Carbocyclic nucleoside-containing oligonucleotide

Abstract

The present application discloses, as one embodiment of the present invention, an oligonucleotide or a salt thereof that demonstrates an excellent effect against target RNA etc. while maintaining a high level of safety. The oligonucleotide is characterized by comprising, in the oligonucleotide sequence, at least one carbocyclic nucleoside derivative residue (B) which is a divalent group represented by formula (B) (in the formula, each symbol is as defined in the description).

Description

Carbocyclic Nucleoside-Containing Oligonucleotides

[0001] As an embodiment of the present invention, there is provided an oligonucleotide containing a carbocyclic nucleoside, etc. More specifically, the oligonucleotide contains a carbocyclic nucleoside that, when introduced into an oligonucleotide, can increase the maximum tolerated dose or therapeutic index of the oligonucleotide in vivo, and is useful, for example, in the field of medicine.

[0002] In recent years, research and development of nucleic acid drugs has been actively pursued. Nucleic acid drugs are drugs based on natural or chemically modified nucleotides. They are chemically synthesized nucleic acids that act directly on living organisms. In addition to their high specificity based on base pairing, nucleic acid drugs can target molecules that cannot be targeted by conventional drugs, such as mRNA and non-coding RNA. Once the platform is established, they are easily standardized relatively quickly. Therefore, nucleic acid drugs are expected to be applied as next-generation drugs following small molecule drugs and antibody drugs. Nucleic acid drugs are classified into various types based on their mechanism of action, including antisense nucleic acids (RNase H-mediated, steric-blocking, splicing-regulating, RNA-editing, etc.), siRNA, aptamers, and antigene nucleic acids. For example, antisense nucleic acids and siRNA act on in vivo RNA, inhibiting or modulating its function to exert their therapeutic effects. Aptamers utilize their higher-order structure to bind to targets such as proteins. Antigene nucleic acids are also expected to exert their therapeutic effects by acting on genomic DNA. Due to these diverse mechanisms of action, they are being applied to many genetic and intractable diseases, including neurodegenerative diseases, metabolic diseases, cancer, and infectious diseases (see Non-Patent Documents 1 to 3).

[0003] Stanley T. Crooke, Xue-Hai Liang, Brenda F. Baker, Rosanne M. Crooke, Antisense technology: A review, Journal of Biological Chemistry, Volume 296, 2021, 100416, https: / / doi.org / 10.1016 / j.jbc.2021.100416Guillermo Aquino-Jarquin, Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing, Molecular Therapy - Nucleic Acids, Volume 19,2020, Pages 1065-1072M. May Zhang, Raman Bahal, Theodore P. Rasmussen, Jose E. Manautou, Xiao-bo ZhongThe growth of siRNA-based therapeutics: Updated clinical studies, Biochemical Pharmacology, Volume 189,2021,114432Guideline for preclinical safety assessment of oligonucleotide therapeutics, PSEHB / PED Notification No. 0330-1, Mar. 30, 2020, https: / / www.pmda.go.jp / english / review-services / regulatory-info / 0003.htmlTerada C, Kawamoto S, Yamayoshi A, Yamamoto T. Chemistry of Therapeutic Oligonucleotides That Drives Interactions with Biomolecules. Pharmaceutics. 2022 Nov 29;14(12):2647.Hu, B., Zhong, L., Weng, Y. et al. Therapeutic siRNA: state of the art.Sig Transduct Target Ther 5, 101 (2020). https: / / doi.org / 10.1038 / s41392-020-0207-x.

[0004] However, the above-mentioned conventional oligonucleotide therapeutic agents have been associated with safety issues such as side effects and toxicity due to non-specific interactions with proteins or RNA that are not therapeutic targets in vivo (off-target effects) (see Non-Patent Documents 4 to 6). An objective of the present invention is to solve the problems of the prior art and to provide an oligonucleotide therapeutic agent with an increased maximum tolerated dose or an enhanced therapeutic index.

[0005] The present invention relates to a carbocyclic structure in which the oxygen atom at the 4'-position of the furanose ring of 2'-deoxyguanosine is replaced by an exocyclic double bond, or the oxygen atom is replaced by a spiro ring, as in the case of entecavir, which is known for its antiviral activity, i.e., a DNA analogue having a structure in which the oxygen atom at the 4'-position is replaced by an sp2 carbon or spiro carbon. The present inventors have conducted extensive research to solve the above-mentioned problems based on the novel idea that, when entecavir or a derivative thereof is introduced into an oligonucleotide therapeutic agent, the removal of the 4'-oxygen atom, which is generally important for enzymatic nucleic acid recognition, and the introduction of a large exo-olefin structure that may cause steric hindrance in interactions, can reduce interactions with nonspecific proteins (hybridization-independent interactions), which are known to be the main mechanism of side effects of oligonucleotide therapeutic agents, and can also reduce hybridization-dependent toxicity mediated by affecting the activity of key enzymes such as RNase H, RISC (RNA-Induced Silencing Complex), and ADAR (adenosine deaminase RNA specific). As a result, they have found that the toxicity of an oligonucleotide (e.g., an antisense oligonucleotide) is reduced compared to before its introduction by introducing a carbocyclic nucleoside derivative (A) (hereinafter also referred to as "nucleoside (A)") represented by the following formula (A) into the nucleotide sequence constituting the oligonucleotide, thereby completing the present invention.

[0006]

[0007] (In the formula, each group and partial structure has the same meaning as the corresponding group and partial structure defined for the carbocyclic nucleoside derivative residue represented by formula (B) in [1] below.)

[0008] The present invention will be described below by showing specific embodiments, but the present invention is not limited thereto. [1] An oligonucleotide or a salt thereof, wherein the oligonucleotide sequence contains a carbocyclic nucleoside derivative residue (B) (hereinafter also referred to as "nucleoside residue (B)") which is a divalent group represented by the following formula (B):

[0009]

[0010] [wherein Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group, each of which may have one or more optional substituents selected from substituent group (a), wherein substituent group (a) consists of a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an oxo group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom (wherein, when the purin-9-yl group or the 2-oxo-1,2-dihydropyrimidin-1-yl group has an oxo group as a substituent selected from substituent group (a), the bond between the carbon atom to which the oxo group is bonded and the adjacent atom is a single bond); R ３ and R ４ each represents a hydrogen atom; ５ represents a hydrogen atom;

[0011]

[0012] The group represented by the following partial structural formula: (i-1) or (i-2):

[0013]

[0014] (In the formula, R ６ and R ７ each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms, or R ６ and R ７are bonded to each other to form a carbon ring having 3 to 6 carbon atoms together with the adjacent carbon atoms, and R ８ , R ９ , R １０ and R １１ each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms; * represents the position of attachment to the adjacent oligonucleotide component, or when the nucleoside residue (B) is located at the 5'-end of the oligonucleotide sequence, R １ : (where R １ is a hydrogen atom, a protecting group for a hydroxyl group in nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms which may be branched or cyclic, an alkenyl group having 2 to 7 carbon atoms which may be branched or cyclic, an aryl group having 3 to 10 carbon atoms which may have one or more optional substituents selected from substituent group (a) and which may contain a heteroatom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may have one or more optional substituents selected from substituent group (a) and which may contain a heteroatom, an acyl group which may have one or more optional substituents selected from substituent group (a), a silyl group which may have one or more optional substituents selected from substituent group (a), a phosphate group which may have one or more optional substituents selected from substituent group (a), a phosphate group protected by a protecting group in nucleic acid synthesis, -P(R １２ ) R １３ (In the formula, R １２ and R １３ each independently represents a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a dialkylamino group having an alkyl group having 1 to 6 carbon atoms); and, ** represents the bonding position to the adjacent oligonucleotide component, or when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, R ２ : (where R ２ is the R １[2] An oligonucleotide (hereinafter also referred to as "oligonucleotide (I)") or a salt thereof, characterized in that the nucleoside residue (B) is a nucleoside residue represented by partial structural formula (i):

[0015]

[0016] is a group represented by the following partial structural formula: (i-1):

[0017]

[0018] (In the formula, R ６ and R ７ The oligonucleotide or salt thereof according to the above [1], wherein the nucleoside residue (B) is a group represented by the following formula:

[0019]

[0020] [3] In the nucleoside residue (B), partial structural formula (i):

[0021] The group represented by the following partial structural formula: (i-2):

[0022]

[0023] (In the formula, R ８ , R ９ , R １０ and R １１ The oligonucleotide or salt thereof according to the above-mentioned [1], wherein the nucleoside residue (B) is a group represented by the following formula:

[0024]

[0025] [4] The oligonucleotide or salt thereof according to any one of [1] to [3] above, which contains 1 to 10 nucleoside residues (B) in the oligonucleotide sequence. [5] The oligonucleotide or salt thereof according to any one of [1] to [4] above, which is 7 to 30 bases long. [6] The oligonucleotide or salt thereof according to any one of [1] to [5] above, which is 10 to 25 bases long. [7] The oligonucleotide or salt thereof according to any one of [1] to [6] above, which has reduced toxicity (e.g., hepatotoxicity and / or weight loss effect) compared to before the introduction of the nucleoside residues (B). [8] The oligonucleotide or salt thereof according to any one of [1] to [7] above, which is a gapmer consisting of a gap region 2 to 14 bases long, a 5' wing region 2 to 5 bases long, and a 3' wing region 2 to 5 bases long, and the gap region is located between the 5' wing region and the 3' wing region. [9] The oligonucleotide or salt thereof according to [8] above, wherein the gap region comprises at least one nucleoside residue (B).

[10] The oligonucleotide or salt thereof according to [8] or [9] above, wherein the 5' wing region and / or the 3' wing region comprises at least one nucleoside residue (B).

[11] The oligonucleotide or salt thereof according to any of [1] to

[10] above, wherein at least one internucleotide linkage in the oligonucleotide is a phosphorothioate linkage.

[12] The oligonucleotide or salt thereof according to [1] to

[11] above, wherein all internucleotide linkages in the oligonucleotide are phosphorothioate linkages.

[13] In the nucleoside residue (B), Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group, each of which optionally has 1 to 3 substituents selected from substituent group (a), where substituent group (a) consists of a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an oxo group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom; the oligonucleotide or salt thereof according to any of [1] to

[12] above.

[14] In the nucleoside residue (B), R. ６ and R ７ represents a hydrogen atom; * represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 5'-end of the oligonucleotide sequence; and ** represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 3'-end of the oligonucleotide sequence.

[15] The oligonucleotide or salt thereof according to the above [1], [2], and [4] to

[13] , wherein in the nucleoside residue (B), R ８ , R ９ , R １０ and R １１each represent a hydrogen atom; * represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 5'-end of the oligonucleotide sequence; and ** represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 3'-end of the oligonucleotide sequence.

[16] Use of the oligonucleotide or its salt according to any of [1] to

[15] above as an antisense oligonucleotide or an oligonucleotide constituting an siRNA.

[17] A method for reducing the toxicity of an oligonucleotide or its salt, comprising:

[0026]

[0027] (wherein each group and partial structure has the same meaning as the corresponding group defined for "nucleoside residue (B)" in [1] above.)

[18] Use of a carbocyclic nucleoside derivative ("nucleoside (A)") represented by the following formula (A) or a salt thereof for reducing the toxicity of an oligonucleotide:

[0028]

[0029] (wherein each group and partial structure has the same meaning as the corresponding group defined for "nucleoside residue (B)" in [1] above.)

[19] The use according to

[18] above, wherein reducing the toxicity of the oligonucleotide comprises introducing at least one nucleoside (A) into the oligonucleotide sequence.

[20] A pharmaceutical comprising, as an active ingredient, the oligonucleotide according to any one of [1] to

[16] above, or a salt thereof.

[0030] According to one embodiment of the present invention, there is provided an oligonucleotide that exhibits excellent effects on target RNA and the like while maintaining high safety.

[0031] FIG. 1 shows the evaluation results of the target gene expression inhibitory effect in Example 5 described below. FIG. 2 shows the evaluation results of cytotoxicity (cell viability) in Example 6 described below. FIG. 3 shows the evaluation results of cytotoxicity (Caspase 3 / 7 activity) in Example 7 described below. FIG. 4 shows the evaluation results of the target gene expression inhibitory effect in Example 9 described below. FIG. 5 shows the evaluation results of weight change in Example 10 described below. FIG. 6 shows the evaluation results of ALT in Example 10 described below. FIG. 7 shows the evaluation results of total bilirubin in Example 10 described below. FIG. 8 shows the evaluation results of direct bilirubin in Example 10 described below. FIG. 9 shows the evaluation results of indirect bilirubin in Example 10 described below. FIG. 10 shows the evaluation results of the target gene expression inhibitory effect in Example 12 described below. FIG. 11 shows the evaluation results of ALT in Example 13 described below. FIG. 12 shows the evaluation results of total bilirubin in Example 13 described below. FIG. 13 shows the evaluation results of direct bilirubin in Example 13 described below. FIG. 14 shows the evaluation results of indirect bilirubin in Example 13 described below. FIG. 15 shows the evaluation results of the target gene expression inhibitory effect in Example 15 described below. FIG. 16 shows the evaluation results of cytotoxicity (cell viability) in Example 16 described below. FIG. 17 shows the evaluation results of cytotoxicity (cell viability) in Example 17 described below. FIG. 18 shows the evaluation results of cytotoxicity (cell viability) in Example 19 described below. FIG. 19 shows the evaluation results of the target gene expression inhibitory effect in Example 20 described below. FIG. 20 shows the evaluation results of weight change in Example 21 described below. FIG. 21 shows the evaluation results of ALT in Example 21 described below. FIG. 22 shows the evaluation results of AST in Example 21 described below. FIG. 23 shows the evaluation results of total bilirubin in Example 21 described below. Figure 24 shows the results of an evaluation of the double-strand forming ability of siRNA in which an entecavir derivative has been introduced into the antisense strand in Example 41 described below. Figure 25 shows the results of an evaluation of the in vitro target gene expression inhibitory effect of siRNA in which an entecavir derivative has been introduced into the AS strand in Example 42 described below. Figure 26 shows the results of an evaluation of stability in Example 56 described below. Figure 27 shows the results of an evaluation of the in vitro target gene expression inhibitory effect of an oligonucleic acid drug in Example 57 described below.FIG. 28 shows the results of evaluation of the cytotoxicity (cell viability) of oligonucleic acid drugs in Example 58 described below.

[0032] The present invention is now described in detail; unless otherwise defined in the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that may be used in connection with the described invention.

[0033] [Regarding Oligonucleotide (I)] According to one embodiment of the present invention, there is provided an oligonucleotide or a salt thereof, wherein the oligonucleotide sequence contains a carbocyclic nucleoside derivative residue (B) ("nucleoside residue (B)") which is a divalent group represented by the following formula (B):

[0034]

[0035] (wherein each group and partial structure has the same meaning as the corresponding group and partial structure defined for "nucleoside residue (B)" in [1] of the above [Means for Solving the Problems] section), or a salt thereof. Oligonucleotide (I) is described in detail below.

