth in SEQ ID NO: 9 and a heavy chain formed by the amino acid sequence set forth in SEQ ID NO: 10; An antibody comprising a light chain formed from the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain formed from the amino acid sequence set forth in SEQ ID NO: 12; The N297 glycan of the Ab is represented by the following formula: In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain The asterisk indicates binding to the nitrogen atom at the 1-position or 3-position of the 1,2,3-triazole ring in the antibody-drug conjugate structure of the above formula, and n 5 is 3, and, m 2 is 1.

48. An antibody-drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the group consisting of: An antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2; An antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 4; An antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 6; An antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 8; An antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10; An antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO: 12; The N297 glycan of the Ab is represented by the following formula: In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) is amide-bonded to the 2-carboxyl group of the sialic acid at the non-reducing ends of both the 1-3 chain side and the 1-6 chain side of the β-Man of the N297 sugar chain The asterisk indicates binding to the nitrogen atom at the 1-position or 3-position of the 1,2,3-triazole ring in the antibody-drug conjugate structure of the above formula, and n 5 is 3, and, m 2 is 1.

49. The antibody-drug conjugate according to claim 48, wherein the antibody is an antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 1, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO:

2.

50. The antibody-drug conjugate according to claim 48, wherein the antibody is an antibody comprising the following light and heavy chains, wherein the light chain comprises a light chain variable region formed from the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 3, and the heavy chain comprises a heavy chain variable region formed from the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO:

4.

51. The antibody-drug conjugate according to claim 48, wherein The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 5, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO:

6.

52. The antibody-drug conjugate according to claim 48, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 7, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO:

8.

53. The antibody-drug conjugate according to claim 48, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 9, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:

10.

54. The antibody-drug conjugate according to claim 48, wherein, The antibody is an antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region formed by the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 11, and the heavy chain comprises a heavy chain variable region formed by the amino acid sequence of amino acid numbers 1 to 116 of SEQ ID NO:

12.

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

56. The antibody-drug conjugate according to any one of claims 1 to 3, 38 to 55, wherein, One or two amino acids are deleted from the carboxyl terminus of the heavy chain.

57. The antibody-drug conjugate according to any one of claims 1 to 3, 38 to 55, wherein, The antibody or an antigen-binding fragment of the antibody is a protein produced in a suitable host cell using a genetically engineered antibody gene.

58. The antibody-drug conjugate according to any one of claims 47 to 55, which exhibits a stronger anti-tumor effect in the animal described in the following (i) or (ii) than the antibody contained in the antibody-drug conjugate: (i) A BALB / c mouse subcutaneously transplanted with the mouse colon cancer cell line CT26.WT, into which a gene of a human-mouse chimeric antigen in which the epitope site on the antigen bound by the antibody contained in the antibody-drug conjugate is replaced with a human type has been introduced; (ii) A BALB / c-nu mouse subcutaneously transplanted with the human renal cancer cell line A-498 cells.

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

60. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 58.

61. An anti-tumor agent comprising the antibody-drug conjugate according to any one of claims 1 to 58.

62. The anti-tumor agent according to claim 61, wherein the tumor is lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, squamous cell carcinoma, thymic carcinoma, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

63. The anti-tumor agent according to claim 61, wherein the tumor is lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, cervical cancer, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, squamous cell carcinoma, thymic carcinoma or small intestine cancer.

64. The anti-tumor agent according to claim 61, wherein the tumor is glioblastoma multiforme, tongue cancer, pharyngeal cancer and auditory organ cancer.

65. Use of an antibody-drug conjugate according to any one of claims 1 to 58 in the manufacture of a STING agonist, a pharmaceutical composition or an anti-tumor agent for treating cancer.

66. The use according to claim 65, wherein the cancer is lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, adrenal cancer, squamous cell carcinoma, thymic carcinoma, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

67. The use according to claim 65, wherein the cancer is lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, cervical cancer, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, cholangiocarcinoma, squamous cell carcinoma, thymic carcinoma or small intestine cancer.

68. The use according to claim 65, wherein the cancer is glioblastoma multiforme, tongue cancer, pharyngeal cancer and auditory organ cancer.

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