WO2025033545A1 - Nucleoside derivative having 4'-position branched substituent group - Google Patents

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

This specification relates to nucleoside derivatives having a branched substituent at the 4' position (hereinafter simply referred to as nucleoside derivatives), etc.

In recent years, there has been progress in the development of nucleic acid medicines that target pathogenic genes. For example, antisense oligonucleotides (ASOs) are single-stranded DNA of about 15-20 mer that bind complementarily to the target mRNA, thereby inhibiting its translation.

ASOs have two main mechanisms of action. One is the inhibition of splicing by forming a complementary strand with the mRNA precursor. The other is the cleavage of mRNA 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.

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 has been introduced in place of the hydrogen atom at the 4' position of ribose is known (Patent Document 1).

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

Also known as nucleoside derivatives are LNA and BNA.

International Publication No. 2018/110678

However, even with these nucleoside derivatives, there is still a demand for nucleic acid drugs to further promote the inhibition of mRNA translation and improve their therapeutic effectiveness. For this reason, it is necessary to further improve nuclease resistance while maintaining affinity with the target RNA, and at the same time promote the cleavage of mRNA by RNase H.

This specification provides nucleoside derivatives and the like that contribute to making nucleic acid medicines more practical.

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 atom-containing propoxy group is introduced at the 4' position of ribose can contribute to improving the degradation activity of mRNA by RNase H and nuclease resistance while maintaining the affinity of the oligonucleotide with the target RNA. According to this specification, the following means are provided.

（式（１）及び式（２）中、Ｒ¹は、水素原子、水酸基、水素原子がアルキル基又はアルケニル基で置換された水酸基又は保護された基を表し、式（１）及び式（２）中、Ｒ²及びＲ⁴は互いに同一又は異なっていてもよく、水素原子、水酸基の保護基、リン酸基、保護されたリン酸基、又は－Ｐ（＝Ｏ）_nＲ⁵Ｒ⁶（ｎは０又は１を示し、Ｒ⁵及びＲ⁶は、互いに同一又は異なっていてもよく、水素原子、水酸基、保護された水酸基、メルカプト基、保護されたメルカプト基、低級アルコキシ基、シアノ低級アルコキシ基、アミノ基、又は置換されたアミノ基のいずれかを示す。ただし、ｎが１のときには、Ｒ⁵及びＲ⁶が共に水素原子となることはない。）を示し、Ｒ³は、ＮＨＲ⁷（Ｒ⁷は、水素原子、アルキル基、アルケニル基又はアミノ基の保護基を表す。）を表し、Ｂは、プリン－９－イル基、２－オキソ－ピリミジン－１－イル基、置換プリン－９－イル基、又は置換２－オキソ－ピリミジン－１－イル基のいずれかを表す。）
［２］前記式（１）及び式（２）中、Ｒ⁷は水素原子を表す、（１）に記載のヌクレオシド誘導体又はその塩。
［３］［１］又は［２］に記載のヌクレオシド誘導体に由来する構造単位を少なくとも１個備える、オリゴヌクレオチド誘導体又はその塩。
［４］前記オリゴヌクレオチド誘導体の３’末端から４塩基以内の範囲及び前記オリゴヌクレオチド誘導体の５’末端から４塩基の以内の範囲において、前記構造単位を、合わせて少なくとも１個を備える、［３］に記載のオリゴヌクレオチド誘導体又はその塩。
［５］前記オリゴヌクレオチド誘導体の３’末端から４塩基以内の範囲及び前記オリゴヌクレオチド誘導体の５’末端から４塩基以内の範囲に、前記構造単位を合わせて少なくとも３個備える、［４］に記載のオリゴヌクレオチド誘導体又はその塩。
［６］前記オリゴヌクレオチド誘導体の３’末端から４塩基以内の範囲及び前記オリゴヌクレオチド誘導体の５’末端から４塩基以内の範囲において、前記構造単位を合わせて少なくとも５個備える、［５］に記載のオリゴヌクレオチド誘導体又はその塩。
［７］前記オリゴヌクレオチド誘導体の３’末端から４塩基以内の範囲において、前記構造単位を１個又は２個以上備える、［４］に記載のオリゴヌクレオチド誘導体又はその塩。
［８］前記オリゴヌクレオチド誘導体の５’末端から４塩基以内の範囲に、前記構造単位を１個又は２個以上備える、［４］に記載のオリゴヌクレオチド又はその塩。
［９］前記オリゴヌクレオチド誘導体の５’末端から４塩基以内の範囲であって、前記５’末端以外に、前記構造単位を１個又は２個以上備える、［８］に記載のオリゴヌクレオチド誘導体又はその塩。
［１０］前記オリゴヌクレオチド誘導体の３’末端から４塩基以内及び前記オリゴヌクレオチド誘導体の５’末端から４塩基以内を除いた範囲において、少なくとも１個備える、［３］～［９］のいずれかに記載のオリゴヌクレオチド誘導体又はその塩。
［１１］前記オリゴヌクレオチド誘導体は、［１］又は［２］に記載のヌクレオシド誘導体に由来する前記構造単位以外に、デオキシリボヌクレオシドに由来する構造単位を備える、［３］～［１０］のいずれかに記載のオリゴヌクレオチド誘導体又はその塩。
［１２］［３］～［１０］のいずれかに記載のオリゴヌクレオチド誘導体又はその塩を含む、ｍＲＮＡを標的とする遺伝子発現抑制剤。 [1] A nucleoside derivative or a salt thereof represented by the following formula (1) or (2):

(In formulas (1) and (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 formulas (1) and (2), R ² and R ⁴ may be the same or different from each other and represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P ⁽ =O) _nR5R6 (n represents 0 or ¹ ; R ⁵ and R ⁶ may be the same or different from each other 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 are not both hydrogen atoms); and R ³ represents NHR ⁷ (R ⁷ represents a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group; and B represents any one of 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.
[2] The nucleoside derivative or a salt thereof according to (1), wherein in the formulas (1) and (2), R ⁷ 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 [1] or [2].
[4] The oligonucleotide derivative or a salt thereof according to [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 derivative or a salt thereof according to [4], comprising at least three 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.
[6] The oligonucleotide derivative or a salt thereof according to [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 [4], which comprises one or more of the structural units within a range of 4 bases from the 3'-end of the oligonucleotide derivative.
[8] The oligonucleotide or a salt thereof according to [4], which comprises one or more of the structural units within a range of 4 bases from the 5'-end of the oligonucleotide derivative.
[9] The oligonucleotide derivative or a salt thereof according to [8], which has one or more of the structural units at or near the 5' end of the oligonucleotide derivative within a range of 4 bases from the 5' end.
[10] The oligonucleotide derivative or a salt thereof according to any one of [3] to [9], which has at least one in a range excluding within 4 bases from the 3' end of the oligonucleotide derivative and within 4 bases from the 5' end of the oligonucleotide derivative.
[11] The oligonucleotide derivative or a salt thereof according to any one of [3] to [10], which has 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 derivative or a salt thereof according to any one of [3] to [10].

