Method for producing nucleoside derivatives

Abstract

To provide a practical method for producing a nucleoside derivative that is applied to RNA pharmaceuticals or the like.SOLUTION: The present invention provides a method for producing a nucleotide derivative represented by a compound 10 of the following formula or the like, wherein a compound 9 of the following formula, obtained through a number of steps from 1,2: 5,6-di-O-isopropylidene-α-D-glucofuranose, is treated with uracil, and bis(trimethylsilyl)acetamide, to obtain a nucleotide compound 10.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present specification relates to a method for producing a nucleoside derivative. [Background technology]

[0002] Many diseases, including cancer, are known to be caused by or related to gene mutations or abnormal gene expression. RNA medicines such as siRNA, which suppress gene expression, are useful for treating these diseases and have great pharmaceutical potential.

[0003] On the other hand, siRNA and the like have problems such as difficulty in permeating cell membranes and susceptibility to degradation by nucleases. Furthermore, although they have high target selectivity, they have the problem of difficulty in selectively transporting them to target tissues. To overcome these problems, delivery carriers such as lipid nanoparticles (LNPs) are being investigated. Attempts have also been made to modify RNA ribose, such as by introducing an aminomethyl group (Non-Patent Documents 1 to 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] HELVATICA CHIMICA ACTA Vol. 83 (2000) 128-151 [Non-patent document 2] The Journal of Organic Chemistry 2012, 77, 3233-3245 [Non-patent document 3] Bioorganic & Chemistry letters(1999) 2667-2672 [Non-patent document 4] The Journal of Organic Chemistry 2013, 78, 9956-9962 Summary of the Invention [Problem to be solved by the invention]

[0005] However, despite these attempts, further improvements in the efficacy of RNA medicines are required. Delivery carriers also have shortcomings, and even these RNA modifications have not been able to achieve sufficient cell membrane permeability, ribonuclease resistance, and target tissue delivery. For these reasons, even at present, siRNAs and the like have not been able to demonstrate their inherent excellent pharmaceutical potential. Furthermore, even if practical nucleosides and the like exist for use in such RNA medicines and the like, it is desirable to efficiently provide such nucleosides and the like.

[0006] The present specification aims to efficiently provide nucleoside derivatives that are more practical for application to RNA medicines and the like. [Means for solving the problem]

[0007] The present inventors focused on ribose, the sugar moiety of ribonucleotides, and discovered that providing a basic substituent such as an amino group at the 4'-position of ribose or substituting a halogen atom for the 2'-hydroxyl group can improve ribonuclease resistance and cell membrane permeability. Furthermore, the present inventors discovered a method for efficiently producing 4'-aminoalkyl nucleoside derivatives and 2'-halogenyl nucleoside derivatives. Based on these findings, the present specification provides the following means.

[0008] (1) A method for producing a nucleoside derivative or a salt thereof represented by the following formula (1) or (2): A production method comprising any reaction selected from the group consisting of the following formulas (5), (6), and (7): [ka] (In formula (1), R 1 represents a hydrogen atom, a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected group, and in formula (2), X represents a halogen atom. 2 and R4 may be the same or different and are each a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P(=O) n R 5 R 6 (n represents 0 or 1, R 5 and R 6 may be the same or different and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group, provided that when n is 1, R 5 and R 6 and cannot both be hydrogen atoms.) and R 3 are NHR groups each having a linking group. 7 (R 7 represents a protecting group for a hydrogen atom, an alkyl group, an alkenyl group, or an amino group; azido group, amidino group, or guanidino group; and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group. [ka] (In formulas (5) to (7), R 10 and R 11 are each independently or R 10 and R 11 together represent a protecting group for a hydroxyl group, and R 12 represents Equation (8), and R 14 represents an alkylene group, Bn represents a benzyl group, and Tf represents a trifluoromethanesulfonyl group. (2) In formulas (5) to (7), R 10 and R 11 is R 10 and R 11 (1) The method for producing a cyclic acetal group according to (1), (3) The method according to (1) or (2), comprising the reaction of the following formula (5)' subsequent to the reaction of the formula (5): [ka] (4) The production method according to (3), which comprises the reaction of the following formula (5)'' subsequent to the reaction of the formula (5)': [ka] (5) The production method according to any one of (1) to (4), which comprises the following reactions of formulas (7)' and (7)'' prior to the reaction of formula (7): [ka] (6) A method for producing an oligonucleotide derivative, comprising the steps of: A method for producing an oligonucleotide derivative using a nucleoside derivative or a salt thereof represented by the following formula (1) or (2), A production method comprising any reaction selected from the group consisting of the following formulas (5), (6), and (7): [ka] (In formula (1), R 1 represents a hydrogen atom, a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected group, and in formula (2), X represents a halogen atom. 2 and R 4 may be the same or different and are each a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P(=O) n R 5 R 6 (n represents 0 or 1, R 5 and R 6 may be the same or different and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group, provided that when n is 1, R 5 and R 6 and cannot both be hydrogen atoms.) and R 3 are NHR groups each having a linking group. 7 (R 7represents a protecting group for a hydrogen atom, an alkyl group, an alkenyl group, or an amino group; azido group, amidino group, or guanidino group; and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group. [ka] (In formulas (5) to (7), R 10 and R 11 are each independently or R 10 and R 11 together represent a protecting group for a hydroxyl group, and R 12 represents Equation (8), and R 14 represents an alkylene group, Bn represents a benzyl group, and Tf represents a trifluoromethanesulfonyl group. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure relates to a method for producing a practical nucleoside derivative or a salt thereof suitable for use in RNA medicines. This production method enables efficient production of a nucleoside derivative or a salt thereof (hereinafter simply referred to as the present nucleoside derivative) that has the following advantages:

[0010] (1) They are ribonuclease-resistant and have excellent cell membrane permeability, making them suitable for administration without the use of carriers such as LNPs, which have been used for delivery of conventional RNA medicines. (2) The nucleoside derivatives are also useful as reagents for RNA-based detection probes, etc. That is, they can provide oligonucleotides suitable for various RNA reagents. (3) The nucleoside derivatives disclosed herein are based on the discovery of unexpectedly useful features after introducing various aminoalkyl-based substituents into the 4' position of ribose, which had previously been difficult to do, and examining their properties. Ribonuclease resistance has generally been achieved by substitution at the 2' or 3' position of ribose. The nucleoside derivatives disclosed herein combine unexpectedly high ribonuclease resistance with cell membrane permeability, properties useful for RNA medicines and the like. Various embodiments of the disclosures herein are described in detail below.

[0011] (This nucleoside derivative) The present nucleoside derivative may be a nucleoside derivative represented by the following formula (1) or (2) or a salt thereof. The present nucleoside derivative may be incorporated into a partial structure of an oligonucleotide by a method well known to those skilled in the art.

[0012] [ka]

[0013] The present nucleoside derivative has a basic substituent at the 4' position of the ribose and deoxyribose, and thus can have charge control ability, i.e., can neutralize at least a portion of the negative charge caused by phosphate groups, etc., in an oligonucleotide having a partial structure derived from the present nucleoside derivative.

[0014] Furthermore, the cell membrane permeability of an oligonucleotide having this partial structure can be improved.

[0015] Furthermore, in an oligonucleotide having a partial structure derived from the present nucleoside derivative, the ribonuclease resistance can be improved.

[0016] In this specification, the term "lower" in a substituent in a compound represented by a formula or the like means that the number of carbon atoms constituting the substituent is up to 10. For example, typically, the number of carbon atoms is 1 to 6 or 1 to 5, and more preferably, the number of carbon atoms is 1 to 4 or 1 to 3.

[0017] The nucleoside derivatives or salts thereof disclosed in the present specification and their uses will be described below.

[0018] (Nucleoside derivatives and their salts) One embodiment of the present nucleoside derivative or a salt thereof is a nucleoside derivative or a salt thereof represented by the following formula (1):

[0019] [ka]

[0020] Another embodiment of the present nucleoside derivative or a salt thereof is a nucleoside derivative or a salt thereof represented by the following formula (2):

[0021] [ka]

[0022] [R 1 About In formula (1), R 1 represents a hydrogen atom, a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected hydroxyl group. 1 When R is a hydrogen atom, the nucleoside derivative is a deoxyribonucleoside derivative. 1 is a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted with an alkyl or alkenyl group, or a protected hydroxyl group, the nucleoside derivative is a ribonucleoside derivative.

[0023] [About X] In formula (2), X represents a halogen atom. Examples of the halogen atom include, but are not limited to, a chlorine atom, an iodine atom, a fluorine atom, and a bromine atom. R 1 is a halogen atom, the nucleoside derivative is a deoxyribonucleoside derivative. As is clear from formula (2), the bonding direction of the halogen atom to the 2'-carbon atom of ribose is not particularly limited, but it is preferable that the halogen atom be bonded in a manner corresponding to the hydroxyl group of natural ribose.

[0024] (Alkyl group) In this specification, the alkyl group includes saturated hydrocarbon groups that are linear, branched, cyclic, or a combination thereof. Generally, lower alkyl groups are preferred, with lower alkyl groups having 1 to 6 carbon atoms or lower alkyl groups having 1 to 5 carbon atoms being more preferred, and lower alkyl groups having 1 to 4 carbon atoms or lower alkyl groups having 1 to 3 carbon atoms being particularly preferred. Suitable examples of linear alkyl groups having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, and n-butyl groups. Of these, methyl, ethyl, and n-propyl groups are preferred, with methyl and ethyl being preferred, and methyl being preferred. Examples of branched alkyl groups having 1 to 4 carbon atoms include isopropyl, isobutyl, s-butyl, and t-butyl groups, with isopropyl being particularly preferred. Examples of cyclic alkyl groups having 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, and cyclopropylmethyl groups.

[0025] (alkenyl group) In this specification, the alkenyl group includes a saturated hydrocarbon group which is linear, branched, cyclic, or a combination thereof. Usually, a lower alkenyl group is preferred, and examples of the lower alkenyl group include an ethenyl group, a 1-propenyl group, a 2-propenyl group, a 1-methyl-2-propenyl group, a 1-methyl-1-propenyl group, a 2-methyl-1-propenyl group, a 1-butenyl group, and a 2-butenyl group.

[0026] (Hydroxyl protecting group or protected hydroxyl group) In this specification, protecting groups for hydroxyl groups are well known to those skilled in the art, and reference can be made to, for example, Protective Groups in Organic Synthesis (John Wiley and Sons, 2007 edition). Representative examples of protecting groups for hydroxyl groups include aliphatic acyl groups, aromatic acyl groups, lower alkoxymethyl groups, oxycarbonyl groups which may have an appropriate substituent, tetrahydropyranyl groups which may have an appropriate substituent, tetrathiopyranyl groups which may have an appropriate substituent, methyl groups substituted with a total of 1 to 3 substituted or unsubstituted aryl groups (wherein the substituents in the substituted aryl groups mentioned above refer to lower alkyl, lower alkoxy, halogen atoms, or cyano groups), and silyl groups.

[0027] In this specification, the alkoxy group includes saturated alkyl ether groups that are linear, branched, cyclic, or a combination thereof. Lower alkoxy groups are preferred, and examples of lower alkoxy groups include lower alkoxy groups having 1 to 6 carbon atoms or lower alkoxy groups having 1 to 5 carbon atoms. Alkoxy groups having 1 to 4 carbon atoms or 1 to 3 carbon atoms are preferred, with alkoxy groups having 1 to 4 carbon atoms being particularly preferred. Preferred examples of alkoxy groups having 1 to 4 carbon atoms include methoxy, ethoxy, n-propoxy, and n-butoxy. Preferred examples also include isopropoxy, isobutoxy, s-butoxy, and t-butoxy. Cyclopropoxy and cyclobutoxy groups are also preferred, with cyclopropylmethoxy being a preferred example.

[0028] In this specification, the alkylthio group includes saturated alkylthio groups that are linear, branched, cyclic, or a combination thereof. Lower alkylthio groups are preferred, and examples of lower alkylthio groups include lower alkylthio groups having 1 to 6 carbon atoms or lower alkylthio groups having 1 to 5 carbon atoms, with lower alkylthio groups having 1 to 4 carbon atoms or alkylthio groups having 1 to 3 carbon atoms being particularly preferred. Preferred examples of saturated alkylthio groups having 1 to 4 carbon atoms include methylthio, ethiothio, n-propylthio, and n-butylthio. Preferred examples also include isopropylthio, isobutylthio, s-butylthio, and t-butylthio. Preferred examples include cyclopropylthio and cyclobutylthio, with cyclopropylmethylthio being even more preferred.

[0029] Among these, particularly preferred examples include an aliphatic acyl group, an aromatic acyl group, and a silyl group. Also preferred examples include a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups (however, the substituents on the substituted aryl groups are as described above).

[0030] Examples of the aliphatic acyl group include an alkylcarbonyl group, a carboxyalkylcarbonyl group, a halogeno lower alkylcarbonyl group, and a lower alkoxy lower alkylcarbonyl group.

[0031] The alkyl in the alkylcarbonyl group is as explained above. That is, examples of the alkylcarbonyl group include a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a pivaloyl group, a valeryl group, an isovaleryl group, an octanoyl group, a nonanoyl group, a decanoyl group, a 3-methylnonanoyl group, an 8-methylnonanoyl group, a 3-ethyloctanoyl group, a 3,7-dimethyloctanoyl group, an undecanoyl group, a dodecanoyl group, a tridecanoyl group, a tetradecanoyl group, a pentadecanoyl group, a hexadecanoyl group, a 1-methylpentadecanoyl group, a 14-methylpentadecanoyl group, a 13,13-dimethyltetradecanoyl group, a heptadecanoyl group, a 15-methylhexadecanoyl group, an octadecanoyl group, a 1-methylheptadecanoyl group, a nonadecanoyl group, an eicosanoyl group, and a heneycosyl group. Among these, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, and pivaloyl groups are preferred, with acetyl being particularly preferred. The alkyl in the carboxylated alkylcarbonyl group is as described above. The carboxyl substitution position can also be appropriately selected. Examples of the carboxylated alkylcarbonyl group include succinoyl, glutaroyl, and adipoyl groups.

