Cyclic dinucleotide derivative and antibody-drug conjugate thereof

Abstract

It is desired to develop a novel CDN derivative having a STING agonist activity and a therapeutic agent and / or a therapeutic method for a disease associated with a STING agonist activity using the novel CDN derivative. In addition, it is desired to develop a therapeutic agent and / or a therapeutic method for a disease associated with STING agonist activity, which can specifically deliver the novel CDN derivative to a target cell or organ.SOLUTION: The present invention provides novel CDN derivatives having strong STING agonist activity, and antibody CDN derivative conjugates comprising the novel CDN derivatives.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cyclic dinucleotide derivatives having a novel structure and having STING agonist activity, antibody-drug conjugates in which the novel cyclic dinucleotide derivatives are linked to antibodies against target cells via a linker, and pharmaceutical compositions containing the antibody-drug conjugates. [Background technology]

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

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

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

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

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

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

[0008] [Patent Document 1] WO2014 / 099824 [Patent Document 2] WO2014 / 179335 [Patent Document 3] WO2014 / 189805 [Patent Document 4] WO2014 / 189806 [Patent Document 5] WO2015 / 074145 [Patent Document 6] WO2015 / 185565 [Patent Document 7] WO2016 / 096714 [Patent Document 8] WO2016 / 012305 [Patent Document 9] WO2016 / 145102 [Patent Document 10] WO2017 / 027646 [Patent Document 11] WO2017 / 027645 [Patent Document 12] WO2017 / 075477 [Patent Document 13] WO2017 / 093933 [Patent Document 14] WO2017 / 100305 [Patent Document 15] WO2017 / 123669 [Patent Document 16] WO2017 / 161349 [Patent Document 17] WO2017 / 175147 [Patent Document 18] WO2017 / 175156 [Patent Document 19] WO2018 / 009466 [Patent Document 20] WO2018 / 045204 [Patent Document 21] WO2018 / 060323 [Patent Document 22] WO2018 / 067423 [Patent Document 23] WO2018 / 065360 [Patent Document 24] WO2014 / 093936 [Patent Document 25] WO2018 / 009648 [Patent Document 26] WO2018 / 100558 [Non-patent literature]

[0009] [Non-Patent Document 1] Nature 2008, 455, 674-678 [Non-Patent Document 2] Mol.Cell, 2013, 51, 226-235 [Non-Patent Document 3] Science 2015a, 347, aaa2630 [Non-Patent Document 4] J.Virol. 2014, 88, 5328-5341 [Non-Patent Document 5] [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​There is a need for the development of CDN derivatives with novel backbones that have STING agonist activity and activate immune cells by increasing the production of inflammatory proteins such as interferons and cytokines, as well as therapeutic agents and / or methods using the novel CDN derivatives for diseases associated with STING agonist activity, such as diseases treatable by immunostimulation (e.g., cancer). There is also a need for the development of antibody-drug conjugates in which the novel CDN derivatives are linked via a linker to antibodies against target cells, which can be administered systemically and deliver STING agonists specifically to target cells or organs (e.g., tumor sites), and therapeutic agents and / or methods using the antibody-drug conjugates for diseases associated with STING agonist activity, such as diseases treatable by immunostimulation (e.g., cancer). [Means for solving the problem]

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

[0012] That is, the present invention relates to the following: [1] Formula (II):

[0013] [ka] (In the formula, m 1 is in the range of 1 to 10, Ab refers to an antibody or a functional fragment of the antibody, and the sugar chain of the antibody may be remodeled. L represents a linker connecting Ab and D, Ab may be bound to L directly through its amino acid residue, or may be bound to L through a glycan or a remodeled glycan of Ab; D is a group represented by the following formula (I):

[0014] [ka] (where, L is L 1 or L 2 and binds to any -NH2 or hydroxy group contained in L 1 is a group represented by the following formula, which may be substituted at any position with one to three groups selected from the group consisting of a hydroxy group, -NH2, a 2-hydroxyacetylaminomethyl group, and a 2-[(2-hydroxyacetyl)amino]ethyl group:

[0015] [ka] (where, R 6 and R 6’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, —NH2, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group, R 7 and R 7’ each independently represents a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is optionally substituted by one or two substituents selected from the group consisting of a halogen atom and an oxo group; R 8 and R 8’ each independently represents a hydrogen atom or a halogen atom, Z 4 represents -CH2-, -NH- or an oxygen atom, Z 5 represents a nitrogen atom or -CH= represents a group selected from the group consisting of L 2 is either (i) or (ii) below: (i) When combined with L, L 2represents -NHR', a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group, where R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group is optionally substituted with 1 to 6 halogen atoms; or (ii) When not bonded to L, L 2 represents a hydrogen atom or a halogen atom, represents a group selected from Q 1 and Q 1’ are each independently a hydroxy group, a thiol group, or a borano group (BH3 - ) and Q 2 and Q 2’ each independently represents an oxygen atom or a sulfur atom, X 1 and X 2 each independently represents an oxygen atom, a sulfur atom, or -CH-, Y 1 and Y 2 represents an oxygen atom or -CH2-, X 3 and X 4 is either (iii) or (iv) below: (iii) Y 1 When is an oxygen atom, X 3 -X 4 represents -CH2-O-, -CH2-S-, -CH2-CH2- or -CH2-CF2-; or (iv) Y 1 When is -CH2-, X 3 -X 4 represents -O-CH2-, represents a group selected from X 5 and X 6 is (v) or (vi) below: (v) Y 2 When is an oxygen atom, X 5 -X 6 represents -CH2-O-, -CH2-S-, -CH2-CH2- or -CH2-CF2-; or (vi) Y 2 When is -CH2-, X 5 -X 6 represents -O-CH2-, represents a group selected from R 1 ,R 2 and R 3 each independently represents a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N3, -NHR', -NR'R'', or -NHC(=O)R' (wherein R' is as defined above and R'' represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group); W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom, or —CH—, W 2 represents a nitrogen atom or -CH=, R 4 represents a hydrogen atom, a halogen atom, or -NH2, R 5 is (vii) to (x) below: (vii)W 1 When is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group; (viii)W 1 When is an oxygen atom, R 5 does not exist; (ix)W 1 is a sulfur atom, R 5 does not exist; or (x)W 1 When is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxy group, -NH2 or a C1-C6 alkyl group, represents a group selected from Z 1 -Z 2 -Z 3are taken together to form -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- or -CH2-CH2-CH(CH3)- (wherein R''' represents -O- or -CH2-CH2-, and X represents a halogen atom), or a group represented by any of the following formulae:

[0016] [ka] (where the asterisk stands for W 1 and the wavy line indicates that the group is bonded to the carbon atom of =C-. (This indicates a compound represented by the formula: an antibody-drug conjugate represented by: [2] W 1 The antibody-drug conjugate according to [1], wherein [3] W 1 is a nitrogen atom, and R 5 The antibody-drug conjugate according to [2], wherein [4]W 1 The antibody-drug conjugate according to [1], wherein [5]W 1 The antibody-drug conjugate according to [1], wherein [6]W 1 The antibody-drug conjugate according to [1], wherein: [7]W 1 is -CH- and R 5 the antibody-drug conjugate according to [6], wherein [8]Z 1 , Z 2 and Z 3 and [9]Z 1 , Z2 and Z 3 and

[10] Z 1 , Z 2 and Z 3 and R′″ are taken together to form —CH—CH—R′″— (wherein R′″ represents —O— or —CH—CH—);

[11] W 2 The antibody-drug conjugate according to any one of [1] to

[10] , wherein

[12] W 2 The antibody-drug conjugate according to any one of [1] to

[10] , wherein

[13] R 4 represents a hydrogen atom;

[14] R 4 represents a fluorine atom;

[15] L 1 R in 8 and R 8’ and each independently represents a hydrogen atom;

[16] L 1 is a group selected from the group consisting of the following formulas:

[0017] [ka] (where R 9 and R 9’ represents a hydrogen atom, a halogen atom, a hydroxy group, or -NH2, R 10 represents a hydroxy group, -NH, -NHC(=O)CHOH, -CHNHC(=O)CHOH, -CHCHNHC(=O)CHOH, a hydroxy C1-C3 alkyl group or an amino C1-C3 alkyl group, R11 and R 11’ each independently represents a hydrogen atom, a fluorine atom, or a methyl group, or R 11 and R 11’ is bonded to form a cyclopropane, Z 4 represents -CH2-, -NH- or an oxygen atom) The antibody-drug conjugate according to any one of [1] to

[15] , wherein

[17] L 1 is a group selected from the group consisting of the following formulas:

[0018] [ka] (where R 13 and R 13’ each independently represents a hydrogen atom, a hydroxy group, or -NH; R 12 represents a hydroxy group, -NH, -CHOH, -NHC(=O)CHOH, -CHNHC(=O)CHOH or -CHCHNHC(=O)CHOH, Z 4 is as previously defined) The antibody-drug conjugate according to any one of [1] to

[15] , wherein

[18] L 1 is a group selected from the group consisting of the following formulas:

[0019] [ka] (where R 14 represents a hydrogen atom or -NH2, R 15 represents a hydrogen atom or -C(=O)CHOH, R 16 represents a hydroxy group, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 or -CH2CH2NH2) The antibody-drug conjugate according to any one of [1] to

[15] , wherein

[19] L 2is bonded to L and represents -NH2, -CH2NH2, or -CH2OH;

[20] L 2 is not bonded to L and represents a hydrogen atom or a fluorine atom;

[21] Q 1 and Q 1’ each independently represent a hydroxy group or a thiol group;

[22] X 1 and X 2 represents an oxygen atom;

[23] Y 1 and Y 2 represents an oxygen atom;

[24] X 3 and X 4 represents —CH2—O—;

[25] X 5 and X 6 represents —CH2—O—;

[26] R 1 ,R 2 and R 3 are each independently a hydrogen atom, a hydroxy group, or a fluorine atom, [the antibody-drug conjugate according to any one of items 1 to 25]

[27] D is the following two equations:

[0020] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 and Q 2’ is as previously defined, R 17 , R 17’ , R18 and R 18’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, or -NH; W 3 represents -NH-, an oxygen atom, a sulfur atom, or -CH2-, W 4 represents -CH= or a nitrogen atom) The antibody-drug conjugate according to any one of [1] to

[26] , wherein

[28] D is the following two equations:

[0021] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 , Q 2’ , R 17 , R 17’ , R 18 and R 18’ is as previously defined) The antibody-drug conjugate according to

[27] , wherein

[29] D is the following 8 formulas:

[0022] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 and Q 2’ is as previously defined, R 19 , R 19’ , R 20 and R 20’ each independently represents a hydrogen atom or a fluorine atom) The antibody-drug conjugate according to

[27] or

[28] , wherein

[30] D is the following four formulas:

[0023] [ka] (where L 1 is as previously defined) The antibody-drug conjugate according to any one of

[27] to

[29] , wherein

[31] D is the following four formulas:

[0024] [ka] (where L 1 is as previously defined) The antibody-drug conjugate according to any one of

[27] to

[30] , wherein

[32] D is the following four equations:

[0025] [ka] (where L 1 is as previously defined) The antibody-drug conjugate according to any one of

[27] to

[30] , wherein

[33] D is a compound of the formula:

[0026] [ka] (where L 1 is as previously defined, Q 3 and Q 3‘ each independently represents a hydroxy group or a thiol group, R 21 and R 22 each independently represents a hydroxy group or a fluorine atom, W 5 represents -NH- or a sulfur atom) The antibody-drug conjugate according to any one of [1] to

[26] , wherein

[34] D is the following two equations:

[0027] [ka] (where L 1 , Q 3 and Q 3‘ , W 5 is as previously defined) The antibody-drug conjugate according to

[33] , wherein

[35] L 1 But the following four equations:

[0028] [ka] The antibody-drug conjugate according to any one of [1] to

[34] , wherein

[36] L 1 But the following four equations:

[0029] [ka] (wherein the asterisk indicates a bond to L) The antibody-drug conjugate according to any one of [1] to

[34] , wherein

[37] D is the following four formulas:

[0030] [ka] (where the asterisk indicates a bond with L, and Q 3 , Q 3‘ and W 5 is as previously defined) The antibody-drug conjugate according to any one of

[33] ,

[34] , and

[36] , wherein

[38] D is the following four formulas:

[0031] [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of

[33] ,

[34] ,

[36] , and

[37] , wherein

[39] D is the following three formulas:

[0032] [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of

[33] ,

[34] ,

[36] , and

[37] , wherein

[40] D is the following four formulas:

[0033] [ka] (where the asterisk indicates binding to L) The antibody-drug conjugate according to any one of

[33] ,

[34] ,

[36] , and

[37] , wherein

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

[40] ;

[42] The antibody-drug conjugate according to

[41] , wherein Lc is not present;

[43] The antibody-drug conjugate according to

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

[44] Lp is: -GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit- and -ICit- The antibody-drug conjugate according to any one of

[41] to

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

[45] The antibody-drug conjugate according to

[44] , wherein Lp is any one of -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, and -FCit-;

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

[41] to

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

[47] The antibody-drug conjugate according to

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

[48] La states the following: -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, and -(CH2)5-C(=O)- The antibody-drug conjugate according to any one of

[41] to

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

[49] Lb is

[0034] [ka]

[0035] [ka] Or,

[0036] [ka] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain);

[50] Lb is -(succinimide-3-yl-N)-; wherein -(succinimide-3-yl-N)- has the following structural formula:

[0037] [ka] indicates, wherein the asterisk indicates binding to La, and the wavy line indicates binding via thioether formation with the side chain of a cysteine ​​residue of the antibody;

[51] The linker L is represented by -Lb-La-Lp-Lc-*; In the formula, the asterisk indicates binding to drug D, Lp is -GGFG- or -GGPI-; La represents -C(=O)-CHCH-C(=O)-; Lb is expressed by the following formula

[0038] [ka] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain), The antibody-drug conjugate according to any one of

[41] ,

[46] to

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

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

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

[53] The antibody-drug conjugate according to

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

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

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

[55] The antibody-drug conjugate according to

[54] , wherein the N297 glycan is a remodeled glycan;

[56] The antibody-drug conjugate according to

[54] or

[55] , wherein the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG;

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

[56] , wherein the antibody is an anti-HER2 antibody, an anti-HER3 antibody, an anti-DLL3 antibody, an anti-FAP antibody, an anti-CDH11 antibody, an anti-CDH6 antibody, an anti-A33 antibody, an anti-CanAg antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD98 antibody, an anti-TROP2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-Mesothelin antibody, an anti-ENPP3 antibody, an anti-CD47 antibody, an anti-EGFR antibody, an anti-GPR20 antibody, or an anti-DR5 antibody;

[58] The antibody-drug conjugate according to

[57] , wherein the antibody is an anti-HER2 antibody;

[59] The antibody-drug conjugate according to

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

[60] The antibody-drug conjugate according to

[58] , wherein the antibody comprises a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 28 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 29, or a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 28 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 30;

[61] The antibody-drug conjugate according to

[57] , wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 31 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 32, an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 33 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 34, or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 35 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 36;

[62] The following formula (Ia):

[0039] [ka] (where, L 1 is a group represented by the following formula, which may be substituted at any position with one to three groups selected from the group consisting of a hydroxy group, -NH2, a 2-hydroxyacetylaminomethyl group, and a 2-[(2-hydroxyacetyl)amino]ethyl group:

[0040] [ka] (where, R 6 and R 6’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, —NH2, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group, R 7 and R 7’ each independently represents a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is optionally substituted by one or two substituents selected from the group consisting of a halogen atom and an oxo group; R 8 and R 8’ each independently represents a hydrogen atom or a halogen atom, Z 4 represents -CH2-, -NH- or an oxygen atom, Z 5 represents a nitrogen atom or -CH= represents a group selected from the group consisting of L 3 represents a hydrogen atom, a halogen atom, —NH2, a hydroxy C1-C3 alkyl group, or an amino C1-C3 alkyl group, Q 1 and Q 1’ are each independently a hydroxy group, a thiol group, or a borano group (BH3 - ) and Q 2 and Q 2’ each independently represents an oxygen atom or a sulfur atom, X 1 and X 2 each independently represents an oxygen atom, a sulfur atom, or -CH-, Y 1 and Y 2 represents an oxygen atom or -CH2-, X 3 and X 4 is either (iii) or (iv) below: (iii) Y 1 When is an oxygen atom, X 3 -X 4 represents -CH2-O-, -CH2-S-, -CH2-CH2- or -CH2-CF2-; or (iv) Y 1 When is -CH2-, X 3 -X 4 represents -O-CH2-, represents a group selected from X 5 and X 6 is (v) or (vi) below: (v) Y 2 When is an oxygen atom, X 5 -X 6 represents -CH2-O-, -CH2-S-, -CH2-CH2- or -CH2-CF2-; or (vi) Y 2 When is -CH2-, X 5 -X 6 represents -O-CH2-, represents a group selected from R 1 ,R 2 and R 3 each independently represent a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N3, -NHR', -NR'R'', or -NHC(=O)R' (wherein R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, the C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group being optionally substituted with 1 to 6 halogen atoms; and R'' represents a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, or a C3-C6 cycloalkyl group); W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom, or —CH—, W 2represents a nitrogen atom or -CH=, R 4 represents a hydrogen atom, a halogen atom, or -NH2, R 5 is (vii) to (x) below: (vii)W 1 When is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group; (viii)W 1 When is an oxygen atom, R 5 does not exist; (ix)W 1 is a sulfur atom, R 5 does not exist; or (x)W 1 When is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxy group, -NH2 or a C1-C6 alkyl group, represents a group selected from Z 1 -Z 2 -Z 3 are taken together to form -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- or -CH2-CH2-CH(CH3)- (wherein R''' represents -O- or -CH2-CH2-, and X represents a halogen atom), or a group represented by any of the following formulae:

[0041] [ka] (where the asterisk stands for W 1 and the wavy line indicates that the group is bonded to the carbon atom of =C-. or a pharmacologically acceptable salt thereof;

[63] W 1 is a nitrogen atom, or a pharmacologically acceptable salt thereof;

[64] W 1 is a nitrogen atom, and R 5 is a hydrogen atom, or a pharmacologically acceptable salt thereof;

[65] W 1 is an oxygen atom, or a pharmacologically acceptable salt thereof;

[66] W 1 is a sulfur atom, or a pharmacologically acceptable salt thereof;

[67] W 1 the compound according to

[62] or a pharmacologically acceptable salt thereof, wherein

[68] W 1 is -CH- and R 5 is a hydrogen atom, or a pharmacologically acceptable salt thereof;

[69] Z 1 , Z 2 and Z 3 and R are taken together to be -CH2-CH2-CH2- or -CH=CH-CH2-;

[70] Z 1 , Z 2 and Z 3 and R are taken together to be -CH2-CH(CH3)-CH2- or -CH2-CH2-CH(CH3)-; or a pharmacologically acceptable salt thereof;

[71] Z 1 , Z 2 and Z 3 and R′″ are taken together to form —CH—CH—R′″— (wherein R′″ represents —O— or —CH—CH—);

[72] W 2 the compound according to any one of

[62] to

[71] , or a pharmacologically acceptable salt thereof, wherein

[73] W 2 the compound or pharmacologically acceptable salt thereof according to any one of

[62] to

[71] , wherein

[74] R 4represents a hydrogen atom, or a pharmacologically acceptable salt thereof;

[75] R 4 represents a fluorine atom, or a pharmacologically acceptable salt thereof;

[76] L 1 R in 8 and R 8’ are each independently a hydrogen atom, or a pharmacologically acceptable salt thereof;

[77] L 1 is a group selected from the group consisting of the following formulas:

[0042] [ka] (where R 9 and R 9’ represents a hydrogen atom, a halogen atom, a hydroxy group, or -NH2, R 10 represents a hydroxy group, -NH, -NHC(=O)CHOH, -CHNHC(=O)CHOH, -CHCHNHC(=O)CHOH, a hydroxy C1-C3 alkyl group or an amino C1-C3 alkyl group, R 11 and R 11’ each independently represents a hydrogen atom, a fluorine atom, or a methyl group, or R 11 and R 11’ is bonded to form a cyclopropane, Z 4 represents -CH2-, -NH- or an oxygen atom) the compound according to any one of

[62] to

[76] , or a pharmacologically acceptable salt thereof;

[78] L 1 is a group selected from the group consisting of the following formulas:

[0043] [ka] (where R 13 and R 13’each independently represents a hydrogen atom, a hydroxy group, or -NH; R 12 represents a hydroxy group, -NH, -CHOH, -NHC(=O)CHOH, -CHNHC(=O)CHOH or -CHCHNHC(=O)CHOH, Z 4 is as previously defined) the compound according to any one of

[62] to

[76] , or a pharmacologically acceptable salt thereof;

[79] L 1 is a group selected from the group consisting of the following formulas:

[0044] [ka] (where R 14 represents a hydrogen atom or -NH2, R 15 represents a hydrogen atom or -C(=O)CHOH, R 16 represents a hydroxy group, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 or -CH2CH2NH2) the compound according to any one of

[62] to

[76] , or a pharmacologically acceptable salt thereof;

[80] L 3 represents a hydrogen atom, a fluorine atom, —NH2, —CH2OH, or —CH2NH2, or a pharmacologically acceptable salt thereof;

[81] Q 1 and Q 1’ each independently represent a hydroxy group or a thiol group, or a pharmacologically acceptable salt thereof;

[82] X 1 and X 2 represents an oxygen atom, or a pharmacologically acceptable salt thereof;

[83] Y 1 and Y 2 represents an oxygen atom, or a pharmacologically acceptable salt thereof;

[84] X 3 and X 4 represents —CH2—O—; the compound according to any one of

[62] to

[83] , or a pharmacologically acceptable salt thereof;

[85] X 5 and X 6 represents —CH2—O—; the compound according to any one of

[62] to

[84] , or a pharmacologically acceptable salt thereof;

[86] R 1 ,R 2 and R 3 are each independently a hydrogen atom, a hydroxy group, or a fluorine atom, or a compound or a pharmacologically acceptable salt thereof according to any one of

[62] to

[85] ;

[87] The following two equations:

[0045] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 and Q 2’ is as previously defined, R 17 , R 17’ , R 18 and R 18’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, or -NH; W 3 represents -NH-, an oxygen atom, a sulfur atom, or -CH2-, W 4 represents -CH= or a nitrogen atom) the compound according to any one of

[62] to

[86] , or a pharmacologically acceptable salt thereof,

[88] The following two equations:

[0046] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 , Q 2’, R 17 , R 17’ , R 18 and R 18’ is as previously defined) the compound according to

[87] or a pharmacologically acceptable salt thereof,

[89] The following eight equations:

[0047] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 and Q 2’ is as previously defined, R 19 , R 19’ , R 20 and R 20’ each independently represents a hydrogen atom or a fluorine atom) the compound according to

[87] or

[88] , or a pharmacologically acceptable salt thereof,

[90] The following four equations:

[0048] [ka] (where L 1 is as previously defined) the compound according to any one of

[87] to

[89] , or a pharmacologically acceptable salt thereof,

[91] The following four equations:

[0049] [ka] (where L 1 is as previously defined) the compound according to any one of

[87] to

[90] , or a pharmacologically acceptable salt thereof,

[92] The following four equations:

[0050] [ka] (where L 1 is as previously defined) the compound according to any one of

[87] to

[90] , or a pharmacologically acceptable salt thereof,

[93] The following formula:

[0051] [ka] (where L 1 is as previously defined, Q 3 and Q 3‘ each independently represents a hydroxy group or a thiol group, R 21 and R 22 each independently represents a hydroxy group or a fluorine atom, W 5 represents -NH- or a sulfur atom) the compound according to any one of

[62] to

[86] , or a pharmacologically acceptable salt thereof;

[94] The following two equations:

[0052] [ka] (where L 1 , Q 3 and Q 3‘ , W 5 is as previously defined) the compound according to

[93] or a pharmacologically acceptable salt thereof,

[95] L 1 But the following:

[0053] [ka] the compound according to any one of

[62] to

[94] , or a pharmacologically acceptable salt thereof,

[96] L 1 But the following four equations:

[0054] [ka] the compound according to any one of

[62] to

[94] , or a pharmacologically acceptable salt thereof,

[97] D is one of the following four formulas:

[0055] [ka] (where Q 3 , Q 3‘ and W 5 is as previously defined) the compound according to any one of

[93] ,

[94] and

[96] , or a pharmacologically acceptable salt thereof,

[98] D is one of the following four formulas:

[0056] [ka] the compound according to any one of

[93] ,

[94] ,

[96] and

[97] , or a pharmacologically acceptable salt thereof, wherein

[99] D is one of the following three formulas:

[0057] [ka] the compound according to any one of

[93] ,

[94] ,

[96] and

[97] , or a pharmacologically acceptable salt thereof, wherein

[0100] D is the following four equations:

[0058] [ka] the compound according to any one of

[93] ,

[94] ,

[96] and

[97] , or a pharmacologically acceptable salt thereof, wherein

[0101] A STING agonist comprising any one selected from the group consisting of the antibody-drug conjugates according to [1] to

[61] and the compounds according to

[62] to

[0100] or pharmacologically acceptable salts thereof;

[0102] A pharmaceutical composition comprising any one selected from the group consisting of the antibody-drug conjugates according to [1] to

[61] and the compounds according to

[62] to

[0100] or pharmacologically acceptable salts thereof;

[0103] an antitumor agent comprising any one selected from the group consisting of the antibody-drug conjugates according to [1] to

[61] and the compounds according to

[62] to

[0100] or pharmacologically acceptable salts thereof;

[0104] The antitumor agent according to

[0103] , wherein the tumor is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma;

[0105] A method for treating cancer, comprising administering any one selected from the group consisting of the antibody-drug conjugate described in [1] to

[61] , the compound described in

[62] to

[0100] or a pharmacologically acceptable salt thereof, the STING agonist described in

[0101] , the pharmaceutical composition described in

[0102] , and the antitumor agent described in

[0103] or

[0104] ;

[0106] The method according to

[0105] , wherein the cancer is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma; Regarding. [Effects of the Invention]

[0059] The present invention provides novel CDN derivatives. The novel CDN derivatives of the present invention have strong STING agonist activity and exhibit high antitumor activity. The present invention also provides novel antibody-CDN derivative conjugates that can be administered systemically and exhibit antitumor effects on tumors expressing the antigen. [Brief explanation of the drawings]