FIG. 1 shows 4'-(S)-2-aminopropoxythymidine (4'-(S)-2-APoT) and 4'-(R)-2-aminopropoxythymidine (4'-(R)-2-APoT). FIG. 1 shows a synthetic scheme for a phosphoramidite derivative of 4'-(S)-2-APoT. FIG. 1 shows a synthetic scheme for a phosphoramidite derivative of 4'-(R)-2-APoT. FIG. 2 shows the sequences of synthesized oligonucleotides. FIG. 1 shows the results of evaluating the thermal stability of oligonucleotides incorporating 4'-(S)-2-APoT and 4'-(R)-2-APoT. FIG. 1 shows the results of evaluating the RNase H activity of oligonucleotides containing 4'-(S)-2-APoT and 4'-(R)-2-APoT. FIG. 1 shows the results of evaluating the nuclease resistance of oligonucleotides incorporating 4'-(S)-2-APoT and 4'-(R)-2-APoT. FIG. 1 shows a synthetic scheme for 4'-(S)-2-APoT-2'-OMe-5Me. FIG. 1 shows a synthetic scheme for 4'-(R)-2-APoT-2'-OMe-5Me. This figure shows the results of evaluating the gene expression-inhibiting activity of ASOs incorporating 4'-(S)-2-APoT-2'-OMe and 4'-(R)-2-APoT-2'-OMe.

This specification relates to nucleoside derivatives useful in nucleic acid medicines, oligonucleotides having structural units derived from these nucleoside derivatives, and their uses and manufacturing methods.

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

The changes in thermal stability, RNase H decomposition 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 knowledge. However, unexpectedly, this nucleoside derivative was able to improve nuclease resistance while maintaining affinity with RNA and RNase H decomposition activity. Nuclease resistance can suppress decomposition by various nucleases inside and outside the cell, and therefore can greatly contribute to the gene expression inhibitory activity of ASO.

The nucleoside derivatives disclosed in this specification are based on the discovery of more useful features than expected after introducing various aminoalkyl-based substituents into the 4' position of ribose, which was previously difficult to do, and examining their properties. Previously, ribonuclease resistance was generally achieved by substitution at the 2' or 3' position of ribose. The nucleoside derivatives disclosed in this specification combine more-than-expected ribonuclease resistance with cell membrane permeability, properties useful for RNA medicines, etc.

Representative and non-limiting examples of the present disclosure will be described in detail below, with reference to the drawings as appropriate. 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. In addition, the additional features and inventions disclosed below can be used separately from or together with other features and inventions to provide further improved nucleoside derivatives and uses thereof.

Furthermore, the combinations of features and steps disclosed in the following detailed description are not essential to implementing the present disclosure in the broadest sense, but are described only to specifically illustrate representative examples of the present disclosure. Furthermore, the various features of the representative examples described above and below, as well as the various features of those described in the independent and dependent claims, do not have to be combined in the exact manner of the examples described herein, or in the order listed, in order to provide additional and useful embodiments of the present disclosure.

All features described in this specification and/or claims are intended to be disclosed individually and independently of one another as limitations to the original disclosure and the specific features claimed, 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 thereof as limitations to the original disclosure and the specific features claimed. Various embodiments of the disclosure of this specification are described in detail below.

(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.

This nucleoside derivative has a basic substituent branched at the 4' position of the ribose and deoxyribose.

In this specification, the meaning of "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, 1 to 6 carbon atoms or 1 to 5 carbon atoms are exemplified, and more preferably, 1 to 4 carbon atoms or 1 to 3 carbon atoms are exemplified.

The nucleoside derivatives or salts thereof disclosed in this specification and their uses are described below.

(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):

Another embodiment of the nucleoside derivative or salt thereof is a nucleoside derivative or 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.

[Regarding ^R1 ]
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. When R ^{1 is a hydroxyl group, the present nucleoside derivative can be said to have ribose as a sugar. When R 1 is} a hydrogen atom, a hydroxyl group in which the hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected hydroxyl group, the present nucleoside derivative can be said to have deoxyribose as a sugar.

(Alkyl group)
In the present specification, examples of the alkyl group include saturated hydrocarbon groups which are linear, branched, cyclic, or a combination thereof. In general, 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, and particularly preferred examples include lower alkyl groups having 1 to 4 carbon atoms or lower alkyl groups having 1 to 3 carbon atoms. Suitable examples of linear alkyl groups having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, and n-butyl groups, and among these, methyl, ethyl, and n-propyl groups are preferred, and also, for example, methyl groups are preferred. Examples of branched alkyl groups having 1 to 4 carbon atoms include isopropyl, isobutyl, s-butyl, and t-butyl groups, and among these, isopropyl is a particularly preferred example. Examples of cyclic alkyl groups having 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, and cyclopropylmethyl groups.

(Alkenyl group)
In the present specification, the alkenyl group includes a saturated hydrocarbon group which is linear, branched, cyclic, or a combination thereof. In general, 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.

(Hydroxyl-protecting group or protected hydroxyl group)
In this specification, the protective group for the hydroxyl group is well known to those skilled in the art, and can be referred to, for example, Protective Groups in Organic Synthesis (John Wiley and Sons, 2007 edition). Representative examples of the protective group for the hydroxyl 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 means a lower alkyl, a lower alkoxy, a halogen atom, or a cyano group), or a silyl group.

In the present specification, examples of alkoxy groups include 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, and furthermore, alkoxy groups having 1 to 4 carbon atoms or 1 to 3 carbon atoms are preferred, and alkoxy groups having 1 to 4 carbon atoms are particularly preferred. Preferred examples of alkoxy groups having 1 to 4 carbon atoms include methoxy groups, ethoxy groups, n-propoxy groups, and n-butoxy groups. Other preferred examples include isopropoxy groups, isobutoxy groups, s-butoxy groups, and t-butoxy groups. Other preferred examples include cyclopropoxy groups and cyclobutoxy groups, and cyclopropylmethoxy groups.

