Novel method for producing antibody-drug conjugate having antineoplastic effect

Abstract

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, and to provide methods for producing a cyclic dinucleotide-linker and antibody-immunostimulator conjugate using the production method.SOLUTION: The present invention relates to a method that makes it possible to produce, in large amounts and with high yield, a cyclic dinucleotide derivative having a desired steric configuration, by using an optically active phosphitylation agent twice, during coupling and cyclization.SELECTED DRAWING: None

Description

[Technical Field]

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

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

[0003] The CDNs studied to date contain two sulfur-modified pentavalent phosphate bonds called phosphorothioate bonds when linking two types of nucleotides into a ring. When asymmetric nucleotides are cyclized, an asymmetric center is generated at the phosphorus atom of each phosphorothioate moiety. Previously reported CDN synthesis methods involve synthesis without controlling the asymmetry at the phosphorus, and the resulting four diastereomers are purified to obtain CDNs with the desired absolute configuration (e.g., Non-Patent Document 3, Patent Documents 1-7). These synthesis methods result in low yields due to the production of four diastereomers. Furthermore, because the diastereomers have similar physical properties, their separation and purification requires strict preparative purification by HPLC at low loads, making the synthesis of large quantities of the desired product a significant burden. Three main methods have been reported for the synthesis of stereoselective CDNs. Methods using key compounds combining pentavalent phosphorus and chiral auxiliary groups require the use of thiophenol derivatives, which have a strong odor, and several steps during the synthesis of the key compounds, resulting in low to moderate yields during cyclization to CDNs (Non-Patent Documents 4-5). Methods using optically active phosphoric acid as a catalyst also exhibit variable selectivity and yield depending on the substrate structure, resulting in low cyclization yields (Non-Patent Document 6). Methods using modified cyclic guanosine-adenosine synthase (cGAS), an enzyme that constructs CDN backbones, also exhibit good yields and selectivity, but because they are based on cGAS, the applicable nucleobase structures are thought to be limited to guanosine and adenosine derivatives (Non-Patent Document 7). Furthermore, there have been several studies on methods for stereoselectively synthesizing phosphorothioate moieties, a characteristic functional group contained in CDN backbones, in relation to stereocontrolled oligonucleotides combining trivalent phosphorus and chiral auxiliary groups (e.g., Non-Patent Documents 8-12, Patent Documents 8-9). On the other hand, there are no examples of dinucleotide bond formation using compounds that can be applied to cyclic dinucleotide synthesis based on this method, and there are no examples of CDNs being synthesized with high stereoselectivity. [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] J.Org.Chem. 2021.86.8851-8861 [Non-patent document 6] Science 2021.371.702-707 [Non-Patent Document 7] Nature 2022.603.439-447 [Non-patent document 8] Tetrahedron Lett.1998.39.2491-2494 [Non-Patent Document 9] Bioorg.Med.Chem.Lett.1998.8.2539-2544 [Non-Patent Document 10] J.Am.Chem.Soc.2002.124.4962-4963 [Non-Patent Document 11] J.Am.Chem.Soc.2003.125.8307-8317 [Non-Patent Document 12] J.Am.Chem.Soc.2008.130.16031-16037 Summary of the Invention [Problem to be solved by the invention]

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

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

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

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

[0010] To solve the above-mentioned problems, the present inventors conducted extensive research and discovered a highly stereoselective synthesis method for cyclic dinucleotide derivatives by using an optically active phosphitylating agent twice, once during coupling and once during cyclization. This reduced the burden of diastereomeric purification and resulted in the completion of a novel production method with improved overall yield. Furthermore, the inventors constructed CDN-linkers using the CDNs obtained by this production method, and also constructed antibody-immunostimulant conjugates using these CDN-linkers. Furthermore, the inventors discovered a novel production method for preparing raw materials used in the production of cyclic dinucleotides that reduces the number of steps and improves yield compared to conventional production methods, thereby completing the present invention.

[0011] That is, the present invention relates to the following: [1] Formula (Rp, Rp-9): [ka] [In the formula, A1 is [ka] and PG1 is a protecting group for the hydroxy group, and PG3 is a protecting group for the amino group. A method for producing a compound represented by the formula: (Step a1) Formula (1A): [ka] [In the formula, PG1 and PG3 are as defined above, and PG2 is a trityl-type protecting group for the hydroxy group.] with an optically active phosphitylating agent (Rc-2) selected from the group consisting of the following formulae (Rc-2-1) and (Rc-2-2): [ka] [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. and reacting with the compound of formula (Rc-3A): [ka] [In the formula, PG1, PG2 and PG3 are as defined above, and B1 is [ka] where R1 and R2 are as defined above. obtaining a compound represented by the formula: (Step a2) The obtained compound of formula (Rc-3A) is reacted with a compound of formula (4A): [ka] [In the formula, A2 is [ka] PG6 is a protecting group for the amino group, and PG4 is a protecting group for the hydroxy group. in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and then further reaction with a thiolating agent to obtain a compound represented by formula (Rc-5A): [ka] [In the formula, A2, PG1, PG2, PG3 and PG4 are as defined above; B2 is [ka] wherein R1 and R2 are as defined above, and PG7 is a protecting group for the amino group. obtaining a compound represented by the formula: (Step a3-1) PG2 of the obtained compound of formula (Rc-5A) is deprotected to give a compound of formula (Rc-6A- 01 ): [ka] [In the formula, A2, B2, PG1, PG3 and PG4 are as defined above.] obtaining a compound represented by the formula: (Step a3-2) The obtained formula (Rc-6A- 01 ) with an acylating agent or an alkoxycarbonylating agent to obtain a compound of formula (Rc-6A): [ka] [In the formula, A2, B2, PG1, PG3, and PG4 are as defined above, and PG5 is an ester-type or carbonate-type protecting group for a hydroxy group.] obtaining a compound represented by the formula: (Step a4) PG4 of the obtained compound of formula (Rc-6A) is deprotected to obtain a compound of formula (Rc-7A): [ka] [In the formula, A2, B2, PG1, PG3 and PG5 are as defined above.] obtaining a compound represented by the formula: (Step a5) The obtained compound of formula (Rc-7A) is treated with an optically active phosphitylating agent (Rc-2) selected from the group consisting of the following formulae (Rc-2-1) and (Rc-2-2), which may be the same as or different from the optically active phosphitylating agent (Rc-2) of Step a1: [ka] wherein R1 and R2 are as defined above. PG5 is deprotected by reaction with an optically active phosphitylation agent (Rc-2), then cyclized in the presence of an activating agent, further treated with an acylating agent or an alkoxycarbonylating agent, and subsequently reacted with a thiolation agent to obtain a compound of the formula (Rc, Rc-8): [ka] [In the formula, A2, B2, PG1 and PG3 are as defined above; B2' is [ka] wherein R1, R2, and PG7 are as defined above. and obtaining a compound represented by the formula: (Step a6) deprotecting B2 and B2′, which are protecting groups at the thiophosphate moiety of the obtained compound of Formula (Rc, Rc-8), and PG6, which is a protecting group in A2, to obtain a compound of Formula (Rp, Rp-9) or a salt thereof; A method comprising: [2] The method according to [1], wherein the activating agents in steps a2 and a5 are independently at least one selected from the group consisting of 1-phenylimidazolium triflate, 1-methylbenzimidazolium triflate, 1-(cyanomethyl)piperidinium triflate, 1-(cyanomethyl)pyrrolidinium triflate, and 1-(cyanomethyl)imidazolium triflate. [3] The acylating agent or alkoxycarbonylating agent in step a2 and step a5 is independently selected from the group consisting of 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-fluorenylmethyl pentafluorophenyl carbonate, 1-[(9 [1] The method according to [2], wherein the compound is at least one selected from the group consisting of N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, and methylimidazole-1-carboxylate. [4] The method according to any one of [1] to [3], wherein the thiolating agents in steps a2 and a5 are independently at least one selected from the group consisting of xanthan hydride, bis(phenylacetyl)disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione. [5] PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl, and The method according to any one of [1] to [4], wherein PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, or allyloxycarbonyl. [6] PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG6 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl, and The method according to any one of [1] to [5], wherein PG7 is acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl. [7] The method according to any one of [1] to [6], wherein PG4 is levulinoyl. [8] The method according to any one of [1] to [7], wherein PG5 is allyloxycarbonyl. [9] PG1 is tert-butyldimethylsilyl, PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, or trityl; PG4 is levulinoyl, and The method according to any one of [1] to [8], wherein PG5 is allyloxycarbonyl.

[10] moreover (Step a7) The protecting groups PG1 and PG3 of the obtained compound of formula (Rp, Rp-9) are deprotected to obtain a compound of formula (Rp, Rp-10): [ka] [In the formula, A1 is as defined in [1]. or a salt thereof; and (Step a8) The obtained compound of formula (Rp, Rp-10) or a salt thereof is Equation (11): [ka] or an activated ester thereof, to form a compound represented by the formula (Rp, Rp-12): [ka] [In the formula, A1 is as defined above.] or a salt thereof The method according to any one of [1] to [9], comprising:

[11] moreover, (Step a9) The obtained compound of formula (Rp, Rp-12) or a salt thereof is bound to an antibody or a functional fragment of the antibody (hereinafter referred to as Ab) to obtain a compound of formula (Rp, Rp-13): [ka] [In the formula, m ranges from 1 to 10, The glycans of Abs are arbitrarily remodeled, Ab is bound to the compound of formula (Rp, Rp-12) directly from the side chain of an optionally modified amino acid residue, or is bound to the compound of formula (Rp, Rp-12) from a sugar chain or a remodeled sugar chain of Ab; A1 is [ka] ] or a mixture thereof. The method according to any one of [1] to

[10] , comprising:

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

[11] , wherein in step a9, the compound of formula (Rp, Rp-12) or a salt thereof and Ab are bonded by a strain-promoted azide-alkyne cycloaddition reaction.

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

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

[14] The compound of formula (4A) (wherein PG4 is levulinoyl) can be obtained by the following steps: (Step a0-1) Formula (XXV'): [ka] or a salt thereof, by protecting the 5'-hydroxy group of the compound of the formula (4A- 01 ): [ka] [In the formula, PG8 is a trityl-type protecting group for a hydroxy group.] or a salt thereof, and (Step a0-2) The obtained formula (4A- 01 a step of protecting the hydroxy group at the 2'-position of the compound of formula (4A) or a salt thereof with levulinic acid, reacting the compound with an acylating agent or an alkoxycarbonylating agent, and then detritylation to obtain the compound of formula (4A). The method according to any one of [1] to

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

[13] .

[15] Formula (4A'): [ka] [In the formula, PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl, and PG6 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl. A compound represented by the formula:

[16] Formula (Rc-5A): [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, and PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl, and PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl. A compound represented by the formula:

[17] Formula (Rc-6A): [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; PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl, and PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, or allyloxycarbonyl. A compound represented by the formula:

[18] Formula (Rc-7A): [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, and PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, or allyloxycarbonyl. A compound represented by the formula:

[19] Formula (Rc,Rc-8): [ka] [In the formula, A2, B2 and B2' are as defined in [1], PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl, and PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. Compound. [Effects of the Invention]

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

[0013] In one aspect, the present invention provides a novel method for producing a CDN derivative, which is an intermediate for an antibody-immunoactivator conjugate that has STING agonist activity and activates immune cells. Such an antibody-immunoactivator conjugate is disclosed in International Publication No. 2020 / 050406 (Patent Document 6). One embodiment of the present invention is a method for stereoselectively synthesizing a CDN derivative having a structure represented by the following formula (Rp, Rp-9):

[0014] [ka] A representative scheme of the present invention, which is one embodiment and focuses on a novel method for producing a CDN derivative of formula (Rp, Rp-9), is outlined below.

[0015] [ka] Preferred embodiments for carrying out the present invention will be described below. The embodiment described below is merely an example of a typical embodiment of the present invention, and is not intended to narrow the scope of the present invention. In this specification, the compound numbers shown in each reaction formula are used to indicate the compounds. That is, they are referred to as "compound of formula (1A)," "compound (1A)," etc. Compounds with other numbers will also be described in the same manner.

[0016] <1. Production of cyclic dinucleotides (CDNs) and CDN - linkers> <1 - 1. Method for producing cyclic dinucleotide (compound (Rp,Rp - 9)) and its intermediates>

[0017] In the conventional production methods, four types of cyclic dinucleotides shown below were synthesized stereonon - selectively (see Patent Document 6, etc.). The configurations of these four types of cyclic dinucleotides are represented in the order of the absolute configurations on the two phosphorus atoms, (absolute configuration of phosphorus at the 2 - position, absolute configuration of phosphorus at the 10 - position), as (Rp,Rp), (Sp,Rp), (Rp,Sp), (Sp,Sp).

[0018]

Chemical formula

[0019] In the production of the CDN of the present invention, in one embodiment, the cyclic dinucleotide of formula (Rp,Rp - 9) can be produced according to the following synthetic scheme <Method A>.

Chemical formula

[0020] In the above synthetic scheme <Method A>, PG1 is a protecting group for a hydroxy group, PG2 is a trityl - type protecting group for a hydroxy group, PG3 is a protecting group for an amino group, PG4 is a protecting group for a hydroxy group, PG5 is an ester - type or carbonate - type protecting group for a hydroxy group, PG8 is a trityl - type protecting group for a hydroxy group, A1 is

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

[0021] In this production method, PG1 is, for example, a silyl-type protecting group for a hydroxy group, and examples thereof include tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl. Preferably, they are tert-butyldimethylsilyl and trimethylsilyl. More preferably, it is tert-butyldimethylsilyl.

[0022] PG2 is a trityl-type protecting group for a hydroxy group, and examples thereof include 4,4’-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. Preferably, they are 4,4’-dimethoxytrityl, 4-methoxytrityl or trityl. More preferably, they are 4-methoxytrityl or 4,4’-dimethoxytrityl.

[0023] PG3 is, for example, a carbamate-type protecting group for an amino group, and examples thereof include 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, and benzyloxycarbonyl. 2-(trimethylsilyl)ethoxycarbonyl or allyloxycarbonyl is preferred. 2-(trimethylsilyl)ethoxycarbonyl is more preferred.

[0024] PG4 is, for example, formyl, or an ester-type protecting group for a hydroxy group, a carbonate-type protecting group, or a benzyl-type protecting group, and examples thereof include formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, and fluorenylmethyloxycarbonyl. Chloroacetyl, levulinoyl, or allyloxycarbonyl is preferred. Levulinoyl is more preferred.

[0025] PG5 is an ester-type protecting group or a carbonate-type protecting group for a hydroxy group. Examples of ester-type protecting groups include acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, and levulinoyl. Examples of carbonate-type protecting groups include fluorenylmethyloxycarbonyl and allyloxycarbonyl. Chloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, and allyloxycarbonyl are preferred. Fluorenylmethyloxycarbonyl and allyloxycarbonyl are more preferred.

[0026] Examples of PG6 include benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, and ethoxycarbonyl. Preferred are benzoyl, 2,2,2-chloroethoxycarbonyl, benzyloxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl. More preferred are benzoyl and 2-(trimethylsilyl)ethoxycarbonyl.

[0027] Examples of PG7 include acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, and ethoxycarbonyl. Preferred are acetyl, trifluoroacetyl, benzoyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl. More preferred are acetyl, trifluoroacetyl, allyloxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl.

[0028] Examples of PG8 include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, and trityl. Preferred are 4,4'-dimethoxytrityl, 4-methoxytrityl, and trityl. More preferred are 4-methoxytrityl and 4,4'-dimethoxytrityl.

[0029] Each step will be described below. (Step a1) In one embodiment, this step is a step of reacting a compound of formula (1A) with an optically active phosphitylating agent (Rc-2) to obtain a compound of formula (Rc-3A).

[0030] In one embodiment, the optically active phosphitylating agent (Rc-2) used in this step is a compound represented by the following formula (Rc-2-1) or (Rc-2-2): [ka] [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. The optically active phosphitylating agent is a reagent for stereoselectively condensing a compound of formula (1A) with a compound of formula (4A) to produce a compound of formula (Rc-5A), and reacts stereoselectively with the compound of formula (1A) to produce an intermediate of formula (Rc-3A).

[0031] Examples of the optically active phosphitylating agent (Rc-2) include (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aR)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, and (3S,3aR)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3 ,2]oxazaphosphole, (3R,3aR)-1-chloro-3-[(4-methylbenzene-1-sulfonyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-[(4-nitrophenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aR)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3a R)-1-chloro-3-[(4-methoxyphenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-(diphenylmethyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aR)-1-chloro-3a,4-dihydro-1H,3H-[1,3,2]oxazaphosphoro[3,4-a]indole, etc. can be used, and preferably (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (3aR)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aR)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used, and more preferably, (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aR)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aR)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, or (3R,3aR)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used. The amount of the optically active phosphitylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 3 equivalents relative to the compound represented by formula (1A).

[0032] The structure of the optically active phosphitylating agent (Rc-2) exemplified above is shown below. [Table 1]

[0033] In this step, the reaction proceeds stereoselectively to give a compound of formula (Rc-3A) with the desired absolute configuration. Here, the absolute configuration of the compound of formula (Rc-3A) is indicated by (Rc), which is the absolute configuration of the asymmetric carbon atom contained in the prolinol skeleton, as shown in the following example.

[0034] [ka]

[0035] In one embodiment, when an optically active phosphitylating agent (Rc-2) in which R1 and R2 are hydrogen atoms is used, a compound of formula (Rc-3A) having the absolute configuration at the phosphorus atom of (Sp) is produced, as shown below. [ka]

[0036] That is, by using an optically active phosphitylating agent with the absolute configuration (Rc), the desired diastereomer can be obtained with high selectivity of 90% or more, preferably 95% or more, and more preferably 98% or more. In conventional production methods, phosphorothioate bonds are formed stereo-nonselectively, and the (Rp) and (Sp) forms are produced in a ratio of approximately 50:50, so the yield of the compound with the desired absolute configuration was less than half.

[0037] This step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate; and sodium acetate. Examples of suitable bases include sodium, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, and 1,4-diazabicyclo[2.2.2]octane. More preferred examples include triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by Formula (1A).

[0038] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include acetonitrile, dichloromethane, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, and toluene, and more preferred examples include dichloromethane.

[0039] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −78° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 10 hours, more preferably 5 to 180 minutes.

[0040] (Step a2) In one embodiment, this step involves reacting a compound of Formula (Rc-3A) with a compound of Formula (4A) or a salt thereof in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and then reacting with a thiolating agent (also referred to as a sulfurizing agent or a thiolating agent) to obtain a compound of Formula (Rc-5A).

[0041] In one embodiment, the reaction in this step proceeds stereoselectively to give a compound of formula (Rc-5A) having the desired absolute configuration. The absolute configuration of formula (Rc-5A) is expressed using the absolute configuration of the asymmetric carbon atom contained in the prolinol skeleton in B2, as in the compound of formula (Rc-3A) above. [ka]

[0042] In this step, the stereoinversion reaction proceeds with selectivity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more while maintaining the asymmetric environment established by formula (Rc-3A). This allows the desired diastereomer to be obtained with high selectivity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. In conventional methods, the compound of formula (1A) and the compound of formula (4A) are condensed stereoselectively, resulting in a mixture of the difficult-to-separate (Rp) and (Sp) isomers in a ratio of approximately 50:50, corresponding to the compound of formula (5A). Therefore, the yield of the desired compound (Rc-5A) isomer is not only improved by approximately 2-fold from the perspective of stereoselectivity, but also, considering the burden of separating the large amount of diastereomers at the phosphorus, which are difficult to separate, productivity is expected to increase by approximately 3-10-fold.

[0043] The activating agent used in this step is not particularly limited as long as the reaction proceeds without damaging the absolute stereochemistry of the compound represented by Formula (Rc-3A). For example, examples of the base component of the counter anion include 1-phenylimidazole, benzimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-pyrrolidineacetonitrile, and 1-(cyanomethyl)imidazole. Examples of the acid component of the counter anion include tetrafluoroboric acid, hexafluorophosphoric acid, perchloric acid, trifluoroacetic acid, methanesulfonic acid, and trifluoromethanesulfonic acid. Preferred examples of the activator include 1-phenylimidazolium triflate, 1-methylbenzimidazolium triflate, 1-(cyanomethyl)piperidinium triflate, 1-(cyanomethyl)pyrrolidinium triflate, and 1-(cyanomethyl)imidazolium triflate, and more preferred examples include 1-phenylimidazolium triflate and 1-methylbenzimidazolium triflate. The amount of the activator used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 3 equivalents relative to the compound represented by formula (Rc-3A).

[0044] The amount of compound (4A) used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 3 equivalents, more preferably 0.8 to 1.2 equivalents, relative to the compound represented by formula (Rc-3A).

[0045] The acylating agent or alkoxycarbonylating agent used in this step is not particularly limited as long as it allows the reaction to proceed, but examples thereof include acetic anhydride, acetyl chloride, N-succinimidyl acetate, pentafluorophenyl acetate, 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, N-methoxydiacetamide, N-acetylimidazole, trifluoroacetic anhydride, bistrifluoroacetamide, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl acetate, and the like. Fluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, S-ethyl trifluorothioacetate, N-methylbistrifluoroacetamide, trifluoroacetyl triflate, 1-trifluoroacetylimidazole, benzoic anhydride, benzoyl chloride, pentafluorophenyl benzoate, benzoyl trifluoromethanesulfonate, 3-benzoylthiazolidine-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy) (iii) Phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butyl carbazate, 2-(tert-butoxycarbonyloximino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole, N-tert-butoxycarbonylimidazole, di-tert-butyl dicarbonate, 9-fluorenylmethyl pentafluorophenyl carbonate, 9-fluorochloroformate oleylmethyl, 9-fluorenylmethyl carbazate, 9-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, chloroformic acid 2,2,2-Trichloroethyl, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethyl imidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, Examples of suitable acylating agents include dimethyl carbonate, dimethyl dicarbonate, and methylimidazole-1-carboxylate. Preferred examples include acetic anhydride, 1-trifluoroacetylimidazole, benzoic anhydride, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(allyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. More preferred examples include acetic anhydride, 1-trifluoroacetylimidazole, N-(allyloxycarbonyloxy)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-3A).

[0046] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 5 hours, more preferably 5 to 90 minutes.

[0047] The reaction of the activating agent, acylating agent or alkoxycarbonylating agent, and thiolating agent in this step is preferably carried out in the presence of a dehydrating agent. The dehydrating agent used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, magnesium sulfate, sodium sulfate, and calcium chloride. Preferred examples include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, and sodium sulfate, and more preferred examples include molecular sieve 3A and molecular sieve 4A. The amount of dehydrating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.01 to 3 times, and more preferably 0.01 to 1 times, the mass of the compound represented by Formula (Rc-3A).

[0048] The thiolation agent used in this step is not particularly limited as long as it allows the reaction to proceed. For example, xanthan hydride, bis(phenylacetyl) disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione can be used; preferably, xanthan hydride, bis(phenylacetyl) disulfide, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione can be used; more preferably, xanthan hydride and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione can be used. The amount of the thionating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 1 to 2 equivalents, relative to the compound represented by Formula (Rc-3A).

[0049] The reaction temperature with the thionating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 5 hours, more preferably 5 to 90 minutes.

[0050] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, and examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; and bases such as potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine. More preferred examples include triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 20 equivalents, more preferably 1 to 10 equivalents, relative to the compound represented by formula (Rc-3A).

[0051] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof can be used. Preferably, acetonitrile and dichloromethane can be mentioned, and more preferably, acetonitrile can be mentioned.

[0052] (Step a3) In one embodiment, step a3 includes step a3-1 and step a3-2. In step a3-1, PG2 of the compound of formula (Rc-5A) is deprotected, and in step a3-2, the resulting hydroxyl group is protected with PG5 to obtain the compound of formula (Rc-6A). By the deprotection reaction of step a3-1, a compound represented by formula (Rc-6A 01 ): [Chemical formula] is obtained (where A2, B2, PG1, PG3, and PG4 are synonymous with the groups described in the synthetic scheme <Method A>).

[0053] In step a3-1, examples of the acid used in the deprotection reaction of PG2 include hydrochloric acid, sulfuric acid, formic acid, oxalic acid, acetic acid, trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid. Preferred examples include hydrochloric acid, acetic acid, monochloroacetic acid, and dichloroacetic acid, and more preferred examples are hydrochloric acid and dichloroacetic acid. The pH used in the deprotection reaction is not limited as long as the reaction proceeds, but is preferably 1 to 4. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from −30° C. to the boiling point of the solvent used in the reaction, more preferably from −20° C. to 30° C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours.

[0054] The solvent used in the deprotection reaction of PG2 in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include dichloromethane.