[0060] [Figure 1] Figure 1 shows a schematic representation of the drug conjugates (molecule (II)) of the present invention, obtained from an SG-type glycosylation remodeling antibody (molecule (II) in Figure 1A) and an MSG-type glycosylation remodeling antibody (molecule (II) in Figure 1B). (a) represents drug D, (b) represents linker L, (c) represents PEG linker (L(PEG)), and (d) represents N297 glycan (wherein the white circle represents NeuAc (Sia), the white hexagon represents Man, the black hexagon represents GlcNAc, the white diamond represents Gal, and the white inverted triangle represents Fuc). The white pentagon represents a triazole ring generated by the reaction of the alkyne derived from linker L with the azide group derived from the PEG linker. The Y-shape represents the antibody Ab. The PEG linker is linked to the carboxyl group at the 2-position of the sialic acid located at the non-reducing end via an amide bond. This notation method is applied throughout this specification unless otherwise stated. [Figure 2]2A and 2B are schematic diagrams showing the structures of (Fucα1,6)GlcNAc-antibody (molecule (III) in A of FIG. 2), SG-type glycosylation remodeling antibody (molecule (IV) in B of FIG. 2), and MSG-type glycosylation remodeling antibody (molecule (IV) in C of FIG. 2), which are production intermediates of the drug conjugates of the present invention. In all figures, the Y-shape represents antibody Ab, as in FIG. 1. In FIG. 2A, (e) represents the N297 oligosaccharide consisting of a disaccharide α-glycosidically linked at position 1 of Fuc and position 6 of GlcNAc. In FIG. 2B and C, (d) represents the same N297 oligosaccharide as in FIG. 1, and (f) represents a PEG linker having an azide group, which is used for binding to linker L at its terminal. The binding mode of the PEG linker having an azide group is the same as that of the PEG linker in FIG. 1. [Figure 3] This is a schematic diagram of the process for producing SG-type glycosylation-remodeled antibodies and MSG-type glycosylation-remodeled antibodies from antibodies produced in animal cells. Molecules (III) and (IV) in the diagram represent (Fucα1,6)GlcNAc-antibodies and SG-type glycosylation-remodeled antibodies or MSG-type glycosylation-remodeled antibodies, respectively, as in Figure 2. Molecule (V) is an antibody produced in animal cells and is a mixture of molecules with heterogeneous N297 glycans. Figure 3A shows the process for producing homogeneous (Fucα1,6)GlcNAc-antibody (III) by treating the heterogeneous N297 glycan of (V) with a hydrolase such as EndoS. Figure 3B shows the process for producing SG-type glycosylation-remodeled antibodies (IV) by glycosylation-transferring an SG-type glycosylation-remodeled antibody to the GlcNAc of the N297 glycan of antibody (III) using a glycosyltransferase such as the EndoS D233Q / Q303L mutant. Figure 3C, like Figure 3B, shows the process for preparing an MSG-type glycan remodeled antibody (IV) by glycosylation of an MSG-type glycan donor molecule to antibody (III). The SG-type glycan donor molecule and MSG-type glycan donor molecule used here have the sialic acid at their non-reducing ends modified with a PEG linker having an azide group, and the SG-type N297 glycan remodeled antibody and MSG-type N297 glycan remodeled antibody to be prepared also have the sialic acid at their non-reducing ends similarly modified, as shown in Figure 2B and C. [Figure 4] The amino acid sequences of the light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2) of trastuzumab are shown. [Figure 5] The amino acid sequence of the light chain (SEQ ID NO: 1) and the heavy chain (SEQ ID NO: 3) of the modified anti-HER2 antibody are shown. [Figure 6] (a) The amino acid sequence of human STING wild-type, (b) the amino acid sequence of human STING REF mutant (R232H), and (c) the amino acid sequence of human STING HAQ mutant (R71H, G230A, R293Q). [Figure 7] This figure shows the antitumor effect of intratumoral administration of CDN derivatives. In the figure, the black square line represents the vehicle group, the white square line represents the compound 6a-administered group, the white inverted triangle line represents the compound 8b-administered group, and the white circle line represents the compound 9b-administered group. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 8] The figure shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugate (1) and anti-LPS antibody-CDN conjugate (1). In the figure, the black squares represent the vehicle group, the white triangles represent the group administered with anti-HER2 antibody-CDN conjugate (1) in which the modified anti-HER2 antibody prepared in Reference Example 1 was conjugated with the compound of Example 8b, and the black triangles represent the group administered with anti-LPS antibody-CDN conjugate (1) in which the modified anti-LPS antibody prepared in Reference Example 2 was also conjugated with the compound of Example 8b. The vertical axis represents tumor volume (mm), and the horizontal axis represents the number of days after tumor implantation. [Figure 9] This figure shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugates (2) and (3). In the figure, the black squares represent the vehicle group, the white squares represent the anti-HER2 antibody-CDN conjugate (2) administration group, and the white triangles represent the anti-HER2 antibody-CDN conjugate (3) administration group. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 10]This figure shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugate (19). The black squares in the figure represent the vehicle group, and the white triangles represent the anti-HER2 antibody-CDN conjugate (19) administration group. In anti-HER2 antibody-CDN conjugate (19), the drug linker is bound to the antibody via cysteine ​​conjugation. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 11] The figure shows the antitumor effect of intravenous administration of anti-HER2 antibody-CDN conjugates (1) and (9) to (12). The black squares in the figure represent the vehicle group, the white triangles represent the anti-HER2 antibody-CDN conjugate (9) administration group, the white inverted triangles represent the anti-HER2 antibody-CDN conjugate (10) administration group, the white diamonds represent the anti-HER2 antibody-CDN conjugate (11) administration group, the white circles represent the anti-HER2 antibody-CDN conjugate (12) administration group, and the white squares represent the anti-HER2 antibody-CDN conjugate (1) administration group. Anti-HER2 antibody-CDN conjugates (9), (10), (11), (12), and (1) were each conjugated to the compound of Example 8b using a different linker. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 12] This figure shows the antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugate (1), anti-HER2 antibody 2, and compound No. 8b. In the figure, the black square line represents the vehicle group, the white triangle line represents the group administered 60 μg of anti-HER2 antibody 2-CDN conjugate (1), the black inverted triangle line represents the group administered 59 μg of anti-HER2 antibody 2, and the black circle line represents the group administered 1.2 μg of compound No. 8b. The doses of anti-HER2 antibody 2 and compound No. 8b are equivalent to the amounts of each component constituting anti-HER2 antibody 2-CDN conjugate (1). The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 13(a)](a) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (2) and (3). (b) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (4), (5), (7), and (8). (c) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugate (6). The black squares in the figure represent the vehicle group, and the white symbol lines represent the groups evaluated treated with each of the anti-HER2 antibody 2-CDN conjugates (2) to (8). The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 13(b)] (a) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (2) and (3). (b) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (4), (5), (7), and (8). (c) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugate (6). The black squares in the figure represent the vehicle group, and the white symbol lines represent the groups evaluated treated with each of the anti-HER2 antibody 2-CDN conjugates (2) to (8). The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 13(c)] (a) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (2) and (3). (b) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (4), (5), (7), and (8). (c) Antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugate (6). The black squares in the figure represent the vehicle group, and the white symbol lines represent the groups evaluated treated with each of the anti-HER2 antibody 2-CDN conjugates (2) to (8). The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 14]This figure shows the antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (9) and (10). In the figure, the black squares represent the vehicle group, the white triangles represent the anti-HER2 antibody 2-CDN conjugate (9) administration group, and the white circles represent the anti-HER2 antibody 2-CDN conjugate (10) administration group. Anti-HER2 antibody 2-CDN conjugates (9) and (10) are antibody-CDN conjugates that use an MSG-type glycan remodeling antibody with an average drug binding number of approximately 2. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 15] Figure 1 shows the antitumor effect of intravenous administration of anti-EphA2 antibody and anti-EphA2 antibody-CDN conjugate (1). In the figure, the black squares represent the vehicle group, the white circles represent the anti-EphA2 antibody-administered group, and the white triangles represent the anti-EphA2 antibody-CDN conjugate (1)-administered group. The vertical axis represents tumor volume (mm), and the horizontal axis represents the number of days after tumor implantation. [Figure 16] Figure 1 shows the antitumor effect of intravenous administration of anti-CD33 antibody and anti-CD33 antibody-CDN conjugate (1). In the figure, the black squares represent the vehicle group, the white circles represent the anti-CD33 antibody-administered group, and the white triangles represent the anti-CD33 antibody-CDN conjugate (1)-administered group. The vertical axis represents tumor volume (mm3), and the horizontal axis represents the number of days after tumor implantation. [Figure 17] The amino acid sequences of the light chain (SEQ ID NO: 28) and heavy chain (SEQ ID NO: 29) of Pertuzumab are shown. [Figure 18] The amino acid sequence of the light chain (SEQ ID NO: 28) and the heavy chain (SEQ ID NO: 30) of modified anti-HER2 antibody 2 are shown. [Figure 19] The amino acid sequence of the light chain (SEQ ID NO: 31) and the heavy chain (SEQ ID NO: 32) of the anti-CD33 antibody are shown. [Figure 20] The amino acid sequence of the light chain (SEQ ID NO: 33) and the heavy chain (SEQ ID NO: 34) of the anti-EphA2 antibody are shown. [Figure 21] The amino acid sequence of the light chain (SEQ ID NO: 35) and the heavy chain (SEQ ID NO: 36) of the anti-CDH6 antibody are shown. [Figure 22]Figure 1 shows the antitumor effect of intravenous administration of anti-HER2 antibody 2-CDN conjugates (11) and (12). In the figure, the black squares represent the vehicle group, the white triangles represent the anti-HER2 antibody 2-CDN conjugate (11)-administered group, and the white circles represent the anti-HER2 antibody 2-CDN conjugate (12)-administered group. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention relates to novel CDN derivatives with STING agonist activity and antibody-drug conjugates thereof, as well as uses thereof. The novel CDN derivatives of the present invention have STING agonist activity and activate immune cells to induce the production of interferons and cytokines. Furthermore, the novel CDN derivatives of the present invention exert antitumor effects by activating these immune cells. The novel CDN derivatives may be administered directly to the target tissue where immune function is desired to be activated, or may be administered systemically after being linked via an optional linker to an antibody that can recognize and bind to target cells (e.g., tumor cells or immune cells).

[0062] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein localized in the endoplasmic reticulum. Congenital STING polymorphisms are known to occur frequently (PLoS One, 2013 Oct, 21, 8(10), e77846). Examples of STING mutants include the R232H mutation, in which the 232nd amino acid is mutated from arginine (R) to histidine (H), and the HAQ mutation, in which the 71st amino acid is mutated to histidine (H), the 230th amino acid is glycine (G) to alanine (A), and the 293rd amino acid is mutated to glutamine (Q). These STING polymorphisms are known to affect the strength of responses, such as cytokine production, induced by STING agonist stimulation (Genes and Immunity, 2011, 12, 263-269). Therefore, for a STING agonist to act stably in humans, it is desirable that it has activity against each form of STING.

[0063] In this specification, the terms "cancer," "carcinoma," and "tumor" are used interchangeably.

[0064] In the present invention, "immunostimulatory activity" refers to inducing in some way the activation of immune cells involved in anti-tumor immunity, such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, and neutrophils, and refers to causing any structural or functional changes in immune cells, such as the production of cytokines and chemokines, increased expression of immune activation markers, decreased expression of immunosuppressive markers, changes such as phosphorylation in intracellular signaling pathways, and changes in gene expression. It also includes causing changes in tumor cells that induce anti-tumor immunity, such as the production of cytokines and chemokines that activate or induce migration of immune cells, and increased sensitivity to immune cells.

[0065] In the present invention, the term "antitumor effect" refers to the direct or indirect effect of a drug on tumor cells, thereby inducing a reduction or regression of tumors. For example, an antitumor effect can be caused by a drug directly damaging tumor cells, by tumor cells stimulating with the drug to activate antitumor immunity, or by a drug delivered to tumor cells being released extracellularly, thereby activating antitumor immunity around the tumor cells.

[0066] In the present invention, "cytotoxic activity" refers to causing pathological changes in cells in some form, including not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes.

[0067] In the present invention, the term "cells" includes cells in an animal body and cultured cells.

[0068] In this specification, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0069] In this specification, the term "C1-C6 alkyl group" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. The "C1-C6 alkyl group" may have a cyclopropane on the alkyl group as long as the total number of carbon atoms does not exceed 6. Examples of the "C1-C6 alkyl group" include those having the following structure:

[0070] [ka] (The wavy line indicates the substitution position.)

[0071] In this specification, the term "C2-C6 alkenyl group" refers to a straight-chain or branched-chain alkenyl group having 2 to 6 carbon atoms.

[0072] In this specification, the term "C2-C6 alkynyl group" refers to a straight-chain or branched-chain alkynyl group having 2 to 6 carbon atoms.

[0073] In this specification, the term "C3-C6 cycloalkyl group" refers to a saturated cyclic hydrocarbon group having 3 to 6 carbon atoms. The "C3-C6 cycloalkyl group" may be substituted with multiple alkyl groups as long as the total number of carbon atoms does not exceed 6. Examples of the "C3-C6 cycloalkyl group" include groups with the following structure:

[0074] [ka] (The wavy line indicates the substitution position.)

[0075] In this specification, the term "hydroxy C1-C6 alkyl group" refers to an alkyl group in which one or two hydroxy groups are substituted at any position of a straight or branched chain alkyl group having 1 to 6 carbon atoms. The "hydroxy C1-C6 alkyl group" may have a cyclopropane on the alkyl group as long as the total number of carbon atoms does not exceed 6. Examples of the "hydroxy C1-C6 alkyl group" include those having the following structure:

[0076] [ka] (The wavy line indicates the substitution position.)

[0077] In the present invention, the term "amino C1-C6 alkyl group" refers to an alkyl group in which one or two amino groups are substituted at any position of a linear or branched alkyl group having 1 to 6 carbon atoms. The term "amino C1-C6 alkyl group" refers to an alkyl group that may have a cyclopropane on the alkyl group as long as the total number of carbon atoms does not exceed 6. Examples of the "amino C1-C6 alkyl group" include those having the following structure:

[0078] [ka] (The wavy line indicates the substitution position.)

[0079] <1. New CDN derivatives> The novel CDN derivative of the present invention has the following formula (Ia):

[0080] [ka] It has the structure shown below.

[0081] L 1 is a group represented by the following formula, which may be substituted at any position with 1 to 3 groups selected from the group consisting of a hydroxy group, -NH2, a 2-hydroxyacetylaminomethyl group, and a 2-[(2-hydroxyacetyl)amino]ethyl group:

[0082] [ka] (where, R 6 and R 6’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, —NH2, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group, R 7 and R7’ each independently represents a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is optionally substituted by one or two substituents selected from the group consisting of a halogen atom and an oxo group; R 8 and R 8’ each independently represents a hydrogen atom or a halogen atom, Z 4 represents -CH2-, -NH- or an oxygen atom, Z 5 represents a nitrogen atom or -CH= represents a group selected from the group consisting of:

[0083] Also, L 1 is a group represented by the following formula, which may be substituted at any position with 1 to 3 groups selected from the group consisting of a hydroxy group, -NH2, a 2-hydroxyacetylaminomethyl group, and a 2-[(2-hydroxyacetyl)amino]ethyl group:

[0084] [ka] (where, R 6 and R 6’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, —NH2, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group, R 7 and R 7’ each independently represents a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is optionally substituted by one or two substituents selected from the group consisting of a halogen atom and an oxo group; R 8 and R 8’ each independently represents a hydrogen atom or a halogen atom, Z 4 represents -CH2-, -NH- or an oxygen atom, Z 5 represents a nitrogen atom or -CH= represents a group selected from the group consisting of:

[0085] L 1 is preferably a group selected from the group consisting of the following formulae:

[0086] [ka] (where R 9 and R 9’ represents a hydrogen atom, a halogen atom, a hydroxy group, or -NH2, R 10 represents a hydroxy group, -NH, -NHC(=O)CHOH, -CHNHC(=O)CHOH, -CHCHNHC(=O)CHOH, a hydroxy C1-C3 alkyl group or an amino C1-C3 alkyl group, R 11 and R 11’ each independently represents a hydrogen atom, a fluorine atom, or a methyl group, or R 11 and R 11’ is bonded to form a cyclopropane, Z 4 represents -CH2-, -NH- or an oxygen atom) is.

[0087] Also, L 1 is preferably a group selected from the group consisting of the following formulae:

[0088] [ka] (where R 13 and R 13’ each independently represents a hydrogen atom, a hydroxy group, or -NH; R 12 represents a hydroxy group, -NH, -CHOH, -NHC(=O)CHOH, -CHNHC(=O)CHOH or -CHCHNHC(=O)CHOH, Z 4 is as previously defined) is.

[0089] Furthermore, L 1is preferably a group selected from the group consisting of the following formulae:

[0090] [ka] (where R 14 represents a hydrogen atom or -NH2, R 15 represents a hydrogen atom or -C(=O)CHOH, R 16 represents a hydroxy group, -NH2, -CH2OH, -CH2CH2OH, -CH2NH2 or -CH2CH2NH2) is.

[0091] L 1 is more preferably a group selected from the group consisting of the following formulae:

[0092] [ka] is.

[0093] L 3 is selected from a hydrogen atom, a halogen atom, —NH 2 , a hydroxy C 1 -C 3 alkyl group, or an amino C 1 -C 3 alkyl group.

[0094] Q 1 and Q 1’ are each independently a hydroxy group, a thiol group, or a borano group (BH3 - ) indicates Q 1 is preferably a hydroxy group or a thiol group. 1’ is preferably a hydroxy group or a thiol group. More preferably, Q 1 and Q 1’ The combination of 1 and Q 1’ is a thiol group, or Q 1 and Q 1’ is a hydroxy group.

[0095] Q 2 and Q 2’each independently represents an oxygen atom or a sulfur atom. 2 and Q 2’ are both oxygen atoms or both sulfur atoms.

[0096] Q 1 and Q 2 The combination of 1 is a thiol group and Q 2 is an oxygen atom, or Q 1 is a thiol group and Q 2 is a sulfur atom.

[0097] Q 1’ and Q 2’ The combination of 1’ is a thiol group and Q 2’ is an oxygen atom or Q 1’ is a hydroxy group and Q 2’ is an oxygen atom or Q 1’ is a thiol group and Q 2’ is a sulfur atom.

[0098] X 1 and X 2 X each independently represents an oxygen atom, a sulfur atom, or -CH2-. 1 is preferably an oxygen atom. 2 is preferably an oxygen atom. More preferably, X 1 and X 2 is an oxygen atom.

[0099] Y 1 and Y 2 represents an oxygen atom or -CH2-. 1 is preferably an oxygen atom. 2 is preferably an oxygen atom. More preferably, Y 1 and Y 2 is an oxygen atom.

[0100] X 3 and X 4 is either (iii) or (iv) below: (iii) Y1 When is an oxygen atom, X 3 -X 4 represents -CH2-O-, -CH2-S-, -CH2-CH2- or -CH2-CF2-; or (iv) Y 1 When is -CH2-, X 3 -X 4 represents -O-CH2-, X represents a group selected from 3 and X 4 is preferably —CH2—O— as defined in (iii) above.

[0101] X 5 and X 6 is (v) or (vi) below: (v) Y 2 When is an oxygen atom, X 5 -X 6 represents -CH2-O-, -CH2-S-, -CH2-CH2- or -CH2-CF2-; or (vi) Y 2 When is -CH2-, X 5 -X 6 represents -O-CH2-, X represents a group selected from 5 and X 6 is preferably —CH2—O— as defined in (v) above.

[0102] R 1 ,R 2 and R 3 each independently represent a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N3, -NHR', -NR'R'', or -NHC(=O)R' (wherein R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, wherein the C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group is optionally substituted with 1 to 6 halogen atoms; and R'' represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group).

[0103] R 1is preferably a hydrogen atom, a hydroxy group or a fluorine atom.

[0104] R 2 is preferably a hydrogen atom, a hydroxy group or a fluorine atom.

[0105] R 3 is preferably a hydrogen atom, a hydroxy group or a fluorine atom.

[0106] W 1 represents a nitrogen atom, an oxygen atom, a sulfur atom or -CH-.

[0107] R 5 is (vii) to (x) below: (vii)W 1 When is a nitrogen atom, R 5 represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group; (viii)W 1 When is an oxygen atom, R 5 does not exist; (ix)W 1 is a sulfur atom, R 5 does not exist; or (x)W 1 When is -CH-, R 5 represents a hydrogen atom, a halogen atom, a hydroxy group, -NH2 or a C1-C6 alkyl group, W represents a group selected from 1 When is a nitrogen atom, R 5 is preferably a hydrogen atom. 1 When is -CH-, R 5 is preferably a hydrogen atom.

[0108] W 2 represents a nitrogen atom or -CH=. 2 is preferably -CH=.

[0109] R 4 represents a hydrogen atom, a halogen atom, or -NH2. 4 is preferably a hydrogen atom.

[0110] Z 1 -Z 2 -Z 3 are taken together to form -CH2-CH2-CH2-, -CH2-CH2-R'''-, -CH=CH-CH2-, -CH=CX-CH2-, -CX=CH-CH2-, -CX=CX-CH2-, -C(=O)-CH2-CH2-, -CH2-CH2-C(=O)-, -CH2-CH(CH3)-CH2- or -CH2-CH2-CH(CH3)- (wherein R''' represents -O- or -CH2-CH2-, and X represents a halogen atom), or a group represented by any of the following formulae:

[0111] [ka] (where the asterisk stands for W 1 and the wavy line indicates that the bond is to the carbon atom of =C-). 1 , Z 2 and Z 3 are preferably taken together to be -CH-CH-CH-, -CH=CH-CH-, -CH-CH(CH)-CH-, -CH-CH-CH(CH)-, or -CH-CH-R'''- (wherein R''' represents -O- or -CH-CH-).

[0112] Furthermore, the novel CDN derivative of the present invention preferably has the following formula:

[0113] [ka] It has the structure shown below.

[0114] L 1 is as previously defined.

[0115] Q 3 and Q 3‘ each independently represents a hydroxy group or a thiol group. 3 and Q 3‘are both thiol groups.

[0116] R 21 and R 22 R each independently represents a hydroxy group or a fluorine atom. 21 is preferably a hydroxy group. 22 is preferably a fluorine atom.

[0117] W 5 represents -NH- or a sulfur atom.

[0118] The method for producing the novel CDN derivative of the present invention will be described below in <3. Production Method>.

[0119] 2. Antibody-drug conjugates The novel CDN derivatives of the present invention may be administered directly to the tissue of interest (e.g., intratumorally), or may be administered as antibody-drug conjugates linked via an optional linker to an antibody capable of recognizing and binding to target cells (e.g., tumor cells or immune cells).

[0120] The antibody-drug conjugate of the present invention has the following formula (II):

[0121] [ka] It is shown by m 1 indicates the number of drugs bound per antibody molecule in the antibody-drug conjugate, Ab indicates an antibody or a functional fragment of the antibody, L indicates a linker connecting Ab and D, and D indicates the novel CDN derivative described above (in this specification, when the novel CDN derivative is used as a part of the antibody-drug conjugate, it is also simply referred to as a "drug").

[0122] Drug D is a compound that has the activity of activating immune cells, specifically, STING agonist activity. When part or all of the linker is cleaved in target cells (e.g., tumor cells or immune cells), Drug D is released in its original structure, thereby exerting its immunostimulatory effect. The desired function is exerted by increasing the sensitivity of target cells to immune cells or by activating immune cells via the target cells. The desired function is not particularly limited as long as it is related to STING agonist activity, but is preferably antitumor activity. That is, Drug D, linked to a tumor-targeting antibody (e.g., an anti-HER2 antibody) via an optional linker, is delivered to target cells or tissues, where part or all of the linker is cleaved, thereby exerting an antitumor effect through increasing the sensitivity of target cells to immune cells or activating immune cells via the target cells (e.g., production of interferon or cytokines).

[0123] The drug D attached to the antibody-drug conjugate of the present invention has the following formula (I):

[0124] [ka] (where, L is L 1 or L 2 and binds to any -NH2 or hydroxy group contained in L 1 is as defined above in <1. Novel CDN derivatives>, L 2 is either (i) or (ii) below: (i) When combined with L, L 2 represents -NHR', a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group, where R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group is optionally substituted with 1 to 6 halogen atoms; or (ii) When not bonded to L, L 2 represents a hydrogen atom or a halogen atom, represents a group selected from Q 1 , Q 1’ , Q 2 , Q 2’ , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , Y 1 , Y 2 , R 1 , R 2 , R 3 , R 4 , R 5 , W 1 , W 2 , Z 1 , Z 2 and Z 3 is as defined above in <1. Novel CDN derivatives>.

[0125] L 2 When combined with L, L is preferably -NH, -CHNH, or -CHOH. 2 When not bound to L, is preferably a hydrogen atom or a fluorine atom.

[0126] The drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following two formulae:

[0127] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 and Q 2’ is as previously defined, R 17 , R 17’ , R 18 and R 18’ each independently represents a hydrogen atom, a halogen atom, a hydroxy group, or -NH; W 3 represents -NH-, an oxygen atom, a sulfur atom, or -CH2-, W 4is represented by either -CH= or a nitrogen atom.

[0128] Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following two formulae:

[0129] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 , Q 2’ , R 17 , R 17’ , R 18 and R 18’ is as previously defined) It is indicated by either

[0130] Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following formula 8:

[0131] [ka] (where L 1 , Q 1 , Q 1’ , Q 2 and Q 2’ is as previously defined, R 19 , R 19’ , R 20 and R 20’ each independently represents a hydrogen atom or a fluorine atom) It is indicated by either Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following four formulas:

[0132] [ka] (where L 1 is as previously defined) It is indicated by either

[0133] Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following four formulas:

[0134] [ka] (where L 1 is as previously defined) It is indicated by either

[0135] Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following four formulas:

[0136] [ka] (where L 1 is as previously defined) It is indicated by either

[0137] Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following formula:

[0138] [ka] (where L 1 , Q 3 , Q 3‘ , R 21 , R 22 and W 5 is as defined above in <1. Novel CDN derivatives>).

[0139] Furthermore, in the novel CDN derivative of the present invention or the drug D used in the antibody-drug conjugate of the present invention, L 1 is preferably the following:

[0140] [ka] It is indicated by either

[0141] Furthermore, in the novel CDN derivative of the present invention or the drug D used in the antibody-drug conjugate of the present invention, L 1 is preferably represented by the following four formulas:

[0142] [ka] (wherein the asterisk indicates bonding to L).

[0143] Furthermore, the drug D used in the novel CDN derivative of the present invention or the antibody-drug conjugate of the present invention is preferably represented by the following four formulas:

[0144] [ka] (where the asterisk indicates a bond with L, and Q 3 , Q 3‘ and W 5 is as defined in <1. Novel CDN derivatives> above).

[0145] <2.1. Linker structure> The linker structure used to link a drug to an antibody in the antibody-drug conjugates of the present invention is described below. The linker used in the antibody-drug conjugates of the present invention is not particularly limited as long as it is understood by those skilled in the art as a linker that connects an antibody and a drug. Examples of linkers used in the antibody-drug conjugates of the present invention include, but are not limited to, those described in Protein Cell, 2018, 9(1):33-46; Pharm Res, 2015, 32:3526-3540; or Int. J. Mol. Sci., 2016, 17, 561. The linker may be either a linker that is cleaved in vivo or a linker that is not cleaved in vivo, but is preferably a linker that is cleaved in vivo.

[0146] Linkers used in the antibody-drug conjugates of the present invention include, but are not limited to, linkers that link a drug to a sugar chain or remodeled sugar chain of the Fc portion of an antibody (sometimes referred to herein as "sugar chain conjugation") (e.g., as described in WO2018 / 003983), or linkers that link a drug to any amino acid residue of an antibody (e.g., a cysteine ​​residue or a lysine residue) (e.g., as described in WO2014 / 057687). Linkers that link a drug to any amino acid residue of an antibody preferably link via a thioether bond with a sulfhydryl group (SH group) of a cysteine ​​of an Ab (sometimes referred to herein as "cysteine ​​conjugation") or via an amide bond with an amino group (NH group) of a lysine of an Ab (sometimes referred to herein as "lysine conjugation"), with cysteine ​​conjugation being preferred.

[0147] A preferred linker L of the present invention is represented by the following formula: -Lb-La-Lp-Lc-* (where the asterisk indicates the L 1 or L 2 It indicates that the bond is made to any amino group or hydroxy group contained in the compound.

[0148] First, Lp will be described.

[0149] Lp either exhibits a linker consisting of an amino acid sequence that is cleavable in vivo or in target cells (hereinafter also referred to as a peptide linker in this specification), or is absent.

[0150] Lp is cleaved by the action of an enzyme such as peptidase or esterase. Lp is a peptide composed of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond at its N-terminus with the carbonyl group at the right end of La (described below), and forms an amide bond at its C-terminus with the amino group (—NH—) of Lc. The amide bond at the C-terminus of Lp is cleaved by the enzyme such as peptidase.

[0151] The amino acids constituting Lp are not particularly limited, but may be, for example, L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, amino acids with structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may also be used, and non-natural amino acids such as N-methylated amino acids may also be used. The amino acid sequence of Lp is not particularly limited, but examples of the constituting amino acids include glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), methionine (Met; M), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D). Among these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), and citrulline (Cit) are preferred. These amino acids may overlap, and the amino acid sequence may include any amino acid selected. Furthermore, the drug release pattern can be controlled by the type of amino acid.

[0152] Specific examples of Lp include -GGVA-, -VA-, -GGFG-, -FG-, -GGPI-, -PI-, -GGVCit-, -VCit-, -GGVK-, -VK-, -GGFCit-, -FCit-, -GGFM-, -FM-, -GGLM-, -LM-, -GGICit-, and -ICit-. The linker Lp is preferably -GGVA-, -VA-, -GGFG-, -FG-, -GGVCit-, -VCit-, -GGFCit-, or -FCit-. The linker Lp is more preferably -GGVA-, -GGFG, or -GGVCit-. Furthermore, the linker Lp is preferably -GGFG- or -GGPI-.

[0153] Next, La will be described.

[0154] La is as follows: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -(CH2)n 4 -OC(=O)-, and -(CH2)n 9 -C(=O)- (wherein, n 2 represents an integer of 1 to 3 (preferably 1 or 2), and n 3 represents an integer of 1 to 5 (preferably an integer of 2 to 5, more preferably 3 or 4), and n 4 represents an integer of 0 to 2 (preferably 0 or 1), and n 9represents any one selected from the group consisting of integers of 2 to 7 (preferably an integer of 2 to 5, more preferably 2, 3, or 5).

[0155] La is preferably one of the following: -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, -(CH2)5-C(=O)-, and It indicates any one selected from the group consisting of:

[0156] La is more preferably -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, or -(CH2)5-C(=O)- is.

[0157] La is even more preferably -C(=O)-CH2CH2-C(=O)-.

[0158] Next, Lb will be described.

[0159] Lb represents a spacer used for the linker of sugar chain conjugation (also referred to as "the spacer of the linker of sugar chain conjugation" in this specification), or a spacer used for cysteine conjugation (also referred to as "the spacer of the linker of cysteine conjugation" in this specification).

[0160] <When Lb is "the spacer of the linker of sugar chain conjugation"> When Lb is "the spacer of the linker of sugar chain conjugation", Lb is not particularly limited, and examples thereof include spacers represented by the following formulas.

[0161]

Chemical formula

[0162]

Chemical formula

[0163]

Chemical formula

[0164] In each of the structural formulas shown above, the asterisk (*) indicates that it is bonded to the -(C=O)- or -CH2- at the left end of La, and the wavy line indicates that it is bonded to the sugar chain of Ab or the remodeled sugar chain. <(

[0165] When selecting any one of Lb-1, Lb-2 or Lb-3 for Lb, 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 a plurality of drugs to one molecule of the antibody. When binding a plurality of drugs to one molecule of the antibody, a plurality of Lbs will also be present (for example, refer to the schematic diagram (1e) of the antibody-drug conjugate shown in Method E of <3. Production Method> described later). When Lb is selected from any one of Lb-1, Lb-2 or Lb-3 and a plurality of such Lbs are present with respect to one molecule of the antibody (for example, when m 2 described later is 1 or 2), in each Lb, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of the two structures or a mixture thereof.

[0166] <When Lb is the spacer of the linker for cysteine conjugation> When Lb is the spacer of the linker for cysteine conjugation, Lb is not particularly limited, and examples thereof include -(succinimid-3-yl-N)-. In the present invention, "-(succinimid-3-yl-N)-" has the following formula:

[0167]

Chemical formula

[0168] Next, Lc will be described.

[0169] Lc is -NH-CH2-, -NH-phenyl group -CH2-O(C=O)-, -NH-heteroaryl group -CH2-O(C=O)-, or is absent. Here, the phenyl group is preferably a 1,4-phenyl group, and the heteroaryl group is preferably a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group. Lc is preferably -NH-CH2- or is absent.

[0170] A more preferred linker L of the present invention is When the binding mode between the drug and the antibody is "glycoconjugation," -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGVA-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGVCit-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFCit-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGICit-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFM-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGLM-, -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 is the structural formula of Lb shown below:

[0171] [ka] ), or When the binding mode between the drug and the antibody is "cysteine ​​conjugation," -Z L2 -(CH2)5-C(=O)-GGFG-, -Z L2 -(CH2)5-C(=O)-GGVA-, -Z L2 -(CH2)5-C(=O)-GGVCit-, -Z L2 -(CH2)5-C(=O)-GGFCit-, -Z L2 -(CH2)5-C(=O)-GGICit-, -Z L2 -(CH2)5-C(=O)-GGFM-, -Z L2 -(CH2)5-C(=O)-GGPI-, -Z L2 -(CH2)5-C(=O)-GGLM-, -Z L2 -(CH2)5-C(=O)-FG-, -Z L2 -(CH2)5-C(=O)-VA-, -Z L2 -(CH2)5-C(=O)-GGFG-NH-CH2-, -ZL2 -(CH2)5-C(=O)-GGVA-NH-CH2-, -Z L2 -(CH2)5-C(=O)-GGVCit-NH-CH2-, -Z L2 -(CH2)5-C(=O)-GGFCit-NH-CH2-, -Z L2 -(CH2)5-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, or -Z L2 -(CH2)5-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, (where Z L2 is the structural formula of Lb shown below:

[0172] [ka] It represents -(succinimide-3-yl-N)-. is.