[0032] The halogen, lower, and alkyl in the halogeno-lower alkylcarbonyl group are as described above. The position of the halogen can also be selected appropriately. That is, examples of the halogeno-lower alkylcarbonyl group include a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, and a trifluoroacetyl group.

[0033] The alkoxy, alkyl, and lower groups in the lower alkoxy-lower alkylcarbonyl group are as described above. The position at which the lower alkoxy is substituted can also be selected appropriately. That is, an example of a lower alkoxy-lower alkylcarbonyl group is a methoxyacetyl group.

[0034] Examples of the aromatic acyl group include an arylcarbonyl group, a halogenoarylcarbonyl group, a lower alkylated arylcarbonyl group, a lower alkoxylated arylcarbonyl group, a carboxylated arylcarbonyl group, a nitrated arylcarbonyl group, and an arylated arylcarbonyl group.

[0035] Examples of the arylcarbonyl group include a benzoyl group, an α-naphthoyl group, and a β-naphthoyl group, and more preferably a benzoyl group. Examples of the halogenoarylcarbonyl group include a 2-bromobenzoyl group and a 4-chlorobenzoyl group. Examples of the lower alkylated arylcarbonyl group include a 2,4,6-trimethylbenzoyl group, a 4-toluoyl group, a 3-toluoyl group, and a 2-toluoyl group. Examples of the lower alkoxylated arylcarbonyl group include a 4-anisoyl group, a 3-anisoyl group, and a 2-anisoyl group.

[0036] Examples of the carboxylated arylcarbonyl group include a 2-carboxybenzoyl group, a 3-carboxybenzoyl group, and a 4-carboxybenzoyl group. Examples of the nitrated arylcarbonyl group include a 4-nitrobenzoyl group, a 3-nitrobenzoyl group, and a 2-nitrobenzoyl group. Examples of the arylated arylcarbonyl group include a 4-phenylbenzoyl group.

[0037] Examples of lower alkoxymethyl groups include methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, and t-butoxymethyl groups, with methoxymethyl being particularly preferred.

[0038] The optionally substituted oxycarbonyl group includes a lower alkoxycarbonyl group, a lower alkoxycarbonyl group substituted with a halogen or a silyl group, and an alkenyloxycarbonyl group.

[0039] Examples of the lower alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, and a t-butoxycarbonylisobutoxycarbonyl group. Examples of the lower alkoxycarbonyl group substituted with a halogen or a silyl group include a 2,2,2-trichloroethoxycarbonyl group and a 2-(trimethylsilyl)ethoxycarbonyl group.

[0040] The alkenyloxycarbonyl group may be a vinyloxycarbonyl group. Preferred examples of the optionally substituted tetrahydropyranyl group include a tetrahydropyran-2-yl group or a 3-bromotetrahydropyran-2-yl group, and a particularly preferred example is a tetrahydropyran-2-yl group.

[0041] Examples of the tetrathiopyranyl group which may have an appropriate substituent include a tetrahydrothiopyran-2-yl group and a 4-methoxytetrahydrothiopyran-4-yl group, and more preferably a tetrahydrothiopyran-2-yl group. In the case of a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups, the substituents in the substituted aryl groups are lower alkyl, lower alkoxy, halogen, or cyano.

[0042] Examples of methyl groups substituted with a total of 1 to 3 substituted or unsubstituted aryl groups include benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, and α-naphthyldiphenylmethyl groups, preferably benzyl and triphenylmethyl groups. Other examples include 9-anthrylmethyl 4-methylbenzyl, 2,4,6-trimethylbenzyl, and 3,4,5-trimethylbenzyl groups, preferably 2,4,6-trimethylbenzyl and 3,4,5-trimethylbenzyl groups. Other examples include 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, and 4,4'-dimethoxytriphenylmethyl groups, preferably 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, and 4,4'-dimethoxytriphenylmethyl groups. Further examples include 4-chlorobenzyl and 4-bromobenzyl groups. Other preferred examples include a 4-cyanobenzyl group.

[0043] In this specification, examples of the silyl group include a trimethylsilyl group, a triethylsilyl group, an isopropyldimethylsilyl group, a t-butyldimethylsilyl group, a methyldiisopropylsilyl group, a methyldi-t-butylsilyl group, a triisopropylsilyl group, a diphenylmethylsilyl group, a diphenylbutylsilyl group, and a diphenylisopropylsilylphenyldiisopropylsilyl group. Among these, more preferred are a trimethylsilyl group, a t-butyldimethylsilyl group, a triisopropylsilyl group, and a diphenylmethylsilyl group, and particularly preferred are a trimethylsilyl group, a t-butyldimethylsilyl group, and a diphenylmethylsilyl group.

[0044] In this specification, the term "protecting group for a hydroxyl group" may refer to a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, or photolysis) or a biological method (e.g., hydrolysis in the human body, e.g., induction in a microorganism, etc.). Particularly preferred examples of the protecting group for a hydroxyl group include substituents that are eliminated by hydrogenolysis or hydrolysis. A protected hydroxyl group can be said to be a hydroxyl group in which the hydrogen atom has been substituted with such a protecting group.

[0045] [R 2 and R 4 About In formula (1) and formula (2), R 2 and R 4 may be the same or different and are each a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P(=O) n (R 5 )R 6 The protecting group for the hydroxyl group is as explained above.

[0046] (protected phosphate group) Protecting groups for the protected phosphate group are known to those skilled in the art and can be found in the above references and explanations.

[0047] Examples of protecting groups for phosphate groups include lower alkyl groups, lower alkyl groups substituted with a cyano group, ethyl groups substituted with a silyl group, lower alkyl groups substituted with a halogen, lower alkenyl groups, lower alkenyl groups substituted with a cyano group, cycloalkyl groups, lower alkenyl groups substituted with a cyano group, aralkyl groups, aralkyl groups in which the aryl ring is substituted with a nitro group, aralkyl groups in which the aryl ring is substituted with a halogen, aryl groups substituted with a lower alkyl group, aryl groups substituted with a halogen, and aryl groups substituted with a nitro group.

[0048] The lower alkyl group is as described above. Examples of the lower alkyl group substituted with a cyano group include a 2-cyanoethyl group and a 2-cyano-1,1-dimethylethyl group, and a 2-cyanoethyl group is particularly preferred. Examples of the ethyl group substituted with a silyl group include a 2-methyldiphenylsilylethyl group, a 2-trimethylsilylethyl group, and a 2-triphenylsilylethyl group.

[0049] Examples of the halogen-substituted lower alkyl group include 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, and 2,2,2-trichloroethyl groups, and 2,2,2-trichloroethyl is particularly preferred. Examples of the lower alkenyl group include ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl groups.

[0050] Examples of the cyano-substituted lower alkenyl group include a 2-cyanoethyl group, a 2-cyanopropyl group, and a 2-cyanobutenyl group. Examples of the aralkyl group include a benzyl group, an α-naphthylmethyl group, a β-naphthylmethyl group, an indenylmethyl group, a phenanthrenylmethyl group, an anthracenylmethyl group, a diphenylmethyl group, a triphenylmethyl group, a 1-phenethyl group, a 2-phenethyl group, a 1-naphthylethyl group, a 2-naphthylethyl group, a 1-phenylpropyl group, a 2-phenylpropyl group, a 3-phenylpropyl group, a 1-naphthylpropyl group, a 2-naphthylpropyl group, a 3-naphthylpropyl group, a 1-phenylbutyl group, a 2-phenylbutyl group, a 3-phenylbutyl group, and a 4-phenylbutyl group. More preferred are a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 1-phenethyl group, and a 2-phenethyl group. Particularly preferred is a benzyl group.

[0051] Examples of the aralkyl group in which the aryl ring is substituted with a nitro group include a 2-(4-nitrophenyl)ethyl group, a 0-nitrobenzyl group, a 4-nitrobenzyl group, a 2,4-dinitrobenzyl group, and a 4-chloro-2-nitrobenzyl group.

[0052] As used herein, the term "protecting group for phosphate" may refer to a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, e.g., induction in a microorganism, etc.). Preferred examples of the protecting group for phosphate include substituents that are eliminated by hydrogenolysis or hydrolysis.

[0053] (-P(=O) n (R 5 )R 6 ) R of the nucleoside analogs of the present invention 2 and R 4 is -P(=O) n (R 5 )R 6 n represents 0 or 1, and R 5 and R 6 may be the same or different and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group, provided that when n is 1, R 5 and R 6 and cannot both be hydrogen atoms. The protected hydroxyl group and lower alkoxy group are as explained above.

[0054] (protected mercapto group) Protected mercapto groups are well known to those skilled in the art. Examples of protected mercapto groups include those exemplified above as protecting groups for hydroxyl groups, as well as alkylthio groups, arylthio groups, aliphatic acyl groups, and aromatic acyl groups. Preferred examples include aliphatic acyl groups and aromatic acyl groups, with aromatic acyl groups being particularly preferred. Preferred alkylthio groups include lower alkylthio groups, such as methylthio, ethylthio, and t-butylthio groups. Preferred arylthio groups include benzylthio groups. Examples of aromatic acyl groups include benzoyl groups.

[0055] Preferred examples of the cyano lower alkoxy group include alkoxy groups having 1 to 5 carbon atoms (when counting the number of carbon atoms in the cyano group) that are linear, branched, or cyclic and are a combination thereof and substituted with a cyano group. Specific examples include cyanomethoxy, 2-cyanoethoxy, 3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy, and 1-cyanomethyl-1,1-dimethylmethoxy, and a particularly preferred example is 2-cyanoethoxy.

[0056] R 5 and R 6 A substituted amino group can be selected as the R. The substituent of the amino group is either a lower alkoxy group, a lower alkylthio group, a cyano lower alkoxy group, or a lower alkyl group. 5 and R 6 and (iii) are both substituted amino groups, the substituted amino groups may be different from each other. The lower alkoxy group, lower alkylthio group, cyano lower alkoxy group, and lower alkyl group are as described above.

[0057] -P(=O) n (R 5 )R 6More specifically, preferred examples of the alkyl group include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group, and a phosphoramidite group is particularly preferred.

[0058] -P(=O) n (R 5 )R 6 In the formula, n is 0 and R 5 and R 6 When at least one of R is a substituted amino group and the other is not important, it becomes a phosphoramidite group. 5 and R 6 A phosphoramidite group in which one of R is a substituted amino group and the other is a lower alkoxy group or a cyano lower alkoxy group is particularly preferred because it has good reaction efficiency in the condensation reaction. Preferred examples of the substituted amino group include a diethylamino group, a diisopropylamino group, and a dimethylamino group, and a particularly preferred example is a diisopropylamino group. 5 and R 6 A preferred example of the lower alkoxy group in the other substituent is a methoxy group. A preferred example of the cyano lower alkoxy group is a 2-cyanoethyl group. Specific preferred examples of the phosphoramidite group include -P(OC2H4CN)(N(CH(CH3)2) or -P(OCH3)(N(CH(CH3)2).

[0059] -P(=O) n (R 5 )R 6 In this case, n is 1 and R 5 and R 6 When at least one of the groups is a hydrogen atom and the other is any group other than a hydrogen atom, the resulting group is an H-phosphonate group. Examples of the substituent other than hydrogen include a hydroxyl group, a methyl group, a methoxy group, a thiol group, etc., and a particularly preferred example is a hydroxyl group.

[0060] Also, -P(=O) n(R 5 )R 6 In this case, n is 1 and R 5 and R 6 When both R are lower alkoxy groups, the group becomes a phosphonyl group. 5 and R 6 The lower alkoxy groups in may be the same or different. Preferred examples of the lower alkoxy group include a methoxy group and an ethoxy group. Specific examples of the phosphonyl group include -P(=O)(OCH3)2.

[0061] R in the nucleoside derivative 2 For example, -P(=O) n (R 5 )R 6 It is particularly preferred that -P(=O) n (R 5 )R 6 Preferred examples of R include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group. 2 In addition, R is preferably a phosphate group or a protected phosphate group. 2 is also preferably a hydrogen atom or a protecting group for a hydroxyl group.

[0062] R 2 Preferred specific examples of the alkyl group include a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a trimethylsilyl group, a tert-butyldiphenylsilyl group, -P(OCHCN)(N(CH(CH)), -P(OCH)(N(CH(CH)), and a phosphonyl group.

[0063] R in the nucleoside derivative 4 is preferably, for example, a protecting group for a hydrogen atom or a hydroxyl group, or, for example, a phosphate group, a protected phosphate group, or -P(=O) n (R 5 )R 6 It is also preferable that R 4Specific examples of preferred groups include a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a dimethoxytrityl group, a monomethoxytrityl group, a tert-butyldiphenylsilyl group, and a trimethylsilyl group.

[0064] [R 3 About In formula (1) and formula (2), R 3 are NHR groups each having a linking group. 7 , azido group, amidino group, or guanidino group. 7 The azido group, amidino group, or guanidino group is each bonded to the 4'-carbon atom via a linking group.

[0065] The linking group can be, for example, a divalent hydrocarbon group having one or more carbon atoms. That is, examples of the divalent hydrocarbon group include alkylene groups having 1 to 8 carbon atoms and alkenylene groups having 2 to 8 carbon atoms.

[0066] The alkylene group as a linking group may be linear or branched, but is preferably linear. For example, a lower alkyl group is preferred, such as a lower alkyl group having 1 to 6 carbon atoms, or a lower alkyl group having 2 to 6 carbon atoms, and further, for example, a lower alkyl group having 2 to 4 carbon atoms or 2 to 3 carbon atoms is preferred. Examples of linear alkyl groups having 1 to 4 carbon atoms include methylene, ethylene, propane-1,3-diyl, n-butane-1,1-diyl, n-pentyl-1,5-diyl, and n-hexyl-1,6-diyl. Other examples include butane-1,2-diyl. Particularly preferred examples include ethylene, propane-1,3-diyl, and n-butane-1,1-diyl.

[0067] The alkenylene group as a linking group may be linear or branched, preferably linear. For example, a lower alkenylene group is preferred, and examples of the lower alkenylene group include an ethene-1,2-diyl group, a propene-1,3-diyl group, and a butene-1,4-diyl group.

[0068] In the nucleoside derivative represented by formula (1), a divalent hydrocarbon group such as an alkylene group having two or more carbon atoms, such as an ethylene group, is preferred from the viewpoint of nuclease resistance and cell membrane permeability of the oligonucleotide derivative. In addition, in the nucleoside derivative represented by formula (2), a divalent hydrocarbon group such as an alkylene group having one or more carbon atoms, such as an ethylene group, is also preferred from the viewpoint of nuclease resistance and cell membrane permeability.

