Novel manufacturing method for antibody-immunostimulant conjugates

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] This invention relates to a novel stereoselective method for producing cyclic dinucleotide derivatives usable in antibody-immunostimulant conjugates, and to a production intermediate thereof. Furthermore, it relates to a novel method for producing cyclic dinucleotide linkers and antibody-immunostimulant conjugates, including the aforementioned production method. Moreover, it relates to a novel method for producing raw material compounds used in the production of cyclic dinucleotide derivatives, and to a production intermediate thereof. [Background technology]

[0002] Cyclic dinucleotides (CDNs) activate STING (Stimulator of Interferon Genes) (Non-Patent Document 1). This activation has been shown to enhance the STING-mediated antitumor immune response when CDNs are administered to tumor-bearing mice, significantly inhibiting tumor growth and improving 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 1-5). Antibody-drug conjugates (ADCs) in which CDNs exhibiting such activity are linked to antibodies via a linker are also being studied, and antitumor effects have been shown in tumors expressing antigens (Patent Documents 6, 7).

[0003] CDNs studied to date have two sulfur-modified pentavalent phosphate bonds called phosphorothioate bonds when two types of nucleotides are linked in a ring. When asymmetric nucleotides are cyclized, chiral centers are generated on the phosphorus atoms of each phosphorothioate group. In previously reported CDN synthesis methods, synthesis is carried out without controlling the chirality on the phosphorus atoms, and the CDN with the desired absolute configuration is obtained by purifying the four resulting diastereomers (e.g., Non-Patent Document 3, Patent Documents 1-7). In such synthesis methods, the generation of four diastereomers results in low yields, and because the diastereomers have similar physical properties, their separation and purification require strict preparative purification by HPLC at low loads. Therefore, synthesizing the target product in large quantities using such methods is burdensome. As a method for the stereoselective synthesis of CDNs, a method using a compound combining pentavalent phosphorus and a chiral auxiliary has been reported, but the yield at the time of cyclization remains low (Non-Patent Document 4). Furthermore, there have been several studies on stereoselective phosphorothioate synthesis methods related to stereocontrolled oligonucleotides combining trivalent phosphorus with asymmetric auxiliary groups (e.g., Non-Patent Documents 5-9, Patent Documents 8-9). On the other hand, there are no examples of dinucleotide bond generation using compounds applicable to cyclic dinucleotide synthesis, nor are there any 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 [Overview of the Initiative] [Problems that the invention aims to solve]

[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-immunostimulant conjugates that have STING agonist activity and activate immune cells.

[0007] The stereoselective synthesis of cyclic dinucleotides requires a method to combine a nucleotide with a strongly acidic functional group, such as phosphorous acid, with a nucleotide having an optically active amidite moiety that is highly unstable in acidic conditions, in a practical yield and with high stereoselectivity. However, conventional manufacturing methods are stereononselective, resulting in low yields of the target product and consequently, difficulty in purification, leading to extremely low overall yields.

[0008] Therefore, one of the objectives of the present invention is to provide a novel method for producing CDN that is industrially superior, by increasing the production rate of the target product using stereoselective synthesis, thereby reducing the purification load and improving the overall yield. Another objective is to provide a novel method for producing CDN-linker using this production method.

[0009] Furthermore, one of the objectives of the present invention is to provide a novel manufacturing method for preparing raw material compounds used in the synthesis of cyclic dinucleotides, which shortens the number of steps and improves the yield. [Means for solving the problem]

[0010] To solve the above problems, the inventors diligently investigated and discovered a stereoselective synthesis method for cyclic dinucleotide derivatives using an optically active phosphytylate, thereby reducing the burden of diastereomer purification and improving the overall yield, resulting in a novel manufacturing method. Furthermore, they constructed a CDN-linker using the CDN obtained by this manufacturing method, and performed antibody-immunostimulation using the CDN-linker. transformation We constructed a conjugation of the agent. Furthermore, we discovered a novel manufacturing method for preparing the raw material compounds used in the production of cyclic dinucleotides, which shortens the number of steps and improves the yield compared to conventional manufacturing methods, thus completing the present invention.

[0011] In other words, 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 hydroxyl group. PG1 is a protecting group for hydroxyl groups, and PG3 is a protecting group for amino groups. A method for producing a compound represented by or a salt thereof, (Step a1) Formula (I): [ka] [In the formula, PG1 and PG3 are as defined above, and PG2 is a protecting group for hydroxyl groups. The compound represented by is an optically active phosphytylate (Rc-II) selected from the group consisting of the following formulas (Rc-II-1) and (Rc-II-2): [ka] [In the formula, R1 is either hydrogen or methyl. R2 is hydrogen, an alkyl group having 1 to 3 carbon atoms, or a phenyl compound. Here, the alkyl is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl molecules, and The phenyl is either unsubstituted or substituted with nitro or methoxy. Reacting with this, equation (Rc-III): [ka] [In the formula, PG1, PG2 and PG3 are as defined above, and B1 is [ka] And R1 and R2 are as defined above. A step to obtain a compound represented by, (Step a2) The obtained compound of formula (Rc-III) is used for formula (IV): [ka] [In the formula, A2 is [ka] and PG4 is a protecting group for amino groups. The compound represented by or a salt thereof is reacted in the presence of an activator, then treated with an acylating agent or an alkoxycarbonylating agent, further reacted with a thiolating agent, and subsequently PG2 is deprotected to obtain the compound (Rc-V): [ka] [In the formula, A2, PG1, and PG3 are as defined above. B2 is [ka] and PG6 is an amino group protecting group. A step of obtaining a compound represented by or a salt thereof, (Step a3) The obtained compound of formula (Rc-V) or its salt is cyclized in the presence of a condensing agent, and then reacted with a thiolating agent or oxidizing agent to obtain formula (Rc-VI): [ka] [In the formula, A2, B2, Q, PG1, and PG3 are defined above. A step of obtaining a compound represented by or a salt thereof, and (Step a4) Deprotecting B2, which is the protecting group of the thiophosphate moiety of the obtained compound of formula (Rc-VI), and PG4, which is the protecting group in A2, to obtain the compound of formula (Rp-VII) or a salt thereof. Methods that include... [2] In step a2, the intermediate obtained after the reaction with the thiorating agent and before deprotecting PG2 is given by formula (Rc-V0): [ka] [In the formula, A2, B2, PG1, PG2, and PG3 are defined in [1]. The method according to [1], wherein the compound is represented by or a salt thereof. [3] Formula (Sp-VII): [ka] [In the formula, A1 is [ka] And, Q is a thiol group or a hydroxyl group. PG1 is a protecting group for hydroxyl groups, and PG3 is a protecting group for amino groups. A method for producing a compound represented by or a salt thereof, (Step a1) Formula (I): [ka] [In the formula, PG1 and PG3 are as defined above, and PG2 is a protecting group for hydroxyl groups. The compound represented by is an optically active phosphytylate (Sc-II) selected from the group consisting of the following formulas (Sc-II-1) and (Sc-II-2): [ka] [In the formula, R1 is either hydrogen or methyl. R2 is hydrogen, an alkyl group having 1 to 3 carbon atoms, or a phenyl compound. Here, the alkyl is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl molecules, and The phenyl is either unsubstituted or substituted with nitro or methoxy. Reacting with this, equation (Sc-III): [ka] [In the formula, PG1, PG2 and PG3 are as defined above, and B1 S teeth, [ka] And R1 and R2 are as defined above. A step to obtain a compound represented by, (Step a2) The compound of formula (Sc-III) obtained is given formula (IV): [ka] [In the formula, A2 is [ka] and PG4 is a protecting group for amino groups. A compound represented by or a salt thereof is reacted in the presence of an activator, then treated with an acylating agent or an alkoxycarbonylating agent, further reacted with a thiolating agent, and subsequently PG2 is deprotected to obtain the compound represented by formula (Sc-V): [ka] [In the formula, A2, PG1, and PG3 are as defined above. B2 Steeth, [ka] and PG6 is an amino group protecting group. A step of obtaining a compound represented by or a salt thereof, (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 oxidizing agent to obtain formula (Sc-VI): [ka] [In the formula, A2, B2 S Q, PG1, and PG3 are as defined above. A step of obtaining a compound represented by or a salt thereof, and (Step a4) B2 is the protecting group for the thiophosphate moiety of the compound of formula (Sc-VI) obtained. S and the process of deprotecting PG4, which is a protecting group in A2, to obtain a compound of formula (Sp-VII) or a salt thereof. Methods that include... [4] In step a2, the intermediate obtained after the reaction with the thiorating agent and before deprotecting PG2 is given by formula (Sc-V0): [ka] [In the formula, A2, B2 S PG1, PG2 and PG3 are [ 3 As defined in [ ]. A compound represented by or a salt thereof, [ 3 The method used in [ ]. [5] The method according to any one of [1] to [4], wherein the activator 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-butyldicarbonate, 9-fluorenylmethylpentafluorophenyl carbonate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide The method according to any one of [1] to [5], wherein at least one selected from the group consisting of cinimide, N-carbobendyloxysuccinimide, 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 thiorating 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-dithiazoline-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion. [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-dioxaphospholinane 2-oxide, dimethylchlorophosphate, diethylchlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphoran, 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinohexafluorophosphate, bromotripyrrolidinohexafluorophosphate, and propylphosphonic anhydride. [9] The method according to any one of [1] to [8], wherein the thiorating 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-dithiazoline-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion.

[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 is used in the following steps: (Step a5) Formula (VIII): [ka] [In the formula, A2 is as defined in [1], and PG5 is a protecting group for hydroxyl groups. A compound represented by or a salt thereof is reacted with a phosphite ester to obtain formula (IX): [ka] [In the formula, A2 and PG5 are as defined above.] A step of obtaining a compound represented by or a salt thereof, Or, (Step a5') Formula (VIII'): [ka] [In the formula, A1 is as defined in [1], and PG5 is defined as above. A compound represented by or a salt thereof is reacted with a phosphite ester to obtain formula (IX'): [ka] [In the formula, A1 and PG5 are as defined above.] A step of obtaining a compound represented by or a salt thereof, and (Step a5'') The compound of formula (IX') obtained or a salt thereof is reacted with an acylating agent or an alkoxycarbonylating agent to obtain formula (IX): [ka] [In the formula, A2 and PG5 are as defined above.] A step to obtain a compound represented by or a salt thereof. and (Step a6) A step to deprotect the PG5 protecting group of the 5' hydroxyl group of the compound of formula (IX) obtained in step a5 or step a5'' to obtain the compound of formula (IV) or a salt thereof. A method according to any one of [1] to

[12] , manufactured by [1].

[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 、Benzoyltrifluoromethanesulfonate, 3-benzoylthiazolidined-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butylcarbazate, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole 、 Di-tert-butyl dicarbonate, 9-fluorenylmethylpentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethylcarbazate, 19-fluorenylmethylcarbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(alyloxycarbonyloxy)succinimide, allylphenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzylcarbazate, benzylphenyl carbonate, N-carb The method according to

[13] or

[14] , wherein 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, ethylimidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, or methylimidazole-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 described in [1], the thioting agent is used to obtain a compound of formula (Rp-VII) (wherein Q is a thiol group) or a salt thereof.

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

[16] , further comprising obtaining a compound or a salt thereof.

[18] moreover (Step a7) Deprotect the protecting groups PG1 and PG3 of the compound of formula (Rp,Rp-VII') obtained to form formula (Rp,Rp-X): [ka] [In the formula, A1 is defined as in [1]. A step of obtaining a compound represented by or a salt thereof, and (Step a8) The compound of formula (Rp,Rp-X) obtained or a salt thereof, Formula (XI): [ka] It condenses with a compound represented by formula (Rp,Rp-XII) or its active ester to produce the compound (Rp,Rp-XII): [ka] [In the formula, A1 is as defined above.] A step to obtain a compound represented by or a salt thereof. The method described in

[17] , including the method described in

[17] .

[19] moreover, (Step a9) The compound of formula (Rp,Rp-XII) obtained or a salt thereof is combined with an antibody or a functional fragment of the antibody (hereinafter referred to as Ab) to form formula (Rp,Rp-XIII): [ka] [In the formula, m is in the range of 1 to 10. The glycans of Ab are arbitrarily remodeled. Ab is either directly bound to the compound of formula (Rp,Rp-XII) from the side chain of an amino acid residue that may be modified, or bound to the compound of formula (Rp,Rp-XII) from the glycan or remodeled glycan of Ab. A1 is [ka] That is the case. A process to obtain an antibody-immunostimulant conjugate represented by the above, or a mixture thereof. The method described in

[18] , including the method described in

[18] .

[20] The method according to

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

[19] or

[20] , wherein the antibody is selected from the group consisting of 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-CD20 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-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-ENPP3 antibody, anti-CD47 antibody, anti-EGFR antibody, anti-GPR20 antibody, and anti-DR5 antibody. [twenty two] Formula (Rc-III): [ka] [In the formula, B1 is defined as 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 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 or a salt thereof. [twenty five] The compound of formula (I) (wherein PG1 is tert-butyldimethylsilyl) is used in the following steps: (Step b1) Formula (XIV): [ka] [In the formula, PG2 is as defined in [1].] The compound represented by formula (XV): [ka] [In the formula, PG3 is as defined in [1], and X is Cl, Br, or I. When reacted with a compound represented by formula (XVI): [ka] [In the formula, PG2 and PG3 are as defined above.] A step of obtaining a compound represented by, (Step b2) The obtained compound of formula (XVI) is reacted with a silylating agent to form a mixture of the following compound of formula (I') and compound of formula (XVII): [ka] [In the formula, PG2 and PG3 are as defined above.] The process involves obtaining a compound, then, in the presence of a base, converting the compound of formula (XVII) in the mixture to the compound of formula (I') to obtain the compound of formula (I'). A method according to any one of [1] to

[21] , manufactured by [1].

[26] The method according to

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

[27] The method according to

[25] or

[26] , further comprising the step in step b2 of carrying out the reaction of 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] 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) is obtained in the following steps: (Step b3) Formula (XVIII): [ka] [In the formula, PG3 is as defined in [1].] The process involves reacting a compound represented by with 2-haloethanol in the presence of an acid or base to obtain a compound of formula (XV). A method according to any one of

[25] to

[30] , manufactured by

[25] to

[30] .

[32] 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. However, the following steps: (Step c1) Formula (XIX): [ka] The compound represented by formula (XX) is reacted with a benzoylating agent to obtain formula (XX): [ka] A step to obtain a compound represented by, (Step c2) The obtained compound of formula (XX) is hydrolyzed to obtain compound of formula (XXI): [ka] A step to obtain a compound represented by, (Step c3) The compound of formula (XXI) obtained is reacted with a chlorinating agent to obtain the compound of formula (XXII): [ka] A step to obtain a compound represented by, (Step c4) The obtained compound of formula (XXII) is used for formula (XXIII): [ka] When reacted with a compound represented by formula (XXIV): [ka] A step to obtain a compound represented by, (Step c5) The benzoyl group is deprotected from the obtained compound of formula (XXIV) to obtain formula (XXV): [ka] A step of obtaining a compound represented by or a salt thereof, (Step c6) A step in which the obtained compound of formula (XXV) or a salt thereof is reacted with a tritylating agent to obtain the compound of formula (VIII') (wherein A1 and PG5 are as defined above) or a salt thereof. A method according to any one of

[13] -

[21] and

[25] -

[32] , manufactured by [the specified method].

[35] moreover, (Step c7) The resulting 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 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 hydroxyl groups. A step of obtaining a compound represented by, (Step c8) The protecting group PG7 of the obtained compound of formula (XXVII) is deprotected to form formula (VIII): [ka] [In the formula, A2 is [ka] and PG4 and PG5 are defined above. A step to obtain a compound represented by or a salt thereof. The method described in

[34] , including the method described in

[34] .

[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 in step c3 produces tris(2,4-di-tert-butylphenyl) phosphite, tri-o- Ri The method according to

[34] or

[35] , carried out in the presence of at least one selected from the group consisting of triphenylphosphite, triethylphosphite, 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 tritylating 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 benzoylating agents, 2-(trimethylsilyl)ethoxycarbonylating agents, tert-butoxycarbonylating agents, 9-fluorenylmethyloxycarbonylating agents, allyloxycarbonylating agents, 2,2,2-trichloroethoxycarbonylating agents, and benzyloxycarbonylating agents.

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

[34] to

[39] , manufactured by

[34] .

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

[40] , manufactured by

[40] .

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

[34] -

[39] and

[41] , manufactured by [the specified method].

[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 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 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] Equation (I): [ka] [In the formula, PG1 is tert-butyldimethylsilyl, PG2 is a protecting group for hydroxyl groups, and PG3 is a protecting group for amino groups. A method for producing a compound represented by, (Step b1) Formula (XIV): [ka] [In the formula, PG2 is as defined above.] The compound represented by formula (XV): [ka] [In the formula, PG3 is as defined above, and X is Cl, Br, or I. When reacted with a compound represented by formula (XVI): [Chemical formula] [wherein, PG2 and PG3 are as defined above.] A step of obtaining a compound represented by, and (Step b2) Reacting the compound of formula (XVI) obtained in step (b2) with a silylating agent to obtain a mixture of a compound of formula (I') and a compound of formula (XVII): [Chemical formula] [wherein, PG2 and PG3 are as defined above.] and then, in the presence of a base, converting the compound of formula (XVII) in the mixture into the compound of formula (I') to obtain the 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] , which comprises, 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 a compound of formula (I') and a compound of formula (XVII), and then converting the compound of formula (XVII) in the mixture into the compound of formula (I') in the presence of a second base to obtain the compound of formula (I').

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

[57] 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, (Step c1) Formula (XIX): [Chemical formula] React the compound represented by the formula with a benzoylating agent to obtain a compound of formula (XX): [Chemical formula] Step of obtaining a compound represented by the formula: (Step c2) Hydrolyze the obtained compound of formula (XX) to obtain a compound of formula (XXI): [Chemical formula] Step of obtaining a compound represented by the formula: (Step c3) React the obtained compound of formula (XXI) with a chlorinating agent to obtain a compound of formula (XXII): [Chemical formula] Step of obtaining a compound represented by the formula: (Step c4) React the obtained compound of formula (XXII) with a compound represented by formula (XXIII): [Chemical formula] To obtain a compound of formula (XXIV): [Chemical formula] Step of obtaining a compound represented by the formula: (Step c5) Deprotect the benzoyl group from the obtained compound of formula (XXIV) to obtain a compound of formula (XXV) or a salt thereof: [Chemical formula] Step of obtaining a compound represented by the formula or a salt thereof: (Step c6) React the obtained compound of formula (XXV) or a salt thereof with a tritylating agent to obtain a compound of formula (VIII’) (wherein A1 and PG5 are as defined above) or a salt thereof A method comprising:

[59] Furthermore, (Step c7) The obtained compound of formula (VIII’) (wherein A1 and PG5 are 58As defined in [ ], ) or a salt thereof is reacted with a silylating agent, and then reacted with an acylating agent or an alkoxycarbonylating agent to form 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 hydroxyl groups. A step of obtaining a compound represented by, (Step c8) The protecting group PG7 of the obtained compound of formula (XXVII) is deprotected to form formula (VIII): [ka] [In the formula, A2 is [ka] and PG4 and PG5 are defined above. A step to obtain a compound represented by or a salt thereof. The method described in

[58] , including the method described in

[58] .