[0173] In a more preferred embodiment of the linker L of the present invention, the binding mode between the drug and the antibody is "glycoconjugation", -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, or -Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-CH2-, (where Z L1 is the structural formula of Lb shown below:

[0174] [ka] (Indicates

[0175] The right end of the "preferred linker L" and "more preferred linker L" is L of formula (I). 1 or L 2 It is bonded to any -NH2 or hydroxy group contained in

[0176] 2.2. Antibodies and their glycosylation <2.2.1 Antibodies> As used herein, the term "gene" refers to a nucleotide or nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof. For example, the term "gene" includes polynucleotides, oligonucleotides, DNA, mRNA, cDNA, RNA, etc., which are nucleotide sequences containing a nucleotide sequence encoding the amino acids of a protein, or their complementary strands.

[0177] As used herein, "nucleotide," "polynucleotide," or "nucleotide sequence" is synonymous with "nucleic acid," and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence."

[0178] In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably.

[0179] As used herein, the term "functional antibody fragment," also referred to as "antigen-binding fragment of antibody," refers to a partial fragment of an antibody that retains antigen-binding activity, and includes Fab, F(ab'), Fv, scFv, diabody, linear antibody, and multispecific antibody fragments formed from antibody fragments. Also included in the antigen-binding fragment of an antibody is Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab') under reducing conditions. However, the antigen-binding fragment is not limited to these molecules, as long as it retains antigen-binding ability. Furthermore, these antigen-binding fragments include not only those obtained by treating the full-length antibody protein molecule with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0180] The functional fragments of the present invention include functional fragments that retain an asparagine (Asn297) and its surrounding amino acids that are highly conserved in the Fc region of an IgG heavy chain and are modified by N-linked glycans, and that have antigen-binding ability.

[0181] The antibody used in the antibody-drug conjugates of the present invention refers to an immunoglobulin, and is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody of the present invention may be of any class, including IgG, IgE, IgM, IgD, IgA, and IgY, with IgG being preferred. Furthermore, the subclass may be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG1, IgG2, or IgG4 being preferred (including antibodies with mutations in the Fc region of the IgG heavy chain that affect ADCC and ADCP activity).

[0182] When the antibody of the present invention is of the IgG1 isotype, effector function can be adjusted by substituting some of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, and WO94 / 29351). Examples of IgG1 mutants include IgG1 LALA mutations (IgG1-L234A, L235A). L234A and L235A represent substitutions of leucine with alanine at positions 234 and 235, as defined by the EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No. 1 (May 15, 1969), pp. 78-85).

[0183] The antibody may be derived from any species, preferably human, rat, mouse, or rabbit. When derived from a species other than human, it is preferable to chimerize or humanize the antibody using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, and modified versions thereof.

[0184] The antibody is preferably, but not limited to, an antibody that targets tumor cells or immune cells, and more preferably, an antibody that targets tumor cells.

[0185] When an antibody targeting tumor cells is used, the antibody preferably has one or more of the following properties: the ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be taken up and internalized into tumor cells, and the ability to damage tumor cells. The drug conjugated to the antibody of the present invention via a linker has STING agonist activity. The drug of the present invention activates the signaling of interferon regulatory factor-3 (IRF3) to induce interferon. Therefore, when an antibody targeting tumor cells is used in the antibody-drug conjugate of the present invention, the antibody-drug conjugate is administered into the body, delivered to the tumor site, and taken up into tumor cells. After that, the linker moiety is cleaved by peptidase or the like, releasing the drug moiety. The released drug moiety is thought to activate anti-tumor immunity through its STING agonist activity, thereby exerting an anti-tumor effect.

[0186] Antibody binding to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to visualize the antibody uptake by the cell under a fluorescent microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to measure the amount of fluorescence uptake by the cell (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released upon intracellular uptake, suppressing cell proliferation (BioTechniques 28:162-165, January 2000). A recombinant complex protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin.

[0187] In the present invention, "high internalization ability" means that the viability (expressed relative to the cell viability without antibody, which is set to 100%) of target antigen-expressing cells (e.g., HER2-expressing cells when an anti-HER2 antibody is used) to which the antibody and a saporin-labeled anti-mouse or rat IgG antibody have been added is preferably 70% or less, more preferably 60% or less.

[0188] When an antibody targeting tumor cells is used in the antibody-drug conjugate of the present invention, it is preferable, but not essential, that the antibody itself have an antitumor effect. The antibody used in the antibody-drug conjugate of the present invention preferably has the property of internalization, that is, translocation into tumor cells.

[0189] The antitumor activity of a drug or antibody-drug conjugate refers to cytotoxic activity against tumor cells, anticellular effect, and tumor volume reduction. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems.

[0190] The immunostimulatory activity of a drug or antibody-drug conjugate refers to the enhancement of tumor cell sensitivity to immune cells or tumor cell-mediated activation of immune cells. The immunostimulatory activity can be confirmed using known in vitro or in vivo evaluation systems.

[0191] Examples of antibodies used in the present invention include, but are not limited to, anti-HER2 antibodies, anti-HER3 antibodies, anti-DLL3 antibodies, anti-FAP antibodies, anti-CDH11 antibodies, anti-CDH6 antibodies, anti-A33 antibodies, anti-CanAg antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD98 antibodies, anti-TROP2 antibodies, anti-CEA antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUC1 antibodies, anti-GPNMB antibodies, anti-integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-Mesothelin antibodies, anti-ENPP3 antibodies, anti-CD47 antibodies, anti-EGFR antibodies, anti-GPR20 antibodies, and anti-DR5 antibodies. The antibody of the present invention is preferably an anti-HER2 antibody (for example, trastuzumab or pertuzumab), an anti-CDH6 antibody, an anti-CD33 antibody, or an anti-EphA2 antibody, more preferably an anti-HER2 antibody.

[0192] The antibodies of the present invention can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies that can be applied to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0193] Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennett, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)), and a monoclonal antibody can be obtained.

[0194] The antigen can be obtained by genetically manipulating a gene encoding the antigen protein in a host cell to produce it.

[0195] The humanized antibody of the present invention can be obtained according to known methods (for example, Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525, WO90 / 07861).

[0196] For example, anti-HER2 antibodies (US5821337, WO2004 / 008099, etc.), anti-CD33 antibodies (WO2014 / 057687, etc.), anti-CD70 antibodies (WO2004 / 073656, etc.), anti-EphA2 antibodies (WO2009 / 028639, etc.), and anti-CDH6 antibodies (WO2018 / 212136, etc.) can be obtained by known means.

[0197] The anti-HER2 antibody of the present invention is not particularly limited, but preferably has the following properties, for example. (1) An anti-HER2 antibody characterized by the following properties: (a) Binds specifically to HER2.

[0198] (b) It has the activity of being internalized into HER2-expressing cells by binding to HER2. (2) The antibody described in (1) above that binds to the extracellular domain of HER2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that reduces ADCC and ADCP activity. (7) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1. (8) The antibody according to (5) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1. (10) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 29 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (11) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (12) The antibody according to any one of (1) to (11) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (13) An antibody obtained by a method for producing the antibody, comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above, and collecting the antibody of interest from the culture obtained in the step.

[0199] 2.2.2 Antibody Glycosylation Remodeling Recently, a method for remodeling heterogeneous antibody glycans by enzymatic reactions to uniformly introduce functionalized glycans has been reported (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Using this glycan remodeling technique, attempts have also been made to site-specifically introduce drugs and synthesize homogeneous ADCs (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0200] Glycan remodeling begins with the use of hydrolases to remove heterogeneous glycans attached to proteins (such as antibodies), leaving only the terminal GlcNAc, to prepare a homogeneous protein portion with GlcNAc attached (hereafter referred to as the "acceptor"). Next, a separately prepared glycan of your choice (hereafter referred to as the "donor") is prepared, and this acceptor and donor are linked using glycosyltransferases. This allows the synthesis of a homogeneous glycoprotein with a desired glycan structure.

[0201] In the present invention, the term "glycan" refers to a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and glycans may be represented by abbreviations such as "GlcNAc-" or "SG-." When these abbreviations are used in structural formulae, the oxygen or nitrogen atom at the reducing end that forms a glycosidic bond with another structural unit is not included in the abbreviation representing the glycan, unless otherwise defined.

[0202] In the present invention, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described by assuming that the carbon atom bonded to the oxygen atom constituting the ring and directly bonded to a hydroxy group (or an oxygen atom belonging to a glycosidic bond) in the ring structure is at position 1 (position 2 only in sialic acid). The names of the example compounds are given based on the entire chemical structure, and this rule does not necessarily apply.

[0203] In the present invention, when a sugar chain is described as a symbol (e.g., SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon atom at the reducing end, and does not include the N or O attributable to the N- or O-glycosidic bond.

[0204] The antibody-drug conjugate of the present invention has the following formula:

[0205] [ka] The antibody Ab or a functional fragment thereof is bound to L either directly through the side chain of its amino acid residue (e.g., cysteine, lysine, etc.) or through a glycan or a remodeled glycan of the Ab.

[0206] The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

[0207] N-linked glycans are bound to amino acid side chains of antibodies via N-glycosidic bonds, and O-linked glycans are bound to amino acid side chains of antibodies via O-glycosidic bonds.

[0208] The Ab of the present invention is an IgG, preferably an IgG1, IgG2, or IgG4.

[0209] IgG has a well-conserved N-linked glycan at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which is known to contribute to the activity and kinetics of antibody molecules (Eon-Duval, A. et al., Biotechnol. Prog. 2012, 28, 608-622; Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0210] The amino acid sequence in the constant region of IgG is well conserved, and in a report by Edelman et al. (Proc. Natl. Acad. Sci. USA, 63, 78-85, (1969)), each amino acid is identified by an EU number (EU INDEX). For example, Asn297, to which N-linked glycosylation is added in the Fc region, corresponds to position 297 in the EU numbering. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by indicating it by EU numbering.

[0211] The diagram below shows the case where the antibody-drug conjugate of the present invention is bound to L from the N297 sugar chain of the antibody or functional fragment thereof.

[0212] [ka]

[0213] An antibody having such a remodeled sugar chain is called a sugar chain remodeling antibody.

[0214] SGP (α2,6-SGP) is an abbreviation for sialyglycopeptide and is a representative N-linked glycopeptide. SGP can be isolated and purified from chicken egg yolk, for example, according to the method described in WO2011 / 027868. Purified SGP products are also commercially available from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd. In this specification, the glycan portion of SGP is referred to as SG, and a glycan lacking one GlcNAc at the reducing end of SG is referred to as SG(10). SG(10) can be prepared by enzymatic hydrolysis of SGP, for example, according to the report by Umekawa et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209). SG(10) can also be purchased from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd.

[0215] In this specification, the glycan structure in which sialic acid at the non-reducing end of only one of the branched β-Man chains of SG(10) is deleted is referred to as MSG(9), the one with sialic acid only on the 1-3 glycan of the branched chain is referred to as MSG1, and the one with sialic acid only on the 1-6 glycan of the branched chain is referred to as MSG2.

[0216] The remodeled sugar chain of the present invention is 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.

[0217] N297-(Fuc)SG is represented by the following structural formula or sequence formula:

[0218] [ka]

[0219] [ka]

[0220] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; the asterisk indicates bonding to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, particularly the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0221] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula:

[0222] [ka]

[0223] [ka]

[0224] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, which indicates that the amino group at the right end is amide bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain. The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0225] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula:

[0226] [ka]

[0227] [ka]

[0228] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5-CH2-CH2-NH-, which indicates that the amino group at the right end is amide bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of the linker L, particularly Lb in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0229] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which four linkers L and four drugs D are bound (the above m 2 =2). When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which two linkers L and two drugs D are bound (the above m 2 =1) (see Figure 1).

[0230] The N297 sugar chain is preferably N297-(Fuc)SG, N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG.

[0231] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, a highly homogeneous ADC can be obtained.

[0232] <3. Manufacturing method> Representative methods for producing the novel CDN derivatives and antibody-drug conjugates thereof of the present invention, or their production intermediates, are described below. In the following, the compound numbers shown in each reaction scheme are used to indicate the compounds. That is, they are referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will also be referred to in the same manner.

[0233] In the following Methods A to E, the substituent R 1 ~R 5 , L 1 , L 2 , W 1 , W 2 , Z 1 ~Z 3 has the same meaning as above. a , R c , R e and R g represents the side chain of a natural α-amino acid, such as a methyl group, an isopropyl group, a sec-butyl group, an isobutyl group, or a benzyl group. 1 represents a protecting group for a primary alcohol. Preferred examples include a 4,4'-dimethoxytrityl group and a 4-methoxytrityl group. PRO 2 , PRO 3 , PRO 7 , PRO 8 represents a protecting group for a secondary alcohol. Preferred examples include a tert-butyldimethylsilyl group, a triisopropylsilyloxymethyl group, a benzoyl group, a 2-nitrobenzyl group, and a 4-methoxytetrahydropyran-4-yl group. PRO 6 represents a protecting group for carboxylic acid. Preferred is a tert-butyl group, a benzyl group, etc. 5 , PRO 9 indicates an amine protecting group. 5 is preferably a tert-butyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, a benzyloxycarbonyl group, or the like, and PRO 9 is preferably a 9-fluorenylmethyloxycarbonyl group or a 2-(trimethylsilyl)ethoxycarbonyl group. 4represents a protecting group for an alcohol or amine. In the case of an alcohol, preferred protecting groups include a tert-butyldimethylsilyl group, a benzoyl group, etc., and in the case of an amine, preferred protecting groups include a 2-(trimethylsilyl)ethoxycarbonyl group, an allyloxycarbonyl group, a tert-butyloxycarbonyl group, etc. a represents an oxygen atom or a sulfur atom, and Q b indicates a hydroxyl group or a thiol group. a’ and Q b’ are each independently a negatively charged oxygen atom (O - ) or sulfur atom (S - ) indicates R x and R y each independently represents a halogen atom or -O-PRO 2 n is an integer of 1 to 3.

[0234] A method The CDN derivative represented by (1) of the present invention can be produced according to Method A described below.

[0235] [ka]

[0236] This production method is a method for producing a compound represented by general formula (1). Steps A-1 to A-5 of this production method can be performed as a one-pot synthesis, which can be carried out by referring to the report by Gaffney et al. (Org. Lett. 2010, 12, 3269-3271).

[0237] [ka]

[0238] (A-1 process) In this step, the compound of formula (1a) is subjected to hydrolysis and removal of the cyanoethyl group in succession using known organic chemistry techniques to produce the compound of formula (2a).

[0239] Compound (1a) was hydrolyzed by treating it with water and an acid (e.g., pyridine trifluoroacetate, 4,5-dicyanoimidazole, 1H-tetrazole, etc.) in a solvent (acetonitrile, tetrahydrofuran, N,N-dimethylformamide, or a mixture thereof) at a temperature ranging from -10°C to the boiling point of the solvent used, preferably 15°C to 35°C. Water was used in an amount ranging from 2 moles to an excess of 1 mole, preferably 2 to 10 moles, and acid was used in an amount ranging from 1 mole to an excess of 1 mole, preferably 1 to 5 moles, per mole of compound (1a). The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. A base (e.g., tert-butylamine) was then added to the reaction mixture to remove the cyanoethyl group. The base was used in an excess of 1 mole of compound (1a), preferably 30 to 50 moles. The reaction time was 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction mixture was concentrated under reduced pressure to obtain crude compound (2a). The crude compound (2a) can be used in the next step without purification.

[0240] (A-2 process) In this step, the protecting group of the hydroxy group is removed from the compound of formula (2a) using a known organic chemistry method to produce a compound of formula (3a). Before starting the reaction of this step, the crude compound of formula (2a) was dried by azeotropy with acetonitrile one to three times, if necessary.

[0241] PRO 1When 2a is a 4,4'-dimethoxytrityl group, the 4,4'-dimethoxytrityl group was removed by treating compound (2a) with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at temperatures ranging from -10°C to the boiling point of the reaction solvent, preferably 15°C to 35°C. Water was used in excess, preferably 10 to 20 moles, per mole of compound (2a). The acid was diluted with the reaction solvent to 1% to 50% (v / v), preferably 5% to 10% (v / v), and the diluted solution was used in excess, preferably 5 to 15 moles. The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. The reaction was quenched by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to neutralize the acid, preferably 2 to 10 moles per mole of acid. The reaction mixture was concentrated under reduced pressure to obtain crude compound (3a). The crude compound (3a) was azeotroped three to five times with dehydrated acetonitrile. The acetonitrile was left over from the final azeotrope to obtain a 0.01M to 1M acetonitrile solution of compound (3a). The resulting acetonitrile solution was used directly in the next step.

[0242] (A-3 process) This step is a step of producing a compound of formula (5a) by sequentially carrying out a coupling reaction of a compound of formula (3a) with a compound of formula (4a) using known organic chemistry techniques and a sulfurization reaction of the resulting coupling product. Before starting the reaction in this step, compound (4a) was azeotroped three to five times with dehydrated acetonitrile. After the final azeotrope, acetonitrile was left over to prepare a 0.01 M to 1 M acetonitrile solution of compound (4a). A desiccant (powdered or pelleted molecular sieves 3A or molecular sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use.

[0243] A coupling reaction was carried out by adding an acetonitrile solution of compound (4a) dried by azeotropy to an acetonitrile solution of compound (3a) at 5°C to 35°C. The reaction time was 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, a sulfurizing agent (N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, 3H-1,2-benzodithiol-3-one, etc.) was added to the reaction solution to carry out a sulfurization reaction. The sulfurizing agent was used in an amount of 1 mole to 5 moles, preferably 1 mole to 2 moles, per mole of compound (3a). The reaction time was 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure to obtain crude compound (5a). The crude compound (5a) obtained was directly used in the next step.

[0244] (A-4 process) In this step, the protecting group of the hydroxy group is removed from the compound of formula (5a) using a known organic chemistry method to produce a compound of formula (6a).

[0245] PRO 1 When the 4,4'-dimethoxytrityl group is present, compound (5a) is treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at temperatures ranging from -10°C to the boiling point of the reaction solvent, preferably 15°C to 35°C, to remove the 4,4'-dimethoxytrityl group. Water is used in excess, preferably 10 to 20 moles, per mole of compound (5a). The acid is diluted with the solvent to 1% to 50% (v / v), preferably 5% to 10% (v / v), and the diluted solution is used in excess, preferably 5 to 15 moles. The reaction time is 1 minute to 3 hours, preferably 5 to 30 minutes. The reaction was quenched by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to neutralize the acid, preferably 10 to 200 moles per mole of acid. The reaction mixture was concentrated under reduced pressure to give crude compound (6a), which was used directly in the next step.

[0246] (A-5 process) In this step, a compound of formula (7a) is produced by successively carrying out a cyclization reaction and a sulfurization reaction on the compound of formula (6a) using known organic chemistry techniques. Compound (6a) was dissolved in pyridine and concentrated under reduced pressure to prepare a 0.01 M to 0.5 M pyridine solution. This pyridine solution was added with a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) at 5°C to 35°C. 5The cyclization reaction was carried out by adding a sulfurizing agent (e.g., 3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, etc.) to the reaction mixture. The dehydration condensation agent was used in an amount of 1 mole to an excess of 1 mole, preferably 3 to 5 moles, per mole of compound (6a). The reaction time was 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and a sulfurizing agent (e.g., 3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, etc.) were added to the reaction mixture to carry out a sulfurization reaction. The amount of water used was in excess of 1 mole, preferably 30 to 50 moles, per mole of compound (6a), and the amount of sulfurizing agent used was 1 mole to 5 moles, preferably 1 to 2 moles. The reaction time was 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction mixture was added to an aqueous sodium bicarbonate solution (0.1 M to 1 M), and the mixture was stirred for 15 minutes to 24 hours to terminate the reaction. The reaction mixture was extracted one to five times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixture thereof), and the combined extracts were dried over anhydrous sodium sulfate or magnesium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol, ethyl acetate / methanol, hexane / ethyl acetate, etc.), C18 silica gel column chromatography (buffer / acetonitrile), or a combination thereof to obtain compound (7a) as a mixture of two or more diastereomers or as two or more pure diastereomers. This step generally yields two diastereomers, but depending on the starting materials (1a) and (4a), one or two additional diastereomers may also be obtained. Even if the resulting compound (7a) is a mixture of multiple diastereomers, it can be carried on to the next step without further purification.

[0247] (A-6 process) In this step, the cyanoethyl group and all acyl protecting groups are simultaneously removed from the compound of formula (7a) using a known organic chemistry method to produce the compound of formula (8a). This step was carried out in an autoclave or a sealed tube, as necessary.

[0248] PRO 4 When is a benzoyl group, compound (7a) was treated with 28% (v / v) aqueous ammonia in a solvent (methanol, ethanol, tetrahydrofuran, or a mixture thereof) at temperatures between 5°C and the boiling point of the solvent used in the reaction to remove the cyanoethyl and benzoyl groups. Ammonia was used in excess molar amount, preferably 300 to 3000 moles, per mole of compound (7a). The reaction time was 30 minutes to 96 hours, preferably 2 to 48 hours. If necessary, the reaction mixture was concentrated, and the residue was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof to obtain compound (8a). Even if the resulting compound (8a) is a diastereomeric mixture, it can be advanced to the next step without further purification. Alternatively, it can be advanced to the next step without further purification in this step.

[0249] (A-7 process) In this step, all silyl protecting groups are simultaneously removed from the compound of formula (8a) using a known organic chemistry method to produce the compound of formula (9a). PRO 2 and PRO 3When is a tert-butyldimethylsilyl group, compound (8a) was directly treated with triethylamine trihydrofluoride at 5°C to 100°C, preferably 35°C to 60°C, to remove the tert-butyldimethylsilyl group. Triethylamine trihydrofluoride was used in excess, preferably 100 to 200 moles, per mole of compound (8a). The reaction time was 30 minutes to 24 hours, preferably 2 to 12 hours. After cooling the reaction solution to room temperature, the reaction was quenched by gradually pouring into an ice-cold mixture of 1 M aqueous triethylammonium bicarbonate and triethylamine (3:1 to 10:1 (v / v)). If necessary, the reaction solution may be poured into an ice-cold mixture of 1 M aqueous triethylammonium bicarbonate and triethylamine. In this case, the reaction vessel was washed with acetonitrile and water. Triethylamine is used in an amount sufficient to change the pH of the reaction mixture to weakly basic, preferably about 2 moles of triethylamine per mole of triethylamine trihydrofluoride. After the organic solvent component of the reaction mixture was distilled off under reduced pressure, the remaining aqueous solution was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof to obtain compound (9a) as a single diastereomer.

[0250] (A-8 process) This step is a step in which the compound of formula (9a) is subjected to ion exchange using a known organic chemistry method to produce the compound of formula (1). A cation exchange resin (BT AG® 50W-X2 resin, 100-200 mesh, hydrogen type) was suspended in purified water and packed into an empty column cartridge. The amount of cation exchange resin used was 10 to 50 times the weight of compound (9a). After allowing excess purified water to flow by gravity, 3 column volumes of 1 M aqueous sodium hydroxide solution were allowed to flow by gravity, followed by 6 column volumes of purified water. Compound (9a) was dissolved in approximately 3 column volumes of purified water and charged onto the column. If the compound is poorly soluble in purified water, a mixture with a small amount of organic solvent (acetonitrile, methanol, etc.) may be used. The solution that flowed by gravity was collected and then eluted with 6 column volumes of purified water, etc., to obtain fractions. The fractions containing the target product were combined and lyophilized to obtain compound (1) as a single diastereomer.

[0251] A' method The CDN derivative represented by (1') of the present invention can be produced according to Method A' described below.

[0252] [ka]

[0253] This production method is a method for producing a compound represented by general formula (1') by partially modifying Method A. Specifically, the compound of general formula (1') can be produced by modifying step A-5 of Method A to step A'-5 shown below. x and R y When both are halogen atoms, step A-7 can be omitted.

[0254] [ka]

[0255] (A'-5 process) In this step, a compound of formula (7a') is produced by successively carrying out a cyclization reaction and an oxidation reaction on the compound of formula (6a') using known organic chemistry techniques. Compound (6a') was dissolved in pyridine and then concentrated under reduced pressure to prepare a 0.01 M to 0.5 M pyridine solution. This pyridine solution was added with a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) at 5°C to 35°C. 5 A cyclization reaction was carried out by adding an oxidizing agent (e.g., -dioxaphosphinan-2-one) to the reaction mixture. The dehydration condensation agent was used in an amount of 1 mole to an excess mole, preferably 3 to 5 moles, relative to 1 mole of compound (6a'). The reaction time was 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and an oxidizing agent (e.g., iodine) were added to the reaction mixture to carry out an oxidation reaction. Water was used in an amount of 0 mole to an excess mole, preferably 30 to 50 moles, relative to 1 mole of compound (6a'), and the oxidizing agent was used in an amount of 2 moles to 10 moles, preferably 3 to 5 moles. The reaction time was 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction mixture was added to an aqueous sodium bicarbonate solution (0.1 M to 1 M) and stirred for 15 minutes to 24 hours to quench the reaction. The reaction mixture was extracted once to five times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixture thereof), and the combined extracts were dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol, ethyl acetate / methanol, hexane / ethyl acetate, etc.), C18 silica gel column chromatography (buffer / acetonitrile), or a combination thereof to obtain compound (7a').

[0256] A'' method The CDN derivative represented by (1″) of the present invention can be produced according to Method A″ described below.

[0257] [ka]

[0258] This production method is a method for producing a compound represented by general formula (1'') by partially modifying Method A. Specifically, the compound of general formula (1'') can be produced by modifying Step A-3 of Method A to Step A''-3 shown below. In addition, when the substituent R x and R y When both are halogen atoms, step A-7 can be omitted.

[0259] [ka]

[0260] (A''-3 process) This step is a step of producing a compound of formula (5a'') by sequentially carrying out a coupling reaction of a compound of formula (3a'') with a compound of formula (4a'') using known organic chemistry techniques and an oxidation reaction of the resulting coupling product. Before starting the reaction of this step, compound (4a'') was azeotroped three to five times with dehydrated acetonitrile. After the final azeotropic distillation, acetonitrile was left over to prepare a 0.01 M to 1 M acetonitrile solution of compound (4a''). A desiccant (powdered or pelleted molecular sieves 3A or molecular sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use.

[0261] A coupling reaction was carried out by adding an acetonitrile solution of compound (4a'') dried by azeotropy to an acetonitrile solution of compound (3a'') at 5 to 35°C. The reaction time was 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, an oxidizing agent (e.g., tert-butyl hydroperoxide) was added to the reaction solution to carry out an oxidation reaction. The oxidizing agent was used in an amount of 1 to 5 moles, preferably 2 to 3 moles, per mole of compound (3a''). The reaction time was 5 minutes to 24 hours, preferably 30 minutes to 6 hours. A saturated aqueous solution of sodium thiosulfate was added to the reaction solution, and the mixture was stirred for 10 to 12 hours to quench the reaction. The reaction solution was extracted once to five times with an organic solvent (e.g., a mixed solvent of dichloromethane and methanol), and the combined extracts were dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain crude compound (5a''). The crude compound (5a'') obtained was used directly in the next step.

[0262] A''' method The CDN derivative represented by (1''') of the present invention can be produced according to Method A''' described below.

[0263] [ka]

[0264] This production method is a method for producing a compound represented by general formula (1''') by partially modifying Method A. Specifically, the compound of general formula (1''') can be produced by changing Step A-3 of Method A to Step A''-3 and Step A-5 to Step A'-5. In addition, the substituent R x and R y When both are halogen atoms, step A-7 can be omitted.

[0265] [ka]

[0266] Method B: Conjugation precursor (glycan conjugation) The conjugation precursor represented by (2) of the present invention can be produced according to Method B described below.

[0267] [ka]

[0268] This manufacturing method is L 1 This is a method for producing a conjugation precursor (2) in which -NH2 is substituted at any position of the above.

[0269] [ka]

[0270] (B-1 process) In this step, the compound of formula (1b) is subjected to removal of the protecting group by a known organic chemistry method to produce the compound of formula (2b).

[0271] PRO 5 When is a tert-butyloxycarbonyl group, the protecting group was removed by treating compound (1b) with trifluoroacetic acid in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixture thereof) at temperatures ranging from −10°C to the boiling point of the solvent used in the reaction, preferably from 15°C to 35°C. Trifluoroacetic acid was used in excess, preferably 20 to 50 moles, per mole of compound (1b). The reaction time ranged from 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure, suspended in toluene, and then concentrated again under reduced pressure. This procedure was repeated two to five times. A solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate, or a mixture thereof) was added to form a slurry, and the solid was collected by filtration to obtain crude compound (2b). The crude compound (2b) was carried on to the next step without further purification.

[0272] (B-2 process) In this step, a compound of formula (4b) is produced by amidating a compound of formula (2b) with a compound of formula (3b) using a known organic chemistry method.

[0273] Compound (2b) was amidated by reacting it with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (3b) in a solvent (N,N-dimethylformamide, N-methylpyrrolidone, N,N-dimethylacetamide, acetonitrile, etc.) at 5°C to 35°C. Relative to 1 mole of compound (2b), 1 mole to 5 moles of base and 0.5 moles to 1.5 moles of compound (3b) were used. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction solution was poured into a bilayer of an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol, or a mixture thereof) and water or an acidic aqueous solution (0.1 to 1 M hydrochloric acid, citric acid, etc.), followed by extraction with the organic solvent one to five times. The combined extracts were washed with saturated saline and then dried over anhydrous sodium sulfate or anhydrous magnesium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. Alternatively, the above separation step can be omitted and the reaction mixture can be directly concentrated under reduced pressure and then subjected to silica gel column purification. The resulting residue was purified by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, etc.] to obtain compound (4b). If necessary, the purity of the resulting compound (4b) can be increased by dissolving it in a good solvent (ethyl acetate, acetonitrile, dichloromethane, methanol, or a mixture thereof) and then reprecipitating it with a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.). The solid is then filtered off.

[0274] (B-3 process) In this step, a compound of formula (5b) is produced by esterifying the compound of formula (4b) using a known organic chemistry method. Esterification was carried out by reacting compound (4b) with N-hydroxysuccinimide and a condensing agent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) in a solvent (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile) at 5°C to 35°C. N-hydroxysuccinimide and the condensing agent were used in amounts of 1 to 3 moles per mole of compound (4b). The reaction time was 30 minutes to 72 hours, preferably 2 to 24 hours. The reaction solution was diluted with an organic solvent (dichloromethane, chloroform, ethyl acetate, or a mixture thereof) and washed three to five times with ice water. The organic layer was dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain crude compound (5b). If necessary, the resulting compound (5b) may be purified by C18 silica gel column chromatography (acetonitrile only). Furthermore, the purity can be increased by dissolving the obtained compound (5b) in a good solvent (ethyl acetate, acetonitrile, dichloromethane, or a mixture thereof), adding a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) to reprecipitate the compound, and then filtering out the solid.