[0069] R 7 Examples of R include a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group. In addition to the alkyl groups already described, preferred examples of the alkyl group include lower alkyl groups. In addition to the alkenyl groups already described, preferred examples of the alkenyl group include lower alkenyl groups. 7 When is a hydrogen atom or any of these groups, the linking group is suitably an alkylene group having 2 or more, such as 3 or more, such as 4 or more, and for example 6 or less, such as 5 or less, such as 4 or less carbon atoms.

[0070] Also, R 7 When is a hydrogen atom, R 3 is an NH2 (amino group) having a linking group, that is, when the linking group is an alkylene group or an alkenylene group, it becomes an aminoalkyl group or an aminoalkenyl group. In formulas (1) and (2), R 3By using an aminoalkyl group or the like, the present nucleoside derivative and the oligonucleotide derivative comprising a monomer unit derived from the present nucleoside derivative can exhibit charge-donating properties characterized by a change in charge depending on the surrounding pH environment. For example, they are cationic under acidic conditions, and the positive charge can be reduced to zero under neutral physiological conditions. In other words, this charge-adjusting ability allows the charge of the nucleoside derivative to be dynamically changed or a desired charge to be imparted as needed by changing the pH environment. Therefore, with such a nucleoside derivative, the charge of the oligonucleotide can be adjusted in a manner different from conventional methods or with a greater degree of freedom than conventional methods. From the above, R 3 The present nucleoside derivatives, in which the aminoalkyl group is such, are useful as charge (positive charge) imparting agents or charge control agents for oligonucleotides and the like.

[0071] R 3 Examples of R include an azide group, an amidino group, i.e., CH3(NH)C(NH)- (a group in which one hydrogen atom has been removed from the amino group of amidine), and a guanidino group, i.e., NH2(NH)C(NH)- (a group in which one hydrogen atom has been removed from the amino group of guanidine), each of which has a linking group. Among these, a guanidino group is particularly preferred. 3 However, when R has these groups, the linking group can be an alkylene group or alkenylene group having 1 or more carbon atoms, for example, 2 or more carbon atoms. 3 However, when the nucleoside derivative is an amidino group or guanidino group having a linking group, it is always cationic, unlike the aminoalkyl group described above. 3 is useful in combination with the present nucleoside derivatives in which the nucleoside group is an aminoalkyl group or the like.

[0072] Protecting groups for amino groups are well known to those skilled in the art, and the aforementioned references can be referred to. Specific examples of protecting groups for amino groups include, in addition to those exemplified above as protecting groups for hydroxyl groups, benzyl, methylbenzyl, chlorobenzyl, dichlorobenzyl, fluorobenzyl, trifluoromethylbenzyl, nitrobenzyl, methoxyphenyl, methoxymethyl (MOM), N-methylaminobenzyl, N,N-dimethylaminobenzyl, phenacyl, acetyl, trifluoroacetyl, pivaloyl, benzoyl, phthalimido, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, t-butoxycarbonyl (Boc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM), and 2-(trimethylsilyl)ethoxymethyl (SEM). More preferred are benzyl, methoxyphenyl, acetyl, trifluoroacetyl (TFA), pivaloyl, benzoyl, t-butoxycarbonyl (Boc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM), and 2-(trimethylsilyl)ethoxymethyl (SEM), and particularly preferred are benzyl, methoxyphenyl, acetyl, benzoyl, and benzyloxymethyl.

[0073] In the present invention, the term "protecting group for an amino group" may refer to a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, e.g., induction in a microorganism, etc.). In particular, a substituent that is eliminated by hydrogenolysis or hydrolysis is preferred as a protecting group for an amino group.

[0074] [B: About bases] The base B in the present nucleoside derivative includes known natural bases as well as artificial bases. For example, B can be selected from a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, and a substituted 2-oxo-pyrimidin-1-yl group.

[0075] That is, B may be a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group, as well as 2,6-dichloropurin-9-yl or 2-oxo-pyrimidin-1-yl, and may further be 2-oxo-4-methoxy-pyrimidin-1-yl, 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl, or 2,6-dimethoxypurin-9-yl.

[0076] Further examples include 2-oxo-4-amino-pyrimidin-1-yl with a protected amino group, 2-amino-6-bromopurin-9-yl with a protected amino group, 2-amino-6-hydroxypurin-9-yl with a protected amino group, 2-amino-6-hydroxypurin-9-yl with a protected amino group and / or hydroxyl group, 2-amino-6-chloropurin-9-yl with a protected amino group, 6-aminopurin-9-yl with a protected amino group, and 4-amino-5-methyl-2-oxo-pyrimidin-1-yl with a protected amino group. The protecting groups for the hydroxyl group and amino group are as already explained.

[0077] Further examples include 6-aminopurin-9-yl (adenine), 2-amino-6-hydroxypurin-9-yl (guanidine), 2-oxo-4-amino-pyrimidin-1-yl (cytosine), 2-oxo-4-hydroxy-pyrimidin-1-yl (uracil), or 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (thymine).

[0078] Further examples include 4-amino-5-methyl-2-oxo-pyrimidin-1-yl (methylcytosine), 2,6-diaminopurin-9-yl, 6-amino-2-fluoropurin-9-yl, 6-mercaptopurin-9-yl, 4-amino-2-oxo-5-chloro-pyrimidin-1-yl, or 2-oxo-4-mercapto-pyrimidin-1-yl.

[0079] Further examples include 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, and 2-amino-6-bromopurin-9-yl.

[0080] The substituents in the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group are any one of a hydroxyl group, a protected hydroxyl group, a lower alkoxy group, a mercapto group, a protected mercapto group, a lower alkylthio group, an amino group, a protected amino group, an amino group substituted with a lower alkyl group, a lower alkyl group, a lower alkoxymethyl group, and a halogen atom, or any combination of two or more thereof. These substituents are as described above.

[0081] As for B in the present nucleoside derivative, the substituents in the substituted purin-9-yl group or substituted 2-oxo-pyrimidin-1-yl group are preferably the aforementioned substituents, but in addition, it is also preferable that a triazole group or a lower alkoxymethyl group be added.

[0082] Preferred examples of the substituted purin-9-yl group include 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, and 6-mercaptopurin-9-yl. When the above-mentioned substituents contain an amino group or a hydroxyl group, preferred examples include substituents in which the amino group and / or hydroxyl group is protected.

[0083] Substituted 2-oxo-pyrimidin-1-yls include, for example, 2-oxo-4-amino-pyrimidin-1-yl, 1H-(1,2,4-triazol-1-yl)-pyrimidin-1-yl, 4-1H-1,4-amino-2-oxo-5-chloro-pyrimidin-1-yl, 2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidin-1-yl, 2-oxo-4-hydroxy-pyrimidin-1-yl, 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl, or 4-amino-5-methyl-2-oxo-pyrimidin-1-yl. Further preferred examples include 2-oxo-4-methoxy-pyrimidin-1-yl and 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl.

[0084] Among these B groups, when the substituent contains an amino group or a hydroxyl group, a preferred example is a substituent in which the amino group or the hydroxyl group is protected.

[0085] The present nucleoside derivative may be a salt. The form of the salt is not particularly limited, but acid addition salts are generally exemplified, and may take the form of intramolecular counterions. Alternatively, depending on the type of substituent, a base addition salt may be formed. Pharmaceutically acceptable salts are preferred. The types of acids and bases that form pharmaceutically acceptable salts are well known to those skilled in the art, and reference can be made to those described in, for example, J. Pharm. Sci., 1-19 (1977). For example, acid addition salts include mineral acid salts and organic acid salts. Furthermore, when one or more substituents contain an acidic moiety, base addition salts are also preferred.

[0086] Examples of mineral acid salts include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogensulfate, phosphate, and hydrogenphosphate. Typically, hydrochloride and phosphate are preferred. Examples of organic acid salts include acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, and p-toluenesulfonate. Typically, acetate and the like are preferred. Examples of base addition salts include alkali metal salts, alkaline earth metal salts, organic amine salts, and amino acid addition salts.

[0087] Examples of the alkali metal salts include sodium salts and potassium salts. Examples of alkaline earth metal salts include magnesium salts and calcium salts. Examples of organic amine salts include triethylamine salts, pyridine salts, procaine salts, picoline salts, dicyclohexylamine salts, diethanolamine salts, triethanolamine salts, and tris(hydroxymethyl)aminomethane salts. Examples of amino acid addition salts include arginine salts, lysine salts, ornithine salts, serine salts, glycine salts, aspartates, and glutamates.

[0088] The present nucleoside derivative or a salt thereof may exist as a hydrate or solvate, and these substances are also included in the scope of the disclosure of this specification. The present nucleoside derivative or a salt thereof can be easily produced by those skilled in the art according to the synthesis examples described below or known methods.

[0089] By incorporating the present nucleoside derivative into an oligonucleotide as at least a portion of the oligonucleotide, it is possible to improve the nuclease resistance of the oligonucleotide as a single strand or a double strand, and also to improve the cell membrane permeability of mammalian cells and the like. That is, the present nucleoside derivative itself is useful as a nuclease resistance improver and / or a cell membrane permeability imparting agent. Furthermore, the present nucleoside derivative can have a basic substituent at the 4' position. This allows it to function as a charge regulator or positive charge imparting agent, which can adjust the negative charge derived from phosphate groups and the like in oligonucleotides and the like.

[0090] (Oligonucleotide derivatives and their salts) The oligonucleotide derivative disclosed herein (hereinafter also referred to as the present oligonucleotide derivative) can contain at least one partial structure represented by formula (3) or formula (4). The partial structures represented by formula (3) and formula (4) can be obtained based on the nucleoside derivatives or salts thereof represented by formula (1) and formula (2), respectively.

[0091] [ka]

[0092] R in the partial structures represented by formula (3) and formula (4) 1 ,X,R 3 and B have the same meanings as in formula (1) and formula (2), respectively.

[0093] The present oligonucleotide derivative may contain two or more partial structures represented by formula (3) and formula (4). In this case, these partial structures may be the same or different from each other. Furthermore, the entire partial structure contained in the present oligonucleotide derivative may consist solely of the partial structure represented by formula (3), or may consist solely of the partial structure represented by formula (4). Furthermore, the oligonucleotide derivative may contain one or more partial structures represented by formula (3) and one or more partial structures represented by formula (4).

[0094] Furthermore, the partial structures represented by formula (3) and formula (4) may be arranged adjacent to each other or apart from each other. For example, the present oligonucleotide derivative may have at least three of the partial structures. In this case, each partial structure may be arranged approximately evenly at the 5'-terminal side, the central portion, and the 3'-terminal side of the present oligonucleotide derivative. "Almost evenly arranged" partial structures at each of these portions of the present oligonucleotide derivative does not necessarily mean that each portion has the same number of partial structures, but it is sufficient that each portion has at least one partial structure. For example, when each portion has about 1 to 3 partial structures, it can be said that the partial structures are arranged approximately evenly. The present oligonucleotide derivative may have at least six partial structures.

[0095] In the partial structure represented by formula (3), the sugar chain portion is derived from ribose or deoxyribose, and therefore the oligonucleotide derivative may be an oligoribonucleotide or an oligodeoxyribonucleotide. The oligonucleotide derivative may also be a chimera of a ribonucleotide and a deoxyribonucleotide.

[0096] The present oligonucleotide derivatives are themselves single-stranded, but can also take the form of hybrids with oligoribonucleotides, oligodeoxyribonucleotides, and oligodeoxyribo / ribonucleotides (chimeric chains), ie, double-stranded.

[0097] The present oligonucleotide derivative may have partial structures other than those represented by formula (3) and formula (4), such as partial structures corresponding to other natural nucleotides, or known nucleoside derivatives and / or nucleotide derivatives, etc. The partial structures defined herein and other partial structures may be bonded to each other by, for example, a phosphodiester bond, a phosphate monoester bond, a thiophosphate ester bond, etc.

[0098] The present oligonucleotide derivative preferably has at least 2 or more, more preferably 8 or more, and particularly preferably 15 or more, partial structures and other nucleoside derivatives as units of number. The upper limit is not particularly limited, but may be, for example, 100 or less, for example, 80 or less, for example, 60 or less, for example, 50 or less, for example, 40 or less, for example, 30 or less, or for example, 20 or less.

[0099] The oligonucleotide derivative may have one or more asymmetric centers in the partial structures represented by formula (3) and formula (4) or other partial structures, and the same applies to the presence of stereoisomers, and any mixture of stereoisomers or racemates are all included in the scope of the present invention. In addition, the oligonucleotide derivative may also exist as a tautomer.

[0100] The present oligonucleotide derivative may be a salt. The form of the salt is not particularly limited, but pharmaceutically acceptable salts are preferred. The salts may be the same as those described above for the present nucleoside derivative. The present oligonucleotide derivative or its salt may be a hydrate or solvate, which are also included in the scope of the present invention.

[0101] (Production of the Nucleoside Derivatives and the Oligonucleotide Derivatives) The present nucleoside derivatives and the present oligonucleotide derivatives can be easily synthesized by those skilled in the art based on the specific synthesis examples described below as well as on the synthesis techniques for nucleosides and oligonucleotides known at the time of filing of this application.

[0102] The present nucleoside derivatives and the present oligonucleotide derivatives can be produced, for example, by the following method, but the method for producing the nucleoside analogs or oligonucleotide analogs of the present invention is not limited to the following method.

[0103] In each reaction, the reaction time is not particularly limited, but since the progress of the reaction can be easily monitored by the analytical means described below, it may be terminated when the yield of the target product reaches a maximum. Furthermore, each reaction may be carried out, if necessary, under an inert gas atmosphere such as a nitrogen or argon stream. When each reaction requires protection with a protecting group and subsequent deprotection, the reaction can be carried out appropriately using the method described below.

