Novel method for producing antibody-immunostimulator conjugate

Abstract

The purpose of the present invention is to provide: a novel stereoselective method for producing a cyclic dinucleotide derivative that can be used for an antibody-immunostimulator conjugate; and a production intermediate thereof. Another purpose is to provide a method for producing a cyclic dinucleotide-linker and antibody-immunostimulator conjugate, in which said production method is used. Provided is a method for producing a cyclic dinucleotide derivative, the method including a step for carrying out stereoselective condensation of a compound (I) with a compound (IV) by using an optically active phosphitylation agent (Rc-II) or (Sc-II).

Description

[Technical Field]

[0001] The present invention relates to a novel stereoselective method for producing cyclic dinucleotide derivatives that can be used for antibody-immunostimulant conjugates, and to production intermediates thereof. The present invention also relates to novel methods for producing cyclic dinucleotide-linkers and antibody-immunostimulant conjugates, including the production method. Furthermore, the present invention relates to novel methods for producing raw material compounds used in the production of cyclic dinucleotide derivatives, and to production intermediates thereof. [Background technology]

[0002] Cyclic dinucleotides (CDNs) activate STING (Stimulator of Interferon Genes) (Non-Patent Document 1). Administration of CDNs to tumor-bearing mice through this activation enhanced the STING-mediated antitumor immune response, significantly inhibited tumor growth, and improved mouse survival (Non-Patent Document 2). Recent research has led to the development of synthetic small molecule compounds that possess STING agonist activity without being degraded by nucleases in vivo (e.g., Patent Documents 1 to 5). Antibody-drug conjugates (ADCs), in which CDNs exhibiting such activity are linked to antibodies via a linker, have also been studied, and have demonstrated antitumor effects in tumors expressing antigens (Patent Documents 6 and 7).

[0003] Previously studied CDNs contain two sulfur-modified pentavalent phosphate bonds called phosphorothioate bonds, which link two types of nucleotides to form a ring. When asymmetric nucleotides are cyclized, an asymmetric center is generated at the phosphorus atom of each phosphorothioate moiety. Previously reported CDN synthesis methods involve synthesis without controlling the asymmetry at the phosphorus, and the resulting four diastereomers are purified to obtain CDNs with the desired absolute configuration (e.g., Non-Patent Document 3, Patent Documents 1-7). These synthesis methods result in low yields due to the production of four diastereomers. Furthermore, because the diastereomers have similar physical properties, their separation and purification requires strict preparative purification by HPLC at low loads. Therefore, synthesizing large quantities of the desired product using these synthesis methods poses a significant burden. A method for stereoselectively synthesizing CDNs using a compound combining a pentavalent phosphorus and a chiral auxiliary has been reported, but the yield upon cyclization remains low (Non-Patent Document 4). Furthermore, there have been several research examples of stereoselective phosphorothioate synthesis methods related to stereocontrolled oligonucleotides that combine trivalent phosphorus with an asymmetric auxiliary group (e.g., Non-Patent Documents 5 to 9, Patent Documents 8 to 9). However, there have been no examples of dinucleotide bond formation using compounds applicable to cyclic dinucleotide synthesis, and there have also been no examples of actual stereoselective synthesis of cyclic dinucleotides. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2014 / 189805 [Patent Document 2] International Publication No. 2014 / 189806 [Patent Document 3] International Publication No. 2016 / 145102 [Patent Document 4] International Publication No. 2017 / 093933 [Patent Document 5] International Publication No. 2018 / 060323 [Patent Document 6] International Publication No. 2020 / 050406 [Patent Document 7] International Publication No. 2021 / 177438 [Patent Document 8] International Publication No. 2005 / 092909 [Patent Document 9] International Publication No. 2016 / 012305 [Non-patent literature]

[0005] [Non-Patent Document 1] Mol.Cell,2013,51,226-235 [Non-patent document 2] Sci.Rep.2016,6,19049 [Non-patent document 3] J.Med.Chem.2016.59.10253-10267 [Non-patent document 4] Science 2018.361.1234-1238 [Non-patent document 5] Tetrahedron Lett.1998.39.2491-2494 [Non-patent document 6] Bioorg.Med.Chem.Lett.1998.8.2539-2544 [Non-Patent Document 7] J.Am.Chem.Soc.2002.124.4962-4963 [Non-patent document 8] J.Am.Chem.Soc.2003.125.8307-8317 [Non-Patent Document 9] J.Am.Chem.Soc.2008.130.16031-16037 Summary of the Invention [Problem to be solved by the invention]

[0006] In one aspect, the present invention provides a novel method for producing cyclic dinucleotide derivatives that are useful as intermediates for antibody-drug conjugates, particularly antibody-immunoactivator conjugates that have STING agonist activity and activate immune cells.

[0007] The stereoselective synthesis of cyclic dinucleotides requires a method for coupling nucleotides with phosphorous acid, a strong acidic functional group, and nucleotides with optically active amidite moieties that are highly unstable in acidic conditions, with practical yields and high stereoselectivity. However, conventional production methods are stereononselective, resulting in low yields of the target product and, as a result, difficulty in purification, resulting in extremely low overall yields.

[0008] Therefore, one object of the present invention is to provide a novel, industrially superior method for producing CDNs that uses stereoselective synthesis to increase the yield of the target product, thereby reducing the purification load and improving the overall yield, and also to provide a novel method for producing CDN-linkers using said method.

[0009] Another object of the present invention is to provide a novel production method for preparing raw material compounds used in the synthesis of cyclic dinucleotides, which requires fewer steps and has an improved yield. [Means for solving the problem]

[0010] In order to solve the above problems, the present inventors have conducted extensive research and discovered a method for stereoselectively synthesizing cyclic dinucleotide derivatives using an optically active phosphitylating agent, thereby reducing the burden of diastereomer purification and completing a novel production method with improved overall yield. Furthermore, the present inventors have also developed a method for constructing a CDN-linker using the CDN obtained by this production method, and a method for producing an antibody-immunostimulatory compound using the CDN-linker. transformation Furthermore, the present inventors have discovered a new production method for preparing raw material compounds used in the production of cyclic dinucleotides, which reduces the number of steps and improves the yield compared to conventional production methods, thereby completing the present invention.

[0011] That is, the present invention relates to the following: [1] Formula (Rp-VII): [ka] [In the formula, A1 is [ka] and Q is a thiol group or a hydroxy group; PG1 is a protecting group for the hydroxy group, and PG3 is a protecting group for the amino group. A method for producing a compound represented by the formula: (Step a1) Formula (I): [ka] [In the formula, PG1 and PG3 are as defined above, and PG2 is a protecting group for the hydroxy group. with an optically active phosphitylating agent (Rc-II) selected from the group consisting of the following formulae (Rc-II-1) and (Rc-II-2): [ka] [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. to form a compound of formula (Rc-III): [ka] [In the formula, PG1, PG2 and PG3 are as defined above, and B1 is [ka] where R1 and R2 are as defined above. obtaining a compound represented by the formula: (Step a2) The obtained compound of formula (Rc-III) is reacted with a compound of formula (IV): [ka] [In the formula, A2 is [ka] and PG4 is a protecting group for the amino group. or a salt thereof in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and further reaction with a thiolating agent, followed by deprotection of PG2 to obtain a compound represented by formula (Rc-V): [ka] [In the formula, A2, PG1 and PG3 are as defined above; B2 is [ka] and PG6 is a protecting group for the amino group. a step of obtaining a compound represented by the formula: (Step a3) The obtained compound of formula (Rc-V) or a salt thereof is cyclized in the presence of a condensing agent, and then reacted with a thiolating agent or an oxidizing agent to obtain a compound of formula (Rc-VI): [ka] [In the formula, A2, B2, Q, PG1 and PG3 are as defined above.] or a salt thereof; and (Step a4) A step of deprotecting B2, which is a protecting group at the thiophosphate moiety of the obtained compound of Formula (Rc-VI), and PG4, which is a protecting group in A2, to obtain a compound of Formula (Rp-VII) or a salt thereof. A method comprising: [2] In step a2, after reacting with a thiolating agent and before deprotecting PG2, the resulting intermediate has the formula (Rc-V0): [ka] [In the formula, A2, B2, PG1, PG2 and PG3 are as defined in [1].] The method according to [1], wherein the compound is a compound represented by the formula: [3] Formula (Sp-VII): [ka] [In the formula, A1 is [ka] and Q is a thiol group or a hydroxy group; PG1 is a protecting group for the hydroxy group, and PG3 is a protecting group for the amino group. A method for producing a compound represented by the formula: (Step a1) Formula (I): [ka] [In the formula, PG1 and PG3 are as defined above, and PG2 is a protecting group for the hydroxy group. with an optically active phosphitylating agent (Sc-II) selected from the group consisting of the following formulae (Sc-II-1) and (Sc-II-2): [ka] [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. to form a compound of formula (Sc-III): [ka] [In the formula, PG1, PG2 and PG3 are as defined above, and B1 S teeth, [ka] where R1 and R2 are as defined above. obtaining a compound represented by the formula: (Step a2) The obtained compound of formula (Sc-III) is reacted with a compound of formula (IV): [ka] [In the formula, A2 is [ka] and PG4 is a protecting group for the amino group. or a salt thereof in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and further reaction with a thiolating agent, followed by deprotection of PG2 to obtain a compound represented by formula (Sc-V): [ka] [In the formula, A2, PG1 and PG3 are as defined above; B2 Steeth, [ka] and PG6 is a protecting group for the amino group. a step of obtaining a compound represented by the formula: (Step a3) The obtained compound of formula (Sc-V) or a salt thereof is cyclized in the presence of a condensing agent, and then reacted with a thiolating agent or an oxidizing agent to obtain a compound of formula (Sc-VI): [ka] [In the formula, A2, B2 S , Q, PG1 and PG3 are as defined above.] or a salt thereof; and (Step a4) B2, which is a protecting group for the thiophosphate moiety of the obtained compound of formula (Sc-VI), S and deprotecting the protecting group PG4 in A2 to obtain a compound of formula (Sp-VII) or a salt thereof. A method comprising: [4] In step a2, after reacting with a thiolating agent and before deprotecting PG2, the resulting intermediate has the formula (Sc-VO): [ka] [In the formula, A2, B2 S , PG1, PG2 and PG3 are 3 ] as defined in A compound represented by the formula (I) or a salt thereof, 3 ] The method described above. [5] The method according to any one of [1] to [4], wherein the activating agent in step a2 is at least one selected from the group consisting of 1-phenylimidazole, benzimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-pyrrolidineacetonitrile, 1-(cyanomethyl)imidazole, and salts thereof. [6] The acylating agent or alkoxycarbonylating agent in step a2 is acetic anhydride, N-succinimidyl acetate, pentafluorophenyl acetate, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, 1-trifluoroacetylimidazole, benzoic anhydride, pentafluorophenyl benzoate 、 1-tert-butoxycarbonyl-1,2,4-triazole, N-tert-butoxycarbonylimidazole, di-tert-butyl dicarbonate, 9-fluorenylmethylpentafluorophenyl carbonate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide [5] The method according to any one of [1] to [5], wherein the compound is at least one selected from the group consisting of succinimide, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, and methylimidazole-1-carboxylate. [7] The method according to any one of [1] to [6], wherein the thiolating agent in step a2 is at least one selected from the group consisting of xanthan hydride, bis(phenylacetyl) disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione. [8] The method according to any one of [1] to [7], wherein the condensing agent in step a3 is at least one selected from the group consisting of 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide, dimethyl chlorophosphate, diethyl chlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphorane, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinohexafluorophosphate, bromotripyrrolidinohexafluorophosphate, and propylphosphonic anhydride. [9] The method according to any one of [1] to [8], wherein the thiolating agent in step a3 is at least one selected from the group consisting of xanthan hydride, bis(phenylacetyl) disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione.

[10] The method according to any one of [1] to [8], wherein the oxidizing agent in step a3 is at least one selected from the group consisting of iodine, tert-butyl hydroperoxide, 3-chloroperbenzoic acid, hydrogen peroxide, periodic acid, potassium permanganate, and oxygen.

[11] The method according to any one of [1] to

[10] , wherein PG4 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl.

[12] The method according to any one of [1] to

[11] , wherein PG6 is acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl.

[13] The compound of formula (IV) or a salt thereof can be prepared by the following process: (Step a5) Formula (VIII): [ka] [In the formula, A2 is as defined in [1], and PG5 is a protecting group for a hydroxy group. or a salt thereof with a phosphite to obtain a compound represented by formula (IX): [ka] [In the formula, A2 and PG5 are as defined above.] a step of obtaining a compound represented by the formula: Or, (Step a5') Formula (VIII'): [ka] [In the formula, A1 is as defined in [1], and PG5 is as defined above.] or a salt thereof with a phosphite to obtain a compound represented by formula (IX'): [ka] [In the formula, A1 and PG5 are as defined above.] or a salt thereof; and (Step a5″) The obtained compound of formula (IX′) or a salt thereof is reacted with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (IX): [ka] [In the formula, A2 and PG5 are as defined above.] or a salt thereof and (Step a6) A step of deprotecting PG5, which is the protecting group of the 5' hydroxyl group of the compound of formula (IX) obtained in step a5 or step a5″, to obtain a compound of formula (IV) or a salt thereof. The method according to any one of [1] to

[12] , wherein the compound is produced by the method according to any one of [1] to

[12] .

[14] The method according to

[13] , wherein the phosphite ester in Step a5 and Step a5' is at least one selected from the group consisting of diphenyl phosphite, methyl phosphite, diethyl phosphite, and dibutyl phosphite.

[15] The acylating agent or alkoxycarbonylating agent in step a5″ is acetic anhydride, acetyl chloride, N-succinimidyl acetate, pentafluorophenyl acetate, 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, N-methoxydiacetamide, N-acetylimidazole, trifluoroacetic anhydride, bistrifluoroacetamide, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, S-ethyl trifluorothioacetate, N-methylbistrifluoroacetamide, trifluoroacetyl triflate, 1-trifluoroacetylimidazole, benzoic anhydride, benzoyl chloride, pentafluorophenyl benzoate 、Benzoyl trifluoromethanesulfonate, 3-benzoylthiazolidine-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butyl carbazate, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole 、 Di-tert-butyl dicarbonate, 9-fluorenylmethyl pentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl carbazate, 19-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbonyl The method according to

[13] or

[14] , wherein the compound is at least one selected from the group consisting of benzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethyl imidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, and methyl imidazole-1-carboxylate.

[16] The method according to any one of [1], [2], [5] to [9], and

[11] to

[12] , characterized in that in step a3 according to [1], the compound of formula (Rp-VII) (wherein Q is a thiol group) or a salt thereof is obtained by using the thiolating agent.

[17] In step a4 of [1], the obtained compound of formula (Rp-VII) (wherein Q is a thiol group) is treated with silica gel column chromatography before being converted into a salt thereof, and / or is crystallized after being converted into a salt thereof to obtain a compound of formula (Rp, Rp-VII′): [ka] The method according to

[16] , further comprising obtaining a compound of formula (I) or a salt thereof.

[18] moreover (Step a7) The protecting groups PG1 and PG3 of the obtained compound of Formula (Rp, Rp-VII′) are deprotected to obtain a compound of Formula (Rp, Rp-X): [ka] [In the formula, A1 is as defined in [1]. or a salt thereof; and (Step a8) The obtained compound of formula (Rp, Rp-X) or a salt thereof is Formula (XI): [ka] or an activated ester thereof, to form a compound represented by the formula (Rp,Rp-XII): [ka] [In the formula, A1 is as defined above.] or a salt thereof The method according to

[17] , comprising:

[19] moreover, (Step a9) The obtained compound of formula (Rp, Rp-XII) or a salt thereof is bound to an antibody or a functional fragment of the antibody (hereinafter referred to as Ab) to obtain a compound of formula (Rp, Rp-XIII): [ka] [In the formula, m ranges from 1 to 10, The glycans of Abs are arbitrarily remodeled, Ab is bound to the compound of formula (Rp, Rp-XII) directly from the side chain of an optionally modified amino acid residue, or to the compound of formula (Rp, Rp-XII) from a sugar chain or a remodeled sugar chain of Ab; A1 is [ka] ] or a mixture thereof. The method according to

[18] , comprising:

[20] The method according to

[19] , wherein in step a9, the compound of formula (Rp,Rp-XII) or a salt thereof and Ab are bonded by a strain-promoted azide-alkyne cycloaddition reaction. [twenty one] The method according to

[19] or

[20] , wherein the antibody is an antibody selected from the group consisting of 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, and an anti-DR5 antibody. [twenty two] Formula (Rc-III): [ka] [In the formula, B1 is as defined in [1], PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. A compound represented by the formula: [twenty three] Formula (Rc-V0): [ka] [In the formula, A2 and B2 are as defined in [1], PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. A compound represented by the formula (I) or a salt thereof. [twenty four] Formula (Rc-V): [ka] [In the formula, A2 and B2 are as defined in [1], PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. A compound represented by the formula (I) or a salt thereof. [twenty five] The compound of formula (I) (wherein PG1 is tert-butyldimethylsilyl) can be prepared by the following steps: (Step b1) Formula (XIV): [ka] [In the formula, PG2 is as defined in [1].] with a compound represented by formula (XV): [ka] [In the formula, PG3 is as defined in [1], and X is Cl, Br, or I. and reacting the compound represented by formula (XVI): [ka] [In the formula, PG2 and PG3 are as defined above.] obtaining a compound represented by the formula: (Step b2) The obtained compound of formula (XVI) is reacted with a silylating agent to obtain a mixture of the compound of formula (I′) and the compound of formula (XVII): [ka] [In the formula, PG2 and PG3 are as defined above.] and then converting the compound of formula (XVII) in the mixture to a compound of formula (I') in the presence of a base to obtain a compound of formula (I'). The method according to any one of [1] to

[21] , wherein the compound is produced by the method according to any one of [1] to

[21] .

[26] The method according to

[25] , wherein the reaction of step b1 is carried out in the presence of a base, and the base is 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo[5.4.0]-7-undecene.

[27] The method according to

[25] or

[26] , comprising the step of: in step b2, reacting the compound of formula (XVI) with a silylating agent in the presence of a first base to obtain a mixture of the compound of formula (I') and the compound of formula (XVII), and then converting the compound of formula (XVII) in the mixture to the compound of formula (I') in the presence of a second base to obtain the compound of formula (I').

[28]

[27] The method according to

[27] , wherein the first base is at least one selected from the group consisting of 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[29] The method according to

[27] or

[28] , wherein the second base is at least one selected from the group consisting of 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[30] The method according to any one of

[25] to

[29] , wherein the silylating agent in step b2 is tert-butyldimethylchlorosilane or tert-butyldimethylsilyl triflate.

[31] The compound of formula (XV) can be prepared by the following steps: (Step b3) Formula (XVIII): [ka] [In the formula, PG3 is as defined in [1]] with 2-haloethanol in the presence of an acid or a base to obtain a compound of formula (XV). The method according to any one of

[25] to

[30] , wherein the compound is produced by the method according to any one of

[25] to

[30] .

[32]

[31] The method according to

[31] , wherein the base in step b3 is at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[33] Formula (XV): [ka] [In the formula, PG3 is 2-(trimethylsilyl)ethoxycarbonyl, and X is Cl, Br, or I. A compound represented by the formula:

[34] The compound of formula (VIII') [In the formula, A1 is [ka] and PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl. But the following steps: (Step c1) Formula (XIX): [ka] is reacted with a benzoylating agent to obtain a compound represented by formula (XX): [ka] obtaining a compound represented by the formula: (Step c2) The obtained compound of formula (XX) is hydrolyzed to obtain a compound of formula (XXI): [ka] obtaining a compound represented by the formula: (Step c3) The obtained compound of formula (XXI) is reacted with a chlorinating agent to obtain a compound of formula (XXII): [ka] obtaining a compound represented by the formula: (Step c4) The obtained compound of formula (XXII) is reacted with a compound of formula (XXIII): [ka] and reacting the compound represented by formula (XXIV): [ka] obtaining a compound represented by the formula: (Step c5) The benzoyl group is deprotected from the obtained compound of formula (XXIV) to obtain a compound of formula (XXV): [ka] a step of obtaining a compound represented by the formula: (Step c6) Reacting the obtained compound of formula (XXV) or a salt thereof with a tritylation agent to obtain a compound of formula (VIII') (wherein A1 and PG5 are as defined above) or a salt thereof. The method according to any one of

[13] to

[21] and

[25] to

[32] , wherein the compound is produced by the method according to any one of

[13] to

[21] and

[25] to

[32] .

[35] moreover, (Step c7) The obtained compound of formula (VIII') (wherein A1 and PG5 are as defined in

[34] ) or a salt thereof is reacted with a silylating agent, and then with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (XXVII): [ka] [In the formula, PG4 is benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG5 is as defined above, and PG7 is a protecting group for the hydroxy group. and obtaining a compound represented by the formula: (Step c8) The protecting group PG7 of the obtained compound of formula (XXVII) is deprotected to obtain a compound of formula (VIII): [ka] [In the formula, A2 is [ka] and PG4 and PG5 are as defined above.] or a salt thereof The method according to

[34] , comprising:

[36] the chlorinating agent in step c3 is at least one selected from the group consisting of trichloroisocyanuric acid, chloroisocyanuric acid, dichloroisocyanuric acid, N-chlorosuccinimide, 1,3-dichloro-5,5-dimethylhydantoin, and carbon tetrachloride; The reaction of step c3 is to obtain tris(2,4-di-tert-butylphenyl)phosphite, tri-o-tolyl R The method according to

[34] or

[35] , wherein the method is carried out in the presence of at least one selected from the group consisting of phenyl phosphite, triphenyl phosphite, triethyl phosphite, and triphenylphosphine.

[37] The method according to any one of

[34] to

[36] , wherein step c4 is carried out in the presence of at least one selected from the group consisting of cesium carbonate, potassium carbonate, sodium carbonate, lithium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[38] The method according to any one of

[34] to

[37] , wherein the tritylation agent in step c6 is at least one selected from the group consisting of 4,4-dimethoxytrityl chloride, 4-methoxytrityl chloride, 2-chlorotrityl chloride, and trityl chloride.

[39] The method according to any one of

[35] to

[38] , wherein the acylating agent or alkoxycarbonylating agent in step c7 is at least one selected from the group consisting of a benzoylating agent, a 2-(trimethylsilyl)ethoxycarbonylating agent, a tert-butoxycarbonylating agent, a 9-fluorenylmethyloxycarbonylating agent, an allyloxycarbonylating agent, a 2,2,2-trichloroethoxycarbonylating agent, and a benzyloxycarbonylating agent.

[40] The compound of formula (XXIII) can be prepared by the following steps: (Step c9) Formula (XXVIII): [ka] The tert-butoxycarbonyl group is deprotected from the compound represented by formula (XXIX): [ka] obtaining a compound represented by the formula: (Step c10) The obtained compound of formula (XXIX) is subjected to a reduction reaction of an alkyne and then a reductive amination reaction in the presence of a catalyst to obtain a compound of formula (XXX): [ka] obtaining a compound represented by the formula: (Step c11) A step of reacting the obtained compound of formula (XXX) with a benzoylating agent to obtain a compound of formula (XXIII). The method according to any one of

[34] to

[39] , wherein the compound is produced by

[41] The compound of formula (XXVIII) can be prepared by the following steps: (Step c12) Formula (XXXI): [ka] is reacted with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain a compound of formula (XXXII): [ka] obtaining a compound represented by the formula: (Step c13) A step of reacting the obtained compound of formula (XXXII) with propargylaldehyde diethyl acetal to obtain a compound of formula (XXVIII). The method according to

[40] ,

[42] The compound of formula (XXIV) can be obtained by the following steps instead of step c4: (Step c14) Reacting the compound of formula (XXII) with the compound of formula (XXIX) to obtain a compound of formula (XXXIII): [ka] obtaining a compound represented by the formula: (Step c15) A step of subjecting the obtained compound of formula (XXXIII) to an alkyne reduction reaction in the presence of a catalyst, followed by a reductive amination reaction, and then reacting it with a benzoylating agent to obtain a compound of formula (XXIV). The method according to any one of

[34] to

[39] and

[41] , wherein the compound is produced by the method according to any one of

[34] to

[39] and

[41] .