[0055] The reagent for introducing a protecting group (PG5) into the hydroxyl group used in step a3-2 varies depending on the type of protecting group, but examples thereof include acetic anhydride, acetyl chloride, N-succinimidyl acetate, pentafluorophenyl acetate, 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, N-methoxydiacetamide, N-acetylimidazole, chloroacetic anhydride, chloroacetyl chloride, dichloroacetic anhydride, dichloroacetyl chloride, trichloroacetic anhydride, trichloroacetyl chloride, levulinic anhydride, 9 9-Fluorenylmethyl pentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl carbazate, 9-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, and the like. Further examples include a combination of an acid such as acetic acid, chloroacetic acid, or levulinic acid with a condensing agent such as N,N'-dicyclohexylcarbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-carbonyldiimidazole, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate. Preferred examples include chloroacetic anhydride, chloroacetyl chloride, levulinic anhydride, 9-fluorenylmethyl chloroformate, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, and a combination of levulinic acid and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, and more preferred examples include chloroacetyl chloride, 9-fluorenylmethyl chloroformate, allyl chloroformate, and a combination of levulinic acid and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. The amount of the reagent for introducing a protecting group (PG5) into the hydroxyl group is not limited as long as the reaction proceeds, but is preferably a compound represented by the formula (Rc-6A- 01The amount of the compound is preferably 0.7 to 5 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by the formula (I).

[0056] The solvent used for introducing a protecting group (PG5) into the hydroxyl group in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include dichloromethane.

[0057] (Step a4) In one embodiment, this step is a step of deprotecting PG4 of a compound of formula (Rc-6A) to obtain a compound of formula (Rc-7A).

[0058] The reagent used for deprotection in this step varies depending on the type of PG4. For example, when PG4 is a levulinoyl group, examples of suitable reagents include sodium borohydride, hydrazine monohydrate, hydrazine acetate, hydrazine monohydrochloride, and hydrazine dihydrochloride. Preferred examples include hydrazine monohydrate, hydrazine acetate, hydrazine monohydrochloride, and hydrazine dihydrochloride, and more preferred examples are hydrazine monohydrate and hydrazine acetate. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from −30°C to the boiling point of the solvent used in the reaction, more preferably from −20°C to 30°C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours.

[0059] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2 - dimethoxyethane, tetrahydrofuran, 2 - methyltetrahydrofuran, 1,4 - dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2 - butanone, N,N - dimethylformamide, N,N - dimethylacetamide, 1 - methyl - 2 - pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof can be used. Preferably, acetonitrile and dichloromethane can be mentioned, and more preferably, dichloromethane can be mentioned.

[0060] (Step a5) In one embodiment, this step involves reacting a compound of formula (Rc - 7A) with an optically active phosphitylating agent (Rc - 2), then performing deprotection of PG5, carrying out a cyclization reaction in the presence of an activator, then treating with an acylating agent or an alkoxycarbonylating agent, and further reacting with a sulfurizing agent (also referred to as a sulfiding agent or a thiolating agent) to obtain a compound of formula (Rc,Rc - 8).

[0061] In this step, as an intermediate obtained after reacting with an optically active phosphitylating agent (Rc - 2), a compound of formula (Rc - 8 - 01 ), and as an intermediate obtained after further deprotecting PG5, a compound of formula (Rc - 8 - 02 ):

Chemical formula

[0062] The optically active phosphitylating agent (Rc-2) used in this step is the same as the phosphitylating agent (Rc-2) used in the above (Step a1), and the base, solvent, reaction temperature and reaction time used simultaneously are also the same as those in the above (Step a1).

[0063] The reagent used for deprotecting PG5 in this step varies depending on the type of PG5. For example, when PG5 is an allyloxycarbonyl group, examples include zero-valent palladium catalysts such as tetrakis(triphenylphosphine)palladium and tris(dibenzylideneacetone)dipalladium. Alternatively, a divalent palladium catalyst (e.g., palladium chloride, dichlorobis(triphenylphosphine)palladium, palladium acetate, etc.) can be used in situ to reduce the PG5 to zero-valent palladium using a reducing agent (e.g., triphenylphosphine, tributyltin hydride, formic acid, ammonium formate, triethylsilane, etc.). Scavenger for the elimination of the allyl group includes dimedone, piperidine morpholine, and dimethylamine-borane complex. Preferred examples include combinations of reducing agents such as tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)dipalladium, or palladium acetate with triphenylphosphine. More preferred are tetrakis(triphenylphosphine)palladium and a combination of palladium acetate and triphenylphosphine. Preferred allyl group scavengers include dimedone and piperidine morpholine, and more preferred is dimedone. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from -30°C to the boiling point of the solvent used in the reaction, more preferably from -20°C to 30°C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably from 1 minute to 48 hours, more preferably from 1 minute to 24 hours.

[0064] The solvent used for deprotection of PG5 in this step may be the same as that used in the subsequent phosphitylation reaction, but is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, acetonitrile, dichloromethane, and mixed solvents thereof, and more preferred examples include dichloromethane, acetonitrile, and mixed solvents thereof.

[0065] The activating agent for the cyclization reaction, the acylating agent or alkoxycarbonylating agent, and the thiolating agent (also called a sulfurizing agent or thiolating agent) used in this step are the same as those used in the above step a2.

[0066] The reaction temperature with the activating agent for the cyclization reaction in this step is not limited as long as the reaction proceeds, but is preferably from −78° C. to the boiling point of the solvent used in the reaction, more preferably −60° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 5 hours, more preferably 5 minutes to 3 hours.

[0067] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1 minute to 5 hours, more preferably 5 minutes to 90 minutes.

[0068] The reaction temperature with the thionating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 5 hours, more preferably 5 minutes to 90 minutes.

[0069] The solvent used in this step from the cyclization reaction using an activator to the thiolation reaction may be the same as that used in the consecutive reaction after the phosphitylation reaction and the deprotection reaction of PG5, but is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, acetonitrile, dichloromethane, and mixed solvents thereof. More preferred examples include dichloromethane, acetonitrile, and mixed solvents thereof.

[0070] The absolute configuration of the compound of formula (8) is expressed in the order of (absolute configuration of phosphorus at position 2, absolute configuration of phosphorus at position 10) relative to the absolute configurations of the two phosphorus atoms, as shown below: (Rp,Rp), (Sp,Rp), (Rp,Sp), (Sp,Sp). [ka]

[0071] In this step, after deprotection of PG5, a cyclization reaction using an activating agent is carried out to form a compound of the formula (Rc-8- 01While maintaining the asymmetric environment constructed in step (1), the desired diastereomer can be obtained with a high selectivity of 85% or more. For example, as shown in Example 8 of the present specification, in the cyclization reaction from the compound of formula (Rc-7) to the compound of formula (Rp,Rp-9), the (Rp,Rp-8) isomer having the desired absolute configuration was obtained with a selectivity of 89:11 relative to the (Sp,Rp-8) isomer.

[0072] Conventional methods do not stereoselectively control the cyclization reaction, but depending on the type of cyclization reaction, the production of one diastereomer may proceed predominantly based on the three-dimensional structure of the molecule itself. However, because this is not a reaction that specifically controls the stereochemistry, the selectivity is thought to be at most approximately 50:50 to 85:15. For example, although the substrate is slightly different, Example 5 of Patent Document 10 (WO 2018 / 138685) describes the production of 46 mg of Rp cyclized product (5m) and 59 mg of Sp cyclized product (5l) after cyclization, clearly demonstrating low selectivity during cyclization.

[0073] From Example 5 of International Publication No. 2018 / 138685 [ka]

[0074] (Step a6) In one embodiment, this step involves deprotecting the thiophosphate moieties B2 and B2' of the compound (Rc, Rc-8) and the protecting group PG6 in A2 to obtain a compound of formula (Rp, Rp-9). This step optionally includes treating with an organic or inorganic base to convert the compound into a salt thereof.

[0075] The ammonia used in the deprotection reaction in this step is not limited as long as the reaction proceeds, but 28% aqueous ammonia is preferably used.

[0076] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from 30° C. to 65° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 1 to 96 hours, more preferably from 1 to 48 hours.

[0077] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, pyridine, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, ethanol, acetonitrile, dichloromethane, and pyridine, and more preferred examples include methanol and pyridine.

[0078] Examples of organic bases used in converting the compound of formula (Rp, Rp-9) into its salt include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, etc. Examples of inorganic bases include potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate, and more preferred examples include triethylamine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate.

[0079] (Process a0-1) In this step, a compound of formula (XXV') or a salt thereof is reacted with a tritylation agent to give a compound of formula (4A- 01 or a salt thereof.

[0080] Examples of the tritylation agent used in this step include 4,4-dimethoxytrityl chloride, 4-methoxytrityl chloride, 2-chlorotrityl chloride, and trityl chloride, and preferred examples include 4,4-dimethoxytrityl chloride and 4-methoxytrityl chloride. The amount of the tritylation agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, and more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXV').

[0081] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 36 hours.

[0082] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, and dichloromethane, and more preferred examples include pyridine.

[0083] (Process a0-2) This step is carried out by reacting a compound of the formula (4A- 01 ) or a salt thereof with levulinic acid to protect the hydroxy group at the 2'-position, followed by reaction with a benzylating agent, an acylating agent, or an alkoxycarbonylating agent, followed by detritylation to obtain a compound of formula (4A).

[0084] Examples of the benzylating agent, acylating agent, or alkoxycarbonylating agent used in this step include benzyl chloride, benzyl bromide, benzoyl chloride, benzoic anhydride, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy)phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butyl carbazate, 2-(tert-butoxycarbonyloximino)-2-phenyl Nylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole, N-tert-butoxycarbonylimidazole, di-tert-butyl dicarbonate, 9-fluorenylmethyl pentafluorophenyl carbonate, 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl carbazate, 9-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluorene-9 -ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethyl Examples of the ethoxycarbonyl group include 2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethyl imidazole-1-carboxylate, and 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline.Preferred are benzoyl chloride, benzoic anhydride, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, 2,2,2-trichloroethyl chloroformate, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-trimethylsilyl Examples of suitable benzoyl compounds include benzoyl chloride, benzoic anhydride, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, and more preferred examples include benzoyl chloride, benzoic anhydride, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. The amount of the reagent for introducing a protecting group (PG4) into the hydroxyl group is not limited as long as the reaction proceeds. 01 The amount of the reagent for introducing a protecting group (PG6) into the hydroxyl group is not limited as long as the reaction proceeds, but is preferably 0.7 to 5 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (4A- 01 The amount of the compound represented by the formula (I) is preferably 1 to 15 equivalents, and more preferably 4 to 10 equivalents.

[0085] The reaction temperature for the protecting group (PG4) on the hydroxyl group in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 36 hours. The reaction temperature for the protecting group (PG6) on the hydroxyl group in this step is not limited as long as the reaction proceeds, but is preferably from 20° C. to the boiling point of the solvent used in the reaction, more preferably 50° C. to 70° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1 hour to 120 hours, more preferably 30 hours to 60 hours.

[0086] As the solvent used for introducing the protecting group (PG4) to the hydroxyl group in this step, there is no particular limitation as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof can be used. Preferably, acetonitrile and dichloromethane can be mentioned, and more preferably, dichloromethane can be mentioned. As the solvent used for introducing the protecting group (PG6) to the hydroxyl group in this step, there is no particular limitation as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof can be used. Preferably, acetonitrile and dichloromethane can be mentioned, and more preferably, acetonitrile can be mentioned.

[0087] (Novel intermediate) As the novel intermediate in the above synthetic scheme <Method A>, the compound represented by the following formula (4A') can be mentioned. [Chemical formula] Here PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl, preferably chloroacetyl, levulinoyl, or allyloxycarbonyl. More preferably, it is levulinoyl.

[0088] PG6 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl, preferably benzoyl or 2-(trimethylsilyl)ethoxycarbonyl, and more preferably 2-(trimethylsilyl)ethoxycarbonyl.

[0089] In addition, as a novel intermediate in the above synthetic scheme <Method A>, a compound represented by the following formula (Rc-5A) can be mentioned.

Chemical formula

Chemical formula

Chemical formula

[0090] PG7 is a protecting group for an amino group, preferably acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl, more preferably acetyl or trifluoroacetyl.

[0091] PG1 is preferably tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl, more preferably tert-butyldimethylsilyl.

[0092] PG2 is preferably 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl, more preferably 4,4'-dimethoxytrityl, 4-methoxytrityl or trityl, even more preferably 4-methoxytrityl or 4,4'-dimethoxytrityl.

[0093] PG3 is preferably 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl, more preferably 2-(trimethylsilyl)ethoxycarbonyl.

[0094] Also, as a novel intermediate in the above synthetic scheme <Method A>, a compound represented by the following formula (Rc-6A) can be mentioned. [Chemical] Here, A2, B2, PG1, PG3, and PG4 have the same meaning as the compound of (Rc-5A) shown above.

[0095] PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, or allyloxycarbonyl, preferably chloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, or allyloxycarbonyl. More preferably, it is fluorenylmethyloxycarbonyl or allyloxycarbonyl.

[0096] In addition, as a novel intermediate in the above synthetic scheme <Method A>, a compound represented by the following formula (Rc-7A) can be mentioned. [Chemical] [[ID=第十九条]]Here, A2, B2, PG, PG3, and PG5 have the same meaning as the compound of (Rc-6A) shown above.

[0097] In addition, as a novel intermediate in the above synthetic scheme <Method A>, a compound represented by the following formula (Rc,Rc-8) can be mentioned. [Chemical] Here, A2, B2, PG1, and PG3 have the same meaning as the compound of (Rc-7A) shown above, B2' has the same meaning as B2, and B2' may be the same as or different from B2.

[0098] In addition, in the production of the CDN of the present invention, in one embodiment, a cyclic dinucleotide of the formula (Rp,Rp-9) can be produced according to the following synthetic scheme <Method B>. [Chemical]

[0099] In the above synthetic scheme <Method B>, A1, A2, B2, PG1, PG2, PG3, PG4, PG6 and PG7 are synonymous with A1, A2, B2, PG1, PG2, PG3, PG4, PG6 and PG7 as defined for the compounds in the synthetic scheme <Method A>.

[0100] B2” is [Chemical formula] and is PG9 is a protecting group for a hydroxy group and examples thereof include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. Preferably, it is 4,4'-dimethoxytrityl, 4-methoxytrityl or trityl. More preferably, it is 4-methoxytrityl or 4,4'-dimethoxytrityl.

[0101] Each step will be described below. (Step b1) In one embodiment, this step is a step of reacting a compound of formula (4B) with an optically active phosphitylating agent (Rc-2) to obtain a compound of formula (Rc-3B). The optically active phosphitylating agent (Rc-2) used in this step is synonymous with (Rc-2) used in the synthetic scheme <Method A>.

[0102] In this step, the reaction proceeds stereoselectively to obtain a compound of formula (Rc-3B) having the desired absolute configuration. Here, the absolute configuration of the compound of formula (Rc-3B) is shown using the (Rc) of the absolute configuration on the asymmetric carbon atom contained in the prolinol skeleton as exemplified below. [Chemical formula]

[0103] In one embodiment, when an optically active phosphitylating agent (Rc-2) in which R1 and R2 are hydrogen atoms is used, a compound of formula (Rc-3B) in which the absolute configuration on the phosphorus atom is (Sp) is produced as shown below. [Chemical formula] That is, by using an optically active phosphitylating agent having the absolute configuration (Rc), the desired diastereomer can be obtained with high selectivity.

[0104] This step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate; and sodium acetate. Examples of suitable bases include sodium, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, and 1,4-diazabicyclo[2.2.2]octane. More preferred examples include triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (4B).

[0105] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include acetonitrile, dichloromethane, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, and toluene, and more preferred examples include dichloromethane.

[0106] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −78° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 10 hours, more preferably 5 to 180 minutes.

[0107] (Process b0) In one embodiment, this step is a step of protecting the hydroxyl group of the compound of formula (1A) with a PG4 group, and then deprotecting PG2.

[0108] The reagent used in this step to introduce a protecting group (PG4) into a hydroxyl group varies depending on the type of protecting group, 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, chloroacetic anhydride, chloroacetyl chloride, dichloroacetic anhydride, dichloroacetyl chloride, trichloroacetic anhydride, trichloroacetyl chloride, levulinic anhydride, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, 4-methoxybenzyl chloride, 4-methoxybenzyl bromide, and 4-azido-3-chlorobenzyl bromide. Further examples include combinations of acids such as acetic acid, chloroacetic acid, and levulinic acid with condensing agents such as N,N'-dicyclohexylcarbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, N,N'-carbonyldiimidazole, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate. Preferred examples include chloroacetic anhydride, chloroacetyl chloride, levulinic anhydride, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, and combinations of levulinic acid and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, and more preferred examples include chloroacetyl chloride, a combination of levulinic acid and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, and allyl chloroformate. The amount of the reagent for introducing a protecting group (PG4) into the hydroxyl group is not limited as long as the reaction proceeds, but is preferably 0.7 to 5 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (1A).

[0109] The solvent used for introducing a protecting group (PG4) into the hydroxyl group in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include dichloromethane.

[0110] The deprotection of PG2 in this step is carried out under acidic conditions using an acid. Examples of the acid used include hydrochloric acid, sulfuric acid, formic acid, oxalic acid, acetic acid, trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid. Preferred acids include hydrochloric acid, acetic acid, monochloroacetic acid, and dichloroacetic acid, and more preferred are hydrochloric acid and dichloroacetic acid. The pH used for the deprotection is not limited as long as the reaction proceeds, but is preferably 1 to 4. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from -30°C to the boiling point of the solvent used in the reaction, more preferably from -20°C to 30°C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours.

[0111] The solvent used in the deprotection reaction of PG2 in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include dichloromethane.

[0112] (Step b2) In one embodiment, this step involves reacting a compound of Formula (Rc-3B) with a compound of Formula (1B) or a salt thereof in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and further reaction with a thiolating agent (also referred to as a sulfurizing agent or a thiolating agent) to obtain a compound of Formula (Rc-5B).

[0113] In one embodiment, the reaction in this step proceeds stereoselectively to obtain a compound of formula (Rc-5B) having the desired absolute configuration. In this step, the reaction proceeds in a highly selective stereoinversion manner while maintaining the asymmetric environment established by formula (Rc-3B). This allows the desired diastereomer to be obtained with high selectivity.

[0114] The activating agent used in this step is not particularly limited as long as the reaction proceeds without damaging the absolute configuration of the compound represented by Formula (Rc-3B). For example, examples of the base component of the counter anion include 1-phenylimidazole, benzimidazole, 1-methylbenzimidazole, 1-cyanomethylpiperidine, 1-pyrrolidineacetonitrile, and 1-(cyanomethyl)imidazole. Examples of the acid component of the counter anion include tetrafluoroboric acid, hexafluorophosphoric acid, perchloric acid, trifluoroacetic acid, methanesulfonic acid, and trifluoromethanesulfonic acid. Preferred examples of the activator include 1-phenylimidazolium triflate, 1-methylbenzimidazolium triflate, 1-(cyanomethyl)piperidinium triflate, 1-(cyanomethyl)pyrrolidinium triflate, and 1-(cyanomethyl)imidazolium triflate, and more preferred examples include 1-phenylimidazolium triflate and 1-methylbenzimidazolium triflate. The amount of the activator used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 3 equivalents relative to the compound represented by formula (Rc-3B).

[0115] The amount of compound (1B) used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 3 equivalents, more preferably 0.8 to 1.2 equivalents, relative to the compound represented by formula (Rc-3B).

[0116] The acylating agent or alkoxycarbonylating agent used in this step is not particularly limited as long as it allows the reaction to proceed, but examples thereof include acetic anhydride, acetyl chloride, N-succinimidyl acetate, pentafluorophenyl acetate, 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, N-methoxydiacetamide, N-acetylimidazole, trifluoroacetic anhydride, bistrifluoroacetamide, ethyl trifluoroacetate, methyl trifluoroacetate, pentafluorophenyl acetate, and the like. Fluorophenyl trifluoroacetate, trifluoroacetylbenzotriazole, S-ethyl trifluorothioacetate, N-methylbistrifluoroacetamide, trifluoroacetyl triflate, 1-trifluoroacetylimidazole, benzoic anhydride, benzoyl chloride, pentafluorophenyl benzoate, benzoyl trifluoromethanesulfonate, 3-benzoylthiazolidine-2-thione, tert-butylphenyl carbonate, N-(tert-butoxycarbonyloxy) (iii) Phthalimide, 2-(tert-butoxycarbonylthio)-4,6-dimethylpyridine, N-tert-butoxycarbonylimidazole, tert-butyl carbazate, 2-(tert-butoxycarbonyloximino)-2-phenylacetonitrile, 1-tert-butoxycarbonyl-1,2,4-triazole, N-tert-butoxycarbonylimidazole, di-tert-butyl dicarbonate, 9-fluorenylmethyl pentafluorophenyl carbonate, 9-fluorochloroformate oleylmethyl, 9-fluorenylmethyl carbazate, 9-fluorenylmethyl carbamate, 1-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, allyl chloroformate, diallyl dicarbonate, N-(allyloxycarbonyloxy)succinimide, allyl phenyl carbonate, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, chloroformic acid 2,2,2-Trichloroethyl, benzyl chloroformate, benzyl carbazate, benzyl phenyl carbonate, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 4-[2-(trimethylsilyl)ethoxycarbonyloxy]nitrobenzene, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, ethyl chloroformate, diethyl dicarbonate, ethyl imidazole-1-carboxylate, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline, methyl chloroformate, Examples of suitable acylating agents include dimethyl carbonate, dimethyl dicarbonate, and methylimidazole-1-carboxylate. Preferred examples include acetic anhydride, 1-trifluoroacetylimidazole, benzoic anhydride, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(allyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. More preferred examples include acetic anhydride, 1-trifluoroacetylimidazole, N-(allyloxycarbonyloxy)succinimide, and N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide. The amount of the acylating agent or alkoxycarbonylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.7 to 5 equivalents, and more preferably 1 to 3 equivalents, relative to the compound represented by Formula (Rc-3B).

[0117] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 5 hours, more preferably 5 to 90 minutes.

[0118] The reaction of the activating agent, acylating agent or alkoxycarbonylating agent, and thiolating agent in this step is preferably carried out in the presence of a dehydrating agent. The dehydrating agent used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, magnesium sulfate, sodium sulfate, and calcium chloride. Preferred examples include molecular sieve 3A, molecular sieve 4A, molecular sieve 5A, molecular sieve 13X, and sodium sulfate, and more preferred examples include molecular sieve 3A and molecular sieve 4A. The amount of dehydrating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.01 to 3 times, and more preferably 0.01 to 1 times, the mass of the compound represented by Formula (Rc-3B).

[0119] The thiolation agent used in this step is not particularly limited as long as it allows the reaction to proceed. For example, xanthan hydride, bis(phenylacetyl) disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione can be used; preferably, xanthan hydride, bis(phenylacetyl) disulfide, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione can be used; more preferably, xanthan hydride and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione can be used. The amount of the thionating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 1 to 2 equivalents, relative to the compound represented by Formula (Rc-3B).

[0120] The reaction temperature with the thionating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 5 hours, more preferably 5 to 90 minutes.

[0121] This step can be preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, and examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; and bases such as potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine. More preferred examples include triethylamine, diisopropylethylamine, and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 20 equivalents, more preferably 1 to 10 equivalents, relative to the compound represented by formula (Rc-3B).

[0122] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include acetonitrile.

[0123] (Step b3) In one embodiment, this step is a step of deprotecting PG9 of a compound of formula (Rc-5B) to obtain a compound of formula (Rc-6B).

[0124] The deprotection in this step is carried out under acidic conditions using an acid. Examples of the acid used include hydrochloric acid, sulfuric acid, formic acid, oxalic acid, acetic acid, trifluoroacetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid. Preferred acids include hydrochloric acid, acetic acid, monochloroacetic acid, and dichloroacetic acid, and more preferred are hydrochloric acid and dichloroacetic acid. The pH used for the deprotection is not limited as long as the reaction proceeds, but is preferably 1 to 4. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from −30°C to the boiling point of the solvent used in the reaction, more preferably from −20°C to 30°C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours.

[0125] The solvent used in the deprotection reaction of PG9 in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include acetonitrile and dichloromethane, and more preferred examples include dichloromethane.

[0126] (Step b4) In one embodiment, this step is a step of deprotecting PG4 of a compound of formula (Rc-6B) to obtain a compound of formula (Rc-7B).

[0127] The reagent used for deprotection in this step varies depending on the type of PG4. For example, when PG4 is a levulinoyl group, examples of suitable reagents include sodium borohydride, hydrazine monohydrate, hydrazine acetate, hydrazine monohydrochloride, and hydrazine dihydrochloride. Preferred examples include hydrazine monohydrate, hydrazine acetate, hydrazine monohydrochloride, and hydrazine dihydrochloride, and more preferred examples are hydrazine monohydrate and hydrazine acetate. The reaction temperature for the deprotection reaction is not limited as long as the reaction proceeds, but is preferably from −30°C to the boiling point of the solvent used in the reaction, more preferably from −20°C to 30°C. The reaction time for this reaction is not limited as long as the reaction proceeds, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours.