[0036] As described above, the oligonucleotide (I) is composed of (1) a nucleoside residue (B) and (2) a nucleoside (or a nucleoside derivative) selected depending on the target oligonucleotide sequence. (1) Nucleoside Residue (B) First, the "nucleoside residue (B)" will be described in detail. The definitions of each group of the nucleoside residue (B) are as described above, but preferred embodiments are as follows.

[0037] In this specification, the notation "Ca-b" (e.g., C1-6) or "Ca-Cb" (e.g., C1-C6) indicates that the number of carbon atoms constituting the group is a to b (e.g., 1 to 6).

[0038] In the present specification, examples of "linear alkyl groups having 1 to 6 carbon atoms" include linear alkyl groups having 1 to 6 carbon atoms, and specific examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc. On the other hand, examples of "alkyl groups having 1 to 6 carbon atoms" include any linear, branched, or cyclic alkyl groups having 1 to 6 carbon atoms, and specific examples thereof include, in addition to the above, branched alkyl groups such as isopropyl, isobutyl, tert-butyl, and isopentyl, and any cyclic alkyl groups having 3 to 6 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0039] In the present specification, "straight-chain alkoxy groups having 1 to 6 carbon atoms" include straight-chain alkoxy groups having 1 to 6 carbon atoms, and specific examples include methoxy, ethoxy, n-propoxy, etc. On the other hand, "alkoxy groups having 1 to 6 carbon atoms" include any straight-chain, branched-chain, or cyclic alkoxy groups having 1 to 6 carbon atoms, and in addition to the above, examples include branched-chain alkoxy groups such as isopropoxy, isobutoxy, tert-butoxy, and isopentyloxy, and any cyclic alkoxy groups having 3 to 6 carbon atoms such as cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. Furthermore, "straight-chain alkoxy groups having 1 to 6 carbon atoms which may be substituted with straight-chain alkoxy groups having 1 to 6 carbon atoms" refers to the above-mentioned "straight-chain alkoxy groups having 1 to 6 carbon atoms," as well as alkoxy groups in which one or more hydrogen atoms constituting the "straight-chain alkoxy groups having 1 to 6 carbon atoms" are substituted with other "straight-chain alkoxy groups having 1 to 6 carbon atoms," which may be the same or different. Examples of such a "straight-chain alkoxy group having 1 to 6 carbon atoms which may be substituted with a straight-chain alkoxy group having 1 to 6 carbon atoms" include a methoxy group, an ethoxy group, an n-propoxy group, a methoxymethoxy group, an ethoxymethoxy group, an n-propoxymethoxy group, a methoxyethoxy group (for example, a 2-methoxyethoxy group), an ethoxyethoxy group (for example, a 2-ethoxyethoxy group), and an n-propoxyethoxy group.

[0040] In the present specification, examples of the "cyanoalkoxy group having 1 to 6 carbon atoms" include any linear, branched, or cyclic alkoxy group having 1 to 6 carbon atoms in which at least one hydrogen atom has been substituted with a cyano group.

[0041] In the present specification, examples of "straight-chain alkylthio groups having 1 to 6 carbon atoms" include alkylthio groups having any straight-chain alkyl group having 1 to 6 carbon atoms. Examples include a methylthio group, an ethylthio group, and an n-propylthio group. On the other hand, examples of "alkylthio groups having 1 to 6 carbon atoms" include any straight-chain, branched-chain, or cyclic alkylthio groups having 1 to 6 carbon atoms.

[0042] In the present specification, the term "straight-chain alkylamino group having 1 to 6 carbon atoms" includes an amino group having one or two straight-chain alkyl groups having 1 to 6 carbon atoms. Examples include a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, and a diethylamino group.

[0043] In this specification, examples of an "alkyl group having 1 to 7 carbon atoms, which may be branched or cyclic" include any linear alkyl group having 1 to 7 carbon atoms, any branched alkyl group having 3 to 7 carbon atoms, and any cyclic alkyl group having 3 to 7 carbon atoms. Sometimes simply referred to as a "C1-7 alkyl group." For example, examples of any linear alkyl group having 1 to 7 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, and an n-heptyl group; examples of any branched alkyl group having 3 to 7 carbon atoms include an isopropyl group, an isobutyl group, a tert-butyl group, an isopentyl group, and the like; and examples of any cyclic alkyl group having 3 to 7 carbon atoms include a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and the like.

[0044] In the present specification, the "alkenyl group having 2 to 7 carbon atoms which may be branched or cyclic" includes any linear alkenyl group having 2 to 7 carbon atoms, any branched alkenyl group having 3 to 7 carbon atoms, and any cyclic alkenyl group having 3 to 7 carbon atoms. It may also be simply referred to as a "C alkenyl group". For example, any linear alkenyl group having 2 to 7 carbon atoms includes ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, and 1-hexenyl groups. Any branched alkenyl group having 3 to 7 carbon atoms includes isopropenyl, 1-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, and 1-methyl-2-butenyl groups. Any cyclic alkenyl group having 3 to 7 carbon atoms includes cyclobutenyl, cyclopentenyl, and cyclohexenyl groups.

[0045] In the present specification, examples of the "carbon ring having 3 to 6 carbon atoms" include cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

[0046] In this specification, "an aryl group having 3 to 10 carbon atoms which may contain a heteroatom" includes any aryl group having 6 to 10 carbon atoms which is composed solely of hydrocarbon, and any heteroaryl group having 3 to 12 carbon atoms in which at least one carbon atom constituting the ring structure of the aryl group is substituted with a heteroatom (for example, a nitrogen atom, an oxygen atom, a sulfur atom, or a combination thereof). Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group, a naphthyl group, an indenyl group, an azulenyl group, etc., and examples of the heteroaryl group having 3 to 12 carbon atoms include a pyridyl group, a pyrrolyl group, a quinolyl group, an indolyl group, an imidazolyl group, a furyl group, a thienyl group, etc.

[0047] In the present specification, examples of the "aralkyl group having an aryl moiety having 3 to 12 carbon atoms and optionally containing a heteroatom" include a benzyl group, a phenethyl group, a naphthylmethyl group, a 3-phenylpropyl group, a 2-phenylpropyl group, a 4-phenylbutyl group, a 2-phenylbutyl group, a pyridylmethyl group, an indolylmethyl group, a furylmethyl group, a thienylmethyl group, a pyrrolylmethyl group, a 2-pyridylethyl group, a 1-pyridylethyl group, and a 3-thienylpropyl group.

[0048] In this specification, the term "acyl group" includes aliphatic acyl groups and aromatic acyl groups. Specific examples of the aliphatic acyl 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, and a heptadecanoyl group. alkylcarbonyl groups such as a 15-methylhexadecanoyl group, an octadecanoyl group, a 1-methylheptadecanoyl group, a nonadecanoyl group, an eicosanoyl group, and a henaicosanoyl group; carboxylated alkylcarbonyl groups such as a succinoyl group, a glutaroyl group, and an adipoyl group; halogeno C1-6 alkylcarbonyl groups such as a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, and a trifluoroacetyl group; C1-6 alkoxy C1-6 alkylcarbonyl groups such as a methoxyacetyl group; and unsaturated alkylcarbonyl groups such as an (E)-2-methyl-2-butenoyl group. Furthermore, examples of the aromatic acyl group include arylcarbonyl groups such as a benzoyl group, an α-naphthoyl group, and a β-naphthoyl group; halogenoarylcarbonyl groups such as a 2-bromobenzoyl group and a 4-chlorobenzoyl group; C1-6 alkylated arylcarbonyl groups such as a 2,4,6-trimethylbenzoyl group and a 4-toluoyl group; C1-6 alkoxylated arylcarbonyl groups such as a 4-anisoyl group; carboxylated arylcarbonyl groups such as a 2-carboxybenzoyl group, a 3-carboxybenzoyl group, and a 4-carboxybenzoyl group; nitrated arylcarbonyl groups such as a 4-nitrobenzoyl group and a 2-nitrobenzoyl group; C1-6 alkoxycarbonylated arylcarbonyl groups such as a 2-(methoxycarbonyl)benzoyl group; and arylated arylcarbonyl groups such as a 4-phenylbenzoyl group.Preferred are a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a pivaloyl group, and a benzoyl group.

[0049] In the present specification, examples of the "silyl group" include tri-C alkylsilyl groups such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, and triisopropylsilyl; and tri-C alkylsilyl groups substituted by 1 to 2 aryl groups such as diphenylmethylsilyl, butyldiphenylbutylsilyl, diphenylisopropylsilyl, and phenyldiisopropylsilyl. Preferred are trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl groups, and more preferred is trimethylsilyl.

[0050] As used herein, the term "halogen atom" includes, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. A fluorine atom or a chlorine atom is preferred.

[0051] As used herein, the "protecting group" in "protecting group for amino group in nucleic acid synthesis," "protecting group for hydroxyl group in nucleic acid synthesis," "hydroxyl group protected by a protecting group in nucleic acid synthesis," "phosphate group protected by a protecting group in nucleic acid synthesis," and "mercapto group protected by a protecting group in nucleic acid synthesis" is not particularly limited as long as it can stably protect an amino group, hydroxyl group, phosphate group, or mercapto group during nucleic acid synthesis. Specifically, it refers to a protecting group that is stable under acidic or neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis. Examples of such protecting groups include a C1-6 alkyl group, a C1-6 alkenyl group, an acyl group, a tetrahydropyranyl or tetrahydrothiopyranyl group, a tetrahydrofuranyl or tetrahydrothiofuranyl group, a silyl group, a C1-6 alkoxymethyl group, a C1-6 alkoxylated C1-6 alkoxymethyl group, a halogeno-C1-6 alkoxymethyl group, a C1-6 alkoxylated ethyl group, a halogenated ethyl group, a methyl group substituted by 1 to 3 aryl groups, "a C1-6 alkyl group, a C1-6 alkoxy group, a methyl group substituted by 1 to 3 aryl groups in which the aryl ring is substituted by a halogen atom or a cyano group", a C1-6 alkoxycarbonyl group, "an aryl group substituted by a halogen atom, a C1-6 alkoxy group or a nitro group", "a C1-6 alkoxycarbonyl group substituted by a halogen atom or a tri-C1-6 alkylsilyl group", an alkenyloxycarbonyl group, an "aralkyloxycarbonyl group in which the aryl ring is optionally substituted by a C1-6 alkoxy or nitro group" and the like.

[0052] More specifically, examples of tetrahydropyranyl or tetrahydrothiopyranyl groups include tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-4-yl, and 4-methoxytetrahydrothiopyran-4-yl groups. Examples of tetrahydrofuranyl or tetrahydrothiofuranyl groups include tetrahydrofuran-2-yl and tetrahydrothiofuran-2-yl groups. Examples of C1-6 alkoxymethyl groups include methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, and t-butoxymethyl groups. Examples of C1-6 alkoxylated C1-6 alkoxymethyl groups include 2-methoxyethoxymethyl groups. Examples of halogeno C1-6 alkoxymethyl groups include 2,2,2-trichloroethoxymethyl groups and bis(2-chloroethoxy)methyl groups. Examples of C1-6 alkoxylated ethyl groups include 1-ethoxyethyl groups and 1-(isopropoxy)ethyl groups. Examples of halogenated ethyl groups include 2,2,2-trichloroethyl groups. Examples of methyl groups substituted with 1 to 3 aryl groups include benzyl groups, α-naphthylmethyl groups, β-naphthylmethyl groups, diphenylmethyl groups, triphenylmethyl groups, α-naphthyldiphenylmethyl groups, and 9-anthrylmethyl groups. Examples of the "methyl group substituted by 1 to 3 aryl groups in which the aryl ring is substituted by a C1-6 alkyl group, a C1-6 alkoxy group, a halogen atom or a cyano group" include 4-methylbenzyl group, 2,4,6-trimethylbenzyl group, 3,4,5-trimethylbenzyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, 4,4'-dimethoxytriphenylmethyl group, 2-nitrobenzyl group, 4-nitrobenzyl group, 4-chlorobenzyl group, 4-bromobenzyl group, 4-cyanobenzyl group, etc. Examples of the C1-6 alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl group, an isobutoxycarbonyl group, etc.Examples of "aryl groups substituted with a halogen atom, a C1-6 alkoxy group, or a nitro group" include a 4-chlorophenyl group, a 2-chlorophenyl group, a 4-methoxyphenyl group, a 4-nitrophenyl group, and a 2,4-dinitrophenyl group. Examples of "C1-6 alkoxycarbonyl groups substituted with a halogen atom or a tri-C1-6 alkylsilyl group" include a 2,2,2-trichloroethoxycarbonyl group and a 2-trimethylsilylethoxycarbonyl group. Examples of alkenyloxycarbonyl groups include a vinyloxycarbonyl group and an aryloxycarbonyl group. Examples of "aralkyloxycarbonyl groups in which the aryl ring may be substituted with a C1-6 alkoxy or a nitro group" include a benzyloxycarbonyl group, a 4-methoxybenzyloxycarbonyl group, a 3,4-dimethoxybenzyloxycarbonyl group, a 2-nitrobenzyloxycarbonyl group, and a 4-nitrobenzyloxycarbonyl group.

[0053] In one embodiment, examples of "protecting groups for hydroxyl groups in nucleic acid synthesis" include, for example, aliphatic acyl groups, aromatic acyl groups, methyl groups substituted with 1 to 3 aryl groups, "methyl groups substituted with 1 to 3 aryl groups in which the aryl ring is substituted with C1-6 alkyl, C1-6 alkoxy, halogen, or cyano group," and silyl groups. Alternatively, in another embodiment, examples of "protecting groups for hydroxyl groups in nucleic acid synthesis" include, for example, acetyl groups, benzoyl groups, benzyl groups, p-methoxybenzoyl groups, dimethoxytrityl groups, monomethoxytrityl groups, tert-butyldiphenylsilyl groups, tert-butyldimethylsilyl (TBDMS) groups, [(triisopropylsilyl)oxy]methyl (TOM) groups, [(2-nitrobenzyl)oxy]methyl (NBOM) groups, bis(acetoxy ethoxy)methyl ether (ACE) group, tetrahydro-4-methoxy-2H-pyran-2-yl (Mthp) group, 1-(2-cyanoethoxy)ethyl (CEE) group, 2-cyanoethoxymethyl (CEM) group, tert-butyldithiomethyl (DTM) group, 2-(4-tolylsulfonyl)ethoxymethyl (TEM) group, and 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl (4-MABOM) group.