[43] Formula (XX): [ka] A compound represented by the formula:

[44] Formula (XXII): [ka] A compound represented by the formula:

[45] Formula (XXIV): [ka] A compound represented by the formula:

[46] Formula (XXV): [ka] A compound represented by the formula (I) or a salt thereof.

[47] Formula (VIII'-1): [ka] [In the formula, PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl.] A compound represented by the formula (I) or a salt thereof.

[48] Formula (XXVII): [ka] [In the formula, PG4 is benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl, and PG7 is trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or triphenylsilyl. A compound represented by the formula:

[49] Formula (XXVIII): [ka] A compound represented by the formula:

[50] Formula (I): [ka] [In the formula, PG1 is tert-butyldimethylsilyl, PG2 is a protecting group for the hydroxy group, and PG3 is a protecting group for the amino group. A method for producing a compound represented by the formula: (Step b1) Formula (XIV): [ka] [In the formula, PG2 is as defined above.] with a compound represented by formula (XV): [ka] [In the formula, PG3 is as defined above, and X is Cl, Br, or I. and reacting the compound represented by formula (XVI): [ka] [In the formula, PG2 and PG3 are as defined above.] obtaining a compound represented by the formula: (Step b2) The obtained compound of formula (XVI) is reacted with a silylating agent to obtain a mixture of the compound of formula (I′) and the compound of formula (XVII): [ka] [In the formula, PG2 and PG3 are as defined above.] and then converting the compound of formula (XVII) in the mixture to a compound of formula (I') in the presence of a base to obtain a compound of formula (I'). A method comprising:

[51] The method according to

[50] , wherein the reaction of step b1 is carried out in the presence of a base, and the base is 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo[5.4.0]-7-undecene.

[52] The method according to

[50] or

[51] , comprising the step of: in step b2, reacting the compound of formula (XVI) with a silylating agent in the presence of a first base to obtain a mixture of the compound of formula (I') and the compound of formula (XVII), and then converting the compound of formula (XVII) in the mixture to the compound of formula (I') in the presence of a second base to obtain the compound of formula (I').

[53]

[52] The method according to

[52] , wherein the first base is at least one selected from the group consisting of 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[54] The method according to

[52] or

[53] , wherein the second base is at least one selected from the group consisting of 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[55] The method according to any one of

[50] to

[54] , wherein the silylating agent in step b2 is tert-butyldimethylchlorosilane or tert-butyldimethylsilyl triflate.

[56] Formula (XV): [ka] [In the formula, PG3 is a protecting group for the amino group, and X is Cl, Br, or I. A method for producing a compound represented by the formula: (Step b3) Formula (XVIII): [ka] [In the formula, PG3 is as defined above.] with 2-haloethanol in the presence of an acid or a base to obtain a compound of formula (XV). A method comprising:

[57]

[56] The method according to

[56] , wherein the base in step b3 is at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[58] Formula (VIII'): [ka] [In the formula, A1 is [ka] and PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl. A method for producing a compound represented by the formula: (Step c1) Formula (XIX): [ka] is reacted with a benzoylating agent to obtain a compound represented by formula (XX): [ka] obtaining a compound represented by the formula: (Step c2) The obtained compound of formula (XX) is hydrolyzed to obtain a compound of formula (XXI): [ka] obtaining a compound represented by the formula: (Step c3) The obtained compound of formula (XXI) is reacted with a chlorinating agent to obtain a compound of formula (XXII): [ka] obtaining a compound represented by the formula: (Step c4) The obtained compound of formula (XXII) is reacted with a compound of formula (XXIII): [ka] and reacting the compound represented by formula (XXIV): [ka] obtaining a compound represented by the formula: (Step c5) The benzoyl group is deprotected from the obtained compound of formula (XXIV) to obtain a compound of formula (XXV): [ka] a step of obtaining a compound represented by the formula: (Step c6) Reacting the obtained compound of formula (XXV) or a salt thereof with a tritylation agent to obtain a compound of formula (VIII') (wherein A1 and PG5 are as defined above) or a salt thereof. A method comprising:

[59] moreover, (Step c7) The compound of formula (VIII′) obtained (wherein A1 and PG5 are 58) or a salt thereof with a silylating agent, and then with an acylating agent or an alkoxycarbonylating agent to give a compound of formula (XXVII): [ka] [In the formula, PG4 is benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG5 is as defined above, and PG7 is a protecting group for the hydroxy group. and obtaining a compound represented by the formula: (Step c8) The protecting group PG7 of the obtained compound of formula (XXVII) is deprotected to obtain a compound of formula (VIII): [ka] [In the formula, A2 is [ka] and PG4 and PG5 are as defined above.] or a salt thereof The method according to

[58] , comprising:

[60] the chlorinating agent in step c3 is at least one selected from the group consisting of trichloroisocyanuric acid, chloroisocyanuric acid, dichloroisocyanuric acid, N-chlorosuccinimide, 1,3-dichloro-5,5-dimethylhydantoin, and carbon tetrachloride; The reaction of step c3 is to obtain tris(2,4-di-tert-butylphenyl)phosphite, tri-o-tolyl R

[59] The method according to

[59] , wherein the method is carried out in the presence of at least one selected from the group consisting of phenyl phosphite, triphenyl phosphite, triethyl phosphite, and triphenylphosphine.

[61] The method according to

[59] or

[60] , wherein step c4 is carried out in the presence of at least one selected from the group consisting of cesium carbonate, potassium carbonate, sodium carbonate, lithium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, and 1,8-diazabicyclo[5.4.0]-7-undecene.

[62] The method according to any one of

[59] to

[61] , wherein the tritylation agent in step c6 is at least one selected from the group consisting of 4,4-dimethoxytrityl chloride, 4-methoxytrityl chloride, 2-chlorotrityl chloride, and trityl chloride.

[63] The method according to any one of

[59] to

[62] , wherein the acylating agent or alkoxycarbonylating agent in step c7 is at least one selected from the group consisting of a benzoylating agent, a 2-(trimethylsilyl)ethoxycarbonylating agent, a tert-butoxycarbonylating agent, a 9-fluorenylmethyloxycarbonylating agent, an allyloxycarbonylating agent, a 2,2,2-trichloroethoxycarbonylating agent, and a benzyloxycarbonylating agent.

[64] Formula (XXIII): [ka] A method for producing a compound represented by the formula: (Step c9) Formula (XXVIII): [ka] The tert-butoxycarbonyl group is deprotected from the compound represented by formula (XXIX): [ka] obtaining a compound represented by the formula: (Step c10) The obtained compound of formula (XXIX) is subjected to a reduction reaction of an alkyne and then a reductive amination reaction in the presence of a catalyst to obtain a compound of formula (XXX): [ka] obtaining a compound represented by the formula: (Step c11) A step of reacting the obtained compound of formula (XXX) with a benzoylating agent to obtain a compound of formula (XXIII). A method comprising:

[65] Formula (XXVIII): [ka] A method for producing a compound represented by the formula: (Step c12) Formula (XXXI): [ka] is reacted with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain a compound of formula (XXXII): [ka] obtaining a compound represented by the formula: (Step c13) A step of reacting the obtained compound of formula (XXXII) with propargylaldehyde diethyl acetal to obtain a compound of formula (XXVIII). A method comprising:

[66] Formula (XXIV): [ka] A method for producing a compound represented by the formula: (Step c14) Formula (XXII): [ka] with a compound represented by formula (XXIX): [ka] by reacting with a compound represented by formula (XXXIII): [ka] obtaining a compound represented by the formula: (Step c15) A step of subjecting the obtained compound of formula (XXXIII) to an alkyne reduction reaction in the presence of a catalyst, followed by a reductive amination reaction, and then reacting it with a benzoylating agent to obtain a compound of formula (XXIV). A method comprising: Regarding. [Effects of the Invention]

[0012] The present invention provides a method for stereoselectively producing cyclic dinucleotides. This production method reduces the burden of diastereomer purification and enables the production of CDNs having the desired stereoconfiguration in large quantities and at higher yields. As a result, CDN-linkers can be produced in high yields by a CDN-linker production method that includes this stereoselective CDN production method. Furthermore, the present invention also enables the preparation of raw material compounds used in the production of cyclic dinucleotides to be performed in a shorter number of steps and at high yields. [Brief explanation of the drawings]

[0013] [Figure 1] This shows a process for producing a cyclic dinucleotide, in which two types of nucleotides (compound (I) and compound (IV)) are stereoselectively phosphorothioate-bonded using an optically active phosphitylating agent (Rc-II) to obtain a production intermediate (Rc-V). [Figure 2] The amino acid sequence of the anti-CD70 antibody 1 light chain (SEQ ID NO: 1) and the amino acid sequence of the anti-CD70 antibody 1 heavy chain (SEQ ID NO: 2) are shown. [Figure 3] The amino acid sequence of the anti-CD70 antibody 2 light chain (SEQ ID NO: 3) and the amino acid sequence of the anti-CD70 antibody 2 heavy chain (SEQ ID NO: 4) are shown. [Figure 4] The amino acid sequence of the anti-TROP2 antibody 1 light chain (SEQ ID NO: 5) and the amino acid sequence of the anti-TROP2 antibody 1 heavy chain (SEQ ID NO: 6) are shown. [Figure 5] The amino acid sequence of the anti-TROP2 antibody 2 light chain (SEQ ID NO: 7) and the amino acid sequence of the anti-TROP2 antibody 2 heavy chain (SEQ ID NO: 8) are shown. [Figure 6] The amino acid sequence of the anti-EGFR antibody 1 light chain (SEQ ID NO: 9) and the amino acid sequence of the anti-EGFR antibody 1 heavy chain (SEQ ID NO: 10) are shown. [Figure 7] The amino acid sequence of the light chain of anti-EGFR antibody 2 (SEQ ID NO: 11) and the amino acid sequence of the heavy chain of anti-EGFR antibody 2 (SEQ ID NO: 12) are shown. [Figure 8] Shown are the CDRL1 amino acid sequence (SEQ ID NO: 13) of anti-CD70 antibody 1, the CDRL2 amino acid sequence (SEQ ID NO: 14) of anti-CD70 antibody 1, the CDRL3 amino acid sequence (SEQ ID NO: 15) of anti-CD70 antibody 1, the CDRH1 amino acid sequence (SEQ ID NO: 16) of anti-CD70 antibody 1, the CDRH2 amino acid sequence (SEQ ID NO: 17) of anti-CD70 antibody 1, and the CDRH3 amino acid sequence (SEQ ID NO: 18) of anti-CD70 antibody 1. CDR sequences were determined according to the definition of Kabat. [Figure 9] Shown are the CDRL1 amino acid sequence (SEQ ID NO: 19) of anti-CD70 antibody 2, the CDRL2 amino acid sequence (SEQ ID NO: 20) of anti-CD70 antibody 2, the CDRL3 amino acid sequence (SEQ ID NO: 21) of anti-CD70 antibody 2, the CDRH1 amino acid sequence (SEQ ID NO: 22) of anti-CD70 antibody 2, the CDRH2 amino acid sequence (SEQ ID NO: 23) of anti-CD70 antibody 2, and the CDRH3 amino acid sequence (SEQ ID NO: 24) of anti-CD70 antibody 2. CDR sequences were determined according to the definition of Kabat. [Figure 10] Shown are the CDRL1 amino acid sequence (SEQ ID NO: 25) of anti-TROP2 antibody 1, the CDRL2 amino acid sequence (SEQ ID NO: 26) of anti-TROP2 antibody 1, the CDRL3 amino acid sequence (SEQ ID NO: 27) of anti-TROP2 antibody 1, the CDRH1 amino acid sequence (SEQ ID NO: 28) of anti-TROP2 antibody 1, the CDRH2 amino acid sequence (SEQ ID NO: 29) of anti-TROP2 antibody 1, and the CDRH3 amino acid sequence (SEQ ID NO: 30) of anti-TROP2 antibody 1. CDR sequences were determined according to the definition of Kabat. [Figure 11]The amino acid sequence of CDRL1 (SEQ ID NO: 31) of anti-TROP2 antibody 2, the amino acid sequence of CDRL2 (SEQ ID NO: 32) of anti-TROP2 antibody 2, the amino acid sequence of CDRL3 (SEQ ID NO: 33) of anti-TROP2 antibody 2, the amino acid sequence of CDRH1 (SEQ ID NO: 34) of anti-TROP2 antibody 2, the amino acid sequence of CDRH2 (SEQ ID NO: 35) of anti-TROP2 antibody 2, and the amino acid sequence of CDRH3 (SEQ ID NO: 36) of anti-TROP2 antibody 2 are shown. CDR sequences were determined according to the definition of Kabat. [Figure 12] Shown are the CDRL1 amino acid sequence (SEQ ID NO: 37) of anti-EGFR antibody 1, the CDRL2 amino acid sequence (SEQ ID NO: 38) of anti-EGFR antibody 1, the CDRL3 amino acid sequence (SEQ ID NO: 39) of anti-EGFR antibody 1, the CDRH1 amino acid sequence (SEQ ID NO: 40) of anti-EGFR antibody 1, the CDRH2 amino acid sequence (SEQ ID NO: 41) of anti-EGFR antibody 1, and the CDRH3 amino acid sequence (SEQ ID NO: 42) of anti-EGFR antibody 1. CDR sequences were determined according to the definition of Kabat. [Figure 13] Shown are the CDRL1 amino acid sequence (SEQ ID NO: 43) of anti-EGFR antibody 2, the CDRL2 amino acid sequence (SEQ ID NO: 44) of anti-EGFR antibody 2, the CDRL3 amino acid sequence (SEQ ID NO: 45) of anti-EGFR antibody 2, the CDRH1 amino acid sequence (SEQ ID NO: 46) of anti-EGFR antibody 2, the CDRH2 amino acid sequence (SEQ ID NO: 47) of anti-EGFR antibody 2, and the CDRH3 amino acid sequence (SEQ ID NO: 48) of anti-EGFR antibody 2. CDR sequences were determined according to the definition of Kabat. [Figure 14] The amino acid sequence of the trastuzumab light chain (SEQ ID NO: 49) and the amino acid sequence of the trastuzumab heavy chain (SEQ ID NO: 50) are shown. [Figure 15] The amino acid sequence of the light chain of the modified anti-HER2 antibody (SEQ ID NO: 49) and the amino acid sequence of the heavy chain of the modified anti-HER2 antibody (SEQ ID NO: 51) are shown. [Figure 16] The amino acid sequence of the pertuzumab light chain (SEQ ID NO: 52) and the amino acid sequence of the pertuzumab heavy chain (SEQ ID NO: 53) are shown. [Figure 17] The amino acid sequence of the light chain of modified anti-HER2 antibody 2 (SEQ ID NO: 52) and the amino acid sequence of the heavy chain of modified anti-HER2 antibody 2 (SEQ ID NO: 54) are shown. [Figure 18] The amino acid sequence of the light chain of the anti-CDH6 antibody (sequence number 55) and the amino acid sequence of the heavy chain of the anti-CDH6 antibody (sequence number 56) are shown. [Figure 19] 19A) and that obtained from an MSG-type glycoremodeling antibody (molecule (XXXIV) in Figure 19B). (a) represents immunostimulator D, (b) linker L, (c) PEG linker (L(PEG)), and (d) N297 glycan (where 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 formed 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 position 2 of the sialic acid at the non-reducing end via an amide bond. This notation method is applied throughout the specification unless otherwise specified. [Figure 20] The amino acid sequence of CDRL1 of pertuzumab (SEQ ID NO: 57), the amino acid sequence of CDRL2 of pertuzumab (SEQ ID NO: 58), the amino acid sequence of CDRL3 of pertuzumab (SEQ ID NO: 59), the amino acid sequence of CDRH1 of pertuzumab (SEQ ID NO: 60), the amino acid sequence of CDRH2 of pertuzumab (SEQ ID NO: 61), and the amino acid sequence of CDRH3 of pertuzumab (SEQ ID NO: 62) are shown. The CDR sequences were determined according to the definition of IMGT. [Figure 21] The amino acid sequence of CDRL1 (SEQ ID NO: 63), CDRL2 (SEQ ID NO: 64), CDRL3 (SEQ ID NO: 65), CDRH1 (SEQ ID NO: 66), CDRH2 (SEQ ID NO: 67), and CDRH3 (SEQ ID NO: 68) of the anti-CDH6 antibody are shown. The CDR sequences were determined according to the definition of Kabat. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one aspect, the present invention provides a novel method for producing a CDN derivative, which is an intermediate for an antibody-immunoactivator conjugate that has STING agonist activity and activates immune cells. Such an antibody-immunoactivator conjugate is disclosed in International Publication No. 2020 / 050406 (Patent Document 6).

[0015] One embodiment of the present invention is a method for stereoselectively synthesizing a CDN derivative having a structure represented by the following formula (Rp-VII) or (Sp-VII):

[0016] [ka]

[0017] A representative scheme of the present invention, which is one embodiment and focuses on a novel method for producing a CDN derivative of formula (Rp-VII), is outlined below.

[0018] [ka]

[0019] Preferred embodiments for carrying out the present invention will be described below. The embodiment described below is merely an example of a typical embodiment of the present invention, and is not intended to narrow the scope of the present invention. In this specification, the compound numbers shown in each reaction formula are used to indicate the compounds. That is, they are referred to as "compound of formula (I)," "compound (I)," etc. Compounds with other numbers will also be described in the same manner.

[0020] <1. Production of Cyclic Dinucleotide (CDN) and CDN-Linker> <1-1. Method for producing cyclic dinucleotides (compounds (Rp-VII) and (Sp-VII)) and intermediates therefor>

[0021] In conventional manufacturing methods, the following four types of cyclic dinucleotides have been synthesized non-stereoselectively (see Patent Document 6, etc.). The configurations of these four types of cyclic dinucleotides are expressed in terms of the absolute configurations at the two phosphorus atoms as (Rp,Rp), (Sp,Rp), (Rp,Sp), and (Sp,Sp) in the order of (absolute configuration of phosphorus at position 2, absolute configuration of phosphorus at position 10).

[0022] [ka]

[0023] In one embodiment, in the production of the CDN of the present invention, a cyclic dinucleotide of formula (Rp-VII) is synthesized according to the following synthetic scheme: <a1-rp>It can be produced according to the following. [Synthetic Scheme <a1-rp>]

[0024] [ka]

[0025] In another embodiment, the cyclic dinucleotide of formula (Sp-VII) can be prepared according to the following synthesis scheme: <a1-sp>It can be produced according to the following. [Synthetic Scheme <a1-sp>]

[0026] [ka]

[0027] The above [Synthetic Scheme <a1-rp>and <a1-sp>]In, PG1 is a protecting group for the hydroxy group, PG2 is a protecting group for a hydroxy group, PG3 is a protecting group for the amino group, A1 is

[0028] [ka] and A2 is

[0029] [ka] (wherein PG4 is a protecting group for an amino group). and The optically active phosphitylating agent (Rc-II) is

[0030] [ka] wherein R1 is hydrogen or methyl, R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl, the alkyl being unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl groups, and the phenyl being unsubstituted or substituted with nitro or methoxy groups. and the optically active phosphitylating agent (Sc-II) is

[0031] [ka] (wherein R1 and R2 have the same meanings as in the phosphitylating agent (Rc-II) above.) and Synthesis scheme <a1-rp>In this case, B1 is

[0032] [ka] and the synthesis scheme <a1-sp>In B1 S teeth,

[0033] [ka] and Synthesis scheme <a1-rp>In this case, B2 is

[0034] [ka] and the synthesis scheme <a1-sp>In B2 S teeth,

[0035] [ka] (Here, PG6 is a protecting group for the amino group.) and Q is a thiol group or a hydroxy group.

[0036] In this production method, examples of PG1 include tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, and tert-butyldiphenylsilyl. Preferred are tert-butyldimethylsilyl and trimethylsilyl. More preferred is tert-butyldimethylsilyl.

[0037] Examples of PG2 include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, and trityl. Preferred are 4,4'-dimethoxytrityl, 4-methoxytrityl, and trityl. More preferred is 4,4'-dimethoxytrityl.

[0038] Examples of PG3 include 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, and benzyloxycarbonyl. 2-(trimethylsilyl)ethoxycarbonyl and allyloxycarbonyl are preferred. 2-(trimethylsilyl)ethoxycarbonyl is more preferred.

[0039] Examples of PG4 include benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, and ethoxycarbonyl. Benzoyl or 2-(trimethylsilyl)ethoxycarbonyl is preferred.

[0040] Examples of PG6 include acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, and ethoxycarbonyl. Preferred are acetyl, trifluoroacetyl, benzoyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl. More preferred are acetyl, trifluoroacetyl, allyloxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl.

[0041] Each step will be explained below. (Step a1) In one embodiment, this step involves reacting a compound of formula (I) with an optically active phosphitylating agent (Rc-II) to give a compound of formula (Rc-III), or in another embodiment, with an optically active phosphitylating agent (Sc-II) to give a compound of formula (Sc-III).

[0042] In one embodiment, the optically active phosphitylating agent (Rc-II) used in this step is a compound represented by the following formula (Rc-II-1) or (Rc-II-2):

[0043] [ka] [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. The optically active phosphitylation agent is a reagent for stereoselectively condensing a compound of formula (I) with a compound of formula (IV) to produce a compound of formula (Rc-V), and can be reacted stereoselectively with a compound of formula (I) to produce a compound of formula (Rc-V). 、 An intermediate of formula (Rc-III) is produced.

[0044] The optically active phosphitylating agent (Rc-II) includes (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aR)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][ 1,3,2]oxazaphosphole, (3R,3aR)-1-chloro-3-[(4-methylbenzene-1-sulfonyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-[(4-nitrophenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aR)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, ( 3S,3aR)-1-chloro-3-[(4-methoxyphenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-(diphenylmethyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aR)-1-chloro-3a,4-dihydro-1H,3H-[1,3,2]oxazaphosphoro[3,4-a]indole, etc. can be used, and preferably (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1, 3,2]oxazaphosphole, (3aR)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aR)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-Pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used, and more preferably, (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aR)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, or (3R,3aR)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used. The amount of the optically active phosphitylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 3 equivalents relative to the compound represented by formula (I).

[0045] The structure of the optically active phosphitylating agent (Rc-II) exemplified above is shown below. [Table 1]

[0046] In another embodiment, the optically active phosphitylating agent (Sc-II) used includes an optically active phosphitylating agent represented by the following formula (Sc-II-1) or (Sc-II-2):

[0047] [ka] [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. Examples of the compound include compounds represented by the following formula:

[0048] The optically active phosphitylating agent (Sc-II) includes (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aS)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][ 1,3,2]oxazaphosphole, (3S,3aS)-1-chloro-3-[(4-methylbenzene-1-sulfonyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-[(4-nitrophenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aS)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, ( 3R,3aS)-1-chloro-3-[(4-methoxyphenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-(diphenylmethyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aS)-1-chloro-3a,4-dihydro-1H,3H-[1,3,2]oxazaphosphoro[3,4-a]indole, etc. can be used, and preferably (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1, 3,2]oxazaphosphole, (3aS)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aS)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-Pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used, and more preferably, (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aS)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, or (3S,3aS)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used. The amount of the optically active phosphitylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (I).