In the present specification, the alkylthio group includes a saturated alkylthio group which is linear, branched, cyclic, or a combination thereof. A lower alkylthio group is preferred, and examples of the lower alkylthio group include a lower alkylthio group having 1 to 6 carbon atoms or a lower alkylthio group having 1 to 5 carbon atoms, and particularly preferred examples include a lower alkylthio group having 1 to 4 carbon atoms or an alkylthio group having 1 to 3 carbon atoms. Preferred examples of the saturated alkylthio group having 1 to 4 carbon atoms include a methylthio group, an ethiothio group, an n-propylthio group, an n-butylthio group, and the like. Preferred examples include an isopropylthio group, an isobutylthio group, an s-butylthio group, and a t-butylthio group. Preferred examples include a cyclopropylthio group or a cyclobutylthio group, and a more preferred example is a cyclopropylmethylthio group.
(I do.)

Among these, aliphatic acyl groups, aromatic acyl groups, and silyl groups are particularly preferred. Also preferred are methyl groups 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).

Examples of the aliphatic acyl group include an alkylcarbonyl group, a carboxyalkylcarbonyl group, a halogeno lower alkylcarbonyl group, or a lower alkoxy lower alkylcarbonyl group.

The alkyl in the alkylcarbonyl group is as described 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 henycosyl group. Among these, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, and pivaloyl 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. That is, examples of the carboxylated alkylcarbonyl group include succinoyl, glutaroyl, and adipoyl.

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.

The alkoxy, alkyl, and lower groups in the lower alkoxy-lower alkyl carbonyl 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 alkyl carbonyl group is a methoxyacetyl group.

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.

The arylcarbonyl group includes, for example, a benzoyl group, an α-naphthoyl group, and a β-naphthoyl group, and more preferably, a benzoyl group. The halogenoarylcarbonyl group includes, for example, a 2-bromobenzoyl group and a 4-chlorobenzoyl group. The lower alkylated arylcarbonyl group includes, for example, a 2,4,6-trimethylbenzoyl group, a 4-toluoyl group, a 3-toluoyl group, and a 2-toluoyl group. The lower alkoxylated arylcarbonyl group includes, for example, a 4-anisoyl group, a 3-anisoyl group, and a 2-anisoyl group.

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.

Examples of lower alkoxymethyl groups include methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, and t-butoxymethyl groups. Methoxymethyl groups are particularly preferred.

Oxycarbonyl groups which may have an appropriate substituent include lower alkoxycarbonyl groups, lower alkoxycarbonyl groups substituted with halogen or a silyl group, and alkenyloxycarbonyl groups.

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.

The alkenyloxycarbonyl group may be a vinyloxycarbonyl group. Preferred examples of the tetrahydropyranyl group, which may have an appropriate substituent, include, for example, a tetrahydropyran-2-yl group or a 3-bromotetrahydropyran-2-yl group, and particularly preferably, a tetrahydropyran-2-yl group.

Tetrathiopyranyl groups which may have suitable substituents include, for example, tetrahydrothiopyran-2-yl and 4-methoxytetrahydrothiopyran-4-yl groups, and more preferably tetrahydrothiopyran-2-yl groups. In the case of a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups, the substituents in the aforementioned substituted aryl groups are lower alkyl, lower alkoxy, halogen, or cyano groups.

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. Other examples include 4-chlorobenzyl and 4-bromobenzyl groups. Another preferred example is the 4-cyanobenzyl group.

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.

In this specification, the term "protective 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, perhaps induced by a microorganism, etc.). As a protective group for a hydroxyl group, particularly preferred examples 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 replaced by such a protective group.

Although ^R1 is not particularly limited, it may be advantageous that R1 is, for example, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group having 1 to 3 carbon atoms, such as a methyl group, because when a structural unit derived from the present nucleotide derivative constitutes a part of a structural unit of a nucleic acid drug such as ASO, it may be possible to further improve nuclease resistance.

[Regarding ^R2 and ^R4 ]
In formula (1) and formula (2), ^R2 and ^R4 may be the same or different and represent a hydrogen atom, a hydroxyl-protecting group, a phosphoryl group, a protected phosphoryl group, or -O-P(=O) _n ( ^R5 ) ^R6 . The hydroxyl-protecting group is as already explained.

(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 phosphate protecting group, which is known to those skilled in the art and can be referred to the above-mentioned references and explanations.

Protective groups for the phosphate group include, for example, 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.

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 the 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.

Examples of the lower alkyl group substituted with halogen include 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, and 2,2,2-trichloroethyl groups, with 2,2,2-trichloroethyl being 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.

Examples of the lower alkenyl group substituted with the cyano group include 2-cyanoethyl, 2-cyanopropyl, and 2-cyanobutenyl groups. Examples of the aralkyl group include benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, and 4-phenylbutyl groups, and more preferably benzyl, diphenylmethyl, triphenylmethyl, 1-phenethyl, and 2-phenethyl groups, and particularly preferably benzyl groups.

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

In this specification, the term "protective group for phosphate 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, perhaps induced by a microorganism, etc.). As the protective group for phosphate group, particularly preferred examples include substituents that are eliminated by hydrogenolysis or hydrolysis.

(-P(=O) _n (R ⁵ )R ⁶ )
^R2 and ^R4 of the nucleoside derivative of the present invention may be -P(=O) _n ( ^R5 ) ^R6 . n represents 0 or 1, and ^R5 and ^R6 may be the same or different and represent any of 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, and a substituted amino group. However, when n is 1, ^R5 and ^R6 are not both hydrogen atoms. The protected hydroxyl group and the lower alkoxy group are as already explained.

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

Preferred examples of the cyano lower alkoxy group include linear, branched, or cyclic alkoxy groups substituted with cyano groups, or combinations thereof, having 1 to 5 carbon atoms (when counting without including the number of carbon atoms in the cyano group). Specific examples include cyanomethoxy, 2-cyanoethoxy, 3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy, and 1-cyanomethyl-1,1-dimethylmethoxy, with the 2-cyanoethoxy group being particularly preferred.

^R5 and ^R6 can be selected as a substituted 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. When both ^R5 and ^R6 are 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.

More specifically, preferred examples of --P(.dbd.O) _n (R ⁵ )R ⁶ include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group, with a phosphoramidite group being particularly preferred.

In -P(=O) _n ( ^R5 ) ^R6 , when n is 0, at least one of ^R5 and ^R6 is a substituted amino group, and the other may be any group, it becomes a phosphoramidite group. As the phosphoramidite group, a phosphoramidite group in which one of ^R5 and ^R6 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. As the substituted amino group, for example, a diethylamino group, a diisopropylamino group, a dimethylamino group, etc. are preferred, and a diisopropylamino group is particularly preferred. In addition, as a lower alkoxy group in the other substituent of ^R5 and ^R6 , a methoxy group is preferred. In addition, as a cyano lower alkoxy group, a 2-cyanoethyl group is preferred. Specific preferred examples of the phosphoramidite group include --O--P(OC ₂ H ₄ CN)(N(CH(CH ₃ ) ₂ ) and --P(OCH ₃ )(N(CH(CH ₃ ) ₂ ).