[0054] In one embodiment, examples of the protecting group for a "hydroxyl group protected by a protecting group in nucleic acid synthesis" include an aliphatic acyl group, an aromatic acyl group, a "methyl group substituted with 1 to 3 aryl groups," an "aryl group substituted with a halogen atom, a C1-6 alkoxy group, or a nitro group," a C1-6 alkyl group, and a C1-6 alkenyl group. Alternatively, in another embodiment, examples of the protecting group for a "hydroxyl group protected by a protecting group in nucleic acid synthesis" include a benzoyl group, a benzyl group, a 2-chlorophenyl group, a 4-chlorophenyl group, and a 2-propenyl group.

[0055] In one embodiment, the "protecting group for an amino group in nucleic acid synthesis" is, for example, an acyl group, preferably a benzoyl group.

[0056] In one embodiment, examples of the "protecting group" of the "phosphate group protected with a protecting group in nucleic acid synthesis" include a C1-6 alkyl group, a C1-6 alkyl group substituted with a cyano group, an aralkyl group, an "aralkyl group in which the aryl ring is substituted with a nitro group or a halogen atom," and an "aryl group substituted with a C1-6 alkyl group, a halogen atom, or a nitro group." Alternatively, examples of the "protecting group" of the "phosphate group protected with a protecting group in nucleic acid synthesis" include a 2-cyanoethyl group, a 2,2,2-trichloroethyl group, a benzyl group, a 2-chlorophenyl group, and a 4-chlorophenyl group.

[0057] In one embodiment, the "protecting group" of the "mercapto group protected by a protecting group for nucleic acid synthesis" includes, for example, an aliphatic acyl group and an aromatic acyl group, preferably a benzoyl group.

[0058] In this specification, -P(R １２ ) R １３ [In the formula, R １２ and R １３each independently represents a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a dialkylamino group having an alkyl group having 1 to 6 carbon atoms], １２ is OR １２ａ and R １３ NR １３ａ A group where R １２ａ is, for example, a cyanoalkoxy group having 1 to 6 carbon atoms, and R １３ａ is, for example, an alkyl group having 1 to 6 carbon atoms. The phosphoramidite group is preferably a group represented by the formula -P(OC ２ H ４ CN)(N(iPr) ２ ) or a group represented by the formula -P(OCH ３ ) (N(iPr) ２ ) wherein iPr represents an isopropyl group.

[0059] (2) Nucleosides (or nucleoside derivatives) selected depending on the target oligonucleotide sequence: As used herein, "nucleosides" and "nucleoside analogs" are not particularly limited and refer to those commonly used in the art, including unnatural "nucleosides" in which a purine or pyrimidine base is linked to a sugar, and nucleosides in which a sugar is linked to an aromatic heterocycle or aromatic hydrocarbon ring other than purine or pyrimidine that can be used as a substitute for a purine or pyrimidine base. In carrying out the present invention, those skilled in the art can appropriately select and use such nucleosides depending on the sequence of the target oligonucleotide (I).

[0060] As used herein, oligonucleotide (I) also encompasses "artificial oligonucleotides" and "oligonucleotide analogs." Here, "artificial oligonucleotides" and "oligonucleotide analogs" refer to non-naturally occurring derivatives of "oligonucleotides" in which, for example, 2 to 50 identical or different "nucleosides" or "nucleoside analogs" are linked via phosphodiester bonds. Suitable analogs include sugar derivatives in which the sugar moiety is modified; thioate derivatives in which the phosphodiester moiety is thioated; ester derivatives in which the terminal phosphate moiety is esterified; and amide derivatives in which the amino group on the purine base is amidated. More preferred examples include sugar derivatives in which the sugar moiety is modified.

[0061] In the present specification, examples of salts of oligonucleotide (I) include metal salts such as alkali metal salts such as sodium salt, potassium salt, and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts such as ammonium salt, t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, and piperazine salt. inorganic acid salts such as hydrohalogenated salts such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide, nitrate, perchlorate, sulfate and phosphate; organic acid salts such as C1-6 alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate, arylsulfonates such as benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and salts with amino acids such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate and aspartate.

[0062] Preferred embodiments of the nucleoside residue (B) in the oligonucleotide (I) are described in detail below.

[0063] Nucleoside Residue (B) (I) In the nucleoside residue (B), Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group, each of which may have 1 to 3 optional substituents selected from group α, wherein 1) the group α consists of a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom; * represents the bonding position to the adjacent oligonucleotide component, or when the nucleoside residue (B) is located at the 5'-end of the oligonucleotide sequence, R １ : (where R １ represents a hydrogen atom, a protecting group for a hydroxyl group in nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms which may be branched or cyclic, or an alkenyl group having 2 to 7 carbon atoms which may be branched or cyclic); and, ** represents the bonding position to the adjacent oligonucleotide component, or when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, R ２ : (where R ２ is the R １ A nucleoside residue (B) representing

[0064] Nucleoside Residue (B) (II) In the nucleoside residue (B), the partial structure represented by formula (i) is the following partial structural formula: (i-1):

[0065]

[0066] (In the formula, R ６ and R ７ each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms, or R ６ and R ７are bonded to each other to form a carbon ring having 3 to 6 carbon atoms together with the adjacent carbon atoms. ６ and R ７ represents a hydrogen atom; * represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence; and ** represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence.

[0067] Nucleoside Residue (B) (III) In the nucleoside residue (B), the partial structure represented by formula (i) is the following partial structural formula: (i-2):

[0068]

[0069] (In the formula, R ８ , R ９ , R １０ and R １１ and each independently represent a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms. ８ , R ９ , R １０ and R １１ each represents a hydrogen atom; * represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence; and ** represents a bonding position to an adjacent oligonucleotide component, or represents a hydrogen atom when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence.

[0070] The oligonucleotide (I) has been described above in detail from the viewpoint of its constituent nucleic acid monomer, the "nucleoside residue (B)." In the following, other aspects of the oligonucleotide (I) will be described in detail.

[0071] The number of nucleoside residues (B) contained in the oligonucleotide (I) is not particularly limited. One embodiment of the present invention includes, for example, an oligonucleotide (I) containing 1 to 25 nucleoside residues (B) or an oligonucleotide (I) containing 1 to 10 nucleoside residues (B) in the oligonucleotide sequence, or a salt thereof. However, as described above, the number of nucleoside residues (B) introduced is not limited and can be determined appropriately depending on the intended use of the oligonucleotide (I). For example, it is possible for the entire sequence of the oligonucleotide (I) to be composed of nucleoside residues (B). By introducing at least one nucleoside residue (B), for example, 1 to 10 nucleoside residues (B), it is possible to effectively reduce the toxicity of an oligonucleotide (hereinafter also referred to as a "parent oligonucleotide") having a base sequence determined in a sequence-dependent manner with respect to a target RNA. Toxicity that can be reduced includes, for example, hepatotoxicity and weight loss effects of the parent oligonucleotide. Such a "method for reducing the toxicity of an oligonucleotide or a salt thereof" is another embodiment of the present invention. A suitable number of nucleoside residues (B) to be introduced into a sequence can be appropriately determined by a person skilled in the art depending on the sequence of the parent oligonucleotide and the type and severity of toxicity. For example, the number may be 1 to 30, with 1 to 10 being preferred, and 1 to 6 being more preferred.

[0072] The position at which the nucleoside residue (B) is introduced into the oligonucleotide (I) is not particularly limited, and can be appropriately determined by those skilled in the art depending on the purpose.

[0073] The total base length of the oligonucleotide (I) is not particularly limited, but one embodiment of the present invention is an oligonucleotide (I) or a salt thereof having a length of 7 to 30 bases. The base length of the oligonucleotide (I) can be appropriately determined by a person skilled in the art depending on the intended use, etc., and the oligonucleotide (I) can be designed accordingly. When the oligonucleotide (I) or a salt thereof is used, for example, as an antisense oligonucleotide, a length of 7 to 30 bases is one preferred embodiment. Furthermore, a length of 10 to 25 bases or 10 to 20 bases is more preferred.

[0074] In one embodiment of the present invention, oligonucleotide (I) may be a gapmer consisting of a gap region 2 to 10 bases long, a 5' wing region 2 to 5 bases long, and a 3' wing region 2 to 5 bases long, with the gap region positioned between the 5' wing region and the 3' wing region. In this embodiment, the wing region may contain a modified nucleic acid with strong RNA binding ability. In this embodiment, the number and position of nucleoside residues (B) introduced into oligonucleotide (I) are not particularly limited. For example, the gap region may contain at least one nucleoside residue (B), and the 5' wing region and / or the 3' wing region may contain at least one nucleoside residue (B). Furthermore, the gap region, the 5' wing region, and / or the 3' wing region may all contain at least one nucleoside residue (B). Those skilled in the art can appropriately determine the number and positions of nucleoside residues (B) to be introduced into oligonucleotide (I) depending on, for example, the sequence of the target RNA and the sequence of the parent oligonucleotide.

[0075] In one embodiment of the present invention, the oligonucleotide (I) may be an oligonucleotide that constitutes the antisense strand (guide strand) and / or sense strand (passenger strand) of an siRNA composed of a double-stranded RNA approximately 21 bases in length. In this embodiment, the base sequences of the antisense strand and sense strand in the oligonucleotide (I) are designed based on the sequence of the target RNA, and 2'-O-methyl modifications (2'OMe) or 2'-F-RNA (2'-F) may be introduced for the purposes of reducing off-target effects, controlling pharmacokinetics, immune response, etc. Furthermore, in this embodiment, the number and positions of the nucleoside residues (B) introduced into the oligonucleotide (I) are not particularly limited; for example, nucleoside modifications may be introduced into the overhang region, seed region, or cleavage region. Those skilled in the art can appropriately determine the number and positions of modifications to the oligonucleotide (I) depending on the sequence of the target RNA, the sequence of the parent oligonucleotide, etc.

[0076] As one embodiment of the present invention, in oligonucleotide (I), at least one of the internucleotide linkages in the nucleic acid sequence may be a phosphorothioate linkage. Furthermore, all of the internucleotide linkages may be phosphorothioate linkages. In oligonucleotide (I), phosphorothioate linkages may be used instead of normal phosphodiester linkages, as necessary, from the viewpoint of improving the resistance of the oligonucleotide to nucleases. The number and positions of phosphorothioate linkages can be appropriately determined by those skilled in the art, but one preferred embodiment is one in which all internucleotide linkages are phosphorothioate linkages.

[0077] In an embodiment of the present invention, the oligonucleotide (I) itself can be used as a single-stranded oligonucleotide (e.g., an antisense oligonucleotide), but it can also be used as an oligonucleotide constituting a double-stranded oligonucleotide (e.g., an siRNA). In this case, the oligonucleotide (I) may constitute one or both of the double-stranded oligonucleotides.

[0078] [Regarding the method for producing oligonucleotide (I)] Oligonucleotide (I) or a salt thereof can be produced by using the "nucleoside (A)" as one of the nucleic acid monomers constituting the oligonucleotide together with other nucleic acid monomers and appropriately incorporating it into the oligonucleotide sequence. Depending on the sequence of the desired oligonucleotide, one or more nucleotides (A) can be introduced not only into the sequence but also at the desired position, including the 3' or 5' end. When two or more nucleotides are introduced, they may be introduced consecutively into the sequence or interposed by one or more other nucleic acid monomers. The specific production of oligonucleotide (I) can be carried out by a method commonly used in the art. For example, when solid-phase synthesis is carried out, it can be carried out by the following procedure. However, the method is not limited thereto.

[0079] Solid-phase synthesis using the phosphoramidite method is widely used in the chemical synthesis of oligonucleotides. In the first step of the synthesis cycle, the 3'-terminal nucleoside of the oligonucleotide (nucleic acid) sequence to be synthesized is supported on a solid support. If commercially available, phosphoramidites of nucleic acid monomers can be purchased and used. Those skilled in the art can also synthesize and obtain phosphoramidites, including nucleoside (A), as appropriate, using, for example, the method described in Example 1 below, or methods similar to this method. In recent years, universal linkers capable of coupling with any nucleoside have become widely used in solid-phase synthesis, as they allow the introduction of any nucleoside at the 3'-terminal of an oligonucleotide, effectively synthesizing oligonucleotides having any sequence regardless of the 3'-terminal nucleoside. More specifically, a universal linker is preliminarily supported on a solid support via a cleavable linker (spacer) such as a succinyl group, and any 3'-terminal nucleoside is coupled thereto. Thereafter, an oligonucleotide elongation reaction is carried out in a reaction column according to a synthesis program of an automatic nucleic acid synthesizer, generally comprising the following steps: (1) deprotecting the 5'-OH group of the protected nucleoside with an acid such as trichloroacetic acid / dichloromethane solution; (2) coupling a nucleoside phosphoramidite (also referred to as a "nucleic acid monomer") to the deprotected 5'-OH group in the presence of an activator (such as tetrazole); (3) capping unreacted 5'-OH groups with acetic anhydride or the like; and (4) oxidizing the phosphite with aqueous iodine or the like, or sulfurizing the phosphite with 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione or the like. The above synthesis cycle is repeated according to the desired sequence, and the oligonucleotide is extended from the 3' end to the 5' end to synthesize oligonucleotide (I) having the desired sequence. Finally, the cleavable linker is hydrolyzed with aqueous ammonia or methylamine solution, and the synthesized oligonucleotide is cleaved from the solid support and the universal linker to obtain oligonucleotide (I).

[0080] (Regarding Nucleoside (A)) Each group and partial structure of nucleoside (A) has the same meaning as the corresponding group and partial structure defined for "nucleoside residue (B)" in [1] of the above-mentioned section [Means for Solving the Problems]. Therefore, for preferred aspects of each group and partial structure, and preferred aspects of nucleoside (A), reference can be made to the corresponding detailed descriptions of "nucleoside residue (B)." A preferred embodiment of nucleoside (A) is shown below by its structural formula. Hereinafter, nucleosides having this structure will also be referred to as nucleoside (A-i-1) and nucleoside (A-i-2), respectively.

[0081]

[0082] Nucleoside (A) can be used to reduce the toxicity of oligonucleotides. More specifically, by introducing at least one nucleoside (A) into a target oligonucleotide sequence, the toxicity of the oligonucleotide can be reduced. The use of nucleoside (A) in this manner is another embodiment of the present invention.

[0083] (Regarding the Production Method of Nucleoside Derivative (A)) A person skilled in the art can appropriately produce the nucleoside derivative (A) starting from a known compound using a known method, for example, by referring to the methods specifically described in the Examples below. A person skilled in the art can also appropriately obtain the raw material compounds starting from known compounds.