[0049] The structure of the optically active phosphitylating agent (Sc-II) exemplified above is shown below. [Table 2]

[0050] In this step, the reaction proceeds stereoselectively to give a compound of formula (III) having the desired absolute configuration. Here, the absolute configuration of the compound of formula (III) is indicated by (Rc) or (Sc), which indicates the absolute configuration at the asymmetric carbon atom contained in the prolinol skeleton, as shown in the following example.

[0051] [ka]

[0052] In one embodiment, when an optically active phosphitylating agent (Rc-II) in which R and R are hydrogen atoms is used, a compound of formula (Rc-III) having the absolute configuration at the phosphorus atom of (Sp) is produced, as shown below.

[0053] [ka] In another embodiment, when an optically active phosphitylating agent (Sc-II) is used, diastereomers having the opposite absolute configuration with respect to the asymmetric center on phosphorus can be obtained.

[0054] [ka]

[0055] That is, by using an optically active phosphitylating agent having the absolute configuration (Rc) or (Sc), the desired diastereomer can be obtained with high selectivity of 90% or more, preferably 95% or more, and more preferably 98% or more. In conventional production methods, phosphorothioate bonds are formed stereo-nonselectively, and the (Rp) and (Sp) forms are produced in a ratio of approximately 50:50, so the yield of the compound having the desired absolute configuration was less than half.

[0056] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of the base include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, and bases such as potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include methylpyridine, 4-dimethylaminopyridine, and 1,4-diazabicyclo[2.2.2]octane, and more preferably, triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by formula (I).

[0057] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include acetonitrile, dichloromethane, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, and toluene. More preferred examples include dichloromethane and tetrahydrofuran.

[0058] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −78° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 10 hours, more preferably 5 to 180 minutes.

[0059] (Step a2) In one embodiment, this process involves reacting a compound of formula (Rc-III) with a compound of formula (IV) or a salt thereof in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and then with a thiolating agent (also known as a sulfurizing agent or a thiolating agent), followed by deprotection of PG2 to obtain a compound of formula (Rc-V). Optionally, the product may be isolated before deprotection of PG2. In another embodiment, a compound of formula (Sc-V) is obtained from a compound of formula (Sc-III) by a similar reaction. Optionally, this process includes treatment with an organic or inorganic base to convert to a salt thereof.

[0060] In this step, after the reaction with the thiolating agent and before the deprotection of PG2, an intermediate of the formula (Rc-V0): [ka] or a salt thereof (wherein A2, B2, PG1, PG2 and PG3 are represented by the synthetic scheme <a1-rp>) is obtained. In another embodiment, an intermediate having the formula (Sc-VO) may be obtained after the reaction with a thiolating agent and before deprotection of PG2: [ka] or a salt thereof (wherein A2, B2 S , PG1, PG2 and PG3 are represented by the synthetic scheme <a1-sp>) is obtained.

[0061] In one embodiment, the reaction in this step proceeds stereoselectively to obtain a compound of formula (Rc-V) having the desired absolute configuration via an intermediate of formula (Rc-V0). The absolute configurations of formulas (V) and (V0) are indicated using the absolute configuration at the asymmetric carbon atom contained in the prolinol skeleton in B2, as in the compound of formula (III) above.

[0062] [ka]

[0063] In this process, the stereoinversion reaction proceeds with selectivity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more while maintaining the asymmetric environment established by formula (Rc-III). This allows the desired diastereomer to be obtained with high selectivity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more. Conventional methods involve the non-stereoselective condensation of compounds of formula (I) and formula (IV), resulting in a roughly 50:50 mixture of the difficult-to-separate (Rp) and (Sp) isomers, corresponding to compound (V). Therefore, the yield of the desired compound (Rc-V) isomer is not only improved by approximately 2-fold from the perspective of stereoselectivity, but also, considering the burden of separating the large amount of diastereomers at the phosphorus, which are difficult to separate, productivity is expected to increase by approximately 3-10-fold.

[0064] On the other hand, when the compound of formula (Sc-III) is used, the compound of formula (Sc-V), which is a similarly inverted diastereomer, can be obtained via the intermediate of formula (Sc-VO). Thus, depending on the absolute configuration of the asymmetric carbon on the prolinol skeleton of the optically active phosphitylating agent used, a compound having the desired absolute configuration can be stereoselectively obtained.

[0065] The activating agent used in this step is not particularly limited as long as it allows the reaction to proceed without affecting the absolute configuration of the compound represented by Formula (Rc-III) or Formula (Sc-III). Examples of the activating agent include 1-phenylimidazole, benzimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-pyrrolidineacetonitrile, 1-(cyanomethyl)imidazole, and salts thereof. Preferred examples include 1-phenylimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-(cyanomethyl)imidazole, and salts thereof. More preferred examples include 1-phenylimidazole, 1-methylbenzimidazole, and salts thereof. The amount of activating agent used in this step is not limited as long as the reaction proceeds. Preferably, the amount is 1 to 3 equivalents relative to the compound represented by Formula (Rc-III) or Formula (Sc-III).

[0066] The amount of compound (IV) or a salt thereof used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 3 equivalents, more preferably 0.8 to 1.2 equivalents, relative to the compound represented by formula (Rc-III) or formula (Sc-III).

[0067] The acylating agent or alkoxycarbonylating agent used in this step is not particularly limited as long as it allows the reaction to proceed, and examples thereof include acetic anhydride, acetyl chloride, N-succinimidyl acetate, pentafluorophenyl acetate, 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, N-methoxydiacetamide, N-acetylimidazole, trifluoroacetic anhydride, bistrifluoroacetamide, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, S-ethyl trifluorothioacetate, N-methylbistrifluoroacetamide, trifluoroacetyl triflate, 1-trifluoroacetylimidazole, benzoic anhydride, benzoyl chloride, and pentafluorophenyl benzoate. 、 Benzoyl trifluoromethanesulfonate, 3-benzoylthiazolidine-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butyl carbazate, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole 、 Di-tert-butyl dicarbonate, 9-fluorenylmethyl pentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl carbazate, 19-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide Benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate silyl, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethyl imidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, or methyl imidazole-1-carboxylate, and preferred examples include acetic anhydride, 1-trifluoroacetyl Examples of suitable acylating agents include imidazole, benzoic anhydride, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(allyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, and more preferred examples include acetic anhydride, 1-trifluoroacetylimidazole, N-(allyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. The amount of the acylating agent or alkoxycarbonylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.7 to 5 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by Formula (Rc-III) or Formula (Sc-III).

[0068] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 5 hours, more preferably 5 to 90 minutes.

[0069] The reaction of the activating agent, acylating agent or alkoxycarbonylating agent, and thiolating agent in this step is preferably carried out in the presence of a dehydrating agent. The dehydrating agent used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, magnesium sulfate, sodium sulfate, and calcium chloride. Preferred examples include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, and sodium sulfate, and more preferred examples include molecular sieve 3A and molecular sieve 4A. The amount of dehydrating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.01 to 3 times, and more preferably 0.01 to 1 times, the mass of the compound represented by Formula (Rc-III) or Formula (Sc-III).

[0070] The thionating agent used in this step is not particularly limited as long as it allows the reaction to proceed, but examples thereof include xanthan hydride, bis(phenylacetyl)disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione. Xanthan hydride, bis(phenylacetyl)disulfide, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione are preferred. The amount of the thionating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 1 to 2 equivalents, relative to the compound represented by Formula (Rc-III) or Formula (Sc-III).

[0071] The reaction temperature with the thionating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 5 hours, more preferably 5 to 90 minutes.

[0072] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, and more preferred bases are triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 20 equivalents, more preferably 1 to 10 equivalents, relative to the compound represented by Formula (Rc-III) or Formula (Sc-III).

[0073] Examples of acids used in the deprotection reaction in this step include hydrochloric acid, sulfuric acid, formic acid, oxalic acid, acetic acid, trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid. Preferred examples include hydrochloric acid, acetic acid, monochloroacetic acid, and dichloroacetic acid, and more preferred examples are hydrochloric acid and dichloroacetic acid. The pH used in the deprotection reaction is not limited as long as the reaction proceeds, but is preferably 1 to 4. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from −30°C to the boiling point of the solvent used in the reaction, more preferably from −20°C to 30°C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours.

[0074] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include acetonitrile.

[0075] Examples of the organic base used for converting the compound of formula (Rc-V) or formula (Sc-V) into a salt thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable inorganic bases include methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene. Examples of suitable inorganic bases include potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate. More preferred examples include triethylamine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate.

[0076] (Step a3) In one embodiment, this step involves cyclizing a compound of formula (Rc-V) or a salt thereof in the presence of a condensing agent, followed by reaction with a thiolating agent or an oxidizing agent to obtain a compound of formula (Rc-VI). In another embodiment, this step involves obtaining a compound of formula (Sc-VI) from a compound of formula (Sc-V) by a similar reaction.

[0077] In this step, when reacting with a thiolating agent, a compound of formula (VI) in which Q is a thiol group can be obtained. In addition, when reacting with an oxidizing agent in this step, a compound of formula (VI) in which Q is a hydroxy group can be obtained.

[0078] The condensing agent used in this step is not particularly limited as long as it allows the reaction to proceed, and examples thereof include 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide, dimethyl chlorophosphate, diethyl chlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphorane, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinohexafluorophosphate, bromotripyrrolidinohexafluorophosphate, propylphosphonic anhydride, diisopropyl chlorophosphate, bis(2,6-dimethylphenyl)chlorophosphate, bis(dimethylamino)phosphoryl chloride, bis(2-oxo-3-oxo) sa Zolidinyl)phosphonic acid chloride, diphenylphosphinic acid chloride, diethyl chlorothiophosphate, (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 6-trifluoromethyl-1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, diphenylphosphorochloridate, bis(2,2,2-trichloroethyl)phosphorochloridate, ethyl dichlorophosphate, o-phenylenephosphorochloridate, 3-(diphenyl) Examples of suitable benzotriazin-4(3H)-one include (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, 1-adamantanecarbonyl chloride, and pivaloyl chloride, and preferably 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane. Examples of suitable condensing agents include 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide, 2-chloro-2-oxo-1,3,2-dioxaphosphorane, and propylphosphonic anhydride, and more preferred are 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide, 2-chloro-2-oxo-1,3,2-dioxaphosphorane, and propylphosphonic anhydride. The amount of the condensing agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by Formula (Rc-V) or Formula (Sc-V).

[0079] The reaction temperature with the condensing agent in this step is not limited as long as the reaction proceeds, but is preferably from −78° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 0° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 10 hours, more preferably 5 minutes to 2 hours.

[0080] The thionating agent used in this step is not particularly limited as long as it allows the reaction to proceed, but examples thereof include xanthan hydride, bis(phenylacetyl)disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione. Xanthan hydride, bis(phenylacetyl)disulfide, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione are preferred. The amount of the thionating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 1 to 2 equivalents, relative to the compound represented by Formula (Rc-V) or Formula (Sc-V).

[0081] The reaction temperature with the thionating agent in this step is not limited as long as the reaction proceeds, but is preferably from −40° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 10 hours, more preferably 5 minutes to 3 hours.

[0082] The oxidizing agent used in this step is not particularly limited as long as it allows the reaction to proceed, but examples thereof include iodine, bromine, tert-butyl hydroperoxide, 3-chloroperbenzoic acid, hydrogen peroxide, periodic acid, potassium permanganate, oxygen, and ozone, and preferably iodine, 3-chloroperbenzoic acid, hydrogen peroxide, and oxygen. The amount of the oxidizing agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by Formula (Rc-V) or Formula (Sc-V).

[0083] The reaction temperature with the oxidizing agent in this step is not limited as long as the reaction proceeds, but is preferably from −40° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 10 hours, more preferably 5 minutes to 3 hours.

[0084] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, and mixed solvents thereof, and more preferred examples include pyridine.

[0085] The absolute configuration of the compound of formula (VI) is represented by the absolute configuration of the asymmetric carbon atom on the prolinol skeleton in B2. In the compound of formula (VI) where Q is a thiol group, the absolute configurations of the two phosphorus atoms are expressed in the order of (absolute configuration of phosphorus at position 2, absolute configuration of phosphorus at position 10), as shown below. Together with the configurations of the asymmetric carbon atoms on the prolinol skeleton, they are expressed as (Rc,Rp,Rp), (Rc,Sp,Rp), (Rc,Rp,Sp), (Rc,Sp,Sp), (Sc,Rp,Rp), (Sc,Sp,Rp), (Sc,Rp,Sp), (Sc,Sp,Sp).

[0086] [ka]

[0087] Although the cyclization reaction in this step is not stereoselective, the production of one diastereomer proceeds predominantly based on the steric structure of the molecule itself. For example, as shown in Examples 3, 8, and A5-4 of the present specification, in the cyclization reaction from a compound of formula (Rc-V) to a compound of formula (Rc-VI), the (Rc,Rp,Rp-VI) isomer having the desired absolute configuration was obtained with a selectivity of about 70:30 to 85:15 relative to the (Rc,Sp,Rp-VI) isomer.

[0088] (Step a4) In one embodiment, this step is a step of deprotecting B2, which is a protecting group of the thiophosphate moiety in compound (Rc-VI), and PG4, which is a protecting group in A2, to obtain a compound of formula (Rp-VII). In another embodiment, this step is a step of obtaining a compound of formula (Sp-VII) from a compound of formula (Sc-VI) by a similar reaction. This step optionally includes a step of treating with an organic or inorganic base to convert it into a salt thereof.

[0089] The ammonia used in the deprotection reaction in this step is not limited as long as the reaction proceeds, but 28% aqueous ammonia is preferably used.

[0090] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from 30° C. to 65° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 1 to 96 hours, more preferably from 5 to 48 hours.

[0091] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, pyridine, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, ethanol, acetonitrile, dichloromethane, and pyridine, and more preferred examples include methanol and pyridine.

[0092] Examples of the organic base used for converting the compound of formula (Rp-VII) or formula (Sp-VII) into a salt thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable inorganic bases include methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene. Examples of suitable inorganic bases include potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate, and more preferred examples include triethylamine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate.

[0093] (New intermediate) The above [Synthetic Scheme <a1-rp>Examples of novel intermediates in the above process include compounds represented by the following formula (Rc-III):

[0094] [ka] Here, B1 is

[0095] [ka] wherein R1 is hydrogen or methyl, R2 is hydrogen, alkyl having 1 to 3 carbon atoms or phenyl, the alkyl being unsubstituted or substituted with one or more phenyl, tosyl or diphenylmethylsilyl, and the phenyl being unsubstituted or substituted with nitro or methoxy is preferred. More preferably, B1 is

[0096] [ka] and more preferably, B1 is

[0097] [ka] is. PG1 is preferably tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldimethylsilyl, more preferably tert-butyldimethylsilyl. PG2 is preferably 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl, more preferably 4,4'-dimethoxytrityl. PG3 is preferably 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl, and more preferably 2-(trimethylsilyl)ethoxycarbonyl.

[0098] In addition, the above [Synthetic Scheme <a1-rp>

[0033] Examples of novel intermediates in the above process include compounds represented by the following formula (Rc-V0):

[0099] [ka] wherein A2 is preferably

[0100] [ka] and more preferably,

[0101] [ka] is. PG4 is an amino-protecting group, preferably benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl, and more preferably benzoyl or 2-(trimethylsilyl)ethoxycarbonyl. PG1, PG2, and PG3 are the same as the groups in the compound of formula (Rc-III) shown above. B2 has the same meaning as B2 in the compound of formula (Rc-V) shown below. do.

[0102] Furthermore, the above [Synthetic Scheme <a1-rp>Examples of novel intermediates in the above process include compounds represented by the following formula (Rc-V):

[0103] [ka] Here, A2 has the same meaning as A2 in the compound of formula (Rc-V0) shown above. B2 is preferably

[0104] [ka] wherein R1 is hydrogen or methyl, R2 is hydrogen, alkyl having 1 to 3 carbon atoms or phenyl, the alkyl being unsubstituted or substituted with one or more phenyl, tosyl or diphenylmethylsilyl groups, and the phenyl being unsubstituted or substituted with nitro or methoxy groups. More preferably, B2 is

[0105] [ka] and more preferably, B2 is

[0106] [ka] is. PG6 is a protecting group for an amino group, and is preferably acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl, more preferably acetyl or trifluoroacetyl. PG1 is preferably tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl, more preferably tert-butyldimethylsilyl. PG3 is preferably 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl, and more preferably 2-(trimethylsilyl)ethoxycarbonyl.

[0107] In addition, in Step a4, a compound of Formula (Rp-VII) in which Q is a thiol group is treated with silica gel column chromatography before being converted into a salt thereof, and / or is crystallized after being converted into a salt thereof to obtain a compound of Formula (Rp, Rp-VII′):

[0108] [ka] or a salt thereof.

[0109] The compounds of formula (Rp-VII) or (Sp-VII), particularly the compounds of formula (Rp, Rp-VII'), obtained by the production method of the present invention can be preferably used for the CDN-linker of formula (Rp, Rp-XII) (hereinafter also referred to as a CDN conjugate precursor) and for antibody-immunostimulant conjugates in which an antibody is further bound to the CDN conjugate precursor. Furthermore, the compounds of formula (Rp-VII) or (Sp-VII) obtained by the production method of the present invention can also be used for the production of antibody-immunostimulant conjugates having other chemical structures, or for other applications, without being limited thereto.

[0110] <1-2. Comparison of the Production Method of Compound (Rp-VII) of the Present Invention with Conventional Methods (Patent Document 6, etc.)> In the conventional method, cyclic dinucleotides are synthesized in approximately equal proportions of four dinucleotides. Te However, because a mixture of dimers is produced, it is necessary to obtain a cyclic dinucleotide having the desired configuration by column purification, resulting in a very low yield. The method for producing the compound of formula (Rp-VII) of the present invention makes it possible to selectively synthesize the desired configuration, and as a result, it is possible to provide a production method that has excellent effects, such as an improved total yield and the elimination of column purification, compared to the synthesis scheme of Patent Document 6 shown below.

[0111] [Conventional synthesis scheme (from the synthesis scheme in Example 77 of Patent Document 6)]

[0112] [ka]

[0113] [Advantages of the production method of formula (Rp-VII) of the present invention] In the conventional synthesis route (Patent Document 6), the configuration of phosphorus is not controlled by the reaction, but by four types of diastereoisomers. Te The synthesis involved a mixture of mers, and then finally obtaining the desired conformation through column purification, resulting in an extremely low yield (total yield of 7.5% for the eight reaction steps (it was stated that impurities were included)). On the other hand, the production method of the present invention makes it possible to stereoselectively form the phosphorothioate bond in the first step, resulting in an improvement in the overall yield from the compound of formula (I) by approximately 20 to 40%, and the quality was also high, ranging from 92.4 to >99% by HPLC analysis.

[0114] In addition, the above synthesis scheme <a1-rp>or <a1-sp>In (step a1), the compound of formula (I) is the compound of formula (I-1) or (I-2):

[0115] [ka] [In the formula, A3 is a substituted or unsubstituted deazapurine group, a substituted or unsubstituted purine base, or a substituted or unsubstituted pyrimidine base; and Each Y is independently H, F, or —O-PG1, where PG1 is a protecting group for a hydroxy group. A compound represented by the formula: In (step a2), the formula (IV) is represented by the following formula (IV-1) or formula (IV-2):

[0116] [ka] [In the formula, A4 is a substituted or unsubstituted deazapurine group, a substituted or unsubstituted purine base, or a substituted or unsubstituted pyrimidine base; and Each Z is independently H, F, or -O-PG1, where PG1 is a protecting group for a hydroxy group. By using the compound represented by the formula (Rp-VII) or a salt thereof, it is also possible to synthesize, depending on the combination, four types of cyclic dinucleotide derivatives having the following 12- to 14-membered ring structure corresponding to formula (Rp-VII), or four types of cyclic dinucleotides (not shown) having the following 12- to 14-membered ring structure corresponding to formula (Sp-VII).

[0117] [ka]

[0118] <1-3. Method for producing the compound of formula (IV)> The compound of formula (IV) or a salt thereof used in the production of the above cyclic dinucleotide can be produced according to the following synthesis scheme. [Synthetic Scheme <a2>]

[0119] [ka]

[0120] The above [Synthetic Scheme <a2>]In, A1 and A2 are each independently selected from the group consisting of the compounds shown in the above [Synthetic Scheme] <a1>] is synonymous with PG5 is a protecting group for the hydroxy group.

[0121] In this production method, examples of PG5 include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, and trityl, and preferably 4,4'-dimethoxytrityl or 4-methoxytrityl.

[0122] Each step will be explained below. (Step a5) and (Step a5') In this step, a compound of formula (VIII) or formula (VIII') is reacted with a phosphite to obtain a compound of formula (IX) or formula (IX') or a salt thereof.

[0123] The phosphite ester used in this step may include diphenyl phosphite, methyl phosphite, diethyl phosphite, or dibutyl phosphite, preferably diphenyl phosphite or methyl phosphite, more preferably diphenyl phosphite. Appointment The amount of the phosphite used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 20 equivalents, more preferably 1 to 10 equivalents, relative to the compound represented by formula (VIII) or formula (VIII').

[0124] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −10° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 120 hours, more preferably 5 minutes to 72 hours.

[0125] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane. More preferred examples include pyridine and acetonitrile.

[0126] Reagents used for converting the compound of formula (IX) or formula (IX') into a salt thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, propylamine, isopropyl Pi Examples of suitable amines include amine, butylamine, isobutylamine, tert-butylamine, pentylamine, aniline, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium chloride, and potassium chloride. Preferred are triethylamine, tributylamine, isopropylamine, Pi Examples of the base include triethylamine, butylamine, isobutylamine, tert-butylamine, potassium carbonate, potassium hydrogencarbonate, sodium carbonate, sodium hydrogencarbonate, sodium chloride, and potassium chloride. More preferred examples include triethylamine, tert-butylamine, potassium carbonate, potassium hydrogencarbonate, sodium carbonate, sodium hydrogencarbonate, sodium chloride, and potassium chloride. The amount of base used for conversion to a salt is not limited as long as the reaction proceeds, but is preferably a base represented by the formula (IX) or formula (IX') The amount is preferably 0.5 to 10 equivalents, more preferably 3 to 8 equivalents, relative to the compound represented by the formula:

[0127] (Step a5'') In this step, a compound of formula (IX') or a salt thereof is reacted with an acylating agent or an alkoxycarbonylating agent. Agent to obtain a compound of formula (IX) or a salt thereof.

[0128] The acylating agent or alkoxycarbonylating agent used in this step is not particularly limited as long as it allows the reaction to proceed, and examples thereof include acetic anhydride, acetyl chloride, N-succinimidyl acetate, pentafluorophenyl acetate, 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, N-methoxydiacetamide, N-acetylimidazole, trifluoroacetic anhydride, bistrifluoroacetamide, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, S-ethyl trifluorothioacetate, N-methylbistrifluoroacetamide, trifluoroacetyl triflate, 1-trifluoroacetylimidazole, benzoic anhydride, benzoyl chloride, and pentafluorophenyl benzoate. 、 Benzoyl trifluoromethanesulfonate, 3-benzoylthiazolidine-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butyl carbazate, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole 、 Di-tert-butyl dicarbonate, 9-fluorenylmethyl pentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl carbazate, 19-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, N-(2 ,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxy Examples of the chloroformate include methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, methyl imidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, and methyl imidazole-1-carboxylate. Preferred examples include acetic anhydride, 1-trifluoroacetylimidazole, benzoic anhydride, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(allyloxycarbonyloxy) ) succinimide, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, and more preferred examples include 1-trifluoroacetylimidazole, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide.The amount of the acylating agent or alkoxycarbonylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 15 equivalents, more preferably 3 to 8 equivalents, relative to the compound represented by formula (IX').