[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 in step c3 produces tris(2,4-di-tert-butylphenyl) phosphite, tri-o- Ri The method according to

[59] , carried out in the presence of at least one selected from the group consisting of triphenylphosphite, triethylphosphite, 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 tritylating 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 benzoylating agents, 2-(trimethylsilyl)ethoxycarbonylating agents, tert-butoxycarbonylating agents, 9-fluorenylmethyloxycarbonylating agents, allyloxycarbonylating agents, 2,2,2-trichloroethoxycarbonylating agents, and benzyloxycarbonylating agents.

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

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

[66] Formula (XXIV): [ka] A method for producing a compound represented by, (Step c14) Formula (XXII): [ka] The compound represented by formula (XXIX): [ka] It reacts with the compound represented by formula (XXXIII): [ka] A step of obtaining a compound represented by, (Step c15) The obtained compound of formula (XXXIII) is subjected to an alkyne reduction reaction, followed by a reductive amination reaction, in the presence of a catalyst, and then reacted with a benzoylating agent to obtain the compound of formula (XXIV). Methods that include... Regarding. [Effects of the Invention]

[0012] The present invention provides a stereoselective method for producing cyclic dinucleotides. This method reduces the burden of diastereomer purification and allows for the production of CDNs with the desired configuration in higher yields and in larger quantities. As a result, CDN linkers can be produced in high yields by CDN linker production methods including this stereoselective CDN production method. Furthermore, the present invention allows for the preparation of raw material compounds used in the production of cyclic dinucleotides in fewer steps and in higher yields. [Brief explanation of the drawing]

[0013] [Figure 1] This invention relates to a method for producing cyclic dinucleotides, and describes a step in which two types of nucleotides (compound (I) and compound (IV)) are stereoselectively linked via phosphorothieate using an optically active phosphytylate agent (Rc-II) to obtain a production intermediate (Rc-V). [Figure 2] The amino acid sequences of the single light chain of the anti-CD70 antibody (SEQ ID NO: 1) and the single heavy chain of the anti-CD70 antibody (SEQ ID NO: 2) are shown. [Figure 3] The amino acid sequences of the anti-CD70 antibody double chain (SEQ ID NO: 3) and the anti-CD70 antibody double chain (SEQ ID NO: 4) are shown. [Figure 4] The amino acid sequences of the anti-TROP2 antibody single light chain (SEQ ID NO: 5) and the anti-TROP2 antibody single heavy chain (SEQ ID NO: 6) are shown. [Figure 5] The amino acid sequences of the anti-TROP2 antibody double chain (SEQ ID NO: 7) and the anti-TROP2 antibody double chain (SEQ ID NO: 8) are shown. [Figure 6] The amino acid sequences of the anti-EGFR antibody single light chain (SEQ ID NO: 9) and the anti-EGFR antibody single heavy chain (SEQ ID NO: 10) are shown. [Figure 7] The amino acid sequences of the light chain (SEQ ID NO: 11) and heavy chain (SEQ ID NO: 12) of anti-EGFR antibody 2 are shown. [Figure 8] The following are the amino acid sequences of anti-CD70 antibody 1: CDRL1 (SEQ ID NO: 13), CDRL2 (SEQ ID NO: 14), CDRL3 (SEQ ID NO: 15), CDRH1 (SEQ ID NO: 16), CDRH2 (SEQ ID NO: 17), and CDRH3 (SEQ ID NO: 18). The CDR sequences were determined according to the Kabat definition. [Figure 9] The following are the amino acid sequences of anti-CD70 antibody 2: CDRL1 (SEQ ID NO: 19), CDRL2 (SEQ ID NO: 20), CDRL3 (SEQ ID NO: 21), CDRH1 (SEQ ID NO: 22), CDRH2 (SEQ ID NO: 23), and CDRH3 (SEQ ID NO: 24). The CDR sequences were determined according to the Kabat definition. [Figure 10] The following are the amino acid sequences of anti-TROP2 antibody 1: CDRL1 (SEQ ID NO: 25), CDRL2 (SEQ ID NO: 26), CDRL3 (SEQ ID NO: 27), CDRH1 (SEQ ID NO: 28), CDRH2 (SEQ ID NO: 29), and CDRH3 (SEQ ID NO: 30). The CDR sequences were determined according to the Kabat definition. [Figure 11]The following are the amino acid sequences of anti-TROP2 antibody 2: CDRL1 (SEQ ID NO: 31), CDRL2 (SEQ ID NO: 32), CDRL3 (SEQ ID NO: 33), CDRH1 (SEQ ID NO: 34), CDRH2 (SEQ ID NO: 35), and CDRH3 (SEQ ID NO: 36). The CDR sequences were determined according to the Kabat definition. [Figure 12] The following are the amino acid sequences of anti-EGFR antibody 1: CDRL1 (SEQ ID NO: 37), CDRL2 (SEQ ID NO: 38), CDRL3 (SEQ ID NO: 39), CDRH1 (SEQ ID NO: 40), CDRH2 (SEQ ID NO: 41), and CDRH3 (SEQ ID NO: 42). The CDR sequences were determined according to the Kabat definition. [Figure 13] The following are the amino acid sequences of anti-EGFR antibody 2: CDRL1 (SEQ ID NO: 43), CDRL2 (SEQ ID NO: 44), CDRL3 (SEQ ID NO: 45), CDRH1 (SEQ ID NO: 46), CDRH2 (SEQ ID NO: 47), and CDRH3 (SEQ ID NO: 48). The CDR sequences were determined according to the Kabat definition. [Figure 14] The amino acid sequences of the trastuzumab light chain (SEQ ID NO: 49) and the trastuzumab heavy chain (SEQ ID NO: 50) are shown. [Figure 15] The amino acid sequences of the light chain (SEQ ID NO: 49) and the heavy chain (SEQ ID NO: 51) of the modified anti-HER2 antibody are shown. [Figure 16] The amino acid sequences of the pertuzumab light chain (SEQ ID NO: 52) and the pertuzumab heavy chain (SEQ ID NO: 53) are shown. [Figure 17] The amino acid sequences of the light chain (SEQ ID NO: 52) and the heavy chain (SEQ ID NO: 54) of the modified anti-HER2 antibody 2 are shown. [Figure 18] The amino acid sequences of the light chain (SEQ ID NO: 55) and heavy chain (SEQ ID NO: 56) of the anti-CDH6 antibody are shown. [Figure 19] This diagram schematically represents antibody-immunostimulant conjugates (molecule of (XXXIV)) obtained from SG-type glycan remodeling antibodies (molecule of (XXXIV) in Figure 19A) and antibody-immunostimulant conjugates obtained from MSG-type glycan remodeling antibodies (molecule of (XXXIV) in Figure 19B). (a) represents immunostimulant D, (b) represents linker L, (c) represents PEG linker (L(PEG)), and (d) represents N297 glycan (where the white circle represents NeuAc(Sia), the white hexagon represents Man, the black hexagon represents GlcNAc, the white rhombus represents Gal, and the white inverted triangle represents Fuc). The white pentagon represents the triazole ring produced by the reaction of the alkyne derived from linker L and the azide group derived from the PEG linker. The Y-shape represents antibody Ab. The PEG linker is linked to the carboxyl group at position 2 of the sialic acid, which is located at the non-reducing end, via an amide bond. This method of representation applies throughout this specification unless otherwise specified. [Figure 20] The amino acid sequences of pertuzumab CDRL1 (SEQ ID NO: 57), CDRL2 (SEQ ID NO: 58), CDRL3 (SEQ ID NO: 59), CDRH1 (SEQ ID NO: 60), CDRH2 (SEQ ID NO: 61), and CDRH3 (SEQ ID NO: 62) are shown. The CDR sequences were determined according to the definition of IMGT. [Figure 21] The following are the amino acid sequences of the anti-CDH6 antibody: 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). The CDR sequences were determined according to the Kabat definition. [Modes for carrying out 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-immunostimulant conjugate having STING agonist activity and activating immune cells. Such an antibody-immunostimulant 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 CDN derivatives having the structure of the following formula (Rp-VII) or formula (Sp-VII).

[0016] [ka]

[0017] The following outlines a representative scheme of the present invention, focusing on a novel method for producing a CDN derivative of formula (Rp-VII), which is one embodiment of the invention.

[0018] [ka]

[0019] The following describes preferred embodiments for carrying out the present invention. The embodiments described below are merely examples of representative embodiments of the present invention and should not be interpreted as narrowing the scope of the invention. In this specification, the compound numbers shown in each reaction formula are used to indicate compounds. That is, they will be referred to as "compound of formula (I)," "compound (I)," etc. Compounds with other numbers will also be described similarly.

[0020] <1. Manufacturing of cyclic dinucleotides (CDNs) and CDN-linkers> <1-1. Method for producing cyclic dinucleotides (compounds (Rp-VII) and (Sp-VII)) and intermediates thereof>

[0021] In conventional manufacturing methods, the following four types of cyclic dinucleotides were synthesized stereononselectively (see Patent Document 6, etc.). The stereochemistry of these four types of cyclic dinucleotides is denoted as (Rp,Rp), (Sp,Rp), (Rp,Sp), and (Sp,Sp) in the order of (absolute stereochemistry of phosphorus at position 2, absolute stereochemistry of phosphorus at position 10) for the absolute stereochemistry of the two phosphorus atoms.

[0022] [ka]

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

[0024] [ka]

[0025] In another embodiment, the cyclic dinucleotide of formula (Sp-VII) is synthesized in the following synthesis scheme <a1-sp>It can be manufactured according to the following. [Synthesis scheme] <a1-sp>]

[0026] [ka]

[0027] The above [composite scheme] <a1-rp>and <a1-sp>]In, PG1 is a protecting group for hydroxyl groups, PG2 is a protecting group for hydroxyl groups, PG3 is an amino group protecting group, A1 is

[0028] [ka] And, A2 is

[0029] [ka] (Here, PG4 is a protecting group for the amino group.) And, Optically active phosphytylating agents (Rc-II) are,

[0030] [ka] (Here, R1 is hydrogen or methyl, R2 is hydrogen, a C1-C3 alkyl group, or phenyl, the alkyl group is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl groups, and the phenyl group is either unsubstituted or substituted with nitro or methoxy groups.) Therefore, optically active phosphytylating agents (Sc-II) are,

[0031] [ka] (Here, R1 and R2 are synonymous with the phosphytylate agent (Rc-II) mentioned above.) And, Synthesis scheme <a1-rp>In this case, B1 is,

[0032] [ka] The composition scheme <a1-sp>In B1 S teeth,

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

[0034] [ka] The composition scheme <a1-sp>In B2 S teeth,

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

[0036] In this manufacturing method, examples of PG1 include tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl. Preferably, tert-butyldimethylsilyl and trimethylsilyl are used. More preferably, tert-butyldimethylsilyl is used.

[0037] Examples of PG2 include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. Preferably, 4,4'-dimethoxytrityl, 4-methoxytrityl, or trityl is preferred. More preferably, 4,4'-dimethoxytrityl is preferred.

[0038] Examples of PG3 include 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. Preferably, 2-(trimethylsilyl)ethoxycarbonyl or allyloxycarbonyl is preferred. More preferably, 2-(trimethylsilyl)ethoxycarbonyl is preferred.

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

[0040] Examples of PG6 include acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl. Preferably, acetyl, trifluoroacetyl, benzoyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, or 2-(trimethylsilyl)ethoxycarbonyl. More preferably, acetyl, trifluoroacetyl, allyloxycarbonyl, or 2-(trimethylsilyl)ethoxycarbonyl.

[0041] The following describes each step. (Step a1) In one embodiment, this step involves reacting the compound of formula (I) with an optically active phosphytylate (Rc-II) to obtain the compound of formula (Rc-III). In another embodiment, this step involves reacting the compound with an optically active phosphytylate (Sc-II) to obtain the compound of formula (Sc-III).

[0042] In one embodiment, the optically active phosphytylate (Rc-II) used in this process is defined by the following formula (Rc-II-1) or formula (Rc-II-2):

[0043] [ka] [In the formula, R1 is either hydrogen or methyl. R2 is hydrogen, an alkyl group having 1 to 3 carbon atoms, or a phenyl compound. Here, the alkyl is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl molecules, and The phenyl is either unsubstituted or substituted with nitro or methoxy. Examples of compounds represented by can be given. The optically active phosphytylate is a reagent for stereoselectively condensing the compound of formula (I) and the compound of formula (IV) to produce the compound of formula (Rc-V), and reacts stereoselectively with the compound of formula (I) 、 An intermediate of equation (Rc-III) is generated.

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

[0045] The structures of the optically active phosphytylates (Rc-II) exemplified above are shown below. [Table 1]

[0046] In addition, the optically active phosphytylate (Sc-II) used in another embodiment may be the following formula (Sc-II-1) or formula (Sc-II-2):

[0047] [ka] [In the formula, R1 is either hydrogen or methyl. R2 is hydrogen, an alkyl group having 1 to 3 carbon atoms, or a phenyl compound. Here, the alkyl is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl molecules, and The phenyl is either unsubstituted or substituted with nitro or methoxy. Examples of compounds represented by [formula] can be given.

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

[0049] The structures of the optically active phosphytylates (Sc-II) exemplified above are shown below. [Table 2]

[0050] In this process, the reaction proceeds stereoselectively, and a compound of formula (III) having the desired absolute configuration can be obtained. Here, the absolute configuration of the compound of formula (III) is indicated using the absolute configuration (Rc) or (Sc) of the chiral carbon atom contained in the prolinol skeleton, as illustrated below.

[0051] [ka]

[0052] In one embodiment, when an optically active phosphytylate (Rc-II) in which R1 and R2 are hydrogen atoms is used, a compound of formula (Rc-III) with an absolute stereochemistry (Sp) on the phosphorus atom is produced, as shown below.

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

[0054] [ka]

[0055] In other words, by using an optically active phosphytylate agent with an absolute configuration of (Rc) or (Sc), the desired diastereomer can be obtained with a high selectivity of 90% or more, preferably 95% or more, and more preferably 98% or more. In conventional manufacturing methods, phosphorothioate bonds are formed stereoselectively, and (Rp) and (Sp) isomers are produced in approximately a 50:50 ratio, resulting in a yield of less than half of the compound having the desired absolute configuration.

[0056] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, these include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, and 2,6- Ji Examples 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 preferably it is 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 process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, acetonitrile, dichloromethane, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, and toluene can be used, and more preferably, dichloromethane and tetrahydrofuran can be used.

[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, and more preferably from -20°C to 30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 10 hours, and more preferably from 5 to 180 minutes.

[0059] (Step a2) In one embodiment, the step involves reacting the compound of formula (Rc-III) with the compound of formula (IV) or a salt thereof in the presence of an activator, then treating with an acylating agent or an alkoxycarbonylating agent, further reacting with a thiolating agent (also called a sulfurizing agent or thioling agent), and subsequently deprotecting PG2 to obtain the compound of formula (Rc-V). Optionally, the product may be isolated before deprotecting PG2. In another embodiment, the step involves obtaining the compound of formula (Sc-V) from the compound of formula (Sc-III) by a similar reaction. This step optionally includes treating with an organic or inorganic base to convert it to a salt thereof.

[0060] In this process, the intermediate obtained after reacting with the thiorating agent and before deprotecting PG2 is given by formula (Rc-V0): [ka] A compound represented by or a salt thereof (where A2, B2, PG1, PG2, and PG3 are the synthetic scheme) <a1-rp>This is synonymous with the base explained earlier. ) is obtained. In another embodiment, the intermediate obtained after reacting with the thiorating agent and before deprotecting PG2 is given by formula (Sc-V0): [ka] A compound represented by (where A2, B2) or a salt thereof (where A2, B2 S PG1, PG2, and PG3 are synthesized schemes <a1-sp>This is synonymous with the base explained earlier. ) is obtained.

[0061] In one embodiment, the reaction proceeds stereoselectively in this step, and a compound of formula (Rc-V) having the desired absolute configuration can be obtained via an intermediate of formula (Rc-V0). The absolute configurations of formulas (V) and (V0) are expressed using the absolute configuration of the chiral carbon atom contained in the prolinol skeleton within B2, similar to the compound of formula (III) described above.

[0062] [ka]

[0063] In this process, the reaction proceeds stereoinverted 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 constructed by formula (Rc-III). As a result, the desired diastereomer can 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. In conventional methods, the compound of formula (I) and the compound of formula (IV) condense stereononselectively, resulting in a mixture of the difficult-to-separate (Rp) and (Sp) isomers corresponding to the compound of formula (V) in approximately a 50:50 ratio. Therefore, regarding the yield of the desired compound (Rc-V), not only is there an improvement of about 2x from the standpoint of stereoselectivity, but considering the separation burden of a large amount of diastereomers on phosphorus that are difficult to separate, productivity is expected to improve by about 3 to 10 times.

[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-V0). Therefore, depending on the absolute configuration of the chiral carbon on the prolinol skeleton of the optically active phosphytylating agent used, a compound having the desired absolute configuration can be obtained stereoselectively.

[0065] The activator used in this step is not particularly limited as long as it allows the reaction to proceed without impairing the absolute stereochemistry of the compound represented by formula (Rc-III) or formula (Sc-III). Examples include 1-phenylimidazole, benzimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-pyrrolidineacetonitrile, 1-(cyanomethyl)imidazole, or salts thereof. Preferably, 1-phenylimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-(cyanomethyl)imidazole, or salts thereof are used, and more preferably, 1-phenylimidazole, 1-methylbenzimidazole, or salts thereof are used. The amount of activator used in this step is not limited as long as the reaction proceeds, but preferably it 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 its salt used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 3 equivalents, and 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 process is not particularly limited as long as the reaction proceeds, but examples 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. 、 Benzoyltrifluoromethanesulfonate, 3-benzoylthiazolidined-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butylcarbazate, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole 、 Di-tert-butyl dicarbonate, 9-fluorenylmethylpentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethylcarbazate, 19-fluorenylmethylcarbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(alyloxycarbonyloxy)succinimide Ianimide, allylphenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzylcarbazate, benzylphenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxy Examples of suitable materials include silates, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethylimidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, or methylimidazole-1-carboxylate. Preferably, acetic anhydride and 1-trifluoroacetyl Examples of suitable agents include imidazole, benzoic anhydride, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(alyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. More preferably, these include acetic anhydride, 1-trifluoroacetylimidazole, N-(alyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. The amount of 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, and 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, and more preferably from -20°C to 30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 5 hours, and more preferably from 5 to 90 minutes.