[0084] [Use of Oligonucleotide (I)] As demonstrated in the examples below, oligonucleotide (I) or a salt thereof (hereinafter also collectively referred to as "the present oligonucleotide") can exhibit the excellent effect of improving or maintaining its efficacy while reducing toxicity by introducing a nucleoside derivative (A) into the original antisense oligonucleotide sequence. Furthermore, the present oligonucleotide itself can also be used, for example, as a novel antisense oligonucleotide. Therefore, the present oligonucleotide is useful, for example, as a pharmaceutical for the prevention or treatment of disease as an antisense oligonucleotide. Such use of the present oligonucleotide as a pharmaceutical is yet another embodiment of the present invention. Hereinafter, an embodiment in which the present oligonucleotide is used as a pharmaceutical will be described in detail.

[0085] As used herein, "prevention" includes preventing the onset of a disease (all pathologies or one or more pathologies) and delaying the onset of the disease. A "prophylactically effective amount" refers to a dose of the present oligonucleotide sufficient to achieve this purpose. As used herein, "treatment" includes curing a disease (all pathologies or one or more pathologies), ameliorating the disease, and suppressing the progression of the severity of the disease. A "therapeutically effective amount" refers to a dose of the present oligonucleotide sufficient to achieve this purpose.

[0086] In carrying out the present invention, the present oligonucleotide can be used either alone or in the form of a pharmaceutical composition containing the present oligonucleotide as an active ingredient together with a pharmaceutically acceptable carrier.

[0087] Examples of such pharmaceutical compositions include tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules), syrups, liquids, emulsions, suspensions, controlled-release preparations (e.g., immediate-release preparations, sustained-release preparations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosal patch films), injections (e.g., subcutaneous injections, intravenous injections (e.g., bolus), intramuscular injections, intraperitoneal injections, intrathecal injections, intraventricular injections), drip infusions, transdermal preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), and eye drops.

[0088] As used herein, the term "pharmaceutically acceptable carrier" refers to any of a variety of carriers commonly used in the field of pharmaceutical formulation technology.

[0089] Specific examples of "pharmaceutically acceptable carriers" that can be used in solid preparations include excipients (e.g., lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc.), lubricants (e.g., magnesium stearate, talc, colloidal silica, etc.), binders (e.g., crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose, etc.), and disintegrants (e.g., starch, carboxymethylcellulose, carboxymethylcellulose calcium, sodium carboxymethylstarch, L-hydroxypropylcellulose, etc.).

[0090] Liquid preparations may contain solvents (e.g., water for injection, isotonic saline, alcohol, propylene glycol, macrogol, sesame oil, etc.), solubilizing agents (e.g., polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, etc.), suspending agents (e.g., surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, glycerin monostearate, etc.; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, etc.), isotonic agents (e.g., glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, etc.), buffers (e.g., buffer solutions such as phosphates and citrates, etc.), and soothing agents (e.g., benzyl alcohol, etc.).

[0091] If necessary, formulation additives such as preservatives (e.g., paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, sorbic acid, etc.), antioxidants (e.g., sulfites, ascorbic acid, α-tocopherol, etc.), colorants, sweeteners, etc. may be further added.

[0092] The pharmaceutical compositions of the present invention may be prepared by adding the oligonucleotides of the present invention in an amount of typically 0.01 to 99% (w / w), preferably 0.1 to 85% (w / w), based on the total amount of the formulation, although this may vary depending on the dosage form, administration method, carrier, etc. The pharmaceutical compositions may be prepared by conventional methods in the field of formulation technology, depending on the form. The pharmaceutical compositions of the present invention may also be formed into sustained-release formulations containing the active ingredient.

[0093] (Regarding the Subject of Administration) The present oligonucleotide is expected to have low toxicity and few side effects, and also has excellent properties as a pharmaceutical. Therefore, the present oligonucleotide can be safely administered to mammals (especially humans). (Regarding the Administration Route) In carrying out the present invention, the present oligonucleotide, either alone or as a pharmaceutical composition, can be administered orally or parenterally (e.g., intravenously, intramuscularly, subcutaneously, intraorgan, intranasally, intradermally, by eye drop, intracerebrally (intraventricularly), intrathecally, rectally, intravaginally, intraperitoneally, and into a lesion). (Regarding the Dosage) The dosage of the pharmaceutical composition of the present invention varies depending on the purpose of administration, the administration method, the type and severity of the target disease, and the condition of the recipient (e.g., gender, age, body weight), and is not particularly limited. For example, when administered systemically to an adult, a single dose of the complex of the present invention is generally 0.01 mg / kg to 1000 mg / kg, and when administered topically, 0.001 mg / body to 100 mg / body is desirable. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times. The pharmaceutical composition of the present invention can also be used in combination with, for example, a therapeutic drug for a disease that is already on the market. These concomitant drugs can be formulated together with the pharmaceutical composition of the present invention and administered as a single formulation, or they can be formulated separately from the pharmaceutical composition of the present invention and administered simultaneously or at different times via the same or a different route as the pharmaceutical composition of the present invention. The dosage of these concomitant drugs may be the amount normally used when the drug is administered alone, or it may be reduced from the amount normally used.

[0094] The present invention will be further illustrated by the following examples, which are not intended to limit the invention and may be modified without departing from the scope of the invention.

[0095] Various examples are shown below, including the results of synthesizing oligonucleotide (I) using entecavir as nucleoside (A) and conducting pharmacological evaluations (efficacy / toxicity).

[0096] Example 1 Synthesis of Amidite Derivatives A phosphoramidite derivative of entecavir was synthesized according to the following synthesis scheme (previously reported).

[0097]

[0098] Compound 1 (entecavir) (500 mg, 1.80 mmol) was added to dry MeOH (17 mL) under a nitrogen atmosphere, followed by the addition of N,N-dimethylformamide dimethyl acetal (905 μL, 6.77 mmol) and stirring at room temperature for 12 hours. After confirming the consumption of the starting material by TLC, the solvent was concentrated under reduced pressure to give compound 2 (581 mg, 98%). Compound 2 (658 mg, 1.98 mmol) was added to dry pyridine (19 mL) and dry DMF (5 mL) under a nitrogen atmosphere, followed by the addition of 4,4-dimethoxytritylchloride (470 mg, 1.39 mmol) and stirring at room temperature for 1 hour. 4,4-Dimethoxytritylchloride (470 mg, 1.39 mmol) was added and the mixture was stirred at room temperature for 3 hours. After confirming the consumption of the starting material by TLC, the solvent was concentrated under reduced pressure. Saturated NaHCO3 solution (100 mL) was added to the residue, which was extracted three times with EtOAc (100 mL). The organic layer was washed with brine and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: DCM / MeOH = 98:2 + 0.5% TEA) to give the desired compound 3 (1.01 g, 81%). Compound 3 (563 mg, 0.89 mmol) and 1H-tetrazole (93.5 mg, 1.34 mmol) were added to a heat-dried reaction vessel, and dry DCM (19 mL) and DIPEA (303 μL, 1.34 mmol) were added under a nitrogen atmosphere. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (367 μL, 1.16 mmol) was added, and the mixture was stirred at room temperature for 2 h. After confirming the consumption of the starting material by TLC, saturated NaHCO3 solution (150 mL) was added, and the mixture was extracted three times with DCM (150 mL).The organic layer was washed with brine, dried over NaSO, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM / MeOH = 100:0 to 95:5 + 1% TEA) to obtain the target compound 4 (514 mg, 0.616 mmol, 69%).

[0099] Example 2 Objective: Oligonucleotide Synthesis Method and Results: (1) Synthesis Scheme Oligonucleotide synthesis was performed using a DNA / RNA synthesizer (NTS M-2-TRS, Nippon Techno Service) according to the standard phosphoramidite method. The oligonucleotides were prepared using commercially available phosphoramidites of dA(Bz), dG(iBu), dC(Bz), dT, LNA-A(Bz), LNA-G(DMF), LNA-mC(Bz), and LNA-T, as well as the entecavir phosphoramidite synthesized in Example 1, in 0.1 M anhydrous acetonitrile solutions. The synthesis was performed on a 1 μmol scale under trityl-on conditions. 5-benzylthio-1H-tetrazole (0.25 M anhydrous acetonitrile) was used as the activator. The condensation time was 3 minutes for the LNA amidite block and 30 seconds for the natural amidite block. After the synthesis was completed, the product was treated with 28% aqueous ammonia at 55°C for 13 hours to remove the base and phosphate diester moieties from the column support. TM The fragment was purified using a DNA Purification Cartridge (Glen Research) and then further purified by reversed-phase HPLC. The HPLC measurement conditions are as follows: (Eluent) Solution A: 100 mM hexafluoro-2-propanol, 8.6 mM triethylamine (pH 8.36) Solution B: Methanol (Gradient) Solution B concentration: 1) 5-30% (30 min) (purification) 2) 5-40% (20 min) (purity confirmation) (Column) 1) Nacalai 5C 18 -MS-II (10 × 250 mm) (purification) 2) nacalai 5C 18-MS-II (4.6 × 50 mm) (purity check) 3) Column temperature 60°C (Flow rate) 1) 2.0 mL / min (purification) 2) 0.5 mL / min (purity check) (Detection) UV (260 nm)

[0100] (2) Synthesized Oligonucleotides According to the above synthesis scheme, oligonucleotides (internucleotide linkages: all phosphorothioate linkages) having the sequences shown in Table 1 below were obtained. In the table, E represents entecavir. In Table 1 and the following Tables 2 to 9, 12 to 14, and 16, unless otherwise specified, nucleosides written in capital letters represent LNA, and nucleosides written in lowercase represent DNA. Entecavir may also be represented by the subscript E. In hApo1-cRNA and hApo1-cDNA, capital letters represent RNA and DNA, respectively.

[0101]

[0102] Discussion: From the above, it has become clear that nucleosides (A) can be introduced into oligonucleotide sequences according to standard methods, and that multiple nucleosides (A) can also be introduced into one sequence, both consecutively.

[0103] [Example 3] Objective: To determine the double-strand melting temperature (T m ) Measurement Method and Results: A sample solution (150 μL) containing 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, and 4 μM of each oligonucleotide and complementary strand RNA (hApo1-cRNA, Table 1) listed in Table 2 below was heated to 95°C for 3 minutes, then gradually cooled to 20°C at a rate of 1°C per minute to allow annealing, after which measurement began. The temperature was raised to 95°C at a rate of 0.5°C per minute, and the absorbance at 260 nm was plotted at 1°C intervals. T m All values ​​were calculated using the midline method. The measurement results are shown in Table 2 below. In the table, E stands for entecavir.

[0104]

[0105] Discussion: The above results demonstrate that nucleoside (A) is a unique non-natural nucleotide that does not impair the thermodynamic stability of ASOs toward their target RNA.

[0106] [Example 4] Objective: To determine the double-strand melting temperature (T m ) Measurement Method and Results: A sample solution (150 μL) with final concentrations of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, and 4 μM of each oligonucleotide and complementary DNA (hApo1-cDNA, Table 1) was heated to 95°C for 3 minutes, then gradually cooled to 20°C at a rate of 1°C per minute to allow annealing, after which measurement began. The temperature was raised to 95°C at a rate of 0.5°C per minute, and the absorbance at 260 nm was plotted at 1°C intervals. T m All values ​​were calculated using the midline method. The measurement results are shown in Table 3 below. In the table, E stands for entecavir.

[0107]

[0108] These results demonstrate that nucleoside (A) is a unique unnatural nucleotide that does not impair the thermodynamic stability of complementary DNA strands.

[0109] [Example 5] Objective: Evaluation of the in vitro inhibitory effect of oligonucleotide drugs with nucleoside (A) introduced into the chain on target gene expression. Methods and results: Cell culture medium (9 mM CaCl 2 ASO was diluted with PBS (added) to a final concentration of 2 μM and added to a 96-well plate. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 24 hours. cDNA was then prepared from the cell lysate using the SuperPrep® II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's protocol. ApoB mRNA expression was analyzed using a CFX Real-Time PCR System (BIO RAD). For analysis of human ApoB, the primer sets hApoB-F: 5'-TTCTCAAGAGTTACAGCAGATCCA-3' (SEQ ID NO: 1) and hApoB-R: 5'-TGGAAGTCCTTAAGAGCAACTAACA-3' (SEQ ID NO: 2) were used. For analysis of the housekeeping gene human Gapdh, the primer sets hGAPDH-F: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 3) and hGAPDH-R: 5'-TGGTGAAGACGCCAGTGGA-3' (SEQ ID NO: 4) were used. The difference in Ct values ​​was converted into the difference in expression level to calculate the KD activity based on relative expression levels. The evaluation results are shown in Figure 1. Discussion: Figure 1 shows that gap2, 2mod, and 3mod showed slightly reduced activity, while gap3 showed activity equivalent to or slightly higher than that of hApo1 (parent strand), indicating that oligonucleotide (I) (oligonucleotide drug) with nucleoside (A) introduced into the strand has gene suppression activity.

[0110] [Example 6] Objective: Evaluation of the cytotoxicity (cell viability) of an oligonucleotide drug incorporating nucleoside (A) into the chain Methods and results: Cell culture medium (9 mM CaCl 2 ASO was diluted with PBS (addition) to a final concentration of 1 or 2 μM and added to a 96-well plate. Huh-7 cells diluted with the above-mentioned medium were seeded at 10,000 cells per well and cultured in an incubator for 72 hours. After 10 μL (per well) of Cell Counting Kit-8 (Dojindo Laboratories) was added, the cells were incubated in the incubator for 2 hours. Absorbance at 450 nm was measured using a microplate reader. The evaluation results are shown in Figure 2. Discussion: Figure 2 shows that gap2, gap3, 2mod, and 3mod inhibited cell death and cytotoxicity. Oligonucleotide (I) (oligonucleotide drug) with nucleoside (A) introduced at the position shown in Table 3 demonstrated low cytotoxicity.

[0111] [Example 7] Objective: Evaluation of cytotoxicity (Caspase 3 / 7 activity) of oligonucleotide drugs incorporating nucleoside (A) into the chain Methods and results: Cell culture medium (9 mM CaCl 2 ASO was diluted with 1000kJ / ml (addition) to a final concentration of 1 or 2 μM and added to a 96-well plate. Huh-7 cells diluted with the above-mentioned medium were seeded at 10,000 cells per well and cultured in an incubator for 72 hours. Caspase 3 / 7 activation was measured using the Caspase-Glo® 3 / 7 Assay Systems, Caspase-Glo® (Promega), according to the manufacturer's protocol. The results are shown in Figure 3. Discussion: Figure 3 shows that the increase in caspase 3 / 7 activity was significantly suppressed with gap2, 2mod, and 3mod. It was demonstrated that the introduction of nucleoside (A) into the chain at the position shown in Table 3 can reduce the toxicity of oligonucleotide drugs.