[0129] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, and more preferred bases include triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 20 equivalents, more preferably 4 to 10 equivalents, relative to the compound represented by formula (IX').

[0130] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 40° C. to 80° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1 hour to 96 hours, more preferably 24 hours to 72 hours.

[0131] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 4-methylpyridine, 2-methyltetrahydrofuran, acetonitrile, and 1-methyl-2-pyrrolidone. More preferred examples include 2-methyltetrahydrofuran and acetonitrile.

[0132] The reagents and amounts used in converting the obtained compound of formula (IX) into its salt can be the same as those used in step a5 above.

[0133] (Step a6) This step is a step of deprotecting PG5, which is the protecting group for the 5' hydroxyl group of the compound of formula (IX), to obtain the compound of formula (IV).

[0134] Examples of the acid used in the deprotection reaction in this step include acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, methanesulfonic acid, toluenesulfonic acid, and sulfuric acid, and preferred examples include acetic acid, chloroacetic acid, dichloroacetic acid, and trichloroacetic acid, and more preferred example is dichloroacetic acid. The amount of the acid used in this step is not limited as long as the reaction proceeds, but is preferably 0.05 to 500 equivalents, more preferably 0.1 to 200 equivalents, relative to the compound represented by formula (IX).

[0135] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1 hour to 96 hours, more preferably 5 hours to 48 hours.

[0136] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, acetonitrile, and dichloromethane, and more preferred examples include dichloromethane.

[0137] <1-4. Method for producing CDN-linker (compound (Rp, Rp-XII))> The compound of formula (Rp,Rp-XII) or a salt thereof can be prepared according to the following synthetic scheme: The compound of formula (Rp,Rp-XII) is a precursor for preparing an antibody-immunostimulant conjugate. [Synthetic Scheme <a3>]

[0138] [ka]

[0139] The above [Synthetic Scheme <a3>]In, PG1, PG3, and A1 are synthesized as described above in the synthetic scheme <a1-rp>] is synonymous with.

[0140] Each step will be explained below. (Step a7) This step is a step of deprotecting the protecting groups PG1 and PG3 of the compound of formula (Rp, Rp-VII') to obtain a compound of formula (Rp, Rp-X).

[0141] Examples of the deprotecting agent used in this step include ammonium fluoride, tetra-n-butylammonium fluoride, pyridine hydrogen fluoride, and triethylamine trihydrofluoride, and preferred examples include ammonium fluoride and tetra-n-butylammonium fluoride, and more preferred examples include tetra-n-butylammonium fluoride. The amount of the deprotecting agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 60 equivalents, more preferably 1 to 30 equivalents, relative to the compound represented by formula (Rp, Rp-VII').

[0142] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from 10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 30 minutes to 240 hours, more preferably from 1 hour to 120 hours.

[0143] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples of the solvent include methanol, acetonitrile, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide, and more preferred examples of the solvent include tetrahydrofuran and dimethyl sulfoxide.

[0144] (Step a8) This step is a step of condensing a compound of formula (Rp,Rp-X) with a compound of formula (XI) to obtain a compound of formula (Rp,Rp-XII) or a salt thereof.

[0145] The compound represented by formula (XI) can be condensed with the compound represented by formula (Rp,Rp-X), preferably by converting it into an activated ester. The amount of the compound represented by formula (XI) used in this step is not limited as long as the reaction proceeds, but is preferably 0.3 to 3 equivalents, more preferably 0.7 to 1.3 equivalents, relative to the compound represented by formula (Rp,Rp-X).

[0146] The method for derivatizing the activated ester in this step is not limited as long as the reaction proceeds. For example, the method can be carried out using a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD HCl) or N,N'-dicyclohexylcarbodiimide (DCC) with an additive such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, ethyl cyanohydroxyiminoacetate, or p-nitrophenol. Alternatively, the method can be carried out using a condensing agent such as 1-[bis(dimethylamino)methylene ]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), or (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), or other condensing agents. Preferably, this reaction can be carried out using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). The amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 0.7 to 2 equivalents, relative to the compound represented by formula (Rp,Rp-X). The amount of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole used in this step is not limited as long as the reaction proceeds, but is preferably 0.05 to 4 equivalents, more preferably 0.1 to 2 equivalents, relative to the compound represented by formula (Rp, Rp-X).The amount of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride used in this step is not limited as long as the reaction proceeds, but is preferably 0.3 to 5 equivalents, more preferably 0.7 to 2 equivalents, relative to the compound represented by formula (Rp,Rp-X).

[0147] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and sodium acetate, and more preferred bases are triethylamine and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (Rp,Rp-X).

[0148] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from −10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 72 hours, more preferably from 1 hour to 24 hours.

[0149] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include water, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and dimethyl sulfoxide, as well as mixed solvents thereof.

[0150] Examples of the base used for converting the compound of formula (Rp, Rp-XII) into its salt include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Methylpyridine, 4-dimethylaminopyridine, propylamine, isopropyl Pi Examples of suitable bases include organic bases such as butylamine, isobutylamine, tert-butylamine, pentylamine, and aniline, and inorganic bases such as potassium 2-ethylhexanoate, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium 2-ethylhexanoate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium chloride, and potassium chloride. Preferred bases include triethylamine, tert-butylamine, potassium 2-ethylhexanoate, potassium carbonate, potassium bicarbonate, sodium 2-ethylhexanoate, sodium carbonate, sodium bicarbonate, sodium chloride, and potassium chloride. More preferred bases include triethylamine, potassium 2-ethylhexanoate, and sodium carbonate.

[0151] Examples of a solvent that can be used in converting a compound of Formula (Rp,Rp-XII) into a salt thereof include water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof. Preferred examples of the solvent include water, 2-propanol, acetonitrile, cyclopentyl methyl ether, and ethyl acetate, as well as mixed solvents thereof.

[0152] <2. Production of starting compound (I) used in the production of cyclic dinucleotide> <2-1. Method for producing the compound of formula (I)> The compound of formula (I) used in the production of the above cyclic dinucleotide can be produced according to the following synthesis scheme. [Synthetic Scheme <b1>]

[0153] [ka]

[0154] The above [Synthetic Scheme <b1>]In, PG1, PG2, and PG3 can be synthesized by the synthesis scheme described above. <a1-rp>] is synonymous with X is Cl, Br, or I.

[0155] In this manufacturing method, PG1 represents a protecting group for a hydroxy group, and examples of the protecting group include tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, and tert-butyldiphenylsilyl. Preferred are tert-butyldimethylsilyl and trimethylsilyl. More preferred is tert-butyldimethylsilyl.

[0156] PG2 represents a protecting group for a hydroxy group, and examples of the protecting group include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, and trityl. Preferred are 4,4'-dimethoxytrityl, 4-methoxytrityl, and trityl. More preferred is 4,4'-dimethoxytrityl.

[0157] PG3 represents an amino-protecting group, and examples of the protecting group include 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, and benzyloxycarbonyl. 2-(trimethylsilyl)ethoxycarbonyl and allyloxycarbonyl are preferred. 2-(trimethylsilyl)ethoxycarbonyl is more preferred.

[0158] Each step will be explained below. (Step b1) In this step, a compound of formula (XIV) is reacted with a compound of formula (XV) to obtain a compound of formula (XVI).

[0159] The reaction of this step is carried out in the presence of a base, and the base may be 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo[5.4.0]-7-undecene, preferably 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo[5.4.0]-7-undecene, more preferably 1,1,3,3-tetramethylguanidine. The amount of the base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XIV).

[0160] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 10° C. to the boiling point of the solvent used in the reaction, more preferably from 20° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 30 minutes to 72 hours, more preferably from 5 hours to 36 hours.

[0161] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include 1,3-dimethyl-2-imidazolidinone.

[0162] (Step b2) In this step, a compound of formula (XVI) is reacted with a silylating agent to obtain a mixture of a compound of formula (I') and a compound of formula (XVII):

[0163] [ka] and then converting the compound of formula (XVII) in the mixture to a compound of formula (I') in the presence of a base to obtain a compound of formula (I').

[0164] This process also includes a step of reacting the compound of formula (XVI) with a silylating agent in the presence of a first base to obtain a mixture of the compound of formula (I') and the compound of formula (XVII), and then converting the compound of formula (XVII) in the mixture to the compound of formula (I') in the presence of a second base to obtain the compound of formula (I').

[0165] The conversion of the compound of formula (XVII) to the compound of formula (I') is carried out by crystallizing the compound of formula (I') from the solution of the mixture, utilizing the equilibrium reaction in the solution of the mixture and the difference in solubility between the compound of formula (XVII) and the compound of formula (I').

[0166] Examples of the first base include 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and preferred examples include 1,1,3,3-tetramethylguanidine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferred examples include 1,1,3,3-tetramethylguanidine. The amount of the first base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by formula (XVI).

[0167] The reaction temperature for the reaction using the first base in this step is not limited as long as the reaction proceeds, but is preferably from 10° C. to the boiling point of the solvent used in the reaction, more preferably 40° C. to 70° C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 30 minutes to 72 hours, more preferably 5 hours to 36 hours.

[0168] The solvent for the reaction using the first base in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include 1,3-dimethyl-2-imidazolidinone.

[0169] Examples of the silylating agent used in this step include tert-butyldimethylchlorosilane and tert-butyldimethylsilyl triflate, and preferably tert-butyldimethylchlorosilane. The amount of the silylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 2 to 4 equivalents, relative to the compound represented by formula (XIV).

[0170] Examples of the second base include 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and preferred examples include 1,1,3,3-tetramethylguanidine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferred examples include 1,1,3,3-tetramethylguanidine. The amount of the second base used in this step is not limited as long as the reaction proceeds, but is preferably 0.01 to 3 equivalents, and more preferably 0.05 to 1 equivalent, relative to the compound represented by formula (XIV).

[0171] The reaction temperature for the reaction using the second base in this step is not limited as long as the reaction proceeds, but is preferably from 10° C. to the boiling point of the solvent used in the reaction, more preferably from 10° C. to 40° C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 10 hours, more preferably from 5 minutes to 90 minutes.

[0172] The first solvent for the reaction using the second base in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include tetrahydrofuran.

[0173] The second solvent for the reaction using the second base in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, heptane, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include heptane.

[0174] <2-2. Method for producing the compound of formula (XV) and intermediates therefor> The compound of formula (XV) used in the production of the above formula (I) can be produced according to the following synthesis scheme. [Synthetic Scheme <b2>]

[0175] [ka]

[0176] The above [Synthetic Scheme <b2>]In, PG3 can be synthesized by the synthesis scheme <a1>] is synonymous with X is the same as in the above [Synthetic Scheme <b1>] is synonymous with.

[0177] Step b3 will be described below. (Step b3) In this step, a compound of formula (XVIII) is reacted with 2-haloethanol in the presence of an acid or a base to obtain a compound of formula (XV).

[0178] The base used in this step includes sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene, preferably sodium hydroxide, potassium hydroxide, triethylamine, and diisopropylethylamine, and more preferably sodium hydroxide. 。 The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 equivalents relative to glycylglycine, the starting material for compound (XVIII). Compound (XVIII) can be obtained from glycylglycine by protecting the amino group and converting the carboxy group to an acetyloxy group.

[0179] The 2-haloethanols used in this step include 2-chloroethanol, 2-bromoethanol, and 2 The amount of 2-haloethanol used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 4 equivalents, relative to the amount of glycylglycine, the starting material for compound (XVIII).

[0180] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −10° C. to 20° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 36 hours, more preferably 1 hour to 10 hours.

[0181] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include 1,2-dimethoxyethane, 2-methyltetrahydrofuran, and 1,4-dioxane. More preferred examples include 1,2-dimethoxyethane.

[0182] (New intermediate) The above synthesis [Scheme <b1>A novel intermediate in the above process may be a compound represented by the following formula (XV):

[0183] [ka] where PG3 is 2-(trimethylsilyl)ethoxycarbonyl, and X is preferably Cl, Br, or I, more preferably Br.

[0184] <2-3. Comparison of the production method of compound (I) of the present invention with conventional methods (Patent Document 6, etc.)> The method for producing the compound of formula (I) of the present invention makes it possible to synthesize a compound in which the 3'-hydroxyl group is selectively tert-butyldimethylsilylated, among the 2'- and 3'-hydroxyl groups, which was a problem in conventional methods. As a result, it is possible to provide a production method that has excellent effects such as an improved total yield, a reduced number of steps, and the elimination of column purification, compared to the synthesis scheme of Patent Document 6 shown below.

[0185] [Conventional synthesis scheme (from the synthesis scheme in Example 77 of Patent Document 6)]

[0186] [ka]

[0187] [Advantages of the production method of formula (I) of the present invention]

[0188] (Effect 1) Synthesis of the 3'-hydroxyl group selectively tert-butyldimethylsilylated derivative (I) among the 2'- and 3'-hydroxyl groups In the conventional synthesis route (Patent Document 6), the tert-butyldimethylsilylation step (Step 5) fails to selectively tert-butyldimethylsilylate the 3'-position of the hydroxyl groups at the 2'- and 3'-positions, resulting in the generation of a by-product in which the 2'-position is tert-butyldimethylsilylated. This resulted in a low yield of approximately 35% of the target compound of formula (I). Furthermore, a method for selectively silylating the hydroxyl groups at the 2'- and 3'-positions using a chiral imidazole to selectively triethylsilylate the 3'-position is known (see Org. Lett., Vol. 15, No. 18, 2013, pp. 4710-4713). However, this method has the drawback of being a special chiral imidazole that is not readily available. Furthermore, selective tert-butyldimethylsilylation using a silver reagent is also known (see Tetrahedron Lett., Vol. 22, No. 52, 1981, pp. 5423-5246), but this is undesirable because it requires the use of an expensive silver reagent in an amount equal to or greater than the amount of the reaction substrate equivalent. Furthermore, although this method was carried out under similar conditions using this substrate, the desired selectivity could not be obtained.

[0189] On the other hand, in the production method of the present invention, conditions were found under which TBS groups were transferred between the 2'- and 3'-positions after non-selective introduction of TBS groups into the 2'- and 3'-hydroxyl groups, and conditions were also found under which only the target compound represented by formula (I) in which the 3'-hydroxyl group was modified with TBS groups was crystallized, making it possible to preferentially obtain the target compound of formula (I).

[0190] (Effect 2) Reduced number of steps, improved yield, avoided column purification In the conventional synthesis route (Patent Document 6), an extra deprotection step was required during N-alkylation of the amide group of a nucleic acid base because the hydroxyl groups at the 2'- and 3'-positions of the reaction substrate were protected.

[0191] On the other hand, in the production method of the present invention, an alkylating agent (compound of formula (XV)) that allows N-alkylation of the amide group of a nucleic acid base to proceed while the hydroxyl groups at the 2'- and 3'-positions remain unprotected has been found, which eliminates the need for steps of protecting and deprotecting the hydroxyl groups at the 2'- and 3'-positions, making it possible to provide a production method that is shorter by two steps.

[0192] (Effect 3) Improved yield throughout the entire scheme, avoiding column purification In the conventional synthesis route (Patent Document 6), the total yield from the starting material 2',3',5'-tris-O-[tert-butyl(dimethyl)silyl]inosine to the compound of formula (I) was approximately 13% (5 steps), whereas the production method of the present invention makes it possible to synthesize the compound of formula (I) from the starting material inosine in a yield of approximately 60% (3 steps). Furthermore, in the conventional synthesis route (Patent Document 6), the product was obtained through purification by silica gel column chromatography in every step, which increases waste (silica gel, hexane, eluent such as ethyl acetate, etc.) and lengthens lead time. However, the production method of the present invention avoids silica gel column chromatography, making it possible to reduce environmental impact and improve productivity.

[0193] <3. Production of starting compound (VIII) and compound of formula (VIII') used in the production of cyclic dinucleotide> <3-1. Method for producing compounds of formula (VIII) and formula (VIII') and intermediates thereof> The compounds of formula (VIII) and formula (VIII') used in the production of the above cyclic dinucleotide can be produced according to the following synthesis scheme. [Synthetic Scheme <c1>]

[0194] [ka]

[0195] The above [Synthetic Scheme <c1>]In, PG4 is benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl, and PG7 is trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or triphenylsilyl.

[0196] Each step will be explained below. (Step c1) In this step, a compound of formula (XIX) is reacted with a benzoylating agent to obtain a compound of formula (XX).

[0197] The benzoylating agent used in this step includes benzoyl chloride, benzoyl bromide, benzoic anhydride, benzoyl trifluoromethanesulfonate, etc., preferably benzoyl chloride or benzoyl bromide, more preferably benzoyl chloride. The amount of the benzoylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 1 to 2 equivalents, relative to the compound represented by formula (XIX).

[0198] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, and the like; and bases such as potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide, and mixtures thereof. Preferred bases include triethylamine, 2,6- J Examples of the base include methylpyridine, 4-dimethylaminopyridine, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, and sodium acetate, as well as mixtures thereof, and more preferably a mixture of triethylamine and 4-dimethylaminopyridine. The amount of the base used in this step is not limited as long as the reaction proceeds, but is preferably 0.1 to 10 equivalents, more preferably 0.2 to 5 equivalents, relative to the compound represented by formula (XIX).

[0199] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −40° C. to the boiling point of the solvent used in the reaction, more preferably −10° C. to 35° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 0.5 to 10 hours.

[0200] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, acetonitrile or N,N-dimethylformamide can be used, and more preferably, acetonitrile can be used.

[0201] (Step c2) In this step, a compound of formula (XX) is hydrolyzed to obtain a compound of formula (XXI).

[0202] Examples of the acid used in this step include hydrochloric acid, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and sulfuric acid, and preferred examples include hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, and sulfuric acid, and more preferred examples include p-toluenesulfonic acid. The amount of the acid used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 20 equivalents, and more preferably 1 to 10 equivalents, relative to the compound represented by formula (XX).

[0203] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably 40° C. to 80° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0204] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof, are usable. Preferred are 1,2-dimethoxyethane, 2-methyltetrahydrofuran, tetrahydrofuran, cyclopentyl methyl ether, and mixed solvents thereof, and more preferred is a mixed solvent of cyclopentyl methyl ether and water.

[0205] (Step c3) In this step, a compound of formula (XXI) is reacted with a chlorinating agent to obtain a compound of formula (XXII).

[0206] Examples of the chlorinating agent used in this step include trichloroisocyanuric acid, chloroisocyanuric acid, dichloroisocyanuric acid, N-chlorosuccinimide, 1,3-dichloro-5,5-dimethylhydantoin, N-chlorosaccharin, N-N-dichloro-p-toluenesulfonamide, N-N-dichlorobenzenesulfonamide, and carbon tetrachloride, and preferred examples include trichloroisocyanuric acid, chloroisocyanuric acid, and dichloroisocyanuric acid, and more preferred examples include trichloroisocyanuric acid. The amount of the chlorinating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.1 to 6 equivalents, and more preferably 0.3 to 3 equivalents, relative to the compound represented by formula (XXI).

[0207] The reaction of this step can be preferably carried out in the presence of a phosphorus reagent. The phosphorus reagent used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include tris(2,4-di-tert-butylphenyl)phosphite, tri-o-tolylphosphite, and the like. R Examples of suitable phosphine include tris(2,4-di-tert-butylphenyl)phosphine, triphenylphosphine, triethylphosphine, tributylphosphine, triphenylphosphine, tris(dimethylamino)phosphine, and tris(diethylamino)phosphine. o -to R The amount of the phosphorus reagent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXI).

[0208] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −80° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0209] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof can be used. Preferred examples include dichloromethane, 1,2-dimethoxyethane, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, and mixed solvents thereof. More preferred examples include dichloromethane, cyclopentyl methyl ether, and mixed solvents thereof.

[0210] (Step c4) In this step, a compound of formula (XXII) is reacted with a compound of formula (XXIII) to obtain a compound of formula (XXIV).

[0211] The reaction of this step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as cesium carbonate, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide, and mixtures thereof. Preferred examples include cesium carbonate, potassium carbonate, and sodium carbonate, and more preferred examples include cesium carbonate. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by Formula (XXII).

[0212] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 10° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0213] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide. More preferred examples include dimethyl sulfoxide.

[0214] (Step c5) This step is a step of deprotecting the benzoyl group from the compound of formula (XXIV) to obtain a compound of formula (XXV) or a salt thereof.

[0215] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene; bases such as cesium carbonate, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide; and mixtures thereof. Preferred examples include 1,8-diazabicyclo[5.4.0]undec-7-ene, sodium methoxide, sodium ethoxide, potassium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide. More preferred examples include sodium hydroxide, sodium methoxide, and sodium ethoxide. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.001 to 10 equivalents, more preferably 0.01 to 5 equivalents, relative to the compound represented by formula (XXIV).

[0216] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0217] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred are water, methanol, ethanol, tetrahydrofuran, and mixed solvents thereof, and more preferred are ethanol or a mixed solvent of water and tetrahydrofuran.

[0218] Examples of acids used in converting the compound of formula (XXV) into its salt include formic acid, acetic acid, propionic acid, oxalic acid, maleic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid. Preferred examples include acetic acid, p-toluenesulfonic acid, and hydrochloric acid. More preferred examples include p-toluenesulfonic acid and hydrochloric acid. The amount of acid used for conversion into the salt is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, and more preferably 1 to 3 equivalents, relative to the compound of formula (XXIV).

[0219] (Step c6) In this step, a compound of formula (XXV) or a salt thereof is reacted with a tritylation agent to obtain a compound of formula (VIII') or a salt thereof.

[0220] Examples of the tritylation agent used in this step include 4,4-dimethoxytrityl chloride, 4-methoxytrityl chloride, 2-chlorotrityl chloride, and trityl chloride, and preferred examples include 4,4-dimethoxytrityl chloride and 4-methoxytrityl chloride. The amount of the tritylation agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, and more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXV).

[0221] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 36 hours.

[0222] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane, and more preferred examples include pyridine.

[0223] (Step c7) In this step, a compound of formula (VIII') or a salt thereof is reacted with a silylating agent, and then with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (XXVII).

[0224] Examples of the silylating agent used in this step include trimethylchlorosilane, trimethylsilyl triflate, triethylchlorosilane, triethylsilyl triflate, triisopropylchlorosilane, triisopropylsilyl triflate, tert-butyldimethylchlorosilane, tert-butyldimethylsilyl triflate, tert-butyldiphenylchlorosilane, tert-butyldiphenylsilyl triflate, triphenylchlorosilane, and triphenylsilyl triflate, and preferred is tert-butyldimethylsilyl triflate.

[0225] Examples of the acylating agent or alkoxycarbonylating agent used in this step include a benzoylating agent, a 2-(trimethylsilyl)ethoxycarbonylating agent, a tert-butoxycarbonylating agent, a 9-fluorenylmethyloxycarbonylating agent, an allyloxycarbonylating agent, a 2,2,2-trichloroethoxycarbonylating agent, or a benzyloxycarbonylating agent, and preferred examples include a benzoylating agent and a 2-(trimethylsilyl)ethoxycarbonylating agent. The amount of the acylating agent or alkoxycarbonylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (VIII') or a salt thereof.

[0226] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0227] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane, and more preferred examples include pyridine.

