Nucleoside derivative having 4'-position branched substituent group

Abstract

Provided is a nucleoside derivative or a salt thereof, which is represented by formula (1) or (2). (In formula (1) and formula (2), R1 denotes a hydrogen atom, a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted by an alkyl group or an alkenyl group, or a protected group. In formula (1) and formula (2), R2 and R4 may be the same as, or different from, each other and each denote a hydrogen atom, a protecting group for a hydroxyl group, a sulfonyl group, a protected sulfonyl group or -P(=O)nR5R6 (n is 0 or 1, and R5 and R6 may be the same as, or different from, each other and each denote 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, if n is 1, R5 and R6 cannot both be hydrogen atoms. R3 denotes NHR7 (R7 denotes a hydrogen atom, an alkyl group, an alkenyl group or a protecting group for an amino group, and B denotes 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.)

Description

Nucleoside derivatives having a branched substituent at the 4' position

[0001] The present specification relates to a nucleoside derivative having a branched substituent at the 4'-position (hereinafter simply referred to as a nucleoside derivative), and the like.

[0002] In recent years, progress has been made in the development of nucleic acid drugs targeting pathogenic genes. For example, antisense oligonucleotides (ASOs) are single-stranded DNAs of approximately 15-20 mer that bind complementarily to target mRNA and inhibit its translation.

[0003] ASOs have two main mechanisms of action. One is splicing inhibition by forming a complementary strand with the mRNA precursor. The other is mRNA cleavage by RNase H, which forms a complementary strand with the mRNA and recognizes the DNA / RNA strand. The mRNA cleaved by RNase H is finally cleaved by nucleases.

[0004] Various nucleoside derivatives useful for producing nucleic acid drugs such as ASO have been developed. For example, a nucleoside derivative in which an aminoethyl group is introduced in place of the hydrogen atom at the 4'-position of ribose is known (Patent Document 1).

[0005] This nucleoside derivative is known to have excellent nuclease resistance, and its effectiveness as an ASO has also been confirmed.

[0006] Furthermore, LNA and BNA are also known as nucleoside derivatives.

[0007] International Publication No. 2018 / 110678

[0008] However, even with these nucleoside derivatives, there is still a demand for nucleic acid drugs that further promote mRNA translation inhibition and improve their therapeutic efficacy. To this end, it is necessary to further improve nuclease resistance while maintaining affinity with the target RNA and simultaneously promote mRNA cleavage by RNase H.

[0009] The present specification provides nucleoside derivatives and the like that contribute to making nucleic acid medicines more practical.

[0010] The present inventors focused on modifications at the 4'-position of ribose and attempted a new modification at the 4'-position. They discovered that a structural unit derived from a nucleoside derivative in which a branched nitrogen-containing propoxy group is introduced at the 4'-position of ribose can contribute to improving the activity of an oligonucleotide to degrade mRNA by RNase H and nuclease resistance, while maintaining its affinity for the target RNA. The present specification provides the following means.

[0011] [1] A nucleoside derivative or a salt thereof represented by the following formula (1) or (2): (In formula (1) and formula (2), 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 formulas (1) and (2), 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 is NHR 7 (R 7 represents a protecting group for a hydrogen atom, an alkyl group, an alkenyl group, or an amino 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. [2] In the formula (1) and the formula (2), R 7represents a hydrogen atom. [3] An oligonucleotide derivative or a salt thereof according to (1), comprising at least one structural unit derived from the nucleoside derivative according to [1] or [2]. [4] The oligonucleotide derivative or a salt thereof according to [3], comprising at least one structural unit in total within 4 bases from the 3'-end of the oligonucleotide derivative and within 4 bases from the 5'-end of the oligonucleotide derivative. [5] The oligonucleotide derivative or a salt thereof according to [4], comprising at least three structural units in total within 4 bases from the 3'-end of the oligonucleotide derivative and within 4 bases from the 5'-end of the oligonucleotide derivative. [6] The oligonucleotide derivative or a salt thereof according to [5], comprising at least five structural units in total within 4 bases from the 3'-end of the oligonucleotide derivative and within 4 bases from the 5'-end of the oligonucleotide derivative. [7] The oligonucleotide derivative or a salt thereof according to [4], comprising one or more structural units in total within 4 bases from the 3'-end of the oligonucleotide derivative. [8] The oligonucleotide or salt thereof according to [4], which comprises one or more of the structural units within a range of four bases from the 5'-end of the oligonucleotide derivative. [9] The oligonucleotide or salt thereof according to [8], which comprises one or more of the structural units within a range of four bases from the 5'-end of the oligonucleotide derivative, other than at the 5'-end.

[10] The oligonucleotide or salt thereof according to any of [3] to [9], which comprises at least one structural unit within a range excluding a range of four bases from the 3'-end of the oligonucleotide derivative and a range of four bases from the 5'-end of the oligonucleotide derivative.

[11] The oligonucleotide or salt thereof according to any of [3] to

[10] , which comprises a structural unit derived from a deoxyribonucleoside in addition to the structural unit derived from the nucleoside derivative according to [1] or [2].

[12] A gene expression inhibitor targeting mRNA, comprising the oligonucleotide or salt thereof according to any of [3] to

[10] .

[0012] FIG. 1 shows 4'-(S)-2-aminopropoxythymidine (4'-(S)-2-APoT) and 4'-(R)-2-aminopropoxythymidine (4'-(R)-2-APoT). FIG. 2 shows a synthetic scheme for a phosphoramidite of 4'-(S)-2-APoT. FIG. 3 shows a synthetic scheme for a phosphoramidite of 4'-(R)-2-APoT. FIG. 4 shows the sequences of synthesized oligonucleotides. FIG. 5 shows the results of evaluating the thermal stability of oligonucleotides into which 4'-(S)-2-APoT and 4'-(R)-2-APoT have been introduced. FIG. 6 shows the results of evaluating the RNase H activity of oligonucleotides into which 4'-(S)-2-APoT and 4'-(R)-2-APoT have been introduced. FIG. 7 shows the results of evaluating the nuclease resistance of oligonucleotides into which 4'-(S)-2-APoT and 4'-(R)-2-APoT have been introduced. 1 shows a synthetic scheme for 4'-(S)-2-APoT-2'-OMe-5Me. 2 shows a synthetic scheme for 4'-(R)-2-APoT-2'-OMe-5Me. 3 shows the results of evaluating the gene expression-inhibiting activity of ASOs into which 4'-(S)-2-APoT-2'-OMe and 4'-(R)-2-APoT-2'-OMe have been introduced.

[0013] The present specification relates to nucleoside derivatives useful for nucleic acid medicines, oligonucleotides having structural units derived from these nucleoside derivatives, and their use and production methods.

[0014] The nucleoside derivative disclosed in the present specification (hereinafter also referred to as the present nucleoside derivative) has a 2-aminopropoxy group at the 4'-position of ribose, thereby enabling an oligonucleotide having a structural unit derived from such a nucleoside derivative to maintain its affinity with a target RNA, while improving the activity of mRNA degradation by RNase H and improving nuclease resistance.

[0015] The changes in thermal stability, RNase H degradation activity, and nuclease resistance resulting from the introduction of a 2-aminopropoxy group at the 4' position of ribose were not easily predictable based on previous findings. However, unexpectedly, the present nucleoside derivative was able to improve nuclease resistance while maintaining affinity for RNA and RNase H degradation activity. Nuclease resistance can inhibit degradation by various nucleases inside and outside of cells, thereby greatly contributing to the gene expression-inhibiting activity of ASOs.

[0016] 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 traditionally 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.

[0017] Representative and non-limiting examples of the present disclosure will be described in detail below, with appropriate reference to the drawings. This detailed description is intended simply to provide those skilled in the art with details for implementing preferred examples of the present disclosure, and is not intended to limit the scope of the present disclosure. Furthermore, the additional features and inventions disclosed below can be used separately or together with other features and inventions to provide further improved nucleoside derivatives and uses thereof.

[0018] Furthermore, the combinations of features and steps disclosed in the following detailed description are not essential to implementing the present disclosure in its broadest sense, but are described solely to specifically illustrate exemplary embodiments of the present disclosure. Furthermore, the various features of the exemplary embodiments described above and below, and the various features of those described in the independent and dependent claims, do not necessarily have to be combined in the exact embodiments described herein, or in the exact order listed, to provide additional and useful embodiments of the present disclosure.