[0069] The reactions of the activator, acylating agent or alkoxycarbonylating agent and the thiolating agent in this step can preferably be 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 include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, magnesium sulfate, sodium sulfate, and calcium chloride. Preferably, molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, and sodium sulfate can be used, and more preferably molecular sieve 3A and molecular sieve 4A can be used. The amount of dehydrating agent used in this step is not limited as long as the reaction proceeds, but preferably it is 0.01 to 3 times the mass of the compound represented by formula (Rc-III) or formula (Sc-III), and more preferably it is 0.01 to 1 time the mass.

[0070] The thiolating agent used in this step is not particularly limited as long as the reaction proceeds, but for example, xanthan hydride, bis(phenylacetyl) disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazoline-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion can be used, and preferably xanthan hydride, bis(phenylacetyl) disulfide, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion can be used. The amount of thiolating agent used in this step is not limited as long as the reaction proceeds, but preferably it is 0.5 to 5 equivalents, and 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 thiolating 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, and more preferably from 0°C to 30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 5 hours, and more preferably from 5 to 90 minutes.

[0072] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji Examples of 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. Preferably, examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, 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 preferably it is 0.5 to 20 equivalents, and 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 of 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. Preferably, hydrochloric acid, acetic acid, monochloroacetic acid, and dichloroacetic acid are used, and more preferably, hydrochloric acid and dichloroacetic acid are used. The pH used for deprotection is not limited as long as the reaction proceeds, but preferably it is 1 to 4. The reaction temperature of the deprotection reaction is not limited as long as the reaction proceeds, but preferably it is from -30°C to the boiling point of the solvent used in the reaction, and more preferably from -20°C to 30°C. The reaction time of this reaction is not limited as long as the reaction proceeds, but preferably it is 0.5 to 48 hours, and more preferably it is 1 to 24 hours.

[0074] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, acetonitrile and dichloromethane can be mentioned, and more preferably, acetonitrile can be mentioned.

[0075] Organic bases used to convert compounds of formula (Rc-V) or formula (Sc-V) to their salts include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, and 2,6- Ji Examples include methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene. 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, the step involves cyclizing a compound of formula (Rc-V) or a salt thereof in the presence of a condensing agent, and then reacting it with a thioting agent or an oxidizing agent to obtain a compound of formula (Rc-VI). In another embodiment, the step involves obtaining a compound of formula (Sc-VI) from a compound of formula (Sc-V) by a similar reaction.

[0077] In this process, when reacted with a thioting agent, a compound of formula (VI) in which Q is a thiol group can be obtained. Also, in this process, when reacted with an oxidizing agent, a compound of formula (VI) in which Q is a hydroxyl group can be obtained.

[0078] The condensing agent used in this process is not particularly limited as long as it allows the reaction to proceed, but examples include 2-chloro-5,5-dimethyl-1,3,2-dioxaphospholinane 2-oxide, dimethylchlorophosphate, diethylchlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphoran, 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinohexafluorophosphate, bromotripyrrolidinohexafluorophosphate, propylphosphonic anhydride, diisopropylchlorophosphate, bis(2,6-dimethylphenyl)chlorophosphate, bis(dimethylamino)phosphoryl chloride, bis(2-oxo-3-oxy sa Zolidinyl phosphonate chloride, diphenylphosphinate chloride, diethyl chlorothiophosphate, (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 6-trifluoromethyl-1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, diphenyl phosphorochloride, bis(2,2,2-trichloroethyl)phosphochloride, ethyl dichlorophosphate, o-phenylene phosphorochloride, 3-(diethyl Examples of suitable solutions include toxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one, 2-chloro-1-methylpyridinium iodide, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate, (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, 1-adamantane carbonyl chloride, and pivaloyl chloride. Preferably, 2-chloro-5,5-dimethyl-1,3,2-dioxaphospholinane Examples of suitable compounds include 2-oxide, dimethyl chlorophosphate, diethyl chlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphoran, and propylphosphonic anhydride. More preferably, 2-chloro-5,5-dimethyl-1,3,2-dioxaphospholinane 2-oxide, 2-chloro-2-oxo-1,3,2-dioxaphosphoran, and propylphosphonic anhydride are used. The amount of condensing agent used in this step is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from -20°C to 0°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 10 hours, and more preferably from 5 minutes to 2 hours.

[0080] The thiolating agent used in this step is not particularly limited as long as the reaction proceeds, but for example, xanthan hydride, bis(phenylacetyl) disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazoline-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion can be used, and preferably xanthan hydride, bis(phenylacetyl) disulfide, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion can be used. The amount of thiolating agent used in this step is not limited as long as the reaction proceeds, but preferably it is 0.5 to 5 equivalents, and 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 thiolating 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, and more preferably from -20°C to -30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 10 hours, and more preferably from 5 minutes to 3 hours.

[0082] The oxidizing agent used in this process is not particularly limited as long as the reaction proceeds, but for example, iodine, bromine, tert-butyl hydroperoxide, 3-chloroperbenzoic acid, hydrogen peroxide, periodic acid, potassium permanganate, oxygen, and ozone can be used, and preferably iodine, 3-chloroperbenzoic acid, hydrogen peroxide, and oxygen can be used. The amount of oxidizing agent used in this process is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from -20°C to -30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 10 hours, and more preferably from 5 minutes to 3 hours.

[0084] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, and mixed solvents thereof can be used, and more preferably, pyridine can be used.

[0085] The absolute stereochemistry of the compound of formula (VI) is expressed by the absolute stereochemistry of the chiral carbon atom on the prolinol skeleton within B2. For the compound of formula (VI) where Q is a thiol group, the absolute stereochemistry of the two phosphorus atoms is expressed in the order of (absolute stereochemistry of phosphorus at position 2, absolute stereochemistry of phosphorus at position 10), as shown below. Combined with the stereochemistry of the chiral carbon atom on the prolinol skeleton, this is 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 process is not stereoselectively controlled, the formation of one diastereomer proceeds predominantly based on the three-dimensional structure of the molecule itself. For example, as shown in Examples 3, 8 and A5-4 of this specification, in the cyclization reaction from the compound of formula (Rc-V) to the compound of formula (Rc-VI), the (Rc,Rp,Rp-VI) isomer with the desired absolute configuration was obtained with a selectivity of approximately 70:30 to 85:15 compared to the (Rc,Sp,Rp-VI) isomer.

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

[0089] The ammonia used in the deprotection reaction of this process is not limited as long as the reaction proceeds, but preferably, 28% aqueous ammonia can be 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, and 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, and more preferably from 5 to 48 hours.

[0091] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, methanol, ethanol, acetonitrile, dichloromethane, and pyridine can be used, and more preferably, methanol and pyridine can be used.

[0092] Organic bases used to convert compounds of formula (Rp-VII) or formula (Sp-VII) to their salts include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate are used, and more preferably, triethylamine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate are used.

[0093] (Novel intermediate) The above [composite scheme] <a1-rp>A novel intermediate in ] is the compound represented by the following formula (Rc-III).

[0094] [ka] Here, B1 is,

[0095] [ka] The group is such that R1 is hydrogen or methyl, and R2 is hydrogen, a C1-C3 alkyl group or phenyl, wherein the alkyl group is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl groups, and the phenyl group is either unsubstituted or substituted with nitro or methoxy groups. More comfortably, B1 is

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

[0097] [ka] That is the case. PG1 is preferably tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldimethylsilyl, and more preferably tert-butyldimethylsilyl. PG2 is preferably 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl, and 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] Also, the above [composite scheme] <a1-rp>A novel intermediate in ] is the compound represented by the following formula (Rc-V0).

[0099] [ka] Here, A2 is preferably,

[0100] [ka] And, moreover,

[0101] [ka] That is the case. PG4 is an amino group protecting group, and preferably includes benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl. More preferably, it is benzoyl or 2-(trimethylsilyl)ethoxycarbonyl. PG1, PG2, and PG3 are equivalent to the respective groups of the compound of formula (Rc-III) shown above. Therefore, B2 is synonymous with B2 of the compound shown in the formula (Rc-V) below. ru.

[0102] Furthermore, the above [composite scheme] <a1-rp>A novel intermediate in ] is the compound represented by the following formula (Rc-V).

[0103] [ka] Here, A2 is synonymous with A2 in the compound of formula (Rc-V0) shown above. B2 is preferably,

[0104] [ka] R1 is hydrogen or methyl, and R2 is hydrogen, a C1-C3 alkyl group or phenyl, wherein the alkyl group is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl groups, and the phenyl group is either unsubstituted or substituted with nitro or methoxy groups. More preferably, B2 is

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

[0106] [ka] That is the case. PG6 is an amino group protecting group, preferably acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl, and more preferably acetyl or trifluoroacetyl. PG1 is preferably tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl, and 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] Furthermore, in step a4, the compound in formula (Rp-VII) where Q is a thiol group is treated by silica gel column chromatography before being converted to its salt, and / or crystallized after being converted to its salt, to obtain formula (Rp,Rp-VII'):

[0108] [ka] The further method involves obtaining a compound or a salt thereof.

[0109] Compounds represented by formula (Rp-VII) or (Sp-VII), particularly those represented by formula (Rp,Rp-VII'), obtained by the manufacturing method of the present invention, can preferably be used for CDN linkers represented by formula (Rp,Rp-XII) (hereinafter also referred to as CDN conjugate precursors), and antibody-immunostimulant conjugates in which an antibody is further conjugated to the CDN conjugate precursor. Furthermore, compounds of formula (Rp-VII) or (Sp-VII), obtained by the manufacturing method of the present invention, are not limited to these and can also be used for the production of antibody-immunostimulant conjugates having other chemical structures or for other applications.

[0110] <1-2. Comparison of the method for producing the compound (Rp-VII) of the present invention with conventional methods (Patent Document 6, etc.)> In the conventional method, cyclic dinucleotides are divided into four types in roughly equal proportions. Te Because a leomer mixture is generated, it was necessary to obtain the cyclic dinucleotide with the desired stereochemistry by column purification, which resulted in a very low yield. The present invention provides a method for producing the compound of formula (Rp-VII) that allows for the selective synthesis of the desired stereochemistry. As a result, compared to the synthesis scheme of Patent Document 6 shown below, it is possible to provide a production method with excellent effects such as improved total yield and avoidance of column purification.

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

[0112] [ka]

[0113] [Effects of the manufacturing method of formula (Rp-VII) of the present invention] In conventional synthesis routes (Patent Document 6), the stereochemistry on phosphorus is not controlled by the reaction, but rather by four types of dias Te The method involved synthesis using a Leomer mixture, followed by column purification to obtain the desired stereochemistry, resulting in extremely low yields (total yield of 7.5% across 8 reaction steps (indicated as containing impurities)). On the other hand, the manufacturing method of the present invention allows for stereoselective phosphorothioate bonding in the first step, resulting in an improvement of approximately 20-40% in the total yield from the compound of formula (I), and high quality with a yield of 92.4-99% as measured by HPLC analysis.

[0114] Note the above synthesis scheme <a1-rp>or <a1-sp>In step (a1), the compound of formula (I) is either formula (I-1) or formula (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 Y is independently H, F, or -O-PG1, where PG1 is a protecting group for the hydroxyl group. Using a compound represented by, In (step a2), the following equation (IV) is used as equation (IV-1) or equation (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 Z is independently H, F, or -O-PG1, where PG1 is a protecting group for the hydroxyl group. By using the compound represented by or a salt thereof, depending on the combination, it is also possible to synthesize four types of cyclic dinucleotide derivatives having the following 12-membered to 14-membered ring structures corresponding to formula (Rp-VII), or four types of cyclic dinucleotides (not shown) having the 12-membered to 14-membered ring structures 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-mentioned cyclic dinucleotide can be produced according to the following synthesis scheme. [Synthesis scheme] <a2>]

[0119] [ka]

[0120] The above [composite scheme] <a2>]In, A1 and A2 are the above [combination scheme] <a1>] is synonymous with, PG5 is a protecting group for hydroxyl groups.

[0121] In this manufacturing method, examples of PG5 include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. Preferably, 4,4'-dimethoxytrityl or 4-methoxytrityl can be used.

[0122] The following describes each step. (Step a5) and (Step a5') This step involves reacting a compound of formula (VIII) or formula (VIII') with a phosphite ester to obtain a compound of formula (IX) or formula (IX') or a salt thereof.

[0123] Examples of phosphite esters used in this process include diphenyl phosphite, methyl phosphite, diethyl phosphite, or dibutyl phosphite, with diphenyl phosphite and methyl phosphite being preferred, and more preferably diphenyl phosphite. Raise This can be achieved. The amount of phosphite ester 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 (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, and more preferably from -10°C to 30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 120 hours, and more preferably from 5 minutes to 72 hours.

[0125] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane can be used, and more preferably, pyridine and acetonitrile can be used.

[0126] Reagents used to convert compounds of formula (IX) or formula (IX') to their salts include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji Methylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, propylamine, iso-pro Pi Examples include triethylamine, butylamine, iso-butylamine, tert-butylamine, pentylamine, aniline, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium chloride, and potassium chloride. Preferably, triethylamine, tributylamine, iso-butylamine, tert-butylamine, pentylamine, aniline, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium chloride, and potassium chloride. Pi Examples include triethylamine, butylamine, iso-butylamine, tert-butylamine, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, sodium chloride, and potassium chloride. More preferably, triethylamine, tert-butylamine, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, sodium chloride, and potassium chloride can be used. The amount of base used to convert to salt is not limited as long as the reaction proceeds, but preferably, the formula (IX) or formula (IX') For the compound represented by [formula], the amount is 0.5 to 10 equivalents, more preferably 3 to 8 equivalents.

[0127] (Step a5'') This process involves acylating or alkoxycarbonylating a compound of formula (IX') or a salt thereof. Agent This is a step of reacting with to obtain the compound of formula (IX) or a salt thereof.

[0128] The acylating agent or alkoxycarbonylating agent used in this process is not particularly limited as long as the reaction proceeds, but examples 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. 、 Benzoyltrifluoromethanesulfonate, 3-benzoylthiazolidined-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butylcarbazate, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole 、 Di-tert-butyl dicarbonate, 9-fluorenylmethylpentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethylcarbazate, 19-fluorenylmethylcarbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(alyloxycarbonyloxy)succinimide, allylphenyl carbonate, N-(2 ,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzylcarbazate, benzylphenyl 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 include cyclonitrylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethylimidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, dimethyl carbonate, dimethyl dicarbonate, or methylimidazole-1-carboxylate. Preferably, these include acetic anhydride, 1-trifluoroacetylimidazole, benzoic anhydride, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(alyloxycarbonyl Examples include succinimide, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, and more preferably, 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 acylating agent or alkoxycarbonylating agent used in this process is not limited as long as the reaction proceeds, but is preferably 1 to 15 equivalents, and more preferably 3 to 8 equivalents, relative to the compound represented by formula (IX').

[0129] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji Examples of 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. Preferably, examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, 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 preferably it is 1 to 20 equivalents, and 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, and more preferably from 40°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 1 hour to 96 hours, and more preferably from 24 hours to 72 hours.

[0131] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, pyridine, 4-methylpyridine, 2-methyltetrahydrofuran, acetonitrile, and 1-methyl-2-pyrrolidone can be used, and more preferably, 2-methyltetrahydrofuran and acetonitrile can be used.

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

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

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

[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, and 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 1 hour to 96 hours, and more preferably from 5 hours to 48 hours.

[0136] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, methanol, acetonitrile, and dichloromethane can be used, and more preferably, dichloromethane can be used.

[0137] <1-4. Method for producing CDN-linker (compound (Rp,Rp-XII))> Compounds of formula (Rp,Rp-XII) or salts thereof can be prepared according to the following synthesis scheme. Compounds of formula (Rp,Rp-XII) are precursors for the production of antibody-immunostimulant conjugates. [Synthesis scheme] <a3>]

[0138] [ka]

[0139] The above [composite scheme] <a3>]In, PG1, PG3, and A1 are the above [combination scheme] <a1-rp>This is synonymous with ].

[0140] The following describes each step. (Step a7) This step involves deprotecting the protecting groups PG1 and PG3 of the compound of formula (Rp,Rp-VII') to obtain the compound of formula (Rp,Rp-X).

[0141] Examples of deprotective agents used in this process include ammonium fluoride, tetra-n-butylammonium fluoride, pyridine hydrogen fluoride, and triethylamine hydrofluoride. Preferably, ammonium fluoride and tetra-n-butylammonium fluoride are used, and more preferably, tetra-n-butylammonium fluoride is used. The amount of deprotective agent used in this process is not limited as long as the reaction proceeds, but preferably, it is 0.5 to 60 equivalents, and 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, and 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, and more preferably from 1 hour to 120 hours.

[0143] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, methanol, acetonitrile, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide can be used, and more preferably, tetrahydrofuran and dimethyl sulfoxide can be used.

[0144] (Step a8) This process involves 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 preferably be converted to an active ester and condensed with the compound represented by formula (Rp,Rp-X). 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, and more preferably 0.7 to 1.3 equivalents, relative to the compound represented by formula (Rp,Rp-X).

[0146] The conversion to the active ester in this step is not limited as long as the reaction proceeds, but can be done using a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD·HCl) or N,N'-dicyclohexylcarbodiimide (DCC) with additives such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, cyano(hydroxyimino)ethyl acetate, or p-nitrophenol, or 1-[bis(dimethylamino)methylene This can be done using a coupling agent such as ]-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), or 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM). Preferably, the reaction can be carried out using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole (HOBt), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole, or 4-(4,6-dimethoxy-1,3,5-triazine-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 preferably it is 0.5 to 5 equivalents relative to the compound represented by formula (Rp,Rp-X), and more preferably 0.7 to 2 equivalents. The amount of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole used in this process is not limited as long as the reaction proceeds, but is preferably 0.05 to 4 equivalents relative to the compound represented by formula (Rp,Rp-X), and more preferably 0.1 to 2 equivalents.The amount of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride used in this process is not limited as long as the reaction proceeds, but is preferably 0.3 to 5 equivalents relative to the compound represented by formula (Rp,Rp-X), and more preferably 0.7 to 2 equivalents.

[0147] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji Examples of 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. Preferably, examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and sodium acetate. More preferably, examples include triethylamine and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but preferably it is 0.5 to 10 equivalents, and 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, and 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, and more preferably from 1 hour to 24 hours.

[0149] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, water, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used.

[0150] The bases used to convert formula (Rp,Rp-XII) to its salt include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, and 2,6- Ji Methylpyridine, 4-dimethylaminopyridine, propylamine, iso-pro Pi Examples of organic bases include triamine, butylamine, iso-butylamine, tert-butylamine, pentylamine, and aniline, as well as 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. Preferably, examples include triethylamine, tert-butylamine, potassium 2-ethylhexanoate, potassium carbonate, potassium bicarbonate, sodium 2-ethylhexanoate, sodium carbonate, sodium bicarbonate, sodium chloride, and potassium chloride. More preferably, examples include triethylamine, potassium 2-ethylhexanoate, and sodium carbonate.