[0228] (Step c8) In this step, a compound represented by formula (XXVII) of This is the step of deprotecting the protecting group PG7 to obtain the compound of formula (VIII).

[0229] Examples of the deprotecting agent used in this step include ammonium fluoride, tetra-n-butylammonium fluoride, pyridine hydrogen fluoride, and triethylamine trihydrofluoride, and preferably ammonium fluoride and tetra-n-butylammonium fluoride, and more preferably tetra-n-butylammonium fluoride. The amount of the deprotecting agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.1 to 5 equivalents, more preferably 0.2 to 2 equivalents, relative to the compound represented by formula (XXVII).

[0230] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0231] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, acetonitrile, tetrahydrofuran, and dichloromethane, and more preferred examples include tetrahydrofuran.

[0232] (New intermediate) The above [Synthetic Scheme <c1>Examples of novel intermediates in the above process include compounds represented by the following formula (XX):

[0233] [ka]

[0234] The above [Synthetic Scheme <c1>As a novel intermediate in the above process, a compound represented by the following formula (XXII) can be mentioned.

[0235] [ka]

[0236] The above [Synthetic Scheme <c1>As a novel intermediate in the above process, a compound represented by the following formula (XXIV) can be mentioned.

[0237] [ka]

[0238] The above [Synthetic Scheme <c1>Examples of novel intermediates in the above process include a compound represented by the following formula (XXV) or a salt thereof:

[0239] [ka]

[0240] The above [Synthetic Scheme <c1>Examples of novel intermediates in the above process include a compound represented by the following formula (VIII'-1) or a salt thereof:

[0241] [ka] Here, PG5 is preferably 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl.

[0242] The above [Synthetic Scheme <c1>As a novel intermediate in the above process, a compound represented by the following formula (XXVII) can be mentioned.

[0243] [ka] Here, PG4 is preferably benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl or benzyloxycarbonyl, more preferably benzoyl or 2-(trimethylsilyl)ethoxycarbonyl. PG5 is preferably 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl, more preferably 4,4'-dimethoxytrityl or 4-methoxytrityl. PG7 is preferably hydroxy(oxo)-λ 5 -phosphanyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl or triphenylsilyl, more preferably tert-butyldimethylsilyl. <3-2. Method for producing the compound of formula (XXIII) and intermediates thereof> The compound of formula (XXIII) used in the production of the compound of formula (VIII) above can be produced according to the following synthesis scheme. [Synthetic Scheme <c2>]

[0244] [ka]

[0245] Each step will be explained below. (Step c9) In this step, the tert-butoxycarbonyl group is deprotected from the compound of formula (XXVIII) to obtain the compound of formula (XXIX).

[0246] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases include potassium hydroxide, potassium bicarbonate, sodium carbonate, and sodium hydroxide, and more preferred bases are sodium hydroxide. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, and more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXVIII).

[0247] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, and more preferably 1 hour to 10 hours.

[0248] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, ethanol, 1-propanol, and tetrahydrofuran, and more preferred examples include ethanol.

[0249] (Step c10) In this step, a compound of formula (XXIX) is subjected to an alkyne reduction reaction in the presence of a catalyst, followed by a reductive amination reaction, to obtain a compound of formula (XXX).

[0250] A metal catalyst can be used as a catalyst for the reduction reaction of the alkyne in this step. The metal catalyst used in this step is not particularly limited as long as it catalyzes hydrogenation; however, preferred examples include ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, and nickel catalysts, and more preferred examples include palladium catalysts. The amount of metal catalyst used in this step is not limited as long as the reaction proceeds; however, it is preferably 0.01 to 1 times, more preferably 0.02 to 0.4 times, and even more preferably 0.05 to 0.2 times the mass of the compound represented by formula (XXVIII). The hydrogen gas pressure is usually 100 to 1,000 kPa, preferably 100 to 700 kPa, and more preferably 200 to 500 kPa. The reaction temperature in this step is not limited as long as the reaction proceeds; however, it is preferably from 0°C to the boiling point of the solvent used in the reaction, more preferably 20°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, and more preferably 1 hour to 24 hours.

[0251] The solvent used in the reduction reaction of the alkyne in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and mixed solvents thereof, and more preferred examples include 1-methyl-2-pyrrolidone.

[0252] A metal catalyst can be used as a catalyst for the reductive amination reaction in this step. The metal catalyst used in this step is not particularly limited as long as it catalyzes hydrogenation; however, preferred examples include ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, and nickel catalysts, and more preferred examples include palladium catalysts. The amount of metal catalyst used in this step is not limited as long as the reaction proceeds; however, it is preferably 0.01 to 1 times, more preferably 0.02 to 0.4 times, and even more preferably 0.05 to 0.2 times the mass of the compound represented by formula (XXVIII). The hydrogen gas pressure is usually 100 to 1,000 kPa, preferably 100 to 700 kPa, and more preferably 200 to 500 kPa. The reaction temperature in this step is not limited as long as the reaction proceeds; however, it is preferably from 0°C to the boiling point of the solvent used in the reaction, more preferably 20°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, and more preferably 1 hour to 24 hours.

[0253] The solvent used in the reductive amination reaction of this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, water, acetic acid, and mixed solvents thereof. More preferred examples include a mixed solvent of 1-methyl-2-pyrrolidone, water, and acetic acid.

[0254] (Step c11) In this step, a compound of formula (XXX) is reacted with a benzoylating agent, followed by debenzoylation by treatment with a base to obtain a compound of formula (XXIII).

[0255] The benzoylating agent used in this step includes benzoyl chloride, benzoyl bromide, benzoic anhydride, benzoyl trifluoromethanesulfonate, etc., and preferably benzoyl chloride. Zo The amount of the ylating agent is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXX).

[0256] The reaction with the benzoylating agent in this step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, and mixed bases thereof. Preferred examples include triethylamine, diisopropylethylamine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, and mixed bases thereof. More preferred examples include a mixed base of triethylamine, diisopropylethylamine, and 4-dimethylaminopyridine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXVIII).

[0257] The reaction temperature with the benzoylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0258] The solvent used in the reaction with the benzoylating agent in this step is not particularly limited as long as it does not inhibit the reaction. Examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chloro Benzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used, and preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include 1,3-dimethyl-2-imidazolidinone.

[0259] The base used in the base treatment in this step is not particularly limited as long as the reaction proceeds. Examples of the base include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases include potassium hydroxide, potassium bicarbonate, sodium carbonate, and sodium hydroxide, and more preferred is triethylamine. The amount of base used in the base treatment in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by Formula (XXVIII).

[0260] The reaction temperature for the base treatment in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0261] The solvent used in the base treatment in this step is not particularly limited as long as it does not inhibit the reaction, and examples thereof include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, and acetone. , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. More preferred examples include a mixed solvent of methanol and 1,3-dimethyl-2-imidazolidinone.

[0262] The above [Synthetic Scheme <c2>An example of an intermediate in the above process is a compound represented by the following formula (XXVIII).

[0263] [ka]

[0264] <3-3. Method for producing the compound of formula (XXVIII)> The compound of formula (XXVIII) used in the production of the compound of formula (XXIII) above can be produced according to the following synthesis scheme. [Synthetic Scheme <c3>]

[0265] [ka]

[0266] Each step will be explained below. (Step c12) In this step, a compound of formula (XXXI) is reacted with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain a compound of formula (XXXII).

[0267] Examples of the tert-butoxycarbonylating agent used in this step include di-tert-butyl dicarbonate, N-tert-butoxycarbonylimidazole, N-tert-butoxycarbonyl-1,2,4-triazole, N-(tert-butoxycarbonyloxy)phthalimide, etc., and preferably di-tert-butyl dicarbonate. The amount of the tert-butoxycarbonylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXXI).

[0268] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0269] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples of the solvent include acetonitrile, tetrahydrofuran, ethyl acetate, benzene, and toluene, and more preferred examples are toluene.

[0270] (Step c13) In this step, a compound of formula (XXXII) is reacted with propargylaldehyde diethyl acetal to obtain a compound of formula (XXVIII).

[0271] This step is preferably carried out in the presence of a transition metal catalyst, and more preferably in the presence of a palladium catalyst. The palladium catalyst used in this step is not particularly limited as long as it allows the reaction to proceed. For example, divalent palladium salts and complexes thereof, such as palladium(II) acetate, palladium(II) trifluoroacetate, palladium(II) chloride, palladium(II) bromide, palladium(II) iodide, and bis(triphenylphosphine)palladium(II) dichloride, and zerovalent palladium metals and complexes thereof, such as palladium black, palladium carbon, tetrakistriphenylphosphinepalladium(0), and bis(dibenzylideneacetone)palladium(0), are usable. Preferably, bis(triphenylphosphine)palladium(II) dichloride is used. The amount of palladium catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.0001 to 1 equivalent, more preferably 0.005 to 0.05 equivalent, relative to the compound represented by formula (XXXII).

[0272] Furthermore, this step can be preferably carried out in the presence of a copper catalyst in addition to the palladium catalyst. Examples of copper catalysts that can be used in this step include copper(I) chloride, copper(I) bromide, and copper(I) iodide, and copper(I) iodide is preferred. The amount of copper catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.0001 to 1 equivalent, more preferably 0.005 to 0.05 equivalent, relative to the compound represented by formula (XXXII).

[0273] This step is preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide, and mixtures thereof. Preferred bases include triethylamine, diisopropylethylamine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, and more preferred is triethylamine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by Formula (XXXII).

[0274] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably 15° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0275] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, and 1-methyl-2-pyrrolidone, and more preferred examples include N,N-dimethylformamide.

[0276] <3-4. Method for producing the compound of formula (XXIV)> The compound of formula (XXIV) used in the production of the above formula (VIII) and formula (VIII') can be produced according to the following synthesis scheme. [Synthetic Scheme <c4>]

[0277] [ka]

[0278] Each step will be explained below. (Step c14) In this step, a compound of formula (XXII) is reacted with a compound of formula (XXIX) to obtain a compound of formula (XXXIII).

[0279] The reaction of this step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of suitable bases include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as bases such as cesium carbonate, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide, and mixtures thereof. Preferred examples include cesium carbonate, potassium carbonate, and sodium carbonate, and more preferred examples include cesium carbonate. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by Formula (XXII).

[0280] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 10° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0281] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred are acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide. More preferred is dimethyl sulfoxide.

[0282] (Step c15) In this step, a compound of formula (XXXIII) is subjected to a reduction reaction of an alkyne in the presence of a catalyst, followed by a reductive amination reaction, and then reacted with a benzoylating agent to obtain a compound of formula (XXIV).

[0283] A metal catalyst can be used as a catalyst for the reduction reaction of alkyne and the reductive amination reaction in this step. The metal catalyst used in this step is not particularly limited as long as it catalyzes hydrogenation, but preferred examples include ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, and nickel catalysts, and more preferred examples include palladium catalysts. The amount of the metal catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.01 to 1 times, more preferably 0.02 to 1 times, and even more preferably 0.05 to 0.5 times the mass of the compound represented by formula (XXXIII). The hydrogen gas pressure is usually 100 to 1,000 kPa, and preferably 100 to 900 kPa. More preferably, The reaction pressure in this step is 200 to 700 kPa. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0°C to the boiling point of the solvent used in the reaction, more preferably 15°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 96 hours, more preferably 1 hour to 36 hours.

[0284] The solvent used in the reduction reaction of the alkyne in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include ethanol, methanol, 1-propanol, 2-propanol, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and mixed solvents thereof, and more preferred examples include ethanol.

[0285] The solvent used in the reductive amination reaction in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include ethanol, water, acetic acid, and mixed solvents thereof, and more preferred examples include a mixed solvent of water and acetic acid.

[0286] The benzoylating agent used in the benzoylation reaction of this step includes benzoic anhydride, benzoyl chloride, benzoyl bromide, benzoyl trifluoromethanesulfonate, etc., preferably benzoyl chloride and benzoyl bromide, more preferably benzoyl chloride. The amount of the benzoylating agent used in this step is not limited as long as the reaction proceeds, but is preferably a benzoyl compound represented by the formula (XXXIII) The amount is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by the formula:

[0287] The benzoylation in this step is preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- J Examples of the base include organic bases such as methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, and bases such as potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred bases are triethylamine, 2,6- J Examples of suitable bases include methylpyridine, 4-dimethylaminopyridine, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, and sodium acetate, and more preferably 4-dimethylaminopyridine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably a base represented by the formula (XXXIII) The amount is preferably 0.05 to 10 equivalents, and more preferably 0.2 to 5 equivalents, relative to the compound represented by the formula:

[0288] The reaction temperature for the benzoylation in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 90° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0289] The solvent used in the benzoylation in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane, and more preferred examples include pyridine.

[0290] <3-5. Comparison of the production method of compound (VIII) of the present invention with conventional methods (Patent Document 6, etc.)> The method for producing the compound of formula (VIII) of the present invention can provide a production method having superior effects such as an improved total yield, a reduced number of steps, and avoidance of column purification, compared to the synthesis scheme of Patent Document 6 shown below.

[0291] [Conventional synthesis scheme 1 (from the synthesis schemes of Examples 1 and 44 of Patent Document 6)]

[0292] [ka] [Conventional synthesis scheme 2 (from the synthesis scheme in Example 42 of Patent Document 6)]

[0293] [ka]

[0294] [Advantages of the production method of formula (VIII) of the present invention]

[0295] (Effect 1) Improved synthesis route (improved yield, omission of column purification) In the conventional synthesis route (Patent Document 6 / Conventional Synthesis Scheme 1), the compound of formula (VIII) was synthesized in 11 steps from commercially available 5-iodotubercidin. However, because this was a linear synthesis, the overall yield was a low 5%.

[0296] On the other hand, in the production method of the present invention, by using parallel synthesis (convergent synthesis), the number of steps from the available raw materials, compound (XXXI) and 2'-deoxy-2'-fluorouridine, was reduced to 10 steps each, and the overall yield was improved to 17% from compound (XXXI) and 9% from 2'-deoxy-2'-fluorouridine.

[0297] (Effect 2) Synthesis of 2-fluororibose derivatives by deglycosylation of compound (XX) A conventional method is the acid hydrolysis method, in which a dihydrouridine derivative such as 2'-deoxy-2'-fluoro-5,6-dihydrouridine is hydrolyzed to produce a 2-deoxy-2-fluororibose derivative such as compound (XXI) (see Carbohydrate Res., I, 1966, pp. 455-466). However, when this method is applied to compound (XIX) to synthesize compound (XXI), a side reaction, deprotection of the benzoyl group from the hydroxyl group, occurs, resulting in a low yield.

[0298] On the other hand, in the production method of the present invention, when the uridine moiety of compound (XIX) is benzoylated to form compound (XX), the reactivity to hydrolysis is improved, deglycosylation becomes possible under milder conditions, and deprotection of the benzoyl group is suppressed, thereby improving the yield.

[0299] (Effect 3) Synthesis of compound (XXII) by selective α-chlorination of compound (XXI) Conventionally, the chlorination reaction of 2-fluororibose derivatives involves protecting the hydroxyl groups at the 2 and 5 positions with TBDMS groups. Ta A method for treating 2-fluororibose derivatives under mesyl chloride / triethylamine conditions has been reported, but this method yields a mixture of α and β chlorinated sugars (see J. Org. Chem., 2009, 74, pp. 5779-5789 and WO 2011 / 003018). Another method for treating 2-fluororibose derivatives in which the hydroxyl groups at the 2 and 5 positions are protected with benzyl groups with hexamethylphosphorous triamide / carbon tetrachloride has also been reported, but this method produced the β isomer and was not applicable to the synthesis of the α isomer (see WO 2014 / 124430 and WO 2015 / 038596).

[0300] On the other hand, in the production method of the present invention, a method for synthesizing an α-chloro-2-fluororibose derivative was investigated. As a result, it was found that the reaction proceeds with high α-selectivity (α / β=15 / 1) by chlorinating a 2-fluororibose derivative (compound (XXI)) in which the hydroxyl groups at the 2 and 5 positions are protected with benzoyl groups using a combination of triarylphosphite or triarylphosphine and a chlorinating agent such as trichloroisocyanuric acid, and thus an efficient method for synthesizing an α-chloro-2-fluororibose derivative was established.

[0301] Furthermore, the chlorination reaction of alcohols using triarylphosphine and a chlorinating agent has long been known as a conventional method. For example, the chlorination reaction using a combination of triphenylphosphine and carbon tetrachloride is widely known as the Appel reaction (see Angew. Chem. Int. Edit., Vol. 14, No. 12, 1975, pp. 801-811). However, this Appel reaction produces triphenylphosphine oxide after the reaction, which requires purification using a silica gel column for its removal, posing a problem for large-scale synthesis.

[0302] On the other hand, in the production method of the present invention, by using 2,4-di-tert-butylphenyl phosphite as the triaryl phosphite, the by-product phosphate ester can be removed by liquid separation, and column purification has been successfully avoided.

[0303] (Effect 4) Synthesis of Compound (XXIII) via Boc Protection Route In the conventional synthesis route (Patent Document 6 / Conventional Synthesis Scheme 2), the yield in the Sonogashira reaction step for synthesizing compound (XXIX) from compound (XXXI) was low, resulting in a low overall yield of 18%, which was a problem. In the production method of the present invention, the yield in the Sonogashira reaction step was improved by going through compound (XXXII) using a Boc protecting group, and the overall yield was improved to 53%.

[0304] <4. Method for producing antibody-immunostimulant conjugates (Rp, Rp-XIII)> <4-1. Antibodies and their glycoremodeling> <4-1-1. Antibodies>

[0305] 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.

[0306] As used herein, the term "functional fragment" includes a functional fragment that retains an asparagine (Asn297) and its surrounding amino acids that are modified by N-linked glycosylation, which is well conserved in the Fc region of an IgG heavy chain, and that has antigen-binding ability.

[0307] The antibody used in producing the antibody-immunostimulant conjugate of the present invention refers to an immunoglobulin, 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).

[0308] When an IgG1 isotype antibody is used to produce the antibody-immunostimulant conjugate of the present invention, its 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).

[0309] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). CDRs, also known as hypervariable regions, are located in the variable regions of the heavy and light chains of an antibody and are particularly highly variable in their primary structure. They are separated into three regions in the primary structure of the heavy and light chain polypeptide chains. Herein, with regard to antibody CDRs, the heavy chain CDRs are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and the light chain CDRs are referred to as CDRL1, CDRL2, and CDRL3 from the amino-terminal end of the light chain amino acid sequence. These regions are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] When an antibody that targets tumor cells is used in the antibody-immunostimulant conjugate of the present invention, it is preferable, but not essential, that the antibody itself have an anti-tumor effect.

[0314] The antitumor activity of an immunostimulant or an antibody-immunostimulant conjugate refers to cytotoxic activity against tumor cells, anticellular effect, and regression of tumor volume. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems.

[0315] The immunostimulatory activity of an immunostimulator or an antibody-immunostimulator 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.

[0316] Examples of antibodies used in the production of the antibody-immunostimulant conjugates of 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 (e.g., trastuzumab or pertuzumab), an anti-CDH6 antibody, an anti-CD33 antibody, an anti-EphA2 antibody, an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody, and more preferably an anti-HER2 antibody, an anti-CDH6 antibody, an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody.

[0317] The antibodies used in producing the antibody-immunostimulant conjugates 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 applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0318] 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.

[0319] The antigen can be obtained by genetically manipulating a gene encoding the antigen protein in a host cell to produce it.

[0320] The humanized antibody used in producing the antibody-immunostimulant conjugate of the present invention can be obtained according to known methods (e.g., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984); Nature (1986) 321, pp. 522-525; WO90 / 07861).

[0321] For example, anti-HER2 antibodies (US Pat. No. 5,821,337, WO2004 / 008099, WO2020 / 050406, etc.), anti-CD33 antibodies (WO2014 / 057687, WO2020 / 050406, etc.), anti-EphA2 antibodies (WO2009 / 028639, WO2020 / 050406, etc.), anti-CDH6 antibodies (WO2018 / 212136, WO2020 / 050406, etc.), ), anti-CD70 antibodies (WO2004 / 073656, WO2007 / 038637, WO2021 / 177438, etc.), anti-TROP2 antibodies (WO2015 / 098099, WO2021 / 177438, etc.), and anti-EGFR antibodies (WO1998 / 050433, WO2002 / 092771, WO2021 / 177438, etc.) can be obtained by known means.

[0322] The anti-HER2 antibody used in the production of the antibody-immunopotentiator conjugate 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. (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: 50 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 49. (8) The antibody according to (6) 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: 51 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 49. (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: 53 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 52. (11) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 54 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 52. (12) The antibody according to any one of (1) to (3), (6) or (8) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 57, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 58, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 59, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 61, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 62. (13) The antibody according to any one of (1) to (12) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (14) 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 (13) above, and collecting the antibody of interest from the culture obtained in the step.

[0323] The anti-CD70 antibody used to produce the antibody-immunostimulant conjugate of the present invention is not particularly limited, but may be, for example, an antibody having the following properties: It is desirable to have something like this. (1) An anti-CD70 antibody that specifically binds to CD70. (2) The antibody according to (1) above, which binds to the extracellular domain of CD70. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, 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 (6) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (8) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1. (9) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 3. (10) The antibody according to any one of (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 13, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 16, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 18. (11) The antibody according to any one of (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 24. (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.

[0324] The anti-TROP2 antibody used in the production of the antibody-immunostimulant conjugate of the present invention is not particularly limited, but preferably has the following properties, for example: (1) An anti-TROP2 antibody that specifically binds to TROP2. (2) The antibody described in (1) above, which binds to the extracellular domain of TROP2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that reduces ADCC and ADCP activity. (7) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 5. (8) The antibody according to (6) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 7. (10) The antibody according to any one of (1) to (3), (6) or (8) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 25, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 26, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 28, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 29, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 30. (11) The antibody according to any one of (1) to (3), (6) or (8) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 31, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 32, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 34, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 35, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 36. (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.

[0325] The anti-EGFR antibody used in the production of the antibody-immunopotentiator conjugate of the present invention is not particularly limited, but preferably has the following properties, for example. (1) Anti-EGFR antibodies that specifically bind to EGFR. (2) The antibody according to (1) above, which binds to the extracellular domain of EGFR. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, 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 (6) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (8) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9. (9) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 11. (10) The antibody according to any one of (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 42. (11) The antibody according to any one of (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 43, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 44, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 48. (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.

[0326] The anti-CDH6 antibody used in the production of the antibody-immunopotentiator conjugate of the present invention is not particularly limited, but preferably has the following properties, for example. (1) An anti-CDH6 antibody that specifically binds to CDH6. (2) The antibody described in (1) above that binds to the extracellular domain of CDH6. (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 (6) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the leucines at positions 234 and 235 according to the EU index are substituted with alanines. (8) The antibody described in (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 56 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 55. (9) The antibody according to any one of (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 63, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 64, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 66, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 67, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 68. (10) The antibody according to any one of (1) to (9) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (11) 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 (10) above, and collecting the antibody of interest from the culture obtained in the step.

[0327] <4-1-2. 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).

[0328] Glycan remodeling involves first using a hydrolase to remove heterogeneous glycans attached to a protein (e.g., an antibody) leaving only the terminal GlcNAc, thereby preparing a homogeneous protein portion with GlcNAc attached (hereafter referred to as the "acceptor"). Next, a separately prepared glycan of your choice (hereafter referred to as the "donor") is prepared, and this acceptor and donor are linked using a glycosyltransferase. This allows the synthesis of a homogeneous glycoprotein with a desired glycan structure.

[0329] As used herein, "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 atom 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.