[0019] All features described in this specification and / or claims are intended to be disclosed individually and independently of one another as limitations on the original disclosure and claimed particulars, apart from the configuration of features described in the examples and / or claims. Furthermore, all numerical ranges and group or aggregate descriptions are intended to disclose intermediate configurations as limitations on the original disclosure and claimed particulars. Various embodiments of the disclosure of this specification are described in detail below.

[0020] (Nucleoside Derivative of the Present) The nucleoside derivative of the present invention may be a nucleoside derivative represented by the following formula (1) or (2) or a salt thereof. The nucleoside derivative of the present invention may be incorporated into a partial structure of an oligonucleotide by a method well known to those skilled in the art.

[0021]

[0022] The nucleoside derivatives have a basic substituent at the 4'-position of the ribose and deoxyribose.

[0023] 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, a substituent having 1 to 6 carbon atoms or 1 to 5 carbon atoms is exemplified, and more preferably, a substituent having 1 to 4 carbon atoms or 1 to 3 carbon atoms is exemplified.

[0024] The nucleoside derivatives or salts thereof disclosed in the present specification and their uses will be described below.

[0025] (Nucleoside Derivative and Salt Thereof) One embodiment of the present nucleoside derivative or salt thereof is a nucleoside derivative or salt thereof represented by the following formula (1):

[0026]

[0027] 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): The nucleotide derivative represented by formula (1) is an enantiomer based on the asymmetric carbon atom in R.

[0028]

[0029] [R 1 Regarding 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 is a hydroxyl group, the nucleoside derivative has ribose as the sugar. 1 However, when the hydrogen atom is a hydroxyl group substituted with an alkyl or alkenyl group, or a protected hydroxyl group, the nucleoside derivative can be said to have deoxyribose as the sugar.

[0030] (Alkyl Group) In this specification, examples of alkyl groups include saturated hydrocarbon groups that are linear, branched, cyclic, or a combination thereof. Generally, lower alkyl groups are preferred, and more preferred examples include lower alkyl groups having 1 to 6 carbon atoms or lower alkyl groups having 1 to 5 carbon atoms, with 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 groups 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 a particularly preferred example. Examples of cyclic alkyl groups having 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, and cyclopropylmethyl groups.

[0031] (Alkenyl Group) In this specification, the term "alkenyl group" refers to a saturated hydrocarbon group which is linear, branched, cyclic, or a combination thereof. Generally, a lower alkenyl group is preferred, and 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.

[0032] (Hydroxyl group-protecting group or protected hydroxyl group) In this specification, the hydroxyl group-protecting group is 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 the hydroxyl group-protecting group include, for example, an aliphatic acyl group, an aromatic acyl group, a lower alkoxymethyl group, an oxycarbonyl group which may have an appropriate substituent, a tetrahydropyranyl group which may have an appropriate substituent, a tetrathiopyranyl group which may have an appropriate substituent, a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups (wherein the substituent in the substituted aryl group is a lower alkyl, a lower alkoxy, a halogen atom, or a cyano group), or a silyl group.

[0033] 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. Other preferred examples include isopropoxy, isobutoxy, s-butoxy, and t-butoxy. Cyclopropoxy and cyclobutoxy groups are also preferred, with cyclopropylmethoxy being another preferred example.

[0034] In this specification, alkylthio groups include 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.

[0035] 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 (wherein the substituents on the substituted aryl groups are as described above).

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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-trichloroethoxycarbonyl group and a 2-(trimethylsilyl)ethoxycarbonyl group.

[0046] The alkenyloxycarbonyl group includes 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.

[0047] 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 on the substituted aryl groups are lower alkyl, lower alkoxy, halogen, or cyano.

[0048] 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 types 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. In addition, a 4-cyanobenzyl group is also a preferred example.

[0049] In this specification, examples of the silyl group include trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, diphenylmethylsilyl, diphenylbutylsilyl, and diphenylisopropylsilylphenyldiisopropylsilyl. Among these, more preferred are trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyl, and diphenylmethylsilyl, and particularly preferred are trimethylsilyl, t-butyldimethylsilyl, and diphenylmethylsilyl.

[0050] 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.). 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 a hydrogen atom has been substituted with such a protecting group.

[0051] R 1 Although there are no particular limitations on the nucleotide derivative, it may be advantageous to use a hydroxyl group in which the hydrogen atom is substituted with an alkyl group having 1 to 3 carbon atoms, such as a methyl group, because when the structural unit derived from the present nucleotide derivative constitutes part of the structural unit of a nucleic acid drug such as ASO, it may be possible to further improve nuclease resistance.

[0052] [R 2 and R 4 Regarding 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 phosphoryl group, a protected phosphoryl group, or -O-P(=O) n (R 5 ) R 6 The protecting group for the hydroxyl group is as explained above.

[0053] (Protected Phosphoryl Group) The phosphoryl group constitutes the phosphate group in formula (1) and formula (2). Therefore, the protecting group in the protected phosphoryl group is a protecting group for phosphate, and protecting groups for phosphate are well known to those skilled in the art, and the above-mentioned references and explanations can be used as references.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] As used herein, the term "protecting group for a phosphate 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.). As the protecting group for a phosphate group, particularly preferred examples include substituents that are eliminated by hydrogenolysis or hydrolysis.

[0060] (-P(=O) n (R 5 ) R 6 ) R of the nucleoside derivative 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 6may 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 cannot both be hydrogen atoms. The protected hydroxyl group and lower alkoxy group are as explained above.

[0061] (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 are 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. Furthermore, preferred aromatic acyl groups include benzoyl groups.

[0062] Preferred examples of the cyano lower alkoxy group include cyano-substituted, linear, branched, or cyclic alkoxy groups or combinations thereof having 1 to 5 carbon atoms (when counting the number of carbon atoms in the cyano group) 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.

[0063] R 5 and R 6 A substituted amino group can be selected as the substituent of the amino group. 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 6and (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.

[0064] -P(=O) n (R 5 ) R 6 More specifically, preferred examples of the alkyl group include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group, with a phosphoramidite group being particularly preferred.

[0065] -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, as 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 -O-P(OCHCN)(N(CH(CH)) or -P(OCH)(N(CH(CH)).

[0066] -P(=O) n (R 5 ) R 6 In the formula, n is 1 and R 5 and R 6When 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.

[0067] Also, -O-P(=O) n (R 5 ) R 6 In the formula, 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.

[0068] R in the present 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 Other examples include a phosphoryl group (OR 2 a protected phosphoryl group (OR 2 It is also preferred that R is a protected phosphate group. 2 is preferably a hydroxyl group or a protecting group for a hydroxyl group.

[0069] 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)), or a phosphonyl group.

[0070] R in the present nucleoside derivative 4 is preferably, for example, a protecting group for a hydrogen atom or a hydroxyl group. Also, for example, a phosphate group, a protected phosphate group, or -P(=O) n (R 5 ) R 6 It is also preferable that R 4 Specific 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.

[0071] [R 3 Regarding Formula (1) and Formula (2), R 3 are respectively NHR 7 (R 7 represents a protecting group for a hydrogen atom, an alkyl group, an alkenyl group, or an amino group.

[0072] 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. When R is a structural unit in an oligonucleotide, 7 is preferably a hydrogen atom.

[0073] Protecting groups for amino groups are well known to those skilled in the art and can be found in the above mentioned references. Specific examples of the protecting group include, in addition to the groups exemplified above as the protecting group for a hydroxyl group, a benzyl group, a methylbenzyl group, a chlorobenzyl group, a dichlorobenzyl group, a fluorobenzyl group, a trifluoromethylbenzyl group, a nitrobenzyl group, a methoxyphenyl group, a methoxymethyl (MOM) group, an N-methylaminobenzyl group, an N,N-dimethylaminobenzyl group, a phenacyl group, an acetyl group, a trifluoroacetyl group, a pivaloyl group, a benzoyl group, a phthalimide group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, a benzyloxycarbonyl group, a t-butoxycarbonyl (Boc) group, a 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc) group, a 9-fluorenylmethoxycarbonyl group, a benzyloxymethyl (BOM) group, and a 2-(trimethylsilyl)ethoxymethyl (SEM) group. 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.

[0074] In this specification, 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.

[0075] [Regarding B: Base] The B: base 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.

[0076] That is, examples of B include 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 further include 2-oxo-4-methoxy-pyrimidin-1-yl, 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl, or 2,6-dimethoxypurin-9-yl.

[0077] 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.

[0078] 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).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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 above-mentioned substituents, and in addition, it is also preferred that a triazole group or a lower alkoxymethyl group be added.

[0083] 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 the hydroxyl group is protected.

[0084] Substituted 2-oxo-pyrimidin-1-yl includes, 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.