[0128] The solvent used in this process is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof can be used. Preferably, acetonitrile and dichloromethane can be mentioned, and more preferably, dichloromethane can be mentioned.

[0129] (Step b5) In one embodiment, this step involves reacting a compound of formula (Rc-7B) with an optically active phosphitylating agent (Sc-2), then performing a cyclization reaction in the presence of an activator, and then treating with an acylating agent or an alkoxycarbonylating agent, and further reacting with a sulfurizing agent (also referred to as a sulfiding agent or a thiolating agent) to obtain a compound of formula (Rc,Sc-8).

[0130] In this step, after reacting with an optically active phosphitylating agent (Sc-2), as an intermediate obtained before cyclization, a compound represented by formula (Rc-8- 01 ):

Chemical formula

Chemical formula

[0131] In one embodiment, the optically active phosphitylating agent (Sc-2) used in this step is represented by the following formula (Sc-2-1) or formula (Sc-2-2):

Chemical formula

[0132] The optically active phosphitylating agent (Sc-2) includes (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aS)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3 ,2]oxazaphosphole, (3S,3aS)-1-chloro-3-[(4-methylbenzene-1-sulfonyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-[(4-nitrophenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aS)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3a S)-1-chloro-3-[(4-methoxyphenyl)methyl]tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-(diphenylmethyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aS)-1-chloro-3a,4-dihydro-1H,3H-[1,3,2]oxazaphosphoro[3,4-a]indole, etc. can be used, and preferably (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphoro[3,4-a]indole (3aS)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-methyl-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3S,3aS)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used, and more preferably, (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3aS)-1-chloro-3,3-dimethyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, (3R,3aS)-1-chloro-3-phenyltetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole, or (3S,3aS)-1-chloro-3-{[methyl(diphenyl)silyl]methyl}tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole can be used. The amount of the optically active phosphitylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by Formula (Rc-7B).

[0133] The structure of the optically active phosphitylating agent (Sc-2) exemplified above is shown below. [Table 2]

[0134] The activating agent for the cyclization reaction, the acylating agent or alkoxycarbonylating agent, and the thiolating agent (also called a sulfurizing agent or a thiolating agent) used in this step are the same as those used in the above step b2.

[0135] The reaction temperature with the activating agent for the cyclization reaction in this step is not limited as long as the reaction proceeds, but is preferably from −78° C. to the boiling point of the solvent used in the reaction, more preferably −60° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 minutes to 5 hours, more preferably 5 minutes to 3 hours.

[0136] The reaction temperature with the acylating agent or alkoxycarbonylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1 minute to 5 hours, more preferably 5 minutes to 90 minutes.

[0137] The reaction temperature with the thionating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 5 hours, more preferably 5 minutes to 90 minutes.

[0138] The solvent used in the cyclization reaction using an activator in this step through the thiolation reaction may be the same as that used in the subsequent reaction after the phosphitylation reaction, but is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, acetonitrile, dichloromethane, and mixed solvents thereof, and more preferred examples include dichloromethane, acetonitrile, and mixed solvents thereof.

[0139] (Step b6) In one embodiment, this step is a step of deprotecting B2, B2″, which are protecting groups at the phosphorothioate moiety of a compound of formula (Rc,Sc-8), and PG6, which is a protecting group in A2, to obtain a compound of formula (Rp,Rp-9). This step optionally includes a step of treating with an organic or inorganic base to convert into a salt thereof.

[0140] The ammonia used in the deprotection reaction in this step is not limited as long as the reaction proceeds, but 28% aqueous ammonia is preferably used.

[0141] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from 30° C. to 65° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 1 to 96 hours, more preferably from 1 to 48 hours.

[0142] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, pyridine, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, ethanol, acetonitrile, dichloromethane, and pyridine, and more preferred examples include methanol and pyridine.

[0143] Examples of the organic base used when converting the compound of formula (Rp,Rp-9) into its salt include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, and the like. Examples of the inorganic base include potassium carbonate, potassium hydroxide, potassium hydrogen carbonate, sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, 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 hydrogen carbonate, sodium carbonate, and sodium hydrogen carbonate can be mentioned, and more preferably, triethylamine, potassium carbonate, potassium hydrogen carbonate, sodium carbonate, and sodium hydrogen carbonate can be mentioned.

[0144] <1-2. Method for producing CDN-linker (compound (Rp,Rp-12))> The compound of formula (Rp,Rp-12) or its salt can be produced according to the following synthetic scheme. The compound of formula (Rp,Rp-12) is a production precursor of an antibody-immunostimulant conjugate. [Synthetic scheme <A2 method>]

[0145]

Chemical formula

[0146] In the above [Synthetic scheme <A2 method>], PG1, PG3, and A1 have the same meanings as in the above [Synthetic scheme ].

[0147] Each step will be described below. (Step a7) This step is a step of deprotecting the protecting groups PG1 and PG3 of the compound of formula (Rp, Rp-9) to obtain a compound of formula (Rp, Rp-10).

[0148] Examples of the deprotecting agent used in this step include ammonium fluoride, tetra-n-butylammonium fluoride, pyridine hydrogen fluoride, and triethylamine trihydrofluoride, and preferably ammonium fluoride and tetra-n-butylammonium fluoride, and more preferably tetra-n-butylammonium fluoride. The amount of the deprotecting agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 60 equivalents, more preferably 1 to 30 equivalents, relative to the compound represented by formula (Rp, Rp-9).

[0149] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from 10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 30 minutes to 240 hours, more preferably from 1 hour to 120 hours.

[0150] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples of the solvent include methanol, acetonitrile, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide, and more preferred examples of the solvent include tetrahydrofuran and dimethyl sulfoxide.

[0151] (Step a8) This step is a step in which a compound of formula (Rp, Rp-10) is condensed with a compound of formula (11) to obtain a compound of formula (Rp, Rp-12) or a salt thereof.

[0152] The compound represented by formula (11) can be condensed with the compound represented by formula (Rp, Rp-10) preferably by converting it into an activated ester. The amount of the compound represented by formula (11) used in this step is not limited as long as the reaction proceeds, but is preferably 0.3 to 3 equivalents, more preferably 0.7 to 1.3 equivalents, relative to the compound represented by formula (Rp, Rp-10).

[0153] The method for derivatizing the activated ester in this step is not limited as long as the reaction proceeds. For example, the method can be carried out using a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD HCl) or N,N'-dicyclohexylcarbodiimide (DCC) with an additive such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, ethyl cyanohydroxyiminoacetate, or p-nitrophenol. Alternatively, the method can be carried out using a condensing agent such as 1-[bis(dimethylamino)methylene ]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), or (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), or other condensing agents. Preferably, this reaction can be carried out using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). More preferably, this reaction can be carried out using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). The amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 0.7 to 2 equivalents, relative to the compound represented by formula (Rp, Rp-10).The amount of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole used in this step is not limited as long as the reaction proceeds, but is preferably 0.05 to 4 equivalents, more preferably 0.1 to 2 equivalents, relative to the compound represented by formula (Rp, Rp-10). The amount of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride used in this step is not limited as long as the reaction proceeds, but is preferably 0.3 to 5 equivalents, more preferably 0.7 to 2 equivalents, relative to the compound represented by formula (Rp, Rp-10).

[0154] This step can be preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, and examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; and bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and sodium acetate. More preferred examples include triethylamine and N-methylmorpholine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (Rp, Rp-10).

[0155] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably from −10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 5 minutes to 72 hours, more preferably from 1 hour to 24 hours.

[0156] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include water, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and dimethyl sulfoxide, as well as mixed solvents thereof.

[0157] Examples of bases used in converting compounds of formula (Rp, Rp-12) into their salts include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, propylamine, isopropylamine, butylamine, isobutylamine, tert-butylamine, pentylamine, and aniline, and inorganic bases such as potassium 2-ethylhexanoate, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium 2-ethylhexanoate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium chloride, and potassium chloride. Preferred bases include triethylamine, tert-butylamine, potassium 2-ethylhexanoate, potassium carbonate, potassium bicarbonate, sodium 2-ethylhexanoate, sodium carbonate, sodium bicarbonate, sodium chloride, and potassium chloride. More preferred examples include triethylamine, potassium 2-ethylhexanoate, and sodium carbonate.

[0158] Examples of the solvent used in converting compounds of formula (Rp, Rp-12) into their salts include water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof. Preferred examples include water, 2-propanol, acetonitrile, cyclopentyl methyl ether, and ethyl acetate, as well as mixed solvents thereof. More preferred examples include mixed solvents of water, 2-propanol, acetonitrile, and cyclopentyl methyl ether.

[0159] <2. Production of starting compound (1A) used in the production of cyclic dinucleotide> <2-1. Method for producing the compound of formula (1A)> The compound of formula (1A) used in the production of the above cyclic dinucleotide can be produced according to the following synthesis scheme. [Synthetic Scheme <x1>]

[0160] [ka]

[0161] The above [Synthetic Scheme <x1>, medium PG1, PG2, and PG3 are synonymous with the above [Synthesis Scheme <Method A>], X is Cl, Br, or I.

[0162] In this production method, PG1 represents a protecting group for a hydroxy group, and examples of the protecting group include tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl. Preferably, tert-butyldimethylsilyl or trimethylsilyl. More preferably, tert-butyldimethylsilyl.

[0163] PG2 represents a protecting group for a hydroxy group, and examples of the protecting group include 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl. Preferably, 4,4'-dimethoxytrityl, 4-methoxytrityl, or trityl. More preferably, 4,4'-dimethoxytrityl. [[ID=十四]]

[0164] [[ID=十五]] [[ID=十六]]PG3 represents a protecting group for an amino group, and examples of the protecting group include 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. Preferably, 2-(trimethylsilyl)ethoxycarbonyl or allyloxycarbonyl. More preferably, 2-(trimethylsilyl)ethoxycarbonyl. [[ID=十七]] [[ID=十八]]

[0165] [[ID=十九]] [[ID=二十]]The following describes each step. [[ID=二十一]] [[ID=二十二]](Step x1) [[ID=二十三]] [[ID=二十四]]This step is a step of reacting a compound of formula (XIV) with a compound of formula (XV) to obtain a compound of formula (XVI). [[ID=二十五]] [[ID=二十六]]

[0166] [[ID=二十七]] The reaction of this step is carried out in the presence of a base, and the base may be 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, or 1,8-diazabicyclo[5.4.0]-7-undecene, preferably 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo[5.4.0]-7-undecene, more preferably 1,1,3,3-tetramethylguanidine. The amount of the base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XIV).

[0167] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 10° C. to the boiling point of the solvent used in the reaction, more preferably from 20° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably from 30 minutes to 72 hours, more preferably from 5 hours to 36 hours.

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

[0169] (Process x2) In this step, a compound of formula (XVI) is reacted with a silylating agent to obtain a mixture of a compound of formula (1A') and a compound of formula (XVII):

[0170] [ka] and then converting the compound of formula (XVII) in the mixture to a compound of formula (1A') in the presence of a base to obtain a compound of formula (1A').

[0171] This step also includes a step of reacting the compound of formula (XVI) with a silylating agent in the presence of a first base to obtain a mixture of the compound of formula (1A') and the compound of formula (XVII), and then converting the compound of formula (XVII) in the mixture to the compound of formula (1A') in the presence of a second base to obtain the compound of formula (1A').

[0172] The conversion of the compound of formula (XVII) to the compound of formula (1A') is carried out by crystallizing the compound of formula (1A') 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 (1A').

[0173] Examples of the first base include 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and preferred examples include 1,1,3,3-tetramethylguanidine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferred examples include 1,1,3,3-tetramethylguanidine. The amount of the first base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, and more preferably 1 to 5 equivalents, relative to the compound represented by formula (XVI).

[0174] The reaction temperature for the reaction using the first base in this step is not limited as long as the reaction proceeds, but is preferably from 10° C. to the boiling point of the solvent used in the reaction, more preferably 40° C. to 70° C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 30 minutes to 72 hours, more preferably 5 hours to 36 hours.

[0175] The solvent for the reaction using the first base in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include 1,3-dimethyl-2-imidazolidinone.

[0176] Examples of the silylating agent used in this step include tert-butyldimethylchlorosilane and tert-butyldimethylsilyl triflate, and preferably tert-butyldimethylchlorosilane. The amount of the silylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 2 to 4 equivalents, relative to the compound represented by formula (XIV).

[0177] Examples of the second base include 1,1,3,3-tetramethylguanidine, triethylamine, diisopropylethylamine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and preferred examples include 1,1,3,3-tetramethylguanidine, 2,6-lutidine, and 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferred examples include 1,1,3,3-tetramethylguanidine. The amount of the second base used in this step is not limited as long as the reaction proceeds, but is preferably 0.01 to 3 equivalents, and more preferably 0.05 to 1 equivalent, relative to the compound represented by formula (XIV).

[0178] The reaction temperature for the reaction using the second base in this step is not limited as long as the reaction proceeds, but is preferably from 10° C. to the boiling point of the solvent used in the reaction, more preferably from 10° C. to 40° C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably from 2 minutes to 10 hours, more preferably from 5 minutes to 90 minutes.

[0179] The first solvent for the reaction using the second base in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include tetrahydrofuran.

[0180] The second solvent for the reaction using the second base in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include tetrahydrofuran, heptane, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include heptane.

[0181] <2-2. Method for producing the compound of formula (XV) and intermediates therefor> The compound of formula (XV) used in the production of the above formula (1A) can be produced according to the following synthesis scheme. [Synthetic Scheme <x2>]

[0182] [ka]

[0183] The above [Synthetic Scheme <b2>]In, PG3 can be synthesized by the synthesis scheme <a1>] is synonymous with X is the same as in the above [Synthetic Scheme <b1>] is synonymous with.

[0184] Step x3 will be described below. (Process x3) In this step, a compound of formula (XVIII) is reacted with 2-haloethanol in the presence of an acid or a base to obtain a compound of formula (XV).

[0185] Examples of the base used in this step include sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene. Preferred examples include sodium hydroxide, potassium hydroxide, triethylamine, and diisopropylethylamine. More preferred examples include sodium hydroxide. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 equivalents relative to glycylglycine, the starting material for compound (XVIII). Compound (XVIII) can be obtained from glycylglycine by protecting the amino group and converting the carboxy group to an acetyloxy group.

[0186] The 2-haloethanol used in this step includes 2-chloroethanol, 2-bromoethanol, and 3-iodoethanol, 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 is preferably 0.5 to 10 equivalents, more preferably 1 to 4 equivalents, relative to the amount of glycylglycine, the starting material for compound (XVIII).

[0187] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably −10° C. to 20° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 36 hours, more preferably 1 hour to 10 hours.

[0188] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include 1,2-dimethoxyethane, 2-methyltetrahydrofuran, and 1,4-dioxane. More preferred examples include 1,2-dimethoxyethane.

[0189] <3. Production of starting compound (XXV) used in the production of cyclic dinucleotide> <3-1. Method for producing the compound of formula (XXV)> The compound of formula (XXV) used in the production of the above cyclic dinucleotide can be produced according to the following synthesis scheme. [Synthetic Scheme <y1>]

[0190] [ka]

[0191] Each step will be explained below. (Process y1) In this step, a compound of formula (XIX) is reacted with a benzoylating agent to obtain a compound of formula (XX).

[0192] The benzoylating agent used in this step includes benzoyl chloride, benzoyl bromide, benzoic anhydride, benzoyl trifluoromethanesulfonate, etc., preferably benzoyl chloride or benzoyl bromide, more preferably benzoyl chloride. The amount of the benzoylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, more preferably 1 to 2 equivalents, relative to the compound represented by formula (XIX).

[0193] This step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium Examples of suitable bases include bases such as tert-butoxide and mixtures thereof, and preferred examples include triethylamine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, and sodium acetate, and mixtures thereof, and more preferred examples include a mixture of triethylamine and 4-dimethylaminopyridine. 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, more preferably 0.2 to 5 equivalents, relative to the compound represented by formula (XIX).

[0194] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −40° C. to the boiling point of the solvent used in the reaction, more preferably −10° C. to 35° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 0.5 to 10 hours.

[0195] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferably, acetonitrile or N,N-dimethylformamide can be used, and more preferably, acetonitrile can be used.

[0196] (Process y2) In this step, a compound of formula (XX) is hydrolyzed to obtain a compound of formula (XXI).

[0197] Examples of the acid used in this step include hydrochloric acid, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, and sulfuric acid, and preferred examples include hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, and sulfuric acid, and more preferred examples include p-toluenesulfonic acid. The amount of the acid used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 20 equivalents, and more preferably 1 to 10 equivalents, relative to the compound represented by formula (XX).

[0198] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably 40° C. to 80° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0199] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof, are usable. Preferred are water, 1,2-dimethoxyethane, 2-methyltetrahydrofuran, tetrahydrofuran, cyclopentyl methyl ether, and mixed solvents thereof, and more preferred is a mixed solvent of cyclopentyl methyl ether and water.

[0200] (Process y3) In this step, a compound of formula (XXI) is reacted with a chlorinating agent to obtain a compound of formula (XXII).

[0201] Examples of the chlorinating agent used in this step include trichloroisocyanuric acid, chloroisocyanuric acid, dichloroisocyanuric acid, N-chlorosuccinimide, 1,3-dichloro-5,5-dimethylhydantoin, N-chlorosaccharin, N-N-dichloro-p-toluenesulfonamide, N-N-dichlorobenzenesulfonamide, and carbon tetrachloride, and preferred examples include trichloroisocyanuric acid, chloroisocyanuric acid, and dichloroisocyanuric acid, and more preferred examples include trichloroisocyanuric acid. The amount of the chlorinating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.1 to 6 equivalents, and more preferably 0.3 to 3 equivalents, relative to the compound represented by formula (XXI).

[0202] The reaction of this step is preferably carried out in the presence of a phosphorus reagent. The phosphorus reagent used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include tris(2,4-di-tert-butylphenyl)phosphite, tri-o-tolylphosphite, triphenylphosphite, triethylphosphite, tributylphosphine, triphenylphosphine, tris(dimethylamino)phosphine, and tris(diethylamino)phosphine. Preferred examples include tris(2,4-di-tert-butylphenyl)phosphite, tri-o-tolylphosphite, and triphenylphosphine. More preferred examples include tris(2,4-di-tert-butylphenyl)phosphite. The amount of the phosphorus reagent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXI).

[0203] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −80° C. to the boiling point of the solvent used in the reaction, more preferably −20° C. to 30° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

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

[0205] (Process y4) In this step, a compound of formula (XXII) is reacted with a compound of formula (XXIII) to obtain a compound of formula (XXIV).

[0206] The reaction of this step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene, 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, and preferred examples include cesium carbonate, potassium carbonate, and sodium carbonate, and more preferred example is cesium carbonate. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXII).

[0207] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 10° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0208] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide. More preferred examples include dimethyl sulfoxide.

[0209] (Process y5) This step is a step of deprotecting the benzoyl group from the compound of formula (XXIV) to obtain a compound of formula (XXV) or a salt thereof.

[0210] This step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; cesium carbonate, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium Examples of suitable bases include 1,8-diazabicyclo[5.4.0]-7-undecene, sodium methoxide, sodium ethoxide, potassium carbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide, and more preferred are 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).

[0211] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0212] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, ethyl acetate, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred are water, methanol, ethanol, tetrahydrofuran, and mixed solvents thereof, and more preferred are ethanol or a mixed solvent of water and tetrahydrofuran.

[0213] Examples of acids used in converting the compound of formula (XXV) into its salt include formic acid, acetic acid, propionic acid, oxalic acid, maleic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid. Preferred examples include acetic acid, p-toluenesulfonic acid, and hydrochloric acid. More preferred examples include p-toluenesulfonic acid and hydrochloric acid. The amount of acid used for conversion into the salt is not limited as long as the reaction proceeds, but is preferably 0.5 to 5 equivalents, and more preferably 1 to 3 equivalents, relative to the compound of formula (XXIV).

[0214] <3-2. Method for producing the compound of formula (XXIII) and intermediates thereof> The compound of formula (XXIII) used in the production of the compound of formula (XXV) above can be produced according to the following synthesis scheme. [Synthetic Scheme <y2>]

[0215] [ka]

[0216] Each step will be described below. (Process y9) In this step, the tert-butoxycarbonyl group is deprotected from the compound of formula (XXVIII) to obtain the compound of formula (XXIX).

[0217] This step is preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene, as well as bases such as potassium carbonate, potassium hydroxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include potassium hydroxide, potassium bicarbonate, sodium carbonate, and sodium hydroxide, and more preferred example is sodium hydroxide. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXVIII).

[0218] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, and more preferably 1 hour to 10 hours.

[0219] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include methanol, ethanol, 1-propanol, and tetrahydrofuran, and more preferred examples include ethanol.

[0220] (Process y10) In this step, a compound of formula (XXIX) is subjected to an alkyne reduction reaction in the presence of a catalyst, followed by a reductive amination reaction, to obtain a compound of formula (XXX).

[0221] A metal catalyst can be used as a catalyst for the reduction reaction of the alkyne in this step. The metal catalyst used in this step is not particularly limited as long as it catalyzes hydrogenation; however, preferred examples include ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, and nickel catalysts, and more preferred examples include palladium catalysts. The amount of metal catalyst used in this step is not limited as long as the reaction proceeds; however, it is preferably 0.01 to 1 times, more preferably 0.02 to 0.4 times, and even more preferably 0.05 to 0.2 times the mass of the compound represented by formula (XXVIII). The hydrogen gas pressure is usually 100 to 1,000 kPa, preferably 100 to 700 kPa, and more preferably 200 to 500 kPa. The reaction temperature in this step is not limited as long as the reaction proceeds; however, it is preferably from 0°C to the boiling point of the solvent used in the reaction, more preferably 20°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, and more preferably 1 hour to 24 hours.

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

[0223] A metal catalyst can be used as a catalyst for the reductive amination reaction in this step. The metal catalyst used in this step is not particularly limited as long as it catalyzes hydrogenation; however, preferred examples include ruthenium catalysts, rhodium catalysts, palladium catalysts, platinum catalysts, and nickel catalysts, and more preferred examples include palladium catalysts. The amount of metal catalyst used in this step is not limited as long as the reaction proceeds; however, it is preferably 0.01 to 1 times, more preferably 0.02 to 0.4 times, and even more preferably 0.05 to 0.2 times the mass of the compound represented by formula (XXVIII). The hydrogen gas pressure is usually 100 to 1,000 kPa, preferably 100 to 700 kPa, and more preferably 200 to 500 kPa. The reaction temperature in this step is not limited as long as the reaction proceeds; however, it is preferably from 0°C to the boiling point of the solvent used in the reaction, more preferably 20°C to 80°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, and more preferably 1 hour to 24 hours.

[0224] The solvent used in the reductive amination reaction of this step is not particularly limited as long as it does not inhibit the reaction. For example, water, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, water, acetic acid, and mixed solvents thereof. More preferred examples include a mixed solvent of 1-methyl-2-pyrrolidone, water, and acetic acid.

[0225] (Process y11) In this step, a compound of formula (XXX) is reacted with a benzoylating agent, followed by debenzoylation by treatment with a base to obtain a compound of formula (XXIII).

[0226] The benzoylating agent used in this step includes benzoyl chloride, benzoyl bromide, benzoic anhydride, benzoyl trifluoromethanesulfonate, etc., and preferably benzoyl chloride. The amount of the benzoylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXX).

[0227] The reaction with the benzoylating agent in this step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium Examples of bases include tert-butoxide and mixed bases thereof, and preferred examples include triethylamine, diisopropylethylamine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, and mixed bases thereof, and more preferred examples include a mixed base of triethylamine, diisopropylethylamine, and 4-dimethylaminopyridine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXVIII).

[0228] The reaction temperature with the benzoylating agent in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0229] The solvent used in the reaction with the benzoylating agent in this step is not particularly limited as long as it does not inhibit the reaction. Examples include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chloro Benzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used, and preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone, and more preferred examples include 1,3-dimethyl-2-imidazolidinone.

[0230] The base used in the base treatment in this step is not particularly limited as long as the reaction proceeds, and examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; and bases such as potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferred examples include potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, and sodium hydroxide, and more preferred example is triethylamine. The amount of base used in the base treatment in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXVIII).

[0231] The reaction temperature for the base treatment in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0232] The solvent used in the base treatment in this step is not particularly limited as long as it does not inhibit the reaction, and examples thereof include pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,6-lutidine, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, and acetone. , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide, and mixed solvents thereof can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and 1,3-dimethyl-2-imidazolidinone. More preferred examples include a mixed solvent of methanol and 1,3-dimethyl-2-imidazolidinone.

[0233] <3-3. Method for producing the compound of formula (XXVIII)> The compound of formula (XXVIII) used in the production of the compound of formula (XXIII) above can be produced according to the following synthesis scheme. [Synthetic Scheme <y3>]

[0234] [ka]

[0235] Each step will be explained below. (Process y12) In this step, a compound of formula (XXXI) is reacted with a tert-butoxycarbonylating agent in the presence of 1-methylimidazole to obtain a compound of formula (XXXII).