[0330] In this specification, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described, for convenience, with the carbon atom in the ring structure that is bonded to the oxygen atom constituting the ring and that is directly bonded to a hydroxy group (or an oxygen atom belonging to a glycosidic bond) being 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.

[0331] In this specification, when a glycan 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.

[0332] The antibody-immunostimulant conjugate used in the production of the antibody-immunostimulant conjugate of the present invention has the following formula (XXXIV):

[0333] [ka] The antibody Ab or a functional fragment thereof is bound to L directly through the side chain of its amino acid residue (e.g., cysteine, lysine, etc.), or through a sugar chain or a remodeled sugar chain of the Ab. The side chain of the amino acid residue may be modified, for example, with an azide group. where: Ab refers to an antibody or a functional fragment of the antibody, and the sugar chain of the antibody may be remodeled. m 1 is in the range of 1 to 10, L represents a linker connecting Ab and D, The linker L is represented by -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates binding to an immunostimulant D, Lp is -GGFG-, where G represents glycine and F represents phenylalanine; La represents -C(=O)-CHCH-C(=O)-; Lb is expressed by the following formula: [ka] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain), Lc represents -NH-CH2-. D is

[0334] [ka] Shows.

[0335] In the present specification, the sugar chain of Ab is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

[0336] 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.

[0337] As used herein, Ab refers to IgG, preferably IgG1, IgG2 or IgG4.

[0338] IgG has a well-conserved N-linked glycan (hereinafter referred to as "Asn297 glycan or N297 glycan") at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which is known to contribute to the activity and kinetics of antibody molecules (Eon-Duval, A. et al., Biotechnol. Prog. 2012, 28, 608-622; Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0339] 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.

[0340] The diagram below shows the case where the antibody-immunostimulant conjugate of the present invention is bound to L from the N297 sugar chain of the antibody or functional fragment thereof. 2 represents an integer of 1 or 2.

[0341] [ka] An antibody having such a remodeled sugar chain is called a sugar chain remodeling antibody.

[0342] 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.

[0343] 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.

[0344] The remodeled sugar chains used in the antibody-immunostimulant conjugates of the present invention are N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, preferably N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1.

[0345] N297-(Fuc)SG is represented by the following structural formula or sequence formula:

[0346] [ka]

[0347] [ka]

[0348] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0349] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula:

[0350] [ka]

[0351] [ka]

[0352] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of 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.

[0353] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula:

[0354] [ka]

[0355] [ka]

[0356] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0357] When the N297 sugar chain of the antibody in the antibody-immunostimulant conjugate of the present invention is N297-(Fuc)SG, the antibody is a dimer, and therefore the antibody-immunostimulant conjugate is a molecule to which four linkers L and four immunostimulants D are bound (the above m 2 =2).

[0358] When the N297 sugar chain of the antibody in the antibody-immunostimulant 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-immunostimulant conjugate is a molecule in which two linkers L and two immunostimulants D are bound (the above-mentioned m 2 =1) (see Figure 19).

[0359] The N297 sugar chain is preferably N297-(Fuc)SG or N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1, and even more preferably N297-(Fuc)SG.

[0360] When the N297 sugar chain of the antibody in the antibody-immunostimulant conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, a highly homogeneous ADC can be obtained.

[0361] <4-2. Production of Glycosylation Remodeling Antibodies> Glycosylation remodeling antibodies can be produced by the method shown in the following scheme, in accordance with the methods described in, for example, WO2018 / 003983, WO2020 / 050406, WO2021 / 177438, PLos ONE 2018, 13, e0193534, etc.

[0362] [ka]

[0363] (D-1 process) This step involves hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing-end chitobiose structure of the N-linked glycan (N297-linked glycan) attached to asparagine at position 297 of the antibody's amino acid sequence, using a known enzymatic reaction, to produce a glycosylated antibody. The target antibody (1) (10 mg / mL) was placed in a buffer solution (e.g., phosphate buffer) at 0°C to 40°C, and the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing-end chitobiose structure was hydrolyzed using a hydrolase such as wild-type EndoS enzyme. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. The wild-type EndoS enzyme was used in an amount of 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, per 100 mg of antibody (1). After the reaction was completed, affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and / or Doro The antibody was purified using a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (BIO-RAD)) to obtain (Fucα1,6)GlcNAc antibody (2).

[0364] (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 (2) obtained in step D-1 using a known enzymatic reaction to produce a glycan remodeling antibody (3).

[0365] Antibody (2) was reacted with an azido-oligosaccharide oxazoline derivative in a buffer solution (such as phosphate buffer) at 0°C to 40°C in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L) to carry out a glycosyltransferase reaction. The reaction time was 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 mg to 3 mg, per 100 mg of antibody, and the azido-oligosaccharide oxazoline derivative was used in an amount ranging from 2 equivalents to an excess equivalent, preferably 4 to 20 equivalents. After the reaction was completed, the antibody was purified by affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and HiTrap rProtein A FF (5 ml) (GE Healthcare)). Doro The resulting antibody was purified using a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (BIO-RAD)) to obtain the sugar chain remodeling antibody (3).

[0366] 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.

[0367] 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.

[0368] [ka]

[0369] 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.

[0370] [ka]

[0371] (D-3 process) This step involves the production of a glycan-remodeled antibody (3) by a glycosyltransferase reaction using two Endo enzymes on the (Fucα1,6)GlcNAc antibody (2) obtained in step D-1. By using two enzymes simultaneously, glycosyltransferases such as SGP and (SG)Asn, which have inactivated reducing ends, can be used as glycan donors to directly transfer glycosylation to the N297 glycan of the antibody. Regarding the two types of Endo enzymes used, enzyme A (EndoM-like enzyme) and enzyme B (EndoS-like enzyme) can be appropriately combined. Examples of enzyme A include EndoM, EndoOm, EndoCC, and EndoM mutants, EndoOm mutants, and EndoCC mutants with reduced hydrolytic activity. Preferred enzymes A are EndoM N175Q, EndoCC N180H, and EndoOm N194Q. Examples of enzyme B include EndoS, EndoS2 (EndoS49), and EndoS mutants with reduced hydrolytic activity, EndoS2 (EndoS49) mutants, etc. Preferred examples of enzyme B include EndoS D233Q, EndoS D233Q / Q303L, EndoS D233Q / E350A, EndoS D233Q / E350Q, EndoS D233Q / E350D, EndoS D233Q / E350N, EndoS D233Q / D405A, EndoS2 D184M, and EndoS2 T138Q. The sugar chain donors were ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3, [N3-PEG(3)]-MSG1-Asn-PEG(3)-N 3、 [N3-PEG(3)]-MSG2-Asn-PEG(3)-N3, etc. can be used.

[0372] Antibody (2) was reacted with ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 in a buffer solution (such as Tris buffer) in the presence of glycosyltransferases Enzyme A (EndoM-like enzyme) and Enzyme B (EndoS-like enzyme) to carry out a glycosyltransferase reaction. The reaction temperature can be appropriately selected depending on the optimum temperature for the enzyme used, but is usually 15 to 50°C, and preferably 25 to 40°C. The reaction time can be appropriately selected between 2 and 48 hours. After the reaction is complete, purification is performed using a method suitable for the reaction scale (e.g., affinity chromatography, Doro We selected a method using a hydroxyapatite column or an ultrafiltration membrane to obtain a glycan remodeling antibody (3).

[0373] 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.

[0374] ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 was synthesized according to the method described in WO2018 / 003983. In step 1-2A described in WO2018 / 003983, the Fmoc-(SG-)Asn free form prepared from Fmoc-(SG-)Asn (1S2S-11NC-Asn-Fmoc, manufactured by Glycotechnology Institute) was reacted with 11-azido-3,6,9-trioxaundecan-1-amine to obtain ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 (compound 1-13 described in WO2018 / 003983).

[0375] MSG-type glycan donor [N3-PEG(3)]-MSG1-Asn-PEG(3)-N 3、 [N3-PEG(3)]-MSG2-Asn-PEG(3)-N3 can also be synthesized in accordance with the method described in steps 1 to 3 of Example 154 of WO2019065964.

[0376] <4-3. Conjugation of antibodies and immunostimulants> The antibody-immunostimulant conjugate of formula (Rp, Rp-XIII) can be prepared according to the following method.

[0377] [ka] (Here, the two asterisks (*) on the left side of the antibody-immunostimulatory agent conjugate (Rp, Rp-XIII) indicate the CDN-linker moiety indicated by the asterisk on the right side.)

[0378] The antibody-immunostimulant conjugate (Rp,Rp-XIII) can be produced by combining the glycan remodeling antibody (3) with the CDN conjugate precursor (Rp,Rp-XII) via a cycloaddition reaction. Examples of cycloaddition reactions include the Diels-Alder reaction and the 1,3-dipolar cycloaddition reaction, with the 1,3-dipolar cycloaddition reaction being preferred. Examples of 1,3-dipolar cycloaddition reactions include the cycloaddition reaction of an azide with a terminal alkyne and the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction (J. Am. Chem. Soc. 2004, 126, 15046-15047), with the SPAAC reaction being preferred.

[0379] This production method is a method for producing an antibody-immunostimulant conjugate (Rp, Rp-XIII) by binding the glycan remodeling antibody (3) obtained in step D-2 or D-3 above with a CDN conjugate precursor (Rp, Rp-XII) via the SPAAC reaction.

[0380] (Process E-1) (Process a9) The SPAAC reaction was carried out by mixing a buffer solution of the glycosylation-remodeling antibody (3) (e.g., phosphate buffer, acetate buffer, borate buffer, etc.) with a solution of the CDN conjugate precursor (Rp, Rp-XII) dissolved in an appropriate solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixture thereof). The CDN conjugate precursor (Rp, Rp-XII) was used in an amount of 2 molar to a molar excess, preferably 4 to 30 molar, per mole of glycosylation-remodeling antibody (3). 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 to 150 hours, preferably 6 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 antibody-immunopotentiator conjugates (Rp, Rp-XIII).

[0381] The antibody-immunopotentiator conjugate can be identified by buffer exchange, purification, measurement of antibody concentration, and measurement of the average number of immunopotentiators bound per antibody molecule according to common procedures D to G described below.

[0382] Common Procedure A: Concentration of antibody aqueous solution The antibody or antibody-immunostimulant conjugate solution was placed in an Amicon® Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and the antibody or antibody-immunostimulant 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.

[0383] 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.

[0384] 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).

[0385] Common Procedure D: Purification of Antibody-Immunostimulant 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-immunoactivator 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-immunoactivator conjugate from which unbound immunoactivator linker, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-immunoactivator conjugate solution was adjusted using common procedures A and C.

[0386] Common Procedure E: Measurement of antibody concentration and average number of adjuvants bound per antibody molecule in antibody-adjuvant conjugates (UV method) The concentration of bound immunostimulant in the antibody-immunostimulant conjugate can be calculated by measuring the absorbance of an aqueous solution of the antibody-immunostimulant conjugate at two wavelengths, 280 nm and 250 nm, using an absorption spectrophotometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.) in accordance with the methods described in WO2020 / 050406 and WO2021 / 177438.

[0387] Common Procedure F: Measurement of antibody concentration and average number of adjuvants bound per antibody molecule in antibody-adjuvant conjugates (reversed-phase high-performance liquid chromatography: RP-HPLC) The antibody concentration in the antibody-immunopotentiator conjugate and the average number of immunopotentiators bound per antibody molecule can be determined by high performance liquid chromatography analysis in accordance with the methods described in WO2020 / 050406 and WO2021 / 177438, in addition to the above-mentioned common procedure E.

[0388] Common Procedure G: Measurement of antibody concentration and average number of adjuvants bound per antibody molecule in antibody-adjuvant conjugates (hydrophobic interaction-high performance liquid chromatography: HI-HPLC) The antibody concentration in the antibody-immunopotentiator conjugate and the average number of immunopotentiators bound per antibody molecule can be determined by high performance liquid chromatography analysis in accordance with the methods described in WO2020 / 050406 and WO2021 / 177438, in addition to the common procedures E and F described above.

[0389] The antibody-immunopotentiator conjugate produced by the method of the present invention or a production intermediate thereof may contain 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.

[0390] In antibody-immunopotentiator conjugates produced by the methods of the present invention, the number of immunopotentiators bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-immunopotentiator conjugates are produced by specifying reaction conditions, such as the amounts of reacting raw materials and reagents, so that a certain number of immunopotentiators are bound. However, unlike chemical reactions of small molecules, the resulting conjugates are typically mixtures of immunopotentiators with different numbers bound to one another. The number of immunopotentiators bound to one antibody molecule can be determined as an average value, i.e., the average number of immunopotentiators bound (DAR: Drug-to-Antibody Ratio). The number of cyclic dinucleotide derivatives bound to an antibody molecule can be controlled; the average number of cyclic dinucleotide derivatives bound to one antibody can range from 1 to 10, preferably 1 to 8, and more preferably 1 to 5.

[0391] In the antibody-immunopotentiator conjugate produced by the method of the present invention, when the antibody Ab is bound to L via a remodeled sugar chain of the antibody Ab, the number of immunopotentiators bound per antibody molecule in the antibody-immunopotentiator 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).

[0392] Furthermore, a person skilled in the art would be able to design a reaction for binding the required number of immunostimulants 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.

[0393] It should be noted that the antibody-immunostimulant conjugate produced by the method of the present invention or a production intermediate thereof may absorb moisture, become adsorbed water, or become a hydrate when left in the air or upon recrystallization, and such water-containing compounds and salts are also encompassed by the present invention.

[0394] When the antibody-immunostimulant conjugate produced by the method of the present invention or a production intermediate thereof has a basic group such as an amino group, it can be converted into a pharmaceutically acceptable salt, if desired. Examples of such salts include hydrohalides such as hydrochlorides and hydroiodides; inorganic acid salts such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfonates such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; arylsulfonates such as benzenesulfonates and p-toluenesulfonates; and formates. salt , acetic acid salt , malic acid salt Examples of suitable salts include organic acid salts such as fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0395] The antibody-immunostimulant conjugates produced by the methods of the present invention or their intermediates generally contain phosphate and / or thiophosphate groups in their structures, and therefore can generally form base addition salts. Furthermore, when the intermediates contain acidic groups such as carboxyl groups, they can generally form base addition salts. Pharmaceutically acceptable salts include, for example, 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, tert-butylamine 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.

[0396] The antibody-immunostimulant conjugates and their production intermediates produced by the methods 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 specific examples include hydrates, ethanol solvates, and 2-propanol solvates. Furthermore, when nitrogen atoms are present in the antibody-immunostimulant conjugates of the present invention and their production intermediates, they may be in the form of N-oxides, and these solvates and N-oxides are also within the scope of the present invention. Furthermore, when sulfur atoms are present in the antibody-immunostimulant conjugates of the present invention and their production intermediates, they may be in the form of sulfoxides, and these solvates and sulfoxides are also within the scope of the present invention.

[0397] The antibody-immunostimulator conjugates and their intermediates produced by the methods of the present invention also include compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-immunostimulator conjugates and their intermediates 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-immunostimulator conjugates of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0398] <5. Pharmaceuticals> The antibody-immunostimulant conjugates produced by the methods of the present invention exhibit antitumor immune activity or cytotoxic activity against cancer cells, and can therefore be used as pharmaceuticals, particularly as therapeutic and / or preventive agents for cancer, or antitumor agents.

[0399] The types of cancer to which the antibody-immunomodulator conjugates produced by the method 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, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, and gastrointestinal stromal tumor. Examples of cancer cells to be treated include cancers such as gastrointestinal stromal tumors (GIST, GIST), gallbladder cancer, bile duct cancer, adrenal cancer, pharyngeal cancer, tongue cancer, ear cancer, thymus cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, and sarcoma; however, with regard to antibody-immunopotentiator conjugates, the cancer cells to be treated are not limited to these, as long as they express a protein that can be recognized by the antibody in the antibody-immunopotentiator conjugate.

[0400] The antibody-immunostimulant conjugate produced by the method of the present invention can be suitably administered to mammals, more preferably humans.

[0401] Substances used in pharmaceutical compositions containing antibody-immunostimulant conjugates produced by the methods 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.

[0402] The antibody-immunostimulatory agent conjugates produced by the methods of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The compositions 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.

[0403] Various delivery systems are known and can be used to administer the antibody-immunostimulant conjugate of the present invention. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, the antibody-immunostimulant conjugate is administered by infusion. Parenteral administration is a preferred administration route.

[0404] In a representative embodiment, a pharmaceutical composition comprising the antibody-immunostimulant 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. Where 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, e.g., as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the pharmaceutical composition is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical-grade water or saline. When 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.

[0405] A pharmaceutical composition comprising an antibody-immunostimulant conjugate produced by the method of the present invention may be a pharmaceutical composition containing only the antibody-immunostimulant conjugate produced by the method of the present invention, or may be a pharmaceutical composition containing the antibody-immunostimulant conjugate produced by the method of the present invention and another cancer therapeutic agent. The antibody-immunostimulant conjugate produced by the method of the present invention can also be administered together with another cancer therapeutic agent, thereby enhancing the anti-tumor effect. The other cancer therapeutic agent used for this purpose may be administered to an individual simultaneously with the antibody-immunostimulant conjugate, separately, or sequentially, or at different administration intervals. Examples of such cancer therapeutic agents include chemotherapeutic agents such as antimetabolites, alkylating agents, and microtubule inhibitors (e.g., abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, vinblastine, or drugs described in International Publication No. WO 2003 / 038043), hormone modulating agents (e.g., LH-RH analogs such as leuprorelin, goserelin, and estramustine, and estrogen antagonists such as tamoxifen and raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, and exemestane), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, molecular targeted drugs (e.g., anti-EGFR antibodies, anti- Anti-VEGF antibodies, anti-VEGFR antibodies, etc.), immune checkpoint inhibitors (anti-PD-1 antibodies such as nivolumab and pembrolizumab, anti-PD-L1 antibodies such as atezolizumab, avelumab, and durvalumab, anti-PD-L2 antibodies, anti-CTLA4 antibodies such as ipilimumab, anti-A2aR antibodies, A2a receptor antagonists, anti-LAG3 antibodies, anti-TIM3 antibodies, etc.), anti-regulatory T cell drugs (anti-CTLA4 antibodies, anti-CD25 antibodies, anti-GITR antibodies, anti-GARP antibodies, anti-TIGIT antibodies, anti-CCR8 antibodies, etc.), immune activators (anti-4-1BB antibodies, anti-OX40 antibodies, anti-CD40 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, IL-2 analogs, cytokines, TLR agonists, etc.), immunomodulators (anti-CD47 antibodies, anti-SIRPα antibodies, inhibitory myeloid modulators, etc.), ADCC (AntibodyExamples of such antitumor agents include antibody drugs with T-cell dependent cytotoxicity (TDC) activity, ADCP (antibody dependent cellular phagocytosis) activity, or complement activity, BiTEs (bi-specific T-cell engagers), antibody-drug conjugates (ADCs) (e.g., drug conjugates containing deruxtecan, DM1, pyrrolobenzodiazepine, MMAF, etc. (anti-HER2-ADC, anti-TROP2-ADC, anti-HER3-ADC, etc.)), ADCs combined with photodynamic therapy, as well as antitumor vaccines, antitumor cell therapies (CAR-T, TCR-T, dendritic cells, NK cells, etc.), antitumor bacterial therapies, antitumor viral therapies, etc., but are not limited thereto as long as they have antitumor activity. Furthermore, the antibody-immunostimulant conjugates of the present invention can be administered together with other antibody-immunostimulant conjugates of the present invention, thereby enhancing the antitumor effect. Furthermore, the antibody-immunopotentiator conjugate of the present invention can enhance the antitumor effect when used in combination with not only immunopotentiators but also treatments that bring about antitumor effects, such as radiation, quantum beams, surgery, bone marrow transplantation, etc., but is not limited thereto as long as the treatment has an antitumor effect.

[0406] 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.

[0407] Although the composition and concentration of a pharmaceutical composition vary depending on the administration method, the antibody-immunoactivator conjugate contained in a pharmaceutical composition containing an antibody-immunoactivator conjugate produced by the method of the present invention exhibits efficacy even at a smaller dose, as the affinity of the antibody-immunoactivator conjugate for an antigen, i.e., the dissociation constant (Kd value) for the antigen, increases (the lower the Kd value). Therefore, when determining the dose of an antibody-immunoactivator conjugate, the dose can be set based on the affinity between the antibody-immunoactivator conjugate and the antigen. When the antibody-immunoactivator conjugate of the present invention is administered to a human, for example, approximately 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0408] The present invention will be described below with reference to examples, but is not limited thereto. In the following, "compound (I)" or "(I)" in the synthesis scheme indicates that it corresponds to "a compound of formula (I)," and the same applies to the subsequent compound numbers. [Example]

[0409] In the following examples, unless otherwise specified, room temperature refers to 15°C to 35°C. Dehydrated dichloromethane was dichloromethane (ultra-dehydrated) sold by Fujifilm Wako Pure Chemical Industries, Ltd. Dehydrated acetonitrile was acetonitrile (ultra-dehydrated) sold by Fujifilm Wako Pure Chemical Industries, Ltd. Dehydrated pyridine was pyridine (dehydrated) sold by Kanto Chemical Co., Ltd. Silica gel chromatography was performed using Biotage Sfar HC D (20 μm, Biotage), amino silica gel column chromatography was performed using Biotage Sfar Amino D (50 μm, Biotage), and preparative HPLC was performed using an Agilent Preparative HPLC System (Agilent Technology). The preparative column used was an XBridge Prep OBD (5 μm, C18, 130 Å, 250 × 30 mm, Waters).

[0410] The following instruments were used to measure various spectral data. 1 H-NMR and 31 P-NMR spectra were measured using a JEOL ECZ500R. Mass spectra were measured using Shimadzu LCMS-2010 and LCMS-2020 (Shimadzu Corporation). LC / MS measurements were performed under the following conditions: column: XBridge C18, 3.5 μm, 150 × 4.6 mm (Waters), mobile phase: 10 mM ammonium acetate / acetonitrile, acetonitrile: 10%-97.5% (0 min-42 min).

[0411] A. Synthesis of Cyclic Dinucleotides (CDNs) A-1 Synthesis of CDN1 1 Cyclic dinucleotide (CDN1) was synthesized according to the following synthesis scheme A-1: ​​A2 = Bz-tetraazabenzo[cd]azulene group, Q = thiol group, PG1 = TBS, PG2 = DMTr, PG3 = Teoc, PG6 = Ac, and (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole was used as (Rc-II). [Synthetic Scheme A-1]

[0412] [ka]

[0413] Example 1: Synthesis of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-[(1S,3aR)-tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (corresponding to the compound of formula (Rc-III))

[0414] [ka]

[0415] (Process 1) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (10.0 g) was added to anhydrous dichloromethane (150 mL) and cooled to -5 °C, followed by the addition of triethylamine (1.73 mL). A solution of (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (Tetrahedron Letter, 1998, 39, 2491-2494) in anhydrous dichloromethane (50 mL) was added dropwise to the above solution, followed by stirring at the same temperature for 30 minutes. The mixture was warmed to room temperature and stirred for 30 minutes, after which the reaction mixture was concentrated. The concentrated crude product was diluted and dissolved in dichloromethane and purified by aminosilica gel chromatography [dichloromethane / ethyl acetate] to obtain the target compound (10.4 g).