[0085] 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.

[0086] 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 an intramolecular counter ion. Alternatively, a base addition salt may be formed depending on the type of substituent. Pharmaceutically acceptable salts are preferred as the salt. 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.

[0087] Examples of mineral acid salts include hydrochlorides, hydrobromides, hydroiodides, nitrates, sulfates, hydrogensulfates, phosphates, and hydrogenphosphates. Typically, hydrochlorides and phosphates are preferred. Examples of organic acid salts include acetates, trifluoroacetates, gluconates, lactates, salicylates, citrates, tartrates, ascorbates, succinates, maleates, fumarates, formates, benzoates, methanesulfonates, ethanesulfonates, and p-toluenesulfonates. Typically, acetates 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.

[0088] 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.

[0089] 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.

[0090] By incorporating the present nucleoside derivative as at least a partial structural unit of an oligonucleotide, the nuclease resistance of the single-stranded oligonucleotide and the double-stranded oligonucleotide can be improved. Furthermore, even when the present nucleoside derivative is incorporated as at least a partial structural unit of an oligonucleotide, the affinity of the oligonucleotide for its complementary RNA strand and its decomposition activity by RNase H can be maintained.

[0091] (Oligonucleotide derivatives and salts thereof) The oligonucleotide derivatives disclosed herein (hereinafter also referred to as the present oligonucleotide derivatives) can contain at least one structural unit represented by formula (3) and formula (4). The structural units represented by formula (3) and formula (4) can be obtained based on nucleoside derivatives or salts thereof represented by formula (1) and formula (2), respectively. The present oligonucleotide derivatives are called derivatives based on the fact that they have a structural unit represented by formula (3) or formula (4).

[0092]

[0093] R in the structural units represented by formula (3) and formula (4) 1 , R 3 and B have the same meanings as in formula (1) and formula (2), respectively.

[0094] The present oligonucleotide derivative may contain two or more structural units represented by formula (3) and formula (4). In this case, these structural units may be the same or different from each other. In addition, the entire structural units contained in the present oligonucleotide derivative may be composed only of structural units represented by formula (3), or may be composed only of structural units represented by formula (4). In addition, the oligonucleotide derivative may have one or more structural units represented by formula (3) and one or more structural units represented by formula (4).

[0095] Furthermore, the structural units represented by formula (3) and formula (4) may be arranged adjacent to each other or apart from each other. For example, each structural unit may be present at at least one of the 5'-terminal side (e.g., within 4 bases from the 5'-terminal), the central portion, and the 3'-terminal side (e.g., within 4 bases from the 3'-terminal) of the present oligonucleotide derivative. Alternatively, each structural unit may be present at two or more of these locations. Furthermore, these structural units may be present approximately evenly at two or more locations. "Approximately evenly presenting structural units at two or more locations" does not necessarily mean that the same number of structural units are present at each of the two or more locations; it is sufficient that at least one structural unit is present at each of these locations. For example, when about 1 to 3 structural units are present at each location, this can be said to be approximately even. At least six structural units may be present in the present oligonucleotide derivative.

[0096] More specifically, the present oligonucleotide derivative can have one, two, three, or four structural units at the 5'-end. For example, three or more. The present oligonucleotide derivative can also have one, two, three, or four structural units at the 3'-end. For example, three or more. Furthermore, the present oligonucleotide derivative can also have a total of one, two, three, four, or five or more structural units at its 5'-end and 3'-end. This can increase the nuclease resistance at the ends.

[0097] Furthermore, the present oligonucleotide derivative may not have a structural unit at the 5'-end and 3'-end, which are the ends of the oligonucleotide chain, but may have one or more structural units within a range of 2 to 4 bases from the 5'-end and / or 3'-end.

[0098] Furthermore, the oligonucleotide derivative may have at least one structural unit in its central portion, i.e., in the range excluding the range within 4 bases from the 3'-end and the range within 4 bases from the 5'-end of the oligonucleotide derivative. Specifically, it may have one, two, three, four or more structural units. For example, it may have one or two or more structural units in the range from the 5th base to the 10th base from the 3'-end, or in the range from the 5th base to the 10th base, or in the range from the 5th base to the 8th base. This can increase the nuclease resistance of the central portion.

[0099] The structural units in the present oligonucleotide derivative may be either S- or R-configuration, but when they are provided adjacent to each other on the same terminal side, it is preferable to use one of the structural units in either the S-configuration or the R-configuration.

[0100] In the structural units represented by formula (3) and formula (4), the sugar chain moiety 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.

[0101] The present oligonucleotide derivatives are single-stranded per se, but can also take the form of a hybrid with an oligoribonucleotide, an oligodeoxyribonucleotide, or an oligodeoxyribo / ribonucleotide (chimeric chain), ie, a double-stranded form.

[0102] The present oligonucleotide derivative may have structural units other than those represented by formulas (3) and (4) that correspond to other natural nucleotides, or known nucleoside derivatives and / or nucleotide derivatives. For example, in the case of an ASO targeting mRNA, the structural units other than those represented by formulas (3) and (4) may include structural units derived from ribonucleosides or derivatives thereof, and / or structural units derived from deoxyribonucleosides or derivatives thereof. Furthermore, the oligonucleotide derivative may include structural units derived from other modified nucleosides, such as LNA.

[0103] The structural units defined herein and other structural units may be linked to each other by, for example, phosphodiester bonds, phosphomonoester bonds, phosphorothioate bonds (thiophosphate ester bonds), and the like.

[0104] The present oligonucleotide derivative has at least 2 or more structural units and other nucleoside derivatives, for example, 8 or more, for example, 10 or more, for example, 12 or more, or for example, 14 or more. 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.

[0105] When the present oligonucleotide derivative is an ASO, the total number of structural units constituting the oligonucleotide derivative can be, for example, 10 to 30, or, for example, 12 to 26, or 14 to 20, etc.

[0106] The present oligonucleotide derivative may have one or more asymmetric centers in the structural units represented by formula (3) and formula (4) as well as other structural units, 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.

[0107] 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.

[0108] (Production of the present nucleoside derivative and the present oligonucleotide derivative) The present nucleoside derivative and the present oligonucleotide derivative can be easily synthesized by a person 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 this application.

[0109] 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 derivatives or oligonucleotide derivatives of the present invention is not limited to the following method.

[0110] 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.

[0111] 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, THA 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.

[0112] For example, one example of the present nucleoside derivative (S-form) represented by formula (1) can be synthesized according to the scheme shown in Figure 2. The scheme shown in Figure 2 is an example of a scheme for synthesizing a phosphoramidite agent for synthesizing the S-form using thymidine as a starting material.

[0113] Furthermore, for example, an example of the present nucleoside derivative (R-form) represented by formula (2) can be synthesized according to the scheme shown in Figure 3. The scheme shown in Figure 3 is an example of a scheme for synthesizing an R-form phosphoramidite agent from compound 4 in the scheme shown in Figure 2.

[0114] The present oligonucleotide derivatives having structural units represented by formulas (3) and (4) can be easily produced by using various present nucleoside derivatives represented by formula (1) or (2) as amidite agents, etc. That is, by using such nucleoside derivatives, they can be synthesized using a known DNA synthesizer, and the resulting 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.

[0115] The oligonucleotide derivatives can be used, for example, as ASOs that target mRNA. They can also be linked to other compounds to form conjugates. Furthermore, the oligonucleotide derivatives can also be used as components of ribozymes. The oligonucleotide derivatives are also useful as RNA chips and other reagents.

[0116] For this reason, the present oligonucleotide derivatives, taking advantage of characteristics not found in natural oligonucleotides, are expected to be more useful than natural nucleosides and conventional nucleosides as components of nucleic acid drugs as various gene expression inhibitors that treat diseases by inhibiting gene activity, including antitumor and antiviral agents. That is, the present oligonucleotide derivatives are useful not only as such nucleic acid drugs, but also as raw materials or intermediate reagents for such drugs. Furthermore, the present nucleoside derivatives are useful as raw materials or intermediates for such RNA drugs.

[0117] 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.

[0118] <Synthesis of 4'-C-(S)-2-aminopropoxythymidine analog (S-form)> The S-form was synthesized according to the scheme shown in Figure 2. The S-form was obtained through a total of seven reaction steps with a total yield of 10%. As also shown in Figure 2, (S)-2-aminopropanol-TFA (compound (10)) was obtained in a yield of 94% using commercially available (S)-2-aminopropanol (compound (9)) as the starting material.