[0104] In this specification, Bn represents a benzyl group, Ac represents an acetyl group, Bz represents a benzoyl group, PMB represents a p-methoxybenzyl group, Tr represents a triphenylmethyl group, TFA represents a trifluoroacetyl group, TsO represents a tosyloxy group, MMTr represents a 4-methoxytriphenylmethyl group, DMTr represents a 4,4'-dimethoxytriphenylmethyl group, TMS represents a trimethylsilyl group, TBDMS represents a tert-butyldimethylsilyl group, TBDPS represents a tert-butyldiphenylsilyl group, MOM represents a methoxymethyl group, BOM represents a benzyloxymethyl group, and SEM represents a 2-(trimethylsilyl)ethoxymethyl group.

[0105] (Method for producing the present nucleoside derivative) The present nucleoside derivatives can be synthesized, for example, according to the following schemes. The following schemes I (compounds 1 to 11), II (compounds 11 to 20), and III (compounds 15 to 66) are examples of schemes for synthesizing a uracil ribonucleoside derivative using glucose as a starting material, and then synthesizing a phosphoramidite agent for synthesizing the present oligonucleotide derivatives. The protecting groups and linking groups in the compounds in the following schemes are all examples.

[0106] (Scheme I) [ka]

[0107] (Scheme II) [ka]

[0108] (Scheme III) [ka]

[0109] That is, according to a conventional method, the above compound 2 is obtained from glucose 1. From compound 2, compounds 3 to 20 and compound 66 can be obtained based on the description in Bioorganic & Medical Chemistry 11 (2003) 211-2226, Bioorganic & Chemistry letters (1999) 2667-2672, The Journal of Organic Chemistry 2013, 78, 9956-9962, HELVATICA CHIMICA ACTA Vol. 83 (2000) 128-151, etc., as well as Bioorganic & Medicinal Chemistry 11 (2003) 2211-2226, Bioorganic & Chemistry letters (1999) 2667-2672.

[0110] According to the present specification, the present nucleoside derivatives can be efficiently obtained, for example, by substituting a part of the method for producing the present nucleoside derivatives exemplified by the above Schemes I to III to include at least one of the reactions represented by the following formulas (5) to (7).

[0111] [ka]

[0112] In the above formulas (5) to (7), R 10 and R 11 are each independently or R 10 and R 11 together represent a protecting group for a hydroxyl group, and R 12 represents Equation (8), and R 14 represents an alkylene group, Bn represents a benzyl group, and Tf represents a trifluoromethylsulfonyl group.

[0113] The reaction of the above formula (5) can replace, for example, part of the steps from compound 1 to compound 4 exemplified in the above scheme I. Below, alternative scheme I including the reaction of formula (5) is shown. The reaction represented by formula (5) corresponds to the reaction from compound 2 to compound 3 in alternative scheme I. Details of alternative schemes I to III shown below in order will be explained in the examples below.

[0114] [ka]

[0115] According to the reaction represented by formula (5), R 3This method can reduce the number of steps required for introducing a group (e.g., an aldehyde group) for introducing an aminoalkyl group. In conventional Scheme I, in order to selectively protect the diol of the two hydroxymethyl groups linked to the 4' position of ribose, it was necessary to separate the isomers by-produced in the reaction of Compounds A and B in Alternative Scheme I (corresponding to Compounds 2 and 3 in Scheme I) using column chromatography on silica gel or the like. However, by using the reaction represented by formula (5), i.e., by using Compound 2 in Alternative Scheme I and then converting Compound 2 to Compound 3, a hydroxymethyl group can be stereoselectively introduced at the 4' position of ribose to obtain Compound 3, which is a single stereoisomer. Compound 3 can also be crystallized and purified, avoiding the need for separation of unintended isomers and allowing Compound 3 in Alternative Scheme I to be obtained in high yield.

[0116] In addition, the alternative schemes I to III do not include R at the 4' position. 3 In this example, an aminoethyl group (the linking group is an ethylene group) is introduced as the linking group. However, other linking groups such as an aminopropyl group (the same propylene group) or other R 3 Alternatively, a Wittig reaction as shown in Scheme I can be used to introduce other alkyl groups into the R group, such as amino alkyl groups. 3 Alternatively, the Horner-Emmons reaction can be used to introduce an olefin that can be converted to R, such as the intended aminoalkyl. 3 It is also possible to introduce an olefin that can be converted to an aminopropyl group. For example, the 4'-position can be modified to be convertible to an aminopropyl group using the Horner-Emmons reaction as follows. Such a reaction is described, for example, in Bioorganic & Medicinal Chemistry 11 (2003) 2211-2226.

[0117] [ka]

[0118] Furthermore, according to the present specification, the reactions represented by the following formulas (5)' and (5)'' can be carried out following the reaction of formula (5) or instead of the reaction of formula (5). These reactions can partially replace the other steps from Compound 1 to Compound 4 exemplified in the above Scheme I, and when combined, they correspond to the reaction from Compound 3 to Compound 5 in Alternative Scheme I. The reaction of formula (5)' is a reaction for protecting the 5'-hydroxyl group of the compound on the left side of the formula, and the reaction of formula (5)'' is a reaction for protecting the R at the 4'-position of the compound on the left side of the formula. 12 is a reaction that converts the aldehyde group into an aldehyde group.

[0119] [ka]

[0120] According to the reactions represented by formula (5)' and formula (5)'', the hydroxyl groups at the 1'-, 2'-, 3'-, and 5'-positions of ribose are efficiently protected from the compound obtained by the reaction represented by formula (5), and a compound (the compound on the right side of formula (5)'') in which preparation for introduction of an aminoalkyl group at the 4'-position is made can be efficiently obtained in high yield. The reaction of formula (5)' is a reaction in which, for example, a proton is abstracted from the hydroxyl group of the hydroxymethyl group at the 4'-position using a strong base such as sodium hydride, and a benzyl halide such as benzyl bromide is used to introduce a benzyl group as a protecting group to the 5'-position hydroxyl group. The reaction of formula (5)'' is a reaction in which an oxidizing agent such as sodium periodate is used to remove the proton from the hydroxyl group of the hydroxymethyl group at the 4'-position. 12 to an aldehyde group.

[0121] The aldehyde group of the compound on the right side of the reaction (5)'' obtained in this way can be converted into a vinyl group by a Witting reaction using methyltriphenylphosphonium bromide or the like, and then a hydroxyl group can be added to the vinyl group to convert it into a hydroxyethyl group.

[0122] For example, the present inventors have confirmed that by combining the reactions represented by formulas (5), (5)', and (5)'', the number of steps from compound 1 to compound 5 in Alternative Scheme I can be reduced from 8 steps in the corresponding conventional method to 5 steps, and the yield in the corresponding conventional method (typically 28%) can be increased to 48%.

[0123] The reaction of the above formula (6) is carried out by attaching an azide group (R 14 The reaction of formula (6) above can replace, for example, part of the steps from Compound 6 to Compound 10 exemplified in Scheme I above. That is, it can replace part of the step of introducing an azide group at the 4'-position of ribose. Alternative Scheme II including the reaction of formula (6) is shown below. The main part of Alternative Scheme II is the step from Compound 7 to Compound 10 in Alternative Scheme II. The reaction represented by formula (6) corresponds to the reaction from Compound 7 to Compound 8 in Alternative Scheme II.

[0124] [ka]

[0125] According to the reaction represented by formula (6), compound 8 can be obtained from compound 7 in Alternative Scheme II by azidation using the Mitsunobu reaction. Previously, compound 7 was considered unsuitable as a starting material for the Mitsunobu reaction because it was predicted that a side reaction would occur if the protecting group for the 5'-hydroxyl group was a benzyl group. Furthermore, the use of benzyl groups for the 3'- and 5'-hydroxyl protecting groups makes it possible to omit the step of replacing the protecting group (compounds 57 to 64) for deprotection in Scheme III to obtain a 2'-halogenated nucleoside derivative.

[0126] The Mitsunobu reaction typically involves the reaction of a secondary alcohol with diethyl azodicarboxylate, triphenylphosphine (Ph3P), and benzoic acid, resulting in stereoinversion (S NThe hydroxyl group can be azidoted by the Mitsunobu reaction using an azido reagent such as diphenylphosphoryl azide (DPPA).

[0127] Furthermore, according to the present specification, following the reaction of formula (6), reactions from compound 8 to compound 9 and from compound 9 to compound 10 in Alternative Scheme II can be carried out. By carrying out these reactions, compound 10 in Alternative Scheme II (compound 11 in Scheme 1) can be obtained efficiently and in good yield. In particular, the yield of compound 10 can be improved by carrying out hydrolysis and acetylation of the isopropylidene group in compound 8 in Alternative Scheme II in two steps.

[0128] For example, the present inventors have confirmed that the yield in the process from Compound 5 to Compound 10 in Alternative Scheme II by combining the reactions represented by formula (6) can be increased to 65%, compared to the corresponding yield in the conventional method (typically 19%).

[0129] The reaction of formula (7) above is a reaction for introducing a halogen atom into the 2'-position of ribose in the compound on the left side of formula (7). The reaction of formula (7) above can replace, for example, part of the steps from compound 10 to compound 11 in scheme I above, through compound 11 to compound 15 in scheme II above, and from compounds 15 to 61 in scheme III above. That is, it can replace part of the step for introducing a halogen atom into the 2'-position of ribose. Alternative scheme III, which includes the reaction of formula (7), is shown below. The main part of alternative scheme III is the step from compound 12 to compound 17 in alternative scheme III. The reaction represented by formula (7) corresponds to the reaction from compound 15 to compound 16 in alternative scheme III.

[0130] [ka]

[0131] According to the reaction represented by formula (7), a fluorination reaction is carried out using compound 15 in Alternative Scheme III, thereby enabling the production of a 2'-halogenated compound in a higher yield than in conventional Scheme III. Furthermore, as described above, since the protecting groups for the hydroxyl groups at the 3' and 5' positions are both benzyl groups, the step of replacing the protecting groups can be omitted compared to Scheme III, and as a result, the deprotection step can be shortened.

[0132] Furthermore, according to the present specification, reactions represented by the following formulas (7)' and (7)" can be carried out prior to or independently of the reaction of formula (7). These reactions correspond to the steps from compound 12 to compound 15 in Alternative Scheme III. That is, the reaction of formula (7)' is a reaction for deprotecting and inverting the hydroxyl group at the 2'-position of the compound on the left side of the formula, and the reaction of formula (7)" is a reaction for introducing a Tf group into the hydroxyl group at the 2'-position of the compound on the left side of the formula.

[0133] [ka]

[0134] According to the reactions represented by formula (7)' and formula (7)'', the compound used in formula (7) can be obtained efficiently and in high yield.

[0135] For example, the present inventors have confirmed that by combining the reactions represented by formulas (7), (7)', and (7)'', the number of steps from compound 10 to compound 18 in Alternative Scheme III can be reduced to 7 steps, compared to 10 steps in the corresponding conventional method, and that the yield in these steps can be increased to 50%, compared to the yield in the corresponding conventional method (typically 22%).

[0136] After the alternative schemes I to III, a nucleoside derivative can be obtained, for example, according to the following scheme: The following schemes IV and V will also be described in detail in the examples below.

[0137] [ka]

[0138] [ka]

[0139] R in the above formula 10 and R 11 The protecting group for the hydroxyl group in is not particularly limited as long as it is a protecting group for a hydroxyl group, and examples thereof include cyclic acetal-type protecting groups such as a methoxymethylene acetal group, an isopropylidene group, a methylene acetal group, and an ethylidene acetal group, a methyl group, a benzyl group, a benzoyl group, a t-butyl group, an acetyl group, a methoxymethyl group, a 2-methoxyethoxymethyl group, a trimethylsilyl group, a triethylsilyl group, a t-butyldimethylsilyl group, a methylthiomethyl group, a trifluoroacetyl group, a benzyloxycarbonyl group, a t-butoxycarbonyl group, a triphenylmethyl group, a toluoyl group, and a 4-methoxymethylphenyl group.

[0140] R 1 and R 2 The hydroxyl-protecting group in (1) is preferably a hydroxyl-protecting group that can be deprotected by acid hydrolysis. Examples of the hydroxyl-protecting group that can be deprotected by acid hydrolysis include cyclic acetal-type protecting groups such as methoxymethylene acetal, isopropylidene, methylene acetal, and ethylidene acetal, as well as methoxymethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, methylthiomethyl, benzyloxycarbonyl, t-butoxycarbonyl, and triphenylmethyl.

[0141] R10 and R 11 From the viewpoint of reaction efficiency and stereoselectivity, R 10 and R 11 In the case where the above groups are united together, a cyclic acetal type protecting group such as a methoxymethylene acetal group, an isopropylidene group, a methylene acetal group or an ethylidene acetal group is preferred, and an isopropylidene group is particularly preferred.

[0142] R in the above formula 14 has the same meaning as the linking group in R3. 15 is a leaving group, and examples thereof include a methanesulfonyl group, a chloromethanesulfonate group, etc. Among these, a methanesulfonyl group is preferred.

[0143] By using the above alternative schemes I to III, which include the reactions represented by formulas (5) to (7), the number of steps can be reduced to about 70% of the total, and the total yield can be improved by at least two times or more, for example, three times or more, or even up to about four times.

[0144] By using at least one of the reactions represented by formulas (5) to (7), the present nucleoside derivatives can be efficiently obtained, and as a result, the present oligonucleotide derivatives having the partial structures represented by formulas (3) and (4) can also be efficiently obtained. That is, by using such nucleoside derivatives, synthesis can be performed using a known DNA synthesizer, and the obtained oligonucleotide derivatives can be purified using a column, and the purity of the product can be analyzed by reverse-phase HPLC or MALDI-TOF-MS to obtain purified present oligonucleotide derivatives. Note that methods for converting the present oligonucleotide derivatives into acid addition salts are well known to those skilled in the art.

[0145] The present oligonucleotide derivatives have a specific N-containing group at the 4'-position of ribose via a linking group, which allows the effective charge of RNA to be adjusted while maintaining in vivo RNA functions such as RNA interference, thereby improving ribonuclease resistance and cell membrane permeability.

[0146] The present oligonucleotide derivative can have at least two of this partial structure. By having multiple of this partial structure, cell membrane permeability, ribonuclease resistance, etc. can be reliably improved and adjusted. The present oligonucleotide derivative can also have at least three of this partial structure.