[0112] Example 8 Objective: Synthesis of oligonucleotide drugs containing nucleoside (A) carrying a targeting ligand. Methods and Results: (1) Synthesis Scheme: Oligonucleotides were synthesized using commercially available dA(Bz), dG(iBu), dC(Bz), dT, LNA-A(Bz), LNA-G(DMF), LNA-mC(Bz), LNA-T, and the entecavir phosphoramidite synthesized in Example 1, prepared as 0.1 M anhydrous acetonitrile solutions, using a DNA / RNA synthesizer (NTS M-2-TRS, Nippon Techno Service) according to the standard phosphoramidite method. The synthesis scale was 1 μmol, and the reaction was carried out under trityl-on conditions. 5-benzylthio-1H-tetrazole (0.25 M anhydrous acetonitrile) was used as the activator, and the condensation time was 3 minutes for the LNA amidite block and 30 seconds for the natural amidite block. After synthesis, the solid support was transferred to a 1.0 mL gas-tight syringe, and the ligand moiety was elongated manually using the GalNAc amidite block (0.1 M anhydrous acetonitrile) and 5-ethylthio-1H-tetrazole (0.5 M anhydrous acetonitrile) as the activator, following the standard phosphoramidite method. The GalNAc amidite block was prepared according to the report in "Terada C, Wada F, Uchida M, Yasutomi Y, Oh K, Kawamoto S, Kayaba Y, Yamayoshi A, Harada-Shiba M, Obika S, Yamamoto T. Programmed Instability of Ligand Conjugation Manifold for Efficient Hepatocyte Delivery of Therapeutic Oligonucleotides. Nucleic Acid Ther. 2021 Dec;31(6):404-416. doi: 10.1089 / nat.2021.0036." After synthesis was completed, the block was treated with 28% aqueous ammonia at 55°C for 13 hours to remove the block from the column support and to deprotect the base and phosphate diester moieties. Next, the block was loaded onto a simple reversed-phase column (Glen-Pak TMThe fragment was purified using a DNA Purification Cartridge (Glen Research) and then further purified by reversed-phase HPLC. The HPLC measurement conditions are as follows: (Eluent) Solution A: 100 mM hexafluoro-2-propanol, 8.6 mM triethylamine (pH 8.36) Solution B: Methanol (Gradient) Solution B concentration: 1) 5-30% (30 min) (purification) 2) 5-40% (20 min) (purity confirmation) (Column) 1) Nacalai 5C 18 -MS-II (10 × 250 mm) (purification) 2) nacalai 5C 18 -MS-II (4.6 × 50 mm) (purity check) 3) Column temperature 60°C (Flow rate) 1) 2.0 mL / min (purification) 2) 0.5 mL / min (purity check) (Detection) UV (260 nm)

[0113] (2) Synthesized Oligonucleotides According to the above synthesis scheme, oligonucleotides having the sequences shown in Table 4 below (internucleotide linkages: two linkages from the 5' end are phosphodiester linkages, and the remaining linkages are phosphorothioate linkages) were obtained. In the table, E represents entecavir, X represents GalNAc APD Shows.

[0114]

[0115] Discussion: From the above, it has become clear that an oligonucleotide drug (oligonucleotide (I)) containing a nucleoside (A) loaded with a targeting ligand can be constructed according to a conventional method.

[0116] [Example 9] Objective: Verification of the in vivo inhibitory effect of an oligonucleotide drug containing a nucleoside (A) carrying a targeting ligand on target gene expression. Methods and Results: All animal experiment protocols were approved by the Animal Experiment Committee of Nagasaki University. ASO targeting ApoB (human-mouse consensus sequence) was administered subcutaneously in a single dose of 200 nmol / kg to 7-week-old C57Bl / 6J mice (male, Japan SLC). 72 hours after administration, whole blood was collected under isoflurane inhalation anesthesia, and then the liver was harvested. The liver was analyzed using RNAlater. TM The tissue was stored overnight at 4°C and then at -20°C until analysis. Liver total RNA was extracted using the QuickGene RNA tissue kit SII (Fujifilm) according to the attached manual. The extracted total RNA was used as a template for High Capacity RNA-to-cDNA PCR. TM After reverse transcription using a PCR kit (Thermo Fisher Scientific), ApoB mRNA expression levels were analyzed using a CFX Real-Time PCR System (BIO RAD). Mouse ApoB was analyzed using the primer sets mApoB-F: 5'-TCCTCGGTGAGTTCAATGACTTTC-3' (SEQ ID NO: 5) and mApoB-R: 5'-TGGACCTGCTGTAGCTTGTAGGA-3' (SEQ ID NO: 6). Mouse Gapdh, a housekeeping gene, was analyzed using the primer sets mGAPDH-F: 5'-TGTGTCCGTCGTGGATCTGA-3' (SEQ ID NO: 7) and mGAPDH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3' (SEQ ID NO: 8). The difference in Ct values ​​was converted into the difference in expression level, and the KD activity was calculated based on the relative expression level. The results are shown in Figure 4. Discussion: As shown in Figure 4, the target knockdown activity in vivo was similar to the in vitro evaluation results, with gap2 showing a slightly reduced activity but still demonstrating a high gene expression suppression effect. On the other hand, gap1 and gap3 showed high activity equivalent to that of hApo1 (parent strand). Although differences were confirmed depending on the insertion site, it was clear that oligonucleotide (I) generally possessed high activity.

[0117] Example 10 Objective: To examine the effect of nucleoside (A) on the toxicity of oligonucleotide drugs using mice. Methods and Results: All animal experiment protocols were approved by the Nagasaki University Animal Experiment Committee. A single dose of 200 nmol / kg of the ApoB-targeting ASO obtained in Example 8 was administered subcutaneously to 7-week-old C57Bl / 6J mice (male, Japan SLC). Seventy-two hours after administration, whole blood was collected under isoflurane inhalation anesthesia, followed by liver excision. Body weight change was assessed, and serum collected from the inferior vena cava was used for evaluation. ALT, total bilirubin, direct bilirubin, and indirect bilirubin were measured by Oriental Yeast Co., Ltd. The evaluation results are shown in Figures 5 to 9. Discussion: The introduction of nucleoside (A) eliminated the weight loss (Figure 5), liver toxicity (Figure 6), and jaundice symptoms (Figures 7, 8, and 9) caused by side effects of antisense nucleic acid drugs for all of the gap1, gap2, and gap3 oligonucleotides (I). In particular, gap2, which also demonstrated efficacy in cell experiments, significantly improved the abnormal ALT and bilirubin levels seen in hApo1. It was revealed that the introduction of nucleoside (A) into the oligonucleotide (I) of the present invention improves the toxicity of the oligonucleotide compared to before introduction, and that by selecting the position and number of nucleosides introduced, it is possible to obtain oligonucleotide drugs with excellent activity and safety.

[0118] [Example 11] Objective: To determine the double-strand melting temperature (T m ) Measurement Method and Results (1) Synthesis of Oligonucleotides Various oligonucleotides (internucleotide linkages: all phosphorothioate linkages) containing mPCS2 as the parent sequence, as shown in Table 5 below, were synthesized in the same manner as in Example 2, and were subjected to the following evaluations. (2) Double-strand melting temperature (T m) Measurement: A sample solution (150 μL) containing 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, and 4 μM of each oligonucleotide and complementary RNA was heated to 95°C for 3 minutes and then gradually cooled to 20°C at a rate of 1°C per minute to allow annealing, after which measurement was initiated. The temperature was raised to 95°C at a rate of 0.5°C per minute, and absorbance at 260 nm was plotted at 1°C intervals. Tm values ​​were all calculated using the median line method. The measurement results are shown in Table 5 below. In the table, E stands for entecavir.

[0119]

[0120] Discussion: These results demonstrate that nucleoside (A) is a unique non-natural nucleotide that does not impair the thermodynamic stability of complementary RNA strands, even in a different ASO sequence (mPCS2).

[0121] [Example 12] Objective: To verify the in vivo effect of an oligonucleotide drug containing a nucleoside (A) carrying a targeting ligand on inhibiting target gene expression Methods and Results (1) Synthesis of an oligonucleotide drug containing a nucleoside (A) carrying a targeting ligand In a manner similar to that described in Example 8, oligonucleotide drugs containing nucleosides (A) carrying various targeting ligands with mPCS2 as the parent sequence, as listed in Table 6 below, were synthesized and subjected to the following evaluations.

[0122]

[0123] (2) Verification of the in vivo gene expression suppression effect of oligonucleotide drugs containing nucleoside (A) carrying a targeting ligand. All animal experiment protocols were approved by the Nagasaki University Animal Experiment Committee. A single dose of PCSK9-targeting ASO (listed in Table 6) was administered subcutaneously to 7-week-old C57Bl / 6J mice (male, Japan SLC) at a dose of 200 nmol / kg. After 72 hours of administration, whole blood was collected under isoflurane inhalation anesthesia, and the liver was then harvested. The liver was analyzed using RNAlater. TMThe tissue was stored overnight at 4°C and then at -20°C until analysis. Liver total RNA was extracted using the QuickGene RNA tissue kit SII (Fujifilm) according to the attached manual. The extracted total RNA was used as a template for reverse transcription using the High Capacity RNA-to-cDNA™ kit (Thermo Fisher Scientific), and PCSK9 mRNA expression levels were analyzed using a CFX real-time PCR system (BIO RAD). For analysis of mouse PCSK9, the primer sets mPSCK9-F: 5'-TCAGTTCTGCACACCTCCAG-3' (SEQ ID NO: 9) and mPCSK9-R: 5'-GGGTAAGGTGCGGTAAGTCC-3' (SEQ ID NO: 10) were used. For analysis of mouse Gapdh as a housekeeping gene, the primer sets mGAPDH-F: 5'-TGTGTCCGTCGTGGATCTGA-3' (SEQ ID NO: 7) and mGAPDH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3' (SEQ ID NO: 8) were used. The KD activity was calculated based on the relative expression level, converting the difference in Ct value into the difference in expression level. The evaluation results are shown in Figure 10.

[0124] Discussion: It was revealed that the introduction of nucleoside (A) can achieve activity equivalent to or greater than that of the parent sequence (mPCS2).

[0125] [Example 13] Objective: To verify the effect of nucleoside (A) on the toxicity of oligonucleotide drugs using mice Methods and Results Tests were conducted using the oligonucleotides synthesized in Example 12 carrying various targeting ligands in a manner similar to that described in Example 10. Body weight changes were assessed, and serum collected from the inferior vena cava was used for evaluation. ALT, total bilirubin, direct bilirubin, and indirect bilirubin were measured by Oriental Yeast Co., Ltd. The evaluation results are shown in Figures 11 to 14. Discussion: It was revealed that the toxicity observed with the parent sequence (mPCS2) can be improved by adjusting the position of the nucleoside (A) introduction.

[0126] [Example 14] Objective: To determine the double-strand melting temperature (T m ) Measurement Method and Results (1) Synthesis of Oligonucleotides Various oligonucleotides (internucleotide linkages: all phosphorothioate linkages) having mPCS2 as the parent sequence, as shown in Table 7 below, were synthesized in the same manner as in Example 2 and subjected to the following evaluation. Here, as the nucleoside (A), in addition to the entecavir derivative (E), a thymidine derivative of carbocyclic DNA (compound 17 in Example 32 below) (E Ｔ (2) The melting temperature (T m ) Measurement: A sample solution (150 μL) containing 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, and 4 μM of each oligonucleotide and complementary RNA strand was heated to 95°C for 3 minutes, then gradually cooled to 20°C at a rate of 1°C per minute to allow annealing, after which measurement was initiated. The temperature was raised to 90°C at a rate of 0.5°C per minute, and absorbance at 260 nm was plotted at 1°C intervals. Tm values ​​were measured three times and calculated using the median line method. The measurement results are shown in Table 7 below. In the table, E stands for entecavir, E Ｔ denotes the thymidine derivative of carbocyclic DNA.

[0127]

[0128] Discussion: The thermodynamic stability of the ASO-target RNA complex is affected by the thymidine derivative of carbocyclic DNA (E Ｔ ) into ASO did not have a significant effect, as did the guanosine derivative (E: entecavir). This indicates that similar thermodynamic effects are obtained regardless of the type of base.

[0129] Example 15 Objective: To evaluate the in vitro inhibitory effect of oligonucleotide drugs incorporating nucleoside (A) into the chain. Methods and Results: ASOs (mPCS2, mPCS2-gap1, mPCS2-gap2, mPCS2-gap3, mPCS2-gapT1, and mPCS2-gapT2 listed in Table 7) were diluted to a final concentration of 1 μM in cell culture medium (supplemented with 9 mM CaCl2) and added to a 96-well plate. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 24 hours. cDNA was then prepared from the cell lysate using the SuperPrep® II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's protocol. Pcsk9 mRNA expression was analyzed using a CFX Real-Time PCR System (BIO RAD). Human PCSK9 was analyzed using the primer sets hPCSK9-F: 5'-AAGGGAAGGGCACGGTTAG-3' (SEQ ID NO: 11) and hPCSK9-R: 5'-GAGTAGAGGCAGGCATCGTC-3' (SEQ ID NO: 12). Human Gapdh, a housekeeping gene, was analyzed using the primer sets hGAPDH-F: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 3) and hGAPDH-R: 5'-TGGTGAAGACGCCAGTGGA-3' (SEQ ID NO: 4). KD activity was calculated based on relative expression levels, converting the difference in Ct values ​​into differences in expression levels. Significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **p < 0.01, *p < 0.05. "ns" indicates no significant difference (p > 0.05). The results are shown in Figure 15. Discussion: Even when a thymidine derivative of carbocyclic DNA was introduced into the chain, knockdown activity equivalent to that of the parent sequence (mPCS2) was obtained. This indicates that similar efficacy can be obtained regardless of the type of nucleoside (A) base.

[0130] Example 16: Objective: Evaluation of the cytotoxicity (cytotoxicity rate) of oligonucleotide drugs incorporating nucleoside (A) into the chain (LDH assay). Methods and Results: (Cell experiment) ASO was diluted to a final concentration of 2 μM in cell culture medium (containing 9 mM CaCl2) and added to a 96-well plate. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 72 hours. (LDH assay) Lysis solution (10 μL) was added to untreated cells and incubated at 37°C for 30 minutes. Supernatant (80 μL) from each well was transferred to another 96-well plate, and working solution (80 μL) was added to each well. After incubation at room temperature for 30 minutes, stop solution (40 μL) was added to each well. The absorbance at 490 nm was measured using a microplate reader, and the background value was subtracted from the control or assay value. The cytotoxicity rate was calculated by ((experimental sample) - (low control)) / ((high control) - (low control)) x 100. Significance was tested by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **p < 0.01, *p < 0.05. "ns": No significant difference (p > 0.05). The evaluation results are shown in Figure 16. Discussion: Compared to the parent strand mPCS2, the guanosine derivative (E) and thymidine derivative (E) of carbocyclic DNA were significantly increased. Ｔ ASOs incorporating nucleoside (A) had a high probability of reducing or eliminating cytotoxicity. This suggests that there are also introduction positions that do not affect the toxicity of the parent strand. These findings demonstrate that it is possible to obtain nucleic acid drugs with reduced toxicity by incorporating nucleoside (A) into the ASO chain, regardless of the target gene or base sequence.