[0236] Examples of the tert-butoxycarbonylating agent used in this step include di-tert-butyl dicarbonate, N-tert-butoxycarbonylimidazole, N-tert-butoxycarbonyl-1,2,4-triazole, N-(tert-butoxycarbonyloxy)phthalimide, etc., and preferably di-tert-butyl dicarbonate. The amount of the tert-butoxycarbonylating agent used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 6 equivalents, more preferably 1 to 3 equivalents, relative to the compound represented by formula (XXXI).

[0237] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from −20° C. to the boiling point of the solvent used in the reaction, more preferably 0° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 24 hours, more preferably 1 hour to 10 hours.

[0238] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples of the solvent include acetonitrile, tetrahydrofuran, ethyl acetate, benzene, and toluene, and more preferred examples are toluene.

[0239] (Process y13) In this step, a compound of formula (XXXII) is reacted with propargylaldehyde diethyl acetal to obtain a compound of formula (XXVIII).

[0240] This step is preferably carried out in the presence of a transition metal catalyst, and more preferably in the presence of a palladium catalyst. The palladium catalyst used in this step is not particularly limited as long as it allows the reaction to proceed. For example, divalent palladium salts and complexes thereof, such as palladium(II) acetate, palladium(II) trifluoroacetate, palladium(II) chloride, palladium(II) bromide, palladium(II) iodide, and bis(triphenylphosphine)palladium(II) dichloride, and zerovalent palladium metals and complexes thereof, such as palladium black, palladium carbon, tetrakistriphenylphosphinepalladium(0), and bis(dibenzylideneacetone)palladium(0), are usable. Preferably, bis(triphenylphosphine)palladium(II) dichloride is used. The amount of palladium catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.0001 to 1 equivalent, more preferably 0.005 to 0.05 equivalent, relative to the compound represented by formula (XXXII).

[0241] Furthermore, this step can be preferably carried out in the presence of a copper catalyst in addition to the palladium catalyst. Examples of copper catalysts that can be used in this step include copper(I) chloride, copper(I) bromide, and copper(I) iodide, and copper(I) iodide is preferred. The amount of copper catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.0001 to 1 equivalent, more preferably 0.005 to 0.05 equivalent, relative to the compound represented by formula (XXXII).

[0242] This step is preferably carried out in the presence of a base. The base used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium Examples of bases include tert-butoxide and mixed bases thereof, and preferred examples include triethylamine, diisopropylethylamine, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene, and more preferred examples include triethylamine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (XXXII).

[0243] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably from 0° C. to the boiling point of the solvent used in the reaction, more preferably 15° C. to 50° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 5 minutes to 72 hours, more preferably 1 hour to 24 hours.

[0244] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfoxide, as well as mixed solvents thereof, can be used. Preferred examples include N,N-dimethylformamide, N,N-dimethylacetamide, and 1-methyl-2-pyrrolidone, and more preferred examples include N,N-dimethylformamide.

[0245] <4. Method for producing antibody-immunostimulant conjugates (Rp, Rp-13)> <4-1. Antibodies and their glycoremodeling> <4-1-1. Antibodies>

[0246] As used herein, the term "functional antibody fragment," also referred to as "antigen-binding fragment of antibody," refers to a partial fragment of an antibody that retains antigen-binding activity, and includes Fab, F(ab'), Fv, scFv, diabody, linear antibody, and multispecific antibody fragments formed from antibody fragments. Also included in the antigen-binding fragment of an antibody is Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab') under reducing conditions. However, the antigen-binding fragment is not limited to these molecules, as long as it retains antigen-binding ability. Furthermore, these antigen-binding fragments include not only those obtained by treating the full-length antibody protein molecule with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0247] As used herein, the term "functional fragment" includes a functional fragment that retains an asparagine (Asn297) and its surrounding amino acids that are modified by N-linked glycosylation, which is well conserved in the Fc region of an IgG heavy chain, and that has antigen-binding ability.

[0248] The antibody used in producing the antibody-immunostimulant conjugate of the present invention refers to an immunoglobulin, a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody of the present invention may be of any class, including IgG, IgE, IgM, IgD, IgA, and IgY, with IgG being preferred. Furthermore, the subclass may be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG1, IgG2, or IgG4 being preferred (including antibodies with mutations in the Fc region of the IgG heavy chain that affect ADCC and ADCP activity).

[0249] When an IgG1 isotype antibody is used to produce the antibody-immunostimulant conjugate of the present invention, its effector function can be adjusted by substituting some of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, and WO94 / 29351). Examples of IgG1 mutants include IgG1 LALA mutations (IgG1-L234A, L235A). L234A and L235A represent substitutions of leucine with alanine at positions 234 and 235, as defined by the EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No. 1 (May 15, 1969), pp. 78-85).

[0250] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). CDRs, also known as hypervariable regions, are located in the variable regions of the heavy and light chains of an antibody and are particularly highly variable in their primary structure. They are separated into three regions in the primary structure of the heavy and light chain polypeptide chains. Herein, with regard to antibody CDRs, the heavy chain CDRs are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and the light chain CDRs are referred to as CDRL1, CDRL2, and CDRL3 from the amino-terminal end of the light chain amino acid sequence. These regions are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0251] The antibody may be derived from any species, preferably human, rat, mouse, or rabbit. When derived from a species other than human, it is preferable to chimerize or humanize the antibody using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, and modified versions thereof.

[0252] The antibody is preferably, but not limited to, an antibody that targets tumor cells or immune cells, and more preferably, an antibody that targets tumor cells.

[0253] Antibody binding to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to visualize the antibody uptake by the cell under a fluorescent microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to measure the amount of fluorescence uptake by the cell (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released upon intracellular uptake, suppressing cell proliferation (BioTechniques 28:162-165, January 2000). A recombinant complex protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin.

[0254] When an antibody that targets tumor cells is used in the antibody-immunostimulant conjugate of the present invention, it is preferable, but not essential, that the antibody itself have an anti-tumor effect.

[0255] The antitumor activity of an immunostimulant or an antibody-immunostimulant conjugate refers to cytotoxic activity against tumor cells, anticellular effect, and regression of tumor volume. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems.

[0256] The immunostimulatory activity of an immunostimulator or an antibody-immunostimulator conjugate refers to the enhancement of tumor cell sensitivity to immune cells or tumor cell-mediated activation of immune cells. The immunostimulatory activity can be confirmed using known in vitro or in vivo evaluation systems.

[0257] Examples of antibodies used in the production of the antibody-immunostimulant conjugates of the present invention include, but are not limited to, anti-HER2 antibodies, anti-HER3 antibodies, anti-DLL3 antibodies, anti-FAP antibodies, anti-CDH11 antibodies, anti-CDH6 antibodies, anti-A33 antibodies, anti-CanAg antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD98 antibodies, anti-TROP2 antibodies, anti-CEA antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUC1 antibodies, anti-GPNMB antibodies, anti-integrin antibodies, anti-PSMA antibodies, anti-tenascin-C antibodies, anti-SLC44A4 antibodies, anti-Mesothelin antibodies, anti-ENPP3 antibodies, anti-CD47 antibodies, anti-EGFR antibodies, anti-GPR20 antibodies, and anti-DR5 antibodies. The antibody of the present invention is preferably an anti-HER2 antibody (e.g., trastuzumab or pertuzumab), an anti-CDH6 antibody, an anti-CD33 antibody, an anti-EphA2 antibody, an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody, and more preferably an anti-HER2 antibody, an anti-CDH6 antibody, an anti-CD70 antibody, an anti-TROP2 antibody, or an anti-EGFR antibody.

[0258] The antibodies used in producing the antibody-immunostimulant conjugates of the present invention can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body, using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0259] Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennett, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)), and a monoclonal antibody can be obtained.

[0260] The antigen can be obtained by genetically manipulating a gene encoding the antigen protein in a host cell to produce it.

[0261] The humanized antibody used in producing the antibody-immunostimulant conjugate of the present invention can be obtained according to known methods (e.g., Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984); Nature (1986) 321, pp. 522-525; WO90 / 07861).

[0262] For example, anti-HER2 antibodies (US Pat. No. 5,821,337, WO2004 / 008099, WO2020 / 050406, etc.), anti-CD33 antibodies (WO2014 / 057687, WO2020 / 050406, etc.), anti-EphA2 antibodies (WO2009 / 028639, WO2020 / 050406, etc.), anti-CDH6 antibodies (WO2018 / 212136, WO2020 / 050406, etc.), ), anti-CD70 antibodies (WO2004 / 073656, WO2007 / 038637, WO2021 / 177438, etc.), anti-TROP2 antibodies (WO2015 / 098099, WO2021 / 177438, etc.), and anti-EGFR antibodies (WO1998 / 050433, WO2002 / 092771, WO2021 / 177438, etc.) can be obtained by known means.

[0263] <4-1-2. Antibody Glycosylation Remodeling> Recently, a method for remodeling heterogeneous antibody glycans by enzymatic reactions to uniformly introduce functionalized glycans has been reported (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Using this glycan remodeling technique, attempts have also been made to site-specifically introduce drugs and synthesize homogeneous ADCs (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0264] Glycan remodeling involves first using a hydrolase to remove heterogeneous glycans attached to a protein (e.g., an antibody) leaving only the terminal GlcNAc, thereby preparing a homogeneous protein portion with GlcNAc attached (hereafter referred to as the "acceptor"). Next, a separately prepared glycan of your choice (hereafter referred to as the "donor") is prepared, and this acceptor and donor are linked using a glycosyltransferase. This allows the synthesis of a homogeneous glycoprotein with a desired glycan structure.

[0265] As used herein, "glycan" refers to a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and glycans may be represented by abbreviations such as "GlcNAc-" or "SG-". When these abbreviations are used in structural formulae, the oxygen atom or nitrogen atom at the reducing end that forms a glycosidic bond with another structural unit is not included in the abbreviation representing the glycan, unless otherwise defined.

[0266] In this specification, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described, for convenience, with the carbon atom in the ring structure that is bonded to the oxygen atom constituting the ring and that is directly bonded to a hydroxy group (or an oxygen atom belonging to a glycosidic bond) being at position 1 (position 2 only in sialic acid). The names of the example compounds are given based on the entire chemical structure, and this rule does not necessarily apply.

[0267] In this specification, when a glycan is described as a symbol (e.g., SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon atom at the reducing end, and does not include the N or O attributable to the N- or O-glycosidic bond.

[0268] The antibody-immunostimulant conjugate used in the production of the antibody-immunostimulant conjugate of the present invention has the following formula (XXXIV):

[0269] [ka] The antibody Ab or a functional fragment thereof is bound to L directly through the side chain of its amino acid residue (e.g., cysteine, lysine, etc.), or through a sugar chain or a remodeled sugar chain of the Ab. The side chain of the amino acid residue may be modified, for example, with an azide group. where: Ab refers to an antibody or a functional fragment of the antibody, and the sugar chain of the antibody may be remodeled. m 1 is in the range of 1 to 10, L represents a linker connecting Ab and D, The linker L is represented by -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates binding to an immunostimulant D, Lp is -GGFG-, where G represents glycine and F represents phenylalanine; La represents -C(=O)-CHCH-C(=O)-; Lb is expressed by the following formula: [ka] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain), Lc represents -NH-CH2-. D is

[0270] [ka] Shows.

[0271] In the present specification, the sugar chain of Ab is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

[0272] N-linked glycans are bound to amino acid side chains of antibodies via N-glycosidic bonds, and O-linked glycans are bound to amino acid side chains of antibodies via O-glycosidic bonds.

[0273] As used herein, Ab refers to IgG, preferably IgG1, IgG2 or IgG4.

[0274] IgG has a well-conserved N-linked glycan (hereinafter referred to as "Asn297 glycan or N297 glycan") at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which is known to contribute to the activity and kinetics of antibody molecules (Eon-Duval, A. et al., Biotechnol. Prog. 2012, 28, 608-622; Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0275] The amino acid sequence in the constant region of IgG is well conserved, and in a report by Edelman et al. (Proc. Natl. Acad. Sci. USA, 63, 78-85, (1969)), each amino acid is identified by an EU number (EU INDEX). For example, Asn297, to which N-linked glycosylation is added in the Fc region, corresponds to position 297 in the EU numbering. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by indicating it by EU numbering.

[0276] The diagram below shows the case where the antibody-immunostimulant conjugate of the present invention is bound to L from the N297 sugar chain of the antibody or functional fragment thereof. 2 represents an integer of 1 or 2.

[0277] [ka] An antibody having such a remodeled sugar chain is called a sugar chain remodeling antibody.

[0278] SGP (α2,6-SGP) is an abbreviation for sialyglycopeptide and is a representative N-linked glycopeptide. SGP can be isolated and purified from chicken egg yolk, for example, according to the method described in WO2011 / 027868. Purified SGP products are commercially available from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd. In this specification, the glycan portion of SGP is referred to as SG, and a glycan lacking one GlcNAc at the reducing end of SG is referred to as SG(10). SG(10) can be prepared by enzymatic hydrolysis of SGP, for example, according to the report by Umekawa et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209). SG(10) can also be purchased from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd.

[0279] In this specification, the glycan structure in which sialic acid at the non-reducing end of only one of the branched β-Man chains of SG(10) is deleted is referred to as MSG(9), the one with sialic acid only on the 1-3 glycan of the branched chain is referred to as MSG1, and the one with sialic acid only on the 1-6 glycan of the branched chain is referred to as MSG2.

[0280] The remodeled sugar chains used in the antibody-immunostimulant conjugates of the present invention are N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, preferably N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1.

[0281] N297-(Fuc)SG is represented by the following structural formula or sequence formula:

[0282] [ka]

[0283] [ka]

[0284] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0285] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula:

[0286] [ka]

[0287] [ka]

[0288] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0289] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula:

[0290] [ka]

[0291] [ka]

[0292] In the above formula, the wavy line indicates binding to Asn297 of the antibody. L(PEG) is -(CH2-CH2-O)n 5 -CH2-CH2-NH-, and the amino group at the right end of the L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb, particularly in the linker L; where n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0293] When the N297 sugar chain of the antibody in the antibody-immunostimulant conjugate of the present invention is N297-(Fuc)SG, the antibody is a dimer, and therefore the antibody-immunostimulant conjugate is a molecule to which four linkers L and four immunostimulants D are bound (the above m 2 =2).

[0294] When the N297 sugar chain of the antibody in the antibody-immunostimulant conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, the antibody is a dimer, and therefore the antibody-immunostimulant conjugate is a molecule in which two linkers L and two immunostimulants D are bound (the above-mentioned m 2 =1) (see Figure 19).

[0295] The N297 sugar chain is preferably N297-(Fuc)SG or N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1, and even more preferably N297-(Fuc)SG.

[0296] When the N297 sugar chain of the antibody in the antibody-immunostimulant conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, a highly homogeneous ADC can be obtained.

[0297] <4-2. Production of Glycosylation Remodeling Antibodies> Glycosylation remodeling antibodies can be produced by the method shown in the following formula, for example, in accordance with the methods described in WO2018 / 003983, WO2020 / 050406, WO2021 / 177438, WO2022 / 050300, PLos ONE 2018,13,e0193534, etc.

[0298] [ka]

[0299] (D-1 process) This step involves hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing-end chitobiose structure of the N-linked glycan (N297-linked glycan) attached to asparagine at position 297 of the antibody's amino acid sequence, using a known enzymatic reaction, to produce a glycosylated antibody. The target antibody (1) (10 mg / mL) was placed in a buffer solution (e.g., phosphate buffer) at 0°C to 40°C, and the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing-end chitobiose structure was hydrolyzed using a hydrolase such as wild-type EndoS enzyme. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. The wild-type EndoS enzyme was used in an amount of 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, per 100 mg of antibody (1). After the reaction was completed, the antibody was purified by affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and / or a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (BIO-RAD)) to obtain (Fucα1,6)GlcNAc antibody (2).

[0300] (D-2 process) This step involves conjugating an SG-type or MSG (MSG1, MSG2)-type glycan oxazoline having an azide group-containing PEG linker (hereinafter referred to as "azidoglycan oxazoline") to the (Fucα1,6)GlcNAc antibody (2) obtained in step D-1 using a known enzymatic reaction to produce a glycan remodeling antibody (3).

[0301] Antibody (2) was reacted with an azido-glycosylated oxazoline derivative in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L) in a buffer solution (e.g., phosphate buffer) at temperatures ranging from 0°C to 40°C to carry out the glycosylation reaction. The reaction time ranged from 10 minutes to 72 hours, preferably 1 to 6 hours. EndoS enzyme (D233Q / Q303L) was used in an amount of 1 mg to 10 mg, preferably 1 to 3 mg, per 100 mg of antibody. The azido-glycosylated oxazoline derivative was used in an amount ranging from 2 equivalents to an excess, preferably 4 to 20 equivalents. After the reaction, the antibody was purified using affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (Bio-Rad)) to obtain glycosylated remodeling antibody (3).

[0302] In the preparation of the above-mentioned glycosylation-remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the common procedures A to C described below.

[0303] The SG-type azide sugar chain oxazoline compound was synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]2-SG(10)-Ox (compound 1-10 described in WO2018 / 003983) is shown in the following scheme.

[0304] [ka]

[0305] The MSG-type azidoglycan oxazoline derivative was also synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]-MSG1(9)-Ox (compound 1-11 described in WO2018 / 003983) is shown in the following scheme.

[0306] [ka]

[0307] (D-3 process) This step involves the production of a glycan-remodeled antibody (3) by a glycosyltransferase reaction using two Endo enzymes on the (Fucα1,6)GlcNAc antibody (2) obtained in step D-1. By using two enzymes simultaneously, glycosyltransferases such as SGP and (SG)Asn, which have inactivated reducing ends, can be used as glycan donors to directly transfer glycosyltransferase to the N297 glycan of the antibody. Regarding the two types of Endo enzymes used, enzyme A (EndoM-like enzyme) and enzyme B (EndoS-like enzyme) can be appropriately combined. Examples of enzyme A include EndoM, EndoOm, EndoCC, and EndoM mutants, EndoOm mutants, and EndoCC mutants with reduced hydrolytic activity. Preferred enzymes A are EndoM N175Q, EndoCC N180H, and EndoOm N194Q. Examples of enzyme B include EndoS, EndoS2 (EndoS49), and EndoS mutants with reduced hydrolytic activity, EndoS2 (EndoS49) mutants, etc. Preferred examples of enzyme B include EndoS D233Q, EndoS D233Q / Q303L, EndoS D233Q / E350A, EndoS D233Q / E350Q, EndoS D233Q / E350D, EndoS D233Q / E350N, EndoS D233Q / D405A, EndoS2 D184M, and EndoS2 T138Q. The sugar chain donors were ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3, [N3-PEG(3)]-MSG1-Asn-PEG(3)-N 3、 [N3-PEG(3)]-MSG2-Asn-PEG(3)-N3, etc. can be used.

[0308] Antibody (2) was reacted with ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 in a buffer solution (such as Tris buffer) in the presence of glycosyltransferases Enzyme A (EndoM-like enzyme) and Enzyme B (EndoS-like enzyme) to carry out a glycosyltransferase reaction. The reaction temperature can be appropriately selected depending on the optimum temperature for the enzyme used, but is usually 15 to 50°C, and preferably 25 to 40°C. The reaction time can be appropriately selected between 2 and 48 hours. After the reaction was completed, a purification method (affinity chromatography, hydroxyapatite column, etc.) and an ultrafiltration method (ultrafiltration membrane) suitable for the reaction scale were selected to obtain the glycan remodeling antibody (3).

[0309] In the preparation of the above-mentioned glycosylation-remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the common procedures A to C described below.

[0310] ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 was synthesized according to the method described in WO2018 / 003983. In step 1-2A described in WO2018 / 003983, the Fmoc-(SG-)Asn free form prepared from Fmoc-(SG-)Asn (1S2S-11NC-Asn-Fmoc, manufactured by Glycotechnology Institute) was reacted with 11-azido-3,6,9-trioxaundecan-1-amine to obtain ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 (compound 1-13 described in WO2018 / 003983).

[0311] MSG-type glycan donor [N3-PEG(3)]-MSG1-Asn-PEG(3)-N 3、 [N3-PEG(3)]-MSG2-Asn-PEG(3)-N3 can also be synthesized in accordance with the method described in steps 1 to 3 of Example 154 of WO2019065964.

[0312] <4-3. Conjugation of antibodies and immunostimulants> The antibody-immunostimulant conjugate of formula (Rp, Rp-13) can be prepared according to the following method.

[0313] [ka] (Here, the two asterisks (*) on the left side of the antibody-immunostimulatory agent conjugate (Rp, Rp-13) indicate the CDN-linker moiety indicated by the asterisk on the right side.)

[0314] The antibody-immunostimulant conjugate (Rp, Rp-13) can be produced by combining the glycan remodeling antibody (3) with the CDN conjugate precursor (Rp, Rp-12) via a cycloaddition reaction. Examples of cycloaddition reactions include the Diels-Alder reaction and the 1,3-dipolar cycloaddition reaction, with the 1,3-dipolar cycloaddition reaction being preferred. Examples of 1,3-dipolar cycloaddition reactions include the cycloaddition reaction of an azide with a terminal alkyne and the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction (J. Am. Chem. Soc. 2004, 126, 15046-15047), with the SPAAC reaction being preferred.

[0315] This production method involves binding the glycan remodeling antibody (3) obtained in step D-2 or D-3 above with a CDN conjugate precursor (Rp, Rp-12) via the SPAAC reaction to produce an antibody-immunostimulant conjugate (Rp, Rp-13).

[0316] (Process E-1) (Process a9) The SPAAC reaction was carried out by mixing a buffer solution of the glycosylation-remodeling antibody (3) (e.g., phosphate buffer, acetate buffer, borate buffer, etc.) with a solution of the CDN conjugate precursor (Rp, Rp-12) dissolved in an appropriate solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixture thereof). The CDN conjugate precursor (Rp, Rp-12) was used in an amount of 2 molar to a molar excess, preferably 4 to 30 molar, per mole of glycosylation-remodeling antibody (3). The ratio of organic solvent to the antibody buffer solution was preferably 1% to 200% (v / v). The reaction temperature was 0°C to 37°C, preferably 15°C to 25°C, and the reaction time was 1 hour to 150 hours, preferably 6 to 72 hours. The pH of the reaction solution was preferably 5 to 9. The reaction solution was purified according to the method described in Common Procedure D below to obtain antibody-immunopotentiator conjugates (Rp, Rp-13).

[0317] The antibody-immunopotentiator conjugate can be identified by buffer exchange, purification, measurement of antibody concentration, and measurement of the average number of immunopotentiators bound per antibody molecule according to common procedures D to G described below.

[0318] Common Procedure A: Concentration of antibody aqueous solution The antibody or antibody-immunostimulant conjugate solution was placed in an Amicon® Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and the antibody or antibody-immunostimulant conjugate solution was concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000 to 4000 G for 5 to 20 minutes.

[0319] Common Procedure B: Antibody concentration measurement Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The 280 nm extinction coefficient (1.3 mL mg) varied depending on the antibody. -1 cm -1 to 1.8 mL mg -1 cm -1 ) was used.

[0320] Common Procedure C: Antibody Buffer Exchange A buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to the aqueous antibody solution, and the solution was concentrated according to the method described in Common Procedure A. This procedure was repeated several times, and then the antibody concentration was measured according to the method described in Common Procedure B. An appropriate buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to this antibody buffer solution to prepare an antibody buffer solution of the desired concentration (for example, approximately 10 mg / mL).

[0321] Common Procedure D: Purification of Antibody-Immunostimulant Conjugates (Gel Filtration Chromatography) A NAP column (NAP-5, NAP-10, NAP-25 (GE Healthcare)) was equilibrated with acetate buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5; herein referred to as ABS) or another appropriate buffer. The antibody-immunoactivator conjugate reaction solution was charged onto this NAP column, and the manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This fraction was again charged onto the NAP column, and the manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This procedure was repeated two to three times to obtain an antibody-immunoactivator conjugate from which unbound immunoactivator linker, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-immunoactivator conjugate solution was adjusted using common procedures A and C.

[0322] Common Procedure E: Measurement of antibody concentration and average number of adjuvants bound per antibody molecule in antibody-adjuvant conjugates (UV method) The concentration of bound immunostimulant in the antibody-immunostimulant conjugate can be calculated by measuring the absorbance of an aqueous solution of the antibody-immunostimulant conjugate at two wavelengths, 280 nm and 250 nm, using an absorption spectrophotometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.) in accordance with the methods described in WO2020 / 050406 and WO2021 / 177438.