In -P(=O) _n ( ^R5 ) ^R6 , when n is 1, at least one of ^R5 and ^R6 is a hydrogen atom, and the other may be any group other than a hydrogen atom, it becomes 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.

In addition, in -O-P(=O) _n ( ^R5 ) ^R6 , when n is 1 and ^R5 and ^R6 are both lower alkoxy groups, it becomes a phosphonyl group. The lower alkoxy groups in ^R5 and ^R6 may be the same or different. Preferred examples of the lower alkoxy group include a methoxy group and an ethoxy group. A specific example of a phosphonyl group is -P(=O)( _OCH3 ) ₂ .

In the present nucleoside derivative, R ² is particularly preferably, for example, -P(=O) _n (R ⁵ )R ^6. Preferred examples of -P(=O) _n (R ⁵ )R ⁶ include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group. ^{R 2} is also preferably a phosphoryl group (OR ² is a phosphate group) or a protected phosphoryl group (OR ² is a protected phosphate group). Furthermore, R ² is also preferably a hydroxyl group or a protecting group for a hydroxyl group.

Preferred specific examples of ^R2 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( _OC2H4CN )(N(CH( _CH3 ) ₂ ), -P( _OCH3 )(N(CH( _CH3 ) ₂ ), or a phosphonyl group.

R ⁴ in the present nucleoside derivative is preferably, for example, a hydrogen atom or a protecting group for a hydroxyl group. Also preferred is, for example, a phosphate group, a protected phosphate group, or -P(=O) _n (R ⁵ )R ^6. Specific preferred examples of R ⁴ 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, or a trimethylsilyl group.

[Regarding ^R3 ]
In formula (1) and formula (2), R ³ is NHR ⁷ (R ⁷ represents a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group).

^R7 may be a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group. The alkyl group may be the alkyl group already described, or a lower alkyl group may be preferably used. The alkenyl group may be the alkenyl group already described, or a lower alkenyl group may be preferably used. When it is a structural unit in an oligonucleotide, ^R7 is preferably a hydrogen atom.

Protective groups for amino groups are well known to those skilled in the art and references mentioned above may be consulted. Specific examples of the protecting group include, in addition to those 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 phthalimido 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.

In this specification, the term "protective group for amino groups" 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, perhaps induced by a microorganism, etc.). In particular, substituents that are eliminated by hydrogenolysis or hydrolysis are preferred as protective groups for amino groups.

[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 purine-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purine-9-yl group, and a substituted 2-oxo-pyrimidin-1-yl group.

In other words, examples of B include a purine-9-yl group or a 2-oxo-pyrimidin-1-yl group, as well as 2,6-dichloropurine-9-yl or 2-oxo-pyrimidin-1-yl. Further examples include 2-oxo-4-methoxy-pyrimidin-1-yl, 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl, or 2,6-dimethoxypurine-9-yl.

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 described above.

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), and 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (thymine).

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, and 2-oxo-4-mercapto-pyrimidin-1-yl.

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.

The substituents in the substituted purine-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group are either 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, or a halogen atom, or any combination of two or more of these. These substituents are as already explained.

In the present nucleoside derivative, B is preferably a substituted purine-9-yl group or a substituted 2-oxo-pyrimidin-1-yl group having the aforementioned substituents, but is also preferably a triazole group or a lower alkoxymethyl group.

Preferred examples of the substituted purine-9-yl group include, for example, 6-aminopurine-9-yl, 2,6-diaminopurine-9-yl, 2-amino-6-chloropurine-9-yl, 2-amino-6-bromopurine-9-yl, 2-amino-6-hydroxypurine-9-yl, 6-amino-2-methoxypurine-9-yl, 6-amino-2-chloropurine-9-yl, 6-amino-2-fluoropurine-9-yl, 2,6-dimethoxypurine-9-yl, 2,6-dichloropurine-9-yl, and 6-mercaptopurine-9-yl. If the above-mentioned substituents contain amino groups or hydroxyl groups, preferred examples include substituents in which the amino groups and/or hydroxyl groups are protected.

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, and 4-amino-5-methyl-2-oxo-pyrimidin-1-yl. Other preferred examples include 2-oxo-4-methoxy-pyrimidin-1-yl and 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl.

If the substituent of B 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.

The nucleoside derivative may be in the form of a salt. The salt form is not particularly limited, but generally, an acid addition salt is 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. As the salt, a pharma- ceutically acceptable salt is preferred. The types of acids and bases that form pharma- ceutical acceptable salts are well known to those skilled in the art, and those described in, for example, J. Pharm. Sci., 1-19 (1977) can be referred to. For example, examples of acid addition salts include mineral acid salts and organic acid salts. Furthermore, when one or more substituents contain an acidic moiety, a base addition salt is also a preferred example.

Examples of mineral acid salts include hydrochlorides, hydrobromides, hydroiodides, nitrates, sulfates, hydrogen sulfates, phosphates, and hydrogen phosphates. In general, 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. In general, 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.

Examples of the alkali metal salts include sodium salts and potassium salts. Examples of the alkaline earth metal salts include magnesium salts and calcium salts. Examples of the 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 the amino acid addition salts include arginine salts, lysine salts, ornithine salts, serine salts, glycine salts, aspartates, and glutamates.

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

By providing this 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 this nucleoside derivative is provided as at least a partial structural unit of an oligonucleotide, the affinity of the oligonucleotide for the complementary strand of RNA and the decomposition activity by RNase H can be maintained.

(Oligonucleotide derivatives and their salts)
The oligonucleotide derivative disclosed in the present specification (hereinafter also referred to as the present oligonucleotide derivative) 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 the nucleoside derivatives or salts thereof represented by formula (1) and formula (2), respectively. The present oligonucleotide derivative is called a derivative based on the fact that it has a structural unit represented by formula (3) or formula (4).

R ¹ , R ³ and B in the structural units represented by formulae (3) and (4) have the same meanings as those in formulae (1) and (2), respectively.

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 as or different from each other. 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). The oligonucleotide derivative may have one or more structural units represented by formula (3) and one or more structural units represented by formula (4).

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 provided at at least one of the 5'-end side (e.g., within 4 bases from the 5'-end), the center, and the 3'-end side (e.g., within 4 bases from the 3'-end) of the present oligonucleotide derivative. Also, each structural unit may be provided at two or more of these positions. Also, these structural units may be provided approximately evenly at two or more positions. The term "approximately evenly provided structural units at two or more positions" does not necessarily mean that the same number of structural units are provided at each of the two or more positions, but it is sufficient to at least satisfy that at least one structural unit is provided at each of these positions. For example, when about 1 to 3 structural units are provided at each position, it can be said that the structural units are approximately even. At least six structural units can be provided in the present oligonucleotide derivative.