[0275] (B-4 process) This step is a step of producing a compound of formula (2) by carrying out a condensation reaction between a compound of formula (5b) and a compound of formula (6b) using a known organic chemistry method.

[0276] Compound (6b) was reacted with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (5b) in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at temperatures ranging from -10°C to 100°C, preferably from 15°C to 35°C, to carry out a condensation reaction. Two to five moles of base and one to two moles of compound (5b) were used per mole of compound (6b). The reaction time ranged from 5 minutes to 24 hours, preferably from 1 hour to 6 hours. The reaction was terminated by adding benzylamine to the reaction solution. Four to ten moles of benzylamine were used per mole of compound (6b). If necessary, the reaction solution was partially concentrated under reduced pressure, and the remaining solution was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof, to obtain compound (2).

[0277] Method B': Conjugation precursor (cysteine ​​conjugation) B' method The conjugation precursor represented by (2') of the present invention can be produced according to Method B' described below.

[0278] [ka]

[0279] This manufacturing method is L 1 is a method for producing a conjugation precursor (2') in which -NH2 is substituted at any position of the above.

[0280] [ka]

[0281] (B-5 process) In this step, a compound of formula (2b') is amidated with a compound of formula (7b) using a known organic chemistry method to produce a compound of formula (8b). Compound (8b) was obtained according to the method described in Step B-2 of Method B, except that a base was not used.

[0282] (B-6 process) In this step, the compound of formula (9b) is produced by removing the protecting group from the compound of formula (8b) using a known organic chemistry method. 6 When is a tert-butyl group, compound (9b) was obtained according to the procedure described in Step B-1 of Method B, except that silica gel column chromatography [dichloromethane / methanol] was used for purification.

[0283] (B-7 process) In this step, the compound of formula (9b) is esterified using a known organic chemistry method to produce the compound of formula (10b). Compound (10b) was obtained according to the method described in Step B-3 of Method B.

[0284] (B-8 process) In this step, a compound of formula (6b) is condensed with a compound of formula (10b) using a known organic chemistry method to produce a compound of formula (2'). Compound (2') was obtained according to the method described in Step B-4 of Method B.

[0285] C method The conjugation precursor represented by (3) of the present invention can be produced according to Method C described below.

[0286] [ka]

[0287] This manufacturing method is L 1 The present invention relates to a method for producing a conjugation precursor (3) in which a hydroxy group is substituted at any position of the above.

[0288] [ka]

[0289] (C-1 process) In this step, a compound of formula (1c) is amidated with a compound of formula (2c) using a known organic chemistry method to produce a compound of formula (3c). Compound (3c) was obtained according to the method described in Step B-2 of Method B.

[0290] (C-2 process) In this step, the compound of formula (3c) is esterified using a known organic chemistry method to produce the compound of formula (4c). Compound (4c) was obtained according to the method described in Step B-3 of Method B.

[0291] (C-3 process) This step is a step of producing a compound of formula (7c) by sequentially carrying out a coupling reaction (aminomethylenation) of a compound of formula (5c) with a compound of formula (6c) using known organic chemistry techniques and deprotecting the resulting coupling product.

[0292] PRO 9When the fluorenylmethyloxycarbonyl group is a 9-fluorenylmethyloxycarbonyl group, aminomethylenation was carried out by reacting compound (5c) with compound (6c) and an acid (e.g., p-toluenesulfonic acid) in tetrahydrofuran at 5°C to 35°C. Compound (6c) was used in an amount of 1 to 20 moles, preferably 2 to 10 moles, per mole of compound (5c), and acid was used in an amount of 0.05 moles to an excess of molar, preferably 0.1 to 3 moles. The reaction time was 30 minutes to 72 hours, preferably 2 to 24 hours. Next, a base (e.g., 1,8-diazabicyclo[5.4.0]-7-undecene) was added to the reaction mixture to carry out deprotection. If the reaction mixture was suspended, a solvent (e.g., N,N-dimethylformamide) could be added to dissolve the mixture before reaction, as necessary. The base was used in an excess of molar amounts, preferably 5 to 20 moles, per mole of compound (5c). The reaction time is 10 minutes to 24 hours, preferably 2 to 12 hours. Water is added to the reaction mixture, which is then purified directly by C18 silica gel column chromatography (buffer solution / acetonitrile, etc.) to obtain compound (7c).

[0293] (C-4 process) In this step, the protecting group is removed from the compound of formula (7c) using a known organic chemistry method to produce a compound of formula (8c). 7 and PRO 8 When is a tert-butyldimethylsilyl group, compound (8c) was obtained according to the procedure described in Step A-7 of Method A.

[0294] (C-5 process) In this step, a compound of formula (8c) is condensed with a compound of formula (4c) using a known organic chemistry method to produce a compound of formula (3). Compound (3) was obtained according to the method described in Step B-4 of Method B.

[0295] C' method The conjugation precursor represented by (3') of the present invention can be produced according to Method C' described below.

[0296] [ka]

[0297] This manufacturing method is L 1 The present invention relates to a method for producing a conjugation precursor (3') in which a hydroxy group is substituted at any position of the above.

[0298] [ka]

[0299] (C'-1 process) In this step, the compound of formula (1c') is subjected to hydrolysis and removal of the cyanoethyl group in succession using known organic chemistry techniques to produce the compound of formula (2c'). Compound (2c') was obtained according to the method described in Method A, Step A-1.

[0300] (C'-2 process) In this step, the protecting group of the hydroxy group of the compound of formula (2c') is removed using a known organic chemistry method to produce a compound of formula (3c'). Compound (3c') was obtained according to the method described in Step A-2 of Method A.

[0301] (C'-3 process) In this step, compound (3c') is subjected to a coupling reaction with compound (4c') using known organic chemistry techniques, followed by a sulfurization or oxidation reaction of the resulting coupling product, to produce compound (5c'). Compound (5c') was obtained according to the method described in Step A-3 of Method A or Step A''-3 of Method A''.

[0302] (C'-4 process) In this step, the protecting group of the hydroxy group of compound (5c') is removed using a known organic chemistry method to produce compound (6c'). Compound (6c') was obtained according to the method described in Step A-4 of Method A.

[0303] (C'-5 process) In this step, compound (6c') is subjected to a cyclization reaction and a sulfurization or oxidation reaction in succession using known organic chemistry techniques to produce compound (7c'). Compound (7c') was obtained according to the method described in Step A-5 of Method A or Step A'-5 of Method A'.

[0304] (C'-6 process) In this step, the cyanoethyl group and all acyl protecting groups are simultaneously removed from compound (7c') using a known organic chemistry method to produce compound (8c'). Compound (8c') was obtained according to the method described in Step A-6 of Method A.

[0305] (C'-7 process) In this step, all silyl protecting groups are simultaneously removed from the compound of formula (8c') using a known organic chemistry method to produce the compound of formula (9c'). 9 When is a 2-(trimethylsilyl)ethoxycarbonyl group, compound (8c') was treated with a tetrahydrofuran solution of tetrabutylammonium fluoride at 5°C to 100°C, preferably 35°C to 60°C, to remove the 2-(trimethylsilyl)ethoxycarbonyl group. Tetrabutylammonium fluoride was used in excess, preferably 10 to 30 moles, per mole of compound (8c'). The reaction time was 1 to 48 hours, preferably 4 to 24 hours. After diluting the reaction mixture with a buffer solution, the organic solvent was removed under reduced pressure, if necessary. The residue was purified by preparative HPLC (buffer / acetonitrile, buffer / methanol, etc.), C18 silica gel column chromatography (buffer / acetonitrile, buffer / methanol, etc.), or a combination thereof, to obtain compound (9c').

[0306] (C'-8 process) In this step, a compound of formula (9c') is condensed with a compound of formula (4c) using a known organic chemistry method to produce a compound of formula (3'). Compound (3') was obtained according to the method described in Step B-4 of Method B.

[0307] Method D: Production of glycosylation remodeling antibodies The glycan remodeling antibody can be produced by the method shown in the following scheme, for example, in accordance with the method described in WO2018 / 003983.

[0308] [ka]

[0309] (D-1 process) This process involves hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing end chitobiose structure of the N-linked glycan (N297-linked glycan) attached to asparagine at position 297 of the antibody's amino acid sequence using a known enzymatic reaction to produce a glycosylated antibody. The target antibody (1d) (10 mg / mL) was hydrolyzed at 0°C to 40°C in a buffer solution (e.g., phosphate buffer) using a hydrolase such as wild-type EndoS enzyme to hydrolyze the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the chitobiose structure at the reducing end. The reaction time ranged from 10 minutes to 72 hours, preferably 1 to 6 hours. The wild-type EndoS enzyme was used in an amount of 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, per 100 mg of antibody (1d). After the reaction, the antibody was purified using affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and / or a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (Bio-Rad)) to obtain (Fucα1,6)GlcNAc antibody (2d).

[0310] (D-2 process) This step involves conjugating an SG-type or MSG (MSG1, MSG2)-type glycan oxazoline having an azide group-containing PEG linker (hereinafter referred to as "azidoglycan oxazoline") to the (Fucα1,6)GlcNAc antibody (2d) obtained in step D-1 using a known enzymatic reaction to produce a glycan remodeling antibody (3d).

[0311] Antibody (2d) was reacted with an azido-glycosylated oxazoline derivative in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L) in a buffer solution (e.g., phosphate buffer) at temperatures ranging from 0°C to 40°C to carry out the glycosylation reaction. The reaction time ranged from 10 minutes to 72 hours, preferably 1 to 6 hours. EndoS enzyme (D233Q / Q303L) was used in an amount of 1 mg to 10 mg, preferably 1 to 3 mg, per 100 mg of antibody. The azido-glycosylated oxazoline derivative was used in an excess amount, preferably 4 to 20 equivalents. After the reaction, the antibody was purified using affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (Bio-Rad)) to obtain glycosylated remodeling antibody (3d).

[0312] In the preparation of the above-mentioned glycosylation-remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the common procedures A to C described below. The SG-type azide sugar chain oxazoline compound was synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]2-SG(10)-Ox (compound 1-10 described in WO2018 / 003983) is shown in the following scheme.

[0313] [ka]

[0314] The MSG-type azidoglycan oxazoline derivative was also synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]-MSG1(9)-Ox (compound 1-11 described in WO2018 / 003983) is shown in the following scheme.

[0315] [ka]

[0316] Method E: Antibody-drug conjugation (glycoconjugation 1)

[0317] [ka]

[0318] (Here, the two asterisks (*) on the left side of antibody drug conjugate (1e) indicate the drug linker moiety indicated by the asterisk on the right side.) This production method involves the conjugation of the glycosylated remodeling antibody (3d) obtained in step D-2 of Method D with the conjugation precursor (2) obtained in step B-4 of Method B via the SPAAC (strain-promoted azide-alkyne cycloaddition: J. Am. Chem. Soc. 2004, 126, 15046-15047) reaction to produce the antibody-drug conjugate (1e).

[0319] (E-1 process) The SPAAC reaction was carried out by mixing a buffer solution of the glycosylation-remodeling antibody (3d) (e.g., phosphate buffer, acetate buffer, borate buffer, etc.) with a solution of the conjugation precursor (2) dissolved in an appropriate solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixture thereof). The conjugation precursor (2) was used in an amount of 2 moles to a molar excess, preferably 4 to 30 moles, per mole of glycosylation-remodeling antibody (3d). The organic solvent ratio relative to the antibody buffer solution was preferably 1% to 200% (v / v). The reaction temperature was 0°C to 37°C, preferably 15°C to 25°C, and the reaction time was 1 hour to 150 hours, preferably 6 hours to 72 hours. The pH of the reaction solution was preferably 5 to 9. The reaction solution was purified according to the method described in Common Procedure D below to obtain the antibody-drug conjugate (1e).

[0320] Method E': Antibody-drug conjugation (cysteine ​​conjugation) The antibody-drug conjugate of the present invention having a cysteine ​​conjugation can be produced in accordance with the method described in WO2014 / 057687, etc., using the antibody of interest prepared according to Reference Example 1, etc., and the conjugation precursor (2') having a maleimide group obtained in Step B-8 of Method B'.

[0321] E'' Method: Antibody-Drug Conjugation (Sugar Conjugation 2) In Method E, the conjugation precursor (2) was converted to the conjugation precursor (3') obtained in Step C'-8 of Method C' to obtain the antibody-drug conjugate (1e'') shown in the following formula.

[0322] [ka]

[0323] (where the two asterisks (*) on the left of the antibody-drug conjugate (1e'') 1) indicates the drug linker moiety indicated by the asterisk on the right.

[0324] The antibody-drug conjugate can be identified by buffer exchange, purification, measurement of antibody concentration, and measurement of the average number of drugs bound per antibody molecule according to the common procedures D to G described below.

[0325] Common Procedure A: Concentration of antibody aqueous solution The antibody or antibody-drug conjugate solution was placed in an Amicon® Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and the antibody or antibody-drug conjugate solution was concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000 to 4000 G for 5 to 20 minutes.

[0326] Common Procedure B: Antibody concentration measurement Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The 280 nm extinction coefficient (1.3 mL mg) varied depending on the antibody. -1 cm -1 to 1.8 mL mg -1 cm -1 ) was used.

[0327] Common Procedure C: Antibody Buffer Exchange A buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to the aqueous antibody solution, and the solution was concentrated according to the method described in Common Procedure A. This procedure was repeated several times, and then the antibody concentration was measured according to the method described in Common Procedure B. An appropriate buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to this antibody buffer solution to prepare an antibody buffer solution of the desired concentration (for example, approximately 10 mg / mL).

[0328] Common Procedure D: Purification of Antibody-Drug Conjugates (Gel Filtration Chromatography) A NAP column (NAP-5, NAP-10, NAP-25 (GE Healthcare)) was equilibrated with acetate buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5; herein referred to as ABS) or another appropriate buffer. The antibody-drug conjugate reaction solution was charged onto this NAP column, and the manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This fraction was again charged onto the NAP column, and the manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This procedure was repeated two to three times to obtain an antibody-drug conjugate from which unbound drug linker, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-drug conjugate solution was adjusted using common procedures A and C.

[0329] Common Procedure E: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (UV method) The bound drug concentration in an antibody-drug conjugate can be calculated by measuring the absorbance of an aqueous antibody-drug conjugate solution at two wavelengths, 280 nm and 260 nm (wavelengths other than 260 nm may also be used), using an absorption spectrophotometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.), and then performing the following calculation: Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (additivity of absorbance), assuming that there is no change in the molar extinction coefficients of the antibody and drug before and after conjugation of the antibody and drug, the antibody concentration and drug concentration in the antibody-drug conjugate are expressed by the following relationship: A 280 =A D , 280 +A A , 280 =ε D , 280 C D +ε A , 280 C A Formula (I) A 260 =A D , 260 +AA , 260 =ε D , 260 C D +ε A , 260 C A Formula (II) where A 280 indicates the absorbance of the antibody-drug conjugate aqueous solution at 280 nm, and A 260 indicates the absorbance of the antibody-drug conjugate aqueous solution at 260 nm, and A A , 280 indicates the absorbance of the antibody at 280 nm, and A A , 260 indicates the absorbance of the antibody at 260 nm, and A D , 280 denotes the absorbance of the conjugate precursor at 280 nm, and A D , 260 denotes the absorbance of the conjugate precursor at 260 nm, and ε A , 280 denotes the molar extinction coefficient of the antibody at 280 nm, and ε A , 260 denotes the molar extinction coefficient of the antibody at 260 nm, and ε D , 280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D , 260 is the molar extinction coefficient of the conjugate precursor at 260 nm, and C A indicates the antibody concentration in the antibody-drug conjugate, and C D denotes the drug concentration in the antibody-drug conjugate, where ε A , 280 , ε A , 260 , ε D , 280 , ε D , 260 is a value prepared in advance (calculated estimated value or actual measured value). For example, ε A , 280 can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A , 260is the difference between the measured value obtained from UV measurement of the antibody and ε A , 280 In the Examples, the molar extinction coefficient of the modified anti-HER2 antibody was calculated from the estimated value of ε A , 280 = 215380 and ε A , 260 = 110117. The molar extinction coefficient of the modified anti-LPS antibody was ε A , 280 = 227300 and ε A , 260 =110710 was used. D , 280 and ε D , 260 The A of the antibody-drug conjugate solution was measured using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length) by dissolving the conjugate precursor to be used at a certain molar concentration. The A of the antibody-drug conjugate solution was measured using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). The molar extinction coefficient of the conjugate precursor in the examples was obtained by UV measurement. 280 and A 260 By measuring these values ​​and substituting them into equations (I) and (II) and solving the simultaneous equations, C A and C D Furthermore, C D C A By dividing by this, the average number of drugs bound per antibody molecule can be calculated.

[0330] Common Procedure F: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (reversed-phase high-performance liquid chromatography: RP-HPLC) The antibody concentration and the average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography analysis using the following method in addition to the above-mentioned common procedure E.

[0331] [F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes to cleave the disulfide bond between the L chain and H chain of the antibody-drug conjugate. This reaction solution was used directly for HPLC analysis.

[0332] [F-2.HPLC analysis] Typical analytical conditions are as follows: HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: Acquity BEH Phenyl (2.1 x 50 mm, 1.7 μm, manufactured by Waters) Column temperature: 75℃ Flow rate: 0.8mL / min Sample injection volume: 10 μL Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropyl alcohol aqueous solution Mobile phase B: 0.075% TFA, 15% isopropyl alcohol in acetonitrile Gradient program (mobile phase B): 14%-36% (0-15 min), 36%-80% (15-17 min), 80%-14% (17-17.1 min), 14%-14% (17.1-23 min) [F-3. Data Analysis] [F-3-1] In contrast to the L chain (L0) and H chain (H0) of an antibody that do not have any drugs bound to them, the H chains bound to drugs (H chains bound to one drug: H1, H chains bound to two drugs: H2) have increased hydrophobicity and longer retention times in proportion to the number of drugs bound to them, and are therefore generally eluted in the order of L0, H0, H1, and H2. By comparing the retention times of L0 and H0, the detected peak can be assigned to L0, H0, H1, or H2.

[0333] [F-3-2] Because the drug linker has UV absorption, the peak area was corrected according to the number of drug linkers bound using the molar extinction coefficients of the H chain and drug linker according to the following formula.

[0334]

number

[0335] Here, the molar extinction coefficients (280 nm) of the L chain and H chain of each antibody were estimated values ​​calculated by the known calculation method described in Common Procedure E. For the modified anti-HER2 antibody, 26213 was used as the molar extinction coefficient of the L chain, and 81478 was used as the molar extinction coefficient of the H chain. Similarly, for the modified anti-LPS antibody, 27703 was used as the molar extinction coefficient of the L chain, and 85948 was used as the molar extinction coefficient of the H chain. For conjugation via SPAAC reaction, the molar extinction coefficient (280 nm) of the drug linker was the measured value of the conjugation precursor. For cysteine ​​conjugation, the measured value of the compound in which each conjugation precursor was reacted with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether was used.

[0336] [F-3-3] The peak area ratio (%) of each chain to the total corrected peak area was calculated according to the following formula.

[0337]

number

[0338] [F-3-4] The average number of drugs bound per antibody molecule (DAR) in the antibody-drug conjugate was calculated according to the following formula.

[0339]

number

[0340] [F-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the following formula.

[0341]

number

[0342] Here, the absorbance (280 nm) of the antibody-drug conjugate was measured using the actual value of the antibody-drug conjugate aqueous solution. The dilution factor indicates how many times the antibody-drug conjugate aqueous solution was diluted when measuring absorbance, and is usually 4-fold. The molar extinction coefficient (280 nm) of the antibody was an estimated value calculated using the known calculation method described in Common Procedure E. The average number of drugs bound was the value obtained in [F-3-4]. For the molar extinction coefficient (280 nm) of the drug linker, the actual measured value of the conjugation precursor was used in the case of conjugation via SPAAC reaction. For cysteine ​​conjugation, the actual measured value of the compound in which each drug linker was reacted with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether was used.

[0343] Common Procedure G: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (hydrophobic interaction-high performance liquid chromatography: HI-HPLC) The antibody concentration and the average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography analysis using the following method in addition to the above-mentioned common procedures E and F.

[0344] [G-1. Preparation of samples for HPLC analysis] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was used directly for HPLC analysis.

[0345] [G-2.HPLC analysis] The typical analytical conditions are as follows: HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: TSK-gel Butyl-NPR (4.6 x 100 mm, 2.5 μm, manufactured by TOSOH) Column temperature: constant temperature around 25°C Mobile phase A: 25 mM phosphate buffer (pH = 7.0) containing 1.5 M ammonium sulfate Mobile phase B: 25 mM phosphate buffer (pH = 7.0) / isopropyl alcohol mixture (3:1) Flow rate: 0.8mL / min Sample injection volume: 15 μL Gradient program (mobile phase B): 10%-15% (0 min-5 min), 15%-65% (5 min-20 min) or HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: PolyPROPYL A (4.6 x 100 mm, 3 μm, 1500 Å, manufactured by PolyLC) Column temperature: constant temperature around 40°C Mobile phase A: 20 mM phosphate buffer (pH 7.4) containing 1.5 M ammonium sulfate Mobile phase B: 20mM phosphate buffer (pH=7.4) Flow rate: 0.8mL / min Sample injection volume: 15 μL Gradient program (mobile phase B): 40%-80% (0 min-20 min)

[0346] [G-3. Data Analysis] [G-3-1] Because hydrophobicity increases in proportion to the number of drugs bound to the antibody and retention time increases, in the case of conjugation by SPAAC reaction, elution generally occurs in the order of DAR=0, DAR=2, and DAR=4. By comparing the retention time with DAR=0, the detected peak can be assigned to either DAR=2 or DAR=4. Depending on the type of antibody and drug linker, peaks of DAR=1 and DAR=3 may also be detected. The DAR of the detected peak may also be estimated by measuring the mass spectrum after fractionating the peak by HI-HPLC. [G-3-2] Because the drug linker has UV absorption, the peak area value was corrected according to the molar absorption coefficients of the antibody and drug linker according to the following formula, depending on the number of drug linkers bound.

[0347]

number

[0348] Here, the molar extinction coefficient (280 nm) of the antibody was an estimated value calculated by the known calculation method described in Common Procedure E. The molar extinction coefficient (280 nm) of the drug linker was the actually measured value of the conjugation precursor.

[0349] [G-3-3] The antibody peak area ratio (%) to the total corrected peak area was calculated according to the following formula.

[0350]

number

[0351] [G-3-4] The average number of drugs bound per antibody molecule in the antibody-drug conjugate was calculated according to the following formula.

[0352]

number

[0353] [G-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the formula described in [F-3-5]. The average number of drugs bound was calculated using the value obtained in [G-3-4].

[0354] The novel CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention may exist as stereoisomers, optical isomers derived from asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers, and all of these isomers, optical isomers, and mixtures thereof are included in the present invention.

[0355] In the antibody-drug conjugates of the present invention, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-drug conjugates are produced by specifying reaction conditions, such as the amounts of raw materials and reagents used, so that a certain number of drugs are bound. However, unlike chemical reactions of small molecules, a mixture of drugs bound to one antibody molecule is usually obtained, with different numbers of drugs bound. The number of drugs bound to one antibody molecule can be specified as an average value, i.e., the drug binding average (DAR). The number of cyclic dinucleotide derivatives bound to an antibody molecule can be controlled, and the average number of drugs bound to one antibody can range from 1 to 10, preferably 1 to 8, and more preferably 1 to 5.

[0356] In the antibody-drug conjugate of the present invention, when the antibody Ab is bound to L via a remodeled sugar chain of the antibody Ab, the number of drugs bound per antibody molecule in the antibody-drug conjugate, m 2 is an integer of 1 or 2. When the sugar chain is an N297 sugar chain, and the sugar chain is N297-(Fuc)SG, m 2 is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.2). When the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1, and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

[0357] Furthermore, a person skilled in the art would be able to design a reaction for binding the required number of drugs to an antibody based on the description of the examples of the present application, and obtain an antibody in which the number of cyclic dinucleotide derivatives bound is controlled.

[0358] The CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention may absorb moisture, become adsorbed water, or become hydrated when left in the air or when recrystallized. Such water-containing compounds and salts are also encompassed by the present invention.

[0359] When the CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention contain a basic group such as an amino group, they can be converted into pharmaceutically acceptable salts, as desired. Examples of such salts include hydrohalides such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0360] The CDN derivatives or antibody-drug conjugates of the present invention generally can form base addition salts because they contain phosphate and / or thiophosphate groups in their structures. Furthermore, when their production intermediates contain acidic groups such as carboxyl groups, they can generally form base addition salts. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

[0361] The CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention may exist as hydrates due to, for example, absorption of moisture from the air. The solvates of the present invention are not particularly limited as long as they are pharmaceutically acceptable, but specifically preferred are hydrates, ethanol solvates, 2-propanol solvates, and the like. Furthermore, when nitrogen atoms are present in the CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention, they may be in the form of N-oxides, and these solvates and N-oxides are also within the scope of the present invention. Furthermore, when sulfur atoms are present in the CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention, they may be in the form of sulfoxides, and these solvates and sulfoxides are also within the scope of the present invention.

[0362] The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the CDN derivatives, antibody-drug conjugates, and their production intermediates of the present invention may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium (H), tritium (H), iodine-125 (I), and carbon-14 (C). The compounds of the present invention may also be radiolabeled with radioactive isotopes such as tritium (H), iodine-125 (I), or carbon-14 (C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo diagnostic imaging agents. All isotopic variants of the antibody-drug conjugates of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0363] <4. Pharmaceuticals> The CDN derivatives or antibody-drug conjugates of the present invention exhibit antitumor immune activity or cytotoxic activity against cancer cells, and therefore can be used as pharmaceuticals, particularly as therapeutic and / or preventive agents for cancer, or antitumor agents.

[0364] The types of cancer to which the CDN derivative or antibody-drug conjugate of the present invention can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer (seminoma, nonseminoma), cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor Examples of cancer cells to be treated include cancers such as gastrointestinal fibronectin (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, and sarcoma; however, the antibody-drug conjugate is not limited to these, as long as the cancer cells to be treated express a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0365] The CDN derivative or antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0366] The substances used in pharmaceutical compositions containing the CDN derivatives or antibody-drug conjugates of the present invention can be appropriately selected from pharmaceutical additives and other substances commonly used in this field in terms of dosage and administration concentration.

[0367] The CDN derivatives or antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, such pharmaceutical compositions typically contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation will correspond to the mode of administration.

[0368] Various delivery systems are known and can be used to administer the CDN derivatives or antibody-drug conjugates of the present invention. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, the CDN derivatives or antibody-drug conjugates are administered by infusion. Parenteral administration is a preferred administration route.

[0369] In a representative embodiment, a pharmaceutical composition comprising the antibody-drug conjugate is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the medicament may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the pharmaceutical composition is to be administered by infusion, it can be dispensed, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration. The pharmaceutical composition may also be provided as a solution.

[0370] The pharmaceutical compositions of the present invention may contain only the CDN derivatives or antibody-drug conjugates of the present application, or may contain the CDN derivatives or antibody-drug conjugates and at least one other cancer therapeutic agent. The CDN derivatives or antibody-drug conjugates of the present invention can also be administered together with other cancer therapeutic agents, thereby enhancing the anti-cancer effect. The other anti-cancer agents used for this purpose may be administered to an individual simultaneously with the CDN derivatives or antibody-drug conjugates, separately, or consecutively, or may be administered at different administration intervals. Examples of such cancer therapeutic agents include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, or the agents described in International Publication No. WO2003 / 038043, as well as LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), and immune checkpoint inhibitors (nivolumab, ipilimumab, etc.), but are not limited thereto as long as they have antitumor activity.

[0371] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0372] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its therapeutic effect even at a smaller dose, as the affinity of the antibody-drug conjugate to the antigen, i.e., the dissociation constant (Kd value) for the antigen, of the antibody-drug conjugate increases (the lower the Kd value). Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and the antigen. When the CDN derivative or antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0373] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Example]

[0374] In the following examples, room temperature refers to 15°C to 35°C. Anhydrous acetonitrile was used as Acetonitrile (Anhydrous)-Super- available from Kanto Chemical or Acetonitrile (Ultra Anhydrous) available from Wako Pure Chemical Industries. Pyridine was used as Pyridine (Anhydrous)-Super- available from Kanto Chemical. Silica gel chromatography was performed using Biotage SNAP Ultra (Biotage), Chromatorex Q-Pack SI (Fuji Silysia Chemical), or Purif-Pack-Ex SI (Shoko Science). DIOL silica gel column chromatography was performed using Chromatorex Q-pack DIOL (Fuji Silysia Chemical). C18 silica gel column chromatography was performed using Biotage SNAP Ultra C18 (Biotage). Amino silica gel column chromatography was performed using Biotage SNAP Isolute NH2 (Biotage). Preparative HPLC was performed using a SHIMADZU SPD-M10A HPLC system (Shimadzu Corporation). The preparative column used was Kinetex (5 μm, C18, 100 Å, 250×30.0 mm, manufactured by Phenomenex) or Kinetex (5 μm, C18, 100 Å, 250×21.2 mm, manufactured by Phenomenex).

[0375] The following instruments were used to measure various spectral data. 1 H-NMR spectra were measured using a JEOL ECS-400 (400 MHz), a Varian 400-MR (400 MHz), or a Varian Unity Inova 500 (500 MHz). 31P-NMR spectra were measured using a JEOL ECS-400 (160 MHz). Mass spectra were measured using an Agilent 6130 Quadrupole LC / MS system (Agilent Technologies). LC / MS measurements were performed under the following conditions: column: Develosil Combi-RP, 5 μm, 50 × 2.0 mm (Nomura Chemical Co., Ltd.); mobile phase: 0.1% formic acid acetonitrile solution / 0.1% formic acid aqueous solution; 0.1% formic acid acetonitrile solution: 2%-100% (0-5 min or 0-10 min).

[0376] Example 1: Synthesis of CDN1 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0377] [ka]

[0378] [Synthetic scheme]

[0379] [ka]

[0380] (Process 1) 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine

[0381] To a solution of 5-iodotubercidin (1.0 g) in N,N-dimethylformamide (10 mL) as previously described (Tetrahedron 2007, 63, 9850-9861), di-tert-butylsilylbis(trifluoromethanesulfonate) (1.24 mL) was slowly added dropwise at 0°C, followed by stirring at the same temperature for 30 minutes. Imidazole (868 mg) was added at 0°C, and the mixture was warmed to room temperature and stirred for 30 minutes. tert-Butyldimethylchlorosilane was added at room temperature, followed by stirring overnight at the same temperature. The reaction mixture was quenched with saturated aqueous sodium bicarbonate and then extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (910 mg). MS(ESI)m / z:647(M+H) + . 1 H-NMR(CDCl3)δ:8.25(1H,s),7.03(1H,s),6.10(1H,s),5.63(2H,brs),4.49-4.44(2H,m),4.26(1H,dd,J=9.7 ,4.8Hz),4.17(1H,m),4.00(1H,t,J=9.7Hz),1.09(9H,s),1.04(9H,s),0.91(9H,s),0.13(3H,s),0.11(3H,s).