[0416] MS(ESI)m / z:1088(M+H) + ,1086(MH) - . 1 H-NMR(MeCN-d3)δ:7.94(1H,s),7.93(1H,s),7.44-7.42(3H,m),7.31(4H,d,J=9.0Hz),7.27(2H,t,J=7.0Hz),7.21(1 H,t,J=7.0Hz),6.84(4H,d,J=7.0Hz),5.88(1H,d,J=6.5Hz),5.83(1H,brm),4.94(1H,m),4.56(2H,m),4.33(1H,m),4 .17-4.02(6H,m),3.74(6H,s),3.69(2H,m),3.61-3.57(3H,m),3.43(1H,dd,J=5.0Hz),3.28-3.19(3H,m),2.48-2.44 (1H,m),1.65-1.49(3H,m)1.26-1.17(1H,m),0.94-0.90(2H,m),0.87(9H,s),0.10(3H,s),0.06(3H,s),0.00(9H,s). 31 P-NMR(MeCN-d3) δ: 151.93.

[0417] Example 2: N,N-Diethylethanaminium (2R,3R,4R,5R)-2-({[(R)-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}{[(2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-5-(hydroxymethyl)oxolan-3-yl]oxy}phosphorothioyl]oxy}methyl)-5-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-4-fluorooxolan-3-yl Synthesis of phosphate (corresponding to the compound of formula (Rc-V))

[0418] [ka]

[0419] (Process 2) 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5

[0044] 260 mg of the triethylamine salt of {2-phosphanyl}-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene was dissolved in 2.7 mL of dehydrated acetonitrile, 12.5 mg of molecular sieve 4A was added, and the mixture was stirred for 30 minutes. In a separate container, 540 mg of the compound obtained in Example 1 was dissolved in 2.7 mL of dehydrated acetonitrile, 25 mg of molecular sieve 4A was added, and the mixture was stirred for 30 minutes to obtain the previously prepared 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 An acetonitrile solution of the triethylamine salt of {-phosphanyl}-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene was added. A 0.4 mol / L solution of 1-phenylimidazolium triflate in anhydrous acetonitrile (2.7 mL) was added and stirred at room temperature for 30 minutes. A 1 mol / L solution of acetic anhydride in anhydrous acetonitrile (0.45 mL) was then added, followed by triethylamine (55 mg) and stirring for 30 minutes. Xanthan hydride (74 mg) was then added and stirred for 30 minutes. The reaction mixture was then filtered to remove the molecular sieves. The filtrate was cooled to 0°C, 20% brine (2.7 mL) was added, the pH was adjusted to 2.5 with hydrochloric acid, and the mixture was stirred overnight at the same temperature. The reaction mixture was separated, and 20% brine (2.7 mL) was added to the resulting organic layer, which was then neutralized with triethylamine to pH 7. The organic layer was then concentrated and crudely purified by silica gel chromatography (acetonitrile / methanol), and concentrated to dryness to obtain the target compound. HPLC analysis revealed that the ratio of diastereomers at the phosphorus atom produced during the reaction was 98.5:1.5.

[0420] MS(ESI)m / z:1336(M+H) + ,1334(MH) - . 31 P-NMR(MeCN-d3)δ:69.41,2.13 (Rotamerδ:68.23,1.91). 31 P-NMR data was measured using the isolated product obtained by reversed-phase HPLC [10 mM aqueous ammonium acetate / acetonitrile].

[0421] Example 3: 2-(Trimethylsilyl)ethyl (2-{[(2-{9-[(5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-10-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2-oxo-2-sulfanyl-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate (corresponding to the compound of formula (Rc-VI)

[0422] [ka]

[0423] (Step 3) The compound obtained in Example 2 (320 mg) was azeotropically dehydrated with dehydrated pyridine (50 mL) to prepare 16 mL of a pyridine solution, which was then cooled to -20°C. 2-Chloro-2-oxo-1,3,2-dioxaphospholane (171 mg) was weighed into a separate container and cooled to -20°C. The pyridine solution was added to the container containing 2-chloro-1,3,2-dioxaphospholane-2-oxide and stirred at -20°C for 1 hour. The diastereomeric ratio at the phosphorus atom generated during cyclization was 75:25. Water (43 mg) was added, and the mixture was stirred at the same temperature for 10 minutes. Xanthan hydride (40 mg) was then added, and the mixture was warmed to room temperature and stirred for 1 hour. The resulting reaction mixture was quenched with 5% aqueous sodium bicarbonate (3 mL), and the mixture was separated by adding ethyl acetate (12 mL) and 20% saline (3 mL). The aqueous layer was re-extracted with ethyl acetate (3 mL), and the organic layer obtained from the first separation was combined with the organic layer obtained from the re-extraction. The combined organic layer was washed with a mixture of 20% brine (1.5 mL) and 5% aqueous sodium bicarbonate (1 mL). The organic layer was then concentrated to dryness, and methanol (30 mL) was added. The mixture was again concentrated to dryness to obtain the target compound.

[0424] MS(ESI)m / z:1350(M+H) + ,1348(MH) - . 31 P-NMR(MeCN-d3)δ:65.93,56.60 (Rotamerδ:65.51,57.12). 31 P-NMR data was measured using the isolated product obtained by reversed-phase HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile].

[0425] Example 4: Bis(N,N-diethylethanaminium)(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (corresponding to the compounds of formula (Rp, Rp-VII'))

[0426] [ka]

[0427] (Step 4) Methanol (12 mL) was added to the compound obtained in Example 2, and 28% aqueous ammonia (4.5 mL) was added to the resulting methanol solution, followed by stirring at 60°C for 30 hours. The resulting reaction mixture was concentrated, and the residue was purified by preparative HPLC [10 mM aqueous triethylammonium acetate solution / acetonitrile] to obtain 84 mg of the target compound as a single diastereomer (Rp,Rp form) at the phosphorus atom. The HPLC purity of the isolated compound was 92.4%.

[0428] MS(ESI)m / z:1121(M+H) + ,1119(MH) - . 1 H-NMR(MeCN-d3)δ:8.61(1H,s),8.10(1H,s),8.07(1H,brs),7.84-7.82(1H,m),7.50(1H,brs),7.03(1H,brs),6.37(1H,dd,J=16.5,3.0Hz),6.10( 1H,d,J=8.0Hz),6.06(1H,brm),5.51(1H,dt,J=52.5,4.0Hz),5.37(1H,m ),5.31(1H,m),4.57(2H,d,J=7.0Hz),4.52(1H,d,J=3.5Hz),4.28(2H,m), 4.19(3H,m),4.07(3H,m),3.91(1H,ddd,J=12.0,5.0,2.0Hz),3.84(1H,d dd,J=12.0,5.0,2.0Hz),3.70(2H,m),3.62(2H,m),3.46(2H,m),2.99(6H, q,J=7.0Hz),2.86(1H,m),2.73(1H,m),1.95(2H,m),1.17(18H,t,J=7.0Hz ),0.95-0.92(2H,m),0.94(9H,s),0.23(3H,s),0.20(3H,s),0.00(9H,s). 31 P-NMR (MeCN-d3) δ: 58.31, 57.22.

[0429] A-2 Synthesis of Compound (IV) (A2 = Bz-tetraazabenzo[cd]azulene group) The starting compound (IV) used in the above synthesis scheme A-1 was synthesized according to the following scheme. [Synthetic Scheme A-2]

[0430] [ka]

[0431] Example 5: 6-Benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 Synthesis of {-phosphanyl}-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene (corresponding to the compound of formula (IX))

[0432] [ka]

[0433] (Process 1) 6-Benzoyl-2-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-2-fluoro-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene (800 mg) was dissolved in pyridine (6 mL) and cooled to 0 °C. Diphenyl phosphite (1.31 g) was added and stirred for 1 hour under cooling. Subsequently, water (3 mL) was added, followed by triethylamine (3 mL), and the mixture was stirred at room temperature for 2 hours. Dichloromethane (8 mL) and 5% aqueous sodium bicarbonate (2 mL) were added to the reaction mixture, and the layers were separated. The organic layer was washed twice with 5% aqueous sodium bicarbonate (4 mL). The organic layer was dried over anhydrous sodium sulfate to obtain the target compound.

[0434] MS(ESI)m / z:779(M+H) + ,777(MH) - .

[0435] Example 6: 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 Synthesis of {-phosphanyl}-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene (corresponding to the compound of formula (IV))

[0436] [ka]

[0437] (Process 2) The solution of the compound obtained in Example 5 was cooled to 0°C, dichloroacetic acid (12 mL) was added, and the mixture was stirred for 24 hours under cooling. The reaction solution was purified by silica gel chromatography [acetonitrile / methanol] and concentrated to prepare an acetonitrile solution, yielding the target compound (358 mg, total quantitative value for two steps by NMR).

[0438] MS(ESI)m / z:477(M+H) + ,475(MH) - .

[0439] A-3 Synthesis of CDN2 Cyclic dinucleotide (CDN2) was synthesized according to the following synthesis scheme A-3: In synthesis scheme A-3, A2 = Bz-adenine group, Q = thiol group, PG1 = TBS, PG2 = DMTr, PG3 = Teoc, and PG6 = Ac. [Synthetic Scheme A-3]

[0440] [ka]

[0441] Example 7: Synthesis of sodium (2R,3R,4R,5R)-2-({[(R)-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}{[(2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-5-(hydroxymethyl)oxolan-3-yl]oxy}phosphorothioyl]oxy}methyl)-5-(6-benzamido-9H-purin-9-yl)-4-fluorooxolan-3-yl phosphonate (corresponding to the compound of formula (Rc-V))

[0442] [ka]

[0443] (Process 1) (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (400 mg) described in Patent Document WO2015 / 185565 was dissolved in anhydrous acetonitrile (4 mL), triethylamine (97 mg) and molecular sieve 4A (20 mg) were added, and the mixture was stirred for 30 minutes. In a separate container, the compound obtained in Example 1 (1.1 g) was dissolved in anhydrous acetonitrile (5.5 mL), molecular sieve 4A (25 mg) was added, and the mixture was stirred for 30 minutes. The acetonitrile solution of the triethylamine salt of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate prepared earlier was added. A 0.4 mol / L solution of 1-phenylimidazolium triflate in dehydrated acetonitrile (5.5 mL) was added and stirred at room temperature for 30 minutes. A 1 mol / L solution of acetic anhydride in dehydrated acetonitrile (1.14 mL) was then added, followed by triethylamine (139 mg) and stirring for 30 minutes. Xanthan hydride (154 mg) was then added and stirred for 30 minutes. The reaction mixture was then filtered to remove the molecular sieves. The filtered molecular sieves were washed with dehydrated acetonitrile (5 mL). The filtrate and washings were combined, cooled to 0°C, 20% brine (6 mL) was added, and the pH was adjusted to 2.5 with hydrochloric acid. The mixture was stirred overnight at the same temperature. The reaction mixture was separated, and the resulting organic layer was added with 20% brine (6 mL) and neutralized with triethylamine to pH 7. The organic layer obtained by separation was then concentrated and crudely purified by silica gel chromatography [ethyl acetate / methanol / 0.4% triethylamine]. The collected fraction was concentrated and purified by preparative HPLC [10 mM aqueous ammonium acetate / acetonitrile] to remove the acetonitrile by concentration. Salt was added to the resulting aqueous solution, which was then extracted with tetrahydrofuran and concentrated to dryness to obtain the target compound (776 mg). HPLC analysis revealed that the ratio of diastereomers at the phosphorus atom produced during the reaction was 98.5:1.5.

[0444] MS(ESI)m / z:1297(M+H) + ,1295(MH) - . 31 P-NMR (MeOH-d3) δ: 70.20, 4.17.

[0445] Example 8: Sodium (5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-10-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}14-(6-benzamido-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2-oxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3.2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate (corresponding to the compound of formula (Rc-VI))

[0446] [ka]

[0447] (Process 2) The compound obtained in Example 7 (18 mg) was dissolved in anhydrous pyridine (1 mL) and molecular sieves 4A (3 mg) was added. The diastereomeric ratio at the phosphorus atom generated during cyclization was 70:30. Subsequently, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (5.7 mg) was added and stirred for 30 minutes, and the molecular sieves were filtered off. Water (10 mg) was added to the filtrate and stirred for 5 minutes. After that, xanthan hydride (2.6 mg) was added and stirred for 30 minutes. The reaction was quenched by adding 5% aqueous sodium bicarbonate (2 mL), and ethyl acetate was added for liquid separation and extraction. The organic layer was concentrated to dryness and purified by preparative HPLC [10 mM aqueous ammonium acetate / acetonitrile], and the acetonitrile was removed by concentration. NaCl was added to the resulting aqueous solution, extracted with tetrahydrofuran, and concentrated to dryness to obtain the target compound (4 mg) as a single diastereomeric phosphorus atom. MS(ESI)m / z:1311(M+H) + ,1309(MH) - . 31 P-NMR (MeOH-d3) δ: 66.89, 58.93.

[0448] Example 9: Disodium (2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3.2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-bisthiolate (corresponding to the compound of formula (Rp, Rp-VII'))

[0449] [ka]

[0450] (Step 3) The compound obtained in Example 8 (1.6 mg) was dissolved in MeOH (0.1 mL), and 28% aqueous ammonia (0.05 mL) was added thereto, followed by stirring for 24 hours at 50° C. The resulting reaction solution was analyzed by LC-MS, and it was confirmed that the target compound was produced as a single diastereomer (Rp,Rp form) on the phosphorus atom.

[0451] MS(ESI)m / z:1121(M+H) + ,1119(MH) - .

[0452] A-4 Synthesis of Compound (IV) (A2 = Bz-adenine group) The starting compound (IV) used in the above synthesis scheme A-3 was synthesized according to the following scheme. [Synthetic Scheme A-4]

[0453] [ka]

[0454] Example 10: N-Benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-deoxy-2'-fluoro-3'-O-[hydroxy(oxo)-λ 5 Synthesis of [-phosphanyl]adenosine (corresponding to the compound of formula (IX))

[0455] [ka]

[0456] (Process 1) Commercially available N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-deoxy-2'-fluoroadenosine (1 g) was dissolved in pyridine (7.5 mL) and cooled to 0°C. Diphenyl phosphite (1.73 g) was added and stirred for 1 hour under cooling. Subsequently, water (3.7 mL) was added, followed by triethylamine (3.7 mL), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to 5 mL to obtain the target compound.

[0457] MS(ESI)m / z:740(M+H) + ,738(MH) - .

[0458] Example 11: N-Benzoyl-2'-deoxy-2'-fluoro-3'-O-[hydroxy(oxo)-λ 5 Synthesis of -phosphanyl]adenosine (corresponding to the compound of formula (IV))

[0459] [ka]

[0460] (Process 2) Dichloromethane (10 mL) was added to the solution obtained in Example 10 and cooled to 0°C. Under cooling, dichloroacetic acid was added until the pH reached 3, and the mixture was stirred for 24 hours. The reaction solution was purified by silica gel chromatography (ethyl acetate / methanol), concentrated to prepare an acetonitrile solution, and the pH was adjusted to 2 or less using concentrated hydrochloric acid. The white crystals that precipitated upon pH adjustment were filtered, washed with 80% aqueous acetonitrile, and dried to obtain the target compound (yield: 0.24 g, 37%).

[0461] MS(ESI)m / z:438(M+H) + ,436(MH) - . 1 H-NMR(DMSO-d6)δ:11.29(1H,brs),8.78(1H,s),8.73(1H,s),8.05(2H,m), 7.65(2H,t,J=7.5Hz),7.60(1H,d,J=1.5Hz),7.56(2H,t,J=7.5Hz),6.45(1 H,dd,J=16.5,3.5Hz),6.24(1H,d,J=1.5Hz),5.88-5.76(1H,m),5.22(1H,m ),4.25(1H,m),3.80(1H,dd,J=12.5,4.0Hz),3.67(1H,dd,J=12.5,4.0Hz).

[0462] A-5 Synthesis of CDN1 2 Cyclic dinucleotide (CDN1) was synthesized according to the following synthesis scheme A-5: A2 = Teoc-tetraazabenzo[cd]azulene group, Q = thiol group, PG1 = TBS, PG2 = DMTr, PG3 = Teoc, PG6 = TFAc, and (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole was used as (Rc-II). [Synthetic Scheme A-5]

[0463] [ka]

[0464] Example A5-1: Synthesis of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-[(1S,3aR)-tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (corresponding to the compound of formula (Rc-III))

[0465] [ka] (Process 1) A solution of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-(2,3,3-trimethylbutan-2-yl)-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (372.7 mg, 0.39 mmol) and molecular sieves 3A (80 mg) in dichloromethane (0.8 mL) was stirred under nitrogen at room temperature for 2 hours, then cooled to 0°C, and triethylamine (58.7 μL, 0.42 mmol) was added. The mixture was then purified by the method described in the literature (Tetrahedron A solution of (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (64.3 mg, 0.39 mmol) in dichloromethane (0.6 mL) was added. The mixture was stirred at room temperature for 1.5 hours to give a dichloromethane solution of the target compound.

[0466] Example A5-2: N,N-diethylethanaminium (2R,3R,4R,5R)-2-({[(R)-({(2R,3R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1, Synthesis of 6-dihydro-9H-purin-9-yl]oxolan-3-yl}oxy){[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-5-(6-{[2-[trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-4-fluorooxolan-3-yl phosphonate (corresponding to the compound of formula (Rc-V0))

[0467] [ka] (Process 2) A solution of N,N-diethylethanaminium 2-(trimethylsilyl)ethyl 2-{2-deoxy-2-fluoro-3-O-[oxide(oxo)-λ5-phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulene-6-carboxylate (200 mg, 0.32 mmol) in acetonitrile (2.0 mL) was added with molecular sieves 3A (60 mg) and stirred at room temperature for 2 hours. After cooling the solution to -10 ° C, triethylamine (18.1 μL, 0.13 mmol) was added, and the dichloromethane solution of the compound obtained in Example A5-1 was added dropwise, followed by washing in with 0.2 mL of dichloromethane. A 0.8 mol / L solution of 1-methylbenzimidazolium triflate in acetonitrile (809.4 μL, 0.65 mmol) was added, and after stirring for 1.5 hours, a 0.8 mol / L solution of 1-methylbenzimidazolium triflate in acetonitrile (121.4 μL, 0.10 mmol) was added. After confirming the completion of the reaction, triethylamine (158.0 μL, 1.13 mmol) and 1-(trifluoroacetyl)imidazole (66.3 μL, 0.58 mmol) were added at -10 °C, and the mixture was warmed to room temperature. After stirring for approximately 30 minutes, xanthan hydride (63.2 mg, 0.42 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated to approximately 2.5 mL and purified by silica gel column chromatography (0-12% methanol / acetonitrile containing 0.4% triethylamine) to obtain the target compound as a pale yellow solid (536.0 mg, 90% yield). HPLC analysis revealed that the ratio of diastereomers at the phosphorus atom generated during the reaction was 94.7:5.3.

[0468] MS(ESI)m / z:1734(M+H) + ,1732(MH) - . 1H-NMR(500MHz,DMSO-d6)δ:9.22(1H,s),9.10-9.02(1H,m),8.50(1H,s),8.45 (1H,s),8.39(1H,s),7.37-7.28(4H,m),7.26-7.16(7H,m),6.85-6.80(4H,m), 6.42(1H,dd,J=18.6,2.0Hz),6.25(1H,d,J=3.4Hz),6.16(1H,s),5.53-5.45(1 H,m),5.42-5.28(1H,m),4.89-4.78(1H,m),4.73-4.67(1H,m),4.57-4.49(2H, m),4.42-4.33(1H,m),4.28-4.19(3H,m),4.18-4.03(6H,m),4.02-3.84(5H,m) ,3.72(6H,s),3.69-3.56(4H,m),3.53-3.41(2H,m),3.16-3.04(7H,m),2.89-2 .72(2H,m),1.97-1.89(2H,m),1.84-1.65(4H,m),1.17(9H,t,J=7.2Hz),0.98- 0.94(2H,m),0.92-0.86(2H,m),0.71(9H,s),0.03--0.09(マイナス0.09)(24H,m). 31P-NMR(DMSO-d6)δ:69.43,1.45.

[0469] Example A5-3: N,N-Diethylethanaminium (2R,3R,4R,5R)-2-({[(R)-{[(2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-5-(hydrogen Synthesis of {2-[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-5-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-4-fluorooxolan-3-yl phosphonate (corresponding to the compound of formula (Rc-V))

[0470] [ka] (Step 3) Acetonitrile (15 mL) and ethanol (4.1 g, 89.84 mmol) were added to a dichloromethane (100 mL) solution of the compound obtained in Example A5-2 (4.1 g, 2.25 mmol) at room temperature. Dichloroacetic acid (0.9 g, 6.74 mmol) was added, and the mixture was stirred at room temperature for 15 hours. Triethylamine (0.91 g, 8.98 mmol) and water (50 mL) were added, and the mixture was stirred. The aqueous layer was discarded, and acetonitrile (22.5 mL), ethanol (6.2 g, 134.8 mmol), and water (50 mL) were added. The aqueous layer was discarded, and the solvent was distilled off under reduced pressure to approximately 15 mL. Acetonitrile (100 mL) was added, and the solvent was distilled off under reduced pressure to approximately 15 mL. This operation was repeated twice to obtain an acetonitrile solution (13.95 g) of the target compound.

[0471] Example A5-4: Sodium (2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2-oxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purine-9-yl [yl]-10-sulfanylidene-14-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)-10-{[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2-thiolate (corresponding to the compound of formula (Rc-VI))

[0472] [ka] (Step 4) A solution of T3P in acetonitrile (1.7 mol / L, 4.9 mL, 8.30 mmol) was added to a mixture of pyridine (42 mL) and dichloromethane (42 mL) and cooled to -20°C. An acetonitrile solution (13.67 g) of the compound obtained in Example A5-3 was added dropwise, and the mixture was stirred at -20°C for 1 hour. The diastereomeric ratio at the phosphorus atom generated during cyclization was 85:15. Xanthan hydride (0.62 g, 4.15 mmol) was added, and the mixture was warmed to approximately 0°C and stirred for 3 hours. 5% aqueous sodium bicarbonate solution (22.5 mL), ethyl acetate (40 mL), and sodium chloride (1.2 g) were added and stirred. The aqueous layer was discarded and washed with a solution of water (22.5 mL), sodium bicarbonate (1.1 g), and sodium chloride (2.4 g). The solvent in the resulting organic layer was distilled off under reduced pressure until the volume was approximately 15 mL, to obtain a pyridine solution of the target compound.

[0473] Example A5-5: Bis(N,N-diethylethanaminium)(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]-14-(6,7,8,9)-tetrahydro-2H-2,3,5,6-tetraazabene Zo [cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (corresponding to the compounds of formula (Rp, Rp-VII'))

[0474] [ka] (Step 5) A pyridine solution of the compound obtained in Example A5-4 was added with 28% aqueous ammonia (35 mL) and stirred at approximately 45°C for 15 hours. After cooling to room temperature, heptane (35 mL) was added and stirred. The organic layer was discarded, and the resulting aqueous layer was washed with heptane (35 mL). The aqueous layer was purified by preparative HPLC [10 mM aqueous triethylamine acetate / acetonitrile], and the resulting fraction was diluted with water. This solution was subjected to solid-phase extraction using silica gel and eluted with 0.1% triethylamine / acetonitrile solution. The resulting fraction was evaporated under reduced pressure to approximately 15 mL. Ethyl acetate (100 mL) was added, and the solvent was evaporated under reduced pressure to approximately 15 mL twice, followed by the addition of ethyl acetate (10 mL). This ethyl acetate solution was added dropwise to heptane (175 mL) at room temperature and stirred for 30 minutes. The solid was collected from the resulting suspension by filtration and washed with heptane (25 mL). The solid was dried at 40° C. overnight to give the desired compound as a white powder (1.9 g, 55% yield, HPLC: >99%).