[0119] Synthesis of Compound (4) Compound (1) (0.4922 g, 2.00 mmol) was dissolved in 10.5 mL of THF in an ice bath under an Ar atmosphere. Triphenylphosphine (0.6222 g, 2.38 mmol), imidazole (0.288 g, 4.16 mmol), and iodine (0.6096 g, 2.40 mmol) were added and the mixture was stirred at room temperature for 4.5 hours to obtain compound (2) as a crude product. DBU (1.20 mL, 8.00 mmol) was added dropwise to the residue in an ice bath and the mixture was stirred at room temperature for 19 hours. The solvent was then removed under reduced pressure, and the residue was separated by silica gel chromatography (CHCl3:MeOH = 20:1) to obtain compound (3) as a crude product. Compound 3 was then separated under an Ar atmosphere using CHCl2Cl2. 2 The residue was dissolved in 5.00 mL of HCl, and imidazole (0.3404 g, 5.00 mmol) and TBDMSCl (0.5685 g, 3.72 mmol) were added. The mixture was stirred at room temperature for 21 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane: EtOAc = 1:2) to give compound (4) (0.3175 g, 0.94 mmol, 47%).

[0120] 1 H NMR (400 MHz, CDCl3) δ: 8.26 (s, 1H), 6.98 (d, J =1.36, 1H), 6.49 (t, J = 6.44, 1H), 4.74 (t, J =2.72 1H), 4.53 (d, J = 1.4 1H), 4.23 (d, J = 2.32, 1H), 2.32 (dq, J = 13.6, 3.1 Hz, 1H), 2.08 (dt, J = 13.7, 6.4 Hz, 1H), 1.94 (d, J = 1.36, 3H), 0.90 (s, 9H), 0.12 (s,6H)

[0121] Synthesis of Compound (5) Compound (4) (0.907 g, 2.66 mmol) was dissolved in 13.5 ml of CHCl, 45.0 ml of saturated aqueous NaHCO, and 9.00 ml of acetone were added. Oxone® (4.92 g, 7.98 mmol) dissolved in 27.0 ml of HO was added dropwise in an ice bath. After stirring at room temperature for 3.5 hours, the product was extracted from the reaction mixture with CHCl and saturated aqueous NaHCO. The organic layer was washed with saturated aqueous NaCl, and then dehydrated with NaSO. The solvent was removed under reduced pressure to obtain the crude product.

[0122] The crude product and (S)-2-(trifluoroacetyl)aminopropanol (1.38 g, 7.98 mmol) were dissolved in 13.0 mL of THF under an Ar atmosphere and added dropwise with 1 M ZnCl2 in THF (2.70 mL, 2.70 mmol) at -40 °C. After stirring at room temperature for 17 h, the mixture was quenched with 15 mL of saturated aqueous NaHCO3. The precipitated salt was removed by filtration through Celite. The compound was extracted from the filtrate with EtOAc and HO. The organic layer was washed with saturated aqueous NaHCO3 and then dehydrated with Na2SO4. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane / EtOAc = 1:1) to give compound (5) (0.54 g, 1.02 mmol, 38%) as white crystals.

[0123] 1H-NMR (400 MHz, DMSO-D6) δ 11.30 (s, 1H), 9.16 (d, J = 7.8 Hz, 1H), 7.67 (s, 1H), 6.18 (t, J = 5.7 Hz, 1H), 5.32 (t, J = 5.3 Hz, 1H), 4.57 (t, J = 8.0 Hz, 1H), 4.00-3.93 (m, 1H), 3.65-3.58 (m, 2H), 3.52 (t, J = 8.5 Hz, 1H), 3.41 (q, J = 5.8 Hz, 1H), 2.28-2.25 (m, 2H), 1.76 (s, 3H), 1.11 (d, J = 6.9Hz, 3H), 0.85 (s, 9H), 0.05 (s, 6H) 13 C-NMR (151 MHz, CHLOROFORM-D) δ 164.1, 157.0, 150.6, 136.7, 116.0, 111.4 (s, 1C), 107.4, 85.4, 72.0, 65.4, 62.5, 46.6, 39.3, 25.7, 18.1, 16.8, 12.6, -4.8 HRMS (ESI-TOF) m / z Calcd for C 21 H 34 F3N3NaO7Si [M + Na] + 548.20158, Found 548.20168

[0124] Synthesis of Compound (6) Under an Ar atmosphere, compound (5) (0.450 g, 0.850 mmol) was dissolved in 8.50 ml of pyridine, and DMTrCl (0.584 g, 1.71 mmol) was added. After stirring at room temperature for 20 hours, the compound was extracted with EtOAc and saturated aqueous NaHCO3. The organic layer was washed with saturated aqueous NaCl and then dehydrated with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane / EtOAc = 2:3) to give compound (6) (0.556 g, 0.680 mmol, 80%) as yellow crystals.

[0125] 1H-NMR (600 MHz, CHLOROFORM-D) δ 9.05 (s, 1H), 7.62 (d, J = 1.2 Hz, 1H), 7.38-7.36 (m, 2H), 7.31-7.24 (m, 7H) , 6.85-6.82 (m, 4H), 6.71 (d, J = 7.5 Hz, 1H), 6.35 (dd, J = 7.1, 4.6 Hz, 1H), 4.74 (t, J = 7.4 Hz, 1H), 4.09-4.05 (m, 1H), 3.79 (s, 6H), 3.72 (dd, J = 9.9, 4.1 Hz, 1H), 3.49 (d, J = 9.8 Hz, 1H), 3.45 (dd, J = 10.0, 5.3 Hz, 1H), 3.18 (d, J = 9.8 Hz, 1H), 2.48-2.43 (m, 1H), 2.36-2.32 (m, 1H), 1.37 (d, J = 1.0 Hz, 3H), 1.17 (d, J = 6.7 Hz, 3H), 0.81 (s, 9H), 0.01 (s, 3H), -0.05 (s, 3H) 13 C-NMR (151 MHz, CHLOROFORM-D) δ 163.9, 159.0, 156.7, 150.5, 143.9, 135.2, 134.9, 130.2, 128.2, 127.5, 115.9, 113.4, 111.6, 107.2, 87.4, 84.0, 71.8, 65.2, 62.9, 55.4, 46.4, 39.8, 25.7, 18.1, 16.9, 11.8, -4.7, -4.8 HRMS (ESI-TOF) m / z Calcd for C 42 H 52 F3N3NaO9Si [M + Na] + 850.33226, Found 850.32938

[0126] Synthesis of Compound (7) Compound (6) (0.610 g, 0.730 mmol) was dissolved in 7.3 ml of THF under an Ar atmosphere, and 1.1 ml of 1 M TBAF in THF was added. The mixture was stirred at room temperature for 17 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane / EtOAc = 1:1) to obtain compound (7) (0.450 g, 0.630 mmol, 87%) as white crystals.

[0127] 1 H-NMR (500 MHz, CHLOROFORM-D) δ 8.89 (s, 1H), 7.39-7.36 (m, 3H), 7.31-7.23 (m, 6H), 6.84 (dd, J = 8.9, 2.0 Hz, 4H), 6.75 (d, J = 8.0 Hz, 1H), 6.33 (dd, J = 6.9, 5.2 Hz, 1H), 4.70 (q, J = 7.4 Hz, 1H), 4.18-4.13 (m, 1H), 3.79 (s, 6H), 3.67 (q, J = 4.6 Hz, 1H), 3.45 (d, J = 9.7 Hz, 1H), 3.35-3.29 (m, 2H), 2.84 (d, J = 8.0 Hz, 1H), 2.50-2.44 (m, 1H), 2.42-2.37 (m, 1H), 1.74 (s, 1H), 1.48 (s, 3H), 1.21 (d, J = 6.9 Hz, 3H) 13 C-NMR (151 MHz, CHLOROFORM-D) δ 163.9, 158.9, 157.1, 150.4, 144.1, 135.4, 135.0, 130.2, 128.2, 127.4, 115.9, 113.5, 111.8, 105.7, 87.4, 83.8, 72.3, 65.4, 62.3, 55.4, 46.4, 39.0, 16.6, 12.0 HRMS (ESI-TOF) m / z Calcd for C 36 H 38 F3N3NaO9[M + Na] + 736.24578, Found 736.24776

[0128] Synthesis of Compound (8) Compound (7) (0.400 g, 0.560 mmol) was dissolved in 4.00 mL of THF under an Ar atmosphere, and DIPEA (0.50 mL, 2.80 mmol) and CEP-Cl (0.40 mL, 1.68 mmol) were added. After stirring at room temperature for 1 hour, the mixture was quenched with 4 mL of saturated aqueous NaHCO3. The compound was extracted with EtOAc and saturated aqueous NaHCO3. The organic layer was washed with saturated aqueous NaCl, and then dehydrated with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane / EtOAc = 2:3) to give compound (8) (0.430 g, 0.470 mmol, 84%) as white crystals.