[0382] (Process 2) 7-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-5-(3,3-diethoxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine To a solution of the compound obtained in Step 1 (910 mg) in N,N-dimethylformamide (3.0 mL) and tetrahydrofuran (9.0 mL), propargylaldehyde dimethyl acetal (1.01 mL), triethylamine (0.392 mL), tetrakis(triphenylphosphine)palladium(0) (163 mg), and copper(I) iodide (53.6 mg) were added in that order, and the mixture was stirred at 40°C for 18 hours. A saturated aqueous solution of sodium bicarbonate and ethyl acetate were added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to yield the title compound (878 mg). MS(ESI)m / z:647(M+H) + . 1 H-NMR(CDCl3)δ:8.27(1H,s),7.17(1H,s),6.09(1H,s),5.56(2H,brs),5.50(1H ,s),4.48(1H,dd,J=9.1,4.9Hz),4.42(1H,d,J=4.9Hz),4.25(1H,dd,J=9.4,4.6 Hz),4.17(1H,m),4.00(1H,t,J=9.7Hz),3.85-3.77(2H,m),3.66(2H,m),1.28(6 H,t,J=7.3Hz),1.08(9H,s),1.04(9H,s),0.91(9H,s),0.13(3H,s),0.11(3H,s).

[0383] (Step 3) 2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 2 (878 mg) in ethanol (8.8 mL), 10% palladium on carbon (M) wet (500 mg) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 9 hours. The catalyst was removed by filtration, and the mixture was washed with dichloromethane, and the filtrate was concentrated under reduced pressure. To a solution of the residue in acetic acid (8.8 mL), 10% palladium on carbon (M) wet (500 mg) was added, and the mixture was stirred under a hydrogen atmosphere at 40°C for 2 days. The catalyst was removed by filtration, and the mixture was washed with dichloromethane, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (603 mg). MS(ESI)m / z:561(M+H) + . 1 H-NMR(CDCl3)δ:8.47(1H,brs),8.07(1H,s),6.70(1H,s),6.14(1H,s),4.47-4.43(2H,m),4.29(1H,dd,J=9.1,4.8Hz),4.15(1H,m),3. 99(1H,t,J=9.7Hz),3.55(2H,m),2.89(2H,t,J=5.4Hz),2.04(2H,m),1.09(9H,s),1.04(9H,s),0.90(9H,s),0.10(3H,s),0.10(3H,s).

[0384] (Step 4) 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3,5-O-(di-tert-butylsilylidene)-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 3 (2.17 g) in dichloromethane (21.7 mL) was added pyridine (1.56 mL), N,N-dimethylaminopyridine (94.5 mg), and benzoyl chloride (0.898 mL) in that order at room temperature, and the mixture was stirred at 50°C for 15 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution to the reaction mixture. After extraction with dichloromethane, the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (1.91 g). MS(ESI)m / z:665(M+H) + . 1 H-NMR(CDCl3)δ:8.08(1H,s),7.37-7.33(3H,m),7.23(2H,t,J=7.6Hz),6.97(1H,s),6.21(1H,s),4.50-4.46(2H,m),4.37-4.30(2H,m), 4.28-4.09(2H,m),4.02(1H,t,J=10.0Hz),3.03(2H,t,J=6.3Hz),2.29-2.17(2H,m),1.10(9H,s),1.05(9H,s),0.90(9H,s),0.10(6H,s).

[0385] (Step 5) 6-Benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene A mixture of hydrogen fluoride-pyridine (0.30 mL) and pyridine (1.88 mL) prepared at 0°C was added to a dichloromethane (15 mL) solution of the compound obtained in Step 4 (1.91 g) above, and the mixture was stirred at 0°C for 2 hours. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution. The reaction was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in pyridine (15 mL), and 4,4'-dimethoxytrityl chloride (1.17 g) was added. The mixture was stirred at 0°C for 12 hours. Methanol was added and the mixture was stirred for 30 minutes. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution. The reaction was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (1.98 g). MS(ESI)m / z:827(M+H) + . 1 H-NMR(CDCl3)δ:8.07(1H,s),7.47(2H,m),7.37-7.19(13H,m),6.84(4H,m),6 .37(1H,d,J=5.5Hz),4.75(1H,t,J=5.2Hz),4.38-4.20(4H,m),3.80(6H,s),3. 53(1H,dd,J=10.7,2.8Hz),3.40(1H,dd,J=11.0,3.1Hz),2.83(1H,d,J=3.7Hz) ,2.78(2H,t,J=6.4Hz),2.17(2H,m),0.81(9H,s),-0.03(3H,s),-0.21(3H,s).

[0386] (Step 6) 6-Benzoyl-2-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound obtained in Step 5 (1.98 g) in dichloromethane (23.9 mL), N,N-diisopropylethylamine (1.02 mL) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.07 mL) were added, and the mixture was stirred at room temperature for 15 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution to the reaction mixture. The reaction mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (2.06 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 7:3). MS(ESI)m / z:1027(M+H) + . 1 H-NMR(CDCl3)δ:8.06(0.3H,s),8.04(0.7H,s),7.50-7.16(15H,m),6.85-6.79(4H,m),6.35(0.7H,d,J=6.7Hz),6.31(0.3H,d,J=6.1 Hz),4.84(0.7H,dd,J=7.0,4.6Hz),4.78(0.3H,t,J=5.8Hz),4.43-4.17(4H,m),4.04-3.85(1.3H,m),3.80-3.76(6H,m),3.69-3.43(3 H,m),3.50(0.7H,dd,J=10.6,3.3Hz),3.33-3.26(1H,m),2.87-2.76(2H,m),2.74-2.60(1.4H,m),2.31(0.6H,t,J=6.7Hz),2.23-2.11 (2H,m),1.21-1.13(7.8H,m),1.04(4.2H,d,J=6.7Hz),0.73(2.7H,s),0.72(6.3H,s),-0.03(0.9H,s),-0.06(2.1H,s),-0.24(3H,s).

[0387] (Step 7) 6-Benzoyl-2-{2-O-[tert-butyl(dimethyl)silyl]-3-O-[hydroxy(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene To a solution of the compound (1.37 g) obtained in Step 6 above in acetonitrile (6.67 mL), water (48 μL) and pyridine trifluoroacetate (335 mg) were added and stirred at room temperature for 15 minutes. tert-Butylamine (6.67 mL) was added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was azeotroped twice with acetonitrile (5 mL). Water (0.240 mL) was added to a solution of the residue in dichloromethane (16.7 mL), followed by a solution of dichloroacetic acid (0.953 mL) in dichloromethane (16.7 mL) and stirring at room temperature for 15 minutes. Pyridine (1.82 mL) was added to quench the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was azeotroped three times with dehydrated acetonitrile (10 mL), leaving approximately 5 mL of acetonitrile in the final distillation. The resulting acetonitrile solution of the title compound was used directly in the next reaction.

[0388] (Step 8) Commercially available N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (1.31 g) was azeotroped with dehydrated acetonitrile (10 mL) three times. For the final distillation, approximately 5 mL of acetonitrile was left over, and molecular sieves 3A, 1 / 16 (five pellets) were added. The acetonitrile solution prepared in step 7 was added to the above solution and stirred at room temperature for 20 minutes under a nitrogen atmosphere. N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide (300 mg) was added to the reaction mixture, which was stirred at room temperature for 30 minutes. The reaction mixture was then concentrated under reduced pressure. To a solution of the residue in dichloromethane (19.0 mL) was added water (0.240 mL), followed by the addition of a solution of dichloroacetic acid (1.20 mL) in dichloromethane (19.0 mL) and stirring at room temperature for 15 minutes. The reaction was quenched by the addition of pyridine (13.2 mL) and then concentrated under reduced pressure. The resulting crude product was used directly in the next reaction.

[0389] (Step 9) N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide A solution of the crude product obtained in step 8 in pyridine (39.6 mL) was concentrated to about 25 mL, and then 2-chloro-5,5-dimethyl-1,3,2λ 5 3H-Dioxaphosphinan-2-one (908 mg) was added and stirred at room temperature for 30 minutes. Water (0.84 mL) and 3H-1,2-benzodithiol-3-one (336 mg) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction mixture was poured into an aqueous solution (180 mL) of sodium bicarbonate (5.25 g) and stirred at room temperature for 30 minutes, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol] to obtain the title compound (507 mg) as a diastereomeric mixture at the phosphorus atom. MS(ESI)m / z:1219(M+H) + .

[0390] (Step 10) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (507 mg) obtained in Step 9 above in methanol (5 mL) was added 28% aqueous ammonia (5 mL) and the mixture was stirred at room temperature for 14 hours. The reaction mixture was concentrated, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to obtain the title compound (301 mg) as a diastereomeric mixture at the phosphorus atom. MS(ESI)m / z:958(M+H) + .

[0391] (Step 11) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) Triethylamine trihydrofluoride (3.84 mL) was added to the compound obtained in Step 10 (301 mg) and stirred at 45°C for 3 hours. An ice-cooled mixture of 1M aqueous triethylammonium bicarbonate (20 mL) and triethylamine (4 mL) was added to the reaction mixture at room temperature. The reaction mixture was concentrated under reduced pressure and then purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate / acetonitrile, acetonitrile: 0%-25% (0 min-40 min)] to separate the diastereomers at the phosphorus atom. The resulting compound (triethylamine salt) was converted to the sodium salt using the following method.

[0392] [Conversion to sodium salt] BT AG® 50W-X2 Resin (biotechnology grade, 100-200 mesh, hydrogen form) (500 mg) was suspended in purified water and packed into an empty column. After allowing excess purified water to flow by gravity, 1 M aqueous sodium hydroxide (5 mL) and purified water (10 mL) were added sequentially by gravity flow. The compound obtained above was dissolved in purified water (5 mL) and charged onto the column. The solution that flowed by gravity was collected and further eluted with purified water (10 mL). Fractions containing the target product were combined and lyophilized to obtain diastereomer 1 (83.4 mg), diastereomer 2 (44.8 mg), and diastereomer 3 (13.1 mg) of the title compound (HPLC retention time: diastereomer 1 > 2, 3). Diastereomer 1 MS(ESI)m / z:730(M+H) + . 1 H-NMR(CD3OD)δ:8.74(1H,s),8.17(1H,s),8.02(1H,s),7.10(1H,s),6.34(1H ,d,J=8.5Hz),6.30(1H,d,J=4.8Hz),5.41-5.34(1H,m),5.19-5.13(1H,m),4.8 5(1H,d,J=3.6Hz),4.79(1H,t,J=4.5Hz),4.52-4.41(2H,m),4.40-4.31(2H,m ),4.07-3.97(2H,m),3.52-3.47(2H,m),2.90-2.76(2H,m),2.05-1.95(2H,m). 31 P-NMR(CD3OD)δ:57.9(s),54.5(s). Diastereomer 2 MS(ESI)m / z:730(M+H) + . 1H-NMR(CD3OD)δ:8.82(1H,s),8.17(1H,s),8.02(1H,s),7.13(1H,s),6.35(1H,d,J=2.4Hz),6.33(1H,s),5.50-5.43(2H,m),4.80(1H,dd ,J=6.7,4.2Hz),4.52-4.28(5H,m),4.02(1H,d,J=12.1Hz),3.93-3.86(1H,m),3.54-3.47(2H,m),2.95-2.88(2H,m),2.05-1.97(2H,m). 31 P-NMR(CD3OD)δ:63.0(s),60.2(s). Diastereomer 3 MS(ESI)m / z:730(M+H) + . 1 H-NMR(CD3OD)δ:9.16(1H,s),8.17(1H,s),8.02(1H,s),7.12(1H,s),6.35(1H,d,J= 8.5Hz),6.29(1H,d,J=6.7Hz),5.63-5.56(1H,m),5.54-5.46(1H,m),4.79(1H,dd,J =6.7,4.8Hz),4.53-4.43(2H,m),4.36-4.28(2H,m),4.26-4.19(1H,m),4.16-4.09( 1H,m),3.93-3.86(1H,m),3.52-3.47(2H,m),2.92-2.87(2H,m),2.04-1.95(2H,m). 31 P-NMR(CD3OD)δ:62.8(s),58.7(s).

[0393] Example 2: Synthesis of CDN2 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione (Diastereomer 4 of Compound 1 described in Example 1)

[0394] [ka]

[0395] [Synthetic scheme]

[0396] [ka]

[0397] (Process 1) N-Benzoyl-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-[hydroxy(oxo)-λ 5 -phosphanyl]adenosine A reaction was carried out in the same manner as in Step 7 of Example 1 using commercially available N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine (962 mg) (ChemGenes), to give an acetonitrile solution of the title compound. This acetonitrile solution was used directly in the next reaction.

[0398] (Process 2) The compound obtained in Step 1 above and the compound obtained in Step 6 of Example 1 (1.00 g) were used to carry out a reaction in the same manner as in Step 8 of Example 1. The obtained crude product was used as it was in the next reaction.

[0399] (Step 3) N-{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}-2-(2-cyanoethoxy)-10-oxo-10-sulfanyl-2-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}benzamide The crude product obtained in Step 2 above was used in a reaction in the same manner as in Step 9 of Example 1 to give the title compound (367 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1219(M+H) + .

[0400] (Step 4) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-amino-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (367 mg) obtained in Step 3 above was reacted in the same manner as in Step 10 of Example 1 to give Diastereomer 1 (115 mg: containing impurities) and Diastereomer 4 (101 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:958(M+H) + . Diastereomer 4 (high polarity) MS(ESI)m / z:958(M+H) + .

[0401] (Step 5) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 4 of Compound 1) The compound (diastereomer 4) (101 mg: containing impurities) obtained in Step 4 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification conditions] to obtain the title compound as a triethylamine salt. Purification conditions: C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 5%-100% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (28.5 mg). MS(ESI)m / z:730(M+H) + . 1H-NMR(CD3OD)δ:9.11(1H,s),8.19(1H,s),8.02(1H,s),7.08(1H,s),6.35(1H,d,J= 8.5Hz),6.27(1H,d,J=4.8Hz),5.43-5.36(1H,m),5.29-5.21(1H,m),4.95-4.88(1H ,m),4.80(1H,dd,J=4.5,2.3Hz),4.50-4.43(1H,m),4.42-4.33(2H,m),4.30-4.22( 1H,m),4.20-4.03(2H,m),3.52-3.46(2H,m),2.85-2.66(2H,m),2.05-1.90(2H,m). 31 P-NMR(CD3OD)δ:58.1(s),54.1(s).

[0402] Example 3: Synthesis of CDN3 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-2,10,15,16-tetrahydroxy-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0403] [ka]

[0404] [Synthetic scheme]

[0405] [ka]

[0406] (Process 1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(olate) To a solution of the compound (diastereomer 1) (30.0 mg) obtained in Step 11 of Example 1 in acetone (0.5 mL) and water (0.2 mL), triethylamine (0.27 mL) and iodomethane (60 μL) were added and stirred for 1 day. The reaction solution was concentrated under reduced pressure and then purified by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 0%-20% (0 min-40 min)]. The obtained compound was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (21.2 mg). MS(ESI)m / z:698(M+H) + . 1 H-NMR(CD3OD)δ:8.55(1H,s),8.18(1H,s),8.01(1H,s),7.32(1H,s),6.26 (1H,s),6.13(1H,s),5.00-4.85(2H,m),4.68-4.64(1H,m),4.48-4.23(5H, m),4.15-4.04(2H,m),3.49-3.39(2H,m),2.90-2.66(2H,m),1.98-1.83(2H,m). 31 P-NMR(CD3OD)δ:-0.22(s).

[0407] Example 4: Synthesis of CDN4 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0408] [ka]

[0409] [Synthetic scheme]

[0410] [ka]

[0411] (Process 1) The reaction of Step 7 of Example 1 was carried out on the following scale (raw material: 1.01 g). An acetonitrile solution of the obtained compound and commercially available (Wuhu Nuowei Chemistry) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-N-(2-methylpropanoyl)guanosine (954 mg) were used to carry out a reaction in the same manner as in Step 8 of Example 1. The obtained crude product was used directly in the next reaction.

[0412] (Process 2) N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-methylpropanamide The crude product obtained in Step 1 above was used in a reaction in the same manner as in Step 9 of Example 1 to give the title compound (357 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1201(M+H) + .

[0413] (Step 3) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound obtained in Step 3 above (357 mg) was reacted in the same manner as in Step 10 of Example 1 to give the title compound (241 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:974(M+H) + .

[0414] (Step 4) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (241 mg) obtained in Step 3 above was reacted in the same manner as in Step 11 of Example 1, and then the diastereomers at the phosphorus atom were separated under the following [Purification Conditions] to obtain two diastereomers of the title compound, each as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-20% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give diastereomer 1 (56.7 mg) and diastereomer 2 (25.9 mg) of the title compound (HPLC retention time: diastereomer 1>2). Diastereomer 1 (less polar) MS(ESI)m / z:746(M+H) + . 1H-NMR(CD3OD)δ:8.03(1H,s),8.00(1H,s),7.11(1H,s),6.27(1H,d,J=3.0Hz),5. 99(1H,d,J=8.5Hz),5.67-5.61(1H,m),5.27-5.21(1H,m),4.85(1H,d,J=3.6Hz), 4.73(1H,dd,J=3.9,2.0Hz),4.48-4.39(2H,m),4.38-4.30(2H,m),4.18-4.08(2H ,m),3.51-3.45(2H,m),2.80-2.71(1H,m),2.63-2.53(1H,m),2.02-1.84(2H,m). 31 P-NMR(CD3OD)δ:57.6(s),53.5(s). Diastereomer 2 (high polarity) MS(ESI)m / z:746(M+H) + . 1 H-NMR(CD3OD)δ:8.20(1H,s),8.01(1H,s),7.19(1H,s),6.32(1H,d,J=6.0Hz) ,6.05(1H,d,J=8.5Hz),5.67-5.53(1H,m),5.47-5.40(1H,m),4.77-4.71(1H, m),4.51-4.46(1H,m),4.45-4.30(3H,m),4.28-4.25(1H,m),4.19-4.08(1H,m ),3.96-3.89(1H,m),3.53-3.46(2H,m),2.92-2.79(2H,m),2.05-1.93(2H,m). 31 P-NMR(CD3OD)δ:61.7(s),59.5(s).

[0415] Example 5: Synthesis of CDN5 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0416] [ka]

[0417] [Synthetic scheme]

[0418] [ka]

[0419] (Process 1) 2',3',5'-Tri-O-acetyl-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine To a suspension of 2',3',5'-tri-O-acetylinosine (5.00 g) in tetrahydrofuran (90 mL), 2-(trimethylsilyl)ethyl (2-hydroxyethyl)carbamate (3.12 g) and triphenylphosphine (3.99 g) were added, followed by a solution of dipropan-2-yl (E)-diazene-1,2-dicarboxylate (3.05 mL) in tetrahydrofuran (10 mL), and the mixture was stirred at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol] to give the title compound (3.01 g). MS(ESI)m / z:582(M+H) + . 1H-NMR(CDCl3)δ:7.96(1H,s),7.93(1H,s),6.10(1H,d,J=4.8Hz),5.86(1H,t,J=5.4Hz),5.58 (1H,t,J=5.1Hz),4.96(1H,t,J=7.3Hz),4.47-4.40(2H,m),4.36(1H,dd,J=13.0,5.1Hz),4.24 (2H,t,J=5.4Hz),4.15(2H,t,J=8.8Hz),3.55(2H,q,J=6.0Hz),2.15(3H,s),2.13(3H,s),2.10(3H,s),0.97(2H,t,J=8.8Hz),0.03(9H,s).

[0420] (Process 2) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine Potassium carbonate (100 mg) was added to a solution of the compound obtained in Step 1 (3.01 g) in tetrahydrofuran (15 mL) and methanol (15 mL), and the mixture was stirred at room temperature for 2 hours. Acetic acid (83 μL) was added to the reaction mixture, which was then concentrated under reduced pressure. The residue was azeotroped with pyridine. After redissolving the mixture in pyridine (30 mL), 4,4'-dimethoxytrityl chloride (2.10 g) was added at 0°C, the mixture was stirred for 30 minutes, and then stored in a refrigerator overnight. Methanol (1 mL) was added to the reaction mixture, which was then stirred for 30 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol / 0.1% triethylamine] to obtain the title compound (3.61 g). MS(ESI)m / z:758(M+H) + . 1H-NMR(CDCl3)δ:7.92(1H,s),7.76(1H,s),7.37(2H,d,J=7.3Hz),7.29-7.14(7H,m),6.78(4H, d,J=8.5Hz),5.94(1H,d,J=5.4Hz),5.63(1H,br s),4.81-4.74(1H,m),4.46-4.41(1H,m),4.36-4.31(1H,m),4.19-4.0 5(4H,m),3.76(6H,s),3.52-3.44(2H,m),3.44-3.31(2H,m),0.99-0.91 (2H,m),0.02(9H,s).(Only observable peaks are listed)

[0421] (Step 3) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine To a dichloromethane (18 mL) solution of the compound obtained in Step 2 (3.61 g), imidazole (811 mg) and tert-butyl(chloro)dimethylsilane (861 mg) were added, and the mixture was stirred at room temperature for 17 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (1.61 g) and its regioisomer, 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine (1.31 g). MS(ESI)m / z:872(M+H) + . 1H-NMR(CDCl3)δ: 7.98(1H,s),7.85(1H,s),7.39(2H,d,J=7.9Hz),7.32-7.15(7H,m),6.78(4H,d, J=9.1Hz),5.93(1H,d,J=4.8Hz),5.22-5.11(1H,m),4.60(1H,q,J=5.6Hz), 4.47(1H,t,J=4.2Hz),4.28-4.08(5H,m),3.77(6H,s),3.59-3.49(2H,m),3. 45(1H,dd,J=10.3,3.0Hz),3.26(1H,dd,J=10.3,3.9Hz),3.15-3.08(1H,m) ,0.95(2H,t,J=8.5Hz),0.88(9H,s),0.07(3H,s),0.02(9H,s),0.00(3H,s). Positional isomer (2'-O-TBS form) MS(ESI)m / z:872(M+H) + . 1 H-NMR(CDCl3)δ: 7.99(1H,s),7.82(1H,s),7.46-7.41(2H,m),7.35-7.19(7H,m),6.84-6.78(4H,m),5.98(1H,d,J=5.4 Hz),5.06-4.96(1H,m),4.84(1H,t,J=5.4 Hz),4.34-4.08(6H,m),3.78(6H,s),3.54(2H,q,J=5.8Hz),3.48(1H,dd,J=10.6,2.7 Hz),3.39(1H,dd,J=10.6,3.9 Hz),2.71(1H,d, J=4.2Hz),0.99-0.91(2H,m),0.85(9H,s),0.03(9H,s),0.02(3H,s),-0.12(3H,s).

[0422] (Step 4) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]inosine To a solution of the compound obtained in Step 3 (1.61 g) in dichloromethane (18.5 mL), 4,5-dicyanoimidazole (240 mg) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.703 mL) were added and stirred at room temperature for 15 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution. The reaction mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by DIOL silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (1.95 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 61:39). MS(ESI)m / z:1072(M+H) + . 1 H-NMR(CDCl3)δ:8.04(0.39H,s),7.99(0.61H,s),7.83(0.39H,s),7.82(0.61H,s),7.42(2H,d, J=7.3Hz),7.35-7.15(7H,m),6.85-6.77(4H,m),6.15(0.61H,d,J=6.0Hz),6.09(0.39H,d,J=4.8 Hz),5.34-5.24(0.61H,m),5.12-5.03(0.39H,m),4.86-4.76(0.39H,m),4.72-4.62(0.61H,m),4.47-4.4 2(0.39H,m),4.42-4.36(0.69H,m),4.31-4.05(6H,m),3.78(6H,s),3.78-3.65(1H,m),3.61-3.39(7H,m), 3.35 (0.61H,dd,J=10.6,3.9Hz),3.28(0.39H,dd,J=10.9,4.2Hz),2.49(0.78H,t,J=6.0Hz),2.29(1.22H,t,J=5.7Hz),1.30-0.9 4(12H,m),0.85(5.49H,s),0.84(3.51H,s),0.09(1.17H,s),0.08(1.83H,s),0.03(9H,s),0.02(1.83H,s),0.00(1.17H,s).

[0423] (Step 5) The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 910 mg). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 4 above (950 mg), a reaction was carried out in the same manner as in Step 8 of Example 1. The obtained crude product was used as is in the next reaction.

[0424] (Step 6) 2-(Trimethylsilyl)ethyl (2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)carbamate The crude product obtained in Step 5 above was used in a reaction in the same manner as in Step 9 of Example 1 to give the title compound (602 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1303(M+H) + .

[0425] (Step 7) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-7-{6-oxo-1-[2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)ethyl]-1,6-dihydro-9H-purin-9-yl}-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (602 mg) obtained in Step 6 above was reacted in the same manner as in Step 10 of Example 1 to give Diastereomer 1 (205 mg: containing impurities) and Diastereomer 2 (244 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1146(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:1146(M+H) + .

[0426] (Step 8-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) Triethylamine trihydrofluoride (1.31 mL) was added to the compound (diastereomer 1) (145 mg, containing impurities) obtained in Step 7 above, and the mixture was stirred at 45°C for 3 hours. An ice-cooled mixture of 1 M triethylammonium bicarbonate solution (10 mL) and triethylamine (2 mL) was added to the reaction mixture at room temperature. The reaction mixture was concentrated under reduced pressure and purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile]. A solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 2 mL) was added to a solution of the obtained compound in tetrahydrofuran (4 mL), and the mixture was stirred at room temperature for 39 hours. 10 mM aqueous triethylammonium acetate solution (4 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 5%-50% (0 min-40 min)]. The salt exchange of the obtained compound (triethylamine salt) was carried out in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1, except that a mixture of acetonitrile-methanol-pure water (1:1:1) was used as the solvent for dissolving the compound and as part of the eluate, to obtain the title compound (72.5 mg). MS(ESI)m / z:774(M+H) + . 1 H-NMR(CD3OD)δ:8.60(1H,s),8.15(1H,s),8.02(1H,s),7.11(1H,s),6.26(1H, d,J=4.8Hz),6.24(1H,t,J=5.1Hz),5.47(1H,dt,J=8.2,4.2Hz),5.23-5.17(1H, m),4.77-4.73(2H,m),4.52-4.44(2H,m),4.36-4.19(3H,m),4.14-4.02(3H,m), 3.48(2H,t,J=4.8Hz),3.31-3.26(2H,m),2.90-2.74(2H,m),2.01-1.93(2H,m). 31 P-NMR(CD3OD)δ:57.7(s),54.7(s).

[0427] (Step 8-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (diastereomer 2) (133 mg: containing impurities) obtained in the above step 7 was used to carry out reaction and salt exchange in the same manner as in the above step 8-1 to obtain the title compound (55.4 mg). MS(ESI)m / z:774(M+H) + . 1 H-NMR(CD3OD)δ:8.72(1H,s),8.25(1H,s),8.02(1H,s),7.11(1H,s),6.31(1H,d,J=6.7Hz),6.28(1H,d,J=8.5 Hz),5.47-5.38(2H,m),4.77(1H,dd,J=6.7,4.2Hz),4.48(1H,d,J=4.2Hz),4.46-4.37(2H,m),4.37-4.29(3H,m),4.27-4.1 8(1H,m),4.08-4.02(1H,m),3.92-3.85(1H,m),3.53-3.46(2H,m),3.28-3.23(2H,m),2.93-2.86(2H,m),2.04-1.96(2H,m). 31 P-NMR(CD3OD)δ:62.6(s),60.0(s).

[0428] Example 6: Synthesis of CDN6 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0429] [ka]

[0430] [Synthetic scheme]

[0431] [ka]

[0432] (Process 1) 2',3',5'-Tri-O-acetyl-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine To a suspension of commercially available 2',3',5'-tri-O-acetylinosine (10.0 g) in tetrahydrofuran (100 mL), 2-{[tert-butyl(dimethyl)silyl]oxy}ethan-1-ol (5.37 g) and triphenylphosphine (7.69 g) were added, followed by dipropan-2-yl (E)-diazene-1,2-dicarboxylate (6.10 mL) and stirring at room temperature for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane / ethyl acetate / dichloromethane] to give the title compound as a mixture with triphenylphosphine oxide (10.6 g). MS(ESI)m / z:553(M+H) + . 1H-NMR(CDCl3)δ:8.05(1H,s),7.92(1H,s),6.12(1H,d,J=5.4Hz),5.86(1H,t,J=5.4Hz),5.59(1H,dd,J=5.4,4.2Hz),4.47-4.41(2H,m),4. 38-4.31(1H,m),4.22-4.17(2H,m),3.89(2H,t,J=4.8Hz),2.15(3H,s ),2.14(3H,s),2.08(3H,s),0.83(9H,s),-0.06(3H,s),-0.06(3H,s).

[0433] (Process 2) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine The compound obtained in Step 1 above (10.6 g) was used in a reaction similar to that in Step 2 of Example 5 to give the title compound as a mixture with triphenylphosphine oxide (7.21 g). MS(ESI)m / z:729(M+H) + . 1 H-NMR(CDCl3)δ:8.01(1H,s),7.97(1H,s),7.35-7.30(2H,m),7.25-7.17(7H,m),6.81-6.76 (4H,m),5.95(1H,d,J=5.4Hz),5.13(1H,brs),4.68-4.61(1H,m),4.43-4.36(2H,m),4.31-4 .23(1H,m),4.15-4.08(1H,m),3.89(2H,t,J=4.5Hz),3.77(6H,s),3.42(1H,dd,J=10.3,3.6 Hz),3.34(1H,dd,J=10.3,3.6Hz),3.10(1H,brs),0.83(9H,s),-0.06(3H,s),-0.07(3H,s).