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 the 5'-end and 3'-end. This can increase the nuclease resistance at the ends.

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

Furthermore, the oligonucleotide derivative may have at least one structural unit in its central portion, i.e., in the range excluding within 4 bases from its 3' end and 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 for example, in the range from the 5th base to the 10th base, or for example, in the range from the 5th base to the 8th base. This can increase the nuclease resistance of the central portion.

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

Since the sugar chain portion of the structural units represented by formula (3) and formula (4) is derived from ribose or deoxyribose, the present oligonucleotide derivative may be an oligoribonucleotide or an oligodeoxyribonucleotide. The present oligonucleotide derivative may also be a chimera of a ribonucleotide and a deoxyribonucleotide.

The present oligonucleotide derivative is single-stranded by itself, but can also take the form of a hybrid with an oligoribonucleotide, oligodeoxyribonucleotide, or oligodeoxyribo/ribonucleotide (chimeric strand), i.e., a double-stranded form.

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

The structural units defined in this specification and other structural units can be bonded to each other, for example, by phosphate diester bonds, phosphate monoester bonds, phosphorothioate bonds (thiophosphate ester bonds), etc.

The present oligonucleotide derivative has at least 2 or more, for example, 8 or more, for example, 10 or more, for example, 12 or more, or for example, 14 or more, in terms of the number of structural units and other nucleoside derivatives. There is no particular limit to the upper limit, but it 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.

When the 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.

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. The same applies when stereoisomers exist, and any mixture of stereoisomers or racemates are all included in the scope of the present invention. They may also exist as tautomers.

The oligonucleotide derivative may be a salt. The form of the salt is not particularly limited, but pharma- ceutically acceptable salts are preferred. The salt forms of the nucleoside derivative described above can be applied. The oligonucleotide derivative or its salt may be a hydrate or solvate, which are also included in the scope of the present invention.

(Production of the Nucleoside Derivative and the Oligonucleotide Derivative)
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 that were publicly known at the time of filing this application.

The nucleoside derivative and the oligonucleotide derivative of the present invention can be produced, for example, by the method described below, but the method for producing the nucleoside derivative or oligonucleotide derivative of the present invention is not limited to the method described below.

In each reaction, the reaction time is not particularly limited, but since the progress of the reaction can be easily tracked by the analytical means described below, it is sufficient to terminate the reaction at the point where the yield of the target product is maximized. In addition, each reaction can be carried out, if necessary, in an inert gas atmosphere, for example, under a nitrogen or argon stream. In each reaction, if protection with a protecting group and subsequent deprotection are required, the reaction can be carried out appropriately by using the method described below.

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.

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.

Also, 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.

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 formulas (1) or (2) as amidite agents, etc. In other words, 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.

The oligonucleotide derivative can be used, for example, as an ASO that targets mRNA. It can also be linked to other compounds to form conjugates. Furthermore, the oligonucleotide derivative can also be used as a component of a ribozyme. The oligonucleotide derivative is also useful as an RNA chip or other reagent.

As a result, the present oligonucleotide derivatives, taking advantage of characteristics not found in natural oligonucleotides, are expected to be more useful than natural nucleosides and previous nucleosides as components of nucleic acid drugs as various gene expression inhibitors that inhibit the function of genes to treat diseases, including antitumor and antiviral agents. In other words, the present oligonucleotide derivatives are useful not only as such nucleic acid drugs, but also as raw materials or intermediate reagents for them. The present nucleoside derivatives are also useful as raw materials or intermediates for such RNA drugs.

Below, specific examples are provided to more specifically explain the disclosure of this specification. The following examples are provided to explain the disclosure of this specification and are not intended to limit the scope thereof.

<Synthesis of 4'-C-(S)-2-aminopropoxythymidine analogue (S-form)>
The S-isomer was synthesized according to the scheme shown in Figure 2. The S-isomer was obtained through a total of seven reaction steps with a total yield of 10%. As 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.

<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 Ar atmosphere, and 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 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 stirred at room temperature for 19 hours. The solvent was then removed under reduced pressure, and the residue was separated by silica gel chromatography (CHCl ₃ :MeOH = 20:1) to obtain compound (3) as a crude product. Compound 3 was dissolved in 5.00 mL _{of CH2Cl2} under an Ar atmosphere, and imidazole (0.3404 g, 5.00 mmol) and TBDMSCl (0.5685 g, 3.72 mmol) were added and stirred at room temperature for 21 hours. The solvent was then removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane: EtOAc = 1:2) to obtain compound (4) (0.3175 g, 0.94 mmol, 47%).

¹ H NMR (400 MHz, CDCl ₃ ) 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)

<Synthesis of compound (5)>
Compound (4) (0.907 g, 2.66 mmol) was dissolved in 13.5 ml _{of CH2Cl2} , and 45.0 ml of _sat.NaHCO3 aq. and 9.00 ml of acetone were added. Oxone (registered trademark) (4.92 g, 7.98 mmol) dissolved in 27.0 ml of _H2O was added dropwise on an ice bath. After stirring at room temperature for 3.5 hours, the product was extracted from the reaction solution with _CHCl3 and _sat.NaHCO3 aq., the organic layer was washed with sat.NaCl aq., and water was removed with _Na2SO . The solvent was removed under reduced pressure to obtain the crude product.

Under Ar atmosphere, the crude product and (S)-2-(trifluoroacetyl)aminopropanol (1.38 g, 7.98 mmol) were dissolved in 13.0 ml of THF, and 1M ZnCl ₂ in THF (2.70 ml, 2.70 mmol) was added dropwise at -40°C. After stirring at room temperature for 17 hours, the mixture was quenched with 15 ml of sat.NaHCO ₃ aq. The precipitated salt was removed by filtration through Celite, and the compound was extracted from the filtrate with EtOAc and H ₂ O. The organic layer was washed with sat.NaHCO ₃ aq., and then water was removed with Na ₂ SO ₄ , and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc=1:1) to obtain compound (5) (0.54 g, 1.02 mmol, 38%) as white crystals.

¹ H-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 F ₃ N ₃ NaO ₇ Si [M + Na] ⁺ 548.20158, Found 548.20168

<Synthesis of compound (6)>
Under 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 sat.NaHCO ₃ aq., and the organic layer was washed with sat.NaCl aq., and then water was removed with Na ₂ SO _4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane/EtOAc=2:3) to obtain compound (6) (0.556 g, 0.680 mmol, 80%) as yellow crystals.