[0151] The solvents used to convert formula (Rp,Rp-XII) to its salt 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. Preferably, water, 2-propanol, acetonitrile, cyclopentyl methyl ether, and ethyl acetate, as well as mixed solvents thereof, can be used.

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

[0153] [ka]

[0154] The above [composite scheme] <b1>]In, PG1, PG2, and PG3 are the above [composite scheme] <a1-rp>] is synonymous with, X is Cl, Br, or I.

[0155] In this manufacturing method, PG1 represents a hydroxyl group protecting group, and examples of such protecting groups include tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl. Preferably, tert-butyldimethylsilyl and trimethylsilyl are used. More preferably, tert-butyldimethylsilyl is used.

[0156] PG2 represents a protecting group for a hydroxyl group, and examples of such protecting groups include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. Preferably, 4,4'-dimethoxytrityl, 4-methoxytrityl, or trityl is used. More preferably, 4,4'-dimethoxytrityl is used.

[0157] PG3 represents an amino group protecting group, and examples of such protecting groups include 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. Preferably, 2-(trimethylsilyl)ethoxycarbonyl or allyloxycarbonyl is preferred. More preferably, 2-(trimethylsilyl)ethoxycarbonyl is preferred.

[0158] The following describes each step. (Step b1) This process involves reacting the compound of formula (XIV) with the compound of formula (XV) to obtain the compound of formula (XVI).

[0159] The reaction in this step is carried out in the presence of a base, which can 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, and more preferably 1,1,3,3-tetramethylguanidine. 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 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 between 10°C and the boiling point of the solvent used in the reaction, and more preferably between 20°C and 50°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably between 30 minutes and 72 hours, and more preferably between 5 hours and 36 hours.

[0161] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone can be used, and more preferably, 1,3-dimethyl-2-imidazolidinone can be used.

[0162] (Step b2) This step involves reacting the compound of formula (XVI) with a silylating agent to produce a mixture of the compound of formula (I') and the compound of formula (XVII):

[0163] [ka] The next step is to obtain the compound of formula (XVII) in the mixture, and then, in the presence of a base, convert the compound of formula (XVII) in the mixture to the compound of formula (I') to obtain the compound of formula (I').

[0164] Furthermore, this process includes carrying out the reaction of 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 from 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 mixture solution and the difference in solubility between the compound of formula (XVII) and the compound of formula (I').

[0166] Here, the first base can be 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, 2,6-lutidine, or 1,8-diazabicyclo[5.4.0]-7-undecene, preferably 1,1,3,3-tetramethylguanidine, 2,6-lutidine, or 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferably 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 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, and more preferably from 40°C to 70°C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably from 30 minutes to 72 hours, and more preferably from 5 hours to 36 hours.

[0168] The solvent used in the reaction using the first base in this step is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone can be used, and more preferably, 1,3-dimethyl-2-imidazolidinone can be used.

[0169] Examples of silylation agents used in this step include tert-butyldimethylchlorosilane or tert-butyldimethylsilyl triflate, with tert-butyldimethylchlorosilane being preferred. The amount of silylation 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 2 to 4 equivalents, relative to the compound represented by formula (XIV).

[0170] Furthermore, the second base can be 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, 2,6-lutidine, or 1,8-diazabicyclo[5.4.0]-7-undecene, preferably 1,1,3,3-tetramethylguanidine, 2,6-lutidine, or 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferably 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 preferably it is 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, and 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, and 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, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone can be used, and more preferably, tetrahydrofuran can be used.

[0173] The second solvent used in the reaction using the second base in this step is not particularly limited as long as it does not inhibit the reaction, but 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, tetrahydrofuran, heptane, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone can be used, and more preferably, heptane can be used.

[0174] <2-2. Method for producing the compound of formula (XV) and its intermediates> The compound of formula (XV) used in the preparation of formula (I) above can be prepared according to the following synthesis scheme. [Synthesis scheme] <b2>]

[0175] [ka]

[0176] The above [composite scheme] <b2>]In, PG3 is the above [synthesis scheme] <a1>] is synonymous with, X is the above [composite scheme] <b1>This is synonymous with ].

[0177] The following describes step b3. (Step b3) This process involves reacting a compound represented by formula (XVIII) with 2-haloethanol in the presence of an acid or a base to obtain a compound of formula (XV).

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

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

[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, and more preferably from -10°C to 20°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 36 hours, and more preferably from 1 hour to 10 hours.

[0181] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and mixed solvents thereof can be used. Preferably, 1,2-dimethoxyethane, 2-methyltetrahydrofuran and 1,4-dioxane can be used, and more preferably, 1,2-dimethoxyethane can be used.

[0182] (Novel intermediate) The above synthesis [scheme] <b1>A novel intermediate in ] is the compound represented by the following formula (XV).

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

[0184] <2-3. Comparison of the method for producing compound (I) of the present invention with conventional methods (Patent Document 6, etc.)> The present invention provides a method for producing the compound of formula (I) that addresses the limitations of conventional methods, specifically the selective synthesis of the tert-butyldimethylsilylated compound at the 3' position among the 2',3' hydroxyl groups. As a result, compared to the synthesis scheme of Patent Document 6 shown below, this method offers superior effects such as improved overall yield, reduced number of steps, and avoidance of column purification.

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

[0186] [ka]

[0187] [Effects of the manufacturing method of the present invention (I)]

[0188] (Effect 1) Selective synthesis of tert-butyldimethylsilylated compound (I) of the 3'-position hydroxyl group among the 2',3'-position hydroxyl groups. In conventional synthesis routes (Patent Document 6), the yield of the target compound of formula (I) was low, approximately 35%, because the tert-butyldimethylsilylation step (step 5) failed to selectively tert-butyldimethylsilylate the 3' position of the hydroxyl group at the 2' and 3' positions, resulting in the formation of a byproduct where the 2' position is tert-butyldimethylsilylated. Furthermore, while a method for selectively silylation of the 2' and 3' hydroxyl groups using a chiral imidazole to selectively triethylsilylate the 3' position is known (see Org. Lett., Vol. 15, No. 18, 2013, pp. 4710-4713), it has the problem that it requires a special chiral imidazole and 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 amount of expensive silver reagent equal to or greater than the equivalent amount of the reaction substrate. Moreover, when this was carried out under similar conditions using the present substrate, the desired selectivity could not be obtained.

[0189] On the other hand, in the production method of the present invention, after the non-selective introduction of TBS groups to the hydroxyl groups at the 2',3' positions, conditions for the transfer of TBS groups between the 2' and 3' positions were found, and conditions were found for only the compound in which the target 3'-position hydroxyl group represented by formula (I) is TBS-modified to crystallize, thereby making it possible to obtain the target compound of formula (I) in a favorable manner.

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

[0191] On the other hand, in the manufacturing method of the present invention, by discovering an alkylating agent (compound of formula (XV)) in which the N-alkylation of the nucleic acid base amide group proceeds while the hydroxyl groups at the 2' and 3' positions remain unprotected, the protection and deprotection steps for the hydroxyl groups at the 2' and 3' positions are unnecessary, making it possible to provide a manufacturing method that is two steps shorter.

[0192] (Effect 3) Improved yield and avoidance of column purification throughout the entire scheme. 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). However, in the production method of the present invention, it is possible to synthesize the compound of formula (I) from the starting material inosine with a yield of approximately 60% (3 steps). Furthermore, in the conventional synthesis route (Patent Document 6), the product was obtained by performing silica gel column chromatography purification at every step, which resulted in an increase in the amount of waste (such as silica gel, hexane, and developing solvents such as ethyl acetate) and a longer lead time. However, in the production method of the present invention, it is possible to avoid silica gel column chromatography, thereby reducing the environmental impact and improving productivity.

[0193] <3. Preparation of starting material compounds (VIII) and formula (VIII') used in the production of cyclic dinucleotides> <3-1. Method for producing compounds of formula (VIII) and formula (VIII') and their intermediates> The compounds of formula (VIII) and (VIII') used in the production of the above-mentioned cyclic dinucleotide can be prepared according to the following synthesis scheme. [Synthesis scheme] <c1>]

[0194] [ka]

[0195] The above [composite 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] The following describes each step. (Step c1) This process involves reacting the compound of formula (XIX) with a benzoylating agent to obtain the compound of formula (XX).

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

[0198] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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, and mixtures thereof. Preferably, triethylamine, 2,6- Ji Examples of suitable bases include methylpyridine, 4-dimethylaminopyridine, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and sodium acetate, as well as mixtures thereof. More preferably, a mixture of triethylamine and 4-dimethylaminopyridine is used. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.1 to 10 equivalents, and 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, and more preferably from -10°C to 35°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 24 hours, and more preferably from 0.5 hours to 10 hours.

[0200] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and mixed solvents thereof can be used. Preferably, acetonitrile and N,N-dimethylformamide can be used, and more preferably, acetonitrile can be used.

[0201] (Step c2) This process involves hydrolyzing a compound represented by formula (XX) to obtain a compound represented by formula (XXI).

[0202] Examples of acids used in this process include hydrochloric acid, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and sulfuric acid. Preferably, hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, and sulfuric acid are used, and more preferably, p-toluenesulfonic acid is used. The amount of acid used in this process is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from 40°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 24 hours, and more preferably from 1 hour to 10 hours.

[0204] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and mixed solvents thereof can be used. Preferably, 1,2-dimethoxyethane, 2-methyltetrahydrofuran, tetrahydrofuran, cyclopentyl methyl ether, and mixed solvents thereof can be used, and more preferably, a mixed solvent of cyclopentyl methyl ether and water can be used.

[0205] (Step c3) This process involves reacting the compound of formula (XXI) with a chlorinating agent to obtain the compound of formula (XXII).

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

[0207] Furthermore, the reaction in this step can preferably be 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 tris(2,4-di-tert-butylphenyl) phosphite, tri-o- Ri Examples include triphenylphosphine, triethylphosphine, tributylphosphine, triphenylphosphine, tris(dimethylamino)phosphine, or tris(diethylamino)phosphine, preferably tris(2,4-di-tert-butylphenyl)phosphine, tri- o -to Ri Examples include phosphorus phosphite and triphenyl phosphite, and more preferably tris(2,4-di-tert-butylphenyl) phosphite. The amount of phosphorus reagent used in this step is not limited as long as the reaction proceeds, but preferably it is 0.5 to 6 equivalents, and 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, and more preferably from -20°C to -30°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 24 hours, and more preferably from 1 hour to 10 hours.

[0209] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and mixed solvents thereof can be used. Preferably, dichloromethane, 1,2-dimethoxyethane, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, and mixed solvents thereof can be used, and more preferably, dichloromethane, cyclopentyl methyl ether, and mixed solvents thereof can be used.

[0210] (Step c4) This process involves reacting the compound of formula (XXII) with the compound of formula (XXIII) to obtain the compound of formula (XXIV).

[0211] The reaction in this step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, cesium carbonate, potassium carbonate, and sodium carbonate are used, and more preferably, cesium carbonate is used. The amount of base used in this step is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from 10°C to 50°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 24 hours, and more preferably from 1 hour to 10 hours.

[0213] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide can be used, and more preferably, dimethyl sulfoxide can be used.

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

[0215] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, 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 preferably, 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, and 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, and more preferably from 0°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 24 hours, and more preferably from 1 hour to 10 hours.

[0217] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and mixed solvents thereof can be used. Preferably, water, methanol, ethanol, tetrahydrofuran, and mixed solvents thereof can be used, and more preferably, a mixed solvent of ethanol or water and tetrahydrofuran can be used.

[0218] Acids used to convert the compound of formula (XXV) to 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. Preferably, acetic acid, p-toluenesulfonic acid, and hydrochloric acid are used. More preferably, p-toluenesulfonic acid and hydrochloric acid are used. The amount of acid used to convert to the salt is not limited as long as the reaction proceeds, but preferably, it is 0.5 to 5 equivalents, and more preferably, 1 to 3 equivalents, relative to the compound represented by formula (XXIV).

[0219] (Step c6) This process involves reacting a compound of formula (XXV) or a salt thereof with a tritylated agent to obtain a compound of formula (VIII') or a salt thereof.

[0220] Examples of tritylated agents used in this step include 4,4-dimethoxytrityl chloride, 4-methoxytrityl chloride, 2-chlorotrityl chloride, or trityl chloride, with 4,4-dimethoxytrityl chloride and 4-methoxytrityl chloride being preferred. The amount of tritylated 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, and more preferably from 0°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, and more preferably from 1 hour to 36 hours.

[0222] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane can be used, and more preferably, pyridine can be used.

[0223] (Step c7) This process involves reacting a compound represented by formula (VIII') or a salt thereof with a silylating agent, and then reacting it with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (XXVII).

[0224] Examples of silylation agents for this process include trimethylchlorosilane, trimethylsilyl triflate, triethylchlorosilane, triethylsilyl triflate, triisopropylchlorosilane, triisopropylsilyl triflate, tert-butyldimethylchlorosilane, tert-butyldimethylsilyl triflate, tert-butyldiphenylchlorosilane, tert-butyldiphenylsilyl triflate, triphenylchlorosilane, or triphenylsilyl triflate, with tert-butyldimethylsilyl triflate being preferred.

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

[0226] The reaction temperature with the acyling 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, and more preferably from 0°C to 40°C. The reaction time with the acyling agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 24 hours, and more preferably from 1 hour to 10 hours.

[0227] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane can be used, and more preferably, pyridine can be used.

[0228] (Step c8) This process involves the compound represented by formula (XXVII). of This is a step in which the protecting group PG7 is deprotected to obtain the compound of formula (VIII).

[0229] Examples of deprotective agents used in this process include ammonium fluoride, tetra-n-butylammonium fluoride, pyridine hydrogen fluoride, and triethylamine hydrofluoride. Preferably, ammonium fluoride and tetra-n-butylammonium fluoride are used, and more preferably, tetra-n-butylammonium fluoride is used. The amount of deprotective agent used in this process is not limited as long as the reaction proceeds, but preferably, it is 0.1 to 5 equivalents, and 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, and more preferably from 0°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 24 hours, and more preferably from 1 hour to 10 hours.

[0231] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, methanol, acetonitrile, tetrahydrofuran, and dichloromethane can be used, and more preferably, tetrahydrofuran can be used.

[0232] (Novel intermediate) The above [composite scheme] <c1>A novel intermediate in ] is the compound represented by the following formula (XX).

[0233] [ka]

[0234] The above [composite scheme] <c1>A novel intermediate in ] is the compound represented by the following formula (XXII).

[0235] [ka]

[0236] The above [composite scheme] <c1>A novel intermediate in ] is the compound represented by the following formula (XXIV).

[0237] [ka]

[0238] The above [composite scheme] <c1>A novel intermediate in ] is the compound represented by the following formula (XXV) or a salt thereof.

[0239] [ka]

[0240] The above [composite scheme] <c1>Examples of novel intermediates in ] include compounds represented by the following formula (VIII'-1) or salts thereof.

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

[0242] The above [composite scheme] <c1>Examples of the novel intermediate in [ ] include compounds represented by the following formula (XXVII).

[0243] [Chemical formula 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. Process for Producing the Compound of Formula (XXIII) and Its Intermediate> The compound of formula (XXIII) used in the production of the above formula (VIII) can be produced according to the following synthetic scheme. [Synthetic scheme <c2>]

[0244] [ka]

[0245] The following describes each step. (Step c9) This process involves deprotecting the tert-butoxycarbonyl group from the compound represented by formula (XXVIII) to obtain the compound represented by formula (XXIX).

[0246] This step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, potassium hydroxide, potassium bicarbonate, sodium carbonate, and sodium hydroxide are used, and more preferably, sodium hydroxide. The amount of base used in this step is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from 0°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 24 hours, and from 1 hour to 10 hours.

[0248] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, methanol, ethanol, 1-propanol, and tetrahydrofuran can be used, and more preferably, ethanol can be used.

[0249] (Step c10) This process involves performing an alkyne reduction reaction followed by a reductive amination reaction on the compound of formula (XXIX) in the presence of a catalyst to obtain the compound of formula (XXX).

[0250] A metal catalyst can be used as the catalyst for the reduction reaction of the alkyne in this process. The metal catalyst used in this process is not particularly limited as long as it catalyzes hydrogenation, but preferably, ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, or nickel catalysts are used, and more preferably, palladium catalysts are used. The amount of metal catalyst used in this process is not limited as long as the reaction proceeds, but preferably, it is 0.01 to 1 times the mass of the compound represented by formula (XXVIII), more preferably, 0.02 to 0.4 times the mass, and more preferably, 0.05 to 0.2 times the mass. The hydrogen gas pressure is usually 100 to 1000 kPa, preferably 100 to 700 kPa, and more preferably 200 to 500 kPa. The reaction temperature in this process is not limited as long as the reaction proceeds, but preferably, it is from 0°C to the boiling point of the solvent used in the reaction, and more preferably, 20°C to 80°C. The reaction time for this process 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 alkynes in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and mixed solvents thereof can be used, and more preferably, 1-methyl-2-pyrrolidone can be used.

[0252] A metal catalyst can be used as the 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, but preferably, ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, or nickel catalysts are used, and more preferably, palladium catalysts are used. The amount of metal catalyst used in this step is not limited as long as the reaction proceeds, but preferably, it is 0.01 to 1 times the mass of the compound represented by formula (XXVIII), more preferably, 0.02 to 0.4 times the mass, and more preferably, 0.05 to 0.2 times the mass. The hydrogen gas pressure is usually 100 to 1000 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, but preferably, it is from 0°C to the boiling point of the solvent used in the reaction, and more preferably, 20°C to 80°C. The reaction time for this process 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, but 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. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, water, acetic acid, and mixed solvents thereof can be used, and more preferably, a mixed solvent of 1-methyl-2-pyrrolidone, water, and acetic acid can be used.

[0254] (Step c11) This process involves reacting the compound of formula (XXX) with a benzoylating agent, followed by debenzoylation by base treatment to obtain the compound of formula (XXIII).

[0255] Examples of benzoylating agents used in this process include benzoyl chloride, benzoyl bromide, benzoic anhydride, or benzoyl trifluoromethanesulfonate, with benzoyl chloride being preferred. Zo The amount of ylating agent 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 (XXX).

[0256] The reaction with the benzoylating agent in this step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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, and mixed bases thereof. Preferably, 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 preferably, examples include mixed bases 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, and 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, and more preferably from 0°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, and more preferably from 1 hour to 24 hours.

[0258] The solvent used in the reaction with the benzoylating agent in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone can be used, and more preferably, 1,3-dimethyl-2-imidazolidinone can be used.

[0259] The base used in the base treatment of this process is not particularly limited as long as the reaction proceeds, but examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, potassium hydroxide, potassium bicarbonate, sodium carbonate, and sodium hydroxide are used, and more preferably, triethylamine is used. The amount of base used in the base treatment of this step is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from 0°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, and more preferably from 1 hour to 24 hours.

[0261] The solvent used for the base treatment in this process is not particularly limited as long as it does not inhibit the reaction, but 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, chlorobenzene, and 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, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone can be used, and more preferably, a mixed solvent of methanol and 1,3-dimethyl-2-imidazolidinone can be used.