[0131] Example 17 Objective: Evaluation of the cytotoxicity (cell viability) of oligonucleotide drugs incorporating nucleoside (A) into the chain (PI staining / dead cell staining). Methods and Results: (Cell experiment) ASO was diluted to a final concentration of 2 μM in cell culture medium (supplemented with 9 mM CaCl2) and added to a 96-well plate. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 96 hours. (PI staining / dead cell staining) The medium was replaced with cell culture medium (supplemented with 9 mM CaCl2, 100 μL), and PI solution (Dojindo Laboratories) diluted to a final concentration of 1.0 μg / mL was added to each well. The cells were incubated at 37°C for 15 minutes and observed under a fluorescence microscope. The evaluation results are shown in Figure 17. Each figure shows, from left to right, bright field, PI, and merge (bright field / PI), with stained dead cells showing white fluorescence in the PI image. In the control (NT) group, almost no PI-positive cells were observed. Compared to the control (NT) group, many PI-positive cells were confirmed and cell death was observed in gap3, which had parent strand mPCS2 and a guanosine derivative of carbocyclic DNA (E) introduced. On the other hand, gap1, gap2, and gap3, which had a guanosine derivative of carbocyclic DNA (E) introduced, showed many PI-positive cells and cell death. Ｔ The number of PI-positive cells was significantly reduced in gapT1 and gapT2, which are ASOs into which the LDH gene was introduced. Discussion: Similar trends were observed in the PI staining experiment in this Example 17, in accordance with the LDH assay results in Example 16.

[0132] [Example 18] Objective: To determine the double-strand melting temperature (T m ) Measurement Methods and Results: (1) Synthesis of Oligonucleotides Various oligonucleotides (internucleotide linkages: all phosphorothioate linkages) with hApoC3 as the parent sequence, as shown in Table 8 below, were synthesized in the same manner as in Example 2, and were subjected to the following evaluations. (2) Double-strand melting temperature (T m) Measurement (Melting Temperature (Tm) Measurement (Evaluation of Duplex Formation Ability)) A sample solution (150 μL) containing 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, and 4 μM of each oligonucleotide and complementary strand RNA was heated to 95°C for 3 minutes and then gradually cooled to 20°C at a rate of 1°C per minute to allow annealing, after which measurement was initiated. The temperature was raised to 95°C at a rate of 0.5°C per minute, and the absorbance at 260 nm was plotted at 1°C intervals. All Tm values ​​were calculated using the median line method. The measurement results are shown in Table 8 below. In the table, E stands for entecavir.

[0133]

[0134] Discussion: The introduction of nucleoside (A) into ASOs had little effect on the thermodynamic stability of the complex between ASOs and target RNA (SD = 0.52 oC / mod). Considering that a positive correlation between thermodynamic stability and toxicity has also been found (see Reference 1 below), this derivative can be considered an excellent artificial nucleic acid that allows for the adjustment of toxicity parameters without changing the target binding ability of ASOs. 1) Watt AT, Swayze G, Swayze EE, Freier SM. Likelihood of Nonspecific Activity of Gapmer Antisense Oligonucleotides Is Associated with Relative Hybridization Free Energy. Nucleic Acid Ther. 2020 Aug;30(4):215-228. doi: 10.1089 / nat.2020.0847.

[0135] Example 19: Objective: Evaluating the cytotoxicity of oligonucleotide drugs incorporating nucleoside (A) into the chain. Methods and Results (Cell Experiments) The ASOs listed in Table 8 were diluted to a final concentration of 2 μM in cell culture medium (supplemented with 9 mM CaCl2) and added to a 96-well plate. Huh-7 cells diluted with the medium were seeded at 10,000 cells per well and cultured in an incubator for 72 hours before performing each assay. (Cell Viability Assay) 10 μL (per well) of Cell Counting Kit-8 (Dojindo Laboratories) was added to the medium and incubated in an incubator for 2 hours. Absorbance at 450 nm was measured using a microplate reader, and background values ​​were subtracted from control or assay values. Significance was determined using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **p < 0.01, *p < 0.05. "ns": not significant (p > 0.05). The evaluation results are shown in Figure 18. Discussion: Compared to the parent hApoC3, gap1, gap2, and gap3 all showed reduced or absent cytotoxicity. This demonstrates that introducing nucleoside (A) can produce nucleic acid drugs with reduced cytotoxicity, regardless of the target gene or base sequence.

[0136] Example 20: Objective: To evaluate the in vitro inhibitory effect of oligonucleotide drugs incorporating nucleoside (A) into the chain. Methods and Results (Cell Experiments): ASO was diluted to a final concentration of 1 μM in cell culture medium (supplemented with 9 mM CaCl2) and added to a 96-well plate. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 24 hours. cDNA was then prepared from the cell lysate using the SuperPrep® II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's protocol. ApoC3 mRNA expression was analyzed using a CFX Real-Time PCR System (BIO RAD). Human ApoC3 was analyzed using the primer sets hApoC3-F: 5'-CTGCTCCAGGAACAGAGGTG-3' (SEQ ID NO: 13) and hApoC3-R: 5'-GTGCGTGCTTCATGTAACC-3' (SEQ ID NO: 14). Human Gapdh, a housekeeping gene, was analyzed using the primer sets hGAPDH-F: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 3) and hGAPDH-R: 5'-TGGTTGAAGACGCCAGTGGA-3' (SEQ ID NO: 4). Relative expression levels were calculated by converting the difference in Ct values ​​into differences in expression levels. Significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **p < 0.01, *p < 0.05. "ns": not significant (p > 0.05). The evaluation results are shown in Figure 19. Discussion: All gap1-3 ASOs, which showed reduced cytotoxicity compared to hApoC3 (parent chain), demonstrated knockdown activity. Among them, gap1 and gap3 showed activity equivalent to that of the parent chain. These results demonstrate that the introduction of nucleoside (A) can improve safety while maintaining efficacy.

[0137] Example 21 Objective: To verify the effect of nucleoside (A) carrying a targeting ligand on the toxicity of oligonucleotide drugs using mice Methods and Results (1) Synthesis of oligonucleotide drugs containing nucleoside (A) carrying a targeting ligand Oligonucleotides carrying various targeting ligands (internucleotide linkages: two linkages from the 5' end are phosphodiester linkages, and the remaining linkages are phosphorothioate linkages) listed in Table 9 below, with hApoC3 as the parent sequence, were synthesized in a manner similar to that described in Example 8 and subjected to the following evaluations. In the table, E represents entecavir.

[0138]

[0139] (2) Measurement of ASO KD activity in mouse liver. All animal experiment protocols were approved by the Nagasaki University Animal Care and Use Committee. A single dose of 200 nmol / kg of an ASO targeting human ApoC3, which has no homology with mouse ApoC3, was administered subcutaneously to 7-week-old C57Bl / 6J mice (male, Japan SLC). Body weight changes were monitored, and whole blood was collected 72 hours after administration under isoflurane inhalation anesthesia. Serum collected from the inferior vena cava was used to measure ALT, AST, and total bilirubin levels. Statistical analysis was performed using the Smirnoff-Grubbs test followed by one-way analysis of variance (AST, ALT, T-BIL) or two-way analysis of variance (body weight change) followed by Dunnett's multiple comparison test. (**p < 0.01, *p < 0.05. "ns" indicates no significant difference (p > 0.05)). The evaluation results are shown in Figures 20 to 23. (Discussion) A significant decrease in body weight following administration was confirmed only with hApoC3 (parent chain). No weight loss was observed with gap1-3. Furthermore, hepatotoxicity was confirmed with hApoC3 (parent chain). All ASOs incorporating nucleoside (A) suppressed the increase in hepatic enzymes and bilirubin levels compared to the parent chain. The degree and trend of this suppression were consistent with the trend of cytotoxicity. These findings demonstrate that the introduction of nucleoside (A) can improve safety while maintaining efficacy. (However, the ASO used here does not share homology with the mouse ApoC3 gene, and knockdown activity in mice was not confirmed.)

[0140] [Production of Nucleoside (A)] Specific embodiments of nucleosides (A) other than those described in Example 1 are shown below along with their production methods (Examples 22 to 38). First, the synthesis scheme for compound 13, which is a common intermediate in the synthesis of each derivative, is shown below. Note that compounds 8 and onward are novel compounds. Starting from compound 13, various nucleosides (A) can be produced.

[0141]

[0142] [Example 22]

[0143]

[0144] (1S,2S,3S,5S)-3-(benzyloxy)-2-((benzyloxy)methyl)-5-(tert-butoxy)-cyclopentan-1-ol (8). Under a nitrogen stream, tert-BuOH (4.48 mL, 47.2 mmol, 4 eq.) dehydrated with MS4A was added to a solution of compound 7 (3.66 g, 11.8 mmol) in anhydrous dichloromethane (100 mL) at −20 °C with stirring. BF3·OEt2 (148 μL, 1.18 mmol, 0.1 eq) diluted with anhydrous dichloromethane (30 mL) was added dropwise at the same temperature, and the mixture was stirred at room temperature for 23 h. Saturated sodium bicarbonate (200 mL) was added to the reaction solution, which was then extracted three times with dichloromethane (200 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:3) to give compound 8 (3.78 g, 83%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.36 - 7.21 (m, 11H), 4.58 - 4.44 (m, 3H), 4.40 (d, J = 11.9 Hz, 1H), 3.99 (q, J = 7.8 Hz, 1H), 3.79 - 3.62 (m, 3H), 3.53 (dd, J = 9.1, 7.0 Hz, 1H), 2.43 (dd, J = 3.0, 1.0 Hz, 1H), 2.21 - 1.99 (m, 2H), 1.75 (dt, J = 13.9, 7.9 Hz, 1H), 1.19 (s, 9H).; 13 C NMR (101 MHz, CDCl3) δ 138.52, 138.31, 128.48, 127.75, 127.68, 79.44, 77.35, 76.93, 76.41, 73.71, 73.38, 71.11, 70.88, 53.55, 50.34, 38.26, 28.74. HRMS (FAB) m / z: [M + H]+ calcd for C 24 H33 O4, 385.2373; found, 385.2379.

[0145] [Example 23]

[0146]

[0147] (2R,3S,5S)-3-(benzyloxy)-2-((benzyloxy)methyl)-5-(tert-butoxy)-cyclopentan-1-one (9). Under a nitrogen atmosphere, Dess-martin periodinane (4.5 g, 10.63 mmol) was added to a solution of compound 8 (3.40 g, 8.86 mmol) in anhydrous dichloromethane (88 mL) at 0°C with stirring, followed by stirring at room temperature for 1 hour. Aqueous sodium thiosulfate (70 mL) and saturated sodium bicarbonate (10 mL) were added at 0°C with stirring, and the mixture was stirred at the same temperature for 20 minutes. Aqueous sodium thiosulfate:saturated sodium bicarbonate (7:1, 150 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (200 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain Compound 9 (3.25 g, 96%) as a yellow oily substance. 1 H NMR (400 MHz, CDCl3) δ 7.37 - 7.21 (m, 13H), 4.58 - 4.49 (m, 2H), 4.45 (s, 2H), 4.41 - 4.32 (m, 1H), 4.17 (d, J = 5.4 Hz, 1H), 3.67 (dd, J = 9.4, 4.3 Hz, 1H), 3.55 (dd, J = 9.4, 7.2 Hz, 1H), 2.59 (ddd, J = 6.6, 4.4, 2.2 Hz, 1H), 2.41 (ddt, J = 12.9, 8.6, 2.1 Hz, 1H), 1.96 (ddd, J = 13.5, 11.5, 5.5 Hz, 1H), 1.22 (s, 9H).; 13C NMR (101 MHz, CDCl3) δ 215.48, 138.16, 138.03, 128.58, 128.45, 127.84, 127.73, 127.69, 77.45, 77.13, 76.82, 75.80, 74.97, 73.54, 73.31, 70.87, 68.20, 52.38, 36.71, 28.30.

[0148] [Example 24]

[0149]

[0150] (((1R,3S,5S)-5-(benzyloxy)-3-(tert-butoxy)-2-methylenecyclopentyl)methoxy)methyl)benzene (10). Under a nitrogen atmosphere, 0.5 M Tebbe reagent (19.7 mL, 9.87 mmol) was added dropwise to a solution of compound 9 (2.90 g, 7.59 mmol) in anhydrous tetrahydrofuran (50 mL) at 0°C with stirring, and the mixture was stirred at room temperature for 50 minutes. 0.1 M aqueous NaOH (50 mL) was added at 0°C with stirring, and the mixture was quenched by stirring for 30 minutes. HO (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (200 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane=1:3) gave compound 10 (2.22 g, 77%) as a yellow oily substance. 11H NMR (400 MHz, CDCl3) δ 7.35 - 7.22 (m, 13H), 5.17 (td, J = 2.5, 0.9 Hz, 1H), 5.07 - 5.03 (m, 1H), 4.59 - 4.42 (m, 6H), 3.92 (dt, J = 5.8, 1.9 Hz, 1H), 3.55 (dd, J = 9.6, 5.5 Hz, 1H), 3.31 (t, J = 9.6 Hz, 1H), 2.92 - 2.82 (m, 1H), 2.13 (tt, J = 7.3, 1.7 Hz, 1H), 1.61 (ddd, J = 13.4, 10.0, 5.8 Hz, 2H), 1.22 (s, 9H).; 13 13C NMR (101 MHz, CDCl3) δ 152.40, 138.84, 138.43, 128.42, 127.76, 127.65, 127.51, 108.37, 79.10, 77.44, 77.33, 77.12, 76.80, 73.82, 73.09, 72.94, 72.18, 70.53, 47.70, 39.63, 28.​​​​​​​​​​​​(1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentan-1-ol (11). Under a nitrogen atmosphere, trifluoroacetic acid (14 mL) was added to a solution of compound 10 (2.14 g, 5.64 mmol) in anhydrous dichloromethane (37 mL) at 0 °C with stirring, and the mixture was stirred at room temperature for 1 h. Further trifluoroacetic acid (5 mL) was added, and after stirring for 30 min, cold saturated aqueous sodium bicarbonate solution (200 mL) was added to the reaction solution, and the aqueous layer was extracted with dichloromethane (200 mL). The organic layer was washed with saturated brine, dehydrated over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:3). The compound whose secondary hydroxyl group was trifluoroacetylated during the reaction was collected and hydrolyzed with 50 mM potassium carbonate-methanol solution (10 mL). The reaction mixture was mixed with saturated aqueous sodium bicarbonate (100 mL) and extracted three times with ethyl acetate (100 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 11 (1.66 g, 90%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.38 - 7.23 (m, 13H), 5.30 - 5.25 (m, 1H), 5.09 (t, J = 1.8 Hz, 1H), 4.59 - 4.44 (m, 6H), 4.17 - 4.08 (m, 1H), 3.61 (dd, J = 9.2, 4.3 Hz, 1H), 3.51 (dd, J = 9.2, 6.1 Hz, 1H), 2.81 (dtt, J = 6.4, 4.3, 2.3 Hz, 1H), 2.20 (d, J = 6.2 Hz, 1H), 1.99 (t, J = 5.7 Hz, 2H). ; 13C NMR (101 MHz, CDCl3) δ 153.37, 138.61, 138.00, 128.55, 128.47, 127.85, 127.79, 127.66, 110.21, 80.15, 77.48, 77.16, 76.84, 74.04, 73.38, 72.08, 71.17, 49.27, 40.85. HRMS (FAB) m / z: [M + H]+ calcd for C 21 H 25 O3, 325.1798; found, 325.1813.