¹ H-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 F ₃ N ₃ NaO ₉ Si [M + Na] ⁺ 850.33226, Found 850.32938

<Synthesis of compound (7)>
Under Ar atmosphere, compound (6) (0.610g, 0.730mmol) was dissolved in THF (7.3ml), 1M TBAF in THF (1.1ml) was added, and 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) (0.450g, 0.630mmol, 87%) as white crystals.

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 F ₃ N ₃ NaO ₉ [M + Na] ⁺ 736.24578, Found 736.24776

<Synthesis of compound (8)>
Under Ar atmosphere, compound (7) (0.400 g, 0.560 mmol) was dissolved in 4.00 ml of THF, 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 sat. NaHCO ₃ aq. The compound was extracted with EtOAc and sat. NaHCO ₃ aq., and the organic layer was washed with sat. NaCl aq., and then water was removed with Na ₂ SO _4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane/EtOAc=2:3) to obtain compound (8) (0.430 g, 0.470 mmol, 84%) as white crystals.

³¹ P-NMR (162 MHz, CHLOROFORM-D) δ 150.9, 150.0
HRMS (ESI-TOF) m/z Calcd for C ₄₅ H ₅₅ F ₃ N ₅ NaO ₁₀ P [M + Na] ⁺ 936.35363, Found 936.35409

<Synthesis of compound (10)>
Under Ar atmosphere, _CF3COOEt (1.93 ml, 16.2 mmol) was dissolved in MeCN 4.6 ml, and (S)-(+)-2-amino-1-propanol (compound (9)) (0.93 ml, 12.0 mmol) was added on 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.

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

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

<Synthesis of compound (11)>
Compound (4) (1.02 g, 3.00 mmol) was dissolved in 15.5 ml _{of CH2Cl2} , and 51.0 ml of _sat.NaHCO3 aq. and 10.2 ml of acetone were added. Oxone (registered trademark) (5.53 g, 9.00 mmol) dissolved in 30.5 ml of _H2O was added dropwise on an ice bath. After stirring at room temperature for 3.5 hours, the product was extracted from the reaction solution with _CHCl3 and _sat.NaHCO3 aq., the organic layer was washed with sat.NaCl _aq ., and water was removed with _Na2SO4 . The solvent was removed under reduced pressure to obtain the crude product.

Under 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 1M ZnCl ₂ in THF (3.00 ml, 3.00 mmol) was added dropwise at -40°C. After stirring at room temperature for 17 hours, the mixture was quenched with 20 ml of sat.NaHCO ₃ aq. The precipitated salt was removed by filtration through Celite, and the compound was extracted from the filtrate with EtOAc and H ₂ O. The organic layer was washed with sat.NaHCO ₃ aq., and then water was removed with Na ₂ SO ₄ , and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc=1:1) to obtain compound (11) (0.610 g, 1.16 mmol, 39%) as white crystals.

¹ H-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 F ₃ N ₃ NaO ₇ Si [M + Na] ⁺ 548.20158, Found 548.20416

<Synthesis of compound (12)>
Under 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 sat.NaHCO ₃ aq., and the organic layer was washed with sat.NaCl aq., and then water was removed with Na ₂ SO _4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane/EtOAc=2:1) to obtain compound (12) (0.179 g, 0.216 mmol, 54%) as yellow crystals.

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)
¹³ C-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 F ₃ N ₃ NaO ₉ Si [M + Na] ⁺ 850.33226, Found 850.33184

<Synthesis of compound (13)>
Under Ar atmosphere, compound (12) (0.166 g, 0.200 mmol) was dissolved in 2.00 ml of THF, and 0.30 ml of 1M 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.

¹ H-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 F ₃ N ₃ NaO ₉ [M + Na] ⁺ 736.24578, Found 736.24632

<Synthesis of compound (14)>
Under Ar atmosphere, compound (13) (0.620 g, 0.870 mmol) was dissolved in 6.20 ml of THF, 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 sat. NaHCO ₃ aq. The compound was extracted with EtOAc and sat. NaHCO ₃ aq., and the organic layer was washed with sat. NaCl aq., and then water was removed with Na ₂ SO _4. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (hexane/EtOAc=2:3) to obtain compound (14) (0.678 g, 0.740 mmol, 85%) as white crystals.

¹ P-NMR (162 MHz, CHLOROFORM-D) δ 150.8, 149.6
HRMS (ESI-TOF) m/z Calcd for C ₄₅ H ₅₅ F ₃ N ₅ NaO ₁₀ P [M + Na] ⁺ 936.35363, Found 936.35612

<Synthesis of compound (16)>
Under Ar atmosphere, _CF3COOEt (1.45 ml, 12.2 mmol) was dissolved in MeCN 3.5 ml, and (R)-(-)-2-amino-1-propanol (compound (15)) (0.70 ml, 9.0 mmol) was added on 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

Synthesis of oligonucleotides containing 4´-2-APoT analogs
Oligonucleotides were synthesized on a 0.2 μmol scale using an automatic 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. In addition, the CPG resin to which the 3´-terminal nucleoside was bound was packed in a column at 0.2 μmol equivalent based on its activity, and synthesis was performed. Note that unysupport 500 resin was used when synthesizing sequences in which the synthesized 4´-(S)-2-APoT or 4´-(R)-2-APoT was introduced at the 3´-terminus. The sequences of the synthesized oligonucleotides are shown in Figure 4.

After the synthesis was completed, the CPG resin was transferred to a sampling tube, 1.0 ml of NH ₃ soln. was added, and the oligonucleotide was cleaved from the resin and deprotected by incubating at 55°C for 12 hours. When synthesizing oligonucleotides containing 4´-(S)-2-APoT or 4´-(R)-2-APoT, in order to prevent the transfer reaction of the cyanoethyl group, which is the protecting group of the phosphate moiety, to the side chain, 900 μl of MeCN and 100 μl of Et ₂ NH were added to the tube, vortexed for 5 minutes, the supernatant was discarded, the resin was washed twice with 1 ml of MeCN, and then cleavage from the CPG resin and deprotection were performed with 1.0 ml of NH ₃ soln. After deprotection, the sample was transferred to an Eppendorf tube, washed twice with 1 ml of H ₂ O, and dried under reduced pressure. For RNA synthesis, the CPG resin was transferred to a sampling tube, 1.0 ml of NH ₃ soln. was added to the tube, and the tube was incubated at 55°C for 4 hours to cleave the oligonucleotide from the resin and deprotect it. The sample was then transferred to an Eppendorf tube, washed twice with 1 ml of H ₂ O, and dried under reduced pressure. The sample was then dissolved in 100 μl of DMSO, and 125 μl of TFA-3HF was added and incubated for 90 minutes to deprotect the TBDMS group. The solution was then diluted to 10 ml with 0.1 M TEAA buffer (2N TEAA buffer (114.38 ml of acetic acid was added to 277.6 ml of _Et3N , and the mixture was diluted to 1000 ml with _H2O to adjust the pH to 7.0) and diluted 20-fold), and the mixture was passed through an equilibrated Sep-pack tC18 reverse phase column to adsorb the target substance onto the column.After washing with sterile water, the product was eluted with 3 ml of 50% MeCN in _H2O and dried under reduced pressure.