[0262] The above [composite scheme] <c2>As an intermediate in ], the compound represented by the following formula (XXVIII) can be cited.

[0263] [ka]

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

[0265] [ka]

[0266] The following describes each step. (Step c12) This process involves reacting the compound of formula (XXXI) with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain the compound of formula (XXXII).

[0267] Examples of tert-butoxycarbonylating agents used in this step include di-tert-butyl dicarbonate, N-tert-butoxycarbonylimidazole, N-tert-butoxycarbonyl-1,2,4-triazole, and N-(tert-butoxycarbonyloxy)phthalimide, with di-tert-butyl dicarbonate being preferred. The amount of tert-butoxycarbonylating 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 (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, and more preferably from 0°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 24 hours, and more preferably from 1 hour to 10 hours.

[0269] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, acetonitrile, tetrahydrofuran, ethyl acetate, benzene, and toluene can be used, and more preferably, toluene can be used.

[0270] (Step c13) This step involves reacting a compound represented by formula (XXXII) with propargylaldehyde diethyl acetal to obtain a compound represented by formula (XXVIII).

[0271] This process can preferably be 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 process is not particularly limited as long as the reaction proceeds, but for example, divalent palladium salts and their complexes such as palladium(II) acetate, palladium(II) trifluoroacetate, palladium(II) chloride, palladium(II) bromide, palladium(II) iodide, and bis(triphenylphosphine)palladium(II) dichloride can be used, and 0-valent palladium metals and their complexes such as palladium black, palladium carbon, tetrakistriphenylphosphinepalladium(O), and bis(dibenzylideneacetone)palladium(O) can be used, and bis(triphenylphosphine)palladium(II) dichloride can preferably be used. The amount of palladium catalyst used in this process is not limited as long as the reaction proceeds, but preferably it is 0.0001 to 1 equivalent, and more preferably 0.005 to 0.05 equivalents, relative to the compound represented by formula (XXXII).

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

[0273] Furthermore, this step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji Examples of 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, and mixed bases thereof. Preferably, examples include triethylamine, diisopropylethylamine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene, and more preferably, triethylamine. The amount of base used in this step is not limited as long as the reaction proceeds, but preferably it is 0.5 to 10 equivalents, and 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, and more preferably from 15°C to 50°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, and more preferably from 1 hour to 24 hours.

[0275] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, N,N-dimethylformamide, N,N-dimethylacetamide, and 1-methyl-2-pyrrolidone can be used, and more preferably, N,N-dimethylformamide can be used.

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

[0277] [ka]

[0278] The following describes each step. (Step c14) This process involves reacting the compound of formula (XXII) with the compound of formula (XXIX) to obtain the compound of formula (XXXIII).

[0279] The reaction in this step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, cesium carbonate, potassium carbonate, and sodium carbonate are used, and more preferably, cesium carbonate is used. The amount of base used in this step is not limited as long as the reaction proceeds, but preferably it is 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, and more preferably from 10°C to 50°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, and more preferably from 1 hour to 24 hours.

[0281] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide can be used, and more preferably, dimethyl sulfoxide can be used.

[0282] (Step c15) This process involves reacting a compound represented by formula (XXXIII) with an alkyne in the presence of a catalyst, followed by a reductive amination reaction, and then with a benzoylating agent to obtain a compound represented by formula (XXIV).

[0283] In this process, a metal catalyst can be used as a catalyst for the reduction and reductive amination reactions of alkynes. The metal catalyst used in this process is not particularly limited as long as it catalyzes hydrogenation, but preferably, ruthenium, rhodium, palladium, platinum, or nickel catalysts are used, and more preferably, palladium catalysts are used. The amount of metal catalyst used in this process is not limited as long as the reaction proceeds, but preferably, it is 0.01 to 1 times the mass of the compound represented by formula (XXXIII), more preferably 0.02 to 1 time, and more preferably 0.05 to 0.5 times the mass. The hydrogen gas pressure is usually 100 to 1000 kPa, preferably 100 to 900 kPa. More preferably, The pressure is 200-700 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, and more preferably from 15°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 96 hours, and more preferably from 1 hour to 36 hours.

[0284] The solvent used in the reduction reaction of alkynes in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, ethanol, methanol, 1-propanol, 2-propanol, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and mixed solvents thereof can be used, and more preferably, ethanol can be used.

[0285] The solvent used in the reductive amination reaction in this process is not particularly limited as long as it does not inhibit the reaction, but 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. Preferably, ethanol, water, acetic acid, and mixed solvents thereof can be used, and more preferably, a mixed solvent of water and acetic acid can be used.

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

[0287] The benzoylation in this step can preferably be 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 include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6- Ji 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. Preferably, triethylamine, 2,6- Ji Examples of bases include methylpyridine, 4-dimethylaminopyridine, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and sodium acetate, with 4-dimethylaminopyridine being more preferred. The amount of base used in this step is not limited as long as the reaction proceeds, but preferably, formula (XXXIII) For the compound represented by [formula], the amount is 0.05 to 10 equivalents, more preferably 0.2 to 5 equivalents.

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

[0289] The solvent used for benzoylation in this process is not particularly limited as long as it does not inhibit the reaction, but 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, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane can be used, and more preferably, pyridine can be used.

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

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

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

[0293] [ka]

[0294] [Effects of the manufacturing method of the present invention (VIII)]

[0295] (Effect 1) Improvement of the 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 from commercially available 5-Iodotubercidin in 11 steps. However, because it is a linear synthesis, the overall yield was low at 5%, which was a problem.

[0296] On the other hand, in the manufacturing method of the present invention, by using convergent synthesis, the number of steps from the readily available raw materials, compound (XXXI) and 2'-deoxy-2'-fluorouridine, is reduced to 10 steps each, and the total yield is 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) Conventionally, an acid hydrolysis method is known for synthesizing 2-deoxy-2-fluororibose derivatives such as compound (XXI) by hydrolyzing dihydrouridine derivatives such as 2'-deoxy-2'-fluoro-5,6-dihydrouridine (see Carbohydrate Res., I, 1966, pp. 455-466). However, when this method is applied to compound (XIX) to synthesize compound (XXI), a side reaction occurs: deprotection of the benzoyl group of the hydroxyl group, resulting in a low yield.

[0298] On the other hand, in the production method of the present invention, when the uridine portion of compound (XIX) is benzoylated to compound (XX), the reactivity to hydrolysis is improved, deglycosylation can be performed under milder conditions, and the deprotection of the benzoyl group is suppressed, resulting in an improved yield.

[0299] (Effect 3) Synthesis of compound (XXII) by selective α-chloromination reaction of compound (XXI) Conventionally, the chlorolysis reaction of 2-fluororibose derivatives involves protecting the hydroxyl group at the 2,5 position with a TBDMS group. Ta A method has been reported for treating 2-fluororibose derivatives under mesylchloride / triethylamine conditions, but this method yields chlorosaccharides as a mixture of α and β forms (see J.Org.Chem.,2009,74,pp.5779-5789 and International Publication No. 2011 / 003018). Another method has been reported for treating 2-fluororibose derivatives with the hydroxyl groups at the 2,5 positions protected by benzyl groups with hexamethylphosphite / carbon tetrachloride, but this method produces the β form and could not be applied to the synthesis of the α form (see International Publication Nos. 2014 / 124430 and International Publication Nos. 2015 / 038596).

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

[0301] Furthermore, the chlorination reaction of alcohols using triarylphosphine and a chlorinating agent has been known for a long time as a conventional method. For example, the chlorination reaction using 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 generates triphenylphosphine oxide after the reaction, requiring purification using a silica gel column to remove it, which posed a challenge 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 instead of triaryl phosphite, the by-product phosphate ester can be removed by liquid-liquid separation, successfully avoiding column purification.

[0303] (Effect 4) Synthesis of compound (XXIII) via the Boc protection route In the conventional synthesis route (Patent Document 6 / Conventional Synthesis Scheme 2), the yield of the Sonogashira reaction step in 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, by using compound (XXXII) with a Boc protecting group as a step, the yield of the Sonogashira reaction step is improved, and the overall yield is improved to 53%.

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

[0305] In this specification, "functional antibody fragment" is also called "antigen-binding antibody fragment," and refers to a partial fragment of an antibody that has antigen-binding activity, including Fab, F(ab')2, Fv, scFv, diabody, linear antibodies, and polyspecific antibodies formed from antibody fragments. Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions, is also included in antibody antigen-binding fragments. However, the term is not limited to these molecules as long as they possess antigen-binding ability. Furthermore, these antigen-binding fragments include not only full-length antibody proteins treated with appropriate enzymes, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0306] In this specification, "functional fragment" includes a functional fragment that retains asparagine (Asn297) and surrounding amino acids that are modified by well-conserved N-linked glycans in the Fc region of the IgG heavy chain, and that has the ability to bind to an antigen.

[0307] The antibody used in the production of the antibody-immunostimulant conjugate of the present invention refers to an immunoglobulin, which is a molecule containing an antigen-binding site that binds immunospecifically to an antigen. The antibody of the present invention may be any of the classes IgG, IgE, IgM, IgD, IgA, and IgY, but IgG is preferred. Furthermore, any of the subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 may be used, but IgG1, IgG2, or IgG4 are preferred (including antibodies having mutations in the Fc region of the IgG heavy chain that affect ADCC and ADCP activity).

[0308] When IgG1 is used as the antibody isotype for the production of the antibody-immunostimulant conjugate of the present invention, the effector function can be adjusted by substituting some of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, WO94 / 29351). Examples of IgG1 variants include IgG1 LALA mutations (IgG1-L234A, L235A). L234A and L235A represent substitutions of leucine to alanine at positions 234 and 235, respectively, as identified 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 antibody molecules have three complementarity determining regions (CDRs) in both the heavy and light chains. CDRs, also known as hypervariable regions, are areas within the variable regions of the antibody's heavy and light chains that exhibit particularly high variability in their primary structure. They are separated into three distinct locations on the primary structure of the heavy and light chain polypeptide chains. In this specification, the CDRs of the heavy chain are denoted as CDRH1, CDRH2, and CDRH3, starting from the amino-terminal end of the heavy chain amino acid sequence, while the CDRs of the light chain are denoted as CDRL1, CDRL2, and CDRL3, starting from the amino-terminal end of the light chain amino acid sequence. These regions are in close proximity to each other in terms of their three-dimensional structure and determine the specificity for the antigen they bind to.

[0310] The antibodies may originate from any species, but preferably from humans, rats, mice, and rabbits. If they originate from a species other than humans, it is preferable to chimerize or humanize them using well-known techniques. The antibodies of the present invention may be polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are preferred. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, as well as chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, or modified versions thereof.

[0311] The antibody is preferably an antibody that targets tumor cells or immune cells, but is not limited to these. The antibody is more preferably an antibody that targets tumor cells.

[0312] The binding affinity of antibodies to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay that visualizes antibodies taken up into cells using a fluorescence microscope with a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay that measures the amount of fluorescence when taken up into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay (Bio Techniques 28: 162-165, January 2000), which uses an immunotoxin that binds to the therapeutic antibody and suppresses cell proliferation by releasing a toxin upon uptake into cells. Recombinant complex proteins of the catalytic domain of diphtheria toxin and protein G can also be used as immunotoxins.

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

[0314] The antitumor activity of immunostimulants and antibody-immunostimulant conjugates refers to cytotoxic activity against tumor cells, anti-cellular effects, and regression of tumor volume. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems.

[0315] The immunostimulatory activity of immunostimulants and antibody-immunostimulator conjugates refers to the increased sensitivity of tumor cells to immune cells or the activation of immune cells via tumor cells. 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 conjugate of the present invention include, but are not limited to, 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-CD20 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-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-ENPP3 antibody, anti-CD47 antibody, anti-EGFR antibody, anti-GPR20 antibody, or anti-DR5 antibody. Preferably, the antibody of the present invention is 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 the production of the antibody-immunostimulant conjugate of the present invention can be obtained by immunizing animals with antigenic polypeptides using methods commonly practiced in this field, and then collecting and purifying the antibodies produced in vivo. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from non-human animals such as mice and rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity between the obtained antibodies that bind to the heterologous antigens and human antigens.

[0318] Furthermore, monoclonal antibodies can also be obtained by establishing hybridomas and fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells, according to known methods (for example, Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennett, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0319] Antigens can be obtained by genetically modifying host cells to produce the gene that codes for the antigen protein.

[0320] The humanized antibodies used in the production of the antibody-immunostimulant conjugate of the present invention can be obtained by 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 (US5821337, 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-immunostimulant conjugate of the present invention is not particularly limited, but it is preferable to have, for example, the following characteristics. (1) Anti-HER2 antibody characterized by having the following properties; (a) It specifically binds to HER2. (b) It has the activity to be 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 described in (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) An antibody according to any of (1) to (3) above, having antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity. (5) An antibody as described in any of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a mutation that results in a reduction of 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 described in SEQ ID NO: 50 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 49. (8) The antibody described in (6) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucine at positions 234 and 235, as indicated by the EU index, is replaced with alanine. (9) The antibody according to (8) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 51 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 49. (10) The antibody according to (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 53 and a light chain consisting of the amino acid sequence described 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 described in SEQ ID NO: 54 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 52. (12) The antibody according to (1) to (3), (6) or (8) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 57, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 58, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 59, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 60, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 61, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 62. (13) An antibody according to any of (1) to (12) above, wherein one or two amino acids are deleted at the heavy chain carboxyl terminus. (14) An antibody obtained by a method for producing an antibody, comprising the steps of culturing host cells transformed with an expression vector containing a polynucleotide encoding an antibody as described in any of (1) to (13) above, and collecting the antibody of the target from the culture obtained in the said step.

[0323] The anti-CD70 antibody used in the production of the antibody-immunostimulant conjugate of the present invention is not particularly limited, but may have the following characteristics, for example. It would be preferable if it were one of those. (1) An anti-CD70 antibody that specifically binds to CD70. (2) The antibody described in (1) above that binds to the extracellular domain of CD70. (3) The antibody described in (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) An antibody according to any of (1) to (3) above, having antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity. (5) An antibody as described in any of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a mutation that results in a reduction of ADCC and ADCP activity. (7) The antibody described in (6) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucine at positions 234 and 235, as indicated by the EU index, is replaced with alanine. (8) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence described 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 described in SEQ ID NO: 4 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 3. (10) The antibody according to (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 13, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 14, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 15, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 16, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 17, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 18. (11) The antibody according to (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 19, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 20, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 21, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 22, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 23, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 24. (12) An antibody according to any of (1) to (11) above, wherein one or two amino acids are deleted at the heavy chain carboxyl terminus. (13) An antibody obtained by a method for producing an antibody, comprising the steps of culturing host cells transformed with an expression vector containing a polynucleotide encoding an antibody as described in any of (1) to (12) above, and collecting the antibody of the target from the culture obtained in the said step.

[0324] The anti-TROP2 antibody used in the production of the antibody-immunostimulant conjugate of the present invention is not particularly limited, but it is preferable to have, for example, the following characteristics. (1) An anti-TROP2 antibody that specifically binds to TROP2. (2) The antibody described in (1) above that binds to the extracellular domain of TROP2. (3) The antibody described in (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) An antibody according to any of (1) to (3) above, having antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity. (5) An antibody as described in any of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a mutation that results in a reduction of 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 described in SEQ ID NO: 6 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 5. (8) The antibody described in (6) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucine at positions 234 and 235, as indicated by the EU index, is replaced with alanine. (9) The antibody described in (8) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 8 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 7. (10) The antibody according to (1) to (3), (6) or (8) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 25, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 26, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 27, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 28, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 29, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 30. (11) The antibody according to (1) to (3), (6) or (8) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 31, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 32, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 33, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 34, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 35, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 36. (12) An antibody according to any of (1) to (11) above, wherein one or two amino acids are deleted at the heavy chain carboxyl terminus. (13) An antibody obtained by a method for producing an antibody, comprising the steps of culturing host cells transformed with an expression vector containing a polynucleotide encoding an antibody as described in any of (1) to (12) above, and collecting the antibody of the target from the culture obtained in the said step.

[0325] The anti-EGFR antibody used in the production of the antibody-immunostimulant conjugate of the present invention is not particularly limited, but it is preferable to have, for example, the following characteristics. (1) An anti-EGFR antibody that specifically binds to EGFR. (2) The antibody described in (1) above that binds to the extracellular domain of EGFR. (3) The antibody described in (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) An antibody according to any of (1) to (3) above, having antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity. (5) An antibody as described in any of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a mutation that results in a reduction of ADCC and ADCP activity. (7) The antibody described in (6) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucine at positions 234 and 235, as indicated by the EU index, is replaced with alanine. (8) The antibody described in (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 10 and a light chain consisting of the amino acid sequence described 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 described in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 11. (10) The antibody according to (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 39, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 42. (11) The antibody according to (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 43, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 44, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 45, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 46, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 47, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 48. (12) An antibody according to any of (1) to (11) above, wherein one or two amino acids are deleted at the heavy chain carboxyl terminus. (13) An antibody obtained by a method for producing an antibody, comprising the steps of culturing host cells transformed with an expression vector containing a polynucleotide encoding an antibody as described in any of (1) to (12) above, and collecting the antibody of the target from the culture obtained in the said step.

[0326] The anti-CDH6 antibody used in the production of the antibody-immunostimulant conjugate of the present invention is not particularly limited, but it is preferable to have, for example, the following characteristics. (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 described in (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) An antibody according to any of (1) to (3) above, having antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity. (5) An antibody as described in any of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody described in (1) to (3) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1 and contains a mutation that results in a reduction of ADCC and ADCP activity. (7) The antibody described in (6) above, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, and the leucine at positions 234 and 235, as indicated by the EU index, is replaced with alanine. (8) The antibody according to (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 56 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 55. (9) The antibody according to (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 63, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 64, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 65, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 66, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 67, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 68. (10) An antibody according to any of (1) to (9) above, wherein one or two amino acids are deleted at the heavy chain carboxyl terminus. (11) An antibody obtained by a method for producing an antibody, comprising the steps of culturing host cells transformed with an expression vector containing a polynucleotide encoding an antibody as described in any of (1) to (10) above, and collecting the antibody of the target from the culture obtained in the said step.

[0327] <4-1-2. Antibody Glycan Remodeling> In recent years, methods have been reported for remodeling heterogeneous antibody glycans using enzymatic reactions to uniformly introduce functional glycans (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Attempts have also been made to synthesize homogeneous ADCs by introducing drugs site-specifically using this glycan remodeling technology (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0328] Glycan remodeling first involves using hydrolytic enzymes to excise heterogeneous glycans attached to proteins (such as antibodies), leaving only the terminal GlcNAc, thereby preparing a homogeneous protein portion with GlcNAc attached (hereinafter referred to as the "acceptor"). Next, a separately prepared glycan of a desired type is prepared (hereinafter referred to as the "donor"), and this acceptor and donor are linked using glycosyltransferases. This allows for the synthesis of a homogeneous glycoprotein with a desired glycan structure.