[0154] [Example 26]

[0155]

[0156] (1R,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentyl acetate (12). Under a nitrogen stream, DEAD (4.2 mL, 9.05 mmol) was added to a solution of triphenylphosphine (2.37 g, 9.05 mmol) in anhydrous tetrahydrofuran (22 mL) at -20°C with stirring, and the mixture was stirred at the same temperature for 20 minutes. Compound 11 (1.42 g, 4.52 mmol) diluted with anhydrous tetrahydrofuran (10 mL) was added, and the mixture was stirred at the same temperature for 10 minutes. Acetic acid (679 μL, 11.3 mmol) was added, and the mixture was stirred at room temperature for 20 hours. Saturated aqueous sodium bicarbonate (200 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (200 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:4) gave compound 12 (1.20 g, 73%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.37 - 7.23 (m, 11H), 5.48 - 5.39 (m, 1H), 5.24 (t, J = 2.2 Hz, 1H), 5.18 (t, J = 2.2 Hz, 1H), 4.55 - 4.44 (m, 4H), 3.90 (q, J = 6.1 Hz, 1H), 3.59 - 3.45 (m, 2H), 2.94 (tq, J = 5.6, 2.7 Hz, 1H), 2.49 (ddd, J = 13.5, 7.3, 6.2 Hz, 1H), 2.08 (s, 3H), 1.79 (dt, J = 13.1, 6.4 Hz, 1H).; 13 C NMR (101 MHz, CDCl3) δ 171.05, 148.74, 138.53, 138.32, 128.46, 127.77, 127.70, 127.67, 111.95, 78.77, 77.46, 77.14, 76.82, 74.52, 73.23, 71.30, 70.98, 49.64, 37.42, 21.42. HRMS (FAB) m / z: [M + H]+ calcd for C 23 H 27 O4, 367.1904; found, 367.1908.

[0157] [Example 27]

[0158]

[0159] (1R,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentan-1-ol (13). Under a nitrogen atmosphere, compound 12 (1.06 g, 2.89 mmol) was added to 50 mM potassium carbonate-methanol (20 mL) and stirred at room temperature for 1 hour. After adding HO (5 mL), the mixture was concentrated under reduced pressure. Ethyl acetate (100 mL) and saturated sodium bicarbonate (100 mL) were added to the residue, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 13 (935 mg, quant.) as a colorless oil.1 1H NMR (400 MHz, CDCl3) δ 7.36 - 7.26 (m, 10H), 5.37 (t, J = 1.9 Hz, 1H), 5.14 (t, J = 1.8 Hz, 1H), 4.56 - 4.42 (m, 4H), 4.38 (s, 1H), 4.05 (dt, J = 5.6, 3.0 Hz, 1H), 3.49 (dd, J = 9.5, 5.1 Hz, 1H), 3.29 (dd, J = 9.5, 8.1 Hz, 1H), 3.13 - 3.02 (m, 1H), 2.48 (d, J = 9.8 Hz, 1H), 2.09 (ddd, J = 13.8, 6.0, 5.1 Hz, 1H), 1.94 (dtd, J = 13.9, 3.6, 1.5 Hz, 1H).

[0160] [Example 28]

[0161]

[0162] 1-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylene-cyclopentyl)-3-((benzyloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (14). Under a nitrogen atmosphere, DEAD (514 μL, 1.13 mmol) was added to a solution of triphenylphosphine (297 mg, 1.13 mmol) in anhydrous acetonitrile (2 mL) at 0 °C with stirring, and the mixture was stirred at the same temperature for 20 min. N3-(benzyloxymethyl)thymine (209 mg, 0.85 mmol) and compound 13 (184 mg, 0.566 mmol) diluted with anhydrous acetonitrile (2 mL) were added, and the mixture was stirred at room temperature for 17 h. Saturated aqueous sodium bicarbonate (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:3) to give compound 14 (189 mg, 61%) as a white foamy solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.40 - 7.26 (m, 15H), 7.06 (t, J = 1.2 Hz, 1H), 5.74 (t, J = 9.4 Hz, 1H), 5.51 (s, 2H), 5.24 (t, J = 2.5 Hz, 1H), 4.93 (t, J = 2.5 Hz, 1H), 4.71 (s, 2H), 4.57 - 4.44 (m, 4H), 4.12 - 4.06 (m, 1H), 3.79 - 3.65 (m, 2H), 2.90 (s, 1H), 2.34 (dd, J = 13.2, 8.1 Hz, 1H), 1.98 (ddd, J = 13.1, 10.1, 5.1 Hz, 1H), 1.57 (d, J = 1.2 Hz, 3H).; 13 C NMR (101 MHz, CDCl 3 ) δ 163.68, 152.15, 149.17, 138.24, 137.88, 137.23, 128.66, 128.54, 128.46, 128.38, 128.01, 127.76, 127.67, 127.58, 111.32, 110.29, 80.24, 77.33, 73.67, 72.94, 72.31, 70.94, 70.74, 58.30, 49.58, 36.50, 12.95; + calcd for C 34 H 37 N2O5, 553.2697; found, 553.2702.

[0163] [Example 29]

[0164]

[0165] 1-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl)-5-methylpyrimidine-2,4(1H,3H)-dione (15). Under a nitrogen stream, 1M BCl3-dichloromethane solution (3.43 mL, 3.43 mmol) was added to a solution of compound 14 (189 mg, 0.343 mmol) in anhydrous dichloromethane (1.0 mL) at −40°C with stirring, and the mixture was stirred at the same temperature for 1.5 hours. A 2M ammonia-methanol solution (2 mL) was added to the mixture at −40°C with stirring, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 10:1) to give compound 15 (71 mg, 82%) as a colorless oil. 1H NMR (400 MHz, CD3OD) δ 7.37 (d, J = 1.4 Hz, 1H), 5.61 (td, J = 9.2, 4.5 Hz, 1H), 5.26 (t, J = 2.5 Hz, 1H), 4.91 (t, J = 2.5 Hz, 1H), 4.30 (p, J = 2.6 Hz, 1H), 3.73 (qd, J = 11.0, 5.4 Hz, 2H), 2.59 (d, J = 6.8 Hz, 1H), 2.07 (dd, J = 9.4, 3.6 Hz, 2H), 1.83 (d, J = 1.2 Hz, 3H). 13 C NMR (101 MHz, CD3OD) δ149.69, 139.50, 110.51, 109.84, 71.87, 63.46, 57.66, 53.65, 48.44, 48.23, 48.01, 38.17, 11.16. HRMS (FAB) m / z: [M+H] + calcd for C 12 H 17 N2O4, 253.1183; found, 253.1191.

[0166] [Example 30]

[0167]

[0168] Compound 15 (71 mg, 0.282 mmol) was added to anhydrous pyridine (2.8 mL) under a nitrogen atmosphere, followed by the addition of 4,4'-dimethoxytrityl chloride (142 mg, 0.42 mmol) and stirring at room temperature for 20 hours. Saturated aqueous sodium bicarbonate (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 97:3, 0.5% triethylamine added) to obtain compound 16 (122 mg, 78%) as a white foamy solid. 1 H NMR (400 MHz, CDCl 3 ) δ 8.41 (s, 1H), 7.42 - 7.33 (m, 2H), 7.32 - 7.19 (m, 9H), 6.89 (d, J = 1.4 Hz, 1H), 6.87 - 6.76 (m, 4H), 5.71 (t, J = 9.0 Hz, 1H), 4.95 (t, J = 2.6 Hz, 1H), 4.85 (t, J = 2.6 Hz, 1H), 4.40 (d, J = 5.1 Hz, 1H), 3.78 (d, J = 0.9 Hz, 6H), 3.59 (dd, J = 9.3, 4.1 Hz, 1H), 3.21 (dd, J = 9.3, 6.3 Hz, 1H), 2.68 (s, 1H), 2.27 - 2.16 (m, 2H), 2.11 (ddd, J = 13.5, 9.0, 5.7 Hz, 1H), 1.54 (d, J = 1.2 Hz, 3H).; 13 C NMR (at 101 MHz, in CDCl 3 ) δ163.93, 158.73, 151.56, 148.84, 144.53, 138.09, 135.76, 135.48, 130.20, 128.23, 128.08, 127. I8, 113.31, 111.47, 111. [21, 87.09, 77.46, 77.14, 76.83, 73.91, 65.29, 56.90, 55.35, 51.63, 39.40, 12.16. HRMS (FAB) m / z: [M + H] + calculated for C 33 H 35 N2O6, 555.2490; found, 555.2502.

[0169] [Example 31]

[0170]

[0171] Compound 16 (122 mg, 0.220 mmol) and 1H-tetrazole (23 mg, 0.33 mmol) were added to a heat-dried reaction vessel, and anhydrous dichloromethane (2.2 mL) and DIPEA (56 μL, 0.33 mmol) were added under a nitrogen atmosphere. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (105 μL, 0.33 mmol) was added and the mixture was stirred at room temperature for 1.5 hours. After confirming the consumption of the starting material by TLC, saturated aqueous sodium bicarbonate (30 mL) was added and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 97:3, 1% triethylamine added) to obtain compound 17 (121 mg, 73%) as a white foamy solid. 1 H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 7.38 (dt, J = 8.2, 1.5 Hz, 2H), 7.34 - 7.16 (m, 9H), 7.05 - 6.88 (m, 1H), 6.81 (dt, J = 8.9, 2.0 Hz, 4H), 5.72 (t, J = 9.7 Hz, 1H), 4.98 (dt, J = 10.1, 2.6 Hz, 1H), 4.86 (dt, J = 12.3, 2.5 Hz, 1H), 4.53 (d, J = 9.5 Hz, 1H), 3.87 - 3.64 (m, 8H), 3.62 - 3.42 (m, 3H), 3.29 - 3.13 (m, 1H), 2.90 - 2.77 (m, 1H), 2.62 (t, J = 6.3 Hz, 1H), 2.51 (t, J = 6.4 Hz, 1H), 2.41 - 2.26 (m, 1H), 2.19 - 2.03 (m, 1H), 1.43 (dd, J = 7.8, 1.2 Hz, 3H), 1.21 - 1.07 (m, 12H).; 31P NMR (162 MHz, CDCl3) δ 147.97, 147.69. HRMS (FAB) m / z: [M + H] + calcd for C 42 H 52 N4O7P, 755.3574; found, 755.3574.

[0172] [Example 32]

[0173]

[0174] 1,2,4-Triazole (123 mg, 1.78 mmol) was added to a heat-dried reaction vessel, and anhydrous acetonitrile (2 mL) was added under a nitrogen atmosphere. Phosphoryl chloride (37.5 μL, 0.403 mmol) was added dropwise and the mixture was stirred at room temperature for 10 min. Triethylamine (331 μL, 2.385 mmol) was added dropwise and the mixture was stirred at room temperature for an additional 30 min. Compound 17 (40 mg, 0.053 mmol) dissolved in anhydrous acetonitrile (1 mL) was then added dropwise and the mixture was stirred at room temperature for an additional 1 h. After confirming the consumption of the starting material by TLC, saturated aqueous sodium bicarbonate (30 mL) was added and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 18 (41 mg, 96%) as a white foamy solid. 11H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.07 (d, J = 1.0 Hz, 1H), 7.76 - 7.65 (m, 1H), 7.43 - 7.34 (m, 2H), 7.33 - 7.21 (m, 9H), 6.82 (ddd, J = 8.9, 2.1, 1.3 Hz, 4H), 5.93 (m, 1H), 5.05 (d, J = 15.1 Hz, 1H), 4.96 - 4.83 (m, 1H), 4.65 - 4.58 (m, 1H), 3.90 - 3.65 (m, 9H), 3.66 - 3.48 (m, 3H), 3.40 - 3.24 (m, 1H), 2.96 - 2.81 (m, 1H), 2.64 (t, J = 6.2 Hz, 1H), 2.60 - 2.42 (m, 2H), 2.32 - 2.16 (m, 1H), 1.87 (dd, J = 10.2, 0.8 Hz, 3H), 1.26 - 1.11 (m, 12H).; 31 31P NMR (162 MHz, CDCl3) δ 148.06, 147.89. HRMS (FAB) m / z: [M + H] + calcd for C 44 H 53 N7O6P, 806.3789; found, 806.3796.

[0175] [Example 33]

[0176]

[0177] 9-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylene-cyclopentyl)-6-chloro-9H-purine (19) Under a nitrogen atmosphere, DEAD (140 μL, 0.308 mmol) was added to a solution of triphenylphosphine (81 mg, 0.308 mmol) in anhydrous tetrahydrofuran (1 mL) with stirring at 0°C, and the mixture was stirred at the same temperature for 20 min. 6-Chloropurine (35.5 mg, 0.308 mmol) was added, and the mixture was stirred for an additional 10 min. Subsequently, compound 13 (50 mg, 0.154 mmol) diluted with anhydrous tetrahydrofuran (500 μL) was added, and the mixture was stirred at room temperature for 24 h. Saturated aqueous sodium bicarbonate (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:2) to give compound 19 (47 mg, 66%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.4 Hz, 1H), 8.21 (d, J = 1.3 Hz, 1H), 7.42 - 7.26 (m, 10H), 5.76 (t, J = 8.9 Hz, 1H), 5.24 (q, J = 2.1 Hz, 1H), 4.80 (q, J = 2.1 Hz, 1H), 4.57 - 4.48 (m, 4H), 4.25 - 4.15 (m, 1H), 3.76 - 3.66 (m, 2H), 3.05 (s, 1H), 2.45 (ddd, J = 9.3, 4.4, 1.4 Hz, 2H).; 13C NMR (101 MHz, CDCl3) δ 171.05, 148.74, 138.53, 138.32, 128.46, 127.77, 127.70, 127.67, 111.95, 78.77, 77.46, 77.14, 76.82, 74.52, 73.23, 71.30, 70.98, 49.64, 37.42, 21.42. HRMS (FAB) m / z: [M + H]+ calcd for C 26 H 26 ClN4O2, 461.1739; found, 461.1754.