[0147] In the present oligonucleotide derivative, the site(s) containing one or more of the present partial structures is not particularly limited, but may be, for example, on either or both the 5'-end and 3'-end. The 5'-end and 3'-end refer to regions ranging from the respective ends of the polymer chain of the present oligonucleotide, each of which refers to a region consisting of an appropriate number of structural units, for example, not more than 30% of all structural units of the polymer chain. The proportion of the range from the above-mentioned ends varies depending on the total length of the polymer chain, but can be, for example, 25% or less, for example, 20% or less, for example, 10% or less, or for example, 5% or less. More specifically, the 5'-end and 3'-end can refer to regions of, for example, 1 to 30, for example, 1 to 25, for example, 1 to 20, for example, 1 to 15, for example, 1 to 10, for example, 1 to 8, for example, 1 to 6, for example, 1 to 5, for example, 1 to 4, for example, 1 to 3, or for example, 1 to 2 nucleoside derivative-derived building blocks from each end. The present oligonucleotide derivative can have one or more of these partial structures in any of these terminal regions. Preferably, two or more can be included. The present oligonucleotide derivative can also have this partial structure in either or both of the 5'-end and 3'-end (i.e., the first building block from each end).

[0148] The present oligonucleotide derivative may have one or more of the present partial structures in the central portion, i.e., a portion other than the 5'-end and 3'-end portions. By having the present partial structure in the central portion of the present oligonucleotide derivative, it becomes easier to improve and adjust ribonuclease resistance and cell membrane permeability. It also becomes easier to adjust the charge of the entire oligonucleotide.

[0149] The present oligonucleotide derivative may also have this partial structure at either or both of the 5'-end and 3'-end, and in the central portion. Preferably, one or more of this partial structure can be provided at each of the 5'-end, 3'-end, and central portion. In this way, by providing this partial structure approximately evenly or dispersedly as a whole, ribonuclease resistance, cell membrane permeability, and charge controllability can be improved. From the viewpoint of improving properties, it is useful to provide two or more of this partial structure in the central portion of the present oligonucleotide derivative.

[0150] The partial structure in the oligonucleotide derivative can be a partial structure derived from a ribonucleoside derivative represented by formula (3) or a partial structure derived from a deoxyribonucleotide derivative represented by formula (4). The ribonucleoside derivative represented by formula (3) and the partial structure of formula (4) can be used as a substitute for a ribonucleoside derivative by including uracil (U), a base in RNA, as the base of B.

[0151] In addition, this partial structure is R in formulas (3) and (4). 3 The linking group is an alkylene group having 1 or 2 or more carbon atoms, and NHR 7 In view of ribonuclease resistance, cell membrane permeability, and charge controllability, it is preferable that R 7may be a hydrogen atom or an acyl group having an alkyl group having about 1 to 6 carbon atoms. The alkylene group may be an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, or the like. It may also be, for example, an ethylene group, a propylene group, a butylene group, or the like. It may also be, for example, an ethylene group, a propylene group, or the like. By using an ethylene group or a propylene group as a linking group, it is possible to obtain higher ribonuclease resistance, cell membrane permeability, and charge controllability than when a methylene group is used.

[0152] The partial structure may also be an amidino group, an azide group, or a guanidino group having a linking group. High ribonuclease resistance and cell membrane permeability can also be achieved by providing such a functional group. In this case, the linking group may be an alkylene group having one or more carbon atoms.

[0153] In addition, this partial structure is R in formula (3) and formula (4). 3 The linking group is preferably an alkyl group having about 1 to 6 carbon atoms, and more preferably, for example, the lower limit of the number of carbon atoms is 2 or more, for example, 3 or more. Such a structure is effective for ribonuclease resistance and cell membrane permeability.

[0154] The present oligonucleotide derivative preferably has at least six of these partial structures, which is advantageous in terms of ribonuclease resistance, cell membrane permeability, and charge controllability. [Example]

[0155] Examples will be described below as specific examples to more specifically explain the disclosure of this specification. The following examples are intended to illustrate the disclosure of this specification, but are not intended to limit the scope thereof.

[0156] In the following examples, each step in the alternative schemes I to III and additional schemes IV and V already described is described. [Example]

[0157] (Compounds 1 to 3) [ka] 1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose (50.0 g, 0.192 mol) and potassium bromide (2.29 g, 19.2 mmol) were dissolved in dichloromethane (50 mL) and saturated aqueous sodium bicarbonate (130 mL). After cooling to 0 °C, Nor-AZADO (1.3 mg) and aqueous sodium hypochlorite (203 mL) were added dropwise and stirred at the same temperature for 1 hour. Saturated sodium thiosulfate was added, followed by stirring and extraction with chloroform five times. The organic layer was dried over magnesium sulfate and concentrated to dryness under reduced pressure to obtain crude compound 2. Crude compound 2 was then dissolved in tetrahydrofuran (370 mL), and paraformaldehyde (11.1 g) and potassium carbonate (51.0 g) were added. The mixture was stirred at 40 °C for 24 hours. Insoluble materials were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (370 mL) and deionized water (74.5 mL) and cooled to 0°C. Sodium borohydride (14.0 g, 0.370 mol) was added and stirred at the same temperature for 3 hours. The reaction mixture was concentrated under reduced pressure and then extracted five times with chloroform. The organic layer was dried over magnesium sulfate and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and hexane was added and stirred. The precipitated solid was collected by filtration and dried to obtain compound 3 (30.7 g, 0.106 mol, 55.2%). 1H-NMR (CDCl3): δ 5.92 (1H, d, J = 4.0), 4.73 (1H, dd, J = 4.0, 6.5), 4.59 (1H, dd, J = 6.5, 7.5), 4.32 (1H, t, J = 6.5), 4.16 (1H, dd, J = 7.5, 9.5), 3.91 (1H, dd, J = 6.5, 9.5), 3.79 (1H, dd, J = 2.5, 11.5), 3.62 (1H, dd, J = 7.0, 11.5), 2.78 (1H, d, J = 7.0), 2.06 (1H, br.t), 1.63, (3H, s), 1.47 (3H, s), 1.41 (3H, s), 1.35 (3H, s).

[0158] (Compounds 3 to 5) [ka]

[0159] Compound 3 (30.0 g, 0.103 mol) was dissolved in dimethylformamide (343 mL) and cooled to 0°C. Sodium hydride (60% oil, 12.4 g, 0.310 mol) was added and stirred at the same temperature for 1 hour. Benzyl bromide (36.8 mL, 0.309 mol) was then added and stirred at room temperature for 17 hours. Methanol (10 mL) was added and stirred, and the reaction mixture was concentrated under reduced pressure. Ethyl acetate was added to the residue and washed twice with water. The organic layer was dried over magnesium sulfate and concentrated to obtain crude compound 4.

[0160] Crude compound 4 was dissolved in acetonitrile (517 mL) and deionized water (68 mL). Sodium periodate (44.2 g, 0.207 mol) and iodine (7.85 g, 30.9 mmol) were added and the mixture was stirred at 70 °C for 24 hours. After removing insoluble materials by filtration, the filtrate was concentrated under reduced pressure. The concentrate was diluted with ethyl acetate and washed with saturated sodium thiosulfate and saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (silica gel 1000 cc, hexane:ethyl acetate = 10:1 to 3:1) to obtain compound 5 (35.80 g, 89.85 mmol, 87.2%). 1 H-NMR (500 MHz, CDCl3): δ 9.91 (1H, s), 7.37-7.23 (10H, m), 5.84, (1H, d, J = 3.5 Hz), 4.72 (1H, d, J = 12.0 Hz), 4.60 (1H, d, J = 12.0 Hz), 4.60 (1H, t, J = 3.5 Hz), 4.52 (1H, d, J = 12.0 Hz), 4.46 (1H, d, J = 12.0 Hz), 4.37 (1H, d, J = 4.5 Hz), 3.68 (1H, d, J = 11.0 Hz), 3.61 (1H, d, J = 12.0 Hz), 1.60 (3H, s), 1.35 (3H, s).

[0161] (Compounds 5 to 6) [ka]

[0162] Methyltriphenylphosphonium bromide (70.6 g, 0.198 mol) was suspended in tetrahydrofuran (396 mL) and cooled to -78 °C. A hexane solution of n-butyllithium (1.6 M, 112 mL, 0.179 mol) was added dropwise over 25 minutes, followed by warming to 0 °C and stirring for 1 hour. A tetrahydrofuran solution (180 mL) of compound 5 (35.80 g, 89.85 mmol) was added dropwise over 20 minutes at the same temperature (the vessel was washed three times with 20 mL of tetrahydrofuran). The mixture was then returned to room temperature and stirred for 20 hours. Saturated ammonium chloride was added to the reaction mixture, followed by stirring. The solvent was evaporated under reduced pressure. The concentrate was extracted with ethyl acetate, and the organic layer was dried over magnesium sulfate and concentrated. The residue was dissolved in acetone (150 mL), concentrated with silica gel, dried, and purified by silica gel column chromatography (silica gel 1000 cc, hexane:ethyl acetate = 10:1). Compound 6 (30.96 g, 78.09 mmol, 86.91%) was obtained. 1 H-NMR (500 MHz, CDCl3): δ 7.38-7.22 (10H, m), 6.19 (1H, dd, J = 11.0, 17.5 Hz), 5.77, (1H, d, J = 4.0 Hz), 5.52 (1H, dd, J = 2.0, 17.5 Hz), 5.25 (1H, dd, J = 1.5, 11.0 Hz), 4.76 (1H, d, J = 12.0 Hz), 4.59 (1H, d, J = 12.0 Hz), 4.57 (1H, t, J = 4.5 Hz), 4.51 (1H, d, J = 12.0 Hz), 4.40 (1H, d, J = 12.0 Hz), 4.25 (1H, d, J = 5.0 Hz), 3.33 (1H, d, J = 11.0 Hz), 3.31 (1H, d, J = 12.0 Hz), 1.52 (3H, s), 1.29 (3H, s).

[0163] (Compounds 6 to 7) [ka]

[0164] Compound 6 (26.62 g, 67.14 mmol) was dissolved in tetrahydrofuran (260 mL). 9-borabicyclononane tetrahydrofuran solution (0.5 M, 404 mL, 0.202 mol) was added dropwise at 0°C over 70 minutes, followed by stirring at room temperature for 18 hours. The reaction mixture was cooled to 0°C, and deionized water (26.6 mL) was added dropwise over 15 minutes. The mixture was stirred at the same temperature for 15 minutes. 3 mol / L aqueous sodium hydroxide solution (133 mL, 0.399 mol) was added dropwise over 1 hour. The mixture was stirred at the same temperature for 15 minutes, and then 30% aqueous hydrogen peroxide (66.6 mL) was added dropwise over 40 minutes. The reaction mixture was returned to room temperature and stirred for 2 hours. Deionized water and ethyl acetate were added to the reaction mixture, and the mixture was stirred. The organic layer was washed with saturated brine. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with saturated brine. The organic layers were combined, dried over magnesium sulfate, and concentrated. The residue was azeotroped three times with toluene. The residue was purified by silica gel column chromatography (silica gel 1000 cc, hexane:ethyl acetate=3:1 to 1:1) to obtain Compound 7 (26.40 g, 63.69 mmol, 94.86%). 1 H-NMR (500 MHz, CDCl3): δ 7.35-7.24 (10H, m), 5.78, (1H, d, J = 4.0 Hz), 4.77 (1H, d, J = 12.0 Hz), 4.65 (1H, dd, J = 4.0, 5.0 Hz), 4.55 (1H, d, J = 12.0 Hz), 4.52 (1H, d, J = 13.5 Hz), 4.43 (1H, d, J = 12.0 Hz), 4.13 (1H, d, J = 5.5 Hz), 3.84 (1H, m), 3.76 (1H, m), 3.54 (1H, d, J = 10.0 Hz), 3.30 (1H, d, J = 10.5 Hz), 2.85 (1H, dd, J = 3.5, 7.5 Hz), 2.85 (1H, dd, J = 3.5, 7.5 Hz), 2.52 (1H, ddd, J = 4.0, 8.5, 15.5), 1.79 (1H, ddd, J = 3.5, 6.5, 15.5), 1.66 (3H, s), 1.33 (3H, s).

[0165] (Compounds 7 to 8) [ka]

[0166] Compound 7 (30.41 g, 73.37 mmol) and triphenylphosphine (38.5 g, 0.147 mol) were dissolved in tetrahydrofuran (489 mL). Bis(2-methoxyethyl) azodicarboxylate (34.4 g, 0.147 mol) was added at 0 °C and the mixture was stirred at the same temperature for 5 minutes. Diphenylphosphate azide (31.5 mL, 0.146 mol) was added dropwise over 30 minutes at the same temperature and the mixture was stirred at room temperature for 15 hours. 28% aqueous ammonia (30 mL) was added to the reaction mixture and stirred for 1 hour. Saturated aqueous sodium bicarbonate was added and the mixture was extracted twice with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was dissolved in acetone (200 mL), silica gel was added, and the mixture was concentrated. Purification by silica gel column chromatography (silica gel 1000 cc, hexane:ethyl acetate = 10:1 to 3:1) gave compound 8 (29.81 g, 67.83 mmol, 92.00%). 1H-NMR (500 MHz, CDCl3): δ 7.37-7.24 (10H, m), 5.75 (1H, d, J = 4.5 Hz), 4.76 (1H, d, J = 12.0 Hz), 4.61 (1H, dd, J = 4.5, 5.5 Hz), 4.55 (1H, d, J = 12.0 Hz), 4.51 (1H, d, J = 12.0 Hz), 4.42 (1H, d, J = 12.0 Hz), 4.10 (1H, d, J = 5.5 Hz), 3.35 (1H, ddd, J = 5.0, 9.5, 12.5 Hz), 3.45 (1H, d, J = 10.5 Hz), 3.40 (1H, ddd, J = 7.0, 10.0, 12.5 Hz), 3.27 (1H, d, J = 10.0 Hz), 2.50 (1H, ddd, J = 5.0, 9.5, 14.5 Hz), 1.84 (1H, ddd, J = 7.0, 10.0, 14.5 Hz), 1.59 (3H, s), 1.33 (3H, s).