[0129] 31 P-NMR (162 MHz, CHLOROFORM-D) δ 150.9, 150.0 HRMS (ESI-TOF) m / z Calcd for C 45 H 55 F3N5NaO 10 P [M + Na] + 936.35363, Found 936.35409

[0130] Synthesis of Compound (10) Under an Ar atmosphere, CF3COOEt (1.93 ml, 16.2 mmol) was dissolved in 4.6 ml of MeCN, and (S)-(+)-2-amino-1-propanol (Compound (9)) (0.93 ml, 12.0 mmol) was added in an ice bath. After stirring at room temperature for 20 minutes, the solvent was removed under reduced pressure to obtain Compound (10) (1.93 g, 11.3 mmol, 94%) as white crystals.

[0131] 1 H-NMR (500 MHz, CHLOROFORM-D) δ 6.55 (s, 1H), 4.17-4.12 (m, 1H), 3.77 (dd, J = 11.2, 3.7 Hz, 1H), 3.64 (q, J = 5.2 Hz, 1H), 1.82 (s, 1H), 1.29 (d, J = 6.9 Hz, 3H) 13C-NMR (151 MHz, DMSO-D6) δ 155.9, 116.0, 63.5, 48.0, 16.3

[0132] <Synthesis of 4'-C-(R)-2-amipropoxythymidine Analog> The R-isomer was synthesized using compound 4 as an intermediate according to the scheme shown in Figure 3. The R-isomer was obtained through a total of seven reaction steps in a total yield of 5.9%.

[0133] Synthesis of Compound (11) Compound (4) (1.02 g, 3.00 mmol) was dissolved in 15.5 ml of CHCl, 51.0 ml of saturated aqueous NaHCO, and 10.2 ml of acetone were added, and Oxone® (5.53 g, 9.00 mmol) dissolved in 30.5 ml of HO was added dropwise in an ice bath. After stirring at room temperature for 3.5 hours, the product was extracted from the reaction mixture with CHCl and saturated aqueous NaHCO. The organic layer was washed with saturated aqueous NaCl, and then dehydrated with NaSO. The solvent was removed under reduced pressure to obtain the crude product.

[0134] Under an Ar atmosphere, the crude product and (R)-2-(trifluoroacetyl)aminopropanol (1.55 g, 9.00 mmol) were dissolved in 14.5 ml of THF and added dropwise with 1 M ZnCl in THF (3.00 ml, 3.00 mmol) at -40 °C. After stirring at room temperature for 17 h, the mixture was quenched with 20 ml of saturated aqueous NaHCO. The precipitated salt was removed by filtration through Celite. The compound was extracted from the filtrate with EtOAc and HO. The organic layer was washed with saturated aqueous NaHCO and then dehydrated with NaSO. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane / EtOAc = 1:1) to give compound (11) (0.610 g, 1.16 mmol, 39%) as white crystals.

[0135] 1H-NMR (600 MHz, CHLOROFORM-D) δ 9.59 (s, 1H), 7.32 (d, J = 1.2 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.16 (dd, J = 7.7, 4.0 Hz, 1H), 4.65 (t, J = 7.8 Hz, 1H), 4.19-4.13 (m, 1H), 3.81 (dd, J = 11.6, 4.3 Hz, 1H), 3.73-3.66 (m, 3H), 3.09 (dd, J = 6.2, 4.9 Hz, 1H), 2.44-2.39 (m, 1H), 2.35 (ddd, J = 13.5, 8.0, 4.2 Hz, 1H), 1.87 (d, J = 1.1 Hz, 3H), 1.26 (d, J = 6.8 Hz, 3H), 0.88 (s, 9H), 0.09 (d, J = 4.9 Hz, 6H) 13 C-NMR (101 MHz, CHLOROFORM-D) δ 164.0, 156.8, 150.5, 136.7, 117.4, 111.6, 107.1, 85.4, 71.7, 64.9, 62.1, 46.4, 39.0, 25.7, 18.1, 17.1, 12.6, -4.8 HRMS (ESI-TOF) m / z Calcd for C 21 H 34 F3N3NaO7Si [M + Na] + 548.20158, Found 548.20416

[0136] Synthesis of Compound (12) Under an Ar atmosphere, compound (11) (0.210 g, 0.400 mmol) was dissolved in 4.00 mL of pyridine, and DMTrCl (0.154 g, 0.800 mmol) was added. After stirring at room temperature for 15 hours, the compound was extracted with EtOAc and saturated NaHCO3 aqueous solution. The organic layer was washed with saturated NaCl aqueous solution and then dehydrated with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane / EtOAc = 2:1) to give compound (12) (0.179 g, 0.216 mmol, 54%) as yellow crystals.

[0137] 1 H-NMR (600 MHz, CHLOROFORM-D) δ 9.45 (s, 1H), 7.64 (d, J = 1.2 Hz, 1H), 7.38-7.36 (m, 2H), 7.31-7.23 (m, 8H), 7.26 (s, 1H), 6.85-6.82 (m, 4H), 6.77 (d, J = 8.3 Hz, 1H), 6.32 (dd, J = 7.5, 3.3 Hz, 1H), 4.80 (t, J = 8.3 Hz, 1H), 4.16-4.12 (m, 1H), 3.79 (d, J = 1.0 Hz, 6H), 3.62 (dd, J = 9.5, 3.4 Hz, 1H), 3.57 (d, J = 9.8 Hz, 1H), 3.48 (dd, J = 9.6, 3.5 Hz, 1H), 3.15 (d, J = 9.7 Hz, 1H), 2.53-2.48 (m, 1H), 2.30 (ddd, J = 13.5, 8.1, 3.3 Hz, 1H), 1.36 (d, J = 1.1 Hz, 3H), 1.22 (d, J = 6.8 Hz, 3H), 0.80 (s, 9H), 0.02 (s, 3H), -0.07 (s, 3H) 13C-NMR (151 MHz, CHLOROFORM-D) δ 164.2 (s, 1C), 158.9 (s, 1C), 156.5 (q, J = 36.7 Hz, 0C), 150.6 (s, 1C), 143.9 (s, 0C), 135.1 (s, 1C), 135.0 (d, J = 36.1 Hz, 2C), 134.9 (s, 1C), 130.2 (d, J = 6.1 Hz, 3C), 128.2 (d, J = 24.2 Hz, 3C), 127.4 (s, 1C), 115.9 (d, J = 288.1 Hz, 0C), 113.4 (d, J = 8.3 Hz, 4C), 111.6 (s, 1C), 106.6 (s, 1C), 87.3 (s, 1C), 83.6 (s, 1C), 70.8 (s, 1C), 64.4 (s, 1C), 61.6 (s, 1C), 55.3 (s, 3C), 45.9 (s, 1C), 39.2 (s, 1C), 25.6 (s, 4C), 17.9 (s, 1C), 17.2 (s, 1C), 11.8 (s, 1C), -4.6 (s, 1C), -4.9 (s, 1C) HRMS (ESI-TOF) m / z Calcd for C 42 H 52 F3N3NaO9Si [M + Na] + 850.33226, Found 850.33184

[0138] Synthesis of Compound (13) Compound (12) (0.166 g, 0.200 mmol) was dissolved in 2.00 ml of THF under an Ar atmosphere, and 0.30 ml of 1 M TBAF in THF was added and stirred at room temperature for 3.5 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane / EtOAc = 1:1) to obtain Compound (13) (0.101 g, 0.14 mmol, 70%) as white crystals.