[0329] In this specification, "glycan" refers to a structural unit in which two or more monosaccharides are linked by a glycosidic bond. Specific monosaccharides and glycans may be denoted by abbreviations, such as "GlcNAc-" and "SG-". When these abbreviations are used in structural formulas, oxygen or nitrogen atoms belonging to the glycosidic bond with another structural unit at the reducing end are not included in the abbreviation representing the glycan, unless otherwise specified.

[0330] In this specification, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described with, for convenience, the carbon atom bonded to the oxygen atom constituting the ring and directly bonded to the hydroxyl group (or the oxygen atom belonging to the glycosidic bond) in their ring structure designated as position 1 (position 2 only in the case of sialic acid). The names of the example compounds are given based on the overall chemical structure, and this rule does not necessarily apply.

[0331] In this specification, when a sugar chain is described by a symbol (e.g., SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol shall include the carbon atom at the reducing end, and the N or O atoms belonging to the N- or O-glycosidic bond shall not be included in the symbol.

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

[0333] [ka] As shown, the antibody Ab or its functional fragment is either directly bound to L from the side chain of its amino acid residue (e.g., cysteine, lysine, etc.) or bound to L from the glycan of Ab or a remodeled glycan. The side chain of the amino acid residue may be modified with, for example, an azide group. Here, Ab represents an antibody or a functional fragment of the antibody, and the glycans of the antibody may be remodeled. m 1 It is in the range of 1 to 10, L represents the linker connecting Ab and D. Linker L is denoted as -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates that it is bound to immunostimulant D. Lp is -GGFG-, where G represents glycine and F represents phenylalanine. La shows -C(=O)-CH2CH2-C(=O)-, Lb is given by the following equation: [ka] (In the structural formula of Lb shown above, the asterisk indicates that it is bonded to La, and the wavy line indicates that it is bonded to the sugar chain or remodeled sugar chain of Ab.) Lc represents -NH-CH2-. D is

[0334] [ka] This indicates.

[0335] In this specification, the glycan of Ab is an N-linked glycan or an O-linked glycan, and is preferably an N-linked glycan.

[0336] N-linked glycans are bound to the amino acid side chains of antibodies via N-glycosidic bonds, while O-linked glycans are bound via O-glycosidic bonds.

[0337] In this specification, Ab is IgG, preferably IgG1, IgG2, or IgG4.

[0338] IgG possesses a well-conserved N-linked glycan (hereinafter referred to as "Asn297 glycan" or "N297 glycan") at the 297th asparagine residue in the Fc region of its heavy chain (hereinafter referred to as "Asn297" or "N297"), which is known to contribute to the activity and dynamics 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 the report by Edelman et al. (Proc. Natl. Acad. Sci. USA, 63, 78-85, (1969)), each amino acid was identified by its EU number (EU INDEX). For example, Asn297, to which an N-linked glycan is attached in the Fc region, corresponds to position 297 in the EU INDEX. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by representing it with its EU number.

[0340] The figure below shows the case where the antibody-immunostimulant conjugate of the present invention is bound to L from the N297 glycan of the antibody or its functional fragment. 2 This represents an integer of 1 or 2.

[0341] [ka] Antibodies that possess such remodeled glycans are called glycan remodeling antibodies.

[0342] SGP (α2,6-SGP) is an abbreviation for Sialylglycopeptide and is a representative N-linked glycopeptide. SGP can be isolated and purified from chicken egg yolk according to the method described in WO2011 / 027868, for example. Purified SGP products are also sold by Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd. In this specification, the sugar chain portion of SGP is denoted as SG, and a sugar chain in which one GlcNAc is missing from the reducing end of SG is denoted as SG(10). SG(10) can be prepared by enzymatic hydrolysis of SGP, for example, referring 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, a sugar chain structure in which the non-reducing sialic acid is missing from only one of the branched chains of β-Man in SG(10) is denoted as MSG(9), and a structure having sialic acid only in the 1-3 sugar chains of the branched chain is denoted as MSG1, and a structure having sialic acid only in the 1-6 sugar chains of the branched chain is denoted as MSG2.

[0344] The remodeled glycans used in the antibody-immunostimulant conjugate of the present invention are N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, preferably N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, and 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 that the antibody is bound to Asn297. L(PEG) is -(CH2-CH2-O)n 5 It shows -CH2-CH2-NH-, indicating that the rightmost amino group of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at both the 1-3 and 1-6 chain sides of the β-Man branched chain of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of linker L, in particular linker L. Here, n 5 is an integer between 2 and 10, preferably between 2 and 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 that the antibody is bound to Asn297. L(PEG) is -(CH2-CH2-O)n 5 It shows -CH2-CH2-NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of linker L, in particular linker L. Here, n 5 is an integer between 2 and 10, preferably between 2 and 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 that the antibody is bound to Asn297. L(PEG) is -(CH2-CH2-O)n 5 It shows -CH2-CH2-NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-6 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of linker L, in particular linker L. Here, n 5 is an integer between 2 and 10, preferably between 2 and 5.

[0357] In the antibody-immunostimulant conjugate of the present invention, if the N297 glycan of the antibody is N297-(Fuc)SG, then since the antibody is a dimer, the antibody-immunostimulant conjugate is a molecule to which four linkers L and four immunostimulants D are bound (as described above m 2 =2)

[0358] In the antibody-immunostimulant conjugate of the present invention, if the N297 glycan of the antibody is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, since the antibody is a dimer, the antibody-immunostimulant conjugate is a molecule in which two linkers L and two immunostimulants D are linked (as described above m 2 =1) (see Figure 19).

[0359] The N297 glycan is preferably N297-(Fuc)SG, 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 glycan of the antibody in the antibody-immunostimulant conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, highly uniform ADCs can be obtained.

[0361] <4-2. Manufacturing of Glycan Remodeling Antibodies> Glycan remodeling antibodies can be manufactured by the method shown in the following formula, 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 process involves using a known enzymatic reaction to hydrolyze the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing end chitobiose structure of the N-linked glycan (N297-linked glycan) that binds to asparagine at amino acid position 297 of the antibody's amino acid sequence, thereby producing a glycosidic-cleaved antibody. The target antibody (1) (10 mg / mL) was subjected to hydrolysis of the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing end chitobiose structure using a hydrolytic enzyme such as wild-type EndoS enzyme in a buffer (such as phosphate buffer) at a temperature of 0°C to 40°C. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. For every 100 mg of antibody (1), 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, of wild-type EndoS enzyme was used. After the reaction is complete, affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and / or high Doro The antibody (Fucα1,6)GlcNAc was purified using a xyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (manufactured by BIO-RAD)) to obtain antibody (2).

[0364] (D-2 process) This step involves attaching an SG-type or MSG (MSG1, MSG2)-type glycan oxazoline (hereinafter referred to as "azide glycan oxazoline") having an azide group-containing PEG linker to the (Fucα1,6)GlcNAc antibody (2) obtained in step D-1 using a known enzymatic reaction, thereby producing a glycan remodeling antibody (3).

[0365] The glycosylation reaction was carried out by reacting antibody (2) with an azide glycan oxazoline in a buffer (such as phosphate buffer) at a temperature of 0°C to 40°C in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L). The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. For every 100 mg of antibody, 1 mg to 10 mg of EndoS enzyme (D233Q / Q303L), preferably 1 mg to 3 mg, was used, and 2 equivalents to an excess equivalent of the azide glycan oxazoline, preferably 4 to 20 equivalents, was used. After the reaction was complete, affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE Healthcare)) and high-frequency chromatography were performed. Doro The antibody was purified using a xyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (manufactured by BIO-RAD)) to obtain a glycosylation remodeling antibody (3).

[0366] In the preparation of the above-described glycosylation remodeling antibody, the concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be carried out according to common operations A to C described below.

[0367] The SG-type azide oxyzoline compounds were synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]2-SG(10)-Ox (compounds 1-10 described in WO2018 / 003983) is shown in the following formula.

[0368] [ka]

[0369] MSG-type azide oxyazoline compounds were also synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]-MSG1(9)-Ox (compounds 1-11 described in WO2018 / 003983) is shown in the following formula.

[0370] [ka]

[0371] (D-3 process) This process involves producing a glycosylation remodeling antibody (3) using a glycosylation reaction with two types of endo enzymes on the (Fucα1,6)GlcNAc antibody (2) obtained in the D-1 process. By using two types of enzymes simultaneously, it is possible to directly transfer glycosylation from SGP or (SG)Asn, whose reducing ends are not activated, to the N297 glycan of the antibody, using them as glycan donors. 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, EndoOm, and EndoCC mutants with reduced hydrolytic activity. Preferred enzyme As are EndoM N175Q, EndoCC N180H, and EndoOm N194Q. Examples of enzyme B include EndoS, EndoS2 (EndoS49), and EndoS mutants and EndoS2 (EndoS49) mutants with reduced hydrolytic activity. Preferred enzyme Bs 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. As for glycan donors, ([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] The glycosylation reaction was carried out by reacting antibody (2) with ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 in a buffer (such as Tris buffer) in the presence of glycosyltransferases A (EndoM-like enzyme) and B (EndoS-like enzyme). The reaction temperature can be appropriately selected according to the optimal temperature of the enzyme used, but is usually 15 to 50 degrees Celsius, preferably 25 to 40 degrees Celsius. The reaction time can be appropriately selected between 2 and 48 hours. After the reaction is complete, a purification method suitable for the reaction scale (affinity chromatography, high-frequency chromatography) is used. Doro A glycan remodeling antibody (3) was obtained by selecting a xyapatite column or an ultrafiltration method (ultrafiltration membrane).

[0373] In the preparation of the above-described glycosylation remodeling antibody, the concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be carried out according to common operations 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 compound, prepared from Fmoc-(SG-)Asn (1S2S-11NC-Asn-Fmoc, manufactured by the Institute of Glycotechnology), was reacted with 11-azido-3,6,9-trioxaundecane-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 according to 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 manufactured according to the following method.

[0377] [ka] (Here, the two asterisks (*) on the left of the antibody-immunostimulant conjugate (Rp,Rp-XIII) indicate the CDN-linker portion shown by the asterisk on the right.)

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

[0379] This manufacturing method involves conjugating the glycosylation remodeling antibody (3) obtained in step D-2 or D-3 above with the CDN conjugate precursor (Rp,Rp-XII) via a SPAAC reaction to produce an antibody-immunostimulant conjugate (Rp,Rp-XIII).

[0380] (Process E-1) (Process a9) The SPAAC reaction was carried out by mixing a buffer solution of the glycosylation remodeling antibody (3) (phosphate buffer, acetate buffer, borate buffer, etc.) with a solution of the CDN conjugate precursor (Rp,Rp-XII) dissolved in a suitable solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixture thereof). The amount of CDN conjugate precursor (Rp,Rp-XII) was 2 moles to an excess mole, preferably 4 moles to 30 moles, per mole of glycosylation remodeling antibody (3), and the ratio of the organic solvent was preferably 1% to 200% (v / v) relative to the antibody buffer solution. The reaction temperature was 0°C to 37°C, preferably 15°C to 25°C, and the reaction time was 1 hour to 150 hours, preferably 6 hours to 72 hours. The pH of the reaction solution was preferably 5 to 9. The reaction solution was purified according to the method described in Common Procedure D below to obtain an antibody-immunostimulant conjugate (Rp,Rp-XIII).

[0381] Antibody-immunostimulant conjugates can be identified by performing buffer exchange, purification, measurement of antibody concentration, and measurement of the average number of immunostimulants bound per antibody molecule using the common procedures D to G described below.

[0382] Common procedure A: Concentration of antibody aqueous solution Antibodies or antibody-immunostimulant conjugate solutions were placed in an Amicon® Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000G to 4000G for 5 to 20 minutes.

[0383] Common procedure B: Measurement of antibody concentration Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's specified method. At that time, different 280nm extinction coefficients (1.3 mL mg) were used for each antibody. -1 cm -1 From 1.8 mL mg -1 cm -1 ) was used.

[0384] Common operation C: Antibody buffer exchange A buffer solution (such as phosphate-buffered saline (pH 6.0) or phosphate buffer (pH 6.0)) was added to the antibody aqueous solution, and the solution was concentrated according to the method described in Common Procedure A. After repeating this procedure several times, the antibody concentration was measured according to the method described in Common Procedure B. A buffer solution of the desired concentration (for example, approximately 10 mg / mL) was prepared by adding an appropriate buffer solution (such as phosphate-buffered saline (pH 6.0) or phosphate buffer (pH 6.0)) to this antibody buffer solution.

[0385] Common procedure D: Purification of antibody-immunostimulant conjugates (gel filtration chromatography) NAP columns (NAP-5, NAP-10, NAP-25 (GE Healthcare)) were equilibrated with acetate buffer (10 mM Acetate Buffer, 5% Sorbitol, pH 5.5; referred to herein as ABS) or other suitable buffer. The antibody-immunostimulant conjugate reaction solution was charged onto the NAP column, and the manufacturer's specified amount of buffer was allowed to flow by gravity to separate the antibody fraction. This fraction was then charged onto the NAP column again, and the manufacturer's specified amount of buffer was allowed to flow by gravity to separate the antibody fraction. This procedure was repeated a total of two to three times to obtain antibody-immunostimulant conjugates free of unbound immunostimulant linkers, dimethyl sulfoxide, and propylene glycol. If necessary, the concentration of the antibody-immunostimulant conjugate solution was adjusted using common procedures A and C.

[0386] Common procedure E: Measurement of antibody concentration and average number of immunostimulants bound per antibody molecule in antibody-immunostimulant conjugates (UV method) The concentration of the conjugated immunostimulant in the antibody-immunostimulant conjugate can be calculated by measuring the absorbance of the aqueous antibody-immunostimulant conjugate solution at two wavelengths, 280 nm and 250 nm, using a 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 immunostimulants bound per antibody molecule in antibody-immunostimulant conjugates (reverse-phase high-performance liquid chromatography: RP-HPLC) The antibody concentration and the average number of immunostimulants bound per antibody molecule in the antibody-immunostimulant conjugate 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 procedure E described above.

[0388] Common procedure G: Measurement of antibody concentration and average number of immunostimulants bound per antibody molecule in antibody-immunostimulant conjugates (hydrophobic interaction - high-performance liquid chromatography: HI-HPLC) The antibody concentration and the average number of immunostimulants bound per antibody molecule in the antibody-immunostimulant conjugate 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 operations E and F described above.

[0389] Antibody-immunostimulant conjugates or their intermediates produced by the method of the present invention may contain stereoisomers or optical isomers derived from chiral carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers. Any of these isomers, optical isomers, or mixtures thereof are included in the present invention.

[0390] In antibody-immunostimulant conjugates produced by the method of the present invention, the number of immunostimulants bound to one antibody molecule is an important factor affecting their efficacy and safety. The production of antibody-immunostimulant conjugates is carried out by defining reaction conditions, such as the amount of raw materials and reagents used, so that the number of immunostimulants bound is constant. However, unlike the chemical reaction of low molecular weight compounds, it is usually obtained as a mixture with different numbers of immunostimulants bound. The number of immunostimulants bound to one antibody molecule can be specified as an average value, i.e., the average number of immunostimulants bound (DAR: Drug to Antibody Ratio). The number of cyclic dinucleotide derivatives bound to antibody molecules is controllable, and the average number of immunostimulants bound per antibody can range from 1 to 10 cyclic dinucleotide derivatives, but preferably from 1 to 8, and more preferably from 1 to 5.

[0391] In the antibody-immunostimulant conjugate produced by the method of the present invention, when antibody Ab is bound to L from the remodeled sugar chain of antibody Ab, the number of immunostimulant conjugates per antibody molecule in the antibody-immunostimulant conjugate is m. 2 m is an integer of 1 or 2. If the glycan is an N297 glycan and the glycan is N297-(Fuc)SG, then m 2 The value is 2, and the DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.2). When the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, 2 The value of is 1, and the DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

[0392] Furthermore, those skilled in the art can design a reaction to conjugate the required number of immunostimulants to an antibody based on the descriptions in the examples of this application, and obtain antibodies with controlled numbers of cyclic dinucleotide derivatives.

[0393] Furthermore, antibody-immunostimulant conjugates or their intermediates produced by the method of the present invention may absorb moisture when left in the air or recrystallized, resulting in the formation of adsorbed water or hydrates. Compounds and salts containing such water are also included in the present invention.

[0394] If the antibody-immunostimulant conjugate or its intermediate produced by the method of the present invention has a basic group such as an amino group, it can optionally be a pharmaceutically acceptable salt. Examples of such salts include hydrohalides such as hydrochloride and hydroiodide; inorganic salts such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfons such as methanesulfonates, trifluoromethanesulfons, and ethanesulfons; allylsulfons such as benzenesulfons and p-toluenesulfons; and formic acid salt acetic acid salt malic acid salt Examples include organic salts such as fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0395] Since the antibody-immunostimulant conjugate or its manufacturing intermediate produced by the method of the present invention contains a phosphate group and / or a thiophosphate group in its structure, it is generally possible to form a base addition salt. Furthermore, even if the manufacturing intermediate has an acidic group such as a carboxyl group, it is generally possible to form a base addition salt. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; 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 conjugate and its manufacturing intermediate produced by the method of the present invention may exist as a hydrate by absorbing moisture from the air, etc. The solvate of the present invention is not particularly limited as long as it is pharmaceutically acceptable, but specifically, hydrates, ethanol hydrates, 2-propanol hydrates, etc. are preferred. Furthermore, if a nitrogen atom is present in the antibody-immunostimulant conjugate and its manufacturing intermediate of the present invention, it may be in the form of an N-oxide, and these solvates and N-oxides are also included in the scope of the present invention. Furthermore, if a sulfur atom is present in the antibody-immunostimulant conjugate and its manufacturing intermediate of the present invention, it may be in the form of a sulfoxide, and these solvates and sulfoxides are also included in the scope of the present invention.

[0397] Furthermore, the antibody-immunostimulant conjugates and their production intermediates produced by the method of the present invention also include compounds labeled with various radioactive or non-radioactive isotopes. The antibody-immunostimulant conjugates and their production intermediates produced by the method of the present invention may also contain non-natural proportions of atomic isotopes in one or more of the constituent atoms. Examples of atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), or carbon-14 (14C). The compounds of the present invention can also be radiolabeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or carbon-14 (14C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, such as assay reagents, and diagnostic agents, such as in vivo imaging agents. All isotopic variants of the antibody-immunostimulant conjugates of the present invention, whether radioactive or non-radioactive, are included within the scope of the present invention.

[0398] <5. Pharmaceuticals> The antibody-immunostimulant conjugate produced by the method of the present invention exhibits antitumor immune activity or cytotoxic activity against cancer cells, and can therefore be used as a pharmaceutical, particularly as a therapeutic and / or prophylactic agent for cancer, or as an antitumor agent.