[0167] (Compounds 8 to 9) [ka]

[0168] Compound 8 (26.38 g, 60.02 mmol) was dissolved in 70% acetic acid (600 mL) and trifluoroacetic acid (60.0 mL) and stirred at room temperature for 17 hours. The reaction mixture was concentrated, and the residue was azeotropically distilled with toluene five times to obtain a crude hemiacetal. The crude hemiacetal was dissolved in pyridine (300 mL), and acetic anhydride (34.0 mL, 0.360 mol) was added. The mixture was stirred at room temperature for 19 hours. Methanol was added to the reaction mixture, and the mixture was stirred and then concentrated. The residue was dissolved in ethyl acetate and washed with deionized water and then saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (silica gel 1000 cc, hexane:ethyl acetate=3:1) to obtain compound 9 (27.08 g, 56.01 mmol, 93.32%). 1H-NMR (500 MHz, CDCl3): δ 7.38-7.21 (12H, m), 6.34 (0.2H, d, J = 5.0 Hz), 6.10 (1H, s), 5.33 (1H, d, J = 5.0 Hz), 5.26 (0.2H, dd, J = 4.5, 6.5 Hz), 4.62-4.44 (4.8H, m), 4.28 (1H, d, J = 5.0 Hz), 4.22 (0.2H, d, J = 6.5 Hz), 3.49-3.27 (4.8H, m), 2.21-1.82 (2.4H, m), 2.12 (3H, s), 2.11 (0.6H, s), 2.06 (0.6H, s), 1.89 (3H, s).

[0169] (Compounds 9 to 10) [ka]

[0170] Compound 9 (29.55 g, 61.11 mmol), uracil (13.7 g, 0.122 mol), bis(trimethylsilyl)acetamide (89.5 mL, 0.366 mol) and 1,2-dichloroethane (306 mL) were added and heated and stirred at 85 °C for 2 hours. The reaction mixture was cooled to 0 °C, and then trimethylsilyl trifluoromethanesulfonate (22.1 mL, 0.122 mol) was added and stirred at 50 °C for 22 hours. The reaction mixture was cooled to 0 °C, and then saturated aqueous sodium bicarbonate was added and stirred. The resulting insoluble material was removed by filtration through Celite, and the organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (silica gel 1000 cc, chloroform:methanol = 100:1) to give compound 10 (30.24 g, 56.46 mmol, 92.39%). 1H-NMR (500 MHz, CDCl3): δ 8.33 (1H, s), 7.66 (1H, d, J = 8.0 Hz), 7.40-7.26 (10H, m), 6.13 (1H, d, J = 4.5 Hz), 5.35 (1H,dd, J = 5.0, 6.0Hz), 5.31 (1H, dd, J = 2.5, 8.0 Hz), 4.64 (1H, d, J = 12.0 Hz), 4.45 (1H, d, J = 11.0 Hz), 4.42 (1H, d, J = 12.0 Hz), 4.39 (1H, d, J = 11.0 Hz), 4.34 (1H, d, J = 6.0 Hz), 3.69 (1H, d, J = 10.5 Hz), 3.46 (1H, m), 3.37 (1H, m), 3.35 (1H, d, J = 10.5 Hz), 2.16 (1H, m), 2.11 (3H, s), 1.76 (1H, m).

[0171] (Compounds 10 to 14) [ka]

[0172] Compound 10 (27.44 g, 51.24 mmol) was dissolved in methanol (256 mL), 3 mol / L aqueous sodium hydroxide solution (34.2 mL, 0.103 mol) was added, and the mixture was stirred at room temperature for 30 minutes. Saturated ammonium chloride was added to the reaction mixture, and the mixture was concentrated. After dilution with deionized water, the mixture was extracted twice with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated to obtain crude compound 11. Crude compound 11 was azeotroped three times with pyridine, and the residue was dissolved in pyridine (256 mL) and cooled to 0 °C. Methanesulfonyl chloride (7.93 mL, 0.102 mol) was added, and the mixture was stirred at room temperature for 21 hours. The reaction mixture was cooled to 0 °C, deionized water was added, and the mixture was stirred and concentrated. Ethyl acetate was added to the residue, and the mixture was washed (deionized water, then saturated aqueous sodium bicarbonate), after which the organic layer was dried over magnesium sulfate and concentrated. The residue was azeotroped three times with toluene to obtain crude compound 12.

[0173] Crude compound 12 was dissolved in methanol (256 mL), 3 mol / L aqueous sodium hydroxide solution (51.2 mL, 0.154 mol) was added, and the mixture was stirred at 60 °C for 2 hours. Saturated ammonium chloride was added to the reaction mixture, and the mixture was concentrated. After dilution with deionized water, the mixture was extracted twice with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (silica gel 1000 cc, chloroform:methanol = 50:1 to 20:1) to obtain compound 14 (22.11 g, 44.80 mmol, 87.43%). 1 H-NMR (500 MHz, CDCl3): δ 9.75 (1H, s), 7.67 (1H, d, J = 8.0 Hz), 7.38-7.26 (10H, m), 6.10 (1H, d, J = 3.5 Hz), 5.33 (1H, d, J = 8.0 Hz), 4.58 (2H, m), 4.56 (1H, d, J = 12.0 Hz), 4.53 (1H, d, J = 10.5 Hz), 4.46 (1H, d, J = 11.5 Hz), 4.13 (1H, d, J = 2.5 Hz), 3.68 (1H, d, J = 9.5 Hz), 3.50 (1H, d, J = 10.0 Hz), 3.44-3.32 (2H, m), 2.06 (1H, m), 1.90 (1H, m).

[0174] (Compounds 14 to 16) [ka]

[0175] Compound 14 (5.00 g, 10.1 mmol), pyridine (12.2 mL), and N,N-dimethylaminopyridine (4.94 g, 40.4 mmol) were dissolved in dichloromethane (202 mL) and cooled to 0 °C. Trifluoromethanesulfonic anhydride (5.10 mL, 30.3 mmol) was added dropwise over 15 minutes, followed by stirring at the same temperature for 3 hours. Saturated aqueous sodium bicarbonate was added and the mixture was stirred. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (silica gel 200 cc, hexane:ethyl acetate = 3:1, 1:1, ethyl acetate) to give compound 15 (5.23 g, 8.36 mmol, 84.4%). Compound 15 (5.23 g, 8.36 mmol) was dissolved in toluene (83.6 mL), triethylamine trihydrofluoride (16.4 mL, 0.101 mol), and triethylamine (9.32 mL, 66.9 mmol) were added, and the mixture was stirred at 50 °C for 40 hours. The reaction mixture was diluted with ethyl acetate and washed with deionized water and then saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated. The residue was dissolved in acetone, silica gel was added, and the mixture was concentrated. Purification by silica gel column chromatography (silica gel 400 cc, hexane:ethyl acetate = 2:1 to 1:1) gave compound 16 (3.10 g, 6.26 mmol, 62.0%). 1H-NMR (500 MHz, CDCl3): δ 8.69 (1H, s), 7.71 (1H, d, J = 8.5 Hz), 7.40-7.22 (10H, m), 6.16 (1H, dd, J = 2.5, 15.5 Hz), 5.26 (1H, dd, J = 1.5, 8.5 Hz), 5.09 (1H, ddd, J = 3.0, 5.0, 53.0 Hz), 4.81 (1H, d, J = 11.5 Hz), 4.51 (1H, d, J = 11.5 Hz), 4.47 (1H, d, J = 10.5 Hz), 4.44 (1H, d, J = 11.0 Hz), 4.28 (1H, dd, J = 5.0, 17.5 Hz), 3.78 (1H, d, J = 10.5 Hz), 3.49-3.38 (3H, m), 2.21 (1H, m), 1.86 (1H, m).

[0176] (Compounds 16 to 17) [ka]

[0177] Compound 16 (6.90 g, 13.9 mmol) was dissolved in dichloromethane (110 mL) and cooled to -78°C. A solution of boron trichloride in dichloromethane (1.0 mol / L, 153 mL, 0.153 mol) was added dropwise over 40 minutes, and the mixture was stirred at the same temperature for 3 hours and at -20°C for 3 hours. Methanol (20 mL) was added dropwise to the reaction mixture at the same temperature, and the mixture was stirred and concentrated. The residue was azeotroped five times with methanol, then dissolved in methanol, silica gel was added, and the mixture was concentrated. The residue was purified by silica gel column chromatography (silica gel 200 cc, chloroform:methanol = 10:1) to give compound 17 (4.06 g, 12.9 mmol, 92.8%). 1H-NMR (500 MHz, DMSO-d6): δ 7.91 (1H, d, J = 8.0 Hz), 6.05 (1H, dd, J = 4.0, 15.5 Hz), 5.80 (1H, d, J = 6.0 Hz), 5.67 (1H, dd, J = 1.0, 8.0 Hz), 5.37 (1H, t, J = 5.0 Hz), 5.17 (1H, dt, J = 4.5, 53.5 Hz), 4.29 (1H, dt, J = 5.0, 14.0 Hz), 3.55 (1H, dd, J = 5.0, 12.0 Hz), 3.45-3.39 (3H, m), 1.97 (1H, m), 1.77 (1H, m).

[0178] (Compounds 17 to 18) [ka]

[0179] Compound 17 (9.03 g, 28.6 mmol) was dissolved in pyridine (143 mL), dimethoxytrityl chloride (13.6 g, 40.1 mmol) was added, and the mixture was stirred at room temperature for 24 hours. Methanol (5 mL) was added to the reaction mixture, and the mixture was concentrated. The residue was added with ethyl acetate and washed with deionized water and then saturated sodium bicarbonate water. The organic layer was dried over magnesium sulfate and concentrated. The residue was azeotroped three times with toluene and purified by silica gel column chromatography (silica gel 500 cc, chloroform:methanol = 50:1). Compound 18 (17.60 g, 28.5 mmol, 99.7%) was obtained. 1H-NMR (500 MHz, CDCl3): δ 8.53 (1H, s), 7.65 (1H, d, J = 8.0 Hz), 7.36-6.84 (13H, m), 6.14 (1H, dd, J = 3.5, 15.5 Hz), 5.35 (1H, dd, J = 2.5, 8.0 Hz), 5.18 (1H, ddd, J = 4.0, 5.5, 53.5), 4.62 (1H, dt, J = 5.5, 14.5 Hz), 3.80 (3H, s), 3.80 (3H, s), 3.38 (1H, d, J = 10.5 Hz), 3.35 (1H, d, J = 10.5 Hz), 3.33 (1H, m), 3.24 (1H, m), 2.59 (1H, dd, J = 3.0, 6.5 Hz), 2.05 (1H, m), 1.89 (1H, m).

[0180] (Compounds 18 to 20) [ka]

[0181] Compound 18 (1.0 g, 1.62 mmol) was dissolved in tetrahydrofuran (40 mL), followed by the sequential addition of triphenylphosphine (1.06 g, 4.05 mmol) and deionized water (1.2 mL). The mixture was stirred at 45 °C for 5.5 hours. After the reaction was complete, the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (10 mL), followed by the sequential addition of triethylamine (0.34 mL, 2.4 mmol) and ethyl trifluoroacetate (0.58 mL, 4.9 mmol). The mixture was stirred at room temperature for 14 hours. Subsequently, triethylamine (0.34 mL, 2.4 mmol) and ethyl trifluoroacetate (0.58 mL, 4.9 mmol) were added sequentially, followed by the sequential addition of triethylamine (0.34 mL, 2.4 mmol) and ethyl trifluoroacetate (0.58 mL, 4.9 mmol). The mixture was stirred at room temperature for 6 hours. After the reaction was complete, extraction with ethyl acetate was performed. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (ethyl acetate / hexane = 1 / 1 → ethyl acetate / hexane = 2 / 1) to obtain compound 20 (1.12 g, 1.62 mmol, quant. for 2 steps). 1 H-NMR (500 MHz, CDCl3): δ 8.16 (1H, brs), 7.55 (1H, d, J = 8.0 Hz), 7.35-7.24 (9H, m), 6.89 (1H, m), 6.85 (4H, d, J = 8.5 Hz), 6.12 (1H, dd, J = 15.0, 4.0 Hz), 5.41 (1H, dd, J = 8.0, 2.0 Hz), 5.25 (1H, ddd, J = 54, 4.5, 4.5 Hz), 4.54 (1H, m), 3.80 (6H, s), 3.35 (4H, m), 2.66 (1H, m), 2.06 (1H, m), 1.98 (1H, m).

[0182] (Compounds 20 to 21) [ka]

[0183] Compound 20 (1.47 g, 2.14 mmol) was azeotropically dehydrated three times with toluene and then dissolved in dichloromethane (10 mL). Subsequently, N,N-diisopropylethylamine (0.93 mL, 5.4 mmol) was added at room temperature, followed by 2-cyanoethyldiisopropylchlorophosphoramidite (1.1 mL, 4.71 mmol) at 0 °C, and the mixture was stirred at room temperature for 30 min. After completion of the reaction, the mixture was quenched with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1 / 1) to give compound 21 (1.65 g, 1.86 mmol, 87%). 31 P-NMR (202 MHz, CDCl3): δ 152.16 (0.55P, d, J = 12.12 Hz), 151.17 (0.45P, d, J = 12.12 Hz).

[0184] (Compounds 18 to 22) [ka]

[0185] Compound 18 (14.0 g, 22.7 mmol) was dissolved in N,N-dimethylformamide (45 mL), and tert-butyldimethylsilyl chloride (10.2 g, 68.0 mmol) and imidazole (9.26 g, 136.0 mmol) were added. The mixture was stirred at 50°C for 20 hours. After the reaction was completed, saturated aqueous sodium bicarbonate was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 3:1 to 1:1) to obtain compound 22 (16.2 g, 98%). 1 H-NMR (500MHz, CDCl3): δ 8.21 (1H, s), 7.84 (1H, d, J = 8.2 Hz), 7.34-7.22 (9H, m), 6.85 (4H, dd, J = 8.8, 3.5 Hz), 6.10 (1H, dd, J = 16.1, 2.4 Hz), 5.25 (1H, d, J = 8.2 Hz), 4.89 (1H, m), 4.60 (1H, dd, J = 18.1, 5.3 Hz), 3.80 (6H, s), 3.50 (1H, d, J = 10.7 Hz), 3.35 (1H, m), 3.23 (1H, m), 3.18 (1H, d, J = 10.6 Hz), 2.15 (1H, m), 1.67 (1H, m), 0.86 (9H, s), 0.10 (3H, s), 0.01 (3H, s).