[0139] 1H-NMR (500 MHz, CHLOROFORM-D) δ 9.02 (s, 1H), 7.39-7.34 (m, 3H), 7.31-7.24 (m, 6H), 7.13 (d, J = 8.6 Hz, 1H), 6.84 (d, J = 7.4 Hz, 4H), 6.38 (t, J = 6.0 Hz, 1H), 4.72 (q, J = 8.0 Hz, 1H), 4.25 (d, J = 4.6 Hz, 1H), 3.79 (s, 6H), 3.58 (t, J = 8.6 Hz, 1H), 3.46 (d, J = 9.7 Hz, 1H), 3.39 (dd, J = 9.5, 3.7 Hz, 1H), 3.31 (d, J = 9.7 Hz, 1H), 2.66 (d, J = 8.6 Hz, 1H), 2.39-2.36 (m, 2H), 1.63 (s, 1H), 1.49 (s, 3H), 1.19 (d, J = 6.9 Hz, 3H) 13 C-NMR (151 MHz, CHLOROFORM-D) δ 164.3, 158.9, 157.5, 150.7, 144.3, 135.5, 135.1, 130.3, 128.2, 127.4, 116.1, 113.5, 112.1, 105.2, 87.2, 82.9, 71.7, 65.1, 61.7, 55.4, 45.8, 38.7, 16.5, 12.1 HRMS (ESI-TOF) m / z Calcd for C 36 H 38 F3N3NaO9[M + Na] + 736.24578, Found 736.24632

[0140] Synthesis of Compound (14) Compound (13) (0.620 g, 0.870 mmol) was dissolved in 6.20 ml of THF under an Ar atmosphere, and DIPEA (0.75 ml, 4.35 mmol) and CEP-Cl (0.60 ml, 2.61 mmol) were added. After stirring at room temperature for 3 hours, the mixture was quenched with 7 ml of saturated aqueous NaHCO3. The compound was extracted with EtOAc and saturated aqueous NaHCO3. The organic layer was washed with saturated aqueous NaCl, and then dehydrated with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane / EtOAc = 2:3) to give compound (14) (0.678 g, 0.740 mmol, 85%) as white crystals.

[0141] 1 P-NMR (162 MHz, CHLOROFORM-D) δ 150.8, 149.6 HRMS (ESI-TOF) m / z Calcd for C 45 H 55 F3N5NaO 10 P [M + Na] + 936.35363, Found 936.35612

[0142] Synthesis of Compound (16) Under an Ar atmosphere, CF3COOEt (1.45 ml, 12.2 mmol) was dissolved in 3.5 ml of MeCN, and (R)-(-)-2-amino-1-propanol (Compound (15)) (0.70 ml, 9.0 mmol) was added in an ice bath. After stirring at room temperature for 15 minutes, the solvent was removed under reduced pressure to obtain Compound (16) (1.46 g, 8.53 mmol, 95%) as white crystals. 1 H-NMR (400 MHz, CHLOROFORM-D) δ 6.59 (s, 1H), 4.19-4.09 (m, 1H), 3.77 (dd, J = 11.0, 3.7 Hz, 1H), 3.64 (dd, J = 11.0, 4.6 Hz, 1H), 1.95 (s, 1H), 1.28 (d, J = 6.4 Hz, 3H) 13C-NMR (151 MHz, DMSO-D6) δ 155.9, 116.0, 63.5, 48.0, 16.3

[0143] Synthesis of Oligonucleotides Containing 4'-2-APoT Analogs Oligonucleotides were synthesized on a 0.2 μmol scale using an automated nucleic acid synthesizer using the phosphoramidite method. Natural DNA and RNA amidites were diluted to 0.1 M, and the synthesized 4'-(S)-2-APoT and 4'-(R)-2-APoT amidites were diluted to 0.15 M with MeCN. Furthermore, CPG resin bearing a 3'-terminal nucleoside was loaded into a column at 0.2 μmol equivalents based on its activity. Unysupport 500 resin was used for the synthesis of sequences incorporating the synthetic 4'-(S)-2-APoT or 4'-(R)-2-APoT at the 3' terminus. The sequences of the synthesized oligonucleotides are shown in Figure 4.

[0144] After synthesis, the CPG resin was transferred to a sampling tube, 1.0 ml of NH₃ (soln.) was added, and the oligonucleotide was then incubated at 55°C for 12 hours to cleave and deprotect the oligonucleotide. When synthesizing oligonucleotides containing 4´-(S)-2-APoT or 4´-(R)-2-APoT, 900 μl of MeCN and 100 μl of Et₂NH₃ were added to the tube, vortexed for 5 minutes, and the supernatant was discarded. The resin was washed twice with 1 ml of MeCN, and then cleaved from the CPG resin and deprotected with 1.0 ml of NH₃ (soln.). After deprotection, the sample was transferred to an Eppendorf tube, washed twice with 1 ml of HO, and evaporated to dryness under reduced pressure. For RNA synthesis, the CPG resin was transferred to a sampling tube, 1.0 ml of NH₃ (soln.) was added, and the tube was incubated at 55°C for 4 hours to cleave and deprotect the oligonucleotides from the resin. The sample was then transferred to an Eppendorf tube, washed twice with 1 ml of HO, and evaporated to dryness under reduced pressure. The sample was then dissolved in 100 μl of DMSO, and 125 μl of TFA-3HF was added. The TBDMS group was deprotected by incubating for 90 minutes. The solution was then diluted to 10 ml with 0.1 M TEAA buffer (a 20-fold dilution of 2N TEAA buffer (114.38 ml of acetic acid, 277.6 ml of Et₃N, and diluted to 1000 ml with HO to pH 7.0)). The target product was then adsorbed onto the column by passing it through an equilibrated Sep-pack tC18 reverse-phase column. After washing with sterile water, the column was eluted with 3 ml of 50% MeCN in HO and evaporated to dryness under reduced pressure.

[0145] Each dried sample was dissolved in 200 μl of loading buffer (0.1 M Tris-HCl in 90% formamide) and subjected to 20% PAGE (500 V, 20 mA) to separate and purify the target oligonucleotides. 40 ml of 40% acrylamide (19:1) solution, 33.6 ml of urea, and 8 ml of 10× TBE buffer were mixed and then diluted to 80 ml with H2O. 55 mg of APS was then added and dissolved, followed by 40 μl of TEMED. The mixture was poured between two glass plates secured together with a 1.5 mm spacer and allowed to solidify for at least 1 hour. 1× TBE buffer was used as the electrophoresis buffer.

[0146] The band of interest was excised, immersed in 10 ml of 0.1 M TEAA buffer, 1 mM EDTA aqueous solution, and shaken overnight. The solution was then purified using an equilibrated Sep-pack tC18 reverse-phase column as described above and evaporated to dryness under reduced pressure. The purified oligonucleotide was dissolved in 1 ml of HO, and the concentration was calculated by measuring the absorbance at 260 nm of the diluted solution. The yield was then calculated according to standard methods. A 60 pmol equivalent of the oligonucleotide was evaporated to dryness under reduced pressure, mixed with 3 μL each of sterile water and matrix solution (prepared by dissolving 4.85 mg of 3-hydroxypicolinic acid (3-HPA) and 0.8 mg of diammonium hydrogen citrate in 50 μL of 50% MeCN), and then evaporated to dryness on a plate. The compound's molecular weight was measured using MALDI-TOF / MS, allowing identification of the compound.

[0147] <Thermal stability evaluation of oligonucleotides containing 4´-2-APoT analogs> 50% melting temperature (T m 600 pmol of each oligonucleotide was evaporated to dryness under reduced pressure and diluted with assay buffer [10 mM NaHPO 4 －The oligonucleotides were dissolved in 200 μL of a 100 mM NaHPO solution (pH 7.0, 100 mM NaCl). The double-stranded oligonucleotides were then heated at 95°C for 3 minutes to dissociate the double strands, and then allowed to stand at room temperature for at least 1 hour to allow annealing. After 10 minutes of degassing, 175 μL of the solution was transferred to a dedicated cell, and the temperature was raised from 20°C to 90°C (Δ 0.5°C / min) to measure the change in absorbance at 260 nm. The 50% melting temperature was calculated from the sigmoidal curve obtained by the measurement using the midline method. The sequences and Tms of the double-stranded oligonucleotides used for evaluation are shown in Figure 5.

[0148] As shown in FIG. 5, the oligonucleotides into which the S- and R-forms had been introduced showed thermal stability comparable to that of the natural DNA (D1) and the 4'-AE form.

[0149] <Assessment of E. coli RNase H activity> Each oligonucleotide (DNA:RNA = 300 pmol:1500 pmol (1:5)) was evaporated to dryness under reduced pressure for 1 min and dissolved in 37.5 μL of assay buffer [50 mM Tris-HCl (pH 8.0), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol]. The double strands were then dissociated by heating at 100°C for 5 min and allowed to stand at room temperature for at least 1 hour to allow annealing. Each sample was incubated at 37°C, and 35 μL of RNase H solution was added. The reaction was stopped at designated intervals by adding 5 μL of sample solution to an Eppendorf tube containing 15 μL of 100% formamide. The E. coli RNase H solution used was prepared by diluting E. coli RNase H (RNase I, 50 units / μL in 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT, 50% glycerol) 1000 times with the assay buffer.