[0434] (Step 3) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine The compound (7.21 g) obtained in Step 2 above was used in a reaction in the same manner as in Step 3 of Example 5 to give the title compound (2.17 g) and the positional isomer of the title compound, 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)inosine (2.55 g). MS(ESI)m / z:843(M+H) + . 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(4 H,m),5.96(1H,d,J=4.2Hz),4.56-4.50(2H,m),4.33-4.25(1H,m),4.19-4.02(2H,m),3.89(2H, t,J=4.8Hz),3.78(6H,s),3.45(1H,dd,J=10.9,4.2Hz),3.27(1H,dd,J=10.9,4.2Hz),3.03(1H, d,J=6.0Hz),0.88(9H,s),0.82(9H,s),0.07(3H,s),-0.01(3H,s),-0.07(3H,s),-0.07(3H,s). Positional isomer (2'-O-TBS form) MS(ESI)m / z:843(M+H) + . 1H-NMR(CDCl3)δ:7.98(1H,s),7.94(1H,s),7.46-7.42(2H,m),7.35-7.20(7H,m),6.85-6.79(4H,m),5.9 9(1H,d,J=5.4Hz),4.83(1H,t,J=5.1Hz),4.33-4.29(1H,m),4.27-4.24(1H,m),4.24-4.12(2H,m),3.90 (2H,t,J=4.5Hz),3.79(3H,s),3.78(3H,s),3.48(1H,dd,J=10.3,3.0Hz),3.40(1H,dd,J=10.3,3.0Hz), 2.71(1H,d,J=3.6Hz),0.86(9H,s),0.83(9H,s),0.01(3H,s),-0.07(3H,s),-0.07(3H,s),-0.11(3H,s).

[0435] (Step 4) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}inosine The compound (2.17 g) obtained in Step 3 above was used in a reaction in the same manner as in Step 4 of Example 5 to give the title compound (2.65 g) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1043(M+H) + . 1H-NMR(CDCl3)δ:8.03(0.53H,s),8.01(0.47H,s),7.97(0.53H,s),7.93( 0.47H,s),7.45-7.41(2H,m),7.35-7.19(7H,m),6.83-6.78(4H,m),6.17 (0.53H,d,J=4.2Hz),6.05(0.47H,d,J=4.2Hz),4.87-4.80(0.47H,m),4. 64-4.58(0.53H,m),4.46-4.40(1H,m),4.30-4.05(3H,m),3.92-3.87(2H ,m),3.78(6H,s),3.86-3.40(5H,m),3.33-3.24(1H,m),2.54(0.94H,t,J =6.0Hz),2.43(1.06H,t,J=6.7Hz),1.16-1.09(9H,m),1.01-0.97(3H,m) ,0.83(4.23H,s),0.83(4.77H,s),0.82(9H,s),0.07(1.41H,s),0.04(1. 59H,s),-0.02(3H,s),-0.07(1.41H,s),-0.08(1.59H,s),-0.08(3H,s).

[0436] (Step 5) The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 935 mg). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 4 above (950 mg), a reaction was carried out in the same manner as in Step 8 of Example 1. The obtained crude product was used as is in the next reaction.

[0437] (Step 6) 3-({(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-yl}oxy)propanenitrile The crude product obtained in Step 5 above was used in a reaction in the same manner as in Step 9 of Example 1 to give the title compound (494 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1274(M+H) + .

[0438] (Step 7) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (494 mg) obtained in Step 6 above was reacted in the same manner as in Step 10 of Example 1 to give Diastereomer 1 (88.5 mg: containing impurities) and Diastereomer 2 (70.7 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI) m / z: 1003 (M-C6H 15 Si+2H) + . Diastereomer 2 (high polarity) MS(ESI) m / z: 1003 (M-C6H 15 Si+2H) + .

[0439] (Step 8-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) The compound (diastereomer 1) (88.5 mg: containing impurities) obtained in Step 7 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (25.7 mg). MS(ESI)m / z:775(M+H) + . 1 H-NMR(CD3OD)δ:8.63(1H,s),8.22(1H,s),8.02(1H,s),7.11(1H,s),6.30 -6.24(2H,m),5.46-5.37(1H,m),5.23-5.15(1H,m),4.83-4.79(1H,m),4.7 8-4.74(1H,m),4.53-4.42(2H,m),4.35-4.16(3H,m),4.16-3.97(3H,m),3 .83-3.78(2H,m),3.52-3.47(2H,m),2.88-2.81(2H,m),2.03-1.95(2H,m). 31P-NMR(CD3OD)δ:57.8(s),54.4(s).

[0440] (Step 8-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (diastereomer 2) (70.7 mg: containing impurities) obtained in Step 7 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. Purification conditions: C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-40 min)], and preparative HPLC [10 mM triethylammonium acetate aqueous solution / methanol, methanol: 15%-70% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (17.8 mg). MS(ESI)m / z:775(M+H) + . 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=13.6,7.6Hz),5.48-5.39(2H,m),4.78(1H,dd,J=6.7,4.2Hz),4. 51-4.28(5H,m),4.26-4.13(2H,m),4.06-4.00(1H,m),3.93-3.86(1H,m),3 .85-3.80(2H,m),3.52-3.47(2H,m),2.94-2.88(2H,m),2.05-1.97(2H,m). 31 P-NMR(CD3OD)δ:62.9(s),60.0(s).

[0441] Example 7: Synthesis of CDN7 N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide

[0442] [ka]

[0443] [Synthetic scheme]

[0444] [ka]

[0445] (Process 1-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{1-[2-(2-hydroxyacetamido)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) To a solution of the compound (10.0 mg) obtained in Step 8-1 of Example 5 in N,N-dimethylformamide (0.5 mL), triethylamine (8 μL) and 1-[(hydroxyacetyl)oxy]pyrrolidine-2,5-dione (5.3 mg) were added and stirred at room temperature for 2 hours. The reaction solution was diluted with 10 mM aqueous triethylammonium acetate and purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate / acetonitrile, acetonitrile: 0%-30% (0 min-40 min)]. The resulting compound (triethylamine salt) was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (10.5 mg). MS(ESI)m / z:832(M+H) + . 1H-NMR(CD3OD)δ:8.57(1H,s),8.04(1H,s),8.03(1H,s),7.13(1H,s),6.26(1H,d,J=4.2Hz),6. 24-6.19(1H,m),5.57-5.49(1H,m),5.26-5.18(1H,m),4.80(1H,d,J=3.6Hz),4.76(1H,t,J=4.5 Hz),4.51-4.41(2H,m),4.35-4.17(3H,m),4.11-3.95(3H,m),3.91(2H,s), 3.62-3.55(2H,m),3.52-3.45(2H,m),2.89-2.65(2H,m),2.02-1.91(2H,m). 31 P-NMR(CD3OD)δ:57.6(s),54.3(s).

[0446] (Step 1-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{1-[2-(2-hydroxyacetamido)ethyl]-6-oxo-1,6-dihydro-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (30.0 mg) obtained in Step 8-2 of Example 5 was reacted in the same manner as in Step 1-1 above, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)].

[0447] The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (23.6 mg). MS(ESI)m / z:832(M+H) + . 1 H-NMR(CD3OD)δ:8.69(1H,s),8.14(1H,s),8.02(1H,s),7.11(1H,s),6.31(1H,d,J=6.7H z),6.26(1H,d,J=7.9Hz),5.49-5.40(2H,m),4.77(1H,dd,J=6.7,4.8Hz),4.48(1H,d,J= 4.2Hz),4.46-4.28(4H,m),4.22(2H,t,J=5.4Hz),4.06-4.00(1H,m),3.94(2H,s),3.92- 3.86(1H,m),3.70-3.55(2H,m),3.52-3.47(2H,m),2.92-2.86(2H,m),2.04-1.96(2H,m). 31 P-NMR(CD3OD)δ:62.7(s),59.9(s).

[0448] Example 8: Synthesis of CDN8 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0449] [ka]

[0450] [Synthetic scheme]

[0451] [ka]

[0452] (Process 1) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2',3'-bis-O-[tert-butyl(dimethyl)silyl]-2-chloroadenosine Imidazole (16.4 g) and tert-butyldimethylchlorosilane (18.2 g) were added to a solution of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-chloroadenosine (29.1 g) in N,N-dimethylformamide (145 mL), as previously described (J. Med. Chem. 1989, 32, 1135-1140), and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction mixture to quench the reaction, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (34.9 g). MS(ESI)m / z:832(M+H) + . 1 H-NMR(CDCl3)δ:8.02(1H,s),7.47-7.42(2H,m),7.36-7.32(4H,m),7.31-7.18(3H,m),6.84-6 .79(4H,m),5.90(1H,d,J=4.8Hz),5.72(2H,brs),4.74(1H,dd,J=4.5,2.3Hz),4.25(1H,dd,J=4 .2,2.3Hz),4.21(1H,q,J=4.2Hz),3.78(6H,s),3.58(1H,dd,J=10.9,4.2Hz),3.33(1H,dd,J=1 0.9,4.2Hz),0.84(9H,s),0.82(9H,s),0.04(3H,s),-0.01(3H,s),-0.02(3H,s),-0.17(3H,s).

[0453] (Process 2) N-Acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2',3'-bis-O-[tert-butyl(dimethyl)silyl]-2-chloroadenosine To a solution of the compound obtained in Step 1 (34.9 g) in pyridine (210 mL), acetic anhydride (140 mL) and 4-dimethylaminopyridine (515 mg) were added, and the mixture was stirred under a nitrogen atmosphere at room temperature for 21 hours. The reaction mixture was diluted with dichloromethane (100 mL), followed by the addition of saturated aqueous sodium bicarbonate and extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. Dichloromethane (210 mL) and morpholine (7.30 mL) were added to the residue, and the mixture was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride was added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (45.4 g, containing impurities). The compound obtained was used in the next reaction without further purification. 1 H-NMR(CDCl3)δ:8.95(1H,brs),8.22(1H,s),7.45-7.42(2H,m),7.34-7.20(7H,m), 6.82(4H,dq,J=9.4,2.7Hz),5.96(1H,d,J=4.8Hz),4.72-4.69(1H,m),4.23(2H,brs) ,3.79(6H,s),3.59(1H,dd,J=10.9,3.6Hz),3.35(1H,dd,J=10.3,3.6Hz),2.73(3H, s),0.83(9H,s),0.82(9H,s),0.04(3H,s),0.00(3H,s),-0.03(3H,s),-0.18(3H,s).

[0454] (Step 3) N-acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-chloroadenosine A solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1.0 M, 100 mL) was added to a solution of the compound obtained in Step 2 (45.4 g, containing impurities) in tetrahydrofuran (200 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. A saturated aqueous solution of ammonium chloride was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane / acetone / 0.1% triethylamine] to give the title compound (23.0 g). 1 H-NMR(CDCl3)δ:8.71(1H,brs),8.16(1H,s),7.28-7.16(9H,m),6.78-6.73(4H,m),5.99(1H,d,J=5.4Hz),4.86(1H,t,J=5.1Hz),4.49(1H,d d,J=5.1,2.7Hz),4.39(1H,q,J=3.2Hz),3.78(3H,s),3.77(3H,s),3.42(1H,dd,J=10.9,3.6Hz),3.35(1H,dd,J=10.6,3.3Hz),2.66(3H,s).

[0455] (Step 4) N-Acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-chloroadenosine To a solution of the compound (23.0 g) obtained in Step 3 above in N,N-dimethylformamide (178 mL), imidazole (5.94 g) and tert-butyldimethylchlorosilane (6.44 g) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (9.01 g). 1H-NMR(CDCl3)δ:8.42(1H,brs),8.14(1H,s),7.38-7.35(2H,m),7.29-7.18(7 H,m),6.80-6.76(4H,m),5.99(1H,d,J=4.2Hz),4.71-4.66(2H,m),4.17-4.14 (1H,m),3.78(6H,s),3.49(1H,dd,J=10.6,3.3Hz),3.29(1H,dd,J=10.9,4.2H z),3.02(1H,d,J=5.4Hz),2.67(3H,s),0.89(9H,s),0.11(3H,s),0.02(3H,s).

[0456] (Step 5) N-Acetyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-chloro-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine The compound (9.01 g) obtained in Step 4 above was reacted in the same manner as in Step 6 of Example 1 to give the title compound (10.6 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 65:35). 1H-NMR(CDCl3)δ:8.40(1H,brs),8.31(0.35H,s),8.25(0.65H,s),7.38(2H,d,J=7.3Hz),7.29-7.19(7H,m),6.80(4H,dd,J=9.1,2.4Hz),6.27(0.3 5H,d,J=3.0Hz),6.13(0.65H,d,J=3.6Hz),4.88-4.83(0.65H,m),4.69-4 .65(0.35H,m),4.56(1H,t,J=4.8Hz),4.23-4.17(1H,m),3.93-3.78(1H,m ),3.78(6H,s),3.64-3.54(4H,m),3.33-3.28(1H,m),2.67(3H,s),2.56( 1.3H,t,J=6.3Hz),2.52(0.7H,t,J=6.3Hz),1.16(2.1H,d,J=7.3Hz),1.14 (3.9H,d,J=6.0Hz),1.12(3.9H,d,J=6.0Hz),1.02(2.1H,d,J=6.7Hz),0. 83(5.9H,s),0.82(3.1H,s),0.10(1.9H,s),0.07(1.1H,s),0.01H(3H,s).

[0457] (Step 6) N,N-Diethylethanaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(6-acetamido-2-chloro-9H-purin-9-yl)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate The same reaction as in Step 7 of Example 1 was carried out on the following scale (starting material: 2.06 g). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 5 above (1.98 g), a reaction was carried out in the same manner as in Steps 8 and 9 of Example 1 to give Diastereomer 1 (180 mg) and Diastereomer 2 (167 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1191(M+H) + . 1 H-NMR(CD3OD)δ:9.10(1H,s),8.00(1H,s),7.40-7.36(2H,m),7.30-7.23(4H,m),6.38(1H,d,J=8.5Hz),6.35(1H,d,J= 3.0Hz),5.67-5.60(1H,m),5.09-5.04(1H,m),4.72(1H,d,J=3.6Hz),4.50-4.29(8H,m),4.07(1H,dd,J=12.4,4.5Hz), 3.91-3.83(1H,m),3.54-3.45(1H,m),3.17(6H,q,J=7.3Hz),3.08(2H,t,J=6.0Hz),2.45-2.42(2H,m),2.42(3H,s),2. 28-2.23(2H,m),1.29(9H,t,J=7.3Hz),1.01(9H,s),0.90(9H,s),0.29(3H,s),0.28(3H,s),0.25(3H,s),0.10(3H,s). Diastereomer 2 (high polarity) MS(ESI)m / z:1191(M+H) + .

[0458] (Step 7-1) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione Ethylenediamine (256 μL) was added to a solution of the compound (diastereomer 1) (49.6 mg) obtained in Step 6 above in methanol (1.28 mL), and the mixture was stirred at 60° C. for 2 hours, followed by a microwave reaction at 120° C. for 2 hours. Purification by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 40%-70% (0 min-30 min)] gave the title compound (37.2 mg). MS(ESI)m / z:1016(M+H) + . 1 H-NMR(CD3OD)δ:8.27(1H,s),7.99(1H,s),7.16(1H,s),6.25(1H,d,J=4.2Hz),6.09(1H,d,J=8.5H z),5.41-5.35(1H,m),5.12-5.08(1H,m),4.86-4.80(2H,m),4.69(1H,t,J=4.5Hz),4.51-4.45(1H, m),4.29-4.23(2H,m),4.11-4.03(2H,m),3.51-3.44(4H,m),3.13-3.04(2H,m),2.80-2.76(2H,m) ,2.02-1.92(2H,m),0.99(9H,s),0.84(9H,s),0.32(3H,s),0.29(3H,s),0.24(3H,s),0.07(3H,s).

[0459] (Step 7-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) Ethylenediamine (25.8 μL) was added to a solution of the compound (diastereomer 2) (50.0 mg, containing impurities) obtained in Step 6 above in methanol (1.29 mL), and the mixture was stirred at 60° C. for 2 hours, followed by a microwave reaction at 120° C. for 2 hours. Purification by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 30%-50% (0 min-30 min)] gave the title compound (23.7 mg). MS(ESI)m / z:1016(M+H) + . 1 H-NMR(CD3OD)δ:8.16(1H,s),8.00(1H,s),7.08(1H,s),6.33(1H,d,J=7.3Hz),6.13(1H,d,J=8.5Hz),5.51-5.48( 1H,m),5.30(1H,t,J=4.8Hz),5.13-5.06(1H,m),4.95(1H,d,J=4.2Hz),4.66-4.55(2H,m),4.24(1H,s),4.08(1H,d d,J=12.4,4.5Hz),3.89-3.83(1H,m),3.69-3.61(1H,m),3.50-3.33(4H,m),3.12-3.01(14H,m),2.89(2H,t,J=5.4 Hz),2.03-1.96(2H,m),1.25(18H,t,J=7.3Hz),0.99(9H,s),0.74(9H,s),0.27(6H,s),0.18(3H,s),-0.08(3H,s).

[0460] (Step 8-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) The compound (37.2 mg) obtained in Step 7-1 above was reacted in the same manner as in Step 11 of Example 1, and then purified by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 2%-20% (0 min-30 min)] to obtain the title compound as a triethylamine salt.

[0461] The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (16.5 mg). MS(ESI)m / z:788(M+H) + . 1 H-NMR(CD3OD)δ:8.21(1H,brs),8.01(1H,s),7.07(1H,s),6.27(1H,d,J=3.6Hz) ,6.10(1H,d,J=8.5Hz),5.51-5.41(1H,m),5.16-5.11(1H,m),4.83(1H,d,J=3.6 Hz),4.73(1H,t,J=4.2Hz),4.50-4.45(2H,m),4.35-4.29(2H,m),4.16-4.04(2H ,m),3.50-3.42(4H,m),3.17-3.05(2H,m),2.82-2.66(2H,m),2.04-1.92(2H,m). 31 P-NMR(CD3OD)δ:57.9(s),54.2(s).

[0462] (Step 8-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (23.7 mg) obtained in Step 7-2 above was reacted in the same manner as in Step 11 of Example 1, and then purified by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 2%-20% (0 min-30 min)] to obtain the title compound as a triethylamine salt.

[0463] The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (14.9 mg). MS(ESI)m / z:788(M+H) + . 1 H-NMR(CD3OD)δ:8.27(1H,brs),8.02(1H,s),7.15(1H,s),6.31(1H,d,J=6.0Hz),6 .12(1H,d,J=8.5Hz),5.45-5.33(2H,m),4.75(1H,dd,J=5.7,4.5Hz),4.50(1H,d,J= 4.2Hz),4.47-4.30(4H,m),4.17-4.13(1H,brm),3.94-3.89(1H,m),3.69-3.59(1H ,brm),3.51-3.44(3H,m),3.21-3.07(2H,m),2.88-2.85(2H,m),2.03-1.97(2H,m). 31 P-NMR(CD3OD)δ:62.2(s),59.8(s).

[0464] Example 9: Synthesis of CDN9 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0465] [ka]

[0466] [Synthetic scheme]

[0467] [ka]

[0468] (Process 1-1) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione To a solution of the compound (diastereomer 1) (50.1 mg) obtained in Step 6 of Example 8 in methanol (1.29 mL) was added 2-aminoethanol (258 μL), and the mixture was stirred at 60° C. for 2 hours, followed by a microwave reaction at 120° C. for 2 hours. Purification by preparative HPLC [10 mM triethylammonium acetate solution / acetonitrile, acetonitrile: 20%-60% (0 min-30 min)] afforded the title compound (39.4 mg) as a mixture containing compounds derived from ethanolamine. MS(ESI)m / z:1017(M+H) + . 1 H-NMR(CD3OD)δ:8.32(1H,s),8.01(1H,s),7.16(1H,s),6.28(1H,d,J=5.4Hz),6.13(1H,d,J=9.1Hz),5.44- 5.38(1H,m),5.19-5.14(1H,m),4.98-4.83(2H,m),4.78-4.75(1H,m),4.45-4.39(1H,m),4.28-4.22(1H,m) ,4.18(1H,s),4.13-4.07(1H,m),4.04-3.99(1H,m),3.67(2H,t,J=5.4Hz),3.51-3.42(4H,m),2.86(2H,t,J =5.4Hz),2.04-1.98(2H,m),0.98(9H,s),0.82(9H,s),0.31(3H,s),0.27(3H,s),0.22(3H,s),0.05(3H,s).

[0469] (Step 1-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (diastereomer 2) (49.3 mg, containing impurities) obtained in Step 6 of Example 8 in methanol (1.27 mL) was added 2-aminoethanol (254 μL), and the mixture was stirred at 60° C. for 2 hours, followed by a microwave reaction at 120° C. for 3 hours. Purification by preparative HPLC [10 mM triethylammonium acetate solution / acetonitrile, acetonitrile: 20%-60% (0 min-30 min)] gave the title compound (26.1 mg). MS(ESI)m / z:1017(M+H) + . 1 H-NMR(CD3OD)δ:8.25(1H,s),8.01(1H,s),7.10(1H,s),6.36(1H,d,J=7.3Hz),6.17(1H,d,J=7.9Hz),5.59-5.53( 1H,m),5.41(1H,t,J=4.5Hz),5.21-5.14(1H,m),5.02-4.95(2H,m),4.70-4.61(2H,m),4.18(1H,s),4.03(1H,dd, J=12.1,4.8Hz),3.91-3.86(1H,m),3.75-3.69(2H,m),3.52-3.43(4H,m),3.14(12H,q,J=7.3Hz),2.93-2.91(2H, m),2.04-1.99(2H,m),1.28(18H,t,J=7.6Hz),0.99(9H,s),0.75(9H,s),0.27(6H,s),0.21(3H,s),-0.05(3H,s).

[0470] (Process 2-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) The mixture (39.4 mg) obtained in Step 1-1 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] Preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 2%-20% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (16.8 mg). MS(ESI)m / z:789(M+H) + . 1 H-NMR(CD3OD)δ:8.25(1H,s),8.02(1H,s),7.10(1H,s),6.30(1H,d,J=3.6Hz) ,6.15(1H,d,J=8.5Hz),5.49-5.42(1H,m),5.21-5.16(1H,m),4.87-4.85(1H,m ),4.77(1H,t,J=4.2Hz),4.50-4.35(3H,m),4.31(1H,s),4.12-4.10(2H,m),3 .64(2H,t,J=5.4Hz),3.51-3.38(4H,m),2.85-2.70(2H,m),2.02-1.94(2H,m). 31 P-NMR(CD3OD)δ:57.8(s),53.9(s).

[0471] (Step 2-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (26.1 mg) obtained in Step 1-2 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] Preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 2%-20% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (18.6 mg). MS(ESI)m / z:789(M+H) + . 1 H-NMR(CD3OD)δ:8.35(1H,s),8.03(1H,s),7.17(1H,s),6.33(1H,d,J=6.0Hz),6.16(1H,d,J=8.5Hz),5.49-5.41(2H,m),4.80(1H,t,J=5.4Hz),4 .51-4.26(5H,m),4.07(1H,d,J=12.7Hz),3.94-3.89(1H,m),3.67(2H,5 ,J=5.7Hz),3.53-3.39(4H,m),2.89(2H,t,J=5.4Hz),2.03-1.99(2H,m). 31 P-NMR(CD3OD)δ:62.8(s),60.3(s).

[0472] Example 10: Synthesis of CDN10 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-amino-2-methylpropyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0473] [ka]

[0474] [Synthetic scheme]

[0475] [ka]

[0476] (Process 1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[(2-amino-2-methylpropyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (diastereomer 1) (41.0 mg) obtained in Step 6 of Example 8 in methanol (1.10 mL), 1,2-diamino-2-methylpropane (210 μL) was added and the mixture was stirred at 60°C for 2 hours, followed by a microwave reaction at 120°C for 6 hours. The mixture was purified by preparative HPLC [10 mM triethylammonium acetate solution / acetonitrile, acetonitrile: 20%-60% (0 min-30 min)] to give the title compound (16.3 mg, containing impurities). The compound obtained was used in the next reaction without further purification. MS(ESI)m / z:1044(M+H) + .

[0477] (Process 2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[(2-amino-2-methylpropyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound obtained in Step 1 above (16.3 mg: containing impurities) was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] Preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to obtain the title compound (6.4 mg). MS(ESI)m / z:816(M+H) + . 1 H-NMR(CD3OD)δ:8.28(1H,brs),8.01(1H,s),7.05(1H,s),6.27(1H,d,J=4.2Hz),6.15(1 H,d,J=7.9Hz),5.41-5.27(1H,m),5.13-5.08(1H,m),4.84(1H,d,J=3.6Hz),4.73(1H,t,J =4.5Hz),4.50-4.44(2H,m),4.36-4.31(2H,m),4.16-4.00(2H,m),3.49(2H,dd,J=6.3,3. 3Hz),3.31-3.25(2H,m),2.84-2.70(2H,m),2.03-1.91(2H,m),1.34(3H,s),1.30(3H,s). 31P-NMR(CD3OD)δ:57.9(s),54.5(s).

[0478] Example 11: Synthesis of CDN11 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(aminomethyl)-9H-purin-9-yl]-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0479] [ka]

[0480] [Synthetic scheme]

[0481] [ka]

[0482] (Process 1) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-cyanoadenosine As previously reported (J. Am. Chem. Soc. 1989, 111, 8502-8504), 4,4'-dimethoxytrityl chloride (642 mg) was added to a solution of 2-cyanoadenosine (440 mg) in pyridine (8.00 mL) and stirred at room temperature for 4 hours under a nitrogen atmosphere. Methanol (10 mL) was added to the reaction mixture to quench the reaction, followed by concentration under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol / 0.1% triethylamine] to obtain the title compound (528 mg). 1H-NMR(CDCl3)δ:8.21(1H,s),7.31-7.17(9H,m),6.79-6.70(4H,m),5.99(1H,d,J=5.4Hz),5.86(2H,brs),4.86(1H,q,J=4.6Hz),4.65(1H,t,J=3 .6Hz),4.48-4.45(1H,m),4.41(1H,q,J=3.0Hz),3.79(6H,s),3.46(1H,dd,J=10.9,3.6Hz),3.34(1H,dd,J=10.6,3.3Hz),2.93(1H,d,J=2.4Hz).

[0483] (Process 2) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine A solution of lithium aluminum hydride in tetrahydrofuran (approximately 2.5 M, 29.0 mL) was added to a solution of the compound obtained in Step 1 (14.3 g) in tetrahydrofuran (500 mL), and the mixture was stirred at 40°C for 2 hours under a nitrogen atmosphere. The reaction mixture was ice-cooled, saturated aqueous sodium bicarbonate (450 mL) was added, and the mixture was stirred for 10 minutes. 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (25.0 g) was then added, and the mixture was allowed to react at room temperature for 18 hours. A saturated aqueous Rochelle salt solution was added, and the mixture was stirred for 2.5 hours. The mixture was then extracted with a mixture of dichloromethane and methanol. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to give the title compound (10.8 g). 1H-NMR(CDCl3)δ:8.01(1H,s),7.26-7.15(9H,m),6.75-6.71(4H,m),6.37(1H,brs), 5.93(1H,d,J=6.0Hz),5.67(2H,brs),5.59(1H,brs),4.77-4.74(1H,m),4.46-4.37 (4H,m),4.21(2H,t,J=8.5Hz),3.76(3H,s),3.76(3H,s),3.42(1H,dd,J=10.6,3.3H z),3.25(1H,dd,J=10.6,3.3Hz),3.16(1H,brs),1.03(2H,t,J=8.5Hz),0.05(9H,s).

[0484] (Step 3) N-Benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine To a solution of the compound (10.8 g) obtained in Step 2 above in pyridine (70.0 mL) was added chlorotrimethylsilane (15.0 mL), and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. Benzoyl chloride (8.44 mL) was added to the reaction mixture, and the mixture was stirred for an additional 2 hours. The reaction mixture was cooled to 0°C, water (21.0 mL) was added, and the mixture was stirred for 10 minutes. 28% aqueous ammonia (31.4 mL) was added, and the mixture was stirred at the same temperature for an additional 20 minutes. The mixture was warmed to room temperature and stirred for an additional 3 hours, after which the reaction mixture was concentrated under reduced pressure. The residue was suspended in ethyl acetate, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol / 0.1% triethylamine] to give the title compound (9.47 g). MS(ESI)m / z:847(M+H) + . 1H-NMR(CDCl3)δ:9.54(1H,brs),8.17(2H,d,J=6.7Hz),7.91(1H,brs),7.66- 7.52(3H,m),7.35-7.10(9H,m),6.75(4H,d,J=8.5Hz),6.45(1H,brs),6.23( 1H,brs),6.03(1H,d,J=6.7Hz),4.70-4.65(2H,m),4.45-4.19(5H,m),3.73( 6H,s),3.38-3.32(2H,m),2.65(1H,brs),1.05(2H,t,J=8.8Hz),0.00(9H,s).

[0485] (Step 4) N-Benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine The compound obtained in Step 3 above (9.47 g) was used in the same manner as in Step 4 of Example 8 to give the title compound (3.13 g). MS(ESI)m / z:961(M+H) + . 1 H-NMR(CDCl3)δ:8.87(1H,brs),8.24(1H,brs),8.02(2H,d,J=7.3Hz),7.64-7.51(3H,m),7. 40-7.18(9H,m),6.81-6.77(4H,m),6.08(1H,d,J=4.8Hz),5.85(1H,brs),4.70-4.52(4H,m), 4.23-4.17(3H,m),3.77(6H,s),3.50(1H,dd,J=10.9,3.0Hz),3.29(1H,dd,J=10.9,4.2Hz),3 .21(1H,d,J=6.0Hz),1.06-1.02(2H,m),0.89(9H,s),0.09(3H,s),0.05(9H,s),0.01(3H,s).

[0486] (Step 5) N-Benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]adenosine To a solution of the compound obtained in Step 4 (1.49 g) in dichloromethane (15.5 mL), N,N-diisopropylethylamine (1.58 mL) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.04 mL) were added, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] and C18 silica gel column chromatography [acetonitrile: 100%] to obtain the title compound (1.39 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 6:4). 1 H-NMR(CDCl3)δ8.84(1H,s),8.33(0.6H,s),8.28(0.4H,s),8.02-7.99(2H,m),7.64-7.59(1H,m),7.55-7.51(2H,m),7.42-7.20(9H, m),6.82-6.79(4H,m),6.30(0.4H,d,J=4.2Hz),6.25(0.6H,d,J=4.2Hz),5.95-5.88(1H,m),4.89-4.77(1H,m),4.60-4.58(2H,m),4. 51-4.45(1H,m),4.25-4.18(3H,m),3.86-3.46(5H,m),3.78(6H,s),3.35-3.29(1H,m),2.53(1.2H,t,J=6.3Hz),2.38(0.8H,t,J=6.3 Hz),1.16-0.98(14H,m),0.85(3.6H,s),0.84(5.4H,s),0.10(1.8H,s),0.08(1.2H,s),0.05(9H,s),0.01(1.2H,s).-0.01(1.8H,s).