[0323] Common Procedure F: Measurement of antibody concentration and average number of adjuvants bound per antibody molecule in antibody-adjuvant conjugates (reversed-phase high-performance liquid chromatography: RP-HPLC) The antibody concentration in the antibody-immunopotentiator conjugate and the average number of immunopotentiators bound per antibody molecule can be determined by high performance liquid chromatography analysis in accordance with the methods described in WO2020 / 050406 and WO2021 / 177438, in addition to the above-mentioned common procedure E.

[0324] Common Procedure G: Measurement of antibody concentration and average number of adjuvants bound per antibody molecule in antibody-adjuvant conjugates (hydrophobic interaction-high performance liquid chromatography: HI-HPLC) The antibody concentration in the antibody-immunopotentiator conjugate and the average number of immunopotentiators bound per antibody molecule can be determined by high performance liquid chromatography analysis in accordance with the methods described in WO2020 / 050406 and WO2021 / 177438, in addition to the common procedures E and F described above.

[0325] The antibody-immunopotentiator conjugate produced by the method of the present invention or a production intermediate thereof may contain stereoisomers, optical isomers derived from asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers, and all of these isomers, optical isomers, and mixtures thereof are included in the present invention.

[0326] In antibody-immunopotentiator conjugates produced by the methods of the present invention, the number of immunopotentiators bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-immunopotentiator conjugates are produced by specifying reaction conditions, such as the amounts of reacting raw materials and reagents, so that a certain number of immunopotentiators are bound. However, unlike chemical reactions of low-molecular-weight compounds, the resulting conjugates are typically mixtures of immunopotentiators with different numbers bound to one another. The number of immunopotentiators bound to one antibody molecule can be determined as an average value, i.e., the average number of immunopotentiators bound (DAR: Drug-to-Antibody Ratio). The number of cyclic dinucleotide derivatives bound to an antibody molecule can be controlled; the average number of cyclic dinucleotide derivatives bound to one antibody can range from 1 to 10, preferably 1 to 8, and more preferably 1 to 5.

[0327] In the antibody-immunopotentiator conjugate produced by the method of the present invention, when the antibody Ab is bound to L via a remodeled sugar chain of the antibody Ab, the number of immunopotentiators bound per antibody molecule in the antibody-immunopotentiator conjugate, m 2 is an integer of 1 or 2. When the sugar chain is an N297 sugar chain, and the sugar chain is N297-(Fuc)SG, m 2 is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.2). When the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1, and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

[0328] Furthermore, a person skilled in the art would be able to design a reaction for binding the required number of immunostimulants to an antibody based on the description of the examples of the present application, and obtain an antibody in which the number of cyclic dinucleotide derivatives bound is controlled.

[0329] It should be noted that the antibody-immunostimulant conjugate produced by the method of the present invention or a production intermediate thereof may absorb moisture, become adsorbed water, or become a hydrate when left in the air or upon recrystallization, and such water-containing compounds and salts are also encompassed by the present invention.

[0330] When the antibody-immunostimulant conjugate produced by the method of the present invention or a production intermediate thereof contains a basic group such as an amino group, it can be converted into a pharmaceutically acceptable salt, if desired. Examples of such salts include hydrohalide salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0331] The antibody-immunostimulant conjugates produced by the methods of the present invention or their intermediates generally contain phosphate and / or thiophosphate groups in their structures, and therefore can generally form base addition salts. Furthermore, when the intermediates contain acidic groups such as carboxyl groups, they can generally form base addition salts. Pharmaceutically acceptable salts include, for example, alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

[0332] The antibody-immunostimulant conjugates and their production intermediates produced by the methods of the present invention may exist as hydrates due to, for example, absorption of moisture from the air. The solvates of the present invention are not particularly limited as long as they are pharmaceutically acceptable, but specific examples include hydrates, ethanol solvates, and 2-propanol solvates. Furthermore, when nitrogen atoms are present in the antibody-immunostimulant conjugates of the present invention and their production intermediates, they may be in the form of N-oxides, and these solvates and N-oxides are also within the scope of the present invention. Furthermore, when sulfur atoms are present in the antibody-immunostimulant conjugates of the present invention and their production intermediates, they may be in the form of sulfoxides, and these solvates and sulfoxides are also within the scope of the present invention.

[0333] The antibody-immunostimulator conjugates and their intermediates produced by the methods of the present invention also include compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-immunostimulator conjugates and their intermediates may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium (H), tritium (H), iodine-125 (I), and carbon-14 (C). The compounds of the present invention may also be radiolabeled with radioactive isotopes such as tritium (H), iodine-125 (I), or carbon-14 (C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo diagnostic imaging agents. All isotopic variants of the antibody-immunostimulator conjugates of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0334] <5. Pharmaceuticals> The antibody-immunostimulant conjugates produced by the methods of the present invention exhibit antitumor immune activity or cytotoxic activity against cancer cells, and can therefore be used as pharmaceuticals, particularly as therapeutic and / or preventive agents for cancer, or antitumor agents.

[0335] The types of cancer to which the antibody-immunomodulator conjugates produced by the method of the present invention can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, and gastrointestinal stromal tumor. Examples of cancer cells to be treated include cancers such as gastrointestinal stromal tumors (GIST, GIST), gallbladder cancer, bile duct cancer, adrenal cancer, pharyngeal cancer, tongue cancer, ear cancer, thymus cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, and sarcoma; however, with regard to antibody-immunopotentiator conjugates, the cancer cells to be treated are not limited to these, as long as they express a protein that can be recognized by the antibody in the antibody-immunopotentiator conjugate.

[0336] The antibody-immunostimulant conjugate produced by the method of the present invention can be suitably administered to mammals, more preferably humans.

[0337] Substances used in pharmaceutical compositions containing antibody-immunostimulant conjugates produced by the methods of the present invention can be appropriately selected from pharmaceutical additives and other substances commonly used in this field in terms of dosage and administration concentration.

[0338] The antibody-immunostimulatory agent conjugates produced by the methods of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation will correspond to the mode of administration.

[0339] Various delivery systems are known and can be used to administer the antibody-immunostimulant conjugate of the present invention. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, the antibody-immunostimulant conjugate is administered by infusion. Parenteral administration is a preferred administration route.

[0340] In a representative embodiment, a pharmaceutical composition comprising the antibody-immunostimulant conjugate is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the medicament may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the pharmaceutical composition is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical-grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration. The pharmaceutical composition may also be provided as a solution.

[0341] A pharmaceutical composition comprising an antibody-immunostimulant conjugate produced by the method of the present invention may be a pharmaceutical composition containing only the antibody-immunostimulant conjugate produced by the method of the present invention, or may be a pharmaceutical composition containing the antibody-immunostimulant conjugate produced by the method of the present invention and another cancer therapeutic agent. The antibody-immunostimulant conjugate produced by the method of the present invention can also be administered together with another cancer therapeutic agent, thereby enhancing the anti-tumor effect. The other cancer therapeutic agent used for this purpose may be administered to an individual simultaneously with the antibody-immunostimulant conjugate, separately, or sequentially, or at different administration intervals. Examples of such cancer therapeutic agents include chemotherapeutic agents such as antimetabolites, alkylating agents, and microtubule inhibitors (e.g., abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, vinblastine, or drugs described in International Publication No. WO 2003 / 038043), hormone modulating agents (e.g., LH-RH analogs such as leuprorelin, goserelin, and estramustine, and estrogen antagonists such as tamoxifen and raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, and exemestane), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, molecular targeted drugs (e.g., anti-EGFR antibodies, anti- Anti-VEGF antibodies, anti-VEGFR antibodies, etc.), immune checkpoint inhibitors (anti-PD-1 antibodies such as nivolumab and pembrolizumab, anti-PD-L1 antibodies such as atezolizumab, avelumab, and durvalumab, anti-PD-L2 antibodies, anti-CTLA4 antibodies such as ipilimumab, anti-A2aR antibodies, A2a receptor antagonists, anti-LAG3 antibodies, anti-TIM3 antibodies, etc.), anti-regulatory T cell drugs (anti-CTLA4 antibodies, anti-CD25 antibodies, anti-GITR antibodies, anti-GARP antibodies, anti-TIGIT antibodies, anti-CCR8 antibodies, etc.), immune activators (anti-4-1BB antibodies, anti-OX40 antibodies, anti-CD40 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, IL-2 analogs, cytokines, TLR agonists, etc.), immunomodulators (anti-CD47 antibodies, anti-SIRPα antibodies, inhibitory myeloid modulators, etc.), ADCC (AntibodyExamples of such antitumor agents include antibody drugs with T-cell dependent cytotoxicity (TDC) activity, ADCP (antibody dependent cellular phagocytosis) activity, or complement activity, BiTEs (bi-specific T-cell engagers), antibody-drug conjugates (ADCs) (e.g., drug conjugates containing deruxtecan, DM1, pyrrolobenzodiazepine, MMAF, etc. (anti-HER2-ADC, anti-TROP2-ADC, anti-HER3-ADC, etc.)), ADCs combined with photodynamic therapy, as well as antitumor vaccines, antitumor cell therapies (CAR-T, TCR-T, dendritic cells, NK cells, etc.), antitumor bacterial therapies, antitumor viral therapies, etc., but are not limited thereto as long as they have antitumor activity. Furthermore, the antibody-immunostimulant conjugates of the present invention can be administered together with other antibody-immunostimulant conjugates of the present invention, thereby enhancing the antitumor effect. Furthermore, the antibody-immunopotentiator conjugate of the present invention can enhance the antitumor effect when used in combination with not only immunopotentiators but also treatments that bring about antitumor effects, such as radiation, quantum beams, surgery, bone marrow transplantation, etc., but is not limited thereto as long as the treatment has an antitumor effect.

[0342] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0343] Although the composition and concentration of a pharmaceutical composition vary depending on the administration method, the antibody-immunoactivator conjugate contained in a pharmaceutical composition containing an antibody-immunoactivator conjugate produced by the method of the present invention exhibits efficacy even at a smaller dose, as the affinity of the antibody-immunoactivator conjugate for an antigen, i.e., the dissociation constant (Kd value) for the antigen, increases (the lower the Kd value). Therefore, when determining the dose of an antibody-immunoactivator conjugate, the dose can be set based on the affinity between the antibody-immunoactivator conjugate and the antigen. When the antibody-immunoactivator conjugate of the present invention is administered to a human, for example, approximately 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0344] The present invention will be described below with reference to examples, but is not limited thereto. In the following, "compound (1A)" or "(1A)" in the synthesis scheme indicates that it corresponds to "a compound of formula (1A)," and the same applies to the subsequent compound numbers. [Example]

[0345] In the following examples, unless otherwise specified, room temperature refers to 15°C to 35°C. Dehydrated dichloromethane was dichloromethane (ultra-dehydrated) sold by Fujifilm Wako Pure Chemical Industries, Ltd. Dehydrated acetonitrile was acetonitrile (ultra-dehydrated) sold by Fujifilm Wako Pure Chemical Industries, Ltd. Dehydrated pyridine was pyridine (dehydrated) sold by Kanto Chemical Co., Ltd. Silica gel chromatography was performed using Biotage Sfar HC D (20 μm, Biotage), amino silica gel column chromatography was performed using Biotage Sfar Amino D (50 μm, Biotage), and preparative HPLC was performed using an Agilent Preparative HPLC System (Agilent Technology). The preparative column used was an XBridge Prep OBD (5 μm, C18, 130 Å, 250 × 30 mm, Waters).

[0346] The following instruments were used to measure various spectral data. 1 H-NMR and 31 P-NMR spectra were measured using a JEOL ECZ500R spectrometer, and mass spectra were measured using Shimadzu LCMS-2010 and LCMS-2020 (Shimadzu Corporation).

[0347] <1. Synthesis of raw material compounds> 1-1. Synthesis of starting compound (1A) The starting compound (1A) used in the CDN synthesis schemes A-1, A-2, and B-1 was synthesized according to the following synthesis scheme 1. [Synthetic Scheme 1]

[0348] [ka]

[0349] Reference example 1: Synthesis of [(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methyl acetate (compound (XVIII)) [ka]

[0350] (Process 1) A solution of glycylglycine (60 g, 0.45 mol), triethylamine (69 g, 0.68 mol), water (600 mL), and tetrahydrofuran (600 mL) was cooled to 0 °C, and then N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide (130 g, 0.50 mol) was added and stirred at 25 °C for 22 hours. The reaction mixture was concentrated to 900 mL under reduced pressure, and then ethyl acetate (600 mL) was added. Triethylamine (69 g, 0.68 mol) was added and stirred, and the organic layer was discarded. Ethyl acetate (600 mL) and concentrated hydrochloric acid (116 g) were added to the resulting aqueous layer. After removing the aqueous layer, the resulting organic layer was washed twice with 10 wt% brine (600 mL). The organic layer was concentrated under reduced pressure to give a solution of N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycylglycine in ethyl acetate (120 mL).

[0351] (Process 2) A solution of lead tetraacetate (302 g, 0.68 mol), acetic acid (360 mL), and tetrahydrofuran (600 mL) was heated to 35°C, and then a solution of N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycylglycine in ethyl acetate (120 mL) in tetrahydrofuran (600 mL) was added dropwise. After stirring at 35°C for 1 hour, the precipitate was collected by filtration and washed with ethyl acetate (600 mL). The filtrate was washed seven times with 20 wt% aqueous trisodium citrate dihydrate (300 mL). 20 wt% brine (180 mL) was added to the resulting organic layer, followed by the addition of an appropriate amount of 25 wt% aqueous sodium hydroxide to adjust the pH to 6.5. The aqueous layer was discarded, and the resulting organic layer was concentrated under reduced pressure to approximately 600 mL. 1,2-Dimethoxyethane (1200 mL) was added, and the mixture was concentrated under reduced pressure to approximately 600 mL. This procedure was repeated to obtain a 1,2-dimethoxyethane solution (approximately 600 mL) of the target compound.

[0352] Reference example 2: N-[(2-bromoethoxy)methyl]-N 2 Synthesis of -{[2-(trimethylsilyl)ethoxy]carbonyl}glycinamide (compound (XV))

[0353] [ka]

[0354] (Step 3) To a 1,2-dimethoxyethane solution (approximately 600 mL) of the compound obtained in Reference Example 1, 1,2-dimethoxyethane (300 mL) was added, followed by the addition of 2-bromoethanol (114 g, 0.91 mol) at room temperature. After cooling to 0°C, 10 mol / L aqueous sodium hydroxide solution (66 g, 0.50 mol) was added and stirred for 3.5 hours. To the reaction solution, acetic acid (41 g, 0.68 mol) was added. Water (300 mL) was then added, and seed crystals of the target compound (60 mg) were added, followed by the addition of water (600 mL). The crystals were collected from the resulting suspension by filtration and washed with 1,2-dimethoxyethane (360 mL) containing 50% water. The crystals were stirred overnight at 40°C to obtain the target compound as white crystals (15 g, 80% yield).

[0355] 1 H-NMR (500MHz,DMSO-d6)δ8.66(1H,t,J=6.6Hz),7.23(1H,t,J=6.0Hz),4.58(2H,d,J=6.9Hz),4.06-4.01(2H, m),3.69(2H,t,J=6.0Hz),3.58(2H,d,J=6.3Hz),3.56(2H,t,J=5.7Hz),0.95-0.90(2H,m),0.02(9H,s).

[0356] Reference example 3: Synthesis of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]inosine (compound (XIV)) [ka]

[0357] (Step 4) To a solution of inosine (5.0 g, 18.4 mmol), pyridine (30 mL), and dimethyl sulfoxide (20 mL) was added 4,4'-dimethoxytrityl chloride (7.0 g, 20.5 mmol) at room temperature under a nitrogen stream. After stirring for 2 hours, additional 4,4'-dimethoxytrityl chloride (1.3 g, 3.7 mmol) was added. After stirring for 4 hours, 5 wt% aqueous sodium bicarbonate solution (50 mL), toluene (50 mL), and 20 wt% saline solution (25 mL) were added to the reaction mixture. The aqueous layer was discarded, and 5 wt% aqueous sodium bicarbonate solution (50 mL) and 20 wt% saline solution (25 mL) were added to the organic layer. After discarding the aqueous layer, the organic layer was washed with 20 wt% saline solution (50 mL). The resulting organic layer was concentrated under reduced pressure to approximately 20 mL, and ethyl acetate (100 mL) was added dropwise to the resulting solution. After stirring at 50°C for 1 hour, the mixture was cooled to room temperature and stirred overnight. The crystals were collected by filtration from the resulting suspension and washed with ethyl acetate (40 mL). The crystals were dried under reduced pressure at 40°C to obtain the target compound as white crystals (9.0 g, yield 85%).

[0358] 1 H-NMR (500MHz,DMSO-d6)δ12.39(1H,brs),8.19(1H,s),8.00(1H,s),7.35(2H,d ,J=7.4Hz),7.26(2H,t,J=7.4Hz),7.24-7.18(5H,m),6.86-6.81(4H,m),5. 91(1H,d,J=4.6Hz),5.60(1H,brs),5.23(1H,brs),4.58(1H,t,J=4.9Hz)4. 24(1H,t,J=5.2Hz),4.06(1H,q,J=4.8Hz),3.73(6H,s),3.23-3.17(2H,m).

[0359] Reference example 4: Synthesis of 1,3-dimethyl-2-imidazolidinone of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (compound (XVI)) [ka]

[0360] (Step 5) The compound obtained in Reference Example 3 (3.0 g, 5.3 mmol), N-[(2-bromoethoxy)methyl]-N- 2 A solution of 1,3-dimethyl-2-imidazolidinone (30 mL) was heated to 35-40°C. 1,1,3,3-Tetramethylguanidine (1.5 mL, 11.8 mmol) was added dropwise. The mixture was stirred at 35-40°C for 16 hours and then cooled to room temperature. Toluene (45 mL) and 10 wt% brine (30 mL) were added and stirred, and the aqueous layer was discarded. 1,3-Dimethyl-2-imidazolidinone (15 mL) and 10 wt% brine (15 mL) were added and stirred, and the aqueous layer was discarded. The resulting organic layer was washed twice with 10 wt% brine (30 mL) and concentrated under reduced pressure to approximately 15 mL. To the concentrated solution was added 1,3-dimethyl-2-imidazolidinone (15 mL), and the mixture was concentrated under reduced pressure to obtain a 1,3-dimethyl-2-imidazolidinone solution (approximately 24 mL) of the target compound.

[0361] Reference example 5: Synthesis of 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 (corresponding to the compound of formula (1A)) [ka]

[0362] (Step 6) To a 1,3-dimethyl-2-imidazolidinone solution (approximately 24 mL) of the compound obtained in Reference Example 4, 1,3-dimethyl-2-imidazolidinone (15 mL) was added, followed by the addition of 1,1,3,3-tetramethylguanidine (4.0 mL, 31.5 mmol) and tert-butyldimethylchlorosilane (2.2 g, 14.5 mmol). The mixture was heated to 55-60°C, stirred for 7 hours, and then cooled to room temperature. Toluene (60 mL) and water (30 mL) were added to the reaction mixture, followed by stirring. The aqueous layer was discarded, and the resulting organic layer was concentrated under reduced pressure to approximately 15 mL. Tetrahydrofuran (30 mL) was added to the concentrated solution, and the mixture was concentrated under reduced pressure to approximately 15 mL. The same procedure was repeated twice to obtain a tetrahydrofuran solution (approximately 15 mL) of a 6:4 mixture 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 (the target compound) and 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-O-(2,3,3-trimethylbutan-2-yl)-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine. Tetrahydrofuran (30 mL), 1,1,3,3-tetramethylguanidine (0.07 mL, 0.5 mmol), and crystalline target compound (1.0 mg) were added. After stirring at 25°C for 30 minutes, n-heptane (30 mL) was added and the mixture was stirred at 25°C overnight. Crystals were collected from the resulting suspension by filtration and washed with tetrahydrofuran / n-heptane (1 / 1, 30 mL). The crystals were dried under reduced pressure to obtain the target compound as white crystals (3.7 g, yield 73%).

[0363] 1 H-NMR (500MHz,CDCl3)δ7.99(1H,s),7.93(1H,s),7.42(2H,d,J=7.7Hz),7.33-7.29(4H,m),7.29-7.24(3H,m),7.23-7.19(1H,m),7. 08-7.01(1H,m),6.83-6.78(4H,m),5.92(1H,d,J=5.2Hz),5.46(1H,brs),4.70-4.65(1H,m),4.64(2H,d,J=6.9Hz),4.50(1H,d d,J=5.4,3.7Hz),4.19(2H,t,J=4.9Hz)4.18-4.12(3H,m),3.81-3.76(8H,m),3.73(2H,d,J=5.7Hz),3.48(1H,dd,J=10.9,3.4H z),3.26(1H,dd,J=10.9,4.0Hz),3.16(1H,d,J=6.9Hz),0.99-0.93(2H,m),0.89(9H,s),0.09(3H,s),0.02(9H,s),0.01(3H,s).

[0364] 1-2. Synthesis of starting compound (XXIII) The starting compound (XXIII) used in Scheme 3 of the synthesis of compound (XXV·TsOH) was synthesized according to the following Scheme 2. [Synthetic Scheme 2]

[0365] [ka]

[0366] Reference example 6: Synthesis of tert-butyl-4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (corresponding to the compound of formula (XXXII)) [ka]

[0367] (Process 1) To a solution of 5-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-amine (318.5 g, 1.225 mol) in toluene (1750 mL), di-tert-butyl dicarbonate (294.1 g, 1.347 mol) and 1-methylimidazole (50.28 g, 0.612 mol) were added and stirred at room temperature for 6 hours. Heptane (7000 mL) was added dropwise to the reaction mixture, which was stirred at room temperature for 10 minutes, then cooled to 0°C and stirred for 1 hour. The precipitated crystals were filtered, washed with a toluene / heptane (280 mL / 1120 mL) mixture, and dried under reduced pressure at 40°C to obtain the target compound (415.7 g, 1.154 mol, 94.2% yield).

[0368] MS(ESI)m / z:361[M+H] + . 1 H-NMR (500MHz, CDCl3) δ8.46(1H,s),7.55(1H,s),5.81(2H,brs),1.65(9H,s).

[0369] Reference example 7: Synthesis of tert-butyl-4-amino-5-(3,3-diethoxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (corresponding to the compound of formula (XXVIII)) [ka]

[0370] (Process 2) A solution of the compound obtained in Reference Example 6 (285.4 g, 0.792 mol) in N,N-dimethylformamide (1500 mL) was degassed under reduced pressure, followed by the addition of copper(I) iodide (1.51 g, 0.0079 mol), bis(triphenylphosphine)palladium(II) dichloride (3.89 g, 0.0055 mol), propargylaldehyde diethyl acetal (182.8 g, 1.426 mol), and triethylamine (240.5 g, 2.377 mol) at room temperature and stirring for 18.5 hours. Toluene (6000 mL) and 10% aqueous ammonium chloride (1500 mL) were added to the reaction mixture, followed by stirring. The mixture was separated and the aqueous layer was removed. The resulting organic layer was washed twice with 10% aqueous ammonium chloride (1500 mL) and once with 20% aqueous sodium chloride (1500 mL). Activated carbon (30 g) was added and stirred at room temperature for approximately 2 hours. The activated carbon was then filtered off and washed with toluene (600 mL). The filtrate was concentrated under reduced pressure to 1500 mL. Heptane (6000 mL) was added dropwise, and the mixture was stirred at room temperature for approximately 10 minutes. The mixture was then cooled to 0°C and stirred for 1 hour. The precipitated crystals were filtered, washed with a toluene / heptane (240 mL / 960 mL) mixture, and dried under reduced pressure at 40°C to obtain the target compound (269.9 g, 0.749 mol, yield 94.6%).

[0371] MS(ESI)m / z:361[M+H] + . 1 H-NMR (500MHz,CDCl3)δ8.47(1H,s),7.60(1H,s),5.71(2H,brs),5.49(1H,s),3.81(2H, dq,J=9.5,7.5Hz),3.67(2H,dq,J=9.5,7.5Hz),1.65(9H,s),1.28(6H,t,J=7.5Hz).

[0372] Reference example 8: Synthesis of 5-(3,3-diethoxyprop-1-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (corresponding to the compound of formula (XXIX)) [ka]

[0373] (Step 3) To a solution of the compound obtained in Reference Example 7 (250.0 g, 0.694 mol) in ethanol (1500 mL), 4 mol / L sodium hydroxide solution (260.1 mL, 1.040 mol) was added and stirred at room temperature for approximately 1.5 hours. Water (1500 mL) was added to the reaction solution, and the pH was adjusted to 7.5 with 2 M hydrochloric acid. The mixture was stirred at room temperature for approximately 45 minutes. Subsequently, water (3000 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The precipitated crystals were filtered and washed with an ethanol / water (250 mL / 750 mL) mixture and acetonitrile (1000 mL) cooled to 0°C. The obtained crystals were dried under reduced pressure at 40°C to obtain the target compound (152.5 g, 0.586 mol, yield 84.4%).