[0399] The types of cancer to which the antibody-immunostimulant conjugate produced by the method of the present invention is applicable include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), kidney cancer, urothelial carcinoma, 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, stomach cancer, esophageal cancer, endometrial cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, and gastrointestinal stromal tumors. Examples of cancers that can be treated include tumors (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, pharyngeal cancer, tongue cancer, auditory organ cancer, thymic cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, and sarcoma. However, antibody-immunostimulant conjugates are not limited to these, as long as the target cancer cells express proteins that can be recognized by the antibody in the antibody-immunostimulant conjugate.

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

[0401] In a pharmaceutical composition containing an antibody-immunostimulant conjugate produced by the method of the present invention, the substance used can be appropriately selected from pharmaceutical additives and others commonly used in this field, in terms of dosage and concentration.

[0402] The antibody-immunostimulant conjugate produced by the method of the present invention may be administered as a pharmaceutical composition containing one or more pharmaceutically compatible components. For example, the pharmaceutical composition typically contains one or more pharmaceutical carriers (e.g., sterile liquids (e.g., water and oils (petroleum, animal, plant, or synthetic oils (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, as well as aqueous dextrose and aqueous glycerol solutions, may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition may also optionally contain trace amounts of wetting or emulsifying agents or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulations 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. Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration may be, for example, by infusion or bolus injection. In certain preferred embodiments, the administration of the antibody-immunostimulant conjugate is by infusion. Parenteral administration is a preferred route of administration.

[0404] In typical embodiments, the pharmaceutical composition comprising the antibody-immunostimulant conjugate described above is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If necessary, the pharmaceutical may also contain a solubilizer and a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the components are supplied either separately as lyophilized powder or anhydrous concentrate in sealed containers, for example, ampoules or sachets indicating the amount of the activator, or mixed together in a unit dosage form. If the pharmaceutical composition is to be administered by infusion, it may be administered, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the pharmaceutical is administered by injection, ampoules of sterile water or saline for injection may be provided, for example, so that the components can be mixed before administration. The pharmaceutical composition may also be supplied as a solution.

[0405] A pharmaceutical composition comprising the antibody-immunostimulant conjugate produced by the method of the present invention may be a pharmaceutical composition comprising only the antibody-immunostimulant conjugate produced by the method of the present invention, or it may be a pharmaceutical composition comprising the antibody-immunostimulant conjugate produced by the method of the present invention and other cancer therapeutic agents. The antibody-immunostimulant conjugate produced by the method of the present invention can also be administered together with other cancer therapeutic agents to enhance the antitumor effect. Other cancer therapeutic agents used for this purpose may be administered to the individual simultaneously with the antibody-immunostimulant conjugate, separately, or consecutively, or with varying administration intervals. Such cancer treatment agents include antimetabolites, alkylating agents, microtubule inhibitors and other chemotherapeutic agents (such as abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, vinblastine, or drugs listed in the international publication WO2003 / 038043), hormone regulators (such as LH-RH analogs like leuprorelin, goserelin, estramustine, and estrogen antagonists like tamoxifen and raloxifene), aromatase inhibitors (such as anastrozole, letrozole, and exemestane), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, and molecular targeted drugs (anti-EGFR antibodies, 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.), immunoactivators (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 modifiers, etc.), ADCC (AntibodyExamples of antitumor drugs include antibody drugs having dependent cellular cytotoxicity (Dependent Cellular Cytotoxicity) activity, ADCP (Antibody Dependent Cellular Phagocytosis) activity, or complement activity; BiTEs (Bi-specific T-cell engagers); Antibody-Drug-Conjugates (ADCs) (e.g., drug conjugates including Deruxtecan, DM1, Pyrrolobenzodiazepine, MMAF, etc. (anti-HER2-ADC, anti-TROP2-ADC, anti-HER3-ADC, etc.)); ADCs combined with photodynamic therapy; and antitumor vaccines, antitumor cell therapies (CAR-T, TCR-T, dendritic cells, NK cells, etc.), antitumor bacterial therapy, antitumor viral therapy, etc., but are not limited to drugs having antitumor activity. Furthermore, the antibody-immunostimulant conjugate 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-immunostimulant conjugate of the present invention can enhance the antitumor effect not only when used as an immunostimulant, but also when used in combination with therapies that produce antitumor effects, such as radiation, heavy particle beam therapy, surgery, bone marrow transplantation, etc., but is not limited to any therapy that has an antitumor effect.

[0406] Such pharmaceutical compositions may be formulated as lyophilized or liquid formulations having the selected composition and required purity. When formulated as a lyophilized formulation, it may contain appropriate pharmaceutical additives used in this field. Similarly, liquid formulations can also be formulated as liquid formulations containing various pharmaceutical additives used in this field.

[0407] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-immunostimulant conjugate contained in the pharmaceutical composition containing the antibody-immunostimulant conjugate produced by the method of the present invention exhibits therapeutic effects even at small doses if the affinity of the antibody-immunostimulant conjugate to the antigen is high (low Kd value), i.e., the dissociation constant (Kd value) to the antigen is high. Therefore, when determining the dosage of the antibody-immunostimulant conjugate, the dosage can also be set based on the affinity between the antibody-immunostimulant conjugate and the antigen. When administering the antibody-immunostimulant conjugate of the present invention to humans, for example, approximately 0.001 to 100 mg / kg can be administered once or multiple times at intervals of 1 to 180 days.

[0408] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. In the following, "compound (I)" or "(I)" in the synthesis scheme refers to "compound of formula (I)", and the same applies to subsequent compound numbers. [Examples]

[0409] In the following examples, room temperature is defined as 15°C to 35°C unless otherwise specified. Anhydrous dichloromethane was dichloromethane (super-anhydrous) sold by Fujifilm Wako Pure Chemical Industries. Anhydrous acetonitrile was acetonitrile (super-anhydrous) sold by Fujifilm Wako Pure Chemical Industries. Anhydrous pyridine was pyridine (anhydrous) sold by Kanto Chemical Co., Ltd. Silica gel chromatography was performed using Biotage Sfar HC D (20 μm, Biotage), aminosilica gel column chromatography was performed using Biotage Sfar Amino D (50 μm, Biotage), and preparative HPLC was performed using the Agilent Preparative HPLC System (Agilent Technology). The preparative column used was 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 CDN1 Synthesis 1 Cyclic dinucleotide (CDN1) was synthesized according to the following synthesis scheme A-1. In 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). [Synthesis 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]oxazaphosphole-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 mixed with anhydrous dichloromethane (150 mL) and cooled to -5°C. Triethylamine (1.73 mL) was then added. To the above solution, a solution of (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphor, known from literature (Tetrahedron Letter, 1998, 39, 2491-2494), in anhydrous dichloromethane (50 mL) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. The mixture was then heated to room temperature, stirred for 30 minutes, and the reaction mixture was concentrated. The concentrated crude product was diluted and dissolved with 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-Diethylethaneaminium(2R,3R,4R,5R)-2-({[(R)-{[(2R)-1-Acetylpyrrolidine-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-silatetetradecane-14-yl)-6-oxo-1,6-dihydro-9H-purine-9-yl]-5-(hydroxymethyl)oxolan-3-yl]oxy}phosphorotioil]oxy}methyl)-5-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)-4-fluorooxolan-3-yl Synthesis of phosphates (corresponding to compounds of formula (Rc-V))

[0418] [ka]

[0419] (Process 2) 6-Benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 The triethylamine salt (260 mg) of phosphanyl]-β-D-ribofuranosyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene was dissolved in anhydrous acetonitrile (2.7 mL), molecular sieve 4A (12.5 mg) was added, and the mixture was stirred for 30 minutes. In a separate container, the compound obtained in Example 1 (540 mg) was dissolved in anhydrous acetonitrile (2.7 mL), molecular sieve 4A (25 mg) was added, and the mixture was stirred for 30 minutes. The previously prepared 6-benzoyl-2-{2-deoxy-2-fluoro-3-O-[hydroxy(oxo)-λ 5 A solution of triethylamine salt of [-phosphanyl]-β-D-ribofuranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene in acetonitrile was added. A solution of 1-phenylimidazolium triflate prepared to 0.4 mol / L (2.7 mL) in dehydrated acetonitrile was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, a solution of acetic anhydride prepared to 1 mol / L (0.45 mL) in dehydrated acetonitrile was added, followed by the addition of triethylamine (55 mg), and the mixture was stirred for 30 minutes. Then, xanthan hydride (74 mg) was added, and the mixture was stirred for 30 minutes. The molecular sieve was removed by filtering the reaction mixture. The filtrate was cooled to 0°C, 20% saline solution (2.7 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 20% saline solution (2.7 mL) was added to the resulting organic layer. The mixture was then neutralized with triethylamine to a pH of 7. Subsequently, the organic layer obtained by separation was concentrated, crudely purified by silica gel chromatography [acetonitrile / methanol], concentrated to dryness, and the target compound was obtained. The diastereomer ratio on the phosphorus atom produced during the reaction was 98.5:1.5, as determined by HPLC.

[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 were measured using isolated samples obtained by reverse-phase HPLC [10 mM ammonium acetate aqueous solution / acetonitrile].

[0421] Example 3: 2-(trimethylsilyl)ethyl(2-{[(2-{9-[(5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-10-{[(2R)-1-acetylpyrrolidine-2-yl]methoxy}-14-(6-benzoyl-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-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 -Flo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-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 anhydrous pyridine (50 mL) to prepare a 16 mL pyridine solution, which was cooled to -20°C. 2-chloro-2-oxo-1,3,2-dioxaphosphoran (171 mg) was weighed into a separate container and cooled to -20°C. The pyridine solution was added to the container of 2-chloro-1,3,2-dioxaphosphoran-2-oxide and stirred at -20°C for 1 hour. The diastereomer ratio on the phosphorus atom generated during cyclization was 75:25. Water (43 mg) was added and stirred at the same temperature for 10 minutes, then xanthan hydride (40 mg) was added, the temperature was raised to room temperature, and the mixture was stirred for 1 hour. The resulting reaction solution was quenched with 5% sodium bicarbonate solution (3 mL), and ethyl acetate (12 mL) and 20% saline solution (3 mL) were added for liquid-liquid separation. The aqueous layer was re-extracted with ethyl acetate (3 mL), and the organic layer obtained in the first liquid-liquid extraction matched the organic layer obtained in the re-extraction. The mixture was then washed with a mixture of 20% saline solution (1.5 mL) and 5% sodium bicarbonate solution (1 mL). Subsequently, the organic layer was concentrated to dryness, methanol (30 mL) was added, and the layer was concentrated to dryness again 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 were measured using isolated samples obtained by reverse-phase HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile].

[0425] Example 4: Bis(N,N-diethylethaneaminium)(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-purine-9-yl]-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -Flo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (corresponding to the compound of formula (Rp,Rp-VII'))

[0426] [ka]

[0427] (Step 4) In Example 2, methanol (12 mL) was added to the compound to prepare a methanol solution. 28% aqueous ammonia (4.5 mL) was added to this methanol solution, and the mixture was stirred at 60°C for 30 hours. The resulting reaction solution was concentrated, and the residue was purified by preparative HPLC [10 mM triethylammonium acetate aqueous solution / acetonitrile] to obtain 84 mg of the target compound as a single diastereomer (Rp,Rp isomer) on 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. [Synthesis 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 compound (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 the mixture was 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% sodium bicarbonate solution (2 mL) were added to the reaction mixture and separated. The organic layer was washed twice with 5% sodium bicarbonate solution (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 in 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 under cooling for 24 hours. The reaction mixture was purified by silica gel chromatography [acetonitrile / methanol] and concentrated to prepare an acetonitrile solution to obtain the target compound (total quantitative value of 358 mg in two steps by NMR).

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

[0439] A-3 CDN2 synthesis 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. [Synthesis Scheme A-3]

[0440] [ka]

[0441] Example 7: Synthesis of sodium (2R,3R,4R,5R)-2-({[(R)-{[(2R)-1-acetylpyrrolidine-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-silatetetradecane-14-yl)-6-oxo-1,6-dihydro-9H-purine-9-yl]-5-(hydroxymethyl)oxolan-3-yl]oxy}phosphorotioil]oxy}methyl)-5-(6-benzamide-9H-purine-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-purine-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (400 mg) described in Patent Document WO2015 / 185565 was dissolved in dehydrated 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 dehydrated acetonitrile (5.5 mL), molecular sieve 4A (25 mg) was added, and the mixture was stirred for 30 minutes. Then, the acetonitrile solution of the triethylamine salt of the previously prepared (2R,3R,4R,5R)-5-(6-benzamido-9H-purine-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate was added. A 0.4 mol / L solution of 1-phenylimidazolium triflate in anhydrous acetonitrile (5.5 mL) was added and the mixture was stirred at room temperature for 30 minutes. Subsequently, a 1 mol / L solution of acetic anhydride in anhydrous acetonitrile (1.14 mL) was added, followed by the addition of triethylamine (139 mg) and stirring for 30 minutes. Then, xanthan hydride (154 mg) was added and the mixture was stirred for 30 minutes, and the molecular sieves were filtered off by filtration of the reaction mixture. The filtered molecular sieves were washed with anhydrous acetonitrile (5 mL), and after matching the filtrate with the washing solution, the mixture was cooled to 0°C, 20% saline solution (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 20% saline solution (6 mL) was added to the resulting organic layer and neutralized with triethylamine to a pH of 7. Subsequently, the organic layer obtained by liquid-liquid separation was concentrated and crudely purified by silica gel chromatography [ethyl acetate / methanol / 0.4% triethylamine]. The recovered fraction was concentrated and purified by preparative HPLC [10 mM ammonium acetate aqueous solution / acetonitrile], and acetonitrile was concentrated and removed. Sodium chloride was added to the resulting aqueous solution, and the target compound was obtained by extraction with tetrahydrofuran and concentration to dryness (776 mg). The diastereomer ratio on the phosphorus atom produced during the reaction was 98.5:1.5 as measured by HPLC.

[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-acetylpyrrolidine-2-yl]methoxy}14-(6-benzamido-9H-purine-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-purine-9-yl]-10-sulfanylideneoctahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -Flo[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 dehydrated pyridine (1 mL), and molecular sieve 4A (3 mg) was added. The diastereomer ratio on the phosphorus atom generated during cyclization was 70:30. Subsequently, 2-chloro-5,5-dimethyl-1,3,2-dioxaphospholinane 2-oxide (5.7 mg) was added, and the mixture was stirred for 30 minutes, after which the molecular sieve was filtered off. Water (10 mg) was added to the filtrate, and after stirring for 5 minutes, xanthan hydride (2.6 mg) was added and the mixture was stirred for 30 minutes. 5% sodium bicarbonate solution (2 mL) was added to the reaction mixture to stop the reaction, and ethyl acetate was added for liquid-liquid extraction. The organic layer was concentrated to dryness and purified by preparative HPLC [10 mM ammonium acetate aqueous solution / acetonitrile], and acetonitrile was concentrated and removed. Sodium chloride was added to the resulting aqueous solution, and the mixture was extracted with tetrahydrofuran. The target compound was obtained as a single phosphorus-based diastereomer (4 mg) by concentration to dryness. 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-purine-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-purine-9-yl]octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -Flo[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), 28% aqueous ammonia (0.05 mL) was added, and the mixture was stirred at 50°C for 24 hours. The resulting reaction solution was analyzed by LC-MS and confirmed that the target compound was formed as a single diastereomer (Rp,Rp isomer) 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. [Synthesis 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 compound (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 the mixture was 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 compound (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 mixture was purified by silica gel chromatography [ethyl acetate / methanol] and concentrated to prepare an acetonitrile solution, which was then adjusted to a pH of 2 or less using concentrated hydrochloric acid. The white crystals precipitated along with the pH adjustment were filtered, the crystals were washed with an 80% acetonitrile aqueous solution, and the target compound was obtained by drying (yield 0.24 g, yield 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 CDN1 Synthesis 2 Cyclic dinucleotide (CDN1) was synthesized according to the following synthesis scheme A-5. In 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). [Synthesis 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]oxazaphosphole-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 sieve 3A (80 mg) in dichloromethane (0.8 mL) was stirred at room temperature under nitrogen for 2 hours, then cooled to 0°C and triethylamine (58.7 μL, 0.42 mmol) was added, and the known (Tetrahedron) compound was prepared. A solution of (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphor (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 obtain a solution of the target compound in dichloromethane.

[0466] Example A5-2: N,N-Diethylethaneaminium(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-silatetradecane-14-yl)-6-oxo-1, Synthesis of 6-dihydro-9H-purine-9-yl]oxolan-3-yl}oxy){[(2R)-1-(trifluoroacetyl)pyrrolidine-2-yl]methoxy}phosphorotiol]oxy}methyl)-5-(6-{[2-[trimethylsilyl)ethoxy]carbonyl}6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)-4-fluorooxolan-3-yl phosphonate (corresponding to the compound of formula (Rc-V0))

[0467] [ka] (Process 2) N,N-diethylethaneaminium 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) was added to a 2.0 mL acetonitrile solution 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 a dichloromethane solution of the compound obtained in Example A5-1 was added dropwise, followed by washing with 0.2 mL of dichloromethane. 809.4 μL, 0.65 mmol of 0.8 mol / L 1-methylbenzimidazolium triflate in acetonitrile was added, and the mixture was stirred for 1.5 hours. Then, 121.4 μL, 0.10 mmol of 0.8 mol / L 1-methylbenzimidazolium triflate in acetonitrile 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 raised to room temperature. After stirring for about 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 solution was concentrated to approximately 2.5 mL and purified by silica gel column chromatography (0-12% methanol acetonitrile with 0.4% triethylamine) to obtain the target compound as a slightly yellowish solid (536.0 mg, 90% yield). The diastereomer ratio on the phosphorus atom formed during the reaction was 94.7:5.3, as determined by HPLC.

[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-Diethylethaneaminium(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-silatetetradecane-14-yl)-6-oxo-1,6-dihydro-9H-purine-9-yl]-5-(Hydro Synthesis of roxymethyl)oxolan-3-yl]oxy}({2-[(2R)-1-(trifluoroacetyl)pyrrolidine-2-yl]methoxy}phosphorotiol]oxy}methyl)-5-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)-4-fluorooxolan-3-yl phosphonate (corresponding to the compound of formula (Rc-V))

[0470] [ka] (Step 3) To a solution of the compound obtained in Example A5-2 (4.1 g, 2.25 mmol) in dichloromethane (100 mL), acetonitrile (15 mL) and ethanol (4.1 g, 89.84 mmol) were added 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 removed under reduced pressure to approximately 15 mL. The process of adding acetonitrile (100 mL) and removing the solvent under reduced pressure to approximately 15 mL 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-i [L]-10-sulfanylidene-14-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene-2-yl)-10-{[(2R)-1-(trifluoroacetyl)pyrrolidine-2-yl]methoxy}octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -Flo[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 mixture of pyridine (42 mL) and dichloromethane (42 mL) was mixed with acetonitrile solution of T3P (1.7 mol / L, 4.9 mL, 8.30 mmol) and cooled to -20°C. Acetonitrile solution of the compound obtained in Example A5-3 (13.67 g) was added dropwise and stirred at -20°C for 1 hour. The diastereomer ratio on the phosphorus atom generated during cyclization was 85:15. Xanthan hydride (0.62 g, 4.15 mmol) was added, and the mixture was heated to approximately 0°C and stirred for 3 hours. 5% sodium bicarbonate aqueous solution (22.5 mL), ethyl acetate (40 mL), and sodium chloride (1.2 g) were added and stirred. The aqueous layer was discarded and the mixture was washed with a solution of water (22.5 mL), sodium bicarbonate (1.1 g), and sodium chloride (2.4 g). The resulting organic layer was drained under reduced pressure until the solvent was reduced to approximately 15 mL to obtain a pyridine solution of the target compound.