[0487] (Step 6) N,N-Diethylethanaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-benzamido-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]-9H-purin-9-yl}-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate The same reaction as in Step 7 of Example 1 was carried out on the following scale (starting material: 1.94 g). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 5 above (2.19 g), a reaction was carried out in the same manner as in Step 8 and Step 9 of Example 1 to give Diastereomer 1 (138 mg) and Diastereomer 2 (82.8 mg) of the title compound. Diastereomer 1 (less polar) 1H-NMR(CD3OD)δ:9.08(1H,s),8.11(2H,d,J=7.3Hz),7.98(1H,s),7.67(1H,t,J=7.6Hz),7.57(2H,t,J=7.9Hz),7.37(2H,d,J=7.9Hz),7.28-7. 22(4H,m),6.54(1H,d,J=8.5Hz),6.36(1H,d,J=1.8Hz),5.66-5.59(1H ,m),5.07-5.02(1H,m),4.85-4.83(1H,m),4.72(1H,d,J=3.6Hz),4.58- 4.08(12H,m),3.88-3.78(1H,m),3.49-3.38(1H,m),3.21(6H,q,J=7.3 Hz),3.05-3.00(2H,m),2.49-2.40(1H,m),2.34-2.26(1H,m),2.09-2.0 3(2H,m),1.31(9H,t,J=7.6Hz),1.21-1.09(2H,m),1.02(9H,s),0.91(9 H,s),0.30(3H,s),0.29(3H,s),0.26(3H,s),0.12(3H,s),0.05(9H,s). ジアステレオマー2 (high polarity) MS(ESI) m / z: 1392(M+H) + . 1 H-NMR(CD3OD)δ:8.91(1H,s),8.10-8.07(2H,m),7.94(1H,s),7.67-7.63(1H,m),7.5 9-7.54(2H,m),7.41-7.20(6H,m),6.54(1H,d,J=8.5Hz),6.22(1H,d,J=5.4Hz),5.36- 5.30(1H,m),3.20(6H,q,J=7.3Hz),3.04-3.00(2H,m),2.85-2.75(2H,m),2.23-2.13( 2H,m),1.30(9H,t,J=7.3Hz),1.03(9H,s),0.79(9H,s),0.05(9H,s).(Observe the possible records of なピークのみ)

[0488] (Project 7-1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (diastereomer 1) (42.3 mg) obtained in Step 6 above was reacted in the same manner as in Step 10 of Example 1 to give the title compound (25.9 mg). MS(ESI)m / z:1131(M+H) + . 1 H-NMR(CD3OD)δ:8.72(1H,s),8.00(1H,s),7.26(1H,s),6.35(1H,d,J=9.1Hz),6.26(1H,d,J=4.8Hz),5.42-5.36(1H ,m),5.20-5.15(1H,m),4.91-4.87(2H,m),4.80-4.78(1H,m),4.43(1H,t,J=11.2Hz),4.36-4.28(3H,m),4.20-4.15 (3H,m),4.09-3.99(2H,m),3.51(2H,d,J=6.7Hz),3.13(12H,q,J=7.3Hz),2.85(2H,brs),2.01-1.97(2H,m),1.25(1 8H,t,J=7.3Hz),1.07-1.00(2H,m),1.00(9H,s),0.82(9H,s),0.32(3H,s),0.28(3H,s),0.25(3H,s),0.07(12H,s).

[0489] (Step 7-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-amino-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (diastereomer 2) (82.8 mg) obtained in Step 6 above was reacted in the same manner as in Step 10 of Example 1 to give the title compound (45.2 mg). 1 H-NMR(CD3OD)δ:8.61(1H,s),8.01(1H,s),7.08(1H,s),6.34(2H,t,J=7.9Hz),5.49(1H,dd,J=10.6,4.5Hz),5.42(1H,t,J=5. 1Hz),5.24-5.17(1H,m),5.00-4.95(2H,m),4.69-4.57(2H,m),4.36(2H,t,J=17.8Hz),4.22-4.15(3H,m),4.05(1H,dd,J=12.4 ,5.1Hz),3.90-3.85(1H,m),3.51(2H,d,J=9.1Hz),3.17(12H,q,J=7.3Hz),2.92(2H,t,J=5.4Hz),2.04-1.99(2H,m),1.29(18H ,t,J=7.3Hz),1.07-0.98(2H,m),1.00(9H,s),0.74(9H,s),0.28(3H,s),0.28(3H,s),0.21(3H,s),0.07(9H,s),-0.06(3H,s).

[0490] (Step 8-1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) Triethylamine trihydrofluoride (700 μL) was added to the compound (25.9 mg) obtained in step 7-1 above, and the mixture was stirred for 2 hours at 55° C. An ice-cooled mixture of 1 M aqueous triethylammonium carbonate solution (3.5 mL) and triethylamine (1.10 mL) was added to the reaction mixture at room temperature, and the mixture was purified by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 10%-40% (0 min-30 min)] to obtain the title compound (19.1 mg). MS(ESI)m / z:903(M+H) + . 1 H-NMR(CD3OD)δ:8.71(1H,s),8.03(1H,s),7.10(1H,s),6.37(1H,d,J=7.9Hz),6.28(1H,d,J=4 .2Hz),5.38-5.33(1H,m),5.18-5.13(1H,m),4.84-4.80(2H,m),4.50-4.40(2H,m),4.35-4.40( 4H,m),4.21-4.16(2H,m),4.07-4.00(2H,m),3.51-3.49(2H,m),3.07(12H,q,J=7.3Hz),2.85( 2H,t,J=5.4Hz),2.02-1.97(2H,m),1.23(18H,t,J=7.3Hz),1.04(2H,t,J=8.2Hz),0.07(9H,s).

[0491] (Step 8-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-amino-2-[({[2-(trimethylsilyl)ethoxy]carbonyl}amino)methyl]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (45.2 mg) obtained in the above step 7-2 was reacted in the same manner as in the above step 8-1 to give the title compound (37.1 mg). MS(ESI)m / z:903(M+H) + . 1 H-NMR(CD3OD)δ:8.75(1H,s),8.02(1H,s),7.13(1H,s),6.36(1H,d,J=9.1Hz),6.33(1H,d,J=6 .7Hz),5.51-5.42(2H,m),4.81(1H,dd,J=6.7,4.8Hz),4.51-4.28(7H,m),4.18(2H,dt,J=8.3, 2.6Hz),4.02(1H,d,J=12.7Hz),3.92-3.87(1H,m),3.51-3.47(2H,m),3.13(12H,q,J=7.3Hz), 2.93-2.90(2H,m),2.04-1.98(2H,m),1.27(18H,t,J=7.3Hz),1.06-0.99(2H,m),0.06(9H,s).

[0492] (Step 9-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(aminomethyl)-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) To a solution of the compound (19.1 mg) obtained in the above step 8-1 in tetrahydrofuran (576 μL), a solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 288 μL) was added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. After that, purification was carried out under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] Preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 0%-30% (0 min-40 min)] and Sep-Pak® C18 [water / acetonitrile]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (8.4 mg). MS(ESI)m / z:759(M+H) + . 1 H-NMR(CD3OD)δ:8.48(1H,s),8.03(1H,s),7.04(1H,s),6.27(1H,d,J=3.6Hz),6.24(1H,d,J=8.5Hz),5.98-5.93(1H,m),5.04-4.99(1H, m),4.81-4.79(2H,m),4.45-4.39(2H,m),4.31-4.27(2H,m),4.12-3.99(4H,m),3.54-3.44(2H,m),2.88-2.85(2H,m),2.02-1.97(2H,m). 31 P-NMR(CD3OD) δ: 57.6, 55.5.

[0493] (Step 9-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(aminomethyl)-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (37.1 mg) obtained in the above step 8-2 was reacted in the same manner as in the above step 9-1 to give the title compound (12.8 mg). MS(ESI)m / z:759(M+H) + . 1 H-NMR(CD3OD)δ:8.61(1H,s),8.02(1H,s),7.13(1H,s),6.31(1H,d,J=6.0Hz) ,6.28(1H,d,J=8.5Hz),5.61-5.55(1H,m),5.38-5.35(1H,m),4.80(1H,t,J=5. 1Hz),4.54(1H,d,J=4.2Hz),4.48-4.28(4H,m),4.13(2H,s),4.08-4.04(1H,m ),3.94-3.90(1H,m),3.52-3.49(2H,m),2.90-2.88(2H,m),2.03-1.98(2H,m). 31 P-NMR(CD3OD)δ:62.2(s),60.0(s).

[0494] Example 12: Synthesis of CDN12 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0495] [ka]

[0496] [Synthetic scheme]

[0497] [ka]

[0498] (Process 1) 6-chloro-2-iodo-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine To a solution of commercially available 6-chloro-2-iodo-9-β-D-ribofuranosyl-9H-purine (9.65 g) in ethylene glycol dimethyl ether (120 mL) was added N,N-diisopropylethylamine (40.7 mL) and tert-butyldimethylsilyl trifluoromethanesulfonate (26.9 mL) at 0°C. The mixture was warmed to room temperature under a nitrogen atmosphere and stirred for 19 hours. The reaction mixture was cooled to 0°C, and saturated aqueous sodium bicarbonate was added to quench the reaction. The mixture was then extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (13.7 g). 1H-NMR(CDCl3)δ:8.48(1H,s),6.02(1H,d,J=4.2Hz),4.54(1H,t,J=4.5Hz),4.29(1H,t,J=4.5Hz),4.18-4.15(1H,m),4.04(1H,dd,J=11.5,4.2Hz),3 .80(1H,dd,J=11.5,2.4Hz),0.96(9H,s),0.93(9H,s),0.84(9H,s),0.17( 3H,s),0.16(3H,s),0.10(3H,s),0.09(3H,s),0.01(3H,s),-0.16(3H,s).

[0499] (Process 2) 2-[(Benzoyloxy)methyl]-6-chloro-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine To a solution of the compound obtained in Step 1 (13.7 g) in tetrahydrofuran (121 mL) was added tetrakis(triphenylphosphine)palladium(0) (2.10 g) and benzyloxymethylzinc iodide (approximately 0.9 M, 30.2 mL) prepared by the method described below under a nitrogen atmosphere, and the mixture was stirred at room temperature for 20 hours. The reaction was quenched by adding saturated aqueous ammonium chloride solution, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (7.29 g). [Preparation of benzyloxymethyl zinc iodide] Under a nitrogen atmosphere, a suspension of zinc powder (5.99 g) in tetrahydrofuran (17.1 mL) was ultrasonically treated, and then a solution of iodomethyl benzoate (12.0 g) in tetrahydrofuran (21.3 mL) was added at 10 to 15°C. The mixture was stirred at the same temperature for 1.5 hours to obtain a solution of benzyloxymethyl zinc iodide in tetrahydrofuran (approximately 0.9 M, 38.4 mL). MS(ESI)m / z:763(M+H) + . 1H-NMR(CDCl3)δ:8.58(1H,s),8.13(1H,dd,J=8.5,1.2Hz),7.60-7.56(1H,m),7.47-7.43(2H,m),6.09( 1H,d,J=4.8Hz),5.60(1H,d,J=13.9Hz,),5.56(1H,d,J=13.9Hz),4.48(1H,t,J=4.5Hz),4.27(1H,t,J= 4.2Hz),4.15-4.11(1H,m),4.03(1H,dd,J=11.5,3.0Hz),3.80(1H,dd,J=11.5,2.4Hz)0.96(9H,s),0.9 0(9H,s),0.76(9H,s),0.16(3H,s),0.15(3H,s),0.08(3H,s),0.06(3H,s),-0.07(3H,s),-0.27(3H,s).

[0500] (Step 3) 2-[(benzoyloxy)methyl]-6-chloro-9-β-D-ribofuranosyl-9H-purine To a solution of the compound (7.29 g) obtained in Step 2 above in tetrahydrofuran (47.7 mL), a solution of tetrabutylammonium fluoride in tetrahydrofuran (approximately 1 M, 38 mL) was added at 0°C under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 2.5 hours. A saturated aqueous solution of ammonium chloride was added to the reaction mixture to quench the reaction, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (3.69 g). 1H-NMR(CDCl3)δ:8.23(1H,s),8.15(2H,dd,J=8.5,1.2Hz),7.63-7.59(1H,m),7.48(2H ,t,J=7.9Hz),5.89(1H,d,J=6.0Hz),5.61(1H,d,J=13.9Hz),5.56(1H,d,J=14.5Hz),4 .90(1H,q,J=5.6Hz),4.46-4.43(1H,m),4.28(1H,q,J=2.2Hz),4.02(1H,dd,J=10.0,2 .7Hz),3.84-3.79(1H,m),3.71-3.65(1H,m),3.56-3.53(1H,m),2.70(1H,d,J=2.4Hz).

[0501] (Step 4) 2-[(Benzoyloxy)methyl]-9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-ribofuranosyl}-6-chloro-9H-purine To a solution of the compound (2.36 g) obtained in Step 3 above in pyridine (56 mL) was added 4,4'-dimethoxytrityl chloride (2.30 g) and the mixture was stirred at room temperature under a nitrogen atmosphere for 17 hours. Ethanol (20 mL) was added to the reaction mixture, and the mixture was stirred for an additional 10 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to obtain the title compound (1.41 g). MS(ESI)m / z:745(M+Na) + . 1 H-NMR(CDCl3)δ:8.32(1H,s),8.14-8.11(2H,m),7.64-7.59(1H,m),7.49-7.44( 2H,m),7.23-7.12(9H,m),6.72(4H,d,J=7.9Hz),5.94(1H,d,J=5.4Hz),5.64(1H, d,J=15.1Hz),5.59(1H,d,J=14.5Hz),4.83-4.77(2H,m),4.37-4.33(2H,m),3.77 (6H,s),3.35(1H,dd,J=10.6,3.3Hz),3.28(1H,dd,J=10.9,3.6Hz),2.64(1H,s).

[0502] (Step 5) 2-[(Benzoyloxy)methyl]-9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6-chloro-9H-purine To a solution of the compound (2.61 g) obtained in Step 4 above in ethylene glycol dimethyl ether (72.0 mL), N,N-diisopropylethylamine (1.89 mL) and tert-butyldimethylsilyl trifluoromethanesulfonate (1.24 mL) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. The reaction mixture was quenched by adding saturated aqueous sodium bicarbonate solution, followed by extraction with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / 0.1% triethylamine] to obtain the title compound (1.10 g). 1 H-NMR(CDCl3)δ:8.35(1H,s),8.12-8.10(2H,m),7.60-7.56(1H,m),7.46-7.42(2H,m),7.37-7.3 5(2H,m),7.28-7.20(7H,m),6.81-6.75(4H,m),6.00(1H,d,J=4.8Hz),5.50(2H,s),4.69(1H,q,J= 5.6Hz),4.39(1H,dd,J=5.1,3.9Hz),4.16(1H,q,J=3.8Hz),3.77(6H,s),3.45(1H,dd,J=10.6,3.3 Hz),3.31(1H,dd,J=10.9,4.2Hz),3.06(1H,d,J=6.7Hz),0.86(9H,s),0.04(3H,s),-0.02(3H,s).

[0503] (Step 6) 2-[(Benzoyloxy)methyl]-9-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-2-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6-chloro-9H-purine The compound obtained in Step 5 above (511 mg) was reacted in the same manner as in Step 6 of Example 1 to give the title compound (569 mg) as a diastereomeric mixture with respect to the phosphorus atom (diastereomeric ratio = 6:4). 1 H-NMR(CDCl3)δ:8.43(0.6H,s),8.39(0.4H,s),8.10(2H,d,J=7.9Hz),7.58(1H,t,J=7.6Hz),7.47-7.38(4H,m),7.31-7.18(7H,m),6.82-6. 78(4H,m),6.25(0.4H,d,J=4.8Hz),6.21(0.6H,d,J=4.8Hz),5.49(1H,d,J=13.9Hz),5.45(1H,d,13.9Hz),4.98-4.93(0.6H,m),4.83-4.78( 0.4H,m),4.45(0.6H,t,J=4.2Hz),4.36(0.4H,t,J=4.2Hz),4.21-4.17(1H,m),3.78(6H,s),3.76-3.32(6H,m),2.46(1.2H,t,J=6.3Hz),2.3 0(0.8H,t,J=6.3Hz),1.28-0.86(12H,m),0.825(5.4H,s),0.815(3.6 H,s),0.08(1.8H,s),0.04(1.2H,s),-0.01(1.8H,s),-0.02(1.2H,s).

[0504] (Step 7) N,N-Diethylethanaminium (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{2-[(benzoyloxy)methyl]-6-chloro-9H-purin-9-yl}-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate The same reaction as in Step 7 of Example 1 was carried out on the following scale (starting material: 2.72 g). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 6 above (3.03 g), a reaction was carried out in the same manner as in Step 8 and Step 9 of Example 1 to give Diastereomer 1 (351 mg) and Diastereomer 2 (351 mg) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1268(M+H) + . 1 H-NMR(CD3OD)δ:9.30(1H,s),8.14(2H,d,J=7.3Hz),7.99(1H,s),7.64(1H,t,J=7.3Hz),7.52(2H,t,J=7.9Hz),7.40-7.36(2H,m), 7.30-7.23(4H,m),6.43(1H,d,J=8.5Hz),6.37(1H,d,J=3.0Hz),5.62-5.56(1H,m),5.62(2H,s),5.06-5.01(1H,m),4.83(1H,dd,J= 4.5,2.7Hz),4.69(1H,d,J=4.2Hz),4.49-4.28(7H,m),4.08(1H,dd,J=12.1,4.8Hz),3.81-3.70(1H,m),3.46-3.38(1H,m),3.17-3. 10(8H,m),2.29-2.23(4H,m),1.28(9H,t,J=7.3Hz),0.91(9H,s),0.90(9H,s),0.29(3H,s),0.20(3H,s),0.16(3H,s),0.11(3H,s). Diastereomer 2 (high polarity) MS(ESI)m / z:1268(M+H) + . 1H-NMR(CD3OD)δ:8.97(1H,s),8.14(2H,d,J=8.5Hz),7.94(1H,s),7.68-7.63(1H,m),7.55-7.50(2H,m),7.39-7.36(2H,m),7.26-7 .21(4H,m),6.41(1H,d,J=7.9Hz),6.21(1H,d,J=5.4Hz),5.64(1H,d,J=15.1Hz),5.57(1H,d,J=15.1Hz),5.25-5.18(2H,m),5.13-5 .10(1H,m),5.04-5.01(1H,m),4.94-4.78(3H,m),4.51(1H,t,J=10.9Hz),4.33-4.06(6H,m),3.15(6H,q,J=7.3Hz),3.08-2.96(2H ,m),2.84-2.71(2H,m),2.25-2.19(2H,m),1.28(9H,t,J=7.3Hz),0.91(9H,s),0.79(9H,s),0.19(6H,s),0.14(3H,s)-0.07(3H,s).

[0505] (Step 8-1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (diastereomer 1) (37.3 mg) obtained in Step 7 above in methanol (0.500 mL), 28% aqueous ammonia (0.500 mL) was added, and the mixture was stirred in a sealed tube at 60° C. for 3 hours. The reaction solution was purified directly by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 35%-55% (0 min-30 min)] to obtain the title compound (22.0 mg: containing impurities). MS(ESI)m / z:988(M+H) + .

[0506] (Step 8-2) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound (diastereomer 2) (37.7 mg) obtained in Step 7 above in tetrahydrofuran (0.500 mL) was added 28% aqueous ammonia (0.500 mL), and the mixture was stirred at 60°C in a sealed tube for 3 hours. 28% aqueous ammonia (0.500 mL) was added, and the mixture was stirred for an additional 3 hours. 28% aqueous ammonia (0.500 mL) was added, and the mixture was stirred overnight. The reaction solution was purified directly by preparative HPLC [10 mM aqueous triethylammonium acetate / acetonitrile, acetonitrile: 20%-50% (0 min-30 min)] to obtain the title compound (19.3 mg). MS(ESI)m / z:988(M+H) + . 1H-NMR(CD3OD)δ:8.64(1H,s),8.02(1H,s),7.09(1H,s),6.35(2H,d,J=7.9Hz),5.48-5.41(2H,m), 5.26-5.19(1H,m),5.00-4.95(2H,m),4.70-4.53(4H,m),4.22(1H,s),4.06(1H,dd,J=12.1,4.8Hz ),3.91-3.86(1H,m),3.53-3.48(2H,m),3.18(12H,q,J=7.3Hz),2.92(2H,t,J=5.4Hz),2.04-1.99 (2H,m),1.29(18H,t,J=7.3Hz),1.00(9H,s),0.74(9H,s),0.28(6H,s),0.21(3H,s),-0.06(3H,s).

[0507] (Step 9-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound obtained in Step 8-1 above (22.0 mg: containing impurities) was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] Preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 2%-30% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (8.0 mg). MS(ESI)m / z:760(M+H) + . 1H-NMR(CD3OD)δ:8.70(1H,s),8.03(1H,s),7.14(1H,s),6.39(1H,d,J=8.5Hz),6 .29(1H,d,J=3.6Hz),5.40-5.35(1H,m),5.15(1H,dt,J=9.1,3.8Hz),4.84(1H,d, J=3.6Hz),4.80(1H,t,J=4.5Hz),4.57(2H,s),4.51-4.43(2H,m),4.38-4.31(2H ,m),4.09-4.00(2H,m),3.52-3.48(2H,m),2.89-2.76(2H,m),2.01-1.97(2H,m). 31 P-NMR(CD3OD)δ:58.1(s),54.4(s).

[0508] (Step 9-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-[6-amino-2-(hydroxymethyl)-9H-purin-9-yl]-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (19.3 mg) obtained in Step 8-2 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] Preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 2%-30% (0 min-30 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (6.5 mg). MS(ESI)m / z:760(M+H) + . 1H-NMR(CD3OD)δ:8.76(1H,s),8.05(1H,s),7.18(1H,s),6.38(1H,d,J=8.5Hz),6.33(1H,d,J=6.7Hz),5.49-5.43(2H,m),4.81(1H,dd,J=6.3,4 .5Hz),4.58(2H,s),4.51-4.29(5H,m),4.04(1H,d,J=12.1Hz),3.93-3. 88(1H,m),3.54-3.52(2H,m),2.92(2H,t,J=5.4Hz),2.05-2.00(2H,m). 31 P-NMR(CD3OD)δ:63.0(s),60.3(s).

[0509] Example 13: Synthesis of CDN13 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0510] [ka]

[0511] [Synthetic scheme]

[0512] [ka]

[0513] (Process 1) 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6-chloro-9H-purine The reaction was carried out in the same manner as in Step 3 of Example 5 using 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-β-D-ribofuranosyl}-6-chloro-9H-purine (15.3 g), which is known from the literature (J. Org. Chem. 2000, 65, 5104-5113), to give the title compound (8.44 g) and the positional isomer of the title compound, 9-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-6-chloro-9H-purine (5.77 g). MS(ESI)m / z:703(M+H) + . 1 H-NMR(CDCl3)δ:8.71(1H,s),8.37(1H,s),7.40-7.37(2H,m),7.31-7.19(7H,m),6.82 -6.78(4H,m),6.06(1H,d,J=4.9Hz),4.79-4.74(1H,m),4.59(1H,dd,J=4.9,3.9Hz),4. 20(1H,dd,J=3.9,1.9Hz),3.79(3H,s),3.78(3H,s),3.52(1H,dd,J=10.7,3.4Hz),3.29 (1H,dd,J=10.7,3.9Hz),3.08(1H,d,J=6.8Hz),0.90(9H,s),0.10(3H,s),0.03(3H,s). Positional isomer (2'-O-TBS form) MS(ESI)m / z:703(M+H) + . 1 H-NMR(CDCl3)δ:8.67(1H,s),8.36(1H,s),7.46-7.42(2H,m),7.36-7.20(7H,m),6 .84-6.80(4H,m),6.11(1H,d,J=5.4Hz),5.00-4.97(1H,m),4.40-4.35(1H,m),4.3 1-4.28(1H,m),3.79(3H,s),3.79(3H,s),3.52(1H,dd,J=10.7,2.9Hz),3.42(1H,d d,J=10.7,3.9Hz),2.68(1H,d,J=3.9Hz),0.84(9H,s),0.00(3H,s),-0.16(3H,s).

[0514] (Process 2) 9-(5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3-O-[tert-butyl(dimethyl)silyl]-2-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-β-D-ribofuranosyl)-6-chloro-9H-purine The compound (5.39 g) obtained in Step 1 above was used in a reaction similar to that in Step 4 of Example 5 to give the title compound (5.78 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 1:1). MS(ESI)m / z:903(M+H) + . 1 H-NMR(CDCl3)δ:8.69(0.5H,s),8.67(0.5H,s),8.43(0.5H,s),8.41(0.5H,s),7.43-7.37(2H,m),7.32-7.19(7H,m),6.83-6.78(4H,m) ),6.30(0.5H,d,J=4.4Hz),6.21(0.5H,d,J=4.9Hz),5.06-5.00(0.5H,m),4.86-4.80(0.5H,m),4.56-4.50(1H,m),4.27-4.20(1H,m),3 .79(6H,s),3.75-3.62(1H,m),3.57-3.46(4H,m),3.30(1H,dt,J=10.7,3.9Hz),2.50(1H,t,J=6.3Hz),2.37(1H,t,J=6.6Hz),1.13-1. 06(9H,m),0.90(1.5H,s),0.89(1.5H,s),0.86(4.5H,s),0.85(4.5H,s),0.12(1.5H,s),0.08(1.5H,s),0.02(1.5H,s),0.02(1.5H,s).

[0515] (Step 3) The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 1.84 g). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 2 above (1.62 g), a reaction was carried out in the same manner as in Step 8 of Example 1. The obtained crude product was used as is in the next reaction.

[0516] (Step 4) 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-(6-chloro-9H-purin-9-yl)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-10-yl]oxy}propanenitrile The crude product obtained in Step 3 above was used in a reaction in the same manner as in Step 9 of Example 1 to give the title compound (626 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1134(M+H) + .

[0517] (Step 5) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano- 2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) Ethylenediamine (0.352 mL) and triethylamine (0.735 mL) were added to an ethanol (10 mL) solution of the compound obtained in Step 4 above (299 mg: diastereomeric mixture), and the mixture was stirred for 15 hours at 60° C. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to obtain diastereomer 1 (122 mg: containing impurities) and diastereomer 2 (111 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1001(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:1001(M+H) + .

[0518] (Step 6-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) The compound (diastereomer 1) (122 mg: containing impurities) obtained in Step 5 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (29.6 mg). MS(ESI)m / z:773(M+H) + . 1 H-NMR(CD3OD)δ:8.77(1H,s),8.27(1H,s),8.03(1H,s),7.10(1H,s),6.35(1H,d,J=8. 5Hz),6.27(1H,d,J=4.8Hz),5.41(1H,ddd,J=7.9,4.2,2.1Hz),5.21-5.14(1H,m),4.8 4-4.77(2H,m),4.49-4.38(2H,m),4.35-4.26(2H,m),4.09-3.99(2H,m),3.92-3.80(2 H,m),3.51-3.45(2H,m),3.22(2H,t,J=6.0Hz),2.89-2.81(2H,m),2.02-1.94(2H,m). 31 P-NMR(CD3OD)δ:57.8(s),55.0(s).

[0519] (Step 6-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{6-[(2-aminoethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (diastereomer 2) (119 mg: containing impurities) obtained in Step 5 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (15.6 mg). MS(ESI)m / z:773(M+H) + . 1 H-NMR(CD3OD)δ:8.83(1H,s),8.27(1H,s),8.02(1H,s),7.10(1H,s),6.35(1H, d,J=7.9Hz),6.32(1H,d,J=6.7Hz),5.55-5.43(2H,m),4.81(1H,dd,J=7.0,4.5 Hz),4.52-4.29(5H,m),4.06-4.00(1H,m),3.93-3.80(3H,m),3.52-3.47(2H,m),3.21(2H,t,J=5.7Hz),2.94-2.88(2H,m),2.05-1.96(2H,m). 31 P-NMR(CD3OD)δ:63.1(s),60.1(s).

[0520] Example 14: Synthesis of CDN14 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0521] [ka]

[0522] [Synthetic scheme]

[0523] [ka]

[0524] (Process 1) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-7-{6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) To a solution of the compound obtained in Step 4 of Example 13 (313 mg) in ethanol (10 mL), 2-aminoethanol (0.330 mL) and triethylamine (0.769 mL) were added, and the mixture was stirred for 15 hours at 60° C. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] to give Diastereomer 1 (111 mg: containing impurities) and Diastereomer 2 (102 mg: containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1002(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:1002(M+H) + .

[0525] (Process 2-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) The compound (diastereomer 1) (111 mg: containing impurities) obtained in Step 1 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (47.1 mg). MS(ESI)m / z:774(M+H) + . 1 H-NMR(CD3OD)δ:8.70(1H,s),8.22(1H,s),8.03(1H,s),7.10(1H,s),6.34(1H,d,J= 8.5Hz),6.29(1H,d,J=4.8Hz),5.41-5.34(1H,m),5.19-5.13(1H,m),4.84(1H,d,J=4 .2Hz),4.79(1H,dd,J=4.8,2.4Hz),4.52-4.41(2H,m),4.39-4.31(2H,m),4.07-3.96 (2H,m),3.81-3.66(4H,m),3.52-3.47(2H,m),2.90-2.77(2H,m),2.03-1.95(2H,m). 31 P-NMR(CD3OD)δ:57.9(s),54.4(s).

[0526] (Step 2-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-{6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (diastereomer 2) (102 mg: containing impurities) obtained in Step 1 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile] and preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-25% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (27.1 mg). MS(ESI)m / z:774(M+H) + . 1H-NMR(CD3OD)δ:8.78(1H,s),8.22(1H,s),8.02(1H,s),7.12(1H,s),6.34(1H,d,J=1.8Hz),6.32(1H,s),5.52-5.42(2H,m),4.80(1H,dd,J=6.7 ,4.8Hz),4.50-4.28(5H,m),4.05-3.98(1H,m),3.93-3.86(1H,m),3.81 -3.68(4H,m),3.53-3.47(2H,m),2.95-2.88(2H,m),2.05-1.98(2H,m). 31 P-NMR(CD3OD)δ:63.0(s),60.2(s).