[0150] The gel was electrophoresed on a 20% PAGE at 500 V and 20 mA for approximately 3 hours, and a photograph of the gel after electrophoresis was taken using a Luminescent Image Analyzer LAS-4000. The oligonucleotides evaluated and the results are shown in Figure 6.

[0151] As shown in Figure 6, the oligonucleotides containing the S- and R-isomers exhibited RNase H activity comparable to that of the native DNA (D1) and the 4'-AE-isomer. Furthermore, it was found that the R-isomer tends to rapidly and generally fragment the full-length cleavage by RNase H into shorter chains than the S-isomer.

[0152] <Evaluation of Nuclease Resistance> 300 pmol of each single-stranded oligonucleotide used in the nuclease resistance test was evaporated to dryness under reduced pressure and dissolved in 37.5 μl of Opti-MEM. A 1.1 μl aliquot was taken as the 0-min sample and added to 5 μl of loading buffer (10 mM EDTA in formamide). Each sample was incubated at 37°C, and 15.6 μl of bovine serum (BS) was added. The reaction was stopped at designated intervals by adding 2.4 μl of the sample solution to an Eppendorf tube containing 10 μl of loading buffer. Electrophoresis was performed on a 20% PAGE gel at 500 V and 20 mA for approximately 3 hours. After electrophoresis, gel images were taken using a LAS-4000 luminescent image analyzer. The oligonucleotides used and the evaluation results are shown in Figure 7.

[0153] As shown in Figure 7, in the case of native DNA 8, all full-length oligonucleotide chains were degraded 30 minutes after the start of incubation. In contrast, in the case of analog-introduced DNAs 9, 10, and 11, full-length DNA remained even after 4 hours of incubation. This is thought to be due to the side chains of the analogs containing cationic amino groups causing steric hindrance and electrostatic repulsion with the active site of the nuclease, improving resistance.

[0154] These results suggest that the S- and R-isomers of the present nucleoside derivatives exhibited significantly higher nuclease resistance than the natural form and higher than the 4'-AEoT-incorporated oligonucleotide. Therefore, these modified nucleic acids are useful as antisense nucleic acids in terms of nuclease resistance.

[0155] <Synthesis of phosphoramidite derivative of 4'-(S)-2-aminopropoxy-2'-OMe-thymidine analog (S-form)> A thymidine derivative (S-form) having a methoxy group at the 2' position was synthesized according to the scheme shown in Figure 8. Compound (4) shown in Figure 8 was obtained in the same manner as in the scheme shown in Figure 2, except that compound (1) (2'-OMe-thymidine) shown in Figure 8 was used as the starting compound.

[0156] <Synthesis of Compound (6)> Under an Ar atmosphere, compound (4) (0.95 g, 2.6 mmol) was dissolved in CHCl (14 mL), and acetone (9.5 mL) and saturated NaHCO aqueous solution (48 mL) were added. At 0°C, an aqueous solution (28 mL) of oxone (4.8 g, 7.7 mmol) was added and stirred at room temperature for 2 hours. After quenching with 5% NaHSO aqueous solution, the product (compound (5)) was extracted with CHCl and saturated NaHCO aqueous solution. The organic layer was then washed with saturated NaHCO 3 The crude product was then washed with aqueous NaCl and dried over NaSO, and the solvent was removed under reduced pressure to obtain a crude product of compound (5). Subsequently, the crude product was dissolved in THF (12 mL) under an Ar atmosphere, and the solution was diluted with 1 M ZnCl at -40°C. 2 The resulting mixture was stirred overnight at room temperature in THF (2.6 mL, 2.6 mmol) and the synthesized 2,2,2-Trifluoro-N-[(1S)-2-hydroxy-1-methylethyl]acetamide (1.3 g, 7.8 mmol). The mixture was quenched with aqueous saturated sodium bicarbonate (NaHCO3) and filtered through Celite. The product was extracted from the filtrate with aqueous ethyl acetate (EtOAc) and saturated sodium bicarbonate (NaHCO3). The organic layer was washed with aqueous saturated sodium bicarbonate (NaCl). The mixture was dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 50:1) and then further purified with hexane:EtOAc = 3:2 to give compound (6) (0.67 g, 1.2 mmol, 47%).

[0157] 1 H NMR (400 MHz, CDCl3)) δ: 0.13 (s, 3H), 0.15 (s, 3H), 0.93 (s, 9H), 1.26 (d, J = 6.4 Hz, 3H), 1.93 (s, 3H), 2.30 (s, 1H), 3.50 (s, 3H), 3.51-3.54 (m, 1H), 3.65 (dd, J = 7.3, 11.5 Hz, 1H), 3.79-3.85 (m, 2H), 3.92-3.94 (m, 1H), 4.20-4.24 (m, 1H), 4.63 (d, J = 6.4 Hz, 1H), 5.57 (d, J = 2.3 Hz, 1H), 7.28 (s, 1H), 7.39 (d, J = 7.3 Hz, 1H), 8.64 (s, 1H)

[0158] Synthesis of Compound (7) Compound (6) (1.6 g, 2.9 mmol) was dissolved in pyridine (29 mL) under an Ar atmosphere. DMTrCl (2.0 g, 5.9 mmol) was added at 0 °C and the mixture was stirred overnight at room temperature. The product was then extracted with EtOAc and saturated NaHCO3 aqueous solution, and the organic layer was washed with saturated NaCl aqueous solution. The mixture was dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: EtOAc = 3:1) to obtain compound (7) (2.5 g, 2.9 mmol, quant).

[0159] 1 H NMR (400 MHz, CDCl3) δ: -0.07 (s, 3H), 0.07 (s, 3H), 0.80 (s, 9H), 1.17 (d, J = 6.4 Hz, 3H), 1.24 (d, J = 0.9Hz, 3H), 3.19 (d, J= 10.1 Hz, 1H), 3.33 (dd, J = 2.3, 9.2Hz, 1H), 3.55 (s, 3H), 3.59 (d, J = 10.1 Hz, 1H), 3.71-3.75 (m, 1H), 4.12-4.19 (m, 1H), 4.59 (d, J = 6.0Hz, 1H), 5.92 (d, J = 0.9Hz, 1H), 6.82 -6.85 (m, 4H), 7.21-7.36 (m, 9H), 7.46 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 1.4Hz, 1H), 8.25 (s, 1H)

[0160] Synthesis of Compound (8) Compound (7) (0.85 g, 0.99 mmol) was dissolved in THF (10 mL) under an Ar atmosphere, and 1 M TBAF in THF (1.5 mL) was added and stirred at room temperature for 3 hours. The solvent was then evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: EtOAc = 2:1) to give compound (8) (0.62 g, 0.83 mmol, 84%).

[0161] 1H NMR (400 MHz, CDCl3) δ: 1.20 (d, J = 6.9 Hz, 3H), 1.33 (d, J = 0.9 Hz, 3H), 2.86 (d, J = 11.9 Hz, 1H), 3.27 (dd, J = 1.8, 9.2 Hz, 1H), 3.42 (dd, J = 7.6, 37.1 Hz, 2H), 3.66 (s, 3H), 3.73 (dd, J = 3.7, 9.2 Hz, 1H), 3.80 (s, 6H), 3.83 (d, J = 6.4 Hz, 1H), 4.17-4.25 (m, 1H), 4.71 (dd, J = 6.4, 11.9 Hz, 1H), 5.84 (s, 1H), 6.84 (d, J = 8.7 Hz, 4H), 7.13 (d, J = 8.7 Hz, 1H), 7.24-7.31 (m, 9H), 7.51 (d, J = 0.9 Hz, 1H), 8.41 (s, 1H)

[0162] Synthesis of Compound (9) Compound (8) (0.63 g, 0.83 mmol) was dissolved in THF (8.3 mL) under an Ar atmosphere, and DIPEA (0.73 mL, 4.1 mmol) and CEP-Cl (0.40 mL, 2.30 mmol) were added and stirred at room temperature for 3 hours. 3 The product was extracted with aqueous solution of saturated NaCl and washed with aqueous solution of saturated NaCl. The product was dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: EtOAc = 1:1) to give compound (9) (0.64 g, 0.68 mmol, 82%). 31 P NMR (162MHz, CDCl3) δ:151.21, 151.77

[0163] <Synthesis of phosphoramidite derivative of 4'-(R)-2-aminopropoxy-2'-OMe-thymidine analog (R-form)> A thymidine derivative (R-form) having a methoxy group at the 2' position was synthesized according to the scheme shown in Figure 9. Compound (5) shown in Figure 9 was obtained from compound (4) shown in Figure 8 by the same procedure.