[0374] MS(ESI)m / z:261[M+H] + . 1 H-NMR (500MHz,DMSO-d6)δ12.01(1H,brs),8.09(1H,s),7.55(1H,s),6.50(2H,brs),5.58(1H ,s),3.68(2H,dq,J=10.0,7.5Hz),3.58(2H,dq,J=10.0,7.5Hz),1.16(6H,t,J=7.5Hz).

[0375] Reference example 9: Synthesis of 6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene (corresponding to the compound of formula (XXX)) [ka]

[0376] (Step 4) To a mixture of the compound obtained in Reference Example 8 (63.0 g, 0.242 mol) and 1-methylpyrrolidone (189 mL) was added 5% palladium on carbon (13.4 g, water content: 53.2%) and the mixture was stirred under a hydrogen atmosphere (300 kPa) at 40°C for approximately 2 hours. The atmosphere in the reaction system was replaced with nitrogen, and acetic acid (630 mL), water (32 mL), and 5% palladium on carbon (13.4 g, water content: 53.2%) were added, followed by stirring under a hydrogen atmosphere (300 kPa) at 50°C for approximately 21 hours. The atmosphere in the reaction system was replaced with nitrogen, and water (300 mL) was added. The palladium on carbon was then filtered off and washed with a mixture of acetic acid (150 mL) and water (150 mL). The filtrate was then concentrated under reduced pressure to 550 mL. Water (620 mL) and 20 mL of 48% potassium hydroxide aqueous solution were added dropwise at 50°C, cooled to 0-5°C, and stirred for approximately 20 hours. The precipitate was filtered, and the separated crystals were washed with a cooled 1-methylpyrrolidone / water (36 mL / 144 mL) mixture and then with water (320 mL). The obtained crystals were dried under reduced pressure at 50°C to obtain the target compound (37.0 g, 0.212 mol, yield 87.8%).

[0377] MS(ESI)m / z:175[M+H] + . 1 H-NMR (500MHz,DMSO-d6)δ11.24(1H,s),7.34(1H,s),6.86(1H,s)3.34-3.38(2H,m),2.80(2H,brt,J=6.0Hz),2.49-2.51(2H,m),1.85-1.91(2H,m).

[0378] Reference example 10: Synthesis of phenyl(2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulen-6-yl)methanone (corresponding to the compound of formula (XXIII)) [ka]

[0379] (Step 5) To a mixture of the compound obtained in Reference Example 9 (4.0 g, 23.0 mmol) and 1,3-dimethyl-2-imidazolidinone (40 mL), triethylamine (10.6 mL, 75.9 mol) and 4-dimethylaminopyridine (280 mg, 2.3 mmol) were added at room temperature. The mixture was then cooled to 0-5°C and benzoyl chloride (8.3 mL, 72.0 mmol) was added. After stirring for approximately 21 hours, methanol (20 mL) and triethylamine (12.0 mL) were added to the reaction mixture and stirred for approximately 23 hours. Acetic acid (6.7 mL) and water (102 mL) were added and the mixture was stirred at 0-5°C for approximately 2 hours. The precipitate was filtered, and the separated powder was washed with a cooled acetonitrile / water (2 mL / 18 mL) mixture. The resulting powder was suspended in acetonitrile (40 mL) and stirred at 0-5°C for approximately 1 hour. The precipitate was filtered, and the filtered powder was washed with cold acetonitrile (10 mL). The obtained powder was dried under reduced pressure at 50°C to obtain the target compound (5.13 g, 18.4 mmol, yield 80.0%).

[0380] MS(ESI)m / z:279[M+H] + . 1 H-NMR (500MHz,DMSO-d6)δ11.99(1H,s),7.93(1H,s),7.32-7.39(4H,m),4.18(2H,m),2.98(2H,t,J=6.3Hz),2.12-2.18(2H,m).

[0381] 1-3. Synthesis of starting compound (XXV·TsOH) The starting compound (XXV·TsOH) used in the CDN synthesis scheme A-1 was synthesized according to the following scheme 3. [Synthetic Scheme 3] [ka]

[0382] Reference example 11: Synthesis of 2'-deoxy-2'-fluoro-3,4,5,6-tetrahydrouridine [ka]

[0383] (Process 1) 2'-Deoxy-2'-fluorouridine (70.0 g, 28.4 mmol) and methanol (420 mL) were added to a 1 L autoclave, and 5% palladium-carbon (13.57 g, 52.2% water) was added. The mixture was stirred under a hydrogen atmosphere (300 kPa) at 50°C for 7 hours. After purging the reaction system with nitrogen, the palladium-carbon was filtered and washed with a methanol / water (189 mL / 21 mL) mixture. The filtrate was concentrated under reduced pressure to less than 210 mL, and N,N-dimethylacetamide (350 mL) and toluene (350 mL) were added, followed by further concentration under reduced pressure to 350 mL. Toluene (350 mL) was then added, and the mixture was again concentrated under reduced pressure to 350 mL. The same procedure was repeated two more times to obtain the target compound.

[0384] Reference example 12: Synthesis of 3',5'-di-O-benzoyl-2'-deoxy-2'-fluoro-3,4,5,6-tetrahydrouridine (compound (XIX)) [ka]

[0385] (Process 2) Pyridine (76.5 g, 96.7 mmol) was added to the N,N-dimethylacetamide solution (340 mL) obtained in Reference Example 11, and benzoyl chloride (85.4 g, 60.5 mmol) was added dropwise under ice cooling. The mixture was then stirred at room temperature for 2.5 hours. Water (6.8 mL) and 2-propanol (408 mL) were added to the reaction mixture, which was then heated to 50°C and water (102 mL) was added dropwise. After stirring at the same temperature for 30 minutes, water (136 mL) was added and stirred for 30 minutes, then cooled to room temperature and stirred for 22 hours. After cooling to 0°C and stirring for 1 hour, the precipitated crystals were filtered, washed with a 2-propanol / water (204 mL / 122 mL) mixture, and dried under reduced pressure at 40°C to obtain the target compound (117.2 g, 25.7 mmol, yield 93.0%) as a white solid.

[0386] MS(ESI)m / z:457[M+H] + . 1 H-NMR (500MHz,CDCl3)δ8.05(4H,dd,J=23.5,8.0Hz),7.62-5.56(2H,m),7.48-7.41(4H,m),7.38-7.32(1H,m),5.85(1H,dd,J= 22.3,2.3Hz),5.54-5.37(2H,m),4.81(1H,dd,J=12.0,2.9Hz),4.59-4.49(2H,m),3.53-3.44(2H,m),2.69-2.58(2H,m).

[0387] Reference example 13: Synthesis of 3-benzoyl-3',5'-di-O-benzoyl-2'-deoxy-2'-fluoro-3,4,5,6-tetrahydrouridine (compound (XX)) [ka]

[0388] (Step 3) To a suspension of the compound obtained in Reference Example 12 (67.0 g, 14.7 mmol) in acetonitrile (402 mL), 4-dimethylaminopyridine (18.0 g, 14.7 mmol) and triethylamine (26.8 g, 26.5 mmol) were added and the mixture was cooled on ice. Benzoyl chloride (33.1 g, 23.5 mmol) was added dropwise, and the mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was cooled on ice and diluted with cyclopentyl methyl ether (670 mL). 10% brine (670 mL) was added and the mixture was stirred for 5 minutes, followed by separation. The organic layer was washed with 0.5 M aqueous tosylic acid (670 mL) and then with water (335 mL). Cyclopentyl methyl ether (670 mL) was added to the resulting organic layer, and the mixture was concentrated under reduced pressure to 670 mL to obtain the target compound.

[0389] Reference example 14: Synthesis of 3,5-di-O-benzoyl-2-deoxy-2-fluoro-D-ribofuranose (compound (XXI)) [ka]

[0390] (Step 4) To the cyclopentyl methyl ether solution (670 mL) obtained in Reference Example 13, an aqueous solution (134 mL) of p-toluenesulfonic acid monohydrate (139.8 g, 73.5 mmol) was added, and the mixture was heated and stirred at 60°C for 6 hours. The reaction mixture was cooled to room temperature and then separated, and the aqueous layer was extracted with cyclopentyl methyl ether (335 mL). The combined organic layers were washed twice with an 8% aqueous sodium hydrogen carbonate solution (503 mL) and then with water (335 mL), and then the organic layer was concentrated under reduced pressure to 335 mL to obtain the target compound.

[0391] Reference example 15: Synthesis of 3,5-di-O-benzoyl-2-deoxy-2-fluoro-α-D-ribofuranosyl chloride (compound (XXII)) [ka]

[0392] (Step 5) To the cyclopentyl methyl ether solution (50 mL) obtained in Reference Example 14, dichloromethane (50 mL) was added, and under ice cooling, trichloroisocyanuric acid (1.55 g, 6.66 mmol) and tris(2,4-di-tert-butylphenyl)phosphite (8.64 g, 13.35 mmol) were added. The mixture was stirred at the same temperature for 1 hour, and then trichloroisocyanuric acid (1.55 g, 6.66 mmol) and tris(2,4-di-tert-butylphenyl)phosphite (8.64 g, 13.35 mmol) were added and stirred for 1 hour. Cyclopentyl methyl ether (100 mL) was added to the reaction solution, which was then concentrated under reduced pressure to less than 150 mL. 2 M aqueous sodium hydroxide solution (50 mL) and 10% aqueous sodium thiosulfate solution (50 mL) were then added and the mixture was stirred for 20 minutes. The reaction mixture was separated, and the organic layer was washed twice with 2M aqueous sodium hydroxide (100 mL), twice with 6% aqueous sodium bicarbonate (100 mL), and twice with water (100 mL). The organic layer was concentrated under reduced pressure to less than 30 mL, and heptane (100 mL) was added and concentrated under reduced pressure to less than 30 mL. Heptane (100 mL) and acetonitrile (100 mL) were added and the mixture was separated. The acetonitrile layer was further separated and washed with heptane (100 mL). Activated carbon (0.5 g) was added to the acetonitrile layer, stirred for 30 minutes, filtered, and washed with cyclopentyl methyl ether (50 mL). The filtrate was concentrated under reduced pressure to 50 mL, CPME (100 mL) was added, and the mixture was concentrated under reduced pressure to 50 mL. The mixture was heated to 50°C, heptane (50 mL) was added, and concentrated under reduced pressure to 50 mL. Heptane (50 mL) was then added dropwise. The mixture was stirred at the same temperature for 30 minutes, concentrated under reduced pressure to 50 mL, and then heptane (50 mL) was added. The same procedure was repeated two more times, and then cyclopentyl methyl ether (10 mL) was added. After cooling to 0°C, the precipitated crystals were filtered, washed with a mixture of heptane and cyclopentyl methyl ether (45 mL / 5 mL), and dried under reduced pressure at room temperature to obtain the target compound (6.2 g, 16.4 mmol, yield 61.3%) as a white solid.

[0393] MS(ESI) m / z: 396 [M+NH3+H] + . 1 H-NMR (500MHz,CDCl3)δ8.13(2H,d,J=7.4Hz),8.01(2H,d,J=8.0Hz),7.63-7.58(2H,m),7.49-7.43(4H,m),6.38(1H,d,J=4.0Hz),5.61 -5.59(1H,m),5.26(1H,ddd,J=50.4,6.9,4.6Hz),4.86-4.84(1H,m),4.73(1H,dd,J=12.6,2.9Hz),4.62(1H,dd,J=12.0,4.0Hz).

[0394] Reference example 16: Synthesis of 6-benzoyl-2-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene (compound (XXIV)) [ka]

[0395] (Step 6) Cesium carbonate (3.51 g, 10.77 mmol) was added to a solution of the compound obtained in Reference Example 15 (1.0 g, 3.59 mmol) in dimethyl sulfoxide (8 mL) and stirred for 10 minutes. Then, the compound obtained in Reference Example 10 (2.72 g, 7.18 mmol) was added in three portions. The equipment used for addition was rinsed with dimethyl sulfoxide (2 mL) and the mixture was stirred at room temperature for 3 hours. Acetic acid (1.19 g, 19.75 mmol), acetonitrile (15 mL), and methanol (5 mL) were added. The mixture was heated to 40°C and water (5 mL) was added dropwise. Seed crystals of the target compound (0.5 mg) were added at approximately 40°C, and water (7 mL) was added dropwise over 1 hour. After the addition, the mixture was returned to room temperature and stirred overnight. The crystals were collected from the resulting suspension by filtration and washed with acetonitrile / water (7 / 3, 25 mL) and then with ethanol (5 mL). Ethanol (20 mL) was added to the crystals and stirred at room temperature for approximately 30 minutes. The crystals were collected by filtration from the suspension and washed with ethanol (5 mL). The crystals were dried overnight at 40°C to obtain the target compound as white crystals (1.4 g, yield 63%).

[0396] MS(ESI)m / z:621[M+H] + . 1 H-NMR (500MHz,CDCl3)δ8.11-8.08(3H,m),8.03-8.00(2H,m),7.64-7.55(2H,m),7.50-7.44(2 H,m),7.42-7.34(5H,m),7.27-7.23(2H,m),7.09(1H,s),6.47(1H,dd,J=19.5,2.3Hz),5. 93-5.74(2H,m),4.86(2H,dd,J=12.6,3.4Hz),4.75(1H,ddd,J=7.4,4.0,3.4Hz),4.61(1 H,dd,J=12.6,4.0Hz),4.31-4.22(2H,m),2.88(2H,dd,J=6.9,6.9Hz),2.24-2.17(2H,m).

[0397] Reference example 17: Synthesis of 2-(2-deoxy-2-fluoro-β-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene·p-toluenesulfonate (compound (XXV·TsOH)) [ka]

[0398] (Step 7) To a solution of the compound obtained in Reference Example 16 (300 mg, 0.48 mmol) in ethanol (1.5 mL), a solution of sodium ethoxide in ethanol (20%, 0.012 mL, 0.03 mmol) was added and stirred at 65 °C for 6 hours. After cooling to room temperature, p-toluenesulfonic acid monohydrate (184 mg, 0.97 mmol) and ethanol (0.6 mL) were added and stirred at room temperature for 30 minutes. Toluene (6.3 mL) was then added, and the mixture was stirred at room temperature overnight and then cooled to 0 °C. After stirring at 0 °C for 4 hours, the precipitated crystals were filtered and washed with a mixture of ethanol / toluene (0.3 mL / 0.9 mL) cooled to 0 °C. The obtained crystals were dried under reduced pressure at 40 °C to obtain the target compound (215.9 mg, 0.45 mmol, yield 93.0%).

[0399] 1 H-NMR (500MHz,DMSO-d6)δ9.02(1H,brs),8.37(1H,s),7.56(1H,brs),7.48(2H,dd,J=10.3,1.8Hz),7.12(2H,d,J=7.5Hz),6.40(1H,dd,J=16.1,4 .0Hz),5.24(1H,m),4.34(1H,dt,J=13.0,5.2Hz),3.98(1H,brs),3.69-3.58(4H,m),2.87(2H,t,J=5.2Hz),2.29(3H,s),2.00-1.98(2H,m).

[0400] 2. Synthesis of Cyclic Dinucleotides (CDNs) 1-1. Synthesis of CDN A-1 Cyclic dinucleotide (CDN A-1) was synthesized according to the following synthesis scheme A-1, in which A2 is a Teoc-tetraazabenzo[cd]azulene group, PG1 is a TBS group, PG2 is a DMTr group, PG3 is a Teoc group, PG4 is a Lev group, PG5 is an Alloc group, and PG7 is a TFAc group. [Synthetic Scheme A-1]

[0401] [ka]

[0402] Example 1: Synthesis of 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-2'-O-[(1S,3aR)-tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (corresponding to the compound of formula (Rc-3A)) [ka]

[0403] (Process 1) 5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl]-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)inosine (10.0 g) was added to anhydrous dichloromethane (150 mL) and cooled to -5 °C, followed by the addition of triethylamine (1.73 mL). A solution of (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (Tetrahedron Letter, 1998, 39, 2491-2494) in anhydrous dichloromethane (50 mL) was added dropwise to the above solution, followed by stirring at the same temperature for 30 minutes. The mixture was warmed to room temperature and stirred for 30 minutes, after which the reaction mixture was concentrated. The concentrated crude product was diluted and dissolved in dichloromethane and purified by amino silica gel chromatography [dichloromethane / ethyl acetate] to obtain the target compound (10.4 g, yield 91.7%).

[0404] 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.

[0405] Example 2: 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 (formula (4A- 01 Synthesis of the compound [ka] (Process 2) To a solution of p-toluenesulfonate (34.2 g, 71.2 mmol) of the compound obtained in Reference Example 17 in pyridine (171 mL) was added 4-methoxytrityl chloride (26.4 g, 85.4 mmol) at room temperature. After stirring at room temperature for approximately 24 hours, 5% aqueous sodium bicarbonate solution (171 mL) was added dropwise. Toluene (684 mL) was added, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 10% brine (171 mL) to obtain an organic layer. Pyridine hydrochloride (8.2 g, 71.2 mmol) was added and stirred for 1 hour. Further pyridine hydrochloride (12.3 g, 106.8 mmol) was added and stirred for approximately 23 hours. Crystals were collected from the resulting suspension by filtration and washed with toluene (103 mL) to obtain wet crystals of the hydrochloride salt of the target compound. A portion of the obtained wet crystals (corresponding to 6.0 g of the p-toluenesulfonate salt of the compound obtained in Reference Example 17) was suspended in acetonitrile (120 mL) and stirred at room temperature for about 2 hours. The insoluble crystals were collected by filtration and dried at 40°C under reduced pressure to obtain the target compound (7.59 g, yield 98.5%).

[0406] 1 H-NMR(DMSO-D6,D2O) δ:8.35(1H,s),7.39-7.13(14H,m),6.91-6.85(2H,m),6.40(1H,dd,J=17.8,1.7Hz),5.36-5.22(1H,m),4.59 -4.49(1H,m),4.14-4.08(1H,m),3.62-3.55(2H,m),3.32-3.27(2H,m),2.74-2.61(2H,m),2.01-1.88(2H,m).

[0407] Example 3: Synthesis of 2-(trimethylsilyl)ethyl 2-[2-deoxy-2-fluoro3-O-(4-oxopentanoyl)-β-D-ribofuranosyl]-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulene-6-carboxylate (corresponding to the compound of formula (4A)) [ka] (Step 3) The hydrochloride salt (4.00 g, 6.48 mmol) of the compound obtained in Example 2 was suspended in dichloromethane (80 mL), and N,N-diisopropylethylamine (1.69 mL, 9.72 mmol) was added to form a homogeneous solution. To the mixed solution, levulinic acid (796 μL, 7.78 mmol), dimethylaminopyridine (79.2 mg, 648 μmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.49 g, 7.78 mmol) were added sequentially, and the mixture was stirred at room temperature for 3 hours. A 5% aqueous solution of sodium bicarbonate (80 mL) was added to the reaction solution, and after stirring, the aqueous layer was separated. The resulting aqueous layer was extracted with dichloromethane (40 mL). The resulting organic layers were combined, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in acetonitrile (20 mL), and N,N-diisopropylethylamine (8.82 mL, 51.9 mmol) and Teoc-ONP (11.0 g, 38.9 mmol) were added. The mixture was heated to 60°C and stirred for 44 hours. After cooling to room temperature, isopropyl acetate (44 mL) and 10% aqueous ammonium chloride solution (44 mL) were added, and the layers were separated. The aqueous layer was extracted with ethyl acetate (44 mL). The combined organic layers were washed with 10% aqueous ammonium chloride solution (44 mL), 10% aqueous potassium carbonate solution (44 mL), and then 10% aqueous potassium carbonate solution (44 mL). The solvent was then evaporated under reduced pressure. The resulting residue was dissolved in dichloromethane (80 mL), MeOH (10.5 mL) and dichloroacetic acid (2.70 mL, 32.9 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Dichloroacetic acid (2.70 mL, 32.9 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 16 hours. Dichloroacetic acid (680 μL, 8.29 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 5 hours. After cooling to 0°C, triethylamine (11.3 mL, 81.1 mmol) was added dropwise. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (methanol / dichloromethane) to obtain the target compound (2.69 g, 75% yield).

[0408] MS(ESI)m / z:552(M+H)+,610(M+AcO)- 1H-NMR(500MHz,DMSO-d6)δ:8.52(1H,d,J=1.1Hz),7.62(1H,s),6.50(1H,dd,J=15.8,4.9Hz),5. 68(0.5H,t,J=4.9Hz),5.58(0.5H,t,J=5.2Hz),5.46-5.42(1H,m),5.29(1H,t,J=5.2Hz),4.26(2 H,tJ=8.0Hz),4.19(1H,s),4.04-3.91(2H,m),3.68-3.59(2H,m),2.90(2H,s),2.78(2H,t,J=6.6 Hz),2.64-2.62(2H,m),2.13(3H,s),2.00(2H,d,J=8.0Hz),0.99(2H,t,J=8.0Hz),-0.01(9H,s).

[0409] Example 4: (2R,3R,4R,5R)-2-({[(R)-({(2R,3R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro- Synthesis of 9H-purin-9-yl]oxolan-3-yl}oxy){[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-4-fluoro5-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)oxolan-3-yl 4-oxopentanoate (corresponding to the compound of formula (Rc-5A)) [ka]

[0410] (Step 4) To a solution of 2-(trimethylsilyl)ethyl 2-[2-deoxy-2-fluoro-3-O-(4-oxopentanoyl)-β-D-ribofuranosyl]-2,7,8,9-tetrahydro-6H-2,3,5,6-tetraazabenzo[cd]azulene-6-carboxylate (the compound obtained in Example 3) (660 mg, 1.20 mmol) in acetonitrile (4.5 mL), molecular sieves 3A (198 mg) were added and stirred at room temperature for 1 hour. In a separate container, to a solution of the compound obtained in Example 1 (1.57 g, 1.44 mmol) in acetonitrile (6.6 mL), molecular sieves 3A (198 mg) were added and stirred at room temperature for 1 hour. Molecular sieves 3A (132 mg) was added, and a 0.92 mol / L acetonitrile solution of 1-methylbenzimidazolium triflate (2.6 mL, 2.40 mmol) that had been dried overnight was added. After stirring at room temperature for 1 hour, triethylamine (666 μL, 4.79 mmol) and 1-(trifluoroacetyl)imidazole (246 μL, 2.16 mmol) were added and the mixture was stirred at room temperature. After stirring for approximately 1 hour, xanthan hydride (234 mg, 1.56 mmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered to remove the molecular sieves and washed with acetonitrile (3 mL). The filtrate was concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain the target compound (1.15 g, 54% yield). HPLC analysis revealed that the phosphorus diastereomer ratio was 95.7:4.3.

[0411] MS(ESI)m / z:1767(M+H)+ 1 H-NMR(DMSO-D6) δ:8.65-8.60(1H,m),8.52(1H,s),8.24(2H,s),7.44(1H,s),7.32-7.14(9H,m),6.85-6.79(4H,m),6.43(1H,dd,J=18.3, 3.4Hz),6.20(1H,d,J=3.4Hz),5.71-5.58(1H,m),5.56-5.46(2H,m),4.77-4.73(1H,m),4.54-4.48(2H,m),4.33-3.83(13 H,m),3.74-3.71(1H,m),3.71(6H,s),3.65-3.33(7H,m),3.10-3.05(1H,m),2.88-2.80(2H,m),2.76-2.71(2H,m),2.64-2 .58(2H,m),2.10(3H,s),2.01-1.91(2H,m),1.75(4H,q,J=91.1Hz),1.00-0.82(6H,m),0.73(9H,s),0.04--0.03(24H,m). 31P-NMR(500MHz,DMSO-d6)δ:68.8

[0412] Example 5: (2R,3R,4R,5R)-2-({[(R)-({(2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-5-[({[(propen-2-yl)oxy ]carbonyl}oxy)methyl]oxolan-3-yl}oxy){[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-4-fluoro5-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)oxolan-3-yl 4-oxopentanoate (corresponding to the compound of formula (Rc-6A)) [ka]

[0413] (Step 5-1) Ethanol (1.5 mL) and dichloroacetic acid (160 μL, 1.94 mmol) were added to a dichloromethane (20 mL) solution of the compound obtained in Example 4 (1.145 g, 0.65 mmol), and the mixture was stirred overnight at room temperature. Dichloroacetic acid (80 μL, 0.97 mmol) was then added, and the mixture was stirred at room temperature for 5.5 hours. The mixture was then quenched with triethylamine (540 μL, 3.89 mmol), cooled on ice, and 5% aqueous sodium bicarbonate (10 mL) was added. The mixture was transferred to a separatory funnel and separated into organic layer 1 and aqueous layer 1. Organic layer 1 was washed with 5% aqueous sodium bicarbonate (10 mL), and the mixture was separated into organic layer 2 and aqueous layer 2. Next, dichloromethane (10 mL) was added to aqueous layer 1 for extraction, and the mixture was separated into organic layer 3 and aqueous layer 3. Furthermore, organic layer 3 was added to aqueous layer 2 for extraction, and the mixture was separated into organic layer 4 and aqueous layer 4. Organic layer 2 and organic layer 4 were combined and concentrated under reduced pressure, and anhydrous acetonitrile (10 mL) was added to the residue and concentrated under reduced pressure to obtain the crude de-Lev isomer of the formula (Rc-6A- 01 1.1374 g of a compound corresponding to (Step 5-2) Next, N,N,N',N'-tetramethylethylenediamine (86 μL, 0.58 mmol) and allyl chlorocarbonate (61 μL, 0.58 mmol) were added to a solution of the de-Lev form (849 mg, 0.58 mmol) in dichloromethane (18 mL), and the mixture was stirred at room temperature for 1 hour and 20 minutes. N,N,N',N'-Tetramethylethylenediamine (86 μL, 0.58 mmol) and allyl chlorocarbonate (61 μL, 0.58 mmol) were added, and the mixture was stirred for 20 minutes. Further N,N,N',N'-tetramethylethylenediamine (86 μL, 0.58 mmol) and allyl chlorocarbonate (61 μL, 0.58 mmol) were added, and the mixture was stirred at room temperature for 10 minutes. N,N,N',N'-tetramethylethylenediamine (21.5 μL, 0.144 mmol) and allyl chlorocarbonate (15 μL, 0.142 mmol) were added. After stirring at room temperature for 30 minutes, 5% aqueous sodium bicarbonate (10 mL) was added and the mixture was stirred. The mixture was then separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain the target compound (757 mg, 80% yield).