Each dried sample was dissolved in 200 μl of loading buffer (0.1 M Tris-HCl in 90% formamide) and the target oligonucleotide was separated and purified by 20% PAGE (500 V, 20 mA). 40 ml of 40% acrylamide (19:1) solution, 33.6 ml of urea, and 8 ml of 10×TBE buffer were added and mixed, and H ₂ O was added to make up to 80 ml. Next, 55 mg of APS was added and dissolved, and 40 μl of TEMED was added and mixed, and the mixture was poured between two glass plates fixed with a 1.5 mm spacer and left to stand for more than 1 hour to solidify. 1×TBE buffer was used as the electrophoresis buffer.

The band of interest was cut out, soaked in 10 ml of 0.1 M TEAA buffer, 1 mM EDTA aqueous solution, and shaken overnight. The solution was then purified in the same manner as above using an equilibrated Sep-pack tC18 reverse phase column, and dried under reduced pressure. The purified oligonucleotide was dissolved in 1 ml of H ₂ O, and the concentration was calculated by measuring the absorbance at 260 nm of the diluted solution, and the yield was calculated according to the usual method. In addition, 60 pmol of oligonucleotide was dried under reduced pressure, mixed with 3 μl 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 ap.), and then dried on a plate, and the compound was identified by measuring the molecular weight using MALDI-TOF/MS.

<Thermal stability evaluation of oligonucleotides containing 4´-2-APoT analogs>
Measurement of the 50% melting temperature (T _m ) of the double strand Each oligonucleotide was dried under reduced pressure at 600 pmol and dissolved in 200 μL of measurement buffer [10 mM NaH ₂ PO _{4 -} Na ₂ HPO ₄ (pH 7.0), 100 mM NaCl]. The double strand was then dissociated by heating at 95 °C for 3 minutes, and allowed to stand at room temperature for more than 1 hour to allow annealing. After degassing for 10 minutes, 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 by the median method from the sigmoid curve obtained from the measurement. The sequences and Tm of the double stranded oligonucleotides used for evaluation are shown in Figure 5.

As shown in Figure 5, the oligonucleotides incorporating the S- and R-forms showed thermal stability equivalent to that of natural DNA (D1) and the 4'-AE form.

<Evaluation of E. coli RNase H activity>
Each oligonucleotide (DNA:RNA = 300 pmol:1500 pmol (1:5)) was dried under reduced pressure for 1 min and dissolved in 37.5 μL of measurement 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 more than 1 h to allow annealing. Each sample was incubated at 37°C, and 35 μL of RNase H solution was added. The reaction was stopped at a designated time 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-fold with the measurement buffer.

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.

As shown in Figure 6, the oligonucleotides with S and R structures showed RNase H activity equivalent to that of natural DNA (D1) and the 4'-AE structure. It was also found that compared to the S structure, the R structure may tend to rapidly and overall fragment the full length cleavage by RNase H into shorter chains.

<Evaluation of nuclease resistance>
300 pmol of each single-stranded oligonucleotide used in the nuclease resistance test was dried under reduced pressure and dissolved in 37.5 μl of Opti-MEM. 1.1 μl of the 0 min sample was taken and added to 5 μl of loading buffer (10 mM EDTA in formamide). Each sample was incubated at 37°C, 15.6 μl of bovine serum (BS) was added, and the reaction was stopped by adding 2.4 μl of the sample solution to an Eppendorf tube containing 10 μl of loading buffer at specified times. Electrophoresis was performed on 20% PAGE at 500 V and 20 mA for about 3 hours, and gel photographs after electrophoresis were taken using a Luminescent Image Analyzer LAS-4000. The oligonucleotides used for evaluation and the evaluation results are shown in Figure 7.

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

These results show that the S- and R-forms of this nucleoside derivative exhibited significantly higher nuclease resistance than the natural form and higher resistance than the 4´-AEoT-introduced oligonucleotide. Therefore, it has become clear that these modified nucleic acids have useful properties as antisense nucleic acids in terms of nuclease resistance.

<Synthesis of phosphoramidite 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.

<Synthesis of compound (6)>
Compound (4) (0.95 g, 2.6 mmol) was dissolved in _CH2Cl2 (14 mL) under Ar atmosphere, acetone (9.5 mL) and _satNaHCO3 aq. (48 _mL ) were added, and an aqueous solution (28 mL) of Oxone (4.8 g, 7.7 mmol) was added at 0°C, and the mixture was stirred at room temperature for 2 hours. After quenching with 5% NaHSO3 aq., the product (compound (5)) was extracted with _CHCl3 and _satNaHCO3 aq., and the organic layer was washed with sat.NaHCO3 aq. and sat.NaCl _aq . The _mixture was dried over _Na2SO4 , and the solvent was distilled off under reduced pressure to obtain a crude product of compound (5). Next, the crude product was dissolved in THF (12 mL) under Ar atmosphere, and 1 M ZnCl ₂ 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) were added at −40°C. The mixture was stirred at room temperature overnight, quenched with satNaHCO ₃ aq., and filtered through Celite. The product was extracted from the filtrate with EtOAc and satNaHCO ₃ aq., and the organic layer was washed with sat NaCl aq. The mixture was dried with Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 50 : 1) and then purified again with (Hexane: EtOAc = 3 : 2) to obtain compound (6) (0.67 g, 1.2 mmol, 47%).

¹ H NMR (400 MHz, CDCl ₃ )) 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)

<Synthesis of compound (7)>
Compound (6) (1.6 g, 2.9 mmol) was dissolved in pyridine (29 mL) under Ar atmosphere, and DMTrCl (2.0 g, 5.9 mmol) was added at 0°C and stirred at room temperature overnight. The product was then extracted with EtOAc and sat.NaHCO ₃ aq., and the organic layer was washed with sat.NaCl aq. The organic layer was dried over Na ₂ SO ₄ , and the solvent was distilled off 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).