[0164] <Synthesis of Compound (10)> Under an Ar atmosphere, compound (4) (0.95 g, 2.6 mmol) shown in Figure 8 was dissolved in CHCl (14 mL), and acetone (9.5 mL) and saturated NaHCO aqueous solution (48 mL) were added. At 0 °C, an aqueous solution (28 mL) of Oxone (4.8 g, 7.7 mmol) was added and stirred at room temperature for 2 hours. After quenching with 5% NaHSO aqueous solution, the product was extracted with CHCl and saturated NaHCO aqueous solution, and the organic layer was washed with saturated NaHCO 3 The crude product was then washed with aqueous sodium chloride and saturated sodium chloride. The crude product was then dried over Na2SO4, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was then dissolved in THF (12 mL) under an Ar atmosphere, and the solution was diluted with 1 M ZnCl 2 The resulting mixture was stirred overnight at room temperature in THF (2.6 mL, 2.6 mmol) and the synthesized 2,2,2-Trifluoro-N-[(1R)-2-hydroxy-1-methylethyl]acetamide (1.3 g, 7.8 mmol). The mixture was quenched with aqueous saturated sodium bicarbonate (NaHCO3) and filtered through Celite. The product was extracted from the filtrate with aqueous ethyl acetate (EtOAc) and saturated sodium bicarbonate (NaHCO3). The organic layer was washed with aqueous saturated sodium bicarbonate (NaCl). The mixture was dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 50:1) and then further purified with hexane:EtOAc = 3:2 to give compound (10) (0.91 g, 1.6 mmol, 64%).

[0165] 1H NMR (400 MHz, CDCl3)) δ: 0.12 (s, 6H), 0.92 (s, 9H), 1.27 (d, J = 6.9 Hz, 3H), 1.93 (d, J = 0.9 Hz, 3H), 2.56 (dd, J = 4.1, 7.3 Hz, 1H), 3.46 (s, 3H), 3.67-3.73 (m, 2H), 3.75-3.78 (m, 2H), 4.01 (dd, J = 3.7, 6.4 Hz, 1H), 4.09-4.13 (m, 1H), 4.57 (d, J = 6.4Hz, 1H), 5.65 (d, J = 3.2 Hz, 1H) 7.19 (d, J = 7.3 Hz, 1H), 7.23 (d, J = 0.9 Hz, 1H), 8.52 (s, 1H)

[0166] Synthesis of Compound (11) Compound (10) (1.9 g, 3.4 mmol) was dissolved in pyridine (34 mL) under an Ar atmosphere. DMTrCl (2.3 g, 6.7 mmol) was added at 0 °C and the mixture was stirred overnight at room temperature. The product was then extracted with EtOAc and saturated NaHCO3 aqueous solution, and the organic layer was washed with saturated NaCl aqueous solution. The mixture was dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: EtOAc = 3:1) to obtain compound (11) (2.8 g, 3.3 mmol, 98%).

[0167] 1H NMR (400 MHz, CDCl3) δ: -0.03 (s, 3H), 0.05 (s, 3H), 0.78 (s,9H), 1.23 (d, J = 6.9Hz, 3H), 1.30 (d, J = 0.9Hz, 3H), 3.25 (d, J = 10.1Hz, 1H), 3.51-3.64 (m, 3H), 3.54 (s, 3H), 3.70 (dd, J = 1.8, 6.4Hz, 1H), 4.03-4.06 (m, 1H), 4.61 (d, J = 6.9Hz, 1H), 6.09 (d, J = 1.8Hz, 1H), 6.81 -6.84 (m, 4H), 7.22-7.37 (m, 10H), 7.60 (d, J = 0.9Hz, 1H), 8.34 (s, 1H)

[0168] Synthesis of Compound (12) Compound (11) (1.2 g, 1.4 mmol) was dissolved in THF (14 mL) under an Ar atmosphere, and 1 M TBAF in THF (2.0 mL) was added and stirred at room temperature for 3 hours. The solvent was then evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: EtOAc = 3:2) to give compound (12) (9.7 g, 1.3 mmol, 95%).

[0169] Synthesis of Compound (13) Compound (12) (0.97 g, 1.3 mmol) was dissolved in THF (13 mL) under an Ar atmosphere, and DIPEA (1.1 mL, 6.5 mmol) and CEP-Cl (0.58 mL, 2.6 mmol) were added and stirred at room temperature for 3 hours. 3 The product was extracted with aqueous solution of saturated NaCl and washed with aqueous solution of saturated NaCl. The product was dried over NaSO, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane: EtOAc = 1:1) to give compound (13) (0.99 g, 1.1 mmol, 81%). 31 P NMR (162MHz, CDCl3) δ:150.91, 151.67

[0170] <Evaluation of gene expression inhibitory ability> Using the S- and R-forms of the thymidine analogs synthesized in Examples 7 and 8 and LNA, the single-stranded oligonucleotides shown in the table below were synthesized in accordance with Example 3. Unless otherwise specified, structural units are structural units derived from deoxynucleosides.

[0171] These single-stranded oligonucleotides were used to evaluate gene expression silencing ability in NCI-460 cells, targeting KRAS mRNA. NCI-H460 cells were transfected with the oligonucleotides at final concentrations of 2.0, 5.0, or 10 nM and incubated at 37°C for 48 hours. cDNA was then generated by RT-PCR. The generated cDNA was then used for quantitative reverse transcription polymerase chain reaction (qPCR) to quantify KRAS mRNA expression. Gene expression levels were normalized to the endogenous control ACTB gene (#4326315E), and relative levels were calculated using the ΔΔCt model. The results are shown in Figure 10.

[0172] As shown in Figure 10, both the S- and R-isomers of the oligonucleotides exhibited a concentration-dependent gene expression inhibitory effect. LNAs have the problem of inducing hepatotoxicity. The S- and R-isomers have the advantage of having a 2-aminopropoxy group at the 4' position, which is thought to suppress or avoid hepatotoxicity.

Claims

1. A nucleoside derivative or a salt thereof represented by the following formula (1) or (2): (In formula (1) and formula (2), R 1 represents a hydrogen atom, a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected group. In formula (1) and formula (2), 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 sulfolyl group, a protected sulfolyl 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 each represents 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 hydrogen atoms. 3 The N.H.R. 7 (R 7 represents a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino 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.

2. In the above formula (1) and formula (2), R 7 The nucleoside derivative or a salt thereof according to claim 1 , wherein represents a hydrogen atom.

3. An oligonucleotide derivative or a salt thereof, comprising at least one structural unit derived from the nucleoside derivative according to claim 1 or 2.

4. The oligonucleotide derivative or salt thereof according to claim 3, comprising at least one of the structural units in total within a range of 4 bases from the 3' end of the oligonucleotide derivative and within a range of 4 bases from the 5' end of the oligonucleotide derivative.

5. The oligonucleotide or salt thereof according to claim 4, comprising a total of at least three of the structural units within a range of four bases from the 3' end of the oligonucleotide derivative and within a range of four bases from the 5' end of the oligonucleotide derivative.

6. The oligonucleotide derivative or salt thereof according to claim 5, comprising a total of at least five of the structural units in a range of 4 bases from the 3' end of the oligonucleotide derivative and a range of 4 bases from the 5' end of the oligonucleotide derivative.

7. The oligonucleotide derivative or a salt thereof according to claim 4, which comprises one or more of said structural units within a range of 4 bases from the 3'-end of said oligonucleotide derivative.

8. The oligonucleotide or salt thereof according to claim 4, which comprises one or more of said structural units within a range of 4 bases from the 5'-end of said oligonucleotide derivative.

9. The oligonucleotide derivative or a salt thereof according to claim 8, which has one or more of the structural units at a position other than the 5' end within a range of 4 bases from the 5' end of the oligonucleotide derivative.

10. The oligonucleotide derivative or salt thereof according to claim 3, comprising at least one such moiety in the range excluding the range within 4 bases from the 3' end of the oligonucleotide derivative and the range within 4 bases from the 5' end of the oligonucleotide derivative.

11. The oligonucleotide derivative or a salt thereof according to claim 3, which comprises a structural unit derived from a deoxyribonucleoside in addition to the structural unit derived from the nucleoside derivative according to claim 1 or 2.

12. A gene expression inhibitor targeting mRNA, comprising the oligonucleotide derivative or a salt thereof according to any one of claims 3 to 11.

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