[0186] (Compounds 22 to 23) [ka]

[0187] Compound 22 (16.2 g, 22.2 mmol) was dissolved in pyridine (110 mL) and cooled to 0°C. Phosphoryl chloride (10.4 mL, 110.8 mmol) was added dropwise over 15 minutes, and triazole (26.0 g, 376.7 mmol) was added at the same temperature. After returning the temperature to room temperature and stirring for 4 hours, ethyl acetate was added. The organic layer was washed with water and saturated aqueous sodium bicarbonate and then dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 2:1 to 1:1) to obtain compound 23 (14.1 g, 81%). 1 H-NMR (500MHz, CDCl3): δ 9.25 (1H, s), 8.81 (1H, d, J = 7.3 Hz), 8.08 (1H, s), 7.35-7.28 (5H, m), 7.23 (4H, dd, J = 9.1, 6.6 Hz), 6.86 (4H, dd, J = 8.8, 1.9 Hz), 6.38 (1H, d, J = 7.3 Hz), 6.21 (1H, d, J = 16.1 Hz), 4.90 (1H, dd, J = 52.5, 4.9 Hz), 4.66 (1H, dd, J = 23.6, 5.0 Hz), 3.81 (6H, s), 3.70 (2H, d, J = 11.0 Hz), 3.38 (1H, m), 3.29 (1H, m), 3.27 (1H, d, J = 10.7 Hz), 2.25(1H, m), 1.67 (1H, m), 0.84 (9H, s), 0.09 (3H, s), -0.02 (3H, s).

[0188] (Compounds 23 to 24) [ka]

[0189] Compound 23 (14.1 g, 18.0 mmol) was dissolved in 1,4-dioxane (135 mL), 28% aqueous ammonia (45 mL) was added, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 40:1 to 20:1) to give compound 24 (12.7 g, 96%). 1 H-NMR (500MHz, CDCl3): δ 8.08 (1H, d, J = 7.3 Hz), 7.37-7.33 (2H, m), 7.30 (2H, t, J = 7.4 Hz), 7.28-7.21 (5H, m), 6.84 (4H, dd, J = 9.0, 4.6 Hz), 6.12 (1H, d, J = 16.1 Hz), 5.17 (1H, d, J = 7.6 Hz), 4.87 (1H, dd, J = 53.6, 5.7 Hz), 4.61 (1H, dd, J = 22.7, 5.0 Hz), 3.80 (3H, s), 3.80 (3H, s), 3.57 (2H, d, J = 10.4 Hz), 3.35 (1H, m), 3.26 (1H, m), 3.15 (1H, d, J = 10.4 Hz), 2.20 (1H, m), 1.68 (1H, m), 0.82 (9H, s), 0.07 (3H, s), -0.04 (3H, s).

[0190] (Compounds 24 to 25) [ka]

[0191] Compound 24 (12.7 g, 17.4 mmol) was dissolved in pyridine (90 mL), benzoyl chloride (2.4 mL, 20.8 mmol) was added, and the mixture was stirred at room temperature for 14 hours. After the reaction was completed, ethyl acetate was added, and the organic layer was washed with saturated aqueous sodium bicarbonate and then dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 3:1 to 1:1) to obtain compound 25 (14.0 g, 97%). 1 H-NMR (500MHz, CDCl3): δ8.49 (1H, d, J = 7.6 Hz), 7.90 (2H, d, J = 7.6 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.52 (2H, t, J = 7. 7Hz), 7.37-7.30 (5H, m), 7.27-7.24 (6H, m), 6.87 (4H, d, J = 8.5 Hz), 6.20 (1H, d, J = 16.1 Hz), 4.90 (1H, dd, J = 53.0, 5.0 Hz), 4.62 (1H, dd, J = 22.5, 4.9 Hz), 3.82 (6H, s), 3.62 (1H, d, J = 10.7 Hz), 3.37 (1H, m), 3.29 (1H, m), 3.24 (1H, d, J = 10.7 Hz), 2.25 (1H, m), 1.69 (1H, m), 0.84 (9H, s), 0.09 (3H, s), -0.02 (3H, s).

[0192] (Compounds 25 to 26) [ka]

[0193] Compound 25 (14.0 g, 16.8 mmol) was dissolved in tetrahydrofuran (80 mL), tetrabutylammonium fluoride (20.1 mL, 20.1 mmol) was added, and the mixture was stirred at room temperature for 2 hours and then concentrated. The residue was dissolved in ethyl acetate, washed with saturated aqueous sodium bicarbonate, and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1:1 to 1:3) to obtain compound 26 (11.5 g, 95%). 1H-NMR (500 MHz, CDCl3): δ 8.67 (1H, br), 8.23 ​​(1H, s), 7.87 (2H, d, J = 6.9 Hz), 7.62 (1H, m), 7.52 (2H, t, J = 7.72 Hz), 7.41-7.24 (11H, m), 6.88 (4H, dd, J = 9.1, 1.3 Hz), 6.19 (1H, dd, J = 16.1, 1.6 Hz), 5.16 (1H, m), 4.67 (1H, d, J = 20.49 Hz), 3.81 (6H, s), 3.49 (1H, d, J = 10.7 Hz), 3.3 9(1H ,d, J = 10.7 Hz), 3.37 (1H, m), 3.27 (1H, m), 2.12 (1H, m), 1.8 (1H, m).

[0194] (Compounds 26 to 28) [ka]

[0195] Compound 26 (11.5 g, 15.9 mmol) was dissolved in tetrahydrofuran (160 mL) and water (5.3 mL). Triphenylphosphine (10.4 g, 39.7 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The mixture was then concentrated to give crude compound 27. Crude compound 27 was azeotroped three times with toluene, then dissolved in dichloromethane (160 mL). Ethyl trifluoroacetate (5.7 mL, 47.7 mmol) and triethylamine (3.3 mL, 23.9 mmol) were added, and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, chloroform was added, and the organic layer was washed with saturated aqueous sodium bicarbonate and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 80:1 to 5:1) to give compound 28 (9.67 g, 77%). 1H-NMR (500 MHz, CDCl3): δ 8.74 (1H, brs), 8.19 (1H, d, J = 7.6 Hz), 7.85 (2H, d, J = 7.6Hz), 7.61 (1H, t, J = 7.4 Hz), 7.50 (2H, t, J = 7.7 Hz), 7.38 (2H, d, J = 7.3 Hz), 7.35-7.17 (9H, m), 6.86 (4H, dd, J = 9.0, 2.1 Hz), 6.18 (1H, dd, J = 15.6, 2.1 Hz), 5.20 (1H,m), 4.65 (1H, dd, J = 20.0, 5.2 Hz), 3.80 (3H, s), 3.79 (3H, s), 3.44 (2H, s), 3.36 (2H, q, J = 6.7 Hz), 2.14 (1H, m), 2.04 (1H, m).

[0196] (Compounds 28 to 29) [ka]

[0197] Compound 28 (5.67 g, 7.17 mmol) was dissolved in dichloromethane (72 mL), N,N-diisopropylethylamine (3.1 mL, 17.9 mmol), and 2-cyanoethyldiisopropylchlorophosphoramidite (3.5 mL, 15.8 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, chloroform was added, and the organic layer was washed with saturated aqueous sodium bicarbonate and water and then dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 20:1) to give compound 29 (5.96 g, 84%). 31 P-NMR (202 MHz, CDCl3): δ 151.53 (0.52P, d, J = 26.5 Hz), 150.76 (0.48P, d, J = 19.0 Hz). [Example]

[0198] In this embodiment, 2'-Deoxy-3'-O-(2-cyanoethyl-N,N-diisopropylaminophosphino)-5'-O-dimethoxytrityl-2'-fluoro-4'-C-trifluoroacetamidopropyluridine (compound 114) was synthesized.

[0199] Synthesis of 3,5-di-O-Benzyl-4-C-ethoxycarbonylvinyl-1,2-O-isopropylidene-α-D-ribofuranose (Compound 102)

[0200] [ka]

[0201] Triethyl phosphonoacetate (13.44 mL, 67.14 mmol) was dissolved in tetrahydrofuran (149 mL) and cooled to 0°C. Sodium hydride (60% in oil, 2.50 g, 62.5 mmol) was added and stirred for 1 hour. A solution of compound 101 (identical to compound 5 in Example 1) (17.89 g, 44.77 mmol) in tetrahydrofuran (149 mL) was added dropwise, and the mixture was allowed to warm to room temperature and stirred for 5 hours. A saturated aqueous solution of ammonium chloride was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1) to give compound 102 (19.49 g, 41.42 mmol, 92.52%). 1H-NMR (CDCl3): d 7.37-7.21 (11H, m), 6.24 (1H, d, J = 16.0), 5.76 (1H, d, J = 4.0), 4.77 (1H, d, J = 12.5), 4.60 (1H, d, J = 12.0), 4.57 (1H, t, J = 4.0), 4.50 (1H, d, J = 12.0), 4.41 (1H, d, J = 12.0), 4.27 (1H, d, J = 4.5), 4.22-4.15 (2H, m), 3.35 (1H, d, J = 11.0), 3.32 (1H, d, J = 11.0), 1.47 (3H, s), 1.28 (3H, s), 1.27 (3H, t, J = 7.5).

[0202] Synthesis of 3,5-di-O-Benzyl-4-C-ethoxycarbonylethyl-1,2-O-isopropylidene-α-D-ribofuranose (Compound 103)

[0203] [ka]

[0204] Compound 102 (19.49 g, 41.42 mmol) was dissolved in methanol (207 mL), Pd / C(en) (390 mg) was added, and the mixture was stirred under a hydrogen atmosphere for 52 hours. The catalyst was removed by filtration through Celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1) to give compound 103 (19.32 g, 41.06 mmol, 99.13%). 1H-NMR (CDCl3): d 7.34-7.23 (10H, m), 5.75 (1H, d, J = 4.0), 4.76 (1H, d, J = 12.0), 4.62 (1H, dd, J = 4.0, 5.5), 4.56 (1H, d, J = 12.0), 4.49 (1H, d, J = 12.0), 4.40 (1H, d, J = 12.0), 4.13 (1H, d, J = 5.5), 4.10 (2H, q, J = 7.0), 3.40 (1H, d, J = 10.5), 3.25 (1H, d, J = 10.5), 2.58 (2H, m), 2.31 (1H, m), 1.87 (1H, m), 1.62 (3H, s), 1.32 (3H, s), 1.23 (3H, t, J = 7.0).

[0205] Synthesis of 3,5-Di-O-benzyl-4-C-hydroxypropyl-1,2-O-isopropylidene-α-D-ribofuranose (Compound 104)

[0206] [ka]

[0207] Compound 103 (19.32 g, 41.06 mmol) was dissolved in tetrahydrofuran (205 mL) and cooled to 0 °C. Lithium aluminum hydride (3.12 g, 82.2 mmol) was added and stirred for 30 minutes. Deionized water (3.12 mL) and then 15% aqueous sodium hydroxide solution (3.12 mL) were carefully added dropwise, followed by deionized water (9.36 mL) and stirring. Insoluble matter was removed by filtration through Celite, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give compound 104 (17.33 g, 40.44 mmol, 98.49%). 1H-NMR (CDCl3): d 7.36-7.23 (10H, m), 5.76 (1H, d, J = 4.0), 4.75 (1H, d, J = 12.5), 4.62 (1H, dd, J = 4.0, 5.0), 4.57 (1H, d, J = 12.0), 4.51 (1H, d, J = 12.0), 4.41 (1H, d, J = 12.0), 4.16 (1H, d, J = 5.0), 3.70 (1H, m), 3.63 (1H, m), 3.50 (1H, d, J = 10.5), 3.32 (1H, d, J = 10.5), 2.21 (1H, m), 1.77-1.69 (3H, m), 1.87 (1H, m), 1.62 (3H, s), 1.33 (3H, s).

[0208] Synthesis of 4-C-Azidopropyl-3,5-di-O-benzyl-1,2-O-isopropylidene-aD-ribofuranose (Compound 105)

[0209] [ka]

[0210] Compound 104 (17.23 g, 40.21 mmol) and triphenylphosphine (21.1 g, 80.4 mmol) were dissolved in tetrahydrofuran (268 mL) and cooled to 0 °C. Bis(2-methoxyethyl) azodicarboxylate (18.8 g, 80.3 mmol) was added and stirred for 5 minutes. After that, diphenylphosphate azide (17.3 mL, 80.5 mmol) was added dropwise over 10 minutes. The reaction mixture was allowed to warm to room temperature and stirred for 18 hours. 28% aqueous ammonia (15 mL) was added and stirred for 1 hour. Saturated sodium bicarbonate was added to the reaction mixture, followed by stirring. The mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was dissolved in acetone (100 mL), silica gel was added, and the mixture was concentrated. The mixture was purified by silica gel column chromatography (hexane:ethyl acetate = 5:1) to give compound 105 (15.82 g, 34.88 mmol, 86.74%). 1 H-NMR (CDCl3): d 7.35-7.23 (10H, m), 5.74 (1H, d, J = 4.0), 4.76 (1H, d, J = 12.0), 4.61 (1H, dd, J = 4.0, 5.5), 4.56 (1H, d, J = 12.0), 4.51 (1H, d, J = 12.0), 4.41 (1H, d, J = 12.0), 4.12 (1H, d, J = 5.5), 3.43 (1H, d, J = 10.5), 3.28 (1H, d, J = 10.5), 3.27 (2H, m), 2.17 (1H, m), 1.86 (1H, m), 1.68-1.57 (2H, m), 1.60 (3H, s), 1.33 (3H, s).