[0475] A-6 Synthesis of compound (IV) (A2 = Teoc-tetraazabenzo[cd]azulene group) The starting compound (IV) used in the above synthesis scheme A-5 was synthesized according to the following scheme. [Synthetic Scheme A-6]

[0476] [ka]

[0477] Reference example A6-1: Synthesis of 2-{2-deoxy-2-fluoro-5-O-[(4-methoxyphenyl)(diphenyl)methyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene hydrochloride (corresponding to the hydrochloride of the compound of formula (VIII'))

[0478] [ka] (Process 1) To a solution of p-toluenesulfonate (34.2 g, 71.2 mmol) of the compound obtained in Example 25-2 in pyridine (171 mL) was added 4-methoxytrityl chloride (26.4 g, 85.4 mmol) at room temperature. After stirring at room temperature for approximately 24 hours, 5% aqueous sodium bicarbonate solution (171 mL) was added dropwise. Toluene (684 mL) was added, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 10% brine (171 mL) to obtain an organic layer. Pyridine hydrochloride (8.2 g, 71.2 mmol) was added and stirred for 1 hour. Further pyridine hydrochloride (12.3 g, 106.8 mmol) was added and stirred for approximately 23 hours. Crystals were collected from the resulting suspension by filtration and washed with toluene (103 mL) to obtain wet crystals of the hydrochloride salt of the target compound.

[0479] Example A6-1: 2-Methylpropan-2-aminium 2-{2-deoxy-2-fluoro-5-O-[(4-methoxyphenyl)(diphenyl)methyl]-3-O-[oxide(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulene-6-carboxylate (corresponding to the tert-butylamine salt of the compound of formula (IX'))

[0480] [ka] (Process 2) Ethyl acetate (342 mL) and 5% sodium bicarbonate (342 mL) were added to the wet crystals of the hydrochloride salt obtained in Reference Example A6-1 and stirred at room temperature. The aqueous layer was discarded, and the resulting organic layer was washed with 10% brine (171 mL). Acetonitrile (171 mL) was added to the organic layer, and the solvent was distilled off under reduced pressure to approximately 170 mL. Additional acetonitrile (342 mL) was added, and the solvent was distilled off under reduced pressure to approximately 170 mL, and triethylamine (61.2 g, 605.2 mmol) was added. Diphenyl phosphite (43.4 g, 185.1 mmol) was added dropwise, and the mixture was stirred at room temperature for 47 hours. Water (34.2 mL) was added, and the mixture was stirred for approximately 5 hours. Ethyl acetate (513 mL) and 23% potassium bicarbonate (239 mL) were added, and the mixture was stirred, and the aqueous layer was discarded. The organic layer was washed twice with 20% potassium bicarbonate (239 mL), and the solvent was evaporated under reduced pressure to approximately 170 mL. Acetonitrile (342 mL) was added, and the solvent was evaporated under reduced pressure to approximately 170 mL twice, followed by the addition of acetonitrile (342 mL). After the addition of tert-butylamine (2.3 mL, 21.4 mmol), seed crystals of the tert-butylamine salt of the target compound (34.2 mg) were added and stirred overnight. Furthermore, tert-butylamine (42.8 mL, 405.8 mmol) was added dropwise in three portions (5.3 mL, 15.0 mL, 22.5 mL). After stirring at room temperature, the mixture was cooled to -1 °C. After stirring at -1 °C for approximately 6 hours, the resulting suspension was filtered, and the crystals were washed with acetonitrile (102 mL). The crystals were dried overnight at 40°C to obtain the tert-butylamine salt of the target compound as white crystals (35.7 g, yield 70%).

[0481] MS(ESI)m / z:645(M+H) + ,643(MH) - . 1H-NMR(500MHz,DMSO-d6)δ:8.04(1H,s),7.84(2H,brs),7.60(1H,t,J=3.4Hz),7.42-7.37(4H,m ),7.32-7.23(8.5H,m),7.07(1H,s),6.88(2H,td,J=6.0,3.4Hz),6.35(1H,dd,J=17.2,2.3Hz),6. 07(0.5H,d,J=1.7Hz),5.30(0.5H,q,J=2.1Hz),5.20(0.5H,q,J=2.3Hz),4.90-4.79(1H,m),4.16 -4.13(1H,m),3.74(3H,s),3.31-3.26(2H,m),2.66-2.53(2H,m),1.87-1.82(2H,m),1.22(9H,s).

[0482] Example A6-2: Potassium 2-(trimethylsilyl)ethyl 2-{2-deoxy-2-fluoro-5-O-[(4-methoxyphenyl)(diphenyl)methyl]-3-O-[oxide(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulene-6-carboxylate (corresponding to the potassium salt of the compound of formula (IX))

[0483] [ka] (Step 3) A solution of 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene (59.2 g, 209.0 mmol) and N,N-diisopropylethylamine (36.0 g, 278.6 mmol) in acetonitrile (125 mL) was heated to 60°C, and the tert-butylamine salt (25.0 g, 34.8 mmol) obtained in Example A6-1 was added in three portions. After stirring at 60°C for approximately 2 days, the mixture was cooled to room temperature, and isopropylamine (10.3 g, 174.2 mmol) was added dropwise, followed by stirring for 2.5 hours. Isopropyl acetate (250 mL) and 10% aqueous ammonium chloride (250 mL) were added and stirred, after which the aqueous layer was discarded. The organic layer was washed with 10% aqueous ammonium chloride (250 mL) and then with 10% aqueous potassium carbonate (500 mL) twice. The organic layer was further washed with 10% aqueous potassium chloride solution (250 mL), and the solvent was distilled off under reduced pressure until the volume was approximately 125 mL, to obtain an acetonitrile solution (123.9 g) of the potassium salt of the target compound.

[0484] Example A6-3: N,N-Diethylethanaminium 2-(trimethylsilyl)ethyl 2-{2-deoxy-2-fluoro-3-O-[oxide(oxo)-λ 5 -phosphanyl]-β-D-ribofuranosyl}-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulene-6-carboxylate (corresponding to the compound of formula (IV))

[0485] [ka] (Step 4) Dichloromethane (417 mL) and methanol (55.4 mL) were added to an acetonitrile solution (121.2 g) of the potassium salt obtained in Example A6-2, followed by dropwise addition of dichloroacetic acid (22.0 g, 170.7 mmol) at room temperature. After stirring for approximately 27 hours, triethylamine (20.7 g, 204.8 mmol) was added dropwise to obtain a solution (658.1 g) of the target compound. The solvent was distilled off from the solution (26.9 g) of the target compound to approximately 5 mL under reduced pressure, and acetonitrile (3 mL), water (10 mL), and n-heptane (10 mL) were added. After stirring, an aqueous layer was obtained. The aqueous layer was washed with n-heptane (10 mL), and dichloromethane (20 mL) was added. The pH was adjusted to 8.5 with triethylamine (97 μL). Triethylamine hydrochloride (500 mg) was added, and the organic layer was obtained after stirring. Water (10 mL) and triethylamine hydrochloride (500 mg) were added to the organic layer, and after stirring, the aqueous layer was discarded. The resulting organic layer was evaporated to approximately 5 mL under reduced pressure. Acetonitrile (10 mL) was added, and the solvent was evaporated to approximately 5 mL under reduced pressure. This operation was repeated twice to obtain an acetonitrile solution of the triethylamine salt of the target compound (632.6 mg, 74% yield).

[0486] MS(ESI)m / z:517(M+H) + ,515(MH) - . 1H-NMR(500MHz,DMSO-d6)δ:8.51(1H,s),7.56(1H,s),7.30(0.5H,s),6.42(1H,dd,J=17.8 ,1.7Hz),6.12(0.5H,s),5.80(1H,brs),5.31-5.30(0.5H,m),5.20-5.19(0.5H,m),4.81-4. 72(1H,m),4.27-4.23(2H,m),4.02-3.91(3H,m),3.72-3.63(2H,m),3.07(6H,s),2.89(2H,t ,J=6.30Hz),1.99(2H,t,J=4.6Hz),1.17(9H,t,J=7.5Hz),1.00-0.97(2H,m),-0.01(9H,s).

[0487] A-7 Synthesis of CDN-linker The CDN-linker was synthesized according to the following scheme. [Synthetic Scheme A-7]

[0488] [ka]

[0489] Reference example A7-1: Synthesis of N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanine (corresponding to the compound of formula (XI))

[0490] [ka] (Process 1) To a suspension of 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (5.0 g, 12.4 mmol) and glycylglycyl-L-phenylalanine (4.2 g, 14.9 mmol) in acetonitrile (50 mL) and water (50 mL) was added triethylamine (2.3 mL, 16.1 mmol) at room temperature and stirred for approximately 4 hours. After adding 1 mol / L aqueous hydrochloric acid (16.2 mL), seed crystals of the target compound (4.6 mg) were added. After stirring overnight at room temperature, water (100 mL) was added dropwise over 2 hours to the resulting suspension. After stirring at 40 °C for approximately 1.5 hours, 1 mol / L aqueous hydrochloric acid (16.2 mL) was added. After stirring for 30 minutes, the mixture was cooled to room temperature and stirred for an additional 2 hours. The crystals were collected from the suspension by filtration and washed with acetonitrile / water (1 / 3, 50 mL) and dried overnight at 30° C. to obtain the target compound as white crystals (6.7 g, yield 95%).

[0491] 1H-NMR(500MHz,DMSO-d6)δ:12.8(1H,brs),8.15-7.95(3H,m),7.68-7.17(13H,m),5.01(1H,J=14.2Hz),4.41-4.37(1H,m),3.74-3.5 7(5H,m)3.05-3.01(1H,m),2.87(1H,dd,J=14.2,9.3Hz),2.68-2.59(1H,m),2.32-2.25(1H,m),2.09-2.03(1H,m),1.82-1.76(1H,m).

[0492] Example A7-1: N-[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-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 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp, Rp-X)) 1

[0493] [ka] (Process 2) Example A5- 5 To a solution of the compound (1 g, 0.76 mmol) obtained in (2) in dimethyl sulfoxide (3 mL) was added 70% aqueous tetrabutylammonium fluoride (3 mL) at room temperature and stirred at 30°C for 2 days. Acetonitrile (12 mL) and calcium chloride (0.59 g, 5.32 mmol) were added and stirred at room temperature for 14 hours. Triethylamine (1 mL) and water (2 mL) were added and stirred at room temperature for 2 hours. 5 mol / L aqueous hydrochl...

Claims

1. Formula (Rp-VII): 【Chemistry 1】 [In the formula, A1 is, 【Chemistry 2】 and Q is a thiol group or a hydroxyl group; PG1 is a protecting group for a hydroxy group, and PG3 is a protecting group for the amino group. A method for producing a compound represented by the formula: (Step a1) Formula (I): 【Chemistry 3】 [In the formula, PG1 and PG3 are as defined above, and PG2 is a protecting group for a hydroxy group. with an optically active phosphitylating agent (Rc-II) selected from the group consisting of the following formulae (Rc-II-1) and (Rc-II-2): 【Chemistry 4】 [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. to obtain a compound of formula (Rc-III): 【Chemistry 5】 [In the formula, PG1, PG2 and PG3 are as defined above, and B1 is, 【Chemistry 6】 where R1 and R2 are as defined above. obtaining a compound represented by the formula: (Step a2) The obtained compound of formula (Rc-III) is reacted with a compound of formula (IV): 【Chemistry 7】 [In the formula, A2 is, 【Chemistry 8】 and PG4 is a protecting group for the amino group. or a salt thereof in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and then reacting with a thiolating agent, followed by deprotection of PG2 to obtain a compound represented by the formula (Rc-V): 【Chemistry 9】 [In the formula, A2, PG1 and PG3 are as defined above; B2 is, 【Chemistry 10】 and PG6 is a protecting group for the amino group. or a salt thereof, (Step a3) The obtained compound of formula (Rc-V) or a salt thereof is cyclized in the presence of a condensing agent, and then reacted with a thiolation agent or an oxidizing agent to obtain a compound of formula (Rc-VI): 【Chemistry 11】 [In the formula, A2, B2, Q, PG1 and PG3 are as defined above. or a salt thereof; and (Step a4) A step of deprotecting B2, which is a protecting group at the phosphoric acid thiol site of the obtained compound of formula (Rc-VI), and PG4, which is a protecting group in A2, to obtain a compound of formula (Rp-VII) or a salt thereof. A method comprising:

2. In step a2, after the reaction with the thiolating agent and before the deprotection of PG2, the resulting intermediate is represented by the formula (Rc-V ０ ): 【Chemistry 12】 [In the formula, A2, B2, PG1, PG2 and PG3 are as defined in claim 1. The method according to claim 1, wherein the compound is a compound represented by the formula:

3. 3. The method according to claim 1, wherein the activating agent in step a2 is at least one selected from the group consisting of 1-phenylimidazole, benzimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-pyrrolidineacetonitrile, 1-(cyanomethyl)imidazole, and salts thereof.

4. The acylating agent or alkoxycarbonylating agent in step a2 is acetic anhydride, N-succinimidyl acetate, pentafluorophenyl acetate, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, 1-trifluoroacetylimidazole, benzoic anhydride, pentafluorophenyl benzoate, 1-tert-butoxycarbonyl-1,2,4-triazole, N-tert-butoxycarbonylimidazole, di-tert-butyl dicarbonate, 9-fluorenylmethylpentafluorophenyl carbonate, 1-[(9H and / or at least one selected from the group consisting of N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, and methylimidazole-1-carboxylate; and / or the thiolation agent in step a2 is at least one selected from the group consisting of xanthan hydride, bis(phenylacetyl)disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione, and / or the condensing agent in step a3 is at least one selected from the group consisting of 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide, dimethyl chlorophosphate, diethyl chlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphorane, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinohexafluorophosphate, bromotripyrrolidinohexafluorophosphate, and propylphosphonic anhydride; and / or the thiolation agent in step a3 is at least one selected from the group consisting of xanthan hydride, bis(phenylacetyl)disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione, and / or The method according to any one of claims 1 to 3, wherein the oxidizing agent in step a3 is at least one selected from the group consisting of iodine, tert-butyl hydroperoxide, 3-chloroperbenzoic acid, hydrogen peroxide, periodic acid, potassium permanganate, and oxygen.

5. PG4 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl, and / or The method of any one of claims 1 to 4, wherein PG6 is acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl.

6. The compound of formula (IV) or a salt thereof can be prepared by the following process: (Step a5) Formula (VIII): 【Chemistry 13】 [In the formula, A2 is as defined in claim 1, and PG5 is a protecting group for a hydroxy group. or a salt thereof with a phosphite to obtain a compound represented by formula (IX): 【Chemistry 14】 [In the formula, A2 and PG5 are as defined above.] or a salt thereof, Or, (Step a5') Formula (VIII'): 【Chemistry 15】 [In the formula, A1 is as defined in claim 1, and PG5 is as defined above.] or a salt thereof with a phosphite to obtain a compound represented by the formula (IX'): 【Chemistry 16】 [In the formula, A1 and PG5 are as defined above.] or a salt thereof; and (Step a5″) The obtained compound of formula (IX′) or a salt thereof is reacted with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (IX): 【Chemistry 17】 [In the formula, A2 and PG5 are as defined above.] or a salt thereof and (Step a6) A step of deprotecting PG5, which is a protecting group of the 5' hydroxyl group of the compound of formula (IX) obtained in Step a5 or Step a5″, to obtain a compound of formula (IV) or a salt thereof. The method according to any one of claims 1 to 5, wherein the compound is produced by

7. In step a3 according to claim 1, a step of obtaining a compound of formula (Rp-VII) (wherein Q is a thiol group) or a salt thereof by using the thiolation agent; In step a4 according to claim 1, the obtained compound of formula (Rp-VII) (wherein Q is a thiol group) is treated with silica gel column chromatography before being converted into a salt thereof, and / or is crystallized after being converted into a salt thereof to obtain a compound of formula (Rp, Rp-VII'): 【Chemistry 18】 or a salt thereof, (Step a7) The protecting groups PG1 and PG3 of the obtained compound of formula (Rp, Rp-VII′) are deprotected to obtain a compound of formula (Rp, Rp-X): 【Chemistry 19】 [In the formula, A1 is as defined in claim 1. or a salt thereof, (Step a8) The obtained compound of formula (Rp, Rp-X) or a salt thereof is Formula (XI): 【Chemistry 20】 or an active ester thereof, to obtain a compound represented by the formula (Rp,Rp-XII): 【Chemistry 21】 [In the formula, A1 is as defined above.] or a salt thereof; and (Step a9) The compound of formula (Rp, Rp-XII) or a salt thereof is bound to an antibody or a functional fragment of the antibody (hereinafter referred to as Ab) to obtain a compound of formula (Rp, Rp-XIII): 【Chemical 22】 [In the formula, m ranges from 1 to 10; The glycans of the Ab are optionally remodeled, Ab is bound to the compound of formula (Rp, Rp-XII) directly from the side chain of an optionally modified amino acid residue, or is bound to the compound of formula (Rp, Rp-XII) from a glycan or a remodeled glycan of Ab; A1 is, 【Chemistry 23】 It is.] Obtaining an antibody-immunostimulant conjugate represented by the formula: The method according to any one of claims 1 to 5, comprising:

8. The antibody is anti-HER2 antibody, anti-HER3 antibody, anti-DLL3 antibody, anti-FAP antibody, anti-CDH11 antibody, anti-CDH6 antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD2 antibody. 0 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-TROP2 antibody, anti-CEA antibody, anti-Cripto antibody, anti-Ep hA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mes 8. The method according to claim 7, wherein the antibody is selected from the group consisting of othelin antibody, anti-ENPP3 antibody, anti-CD47 antibody, anti-EGFR antibody, anti-GPR20 antibody, and anti-DR5 antibody.

9. Formula (Rc-III): 【Chemistry 24】 [In the formula, B1 is as defined in claim 1, PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. A compound represented by the formula:

10. Formula (Rc-V ０ ): 【Chemistry 25】 [In the formula, A2 and B2 are as defined in claim 1, PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. A compound represented by the formula:

11. Formula (Rc-V): 【Chemistry 26】 [In the formula, A2 and B2 are as defined in claim 1, PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. A compound represented by the formula:

12. The compound of formula (I) (wherein PG1 is tert-butyldimethylsilyl) can be prepared by the following steps: (Step b1) Formula (XIV): 【Chemistry 27】 [In the formula, PG2 is as defined in claim 1.] With the compound represented by formula (XV): 【Chemistry 28】 [In the formula, PG3 is as defined in claim 1, and X is Cl, Br, or I. by reacting with a compound represented by formula (XVI): 【Chemical 29】 [In the formula, PG2 and PG3 are as defined above.] and obtaining a compound represented by the formula: (Step b2) The obtained compound of formula (XVI) is reacted with a silylating agent to obtain a mixture of the compound of formula (I′) and the compound of formula (XVII) shown below: 【Chemistry 30】 [In the formula, PG2 and PG3 are as defined above.] and then converting the compound of formula (XVII) in the mixture to a compound of formula (I') in the presence of a base to obtain a compound of formula (I'). The method according to any one of claims 1 to 8, wherein the compound is produced by

13. The reaction of step b1 is carried out in the presence of a base, and the base is 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo[5.4.0]-7-undecene; and / or 13. The method according to claim 12, comprising the step of: in step b2, reacting the compound of formula (XVI) with a silylating agent in the presence of a first base to obtain a mixture of the compound of formula (I') and the compound of formula (XVII), and then converting the compound of formula (XVII) in the mixture to the compound of formula (I') in the presence of a second base to obtain the compound of formula (I').

14. The compound of formula (XV) can be prepared by the following process: (Step b3) Formula (XVIII): 【Chemistry 31】 [In the formula, PG3 is as defined in claim 1.] with 2-haloethanol in the presence of an acid or a base to obtain a compound of formula (XV). The method according to claim 12 or 13, wherein the compound is produced by

15. Formula (XV): 【Chemistry 32】 [In the formula, PG3 is 2-(trimethylsilyl)ethoxycarbonyl, and X is Cl, Br, or I. A compound represented by the formula:

16. The compound of formula (VIII') [In the formula, A1 is, 【Chemical 33】 and PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl. But the following steps: (Step c1) Formula (XIX): 【Chemical 34】 The compound represented by the formula (XX): 【Chemistry 35】 obtaining a compound represented by the formula: (Step c2) The obtained compound of formula (XX) is hydrolyzed to obtain a compound of formula (XXI): 【Chemical 36】 obtaining a compound represented by the formula: (Step c3) The obtained compound of formula (XXI) is reacted with a chlorinating agent to obtain a compound of formula (XXII): 【Chemical 37】 obtaining a compound represented by the formula: (Step c4) The obtained compound of formula (XXII) is converted into a compound of formula (XXIII): 【Chemical Formula 38】 with a compound represented by formula (XXIV): 【Chemical Formula 39】 obtaining a compound represented by the formula: (Step c5) The benzoyl group is deprotected from the obtained compound of formula (XXIV) to obtain a compound of formula (XXV): 【Chemistry 40】 or a salt thereof, (Step c6) reacting the obtained compound of formula (XXV) or a salt thereof with a tritylation agent to obtain a compound of formula (VIII') (wherein A1 and PG5 are as defined above) or a salt thereof. The method according to any one of claims 6 to 8 and 12 to 14, wherein the compound is produced by

17. moreover, (Step c7) The obtained compound of formula (VIII') (wherein A1 and PG5 are as defined in claim 16) or a salt thereof is reacted with a silylating agent, and then with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (XXVII): 【Chemistry 41】 [In the formula, PG4 is benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG5 is as defined above, and PG7 is a protecting group for a hydroxy group. obtaining a compound represented by the formula: (Step c8) The protecting group PG7 of the obtained compound of formula (XXVII) is deprotected to obtain a compound of formula (VIII): 【Chemistry 42】 [In the formula, A2 is, 【Chemistry 43】 and PG4 and PG5 are as defined above.] or a salt thereof 17. The method of claim 16, comprising:

18. The compound of formula (XXIII) can be prepared by the following process: (Step c12) Formula (XXXI): 【Chemistry 44】 is reacted with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain a compound represented by formula (XXXII): 【Chemistry 45】 obtaining a compound represented by the formula: (Step c13) The obtained compound of formula (XXXII) is reacted with propargylaldehyde diethyl acetal to obtain a compound of formula (XXVIII): 【Chemistry 46】 obtaining a compound represented by the formula: (Step c9) The tert-butoxycarbonyl group is deprotected from the obtained compound of formula (XXVIII) to obtain a compound of formula (XXIX): 【Chemistry 47】 obtaining a compound represented by the formula: (Step c10) The obtained compound of formula (XXIX) is subjected to an alkyne reduction reaction and then a reductive amination reaction in the presence of a catalyst to obtain a compound of formula (XXX): 【Chemistry 48】 and obtaining a compound represented by the formula: (Step c11) reacting the obtained compound of formula (XXX) with a benzoylating agent to obtain a compound of formula (XXIII) 18. The method of claim 16 or 17, wherein the compound is produced by

19. Formula (XX): 【Chemistry 49】 or Formula (XXII): 【Chemistry 50】 or Formula (XXIV): 【Chemistry 51】 or Formula (XXV): 【Chemistry 52】 A compound represented by the formula (I) or a salt thereof.

20. Formula (VIII'-1): 【Chemistry 53】 [In the formula, PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl.] A compound represented by the formula:

21. Formula (XXVII): 【Chemical 54】 [In the formula, PG4 is benzoyl, 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl or trityl; and PG7 is trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or triphenylsilyl. A compound represented by the formula:

22. Formula (XXVIII): 【Chemistry 55】 A compound represented by the formula:

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