[0414] MS(ESI) m / z: 1548.9 (M + H)+ 1 1H-NMR (DMSO-D6) δ: 8.65 - 8.60 (1H, m), 8.51 (1H, s), 8.26 (1H, s), 8.23 (1H, s), 7.45 (1H, s), 7.21 - 7.16 (1H, m), 6.41 (1H, dd, J = 18.9, 3.4 Hz), 6.16 - 6.14 (1H, m), 5.93 - 5.85 (1H, m), 5.70 - 5.57 (1H, m), 5.51 - 5.42 (2H, m), 5.33 - 5.28 (1H, m), 5.25 - 5.20 (1H, m), 4.72 - 4.67 (1H, m), 4.61 - 4.56 (2H, m), 4.54 - 4.49 (H, m), 4.46 - 4.41 (1H, m), 4.32 - 3.90 (15H, m), 3.65 - 3.41 (7H, m), 2.89 - 2.82 (2H, m), 2.78 - 2.73 (2H, m), 2.66 - 2.57 (3H, m), 2.53 - 2.35 (1H, m), 2.12 (3H, s), 2.03 - 1.94 (2H, m), 1.86 - 1.73 (2H, m), 1.00 - 0.95 (2H, m), 0.94 - 0.89 (3H, m), 0.85 (9H, s), 0.10 - 0.07 (6H, m), 0.02 - 0.03 (18H, m). 31P-NMR (500 MHz, DMSO-d6) δ: 68.7

[0415] Example 6:<~ [(2R,3R,4R,5R)-3-{[tert-butyl(dimethyl)silyl]oxy}-5-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-4-{[(R)-({(2R,3R,4R,5R)-4-fluoro-3-hydroxybenzo ...] Synthesis of hydroxy-5-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)oxolan-2-yl}methoxy){[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}oxolan-2-yl]methyl prope-2-en-1-yl carbonate (corresponding to the compound of formula (Rc-7A)) [ka] (Step 6) Hydrazine acetate (53 mg, 0.58 mmol) was added to a solution of the compound obtained in Example 5 (0.746 g, 0.48 mmol) in dichloromethane (15 mL) and stirred overnight at room temperature. 2% aqueous sodium bicarbonate (10 mL) was added and the mixture was stirred and separated. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain the target compound (552 mg, 79% yield) as a solid.

[0416] MS(ESI)m / z:1448.65(MH)- 1 H-NMR(DMSO-D6) δ:8.65-8.60(1H,m),8.51(1H,s),8.26(1H,s),8.24(1H,s),7.37(1H,s),7.23-7.16(1H,m),6.39(1H,dd,J=19.5,2. 3Hz),6.15(1H,d,J=5.2Hz),5.94-5.85(2H,m),5.50-5.44(1H,m),5.35-5.20(3H,m),4.71-4.67(1H,m),4.61-4.58( 2H,m),4.54-4.51(2H,m),4.46-4.35(2H,m),4.31-3.87(16H,m),3.66-3.44(6H,m),2.87-2.81(2H,m),2.05-1.92(2 H,m),1.86-1.65(4H,m),1.02-0.96(2H,m),0.94-0.90(2H,m),0.86(9H,s),0.11-0.09(6H,m),0.03--0.01(18H,m). 31P-NMR(500MHz,DMSO-d6)δ:68.6

[0417] Example 7: 2-(Trimethylsilyl)ethyl [2-({[2-(9-{(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-bis(sulfanylidene)-2,10-bis{[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}-14-(6-{[2-(trimethylsilyl)ethoxy]carbonyl}-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclooctadecin-7-yl}-6-oxo-6,9-dihydro-1H-purin-1-yl)ethoxy]methyl}amino)-2-oxoethyl]carbamate (corresponding to the compounds of formula (Rc, Rc-8)) [ka] (Step 7) A solution of the compound obtained in Example 6 (100 mg, 68.9 μmol) in dichloromethane (1.0 mL) was mixed with molecular sieves 3A (30.0 mg) and stirred at room temperature for 18 hours. The mixture was cooled to 0°C, and a solution of triethylamine (14.4 μL, 103 μmol) and (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (13.7 mg, 82.8 μmol) in dichloromethane (1·0 mL) was added. The mixture was warmed to room temperature and stirred for 30 minutes. Dimedone (19.3 mg, 137 μmol) and tetrakis(triphenylphosphine)palladium (15.9 mg, 13.8 μmol) were then added and stirred for 1 hour. The mixture was cooled to 0°C, and acetonitrile (2.0 mL) and a 0.8 mol / L solution of 1-phenylimidazolium triflate in dehydrated acetonitrile (172 μL) were added. The mixture was stirred at room temperature for 1 hour. Triethylamine (38.4 μL, 275 μmol) and 1-(trifluoroacetyl)imidazole (11.8 μL, 104 μmol) were then added and the mixture was stirred at room temperature for 2 hours. Xanthan hydride (20.7 mg, 138 μmol) was then added and the mixture was stirred for 2 hours. The reaction mixture was filtered to remove the molecular sieves, and water (1.0 mL) was added. After stirring, the aqueous layer was discarded. Of the resulting organic layer (3467 mg), 3084 mg was concentrated under reduced pressure to yield a residue of the target compound (170.6 mg).

[0418] MS(ESI)m / z:1623(M+H)+,1621(MH)-

[0419] Example 8: Bis(N,N-diethylethanaminium)(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1 Synthesis of [Rp,Rp-9]-14-(6,7,8,9)-tetrahydro-2H-2,3,5,6-tetraazabenso[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (corresponding to the compound of formula (Rp,Rp-9)) [ka]

[0420] (Step 8) The residue (170.6 mg) obtained in Example 7 was dissolved in methanol (1.7 mL), and 28% aqueous ammonia (838 μL) was added. The mixture was heated to 45°C and stirred for 20 hours. The reaction mixture was filtered to remove insoluble matter and concentrated under reduced pressure. The residue was dissolved in methanol, and 5020 mg of the 5057 mg methanol solution was concentrated under reduced pressure. 50% aqueous methanol and triethylamine were added to the residue and filtered, yielding 2473 mg of a mixed solution. 2380 mg of this solution was purified by reverse-phase HPLC (acetonitrile / 10 mM aqueous triethylammonium acetate solution), and the fraction was diluted with water. This solution was subjected to solid-phase extraction using silica gel and eluted with 0.06% triethylamine / acetonitrile solution. The obtained fraction was concentrated to obtain the target compound (20.0 mg, 26% yield for the two steps) as a solid. The diastereomeric ratio at the phosphorus atom produced in steps 7 and 8 was 89:11 as determined by HPLC after the reaction and before purification.

[0421] MS(ESI)m / z:1121(M+H)+,1119(MH)- 1H-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). 31P-NMR(MeCN-d3)δ:58.31,57.22.

[0422] 1-2. Synthesis of CDN A-2 Cyclic dinucleotide (CDN A-2) was synthesized according to the following synthesis scheme A-2, in which A2 is a Bz-adenine group, PG1 is a TBS group, PG2 is a DMTr group, PG3 is a Teoc group, PG4 is a Lev group, PG5 is an Alloc group, and PG7 is a TFAc group. [Synthetic Scheme A-2] [ka]

[0423] Example 9: Synthesis of N-benzoyl-2'-deoxy-2'-fluoro-3'-O-(4-oxopentanoyl)adenosine (corresponding to the compound of formula (4A)) [ka] (Process 1) To a solution of N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-deoxy-2'-fluoroadenosine (2.50 g, 3.70 mmol) in dichloromethane (25 mL) was added levulinic acid (568 μL, 5.55 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.06 g, 5.55 mmol), and 4-dimethylaminopyridine (4.5 mg, 0.04 mmol) under nitrogen. After stirring overnight at room temperature, levulinic acid (189 μL, 1.85 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (353 mg, 1.84 mmol) were added, and the mixture was heated to 35 °C. After stirring for 1.5 hours, 4-dimethylaminopyridine (4.5 mg, 0.04 mmol) was added and the mixture was stirred at 35 °C for 4 hours. The mixture was then returned to room temperature, and ethyl acetate (25 mL) and 5% aqueous sodium bicarbonate (50 mL) were added and the mixture was separated. The resulting organic layer was washed with 20% brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. The residue was dissolved in dichloromethane (37.5 mL) and water (0.667 mL), and a 6% solution of dichloroacetic acid in dichloromethane was added under nitrogen. After stirring for 15 minutes at room temperature, the reaction was quenched with methanol (12.5 mL), and 5% aqueous sodium bicarbonate (75 mL) was added in small portions. After stirring, the mixture was separated. The resulting organic layer was washed with 20% brine (75 mL). The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / dichloromethane) to obtain the target compound (1.64 g, 94% yield).

[0424] 1 H-NMR(CDCl3) δ:9.08(1H,s),8.80(1H,s),8.14(1H,s),8.03(2H,d,J=7.4Hz),7.63(1H ,t,J=7.4Hz),7.54(2H,t,J=7.7Hz),6.19(1H,dd,J=12.0,6.3Hz),5.90( 1H,dt,J=50.8,5.7Hz),5.76-5.66(2H,m),4.42(1H,s),4.02-3.96(1H,m ),3.87-3.80(1H,m),2.93-2.84(1H,m),2.83-2.65(3H,m),2.23(3H,s).

[0425] Example 10: 2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-({[(R)-({(2R,3R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]oxolan-3-yl}oxy){[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-4-fluorooxolan-3-yl 4-oxopentanoate Synthesis of (corresponding to the compound of formula (Rc-5A)) [ka]

[0426] (Process 2) A solution of the compound obtained in Example 9 (800 mg, 1.70 mmol) and molecular sieves 4A (160 mg) in acetonitrile (8 mL) and a solution of the compound obtained in Example 1 (2.03 g, 1.87 mmol) and molecular sieves 4A (160 mg) in acetonitrile (8 mL) were stirred under nitrogen at room temperature for 1 hour, and then mixed. 1-phenylimidazolium triflate (749 mg, 2.55 mmol) was added. After stirring at room temperature for approximately 2 hours, triethylamine (1.18 mL, 8.48 mmol) and 1-(trifluoroacetyl)imidazole (271 μL, 2.38 mmol) were added and stirred at room temperature for 1 hour. Xanthan hydride (382 mg, 2.55 mmol) was added, and after stirring for 18 hours, ethyl acetate (30 mL) and 5% aqueous sodium bicarbonate solution (10 mL) were added. After stirring, the aqueous layer was discarded. The organic layer was washed with 20% brine (10 mL), and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (1.34 g, yield 47%).

[0427] MS(ESI)m / z:1688(M+H)+,1686(MH)- 1H-NMR (500 MHz, CDCl3) δ: 9.25 (1H, s), 8.77 (1H, s), 8.25 (1H, s), 8.05 (2H, d, J = 7.4 Hz), 7.94 (1H, s), 7.86 (1H, s), 7.60 (1H, t, J = 7.4 Hz), 7.51 (2H, t, J = 7.7 Hz), 7.40 (2H, d, J = 7.4 Hz), 7.29 (4H, dd, J = 8.6, 1.7 Hz), 7.23 (1H, d, J = 7.4 Hz), 7.17 (1H, t, J = 7.2 Hz), 6.80 (4H, dd, J = 8.9, 1.4 Hz), 6.28 (1H, dd, J = 18.9, 1.7 Hz), 6.11 (1H, d, J = 4.6 Hz), 5.81 - 5.61 (3H, m), 5.43 - 5.38 (1H, m), 4.63 (2H, d, J = 6.9 Hz), 4.50 (1H, t, J = 4.6 Hz), 4.36 (1H, m), 4.28 - 4.25 (3H, m), 4.23 - 4.20 (1H, m), 4.17 - 4.08 (6H m), 4.03 - 4.00 (1H, m), 3.83 (2H, d, J = 6.3 Hz), 3.78 - 3.74 (8H, m), 3.62 (2H, q, J = 6.9 Hz), 3.49 (1H, dd, J = 10.9, 3.4 Hz), 3.24 (1H, dd, J = 10.9, 4.0 Hz), 2.81 (2H, dd, J = 10.9, 6.3 Hz), 2.69 (2H, q, J = 6.3 Hz), 2.20 (3H, s), 2.02 - 1.89 (4H, m), 1.26 (1H, t, J = 7.2), 0.95 - 0.91 (2H, m), 0.80 (9H, s), 0.00 (s, 15H). 31P-NMR (500 MHz, CDCl3) δ: 70.0

[0428] Example 11: (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-({[(R)-{[(2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-5-(hydroxymethyl)oxolan-3-yl]oxy}{[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-4-fluorooxolan-3-yl 4-oxopentanoate (formula (Rc-6A- 01 Synthesis of [ka] (Step 3) The compound obtained in Example 10 (1.24 g, 735 μmol) was dissolved in dichloromethane (25 mL), and purified water (0.13 mL) and dichloroacetic acid (750 μL, 9.07 mmol) were added in this order at room temperature. After stirring for 3 hours, 5% sodium bicarbonate (25 mL) was added dropwise, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (25 mL), and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (569 mg, yield 56%).

[0429] MS(ESI)m / z:1385(M+H)+,1383(MH)- Example 12: (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-({[(R)-({(2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-5-[({[(prop-2-en-1-yl)oxy]carbonyl}oxy)methyl]oxolan-3-yl}oxy){[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}methyl)-4-fluorooxolan-3-yl 4-oxopentanoate Synthesis of (corresponding to compound of formula (Rc-6A)) [ka] (Step 4) The compound obtained in Example 11 (339 mg, 245 μmol) was dissolved in dichloromethane (7 mL), and N,N,N',N'-tetramethylethylenediamine (22 μL, 0.15 mmol) and allyl chloroformate (26 μL, 0.24 mmol) were added in this order at 0°C. After stirring for approximately 3 hours, N,N,N',N'-tetramethylethylenediamine (22 μL, 0.15 mmol) and allyl chloroformate (26 μL, 0.24 mmol) were added in this order at 0°C. The mixture was warmed to room temperature and stirred for approximately 1 hour, repeating this process four times. 5% sodium bicarbonate (7 mL) was added dropwise, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (7 mL), and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (215 mg, 60% yield).

[0430] MS(ESI)m / z:1469(M+H)+,1467(MH)-

[0431] Example 13: Synthesis of {(2R,3R,4R,5R)-4-{[(R)-{[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}{[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}phosphorothioyl]oxy}-3-{[tert-butyl(dimethyl)silyl]oxy}-5-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]oxolan-2-yl}methyl prope-2-en-1-yl carbonate (corresponding to the compound of formula (Rc-7A)) [ka] (Step 5) The compound obtained in Example 12 (107 mg, 72.9 μmol) was dissolved in dichloromethane (2 mL), and hydrazine acetate (6.7 mg, 73 μmol) was added at room temperature and stirred for approximately 3 hours. Hydrazine acetate (1.3 mg, 15 μmol) was added at room temperature and stirred for approximately 3 hours. 5% sodium bicarbonate (1 mL) was then added dropwise, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (1 mL), and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (77.7 mg, 78% yield).

[0432] 31P-NMR (500MHz, CDCl3) δ: 70.4

[0433] Example 14: 2-(Trimethylsilyl)ethyl (2-{[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-benzamido-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-bis(sulfanylidene)-2,10-bis{[(2R)-1-(trifluoroacetyl)pyrrolidin-2-yl]methoxy}octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclooctadecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate (corresponding to compounds of formula (Rc, Rc-8)) [ka] (Step 6) The compound obtained in Example 13 (35.5 mg, 25.9 μmol) was dissolved in tetrahydrofuran (0.5 mL) and triethylamine (7.2 μL) was added. A solution of (3aR)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (6.4 mg, 38.7 μmol) in tetrahydrofuran (178 μL) was added dropwise at −20°C, and the mixture was warmed to room temperature and stirred for 30 minutes. The precipitated solid was filtered using a syringe filter (Millex LH 0.45 μm) and washed with tetrahydrofuran (360 μL). Tetrakistriphenylphosphinepalladium (3.0 mg, 2.6 μmol) and dimedone (7.3 mg, 52 μmol) were added to the filtrate and stirred for 2 hours. Tetrakistriphenylphosphinepalladium (3.0 mg, 2.6 μmol) was added and stirred for 2 hours. A 0.4 mol / L solution of 1-phenylimidazolium triflate in dehydrated acetonitrile (97.5 μL) was added at -50°C, and the mixture was warmed to room temperature and stirred for 2 hours. Subsequently, triethylamine (21.7 μL, 155 μmol) and 1-(trifluoroacetyl)imidazole (4.4 μL, 2.4 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Xanthan hydride (11.6 mg, 77.6 μmol) was added, and after stirring for 16 hours, ethyl acetate (1 mL) and 5% aqueous sodium bicarbonate solution (1 mL) were added. After stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (1 mL), and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (10.2 mg, 26% yield, 96.2% peak area ratio by HPLC (210 nm)).

[0434] MS(ESI)m / z:1545(M+H)+,1543(MH)-

[0435] Example 15: 2-(Trimethylsilyl)ethyl (2-{[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclooctadecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate (corresponding to the compounds of formula (Rp, Rp-9)) [ka]

[0436] (Step 7) The compound obtained in Example 14 (2.0 mg, 1.3 μmol) was dissolved in methanol (0.1 mL), 25% aqueous ammonia (20 μL) was added, and the mixture was heated to 50° C. and stirred. After 2 hours, HPLC (210 nm) measurement was performed, and it was confirmed that the target compound was produced with a peak area ratio of 71.7%.

[0437] MS(ESI)m / z:1082(M+H)+,1080(MH)-

[0438] 1-3.Synthesis of CDN B-1 Cyclic dinucleotide (CDN B-1) was synthesized according to the following synthesis scheme B-1, in which A2 is a Bz-adenine group, PG1 is a TBS group, PG2 is a DMTr group, PG3 is a Teoc group, PG4 is a Lev group, PG5 is an Alloc group, and PG7 is a TFAc group. [Synthetic Scheme B-1] [ka]

[0439] Reference example 18: Synthesis of N-benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-deoxy-2'-fluoro-3'-O-[(1S,3aR)-tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphol-1-yl]adenosine (corresponding to the compound of formula (Rc-3B)) [ka]

[0440] (Process 1) N-Benzoyl-5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2'-deoxy-2'-fluoroadenosine (1.50 g, 2.22 mmol) was dissolved in dichloromethane (26 mL), molecular sieves 4A (623 mg), and triethylamine (371 μL, 2.66 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The mixture was cooled to -16 °C, and R-PROC (440 mg, 2.66 mmol) dissolved in anhydrous dichloromethane (3 mL) was added. After warming to room temperature and stirring for 30 minutes, the reaction mixture was filtered to remove the molecular sieves. The filtrate was concentrated, and the resulting crude product was diluted and dissolved in dichloromethane and purified by aminosilica gel chromatography (dichloromethane) to obtain the target compound (1.25 g, 70% yield).

[0441] 1 H-NMR(CDCl3) δ:8.90(1H,brs),8.79(1H,s),8.29(1Hs),8.02(2H,d,J=7.4Hz),7.64-7.60(1H,m),7.56-7.51(2H,m) ,7.41-7.38(2H,m),7.32-7.18(7H,m),6.82-6.77(4H,m),6.29(1H,dd,J=16.6,2.1Hz),5.62(1H,dq,J =52.1,2.1Hz),5.13-5.04(1H,m),4.45-4.41(1H,m),4.36-4.32(1H,m),3.90-3.84(1H,m),3.80-3.68 (7H,m),3.61-3.49(2H,m),3.40-3.35(1H,m),3.16-3.07(1H,m),1.93-1.70(3H,m),1.44-1.36(1H,m). 31P-NMR(500MHz,CDCl3)δ:155.65(d,J=5.8)

[0442] Reference example 19: Synthesis of 3'-O-[tert-butyl(dimethyl)silyl]-2'-O-(4-oxopentanoyl)-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycine)amino]methoxy}ethyl)inosine (corresponding to the compound of formula (1B)) [ka]

[0443] (Process 2) The compound obtained in Reference Example 5 (2.50 g, 2.61 mmol) was dissolved in dichloromethane (25 mL), and levulinic acid (605 mg, 5.21 mmol), dimethylaminopyridine (31.8 mg, 261 μL), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (809 mg, 5.21 mmol) were added sequentially, followed by stirring at room temperature for 4 hours. A 5% aqueous solution of potassium dihydrogen phosphate (25 mL) was added to the reaction solution, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with a 5% aqueous solution of sodium bicarbonate (25 mL) and 20% brine (25 mL), and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in dichloromethane (50 mL), purified water (470 μL), and dichloroacetic acid (1.50 mL, 18.1 mmol) were added at room temperature, and the mixture was stirred for 3 hours. After ice-cooling and adding methanol (1 mL), the mixture was warmed to room temperature, and 5% aqueous sodium bicarbonate (25 mL) was added. After stirring, the aqueous layer was discarded. The resulting organic layer was washed with 5% aqueous sodium bicarbonate (25 mL) and 20% brine (25 mL), and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (methanol / dichloromethane) to obtain the target compound (1.49 g, 76% yield).

[0444] 1 H-NMR(CDCl3) δ:8.05(1H,s),7.85(1H,s),7.15(1H,d,J=33.2Hz),5.98(1H,brs),5.72(1H,dt,J=6.9,5 .2Hz),5.63(1H,brs),5.42(1H,brs),4.79-4.74(1H,m),4.71-4.69(1H,m),4.67-4.60(1 H,m),4.24-4.11(5H,m),4.00-3.95(1H,m),3.84-3.65(5H,m),2.70-2.65(2H,m),2.59-2 .53(2H,m),2.13(3H,s),1.00-0.95(2H,m),0.94(9H,s),0.13-0.09(6H,m),0.03(9H,s).

[0445] Reference example 20: Synthesis of (2R,3R,4R,5R)-5-({[(R)-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}{[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-fluorooxolan-3-yl]oxy}phosphorothioyl]oxy}methyl)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]oxolan-3-yl 4-oxopentanoate (corresponding to the compound of formula (Rc-5B)) [ka]

[0446] (Step 3) A solution of the compound obtained in Reference Example 19 (1.10 g, 1.46 mmol) and molecular sieves 4A (220 mg) in acetonitrile (5.5 mL) and a solution of the compound obtained in Reference Example 18 (1.29 g, 1.60 mmol) and molecular sieves 4A (220 mg) in acetonitrile (5.5 mL) were stirred under nitrogen at room temperature for 30 minutes, and then mixed. A 0.4 M solution of 1-phenylimidazolium triflate in acetonitrile (5.48 mL, 2.19 mmol) was added. The mixture was stirred at room temperature for 10 minutes, followed by the addition of triethylamine (1.02 mL, 7.28 mmol) and a 1 M solution of acetic anhydride in acetonitrile (1.53 mL, 1.53 mmol), and the mixture was stirred at room temperature for 2 hours. Xanthan hydride (230 mg, 1.53 mmol) was added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was filtered to remove the molecular sieves, and then ethyl acetate (22 mL) and 5% aqueous sodium bicarbonate (11 mL) were added and stirred. The aqueous layer was then discarded. The resulting organic layer was washed with 20% brine (11 mL), and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (1.49 g, 63% yield).