[0211] Synthesis of 1,2-Di-O-acetyl-4-C-azidopropyl-3,5-di-O-benzyl-D-ribofuranose (Compound 106)

[0212] [ka]

[0213] Compound 105 (15.85 g, 34.88 mmol) was dissolved in 70% acetic acid (349 mL), trifluoroacetic acid (35 mL) was added, and the mixture was stirred for 15 hours. The reaction mixture was concentrated, and the residue was azeotroped five times with toluene. The residue was dissolved in pyridine (174 mL), and acetic anhydride (19.8 mL, 0.209 mol) was added, and the mixture was stirred for 24 hours. The reaction mixture was concentrated, and the residue was azeotroped five times with toluene. The residue was dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 10:1 to 5:1) to give compound 106 (14.8 g, 29.7 mmol, 85.1%). 1H-NMR (CDCl3): d7.38-7.25 (10H, m), 6.34 (0.14H, d, J = 4.6), 6.10 (0.87H, s), 5.33 (0.87H, d, J = 5.5), 5.27 (0.14H, dd, J = 5.0, 6.4), 4.62-4.42 (4H, m), 4.30 (0.87H, d, J = 5.0), 4.21 (0.14H, d, J = 6.4), 3.41-3.33 (2H, m), 3.30-3.19 (2H, m), 2.11 (2.61H, s), 2.10 (0.42H, s), 2.05 (0.42H, s), 1.88 (2.61H, s), 1.97-1.59 (4H, m).

[0214] Synthesis of 4'-C-Azidopropyl-3',5'-di-O-benzyl-bD-arabinofuranosyluracil (Compound 108)

[0215] [ka]

[0216] Compound 106 (14.27 g, 28.68 mmol), uracil (6.43 g, 57.4 mmol), and N,O-bis(trimethylsilyl)acetamide (42.5 mL, 0.172 mol) were added to 1,2-dichloroethane (143 mL) and stirred at 85°C for 2 hours. The reaction mixture was cooled to 0°C, and trimethylsilyl trifluoromethanesulfonate (10.4 mL, 57.6 mmol) was added. The mixture was stirred at 85°C for 20 hours. Saturated aqueous sodium bicarbonate was added to the reaction mixture, and the insoluble solids were removed by filtration through Celite. The organic layer of the filtrate was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to give compound 107 (12.28 g, 22.34 mmol, 77.89%).

[0217] Compound 107 (12.28 g, 23.34 mmol) was dissolved in methanol (117 mL), and 3 mol / L aqueous sodium hydroxide solution (15.6 mL, 48.8 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was neutralized with 1 mol / L hydrochloric acid and then concentrated. The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate and concentrated. The residue was azeotroped three times with pyridine and then dissolved in pyridine (117 mL). The solution was cooled to 0°C, and methanesulfonyl chloride (3.61 mL, 46.6 mmol) was added. The mixture was stirred at room temperature for 16 hours. Deionized water was added to the reaction mixture, and the mixture was concentrated. The residue was then dissolved in ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate and concentrated. The residue was azeotroped three times with toluene. The residue was dissolved in methanol (117 mL), 3 mol / L aqueous sodium hydroxide solution (23.3 mL, 69.9 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was neutralized with 1 mol / L hydrochloric acid and then concentrated. The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to obtain compound 108 (10.67 g, 21.02 mmol, 90.06%). 1 H-NMR (CDCl3): d 9.76 (1H, br.s), 7.73 (1H, d, J = 8.2), 7.38-7.24 (10H, m), 6.12 (1H, d, J = 4.6), 5.32 (1H, d, J = 7.8), 4.81 (1H, d, J = 12.4), 4.61-4.43 (5H, m), 4.14 (1H, d, J = 3.7), 3.63 (1H, d, J = 10.1), 3.47 (1H, d, J = 10.1), 3.25 (2H, m), 1.79-1.53 ​​(4H, m).

[0218] Synthesis of 4'-C-Azidopropyl-3',5'-di-O-benzyl-2'-deoxy-2'-fluorouridine (Compound 109)

[0219] [ka]

[0220] Compound 108 (10.50 g, 20.69 mmol), pyridine (25.0 mL), and 4-dimethylaminopyridine (10.1 g) were dissolved in dichloromethane (414 mL) and cooled to 0 °C. Trifluoromethanesulfonic anhydride (10.2 mL, 62.2 mmol) was added and stirred for 90 minutes. After stirring, saturated aqueous sodium bicarbonate was added and stirred. The organic layer was washed with 1 mol / L hydrochloric acid and then saturated aqueous sodium bicarbonate, dried over magnesium sulfate, and concentrated. The residue was azeotroped three times with toluene and purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 to 1:1). The residue was dissolved in toluene (168 mL), and triethylamine trihydrofluoride (33.0 mL) and triethylamine (18.8 mL) were added. The mixture was stirred at 50 °C for 41 hours. The reaction mixture was diluted with ethyl acetate, washed with deionized water, and then saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate. The dried solution was concentrated, and the precipitated solid was collected by filtration. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (hexane:ethyl acetate=2:1 to 1:1) to obtain Compound 109 (7.00 g, 13.7 mmol, 66.2%). 1 H-NMR (CDCl3): d 8.20 (1H, br.s), 7.72 (1H, d, J = 7.8), 7.41-7.20 (10H, m), 6.18 (1H, dd, J = 3.2, 15.1), 5.26 (1H, d, J = 8.2), 5.01 (1H, ddd, J = 3.2, 5.0, 53.1), 4.82 (1H, d, J = 11.9), 4.51 (1H, d, J = 11.9), 4.47 (1H, d, J = 11.0), 4.43 (1H, d, J = 10.5), 4.25 (1H, dd, J = 5.0, 17.4), 3.73 (1H, d, J = 10.5), 3.46 (1H, d, J = 10.1), 3.28 (2H, m), 1.95-1.65 (4H, m).

[0221] Synthesis of 4'-C-Azidopropyl-2'-deoxy-2'-fluorouridine (Compound 110)

[0222] [ka]

[0223] Compound 109 (7.00 g, 13.7 mmol) was dissolved in dichloromethane (108 mL) and cooled to -78°C. A 1 mol / L solution of boron trichloride in dichloromethane (151 mL, 151 mmol) was added, and the mixture was heated to -20°C and stirred for 2 hours. The reaction mixture was cooled to -78°C again, and methanol (20 mL) was added dropwise thereto and then concentrated. The residue was azeotroped five times with methanol and then purified by silica gel column chromatography (chloroform:methanol = 20:1 to 10:1) to give compound 110 (4.15 g, 12.6 mmol, 92.0%). 1 H-NMR (DMSO-d6): d 11.40 (1H, s), 7.91 (1H, d, J = 8.2), 6.04 (1H, dd, J = 4.1, 14.6), 5.69-5.66 (2H, m), 5.30 (1H, t, J = 5.0), 5.14 (1H, dt, J = 5.0, 53.5), 4.27 (1H, dt, 5.5, 12.8), 3.54 (1H, dd, J = 5.0, 11.9), 3.43 (1H, dd, J = 5.0, 11.9), 3.32 (2H, m), 1.73-1.50 (4H, m).

[0224] Synthesis of 4'-C-Azidopropyl-2'-deoxy-5'-O-dimethoxytrityl-2'-fluorouridine (Compound 111)

[0225] [ka]

[0226] Compound 110 (3.50 g, 10.6 mmol) was dissolved in pyridine (53.0 mL), dimethoxytrityl chloride (5.03 g, 14.8 mmol) was added, and the mixture was stirred at room temperature for 14 hours. Methanol (5 mL) was added to the reaction mixture, and the mixture was concentrated. Ethyl acetate was added to the residue, and the mixture was washed with deionized water and then saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated. The residue was azeotroped three times with toluene. The residue was purified by silica gel column chromatography (chloroform:methanol = 50:1) to give compound 111 (6.51 g, 10.3 mmol, 97.2%). 1 H-NMR (CDCl3): d 8.19 (1H, br.s), 7.68 (1H, d, J = 8.2), 7.37-6.84 (13H, m), 6.15 (1H, dd, J = 3.2, 15.1), 5.15 (1H, ddd, J = 3.7, 5.0, 53.5), 4.63 (1H, dt, J = 5.5, 15.1), 3.81 (6H, s), 3.38 (1H, d, J = 10.5), 3.33 (1H, d, J = 10.5), 3.23 (2H, m), 2.34 (1H, dd, J = 3.7, 6.4), 1.82-1.36 (4H, m).

[0227] Synthesis of 4'-C-Aminopropyl-2'-deoxy-5'-O-dimethoxytrityl-2'-fluorouridine (Compound 112)

[0228] [ka]

[0229] Compound 111 (6.30 g, 9.97 mmol) was dissolved in tetrahydrofuran (24.6 mL) and deionized water (7.4 mL), and triphenylphosphine (6.54 g, 24.9 mmol) was added and stirred at 45 °C for 17 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (chloroform:methanol = 10:1 to 5:1 with 1% triethylamine) to give compound 112 (5.86 g, 9.68 mmol, 97.1%). 1 H-NMR (DMSO-d6): d 7.68 (1H, d, J = 8.2), 7.36-6.85 (13H, m), 5.90 (1H, dd, J = 2.7, 18.3), 5.22 (1H, d, J = 8.2), 5.13 (1H, ddd, J = 2.7, 5.5, 54.0), 4.49 (1H, dd, 5.5, 20.1), 3.70 (6H, s), 3.29 (2H, br.s), 3.12 (1H, d, J = 10.1), 3.07 (1H, d, J = 10.5), 2.37 (2H, t, J = 6.9), 1.66-1.49 (2H, m), 1.24-0.97 (2H, m).

[0230] Synthesis of 2'-Deoxy-5'-O-dimethoxytrityl-2'-fluoro-4'-C-trifluoroacetamidopropyluridine (Compound 113)

[0231] [ka]

[0232] Compound 112 (2.50 g, 4.13 mmol) was suspended in dichloromethane (51.0 mL), triethylamine (1.74 mL, 12.5 mmol) and ethyl trifluoroacetate (1.48 mL) were added, and the mixture was stirred at room temperature for 19 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (chloroform:methanol = 20:1) to give compound 113 (2.82 g, 4.02 mmol, 97.3%). 1 H-NMR (CDCl3): d 8.60 (1H, br.s), 7.64 (1H, d, J = 7.6), 7.35-6.84 (13H, m), 6.66 (1H, br.s), 6.17 (1H, dd, J = 4.6, 14.6), 5.35 (1H, d, J = 8.2), 5.20 (1H, dt, J = 4.6, 53.1), 4.59 (1H, dt, J = 5.0, 11.9), 3.80 (6H, s), 3.36-3.25 (4H, m), 2.70 (1H, br.s), 1.79-1.38 (4H, m).

[0233] Synthesis of 2'-Deoxy-3'-O-(2-cyanoethyl-N,N-diisopropylaminophosphino)-5'-O-dimethoxytrityl-2'-fluoro-4'-C-trifluoroacetamidopropyluridine (compound 114)

[0234] [ka]

[0235] Compound 113 (2.77 g, 3.95 mmol) was dissolved in dichloromethane (19.8 mL), N,N-diisopropylethylamine (1.72 mL, 10.1 mmol), and 2-cyanoethyldiisopropylchlorophosphoramidite (2.03 mL) were added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give compound 114 (3.10 g, 3.44 mmol, 87.1%). 31 P-NMR (CDCl3): d 152.25 (0.43P, d, J = 16.8), 151.20 (0.64P, d, J = 16.8).

Claims

1. A method for producing a nucleoside derivative or a salt thereof represented by the following formula (2): A production method including all of the reactions of the following formulas (5), (6), (7)″ and (7). 【Transformation 56】 (In formula (2), X represents a fluorine atom, and R 2 and R 4 may be the same or different, and each represents a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P(=O) n R 5 R 6 (n represents 0 or 1, R 5 and R 6 may be the same or different and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group. However, when n is 1, R 5 and R 6 and R are not both hydrogen atoms. 3 are NHR groups each having a linking group. 7 , azido group, amidino group, or guanidino group, and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group. 7 represents a protecting group for a hydrogen atom, an alkyl group, an alkenyl group, or an amino group, and the linking group is an alkylene group having 1 to 6 carbon atoms. 【Chemistry 57】 (In formulas (5) to (7), R 10 and R 11 are each independently or R 10 and R 11 together represent a protecting group for a hydroxyl group, and R 12 represents formula (8), and R 14 represents an alkylene group, Bn represents a benzyl group, and Tf represents a trifluoromethanesulfonyl group. Furthermore, formula (7) represents a reaction in which a purified product containing the right-hand side compound of formula (7)'' obtained by purifying the reaction solution of formula (7)'' by column chromatography is used as a product containing the left-hand side compound, and triethylamine trihydrofluoride is used to fluorinate the trifluorosulfonyloxy group at the 2'-position of the left-hand side compound.

2. In formulas (5) to (7), R 10 and R 11 is R 10 and R 11 The method according to claim 1 , wherein

3. The method according to claim 1 or 2, further comprising the reaction of the following formula (5)' following the reaction of the formula (5): 【Chemistry 58】

4. The method according to claim 3 , further comprising the reaction of the following formula (5)″ subsequent to the reaction of the formula (5)′: 【Chemistry 59】

5. The method according to any one of claims 1 to 4, comprising the reaction of the following formula (7)' prior to the reaction of the formula (7)'': 【Transformation 60】 (In formula (7)', R 15 represents a leaving group.)

6. A method for producing an oligonucleotide derivative, comprising the steps of: A method for producing an oligonucleotide derivative using a nucleoside derivative or a salt thereof represented by the following formula (2): A production method including all of the reactions of the following formulas (5), (6), (7)″ and (7). 【Chemistry 61】 (In formula (2), X represents a fluorine atom, and R 2 and R 4 may be the same or different, and each represents a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P(=O) n R 5 R 6 (n represents 0 or 1, R 5 and R 6 may be the same or different and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group. However, when n is 1, R 5 and R 6 and R are not both hydrogen atoms. 3 are NHR groups each having a linking group. 7 , azido group, amidino group, or guanidino group, and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group. 7 represents a protecting group for a hydrogen atom, an alkyl group, an alkenyl group, or an amino group, and the linking group is an alkylene group having 1 to 6 carbon atoms. 【Transformation 62】 (In formulas (5) to (7), R 10 and R 11 are each independently or R 10 and R 11 together represent a protecting group for a hydroxyl group, and R 12 represents formula (8), and R 14 represents an alkylene group, Bn represents a benzyl group, and Tf represents a trifluoromethanesulfonyl group. Furthermore, formula (7) represents a reaction in which a purified product containing the right-hand side compound of formula (7)'' obtained by purifying the reaction solution of formula (7)'' by column chromatography is used as a product containing the left-hand side compound, and triethylamine trihydrofluoride is used to fluorinate the trifluorosulfonyloxy group at the 2'-position of the left-hand side compound.

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