[0473] Example A5-5: Bis(N,N-diethylethaneaminium)(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-purine-9-yl]-14-(6,7,8,9)-tetrahydro-2H-2,3,5,6-tetraazaben Zo [cd] Azulene-2-yl) Octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -Flo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (corresponding to the compound of formula (Rp,Rp-VII'))

[0474] [ka] (Step 5) 35 mL of 28% aqueous ammonia was added to the pyridine solution of the compound obtained in Example A5-4, and the mixture was stirred at approximately 45°C for 15 hours. After cooling to room temperature, 35 mL of heptane was added and the mixture was stirred. The organic layer was discarded, and the resulting aqueous layer was washed with 35 mL of heptane. The aqueous layer was purified by preparative HPLC [10 mM triethylamine acetate aqueous solution / acetonitrile], and the resulting fraction was diluted with water. Solid-phase extraction was performed on this solution using a silka gel, and the mixture was eluted with a 0.1% triethylamine / acetonitrile solution. The solvent was removed from the resulting fraction under reduced pressure to approximately 15 mL. 100 mL of ethyl acetate was added, and the solvent was removed under reduced pressure to approximately 15 mL. This process was repeated twice, and 10 mL of ethyl acetate was added. This ethyl acetate solution was added dropwise to 175 mL of heptane at room temperature and stirred for 30 minutes. The solid was filtered from the resulting suspension and washed with 25 mL of heptane. The solid was dried overnight at 40°C to obtain the target compound as a white powder (1.9 g, yield 55%, 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. [Synthesis 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 salt of the compound in 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), 4-methoxytrityl chloride (26.4 g, 85.4 mmol) was added at room temperature. After stirring at room temperature for approximately 24 hours, 5% sodium bicarbonate aqueous 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% saline solution (171 mL) to obtain the organic layer. Pyridine hydrochloride (8.2 g, 71.2 mmol) was added and the mixture was stirred for 1 hour. Further pyridine hydrochloride (12.3 g, 106.8 mmol) was added and the mixture was stirred for approximately 23 hours. Crystals were filtered from the resulting suspension and washed with toluene (103 mL) to obtain wet crystals of the hydrochloride salt of the target compound.

[0479] Example A6-1: 2-methylpropane-2-aminium 2-{2-deoxy-2-fluoro-5-O-[(4-methoxyphenyl)(diphenyl)methyl]-3-O-[oxide(oxo)-λ 5 Synthesis of 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 in formula (IX'))

[0480] [ka] (Process 2) To the wet crystals of the hydrochloride salt obtained in Reference Example A6-1, ethyl acetate (342 mL) and 5% sodium bicarbonate (342 mL) were added and the mixture was stirred at room temperature. The aqueous layer was discarded, and the resulting organic layer was washed with 10% saline solution (171 mL). Acetonitrile (171 mL) was added to the organic layer, and the solvent was removed under reduced pressure to approximately 170 mL. Acetonitrile (342 mL) was added, and the solvent was removed under reduced pressure to approximately 170 mL, after which 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. After adding water (34.2 mL), the mixture was stirred for approximately 5 hours. Ethyl acetate (513 mL) and 23% potassium bicarbonate (239 mL) were added, the mixture was stirred, and then the aqueous layer was discarded. The organic layer was washed twice with 20% potassium bicarbonate (239 mL), and the solvent was removed from the resulting organic layer under reduced pressure to approximately 170 mL. Acetonitrile (342 mL) was added, and the solvent was removed under reduced pressure to approximately 170 mL twice, after which acetonitrile (342 mL) was added again. After adding tert-butylamine (2.3 mL, 21.4 mmol), a seed crystal of the tert-butylamine salt of the target compound (34.2 mg) was added and the mixture was stirred overnight. Further 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. Crystals were filtered from the suspension obtained after stirring at -1°C for approximately 6 hours and 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, 70% yield).

[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 Synthesis of 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 compound (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 about 2 days, it 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, and the aqueous layer was discarded. The organic layer was washed with 10% aqueous ammonium chloride (250 mL), and then washed twice with 10% aqueous potassium carbonate (500 mL). Furthermore, the organic layer, washed with 250 mL of 10% potassium chloride aqueous solution, was subjected to reduced pressure to remove the solvent to approximately 125 mL, yielding 123.9 g of an acetonitrile solution of the potassium salt of the target compound.

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

[0485] [ka] (Step 4) To the acetonitrile solution (121.2 g) of the potassium salt obtained in Example A6-2, dichloromethane (417 mL) and methanol (55.4 mL) were added, and then dichloroacetic acid (22.0 g, 170.7 mmol) was added dropwise at room temperature. After stirring for about 27 hours, triethylamine (20.7 g, 204.8 mmol) was added dropwise to obtain a solution (658.1 g) of the target compound. The solution (26.9 g) of the target compound was concentrated under reduced pressure to about 5 mL, 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), dichloromethane (20 mL) was added, and the pH was adjusted to 8.5 with triethylamine (97 μL). Triethylamine hydrochloride (500 mg) was added, and after stirring, an organic layer was obtained. Water (10 mL) and triethylamine hydrochloride (500 mg) were added to the organic layer, and after stirring, the aqueous layer was discarded. The obtained organic layer was concentrated under reduced pressure to about 5 mL. Acetonitrile (10 mL) was added, and the operation of concentrating the solvent to about 5 mL under reduced pressure was performed twice to obtain an acetonitrile solution of the triethylamine salt of the target compound (632.6 mg, yield 74%).

[0486] MS(ESI)m / z:517(M+H) + ,515(M-H) - . 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] Synthesis of A-7 CDN-Linker The CDN linker was synthesized according to the following scheme. [Synthesis Scheme A-7]

[0488] [ka]

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

[0490] [ka] (Process 1) 1-{[4-(11,12-didehydrodibenzo[b,f]azosin-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) were suspended in acetonitrile (50 mL) and water (50 mL). Triethylamine (2.3 mL, 16.1 mmol) was added at room temperature, and the mixture was stirred for approximately 4 hours. After adding 1 mol / L hydrochloric acid aqueous solution (16.2 mL), a seed crystal of the target compound (4.6 mg) was added. The mixture was stirred overnight at room temperature, and water (100 mL) was added dropwise over 2 hours. After stirring at 40°C for approximately 1.5 hours, 1 mol / L hydrochloric acid aqueous solution (16.2 mL) was added. After stirring for 30 minutes, the mixture was cooled to room temperature and stirred for a further 2 hours. The crystals were filtered from the suspension and washed with acetonitrile / water (1 / 3, 50 mL). The crystals were dried overnight at 30°C to obtain the target compound as white crystals (6.7 g, 95% yield).

[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]azulene-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -Flo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp,Rp-X))

[0493] [ka] (Process 2) Example A5- 5 To a solution of the compound obtained (1 g, 0.76 mmol) in dimethyl sulfoxide (3 mL), 3 mL of 70% tetrabutylammonium fluoride aqueous solution was added at room temperature, and the mixture was stirred at 30°C for 2 days. Acetonitrile (12 mL) and calcium chloride (0.59 g, 5.32 mmol) were added, and the mixture was stirred at room temperature for 14 hours. Triethylamine (1 mL) and water (2 mL) were added, and the mixture was stirred at room temperature for 2 hours, after which 1.2 mL of 5 mol / L hydrochloric acid solution was added in 6 portions. Triethylamine (60 μL) was added, and the insoluble matter was filtered off. The solvent was removed from the obtained filtrate under reduced pressure to approximately 10 mL. The process of adding acetonitrile (20 mL) and removing the solvent under reduced pressure to approximately 10 mL was repeated twice, and then acetonitrile (50 mL) was added and the solvent was removed under reduced pressure to approximately 10 mL was repeated twice. To the obtained solution, 165 μL of 5 mol / L hydrochloric acid solution was added, followed by the addition of 50 mL of acetonitrile. The mixture was then cooled to 0°C and stirred for 2 hours. The solid was filtered from the resulting suspension and washed with 5 mL of acetonitrile. The solid was dried overnight at 25°C to obtain the target compound as white crystals...

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 hydroxyl groups, and PG3 is a protecting group for amino groups. A method for producing a compound represented by or a salt thereof, (Step a1) Formula (I): 【Transformation 3】 [In the formula, PG1 and PG3 are as defined above, and PG2 is a protecting group for hydroxyl groups. The compound represented by is an optically active phosphytylate (Rc-II) selected from the group consisting of the following formulas (Rc-II-1) and (Rc-II-2): 【Chemistry 4】 [In the formula, R1 is hydrogen or methyl, R2 is hydrogen, an alkyl group having 1 to 3 carbon atoms, or a phenyl compound. Here, the alkyl is either unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl molecules, and The phenyl is either unsubstituted or substituted with nitro or methoxy. The reaction is then expressed as equation (Rc-III): 【Transformation 5】 [In the formula, PG1, PG2 and PG3 are as defined above, and B1 is, 【Transformation 6】 And R1 and R2 are as defined above. A step to obtain a compound represented by, (Step a2) The obtained compound of formula (Rc-III) is used for formula (IV): 【Transformation 7】 [In the formula, A2 is, 【Transformation 8】 and PG4 is a protecting group for amino groups. A compound represented by or a salt thereof is reacted in the presence of an activator, then treated with an acylating agent or an alkoxycarbonylating agent, further reacted with a thiolating agent, and subsequently PG2 is deprotected to obtain the compound (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 amino groups. A step of obtaining a compound represented by 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 thiolating agent or an oxidizing agent to obtain formula (Rc-VI): 【Chemistry 11】 [In the formula, A2, B2, Q, PG1, and PG3 are as defined above. A step of obtaining a compound represented by or a salt thereof, and (Step a4) Deprotecting B2, which is the protecting group of the thiophosphate moiety of the obtained compound of formula (Rc-VI), and PG4, which is the protecting group in A2, to obtain the compound of formula (Rp-VII) or a salt thereof. Includes, The compound of formula (IV) or a salt thereof is used in the following steps: (Step a5) Formula (VIII): 【Chemistry 12】 [In the formula, A2 is as defined above, and PG5 is a protecting group for hydroxyl groups. A compound represented by formula (IX) or a salt thereof is reacted with a phosphite ester to obtain the compound represented by formula (IX): 【Chemistry 13】 [In the formula, A2 and PG5 are as defined above.] A step of obtaining a compound represented by or a salt thereof, Or, (Step a5') Formula (VIII'): 【Chemistry 14】 [In the formula, A1 is as defined above, and PG5 is defined as above. A compound represented by formula (IX') or a salt thereof is reacted with a phosphite ester to obtain a compound represented by formula (IX'): 【Chemistry 15】 [In the formula, A1 and PG5 are as defined above.] A step of obtaining a compound represented by or a salt thereof, and (Step a5'') The compound of formula (IX') obtained or a salt thereof is reacted with an acylating agent or an alkoxycarbonylating agent to obtain formula (IX): 【Chemistry 16】 [In the formula, A2 and PG5 are as defined above.] A step to obtain a compound represented by or a salt thereof. and (Step a6) A step of deprotecting the PG5 protecting group of the 5' hydroxyl group of the compound of formula (IX) obtained in step a5 or step a5'' to obtain the compound of formula (IV) or a salt thereof. A method of manufacturing.

2. In step a2, the intermediate obtained after the reaction with the thiorating agent and before deprotecting PG2 is given by formula (Rc-V ０ ): 【Chemistry 17】 [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 represented by or a salt thereof.

3. The method according to claim 1 or 2, wherein the activator 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 At least one selected from the group consisting of -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 thiorating 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-dithiazoline-3-one and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion, 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-dioxaphospholinane 2-oxide, dimethylchlorophosphate, diethylchlorophosphate, 2-chloro-2-oxo-1,3,2-dioxaphosphoran, 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, chlorotripyrrolidinohexafluorophosphate, bromotripyrrolidinohexafluorophosphate, and propylphosphonic anhydride, and / or The thiorating 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-dithiazoline-3-one and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion, 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 according to 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. In step a3 of 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 thiolating agent, In step a4 of claim 1, the obtained compound of formula (Rp-VII) (wherein Q is a thiol group) is treated by silica gel column chromatography before being converted to its salt, and / or crystallized after being converted to its salt to obtain the compound of formula (Rp,Rp-VII'): [Chemistry 18] A step to obtain a compound or salt thereof, (Step a7) The protecting groups PG1 and PG3 of the compound of formula (Rp, Rp-VII') obtained are deprotected to form formula (Rp, Rp-X): 【Chemistry 19】 [In the formula, A1 is as defined in claim 1. A step of obtaining a compound represented by or a salt thereof, (Step a8) The obtained compound of formula (Rp, Rp-X) or a salt thereof, Formula (XI): 【Chemistry 20】 It condenses with a compound represented by formula (Rp, Rp-XII) or its active ester to produce the compound (Rp, Rp-XII): 【Chemistry 21】 [In the formula, A1 is as defined above.] A step of obtaining a compound represented by or a salt thereof, and (Step a9) The obtained compound of formula (Rp, Rp-XII) or a salt thereof is combined with an antibody or a functional fragment of the antibody (hereinafter referred to as Ab) to form formula (Rp, Rp-XIII): 【Chemistry 22】 [In the formula, m is in the range of 1 to 10. The sugar chain of Ab is arbitrarily remodeled. Ab is either directly bound to the compound of formula (Rp,Rp-XII) from the side chain of an amino acid residue that may be modified, or bound to the compound of formula (Rp,Rp-XII) from the glycan of Ab or a remodeled glycan. A1 is, 【Chemistry 23】 That is the case. A process to obtain an antibody-immunostimulant conjugate represented by the above, or a mixture thereof. The method according to any one of claims 1 to 5, including the method described in any one of claims 1 to 5.

7. 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 7. The method according to claim 6, 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.

8. The compound of formula (I) (wherein PG1 is tert-butyldimethylsilyl) is used in the following steps: (Step b1) Formula (XIV): 【Chemistry 24】 [In the formula, PG2 is as defined in claim 1.] The compound represented by formula (XV): 【Chemistry 25】 [In the formula, PG3 is as defined in claim 1, and X is Cl, Br, or I. When reacted with a compound represented by formula (XVI): 【Chemistry 26】 [In the formula, PG2 and PG3 are as defined above.] A step of obtaining a compound represented by, (Step b2) The obtained compound of formula (XVI) is reacted with a silylating agent to obtain a mixture of the following compound of formula (I') and compound of formula (XVII): 【Chemistry 27】 [In the formula, PG2 and PG3 are as defined above.] The process involves obtaining a compound, then, in the presence of a base, converting the compound of formula (XVII) in the mixture to the compound of formula (I') to obtain the compound of formula (I'). The method according to any one of claims 1 to 7, manufactured by

9. The reaction in step b1 is carried out in the presence of a base, wherein the base is 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo[5.4.0]-7-undecene, and / or The method according to claim 8, further comprising the step in step b2 of carrying out the reaction of 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').

10. The compound of formula (XV) is obtained in the following steps: (Step b3) Formula (XVIII): 【Chemistry 28】 [In the formula, PG3 is as defined in claim 1.] The process involves reacting a compound represented by with 2-haloethanol in the presence of an acid or base to obtain a compound of formula (XV). The method according to claim 8 or 9, manufactured by...

11. Formula (XV): 【Chemistry 29】 [In the formula, PG3 is 2-(trimethylsilyl)ethoxycarbonyl, and X is Cl, Br, or I. A compound represented by the formula.

12. The compound of formula (VIII') [In the formula, A1 is, 【Transformation 30】 and PG5 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. However, the following steps: (Step c1) Formula (XIX): 【Chemistry 31】 The compound represented by is reacted with a benzoylating agent to obtain formula (XX): 【Chemistry 32】 A step to obtain a compound represented by, (Step c2) The obtained compound of formula (XX) is hydrolyzed to obtain the compound of formula (XXI): 【Transformation 33】 A step to obtain a compound represented by, (Step c3) The compound of formula (XXI) obtained is reacted with a chlorinating agent to obtain the compound of formula (XXII): 【Transformation 34】 A step to obtain a compound represented by, (Step c4) The compound of formula (XXII) obtained is taken as formula (XXIII): 【Chemistry 35】 When reacted with a compound represented by formula (XXIV): 【Transformation 36】 A step to obtain a compound represented by, (Step c5) The benzoyl group is deprotected from the compound of formula (XXIV) obtained to form formula (XXV): 【Chemistry 37】 A step of obtaining a compound represented by or a salt thereof, (Step c6) A step of reacting the obtained compound of formula (XXV) or a salt thereof with a tritylating agent to obtain the compound of formula (VIII') (wherein A1 and PG5 are as defined above) or a salt thereof. The method according to any one of claims 1 to 10, manufactured by

13. 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 reacted with an acylating agent or an alkoxycarbonylating agent to obtain formula (XXVII): 【Transformation 38】 [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 hydroxyl groups. A step of obtaining a compound represented by, (Step c8) Deprotect the PG7 protecting group of the obtained compound of formula (XXVII) to obtain formula (VIII): 【Chemistry 39】 [In the formula, A2 is, 【Chemistry 40】 and PG4 and PG5 are defined above. A step to obtain a compound represented by or a salt thereof. The method according to claim 12, including the method described in claim 12.

14. The compound of formula (XXIII) is obtained in the following steps: (Step c12) Formula (XXXI): 【Chemistry 41】 The compound is reacted with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain formula (XXXII): 【Chemistry 42】 A step to obtain a compound represented by, (Step c13) The obtained compound of formula (XXXII) is reacted with propargylaldehyde diethyl acetal to obtain compound (XXVIII): 【Chemistry 43】 A step to obtain a compound represented by, (Step c9) The tert-butoxycarbonyl group is deprotected from the compound of formula (XXVIII) obtained to form formula (XXIX): 【Chemistry 44】 A step to obtain a compound represented by, (Step c10) The obtained compound of formula (XXIX) is subjected to an alkyne reduction reaction, followed by a reductive amination reaction, in the presence of a catalyst, to obtain formula (XXX): 【Chemistry 45】 A step of obtaining a compound represented by, (Step c11) A step in which the obtained compound of formula (XXX) is reacted with a benzoylating agent to obtain the compound of formula (XXIII). The method according to claim 12 or 13, manufactured by...

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