[0447] 31P-NMR(500MHz,MeCN-d3)δ:69.1 MS(ESI)m / z:1634(M+H)+,1632(MH)-

[0448] Reference example 21: Synthesis of (2R,3R,4R,5R)-5-({[(R)-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}{[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro2-(hydroxymethyl)oxolan-3-yl]oxy}phosphorothioyl]oxy}methyl)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[1-(2,2-dimethyl-6,9-dioxo-5,12-dioxa-7,10-diaza-2-silatetradecan-14-yl)-6-oxo-1,6-dihydro-9H-purin-9-yl]oxolan-3-yl 4-oxopentanoate (corresponding to the compound of formula (Rc-6B)) [ka]

[0449] (Step 4) The compound obtained in Reference Example 20 (400 mg, 245 μmol) was dissolved in dichloromethane (4 mL), and purified water (44 μL) and dichloroacetic acid (120 μL, 1.45 mmol) were added in this order at room temperature. After stirring for 4 hours, 5% sodium bicarbonate (4 mL) was added dropwise, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (4 mL), and the solvent was evaporated under reduced pressure. The residue was purified by reverse-phase HPLC (acetonitrile / water) to obtain the target compound (103 mg, 32% yield).

[0450] MS(ESI)m / z:1331(M+H)+,1329(MH)-

[0451] Reference example 22: Synthesis of 2-(trimethylsilyl)ethyl (2-{[(2-{9-[(2R,3R,4S,5R)-5-({[(R)-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}{[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro2-(hydroxymethyl)oxolan-3-yl]oxy}phosphorothioyl]oxy}methyl)-4-{[tert-butyl(dimethyl)silyl]oxy}-3-hydroxyoxolan-2-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate (corresponding to the compound of formula (Rc-7B)) [ka]

[0452] (Step 5) The compound obtained in Reference Example 21 (94.6 mg, 71.0 μmol) was dissolved in dichloromethane (2 mL), and hydrazine acetate (6.54 mg, 71.0 μmol) was added at 0°C and then at room temperature, followed by stirring for 6 hours. Water (1 mL) was added, and after stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (1 mL), and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to obtain the target compound (64.8 mg, 74% yield).

[0453] MS(ESI)m / z:1233(M+H)+,1231(MH)- 31P-NMR(500MHz,MeCN-d3)δ:69.6

[0454] Reference example 23: 2-(Trimethylsilyl)ethyl (2-{[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-2-{[(2R)-1-acetylpyrrolidin-2-yl]methoxy}-10-{[(2S)-1-acetylpyrrolidin-2-yl]methoxy}-14-(6-benzamido-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-bis(sulfanylidene)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclooctadecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate (formula (R c, Sc-8) Compound (corresponding to the synthesis) [ka]

[0455] (Step 6) The compound obtained in Reference Example 22 (32.4 mg, 26.3 μmol) was dissolved in dichloromethane (0.65 mL), molecular sieves (6.5 mg) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was cooled to -20°C, and a solution of triethylamine (4.4 μL, 31.6 μmol) and (3aS)-1-chlorotetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (4.4 mg, 26.3 μmol) in dichloromethane (0.32 mL) was added, and the mixture was warmed to room temperature. The reaction solution was stirred for 1 hour, and then a solution of 1-phenylimidazolium triflate in dehydrated acetonitrile (178 μL) adjusted to 0.4 mol / L was added, and the mixture was stirred at room temperature for 1 hour. Triethylamine (4.4 μL, 31.6 μmol) and a 1 M solution of acetic anhydride in acetonitrile (28.0 μL, 28 μmol) were added to the reaction solution, and the mixture was stirred at room temperature for 3 hours. Xanthan hydride (4.1 mg, 27.6 μmol) was added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. The reaction solution was filtered to remove the molecular weight, and then dichloromethane (0.4 mL) and 5% aqueous sodium bicarbonate solution (0.5 mL) were added. After stirring, the aqueous layer was discarded. The resulting organic layer was washed with 20% brine (0.5 mL), and the solvent was evaporated under reduced pressure. The residue was separated by reverse-phase HPLC (acetonitrile / water) and purified to obtain the target compound (2.5 mg, yield 7%, HPLC (210 nm) peak area ratio 67.6%).

[0456] MS(ESI)m / z:1436(M+H)+,1434 (MH)-

[0457] Reference example 24: 2-(Trimethylsilyl)ethyl (2-{[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclooctadecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]amino}-2-oxoethyl)carbamate (corresponding to the compounds of formula (Rp, Rp-9)) [ka]

[0458] (Step 7) The compound (cyclized product) (1.3 mg, 0.87 μmol) obtained in Reference Example 23 was dissolved in methanol (50 μL), 25% aqueous ammonia (10 μL) was added, and the mixture was heated to 50° C. and stirred. After 6 hours, HPLC (210 nm) measurement confirmed that the target compound was produced with a peak area ratio of 21.3%.

[0459] MS(ESI)m / z:1082(M+H)+,1080(MH)-

[0460] 3. Synthesis of CDN-Linker The CDN-linker was synthesized according to the following synthesis scheme 4. [Synthetic Scheme 4]

[0461] [ka]

[0462] Reference example 25: Synthesis of N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanine (compound (11))

[0463] [ka] (Process 1) To a suspension of 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (5.0 g, 12.4 mmol) and glycylglycyl-L-phenylalanine (4.2 g, 14.9 mmol) in acetonitrile (50 mL) and water (50 mL) was added triethylamine (2.3 mL, 16.1 mmol) at room temperature and stirred for approximately 4 hours. After adding 1 mol / L aqueous hydrochloric acid (16.2 mL), seed crystals of the target compound (4.6 mg) were added. After stirring overnight at room temperature, water (100 mL) was added dropwise over 2 hours to the resulting suspension. After stirring at 40 °C for approximately 1.5 hours, 1 mol / L aqueous hydrochloric acid (16.2 mL) was added. After stirring for 30 minutes, the mixture was cooled to room temperature and stirred for an additional 2 hours. The crystals were collected from the suspension by filtration and washed with acetonitrile / water (1 / 3, 50 mL) and dried overnight at 30° C. to obtain the target compound as white crystals (6.7 g, yield 95%).

[0464] 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).

[0465] Example 16: N-[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp, Rp-10)) 1 [ka] (Process 2) Bis(N,N-diethylethanaminium)(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]-14-(6,7,8,9)-tetrahydro-2H-2,3,5, To a solution of 6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) (the compound from Example 8 (Rp, Rp-9)) (1 g, 0.76 mmol) in dimethyl sulfoxide (3 mL) was added 70% aqueous tetrabutylammonium fluoride (3 mL) at room temperature and stirred at 30 °C for 2 days. Acetonitrile (12 mL) and calcium chloride (0.59 g, 5.32 mmol) were added and 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. Then, 5 mol / L aqueous hydrochloric acid (1.2 mL) was added in six portions. Triethylamine (60 μL) was added, and the insoluble matter was filtered off. The solvent was evaporated under reduced pressure to approximately 10 mL from the resulting filtrate. Acetonitrile (20 mL) was added, and the solvent was evaporated under reduced pressure to approximately 10 mL. This procedure was repeated twice, followed by the addition of acetonitrile (50 mL), and the solvent was evaporated under reduced pressure to approximately 10 mL. This procedure was repeated twice. 5 mol / L aqueous hydrochloric acid (165 μL) was added to the resulting solution, and acetonitrile (50 mL) was added. The mixture was then cooled to 0°C and stirred for 2 hours. The solid was collected from the resulting suspension by filtration and washed with acetonitrile (5 mL). The solid was dried overnight at 25°C to obtain the target compound as white crystals (0.46 g, 71% yield).

[0466] MS(ESI)m / z:863(M+H) + ,861(MH) - . 1H-NMR(500MHz,DMSO-d6)δ:9.18(1H,t,J=6.5Hz),9.02(1H,brs),8.69(1H,s),8.37(1H,s),8.20(1H,s), 8.09(2H,brs),7.47(1H,s),6.40(1H,dd,J=15.0,2.0Hz),6.06(1H,d,J=8.0Hz),5.42(1H,d,J=52.5Hz),5. 22(1H,m),5.09(1H,m),4.56(1H,dd,J=10.0,6.5Hz),4.43-4.40(2H,m),4.30(2H,m),4.19-4.08(3H,m),3 .91(2H,m),3.77(1H,dd,J=11.0,3.5Hz),3.66(2H,m),3.54(2H,m),3.41(2H,m),2.58(2H,m),1.86(2H,m). 31P-NMR(DMSO-d6)δ:55.36,51.31.

[0467] Example 17: N-[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp, Rp-10)) 2 [ka] (Step 2') Bis(N,N-diethylethanaminium)(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-15-fluoro-2,10-dioxo-7-[6-oxo-1-(2-{[(N-{[2-(trimethylsilyl)ethoxy]carbonyl}glycyl)amino]methoxy}ethyl)-1,6-dihydro-9H-purin-9-yl]-14-(6,7,8,9)-tetrahydro-2H-2,3,5,6- A solution of tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyne-2,10-bis(thiolate) (compound from Example 8 (Rp, Rp-9)) (0.8 g, 0.60 mmol) in dimethyl sulfoxide (2.4 mL) was added with 70% aqueous tetrabutylammonium fluoride (2.4 mL) at room temperature and stirred for 4 days. Acetonitrile (9.6 mL), calcium carbonate (0.67 g, 6.65 mmol), and DOWEX 50Wx8 (2.24 g, 2.42 mmol) were added and stirred at room temperature for 1.5 hours. After degassing the reaction mixture under reduced pressure, triethylamine (0.8 mL) was added and the mixture was stirred for 1 hour. The insoluble matter was filtered off and washed with acetonitrile (8.0 mL). The solvent was removed from the resulting filtrate under reduced pressure to approximately 4 mL. Acetonitrile was added to adjust the volume to 8 mL, and 5 mol / L aqueous hydrochloric acid (280 μL) was added to the solution (8 mL). After adding acetonitrile (9.6 mL), the mixture was stirred for 1 hour. The solid was collected from the resulting suspension by filtration and washed with acetonitrile (4.0 mL). The solid was dried overnight at room temperature to obtain the target compound as white crystals (0.48 g, yield 91%).

[0468] Example 18: Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6,-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp, Rp-12)) [ka] (Step 3) To a suspension of the compound obtained in Reference Example 25 (132.6 mg, 0.23 mmol) in acetonitrile (1.8 mL) and water (0.44 mL), triethylamine (52.6 mg, 0.52 mmol) was added, followed by the compound obtained in Example 17 (222.0 mg, 0.26 mmol). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (93.5 mg, 0.34 mmol) was added at room temperature, and the mixture was stirred at room temperature for 18 hours. Acetonitrile (2.2 mL), cyclopentyl methyl ether (2.2 mL), and 13.8% aqueous sodium sulfate solution (3.3 mL) were added to the reaction mixture, and the mixture was stirred. The aqueous layer was discarded, and the solvent was evaporated under reduced pressure from the resulting organic layer. The residue was purified by silica gel column chromatography to obtain the target compound as a white solid (222 mg, 59% yield).

[0469] 1H-NMR(500MHz,CD3OD)δ:8.58(1H,s),8.09(1H,s),8.04(1H,s),7.57-7.49(2H,m),7.43-7.34(3H,m),7.32-7.08(9H ,m),6.47(1H,d,J=16.9Hz),6.23(1H,d,J=7.9Hz),5.56-5.37(2H,m),5.31-5.17(1H,m),5.03(1H,d,J=13.9Hz),4.79( 1H,d,J=4.2Hz),4.64-4.38(6H,m),4.36-4.21(4H,m),4.05-3.60(10H,m),3.53-3.42(3H,m),3.18(12H,q,J=7.3Hz), 3.01-2.92(1H,m),2.86-2.73(1H,m),2.70-2.54(2H,m),2.37-2.16(2H,m),2.06-1.77(3H,m),1.28(18H,t,J=7.3Hz).

[0470] Example 19: Dipotassium N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6,-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp, Rp-12)) [ka] (Step 3') To a suspension of the compound obtained in Reference Example 25 (177.3 mg, 0.31 mmol) in acetonitrile (3.9 mL) and water (0.78 mL), triethylamine (70.8 mg, 0.70 mmol) was added, followed by the compound obtained in Example 17 (300.0 mg, 0.35 mmol). 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (125.1 mg, 0.45 mmol) was added at room temperature, and the mixture was stirred at room temperature for 5.5 hours. Acetonitrile (3.9 mL), cyclopentyl methyl ether (2.0 mL), and 13.8% aqueous sodium sulfate solution (5.8 mL) were added to the reaction mixture. After stirring, the aqueous layer was discarded to obtain the organic layer. The organic layer was added dropwise to a solution of potassium 2-ethylhexanoate (380.4 mg, 2.09 mmol) in 2-propanol (19.5 mL) at room temperature, followed by stirring for 1 hour. Cyclopentyl methyl ether (7.8 mL) was added, and the mixture was stirred for approximately 15 hours. The solid was collected from the resulting suspension by filtration and washed with 2-propanol / acetonitrile / cyclopentyl methyl ether (5 / 2 / 2, 3 mL). The solid was dried under reduced pressure at room temperature for approximately 8 hours to obtain the target compound as a white solid (384.6 mg, 74% yield).

[0471] 1H-NMR(500MHz,DMSO-d6)δ:8.68(brs,1H),8.61(dd,J=13.2,6.3Hz,1H),8.35(dt,J=10.9,5.7H z,1H),8.25(brs,1H),8.17(dt,J=16.6,5.7Hz,1H),8.13-7.99(m,3H),7.70-7.62(m,1H),7.60- 7.55(m,2H),7.50-7.46(m,1H),7.46-7.41(m,2H),7.38-7.27(m,3H),7.26-7.20(m,4H),7.18-7 .13(m,1H),7.11(brs,1H),6.37(dd,J=14.6,3.7Hz,1H),6.03(d,J=8.0Hz,1H),5.48(dt,J=51.0 ,3.9Hz,1H),5.22-5.16(m,1H),5.12-5.04(m,1H),5.00(d,J=13.7Hz,1H),4.93(brs,1H),4.62- 4.57(m,1H),4.56-4.50(m,2H),4.49-4.43(m,1H),4.36(brs,1H),4.26-4.17(m,3H),4.06-3.96 (m,3H),3.82-3.51(m,11H),3.04(dd,J=13.7,4.6Hz,1H),2.79(dd,J=13.7,9.7Hz,1H),2.70-2. 57(m,3H),2.29(dt,J=15.5,7.7Hz,1H),2.12-2.02(m,1H),1.91-1.74(m,3H),1.59-1.52(m,1H).

[0472] Example 20: Disodium N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6,-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 Synthesis of -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide (corresponding to the compound of formula (Rp, Rp-12)) [ka] (Step 3'') To a suspension of the compound obtained in Reference Example 25 (18.1 mg, 0.03 mmol) in acetonitrile (0.5 mL), triethylamine (12.1 μL, 0.09 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10.4 mg, 0.04 mmol) were added at room temperature. The compound obtained in Example 17 (25.0 mg, 0.03 mmol) was added at room temperature and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and ethyl acetate (0.75 mL) and 5% aqueous sodium carbonate solution (0.25 mL) were added to the resulting residue. An oil component was separated from the two-layer solution, and acetonitrile (1 mL) was added to the oil component and stirred at room temperature for 6 hours. The supernatant was removed from the resulting suspension to obtain a solid. The solid was dried under reduced pressure for 5 hours to give the desired compound as a white solid (38.2 mg, 91% yield).

[0473] MS(ESI)m / z:1411(M+H) + ,1409(MH) - .

Claims

1. Formula (Rp, Rp-9): 【Chemistry 1】 [In the formula, A1 is, 【Chemistry 2】 and PG1 is a protecting group for a hydroxy group, and PG3 is a protecting group for the amino group. A method for producing a compound represented by the formula: (Step a1) Formula (1A): 【Transformation 3】 [In the formula, PG1 and PG3 are as defined above, and PG2 is a trityl-type protecting group for a hydroxy group.] with an optically active phosphitylating agent (Rc-2) selected from the group consisting of the following formulae (Rc-2-1) and (Rc-2-2): 【Chemistry 4】 [In the formula, R1 is hydrogen or methyl; R2 is hydrogen, alkyl having 1 to 3 carbon atoms, or phenyl; wherein the alkyl is unsubstituted or substituted with one or more phenyl, tosyl, or diphenylmethylsilyl; and The phenyl is unsubstituted or substituted with nitro or methoxy. to form a compound of formula (Rc-3A): 【Transformation 5】 [In the formula, PG1, PG2 and PG3 are as defined above, and B1 is, 【Transformation 6】 where R1 and R2 are as defined above. obtaining a compound represented by the formula: (Step a2) The obtained compound of formula (Rc-3A) is reacted with a compound of formula (4A): 【Transformation 7】 [In the formula, A2 is, 【Transformation 8】 PG6 is a protecting group for the amino group, and PG4 is a protecting group for a hydroxy group. in the presence of an activating agent, followed by treatment with an acylating agent or an alkoxycarbonylating agent, and then further reaction with a thiolating agent to obtain a compound represented by formula (Rc-5A): 【Chemistry 9】 [In the formula, A2, PG1, PG2, PG3 and PG4 are as defined above; B2 is, 【Chemistry 10】 wherein R1 and R2 are as defined above, and PG7 is a protecting group for the amino group. obtaining a compound represented by the formula: (Step a3-1) PG2 of the obtained compound of formula (Rc-5A) is deprotected to give a compound of formula (Rc-6A- ０１ ): 【Chemistry 11】 [In the formula, A2, B2, PG1, PG3 and PG4 are as defined above.] obtaining a compound represented by the formula: (Step a3-2) The obtained compound of formula (Rc-6A- ０１ ) is reacted with an acylating agent or an alkoxycarbonylating agent to give a compound of formula (Rc-6A): 【Chemistry 12】 [In the formula, A2, B2, PG1, PG3, and PG4 are as defined above, and PG5 is an ester-type or carbonate-type protecting group for a hydroxy group.] obtaining a compound represented by the formula: (Step a4) PG4 of the obtained compound of formula (Rc-6A) is deprotected to obtain a compound of formula (Rc-7A): 【Chemistry 13】 [In the formula, A2, B2, PG1, PG3 and PG5 are as defined above.] obtaining a compound represented by the formula: (Step a5) The obtained compound of formula (Rc-7A) is treated with an optically active phosphitylating agent (Rc-2) selected from the group consisting of the following formulae (Rc-2-1) and (Rc-2-2), which may be the same as or different from the optically active phosphitylating agent (Rc-2) of Step a1: 【Chemistry 14】 wherein R1 and R2 are as defined above. PG5 is deprotected by reaction with an optically active phosphitylation agent (Rc-2), then cyclized in the presence of an activating agent, further treated with an acylating agent or an alkoxycarbonylating agent, and subsequently reacted with a thiolation agent to give a compound of the formula (Rc, Rc-8): 【Chemistry 15】 [In the formula, A2, B2, PG1 and PG3 are as defined above; B2' is 【Chemistry 16】 wherein R1, R2, and PG7 are as defined above. and obtaining a compound represented by the formula: (Step a6) deprotecting B2 and B2′, which are protecting groups at the thiophosphate moiety of the obtained compound of formula (Rc, Rc-8), and PG6, which is a protecting group in A2, to obtain a compound of formula (Rp, Rp-9) or a salt thereof; A method comprising:

2. 2. The method of claim 1, wherein the activators in steps a2 and a5 are independently at least one selected from the group consisting of 1-phenylimidazolium triflate, 1-methylbenzimidazolium triflate, 1-(cyanomethyl)piperidinium triflate, 1-(cyanomethyl)pyrrolidinium triflate, and 1-(cyanomethyl)imidazolium triflate.

3. The acylating agent or alkoxycarbonylating agent in step a2 and step a5 is independently selected from 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-fluorenylmethyl pentafluorophenyl carbonate, 1-[(9 3. The method of claim 1, wherein the hydroxybenzotriazole is at least one selected from the group consisting of N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]benzotriazole, N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide, N-(2,2,2-trichloroethoxycarbonyloxy)succinimide, N-carbobenzyloxysuccinimide, dibenzyl dicarbonate, 2-(trimethylsilyl)ethyl-3-nitro-1H-1,2,4-triazole-1-carboxylate, N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, N-ethoxycarbonylphthalimide, and methylimidazole-1-carboxylate.

4. The method according to any one of claims 1 to 3, wherein the thionating agents in steps a2 and a5 are independently at least one selected from the group consisting of xanthan hydride, bis(phenylacetyl)disulfide, 3H-1,2-benzodithiol-3-one-1,1-dioxide, 5-phenyl-3H-1,2,4-dithiazol-3-one, and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione.

5. PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl; and The method of any one of claims 1 to 4, wherein PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, or allyloxycarbonyl.

6. PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG6 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl, and 6. The method of any one of claims 1 to 5, wherein PG7 is acetyl, trifluoroacetyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, methoxycarbonyl, or ethoxycarbonyl.

7. The method according to any one of claims 1 to 6, wherein PG4 is levulinoyl.

8. The method of any one of claims 1 to 7, wherein PG5 is allyloxycarbonyl.

9. PG1 is tert-butyldimethylsilyl, PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, or trityl; PG4 is levulinoyl, and The method of any one of claims 1 to 8, wherein PG5 is allyloxycarbonyl.

10. moreover (Step a7) The protecting groups PG1 and PG3 of the obtained compound of formula (Rp, Rp-9) are deprotected to give a compound of formula (Rp, Rp-10): 【Chemistry 17】 [In the formula, A1 is as defined in claim 1.] or a salt thereof; and (Step a8) The obtained compound of formula (Rp, Rp-10) or a salt thereof is reacted with Formula (11): [Chemistry 18] or an activated ester thereof to form a compound represented by the formula (Rp, Rp-12): 【Chemistry 19】 wherein A1 is as defined above. or a salt thereof The method according to any one of claims 1 to 9, comprising:

11. moreover, (Step a9) The obtained compound of formula (Rp, Rp-12) or a salt thereof is bound to an antibody or a functional fragment of the antibody (hereinafter referred to as Ab) to obtain a compound of formula (Rp, Rp-13): 【Chemistry 20】 [In the formula, m ranges from 1 to 10; The sugar chains of the Ab are optionally remodeled, Ab binds to the compound of formula (Rp, Rp-12) directly from the side chain of an optionally modified amino acid residue, or binds to the compound of formula (Rp, Rp-12) from a sugar chain or a remodeled sugar chain of the Ab; A1 is, 【Chemistry 21】 It is.] or a mixture thereof. The method according to any one of claims 1 to 10, comprising:

12. The method according to any one of claims 1 to 11, wherein in step a9, the compound of formula (Rp, Rp-12) or a salt thereof and Ab are bonded together by a strain-promoted azide-alkyne cycloaddition reaction.

13. 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-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 13. The method of any one of claims 1 to 12, wherein the antibody is selected from the group consisting of an anti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, an anti-SLC44A4 antibody, an anti-mesothelin antibody, an anti-ENPP3 antibody, an anti-CD47 antibody, an anti-EGFR antibody, an anti-GPR20 antibody, and an anti-DR5 antibody.

14. The compound of formula (4A) (wherein PG4 is levulinoyl) can be obtained by the following steps: (Step a0-1) Formula (XXV'): 【Chemistry 22】 or a salt thereof, by protecting the 5'-hydroxy group of the compound of the formula (4A- ０１ ): 【Chemistry 23】 [In the formula, PG8 is a trityl-type protecting group for a hydroxy group.] or a salt thereof, and (Step a0-2) The obtained compound of formula (4A- ０１ a step of protecting the hydroxy group at the 2'-position of the compound of formula (4A) or a salt thereof with levulinic acid, reacting the compound with an acylating agent or an alkoxycarbonylating agent, and then detritylation to obtain the compound of formula (4A). The method according to any one of claims 1 to 13, wherein the compound is produced by

15. Formula (4A'): 【Chemistry 24】 [In the formula, PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl; and PG6 is benzyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, or ethoxycarbonyl. A compound represented by the formula:

16. Formula (Rc-5A): 【Chemistry 25】 [In the formula, A2 and B2 are as defined in claim 1; PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG2 is 4,4'-dimethoxytrityl, 4-methoxytrityl, 2-chlorotrityl, or trityl; and PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl, and PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl. A compound represented by the formula:

17. Formula (Rc-6A): 【Chemistry 26】 [In the formula, A2 and B2 are as defined in claim 1; PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl; PG4 is formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, azidobutyryl, allyloxycarbonyl, chloroazidobenzyl, methoxybenzyl, or fluorenylmethyloxycarbonyl; and PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, or allyloxycarbonyl. A compound represented by the formula:

18. Formula (Rc-7A): 【Chemistry 27】 [In the formula, A2 and B2 are as defined in claim 1; PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl, and PG5 is acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, levulinoyl, fluorenylmethyloxycarbonyl, or allyloxycarbonyl. A compound represented by the formula:

19. Formula (Rc, Rc-8): 【Chemistry 28】 [In the formula, A2, B2 and B2' are as defined in claim 1; PG1 is tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, or tert-butyldiphenylsilyl; and PG3 is 2-(trimethylsilyl)ethoxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or benzyloxycarbonyl. Compound.

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