[0527] Example 15: Synthesis of CDN15 N-[2-({9-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-2,10-dioxo-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-9H-purin-6-yl}amino)ethyl]-2-hydroxyacetamide

[0528] [ka]

[0529] [Synthetic scheme]

[0530] [ka]

[0531] (Process 1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-(6-{[2-(2-hydroxyacetamido)ethyl]amino}-9H-purin-9-yl)-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (10.0 mg) obtained in Step 6-2 of Example 13 was reacted in the same manner as in Step 1-1 of Example 7, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. Purification conditions: C18 silica gel column chromatography [10 mM triethylammonium acetate aqueous solution / acetonitrile], preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile, acetonitrile: 5%-35% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (6.6 mg). MS(ESI)m / z:831(M+H) + . 1 H-NMR(CD3OD)δ:8.78(1H,s),8.24(1H,s),8.02(1H,s),7.12(1H,s),6.33(2H,d,J=6.7Hz),5.52-5.42(2H,m),4.80(1H,dd,J=6.7,4.2Hz),4.5 0-4.27(5H,m),4.04-3.98(1H,m),3.95(2H,s),3.93-3.86(1H,m),3.83 -3.73(2H,m),3.58-3.46(4H,m),2.95-2.88(2H,m),2.05-1.98(2H,m). 31 P-NMR(CD3OD)δ:63.1(s),60.4(s).

[0532] Example 16: Synthesis of CDN16 (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-amino-6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0533] [ka]

[0534] [Synthetic scheme]

[0535] [ka]

[0536] (Process 1) 2-Acetamido-2',3',5'-tri-O-acetyl-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)adenosine To a solution of commercially available 6-chloro-9-β-D-ribofuranosyl-9H-purin-2-amine (5.00 g) (Tokyo Chemical Industry) in ethanol (30 mL), 2-{[tert-butyl(dimethyl)silyl]oxy}ethan-1-amine (3.49 g) and N,N-diisopropylethylamine (4.33 mL) were added and stirred at 80°C for 65 hours. The reaction mixture was concentrated under reduced pressure, and pyridine (15 mL) and acetic anhydride (15 mL) were added to the residue, followed by stirring at 70°C for 4 hours. The reaction mixture was concentrated under reduced pressure, and saturated aqueous sodium bicarbonate was added, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate] to give the title compound (8.91 g). MS(ESI)m / z:609(M+H) + . 1 H-NMR(CDCl3)δ:7.84(1H,s),7.77(1H,s),6.14(1H,brs),6.03(1H,d,J=4.8Hz),5.92(1H,t, J=5.1Hz),5.72(1H,t,J=5.1Hz),4.50-4.40(2H,m),4.35(1H,dd,J=11.8,4.5Hz),3.83(2H,t, J=5.1Hz),3.76-3.63(2H,m),2.54(3H,s),2.14(3H,s),2.10(6H,s),0.91(9H,s),0.07(6H,s).

[0537] (Process 2) 2-Acetamido-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)adenosine A solution of the compound obtained in Step 1 (4.00 g) in dichloromethane (40 mL) was added with a methanol solution of sodium methoxide (1.0 M, 6.64 mL) and stirred at 0°C for 1 hour. Acetic acid (0.413 mL) and pyridine (0.5 mL) were added to the reaction mixture to quench the reaction, and the reaction mixture was then concentrated under reduced pressure. Pyridine was added to the residue, and the mixture was partially concentrated under reduced pressure to prepare a pyridine (approximately 20 mL) solution. 4,4'-Dimethoxytrityl chloride (4.68 g) was added to this solution at 0°C, and the mixture was stirred at the same temperature for 30 minutes and then stored at 4°C overnight. Methanol (2 mL) was added to the reaction mixture, and the mixture was stirred for 30 minutes and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography [hexane / ethyl acetate / methanol / 0.1% triethylamine] to obtain the title compound (4.41 g). MS(ESI)m / z:785(M+H) + . 1 H-NMR(CDCl3)δ:7.97(1H,s),7.90(1H,s),7.21-7.08(9H,m),6.73-6.67(4H,m),6.14(1H, s),5.88(1H,d,J=6.7Hz),4.91-4.85(1H,m),4.46(1H,t,J=3.0Hz),4.32(1H,d,J=5.4Hz), 3.86-3.80 (2H, m), 3.76 (3H, s), 3.76 (3H, s), 3.70-3.66 (1H, m), 3.42-3.34 (2H, m), 3.13 (1H, dd, J = 10.6, 2.7 Hz), 2.24 (3H, s), 0.90 (9H, s), 0.07 (3H, s), 0.07 (3H, s). (Only observable peaks are shown.)

[0538] (Step 3) 2-Acetamido-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)adenosine The compound (4.41 g) obtained in Step 2 above was used to carry out a reaction in the same manner as in Step 3 of Example 5, to give the title compound (1.75 g) and the positional isomer of the title compound, 2-acetamido-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-[tert-butyl(dimethyl)silyl]-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)adenosine (1.31 g). MS(ESI)m / z:899(M+H) + . 1 H-NMR(CDCl3)δ:7.89(1H,s),7.69(1H,brs),7.39-7.34(2H,m),7.29-7.18(7H,m),6.80-6.75(4H,m),6.11( 1H,brs),5.89(1H,d,J=5.4Hz),4.65(1H,dd,J=5.4,2.7Hz),4.43(1H,dd,J=5.1,3.3Hz),4.19-4.15(1H,m), 3.83 (2H, dd, J = 5.4, 2.7 Hz), 3.77 (6H, s), 3.74-3.63 (2H, m), 3.40 (1H, dd, J = 10.9, 3.6 Hz), 3.22 (1H, dd, J = 10.9, 3.9 Hz), 2.43 (3H, s), 0.90 (9H, s), 0.88 (9H, s), 0.10 (3H, s), 0.06 (6H, s), 0.03 (3H, s). (Only observable peaks are listed.) Positional isomer (2'-O-TBS form) MS(ESI)m / z:899(M+H) + . 1H-NMR(CDCl3)δ:7.84(1H,s),7.52(1H,brs),7.48-7.44(2H,m),7.37-7.32(4H,m),7.29-7.18(3H,m),6.83-6.7 8(4H,m),6.12(1H,brs),5.89(1H,d,J=6.0Hz),4.96-4.89(1H,m),4.32-4.27(1H,m),4.24(1H,dd,J=3.2,1.6Hz) ,3.84(2H,dd,J=5.4,2.7Hz),3.78(6H,s),3.75-3.65(2H,m),3.48(1H,dd,J=10.6,2.7Hz),3.35(1H,dd,J=10.6 ,3.6Hz),2.72(1H,d,J=3.0Hz),2.37(3H,s),0.91(9H,s),0.84(9H,s),0.07(6H,s),-0.01(3H,s),-0.17(3H,s).

[0539] (Step 4) 2-Acetamido-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-N-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-2'-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}adenosine The compound (1.75 g) obtained in Step 3 above was used in a reaction similar to that in Step 4 of Example 5 to give the title compound (2.08 g) as a diastereomeric mixture at the phosphorus atom (diastereomeric ratio = 6:4). MS(ESI)m / z:1099(M+H) + . 1H-NMR(CDCl3)δ:7.90(0.6H,s),7.88(0.4H,s),7.61(1H,d,J=7.3 Hz),7.45-7.18(9H,m),6.81(4H,m),6.10(0.6H,d,J=5.4Hz),6.09(1H,brs),6.06(0.4H,d,J=4.8Hz),4.95-4.85(0. 6H,m),4.76-4.69(0.4H,s),4.45-4.41(0.6H,m),4.40-4.36(0.4H,m),4.19-4.13(1H,m),3.86-3.80(2H,m),3.78(6 H,s),3.75-3.41(8H,m),3.32-3.22(1H,m),2.55-2.45(3H,m),2.36-2.30(1H,m),1.30-1.10(9H,m),0.92(1.2H,d,J =6.7Hz),0.90(9H,s),0.85(9H,s),0.76(1.8H,d,J=6.7Hz),0.10(1.8H,s),0.07(1.2H,s),0.06(6H,s),0.00(3H,s).

[0540] (Step 5) The same reaction as in Step 7 of Example 1 was carried out on the following scale (raw material: 981 mg). Using an acetonitrile solution of the obtained compound and the compound obtained in Step 4 above (1.05 g), a reaction was carried out in the same manner as in Step 8 of Example 1. The obtained crude product was used as is in the next reaction.

[0541] (Step 6) N-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-10-(2-cyanoethoxy)-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-[(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)amino]-9H-purin-2-yl}acetamide The crude product obtained in Step 5 above was used in a reaction in the same manner as in Step 9 of Example 1 to give the title compound (413 mg) as a mixture of diastereomers at the phosphorus atom. MS(ESI)m / z:1330(M+H) + .

[0542] (Step 7) Bis(N,N-diethylethanaminium) (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-{2-amino-6-[(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)amino]-9H-purin-9-yl}-15,16-bis{[tert-butyl(dimethyl)silyl]oxy}-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) The compound (413 mg) obtained in step 6 above was dissolved in methanol (5 mL) and 28% aqueous ammonia (5 mL) and stirred at room temperature for 63 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate / acetonitrile] to separate the less polar diastereomer 1 and the more polar diastereomer 2. Each diastereomer was dissolved again in methanol (5 mL) and 28% aqueous ammonia (5 mL) and stirred at 100°C for 2 days. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate / acetonitrile] to obtain diastereomer 1 (70.4 mg, containing impurities) and diastereomer 2 (65.1 mg, containing impurities) of the title compound. Diastereomer 1 (less polar) MS(ESI)m / z:1131(M+H) + . Diastereomer 2 (high polarity) MS(ESI)m / z:1131(M+H) + .

[0543] (Step 8-1) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-amino-6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 1) The compound (diastereomer 1) (70.4 mg: containing impurities) obtained in Step 7 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile] and preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (20.8 mg). MS(ESI)m / z:789(M+H) + . 1H-NMR(CD3OD)δ: 8.24(1H,s),8.02(1H,s),7.09(1H,s),6.29(1H,d,J=4.2Hz),6.12(1H,d,J=8.5H z),5.46-5.39(1H,m),5.22-5.15(1H,m),4.84(1H,d,J=3.6Hz),4.80(1H,t,J=4.5 Hz),4.50-4.32(3H,m),4.32-4.28(1H,m),4.12-3.98(2H,m),3.74(2H,t,J=5.4Hz ),3.68-3.60(2H,m),3.48(2H,t,J=5.4Hz),2.87-2.70(2H,m),2.01-1.93(2H,m). 31 P-NMR(CD3OD)δ:57.8(s),54.1(s).

[0544] (Step 8-2) Disodium (5R,7R,8R,12aR,14R,15R,15aS,16R)-7-{2-amino-6-[(2-hydroxyethyl)amino]-9H-purin-9-yl}-15,16-dihydroxy-2,10-dioxo-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (Diastereomer 2) The compound (diastereomer 2) (65.1 mg: containing impurities) obtained in Step 7 above was reacted in the same manner as in Step 11 of Example 1, and then purified under the following [Purification Conditions] to obtain the title compound as a triethylamine salt. [Purification conditions] C18 silica gel column chromatography [10 mM aqueous triethylammonium acetate solution / acetonitrile], preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile, acetonitrile: 5%-30% (0 min-40 min)], and preparative HPLC [10 mM aqueous triethylammonium acetate solution / methanol, methanol: 0%-60% (0 min-40 min)]. The obtained triethylamine salt was subjected to salt exchange in the same manner as in [Conversion to sodium salt] described in Step 11 of Example 1 to give the title compound (7.9 mg). MS(ESI)m / z:789(M+H) + . 1 H-NMR(CD3OD)δ:8.39(1H,s),8.02(1H,s),7.13(1H,s),6.33(1H,d,J=6.7Hz), 6.13(1H,d,J=8.5Hz),5.51-5.40(2H,m),4.83-4.78(1H,m),4.51-4.29(4H,m), 4.28-4.23(1H,m),4.07-4.00(1H,m),3.94-3.87(1H,m),3.75(2H,t,J=5.7Hz), 3.69-3.62(2H,m),3.53-3.47(2H,m),2.91(2H,t,J=5.7Hz),2.05-1.97(2H,m). 31 P-NMR(CD3OD)δ:62.9(s),60.3(s).

[0545] Example 17: Synthesis of CDN17 (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[6-(hydroxymethyl)-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione

[0546] [ka]

[0547] [Synthetic scheme]

[0548] [ka]

[0549] (Process 1) 6-[(benzoyloxy)methyl]-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine The title compound (10.4 g) was obtained by the same procedure as in Step 2 of Example 12 using 6-chloro-9-{2,3,5-tris-O-[tert-butyl(dimethyl)silyl]-β-D-ribofuranosyl}-9H-purine (10.7 g), which is known in the literature (J. Org. Chem. 1997, 62, 6833-6841). MS(ESI)m / z:729(M+H) + . 1 H-NMR(CDCl3)δ:8.94(1H,s)...

Claims

1. The following formula (Ia): 【Chemistry 1】 (where, L １ is a hydroxy group, -NH ２ , a 2-hydroxyacetylaminomethyl group, and a 2-[(2-hydroxyacetyl)amino]ethyl group, each of which may be substituted at any position by 1 to 3 groups selected from the group consisting of: 【Chemistry 2】 (where, R ６ and R ６’ are each independently a hydrogen atom, a halogen atom, a hydroxy group, or —NH ２ , a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group; R ７ and R ７’ each independently represents a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is optionally substituted by one or two substituents selected from the group consisting of a halogen atom and an oxo group; R ８ and R ８’ each independently represents a hydrogen atom or a halogen atom, Z ４ is -CH ２ represents -, -NH- or an oxygen atom, Z ５ represents a nitrogen atom or -CH=) represents a group selected from the group consisting of L ３ represents a hydrogen atom, a halogen atom, -NH ２ , a hydroxy C1-C3 alkyl group or an amino C1-C3 alkyl group; Q １ and Q １’ are each independently a hydroxy group, a thiol group, or a borano group (BH ３ － ) and Q ２ and Q ２’ each independently represents an oxygen atom or a sulfur atom, X １ and X ２ indicates an oxygen atom, Y １ and Y ２ indicates an oxygen atom, X ３ and X ４ is -CH ２ represents —O—, X ５ and X ６ is -CH ２ represents —O—, R １ , R ２ and R ３ are each independently a hydrogen atom, a halogen atom, —OR′, —OC(═O)R′, —N ３ , -NHR', -NR'R'', or -NHC(=O)R' (wherein R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, the C1-6 alkyl group, the C2-C6 alkenyl group, or the C2-C6 alkynyl group being optionally substituted with 1 to 6 halogen atoms; and R'' represents a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, or a C3-C6 cycloalkyl group), W １ represents a nitrogen atom, an oxygen atom, a sulfur atom, or —CH—, W ２ represents a nitrogen atom or -CH=, R ４ is a hydrogen atom, a halogen atom, or —NH ２ indicates, R ５ is the following (vii) to (x): (vii) W １ is a nitrogen atom, R ５ represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group; (viii) W １ is an oxygen atom, R ５ does not exist; (ix) W １ is a sulfur atom, R ５ is not present; or (x)W １ When is -CH-, R ５ is a hydrogen atom, a halogen atom, a hydroxyl group, -NH ２ or a C1-C6 alkyl group; represents a group selected from Z １ -Z ２ -Z ３ together, -CH ２ -CH ２ -CH ２ -, -CH ２ -CH ２ -R'''-, -CH=CH-CH ２ -, -CH=CX-CH ２ -, -CX=CH-CH ２ -, -CX=CX-CH ２ -, -C(=O)-CH ２ -CH ２ -, -CH ２ -CH ２ -C(=O)-, -CH ２ -CH(CH ３ )-CH ２ - or -CH ２ -CH ２ -CH(CH ３ )-(wherein R''' is -O- or -CH ２ -CH ２ -, and X represents a halogen atom) or a group represented by any of the following formulae: 【Transformation 3】 (where the asterisk stands for W １ and the wavy line indicates that the group is bonded to the carbon atom of =C-. or a pharmacologically acceptable salt thereof.

2. W １ The compound according to claim 1 or a pharmacologically acceptable salt thereof, wherein is a nitrogen atom.

3. W １ is a nitrogen atom, and R ５ The compound according to claim 2 or a pharmacologically acceptable salt thereof, wherein is a hydrogen atom.

4. W １ The compound according to claim 1 or a pharmacologically acceptable salt thereof, wherein is an oxygen atom.

5. W １ The compound according to claim 1 or a pharmacologically acceptable salt thereof, wherein is a sulfur atom.

6. W １ The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein is -CH-.

7. W １ is —CH—, and R ５ The compound according to claim 6 or a pharmacologically acceptable salt thereof, wherein is a hydrogen atom.

8. Z １ , Z ２ and Z ３ But together, -CH ２ -CH ２ -CH ２ - or -CH=CH-CH ２ The compound according to any one of claims 1 to 7, or a pharmacologically acceptable salt thereof, wherein

9. Z １ , Z ２ and Z ３ But together, -CH ２ -CH(CH ３ )-CH ２ - or -CH ２ -CH ２ -CH(CH ３ 8. The compound according to claim 1, wherein R is 1 or 2; or a pharmacologically acceptable salt thereof.

10. Z １ , Z ２ and Z ３ But together, -CH ２ -CH ２ -R'''- (wherein R''' is -O- or -CH ２ -CH ２ 8. The compound according to claim 1, wherein R is 0 or 1; or a pharmacologically acceptable salt thereof.

11. W ２ The compound according to any one of claims 1 to 10, or a pharmacologically acceptable salt thereof, wherein is -CH=.

12. W ２ The compound according to any one of claims 1 to 10, or a pharmacologically acceptable salt thereof, wherein is a nitrogen atom.

13. R ４ The compound according to any one of claims 1 to 12, or a pharmacologically acceptable salt thereof, wherein represents a hydrogen atom.

14. R ４ The compound according to any one of claims 1 to 12, or a pharmacologically acceptable salt thereof, wherein represents a fluorine atom.

15. L １ R in ８ and R ８’ The compound according to any one of claims 1 to 14, or a pharmacologically acceptable salt thereof, wherein each of

16. L １ is a group selected from the group consisting of the following formulas: 【Chemistry 4】 (where R ９ and R ９’ is a hydrogen atom, a halogen atom, a hydroxy group, or —NH ２ indicates, R １０ is a hydroxy group, -NH ２ , -NHC(=O)CH ２ OH, -CH ２ NHC(=O)CH ２ OH, -CH ２ CH ２ NHC(=O)CH ２ OH, a hydroxy C1-C3 alkyl group, or an amino C1-C3 alkyl group; R １１ and R １１’ each independently represents a hydrogen atom, a fluorine atom, or a methyl group, or R １１ and R １１’ is bonded to form a cyclopropane, Z ４ is -CH ２ represents -, -NH- or an oxygen atom) The compound according to any one of claims 1 to 15, or a pharmacologically acceptable salt thereof,

17. L １ is a group selected from the group consisting of the following formulas: 【Transformation 5】 (where R １３ and R １３’ are each independently a hydrogen atom, a hydroxy group, or —NH ２ indicates, R １２ is a hydroxy group, -NH ２ , -CH ２ OH, -NHC(=O)CH ２ OH, -CH ２ NHC(=O)CH ２ OH or -CH ２ CH ２ NHC(=O)CH ２ represents OH, Z ４ is as previously defined) The compound according to any one of claims 1 to 15, or a pharmacologically acceptable salt thereof,

18. L １ is a group selected from the group consisting of the following formulas: 【Transformation 6】 (where R １４ is a hydrogen atom or -NH ２ indicates, R １５ is a hydrogen atom or -C(=O)CH ２ represents OH, R １６ is a hydroxy group, -NH ２ , -CH ２ OH, -CH ２ CH ２ OH, -CH ２ NH ２ or -CH ２ CH ２ NH ２ (Indicates The compound according to any one of claims 1 to 15, or a pharmacologically acceptable salt thereof,

19. L ３ is a hydrogen atom, a fluorine atom, -NH ２ , -CH ２ OH or -CH ２ NH ２ The compound according to any one of claims 1 to 18, or a pharmacologically acceptable salt thereof, which represents:

20. Q １ and Q １’ The compound according to any one of claims 1 to 19, or a pharmacologically acceptable salt thereof, wherein each independently represents a hydroxy group or a thiol group.

21. R １ , R ２ and R ３ The compound according to any one of claims 1 to 20, or a pharmacologically acceptable salt thereof, wherein each of the groups independently represents a hydrogen atom, a hydroxy group, or a fluorine atom.

22. The following two formulas: 【Transformation 7】 (Here, L １ , Q １ , Q １’ , Q ２ and Q ２’ is as previously defined, R １７ , R １７’ , R １８ and R １８’ are each independently a hydrogen atom, a halogen atom, a hydroxy group, or —NH ２ indicates, W ３ represents -NH-, an oxygen atom, a sulfur atom, or -CH ２ - indicates W ４ represents -CH= or a nitrogen atom) The compound according to any one of claims 1 to 21, or a pharmacologically acceptable salt thereof, wherein

23. The following two formulas: 【Transformation 8】 (Here, L １ , Q １ , Q １’ , Q ２ , Q ２’ , R １７ , R １７’ , R １８ and R １８’ is as previously defined) 23. The compound according to claim 22, wherein:

24. The following eight formulas: 【Chemistry 9】 (Here, L １ , Q １ , Q １’ , Q ２ and Q ２’ is as previously defined, R １９ , R １９’ , R ２０ and R ２０’ each independently represents a hydrogen atom or a fluorine atom) 24. The compound according to claim 22 or 23, wherein:

25. The following four formulas: 【Chemistry 10】 (Here, L １ is as previously defined) The compound according to any one of claims 22 to 24, or a pharmacologically acceptable salt thereof, wherein

26. The following four formulas: 【Chemistry 11】 (Here, L １ is as previously defined) The compound according to any one of claims 22 to 25, or a pharmacologically acceptable salt thereof, wherein

27. The following four formulas: 【Chemistry 12】 (Here, L １ is as previously defined) The compound according to any one of claims 22 to 25, or a pharmacologically acceptable salt thereof, wherein

28. The following formula: 【Chemistry 13】 (Here, L １ is as previously defined, Q ３ and Q ３‘ each independently represents a hydroxy group or a thiol group, R ２１ and R ２２ each independently represents a hydroxy group or a fluorine atom, W ５ represents —NH— or a sulfur atom) The compound according to any one of claims 1 to 21, wherein the compound is represented by the formula: or a pharmacologically acceptable salt thereof.

29. The following two formulas: 【Chemistry 14】 (Here, L １ , Q ３ and Q ３‘ , W ５ is as previously defined) 29. The compound according to claim 28, wherein:

30. L １ but the following: 【Chemistry 15】 The compound according to any one of claims 1 to 29, or a pharmacologically acceptable salt thereof, wherein

31. L １ However, the following four equations: 【Chemistry 16】 The compound according to any one of claims 1 to 29, or a pharmacologically acceptable salt thereof, wherein

32. D is a compound of the following four formulas: 【Chemistry 17】 (Here, Q ３ , Q ３‘ and W ５ is as previously defined) 32. The compound according to claim 28, 29 or 31, wherein:

33. D is a compound of the following four formulas: [Chemistry 18] 33. The compound according to any one of claims 28, 29, 31 and 32, wherein:

34. D is a compound of the following three formulas: 【Chemistry 19】 33. The compound according to any one of claims 28, 29, 31 and 32, wherein:

35. A STING agonist comprising any one selected from the group consisting of the compounds according to claims 1 to 34 or pharmacologically acceptable salts thereof.

36. A pharmaceutical composition comprising any one selected from the group consisting of the compounds according to claims 1 to 34 or pharmacologically acceptable salts thereof.

37. An antitumor agent comprising any one selected from the group consisting of the compound according to claims 1 to 34 or a pharmacologically acceptable salt thereof.

38. The antitumor agent of claim 37, wherein the tumor is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

39. The following formula (x): 【Chemistry 20】 (where, L １ is a hydroxy group, -NH ２ , a 2-hydroxyacetylaminomethyl group, and a 2-[(2-hydroxyacetyl)amino]ethyl group, each of which may be substituted at any position by 1 to 3 groups selected from the group consisting of: 【Chemistry 21】 (where, R ６ and R ６’ are each independently a hydrogen atom, a halogen atom, a hydroxy group, or —NH ２ , a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group; R ７ and R ７’ each independently represents a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is optionally substituted by one or two substituents selected from the group consisting of a halogen atom and an oxo group; R ８ and R ８’ each independently represents a hydrogen atom or a halogen atom, Z ４ is -CH ２ represents -, -NH- or an oxygen atom, Z ５ represents a nitrogen atom or -CH=) represents a group selected from the group consisting of L ２ represents a hydrogen atom or a halogen atom, R １ , R ２ and R ３ are each independently a hydrogen atom, a halogen atom, —OR′, —OC(═O)R′, —N ３ , —NHR′, —NR′R″, or —NHC(═O)R′ (wherein R′ represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, the C1-C6 alkyl group, the C2-C6 alkenyl group, or the C2-C6 alkynyl group being optionally substituted with 1 to 6 halogen atoms, and R″ represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group), W １ represents a nitrogen atom, an oxygen atom, a sulfur atom, or —CH—, W ２ represents a nitrogen atom or -CH=, R ４ is a hydrogen atom, a halogen atom, or —NH ２ indicates, R ５ is the following (vii) to (x): (vii) W １ is a nitrogen atom, R ５ represents a hydrogen atom, a C1-C6 alkyl group, a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group; (viii) W １ is an oxygen atom, R ５ does not exist; (ix) W １ is a sulfur atom, R ５ is not present; or (x)W １ When is -CH-, R ５ is a hydrogen atom, a halogen atom, a hydroxyl group, -NH ２ or a C1-C6 alkyl group; represents a group selected from Z １ -Z ２ -Z ３ together, -CH ２ -CH ２ -CH ２ -, -CH ２ -CH ２ -R'''-, -CH=CH-CH ２ -, -CH=CX-CH ２ -, -CX=CH-CH ２ -, -CX=CX-CH ２ -, -C(=O)-CH ２ -CH ２ -, -CH ２ -CH ２ -C(=O)-, -CH ２ -CH(CH ３ )-CH ２ - or -CH ２ -CH ２ -CH(CH ３ )-(wherein R''' is -O- or -CH ２ -CH ２ -, and X represents a halogen atom) or a group represented by any of the following formulae: 【Chemistry 22】 (where the asterisk stands for W １ and the wavy line indicates that the group is bonded to the carbon atom of =C-, R ｅ and R ｇ represents the side chain of a naturally occurring α-amino acid, Q ａ’ and Q ｂ’ are each independently a negatively charged oxygen atom (O － ) or a sulfur atom (S － ), and n represents an integer of 1 to 3) or a pharmaceutically acceptable salt thereof.

40. R １ , R ２ and R ３ 40. The compound of claim 39, or a pharmaceutically acceptable salt thereof, wherein each is independently a hydrogen atom, a hydroxy group, or a fluorine atom.

41. R ４ The compound according to any one of claims 39 to 40, or a pharmaceutically acceptable salt thereof, wherein represents a hydrogen atom.

42. W １ is a nitrogen atom, and R ５ The compound according to any one of claims 39 to 41, or a pharmaceutically acceptable salt thereof, wherein is a hydrogen atom.

43. L １ is expressed by the following formula: 【Chemistry 23】 (where, R ６ indicates a hydrogen atom, R ７ represents a C2 alkyl group, and R ８ indicates a hydrogen atom) The compound according to any one of claims 39 to 42, or a pharmaceutically acceptable salt thereof, represented by:

44. L ２ The compound according to any one of claims 39 to 43, or a pharmaceutically acceptable salt thereof, wherein represents a hydrogen atom or a fluorine atom.

45. W ２ or a pharmaceutically acceptable salt thereof.

46. Z １ , Z ２ and Z ３ But together, -CH ２ -CH ２ -CH ２ - or -CH=CH-CH ２ 46. ​​The compound of any one of claims 39 to 45, or a pharmaceutically acceptable salt thereof, wherein:

47. The following formula: 【Chemistry 24】 【change】 or a pharmaceutically acceptable salt thereof.

48. The following formula (II): 【Chemistry 25】 (In the formula, m １ ranges from 3 to 5, Ab refers to an antibody or a functional fragment of the antibody, and the sugar chain of the antibody has been remodeled; L represents a linker connecting Ab and D; The linker L is represented by -Lb-La-Lp-Lc-*, The asterisk indicates binding to drug D. Lp is -GGFG- or -GGPI-; La is -C(=O)-CH ２ CH ２ represents —C(═O)—, Lb is represented by the following formula: 【Chemistry 26】 In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the remodeled sugar chain of Ab, and Lc is —NH—CH ２ - indicates Ab is linked to L via the sugar chain (N297-(Fuc)SG) linked to Asn297 of the antibody, D is of the following formula: 【Chemistry 27】 where the asterisk in the formula of D shown above indicates the bond to L. A method for producing an antibody-drug conjugate represented by the formula: a step of using a hydrolase to remove heterogeneous sugar chains attached to an antibody or a functional fragment of said antibody, leaving only terminal GlcNAc or (Fucα1,6)GlcNAc, to prepare a homogeneous protein moiety (hereinafter referred to as "acceptor") to which GlcNAc or (Fucα1,6)GlcNAc has been attached; A step of synthesizing a uniform glycoprotein having an arbitrary glycan structure (hereinafter referred to as a "glycan remodeling antibody") by preparing an arbitrary glycan (hereinafter referred to as a "donor") and linking this acceptor and donor using a glycosyltransferase; A glycochain remodeling antibody and a compound of the following formula (X): 【Chemistry 28】 or a pharmaceutically acceptable salt thereof (hereinafter referred to as "conjugation precursor") to obtain an antibody-drug conjugate. A method comprising:

49. 1. A method for producing an antibody drug conjugate, comprising: a step of using a hydrolase to remove heterogeneous sugar chains attached to an antibody or a functional fragment of said antibody, leaving only terminal GlcNAc or (Fucα1,6)GlcNAc, to prepare a homogeneous protein moiety (hereinafter referred to as "acceptor") to which GlcNAc or (Fucα1,6)GlcNAc has been attached; a step of synthesizing a uniform glycoprotein having a desired glycan structure (hereinafter referred to as a "glycan remodeling antibody") by preparing an arbitrary glycan (hereinafter referred to as a "donor") and linking this acceptor and donor using a glycosyltransferase; A step of reacting a sugar chain remodeling antibody with the compound according to any one of claims 39 to 47 or a pharmaceutically acceptable salt thereof (hereinafter referred to as a "conjugation precursor") to obtain an antibody-drug conjugate. A method comprising:

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