¹ H NMR (400 MHz, CDCl ₃ ) 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)

<Synthesis of compound (8)>
Compound (7) (0.85 g, 0.99 mmol) was dissolved in THF (10 mL) under 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 removed 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%).

¹ H NMR (400 MHz, CDCl ₃ ) 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)

<Synthesis of compound (9)>
Compound (8) (0.63 g, 0.83 mmol) was dissolved in THF (8.3 mL) under Ar atmosphere, DIPEA (0.73 mL, 4.1 mmol) and CEP-Cl (0.40 mL, 2.30 mmol) were added, and the mixture was stirred at room temperature for 3 hours. The product was extracted with EtOAc and _sat.NaHCO3 aq. and washed with sat.NaCl aq. 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 = 1:1) to give compound (9) (0.64 g, 0.68 mmol, 82%).
^31P NMR (162MHz, CDCl ₃ ) δ: 151.21, 151.77

<Synthesis of phosphoramidite 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.

<Synthesis of compound (10)>
Compound (4) (0.95 g, 2.6 mmol) shown in Figure 8 was dissolved in _CH2Cl2 (14 mL) under Ar atmosphere, acetone (9.5 mL) and satNaHCO3 _aq . (48 _mL ) were added, and an aqueous solution (28 mL) of Oxone (4.8 g, 7.7 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 2 hours. After quenching with 5% NaHSO3 aq., the product was extracted with _CHCl3 and _satNaHCO3 aq., and the organic layer was washed with _sat.NaHCO3 aq. and sat.NaCl aq. The _mixture was dried with _Na2SO4 , and the solvent was distilled off under reduced pressure to obtain a crude product. Next, the crude product was dissolved in THF (12 mL) under Ar atmosphere, and 1 M ZnCl ₂ 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) were added at −40°C. The mixture was stirred overnight at room temperature, quenched with satNaHCO ₃ aq., and filtered through Celite. The product was extracted from the filtrate with EtOAc and satNaHCO ₃ aq., and the organic layer was washed with sat NaCl aq. The mixture was dried with Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 50 : 1) and then purified again (Hexane: EtOAc = 3 : 2) to obtain compound (10) (0.91 g, 1.6 mmol, 64%).

¹ H NMR (400 MHz, CDCl ₃ )) 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)

<Synthesis of compound (11)>
Compound (10) (1.9 g, 3.4 mmol) was dissolved in pyridine (34 mL) under Ar atmosphere, and DMTrCl (2.3 g, 6.7 mmol) was added at 0°C and stirred at room temperature overnight. The product was then extracted with EtOAc and sat.NaHCO ₃ aq., and the organic layer was washed with sat.NaCl aq. It was also dried with Na ₂ SO ₄ , and the solvent was distilled off 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%).

¹ H NMR (400 MHz, CDCl ₃ ) 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)

<Synthesis of compound (12)>
Compound (11) (1.2 g, 1.4 mmol) was dissolved in THF (14 mL) under 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 removed under reduced pressure. The residue was purified by silica gel column chromatography (Hexane: EtOAc = 3: 2) to obtain compound (12) (9.7 g, 1.3 mmol, 95%).

<Synthesis of compound (13)>
Compound (12) (0.97 g, 1.3 mmol) was dissolved in THF (13 mL) under Ar atmosphere, DIPEA (1.1 mL, 6.5 mmol) and CEP-Cl (0.58 mL, 2.6 mmol) were added, and the mixture was stirred at room temperature for 3 hours. The product was extracted with EtOAc and _sat.NaHCO3 aq. and washed with sat.NaCl aq. 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 = 1:1) to give compound (13) (0.99 g, 1.1 mmol, 81%).
^31P NMR (162MHz, CDCl ₃ ) δ: 150.91, 151.67

<Evaluation of gene expression suppression 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 following table were synthesized in accordance with Example 3. Unless otherwise specified, structural units are structural units derived from deoxynucleosides.

These single-stranded oligonucleotides were used to evaluate the ability to suppress gene expression in NCI-460 cells, with KRAS mRNA as the target RNA. NCI-H460 cells were transfected with the oligonucleotides at final concentrations of 2.0, 5.0, and 10 nM, and incubated at 37°C for 48 hours, after which cDNA was prepared by RT-PCR. The cDNA was then used to perform quantitative reverse transcription polymerase reaction (qPCR) to quantify the expression level of KRAS mRNA. The gene expression level was normalized with the endogenous control ACTB gene (#4326315E), and the relative level was calculated using the ΔΔCt model. The results are shown in Figure 10.

As shown in Figure 10, both the S- and R-form oligonucleotides exhibited a concentration-dependent effect of suppressing gene expression. LNA has the problem of inducing liver toxicity. The S- and R-forms have the advantage that they contain a 2-aminopropoxy group at the 4' position, which is thought to suppress or avoid the induction of liver toxicity.

Claims (12)

A nucleoside derivative or a salt thereof represented by the following formula (1) or (2):

(In formulas (1) and (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 formulas (1) and (2), R ² and R ⁴ may be the same or different from each other and represent a hydrogen atom, a protecting group for a hydroxyl group, a sulfolyl group, a protected sulfolyl group, or -P ⁽ =O) _nR5R6 (n represents 0 or ¹ ; R ⁵ and R ⁶ may be the same or different from each other 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 are not both hydrogen atoms); and R ³ represents NHR ⁷ (R ⁷ represents a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group; and B represents any one of 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. The nucleoside derivative or a salt thereof according to claim 1, wherein in the formulas (1) and (2), R ⁷ represents a hydrogen atom. An oligonucleotide derivative or a salt thereof, comprising at least one structural unit derived from the nucleoside derivative according to claim 1 or 2. 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. The oligonucleotide or salt thereof according to claim 4, comprising at least three 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. The oligonucleotide derivative or salt thereof according to claim 5, comprising at least five 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. The oligonucleotide derivative or salt thereof according to claim 4, which has one or more of the structural units within a range of 4 bases from the 3' end of the oligonucleotide derivative. The oligonucleotide or salt thereof according to claim 4, comprising one or more of the structural units within a range of 4 bases from the 5' end of the oligonucleotide derivative. The oligonucleotide derivative or salt thereof according to claim 8, which has one or more of the structural units in a range of 4 bases from the 5' end of the oligonucleotide derivative, other than the 5' end. The oligonucleotide derivative or salt thereof according to claim 3, which has at least one in a range excluding within 4 bases from the 3' end of the oligonucleotide derivative and within 4 bases from the 5' end of the oligonucleotide derivative. The oligonucleotide derivative or its salt 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. A gene expression inhibitor that targets mRNA, comprising the oligonucleotide derivative or its salt according to any one of claims 3 to 11.