[0178] [Example 34]

[0179]

[0180] 9-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylene-cyclopentyl)-9H-purin-6-amine (20) Compound 19 (180 mg, 0.392 mmol) was added to 7 M ammonia-methanol solution (3 mL) under a nitrogen atmosphere and stirred in a pressure-resistant vial at 110 °C for 2 hours. The reaction solution was returned to room temperature and then concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 2:1 → dichloromethane / methanol = 10:1) gave compound 20 (142 mg, 82%) as a white foamy solid. 1 H NMR (400 MHz, CD3OD) δ 8.15 (s, 1H), 8.03 (s, 1H), 7.37 - 7.19 (m, 10H), 5.64 (td, J = 8.9, 2.6 Hz, 1H), 5.21 (t, J = 2.4 Hz, 1H), 4.70 (t, J = 2.5 Hz, 1H), 4.60 - 4.46 (m, 4H), 4.18 (dt, J = 4.6, 2.7 Hz, 1H), 3.78 - 3.67 (m, 2H), 3.03 (q, J = 4.4 Hz, 1H), 2.51 - 2.37 (m, 2H).; 13C NMR (101 MHz, CD3OD) δ 155.95, 152.33, 150.08, 149.56, 140.26, 138.44, 138.25, 128.16, 128.04, 127.69, 127.58, 127.50, 127.30, 118.58, 110.14, 79.43, 73.00, 71.56, 70.29, 56.53, 49.34, 48.32, 48.11, 47.90, 47.68, 47.47, 47.26, 47.04, 36.77. HRMS (FAB) m / z: [M + H]+ calcd for C 26 H 28 N5O2, 442.2238; found, 442.2243.

[0181] [Example 35]

[0182]

[0183] N-(9-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylene-cyclopentyl)-9H-purin-6-yl)benzamide (21). Under a nitrogen atmosphere, N,N-diisopropylethylamine (76 μL, 0.446 mmol) was added to a solution of compound 20 (131 mg, 0.297 mmol) and 4-dimethylaminopyridine (3.6 mg, 0.030 mmol) in anhydrous N,N-dimethylformamide (2.9 mL). Benzoic anhydride (134 mg, 0.594 mmol) was then added, and the mixture was stirred at 100 °C for 7.5 h. A 2M ammonia-methanol solution (1 mL) was added under ice cooling, and after stirring for 10 min, saturated aqueous sodium bicarbonate (50 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (50 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:1) to give compound 21 (109 mg, 67%) as a white foamy solid. 11H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.77 (s, 1H), 8.09 (s, 1H), 8.08 - 7.98 (m, 2H), 7.67 - 7.56 (m, 1H), 7.56 - 7.46 (m, 2H), 7.42 - 7.27 (m, 11H), 5.76 (t, J = 8.8 Hz, 1H), 5.24 (t, J = 2.4 Hz, 1H), 4.83 (t, J = 2.4 Hz, 1H), 4.63 - 4.44 (m, 4H), 4.21 (q, J = 3.8 Hz, 1H), 3.72 (d, J = 5.3 Hz, 2H), 3.05 (s, 1H), 2.47 (dd, J = 8.7, 3.9 Hz, 2H).; 13 13C NMR (101 MHz, CDCl3) δ164.98, 152.48, 152.33, 149.55, 149.08, 142.70, 138.17, 137.96, 133.83, 132.80, 128.89, 128.62, 128.55, 128.05, 127.96, 127.82, 127.77, 123.22, 111.87, 79.61, 77.51, 77.39, 77.19, 76.87, 73.49, 71.58, 70.95, 56.96, 49.52, 37.63. HRMS (FAB) m / z: [M + H]+ calcd for C 33 H 32 N5O3, 546.2500; found, 546.2506.

[0184] [Example 36]

[0185]

[0186] N-(9-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl)-9H-purin-6-yl)benzamide (22) Under a nitrogen atmosphere, 1 M boron trichloride (1.98 mL, 1.98 mL) was added dropwise to a solution of compound 21 (108 mg, 0.198 mmol) in anhydrous dichloromethane (500 μL) at −40°C with stirring. After stirring at the same temperature for 20 minutes, triethylamine (1 mL) and methanol (1 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 20:1). After concentration under reduced pressure, saturated aqueous ammonium chloride solution (40 mL) was added to the residue, and the mixture was extracted five times with ethyl acetate (40 mL). The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to give compound 22 (60 mg, 83%) as a white foamy solid. 1 H NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 8.49 (s, 1H), 8.12 - 8.01 (m, 2H), 7.67 - 7.60 (m, 1H), 7.55 (dd, J = 8.3, 6.8 Hz, 2H), 5.88 - 5.75 (m, 1H), 5.28 (t, J = 2.5 Hz, 1H), 4.80 (t, J = 2.5 Hz, 1H), 4.45 (dt, J = 5.2, 2.6 Hz, 1H), 3.94 - 3.77 (m, 2H), 2.73 (s, 1H), 2.58 (ddd, J = 13.2, 9.8, 5.0 Hz, 1H), 2.37 - 2.27 (m, 1H).; 13C NMR (101 MHz, CD3OD) δ166.89, 152.14, 151.48, 149.77, 144.13, 133.65, 132.60, 128.45, 128.12, 123.79, 110.83, 71.83, 63.35, 57.16, 54.16, 48.34, 48.13, 47.91, 47.70, 47.48, 47.27, 47.06, 46.54, 39.20. HRMS (FAB) m / z: [M + H]+ calcd for C 19 H 20 N5O3, 366.1561; found, 366.1566.

[0187] [Example 37]

[0188]

[0189] N-(9-((1S,3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylenecyclopentyl)-9H-purin-6-yl)benzamide (23). Compound 22 (60 mg, 0.164 mmol) was added to anhydrous pyridine (1.6 mL) under a nitrogen atmosphere, followed by the addition of 4,4'-dimethoxytrityl chloride (67 mg, 0.197 mmol) and stirring at room temperature for 6 hours. Saturated aqueous sodium bicarbonate (40 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (40 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 3:1 to dichloromethane / methanol = 20:1) to obtain compound 23 (74 mg, 68%) as a white foamy solid. 11H NMR (400 MHz, CDCl3) δ 9.32 (s, 1H), 8.68 (s, 1H), 8.10 - 7.95 (m, 2H), 7.88 (s, 1H), 7.61 - 7.41 (m, 5H), 7.35 - 7.16 (m, 8H), 6.92 - 6.73 (m, 4H), 5.71 (t, J = 8.1 Hz, 1H), 4.99 (t, J = 2.5 Hz, 1H), 4.74 (d, J = 2.5 Hz, 1H), 4.52 (q, J = 5.2 Hz, 1H), 3.78 (s, 6H), 3.56 (ddd, J = 9.6, 5.2, 1.7 Hz, 1H), 3.34 (dd, J = 9.4, 8.1 Hz, 1H), 2.81 (d, J = 6.3 Hz, 1H), 2.36 (qdd, J = 13.6, 7.8, 5.3 Hz, 2H).; 13 13C NMR (101 MHz, CDCl3) δ 175.48, 165.19, 158.68, 152.19, 149.74, 148.37, 144.67, 142.28, 135.91, 135.80, 133.69, 132.85, 130.11, 128.85, 128.22, 128.08, 127.12, 123.19, 113.33, 112.18, 86.86, 77.49, 77.37, 77.17, 76.85, 73.63, 64.76, 56.22, 55.35, 53.56, 51.95, 39.73, 29.79, 21.04. HRMS (FAB) m / z: [M + H] + calculated for C 40 H 38 N5O5, 668.2867; found, 668.2873.

[0190] [Example 38]

[0191]

[0192] Compound 23 (74.1 mg, 0.111 mmol) and 1H-tetrazole (12 mg, 0.167 mmol) were added to a heat-dried reaction vessel, and anhydrous dichloromethane (1.1 mL) and DIPEA (28 μL, 0.167 mmol) were added under a nitrogen atmosphere. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (53 μL, 0.167 mmol) was added and the mixture was stirred at room temperature for 1 hour. After confirming the consumption of the starting material by TLC, saturated aqueous sodium bicarbonate (30 mL) was added and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:1 → 2 / 1, 1% triethylamine added). The residue was dissolved in dichloromethane (1 mL), followed by addition of hexane (49 mL), and the mixture was centrifuged at 25°C and 5000 xg for 5 minutes. The supernatant was removed, and the precipitate was subjected to the same procedure again. The precipitate was redissolved in dichloromethane and concentrated under reduced pressure to give compound 24 (54.6 mg, 57%) as a white foamy solid. 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 1H), 8.68 (d, J = 5.9 Hz, 1H), 8.04 - 7.97 (m, 2H), 7.93 (d, J = 8.4 Hz, 1H), 7.64 - 7.55 (m, 1H), 7.55 - 7.40 (m, 5H), 7.39 - 7.17 (m, 10H), 6.87 - 6.78 (m, 5H), 5.72 (dd, J = 10.2, 7.7 Hz, 1H), 5.06 (dt, J = 6.2, 2.4 Hz, 1H), 4.70 (dt, J = 13.5, 2.4 Hz, 1H), 4.65 - 4.57 (m, 1H), 3.92 - 3.69 (m, 9H), 3.62 (dp, J = 10.3, 6.8 Hz, 2H), 3.47 - 3.34 (m, 2H), 3.01 (d, J = 7.2 Hz, 1H), 2.62 (t, J = 6.3 Hz, 1H), 2.56 - 2.29 (m, 3H), 1.24 - 1.09 (m, 12H).; 31 P NMR (162 MHz, CDCl3) δ 148.18, 148.11.; HRMS (FAB) m / z: [M + H] + calcd for C 49 H 55 N7O6P, 868.3946; found,868.3951.

[0193] [Example 39]

[0194]

[0195] Amidite derivatives of the two cytosine derivatives represented by the above structural formulas can be prepared by those skilled in the art, for example, starting from compound 13 (Example 27) and referring to the methods described in Examples 28 to 32 as well as other methods known in the art.

[0196] [Example 40] Objective: Synthesis of siRNA Methods and Results: (1) Synthesis Scheme: siRNA was synthesized using commercially available phosphoramidites of 2'OMe-A(Bz), 2'OMe-G(iBu), 2'OMe-C(Ac), 2'OMe-U, 2'FA(Bz), 2'FG(iBu), 2'FC(Ac), 2'FU, and entecavir, prepared as a 0.1 M solution in anhydrous acetonitrile, according to the phosphoramidite method. (2) Sequence and Structure of Synthesized siRNA: Based on the structure of Vutrisiran, oligonucleotides with the sequences shown in Table 10 below were prepared (circles indicate PS bonds, and the remaining linkages are phosphodiester bonds). In the table, E represents entecavir, N represents 2'-OMe RNA, and Nf represents 2'-F RNA.

[0197]

[0198] Discussion: The above results demonstrate that oligonucleotides containing entecavir derivatives can be synthesized according to conventional methods and siRNAs can be constructed.

[0199] Example 41 Objective: To evaluate the duplex formation ability of siRNAs containing an entecavir derivative in the antisense strand. Methods and Results: A sample solution (10 μL) containing an equal mixture of the sense strand (Sense) and each antisense strand (AS, AS@6, AS@5, AS@5,6) synthesized in Example 40 was heated to 95°C for 3 minutes and then gradually cooled to 20°C at a rate of 1°C per minute for annealing. A 10 pmol portion of each sample (20 μM) was mixed with Orange DNA Ladder Dye (6X) (Thermo Fisher Scientific) and 10x TBE, applied to a 20% TBE gel, and electrophoresed at 4°C and 200V for 60 minutes. The gel was stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific) and imaged using a FAS-V (Nippon Genetics). The evaluation results are shown in Figure 24. Discussion: It was suggested that any AS strand containing one or more entecavir derivatives can form a duplex similar to unmodified siRNA by mixing with a Sense strand.

[0200] [Example 42] Objective: To evaluate the in vitro target gene expression inhibitory effect and IC of siRNA incorporating an entecavir derivative into the AS chain. 50Evaluation Methods and Results: Equal amounts of the sense strand (Sense) and each antisense strand (AS, AS@6, AS@5, AS@5,6) were mixed and annealed by heating the mixture to 95°C for 3 minutes and then gradually cooling to 20°C at a rate of 1°C per minute. The annealed siRNAs were diluted in cell culture medium (supplemented with CaCl2) to final concentrations of 0, 0.01, 0.1, 1, 10, and 100 nM and added to a 96-well plate. Huh-7 cells diluted with the medium were seeded at 10,000 cells per well and cultured in an incubator for 24 hours. cDNA was prepared from the cell lysates using the SuperPrep® II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's protocol. TTR mRNA expression was analyzed using a QuantStudio3 (Thermo Fisher Scientific). A FAM-labeled TaqMan probe (Hs00174914_m1) was used to analyze human TTR, and a VIC-labeled TaqMan probe (Hs02758991_g1) was used to analyze human Gapdh as a housekeeping gene. The difference in Ct values ​​was converted into the difference in expression level to calculate the KD activity based on the relative expression level. The 50% inhibitory concentration (IC 50 The graph of the evaluation results is shown in Figure 25. 50 The results are shown in Table 11.

[0201]

[0202] Discussion: When one or more entecavir derivatives were introduced into the AS strand of siRNA in cultured cells, it was shown to have a high knockdown activity comparable to that of Vutrisiran.

[0203] [Production of Nucleoside (A)] The synthesis schemes of representative nucleoside (Ai-2) compounds and their phosphoramidite derivatives are shown below.

[0204] Each synthesis step in the above synthesis scheme is described in detail below. [Example 43]

[0205]

[0206] N-(1-((1S,2S,3S,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-hydroxycyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (25). Under a nitrogen stream, diethylzinc (approximately 15% hexane solution, approximately 1 mol / L) (29.5 mL, 29.5 mmol, 3.0 eq.) and diiodomethane (4.75 mL, 59.0 mmol, 6.0 eq.) were added to a solution of compound 13 (3.189 g, 9.84 mmol) in anhydrous diethyl ether (98.4 mL) at 0°C with stirring. After stirring at 40°C for 6.5 hours, saturated sodium bicarbonate (100 mL) was added to the reaction solution with stirring at 0°C, and the mixture was extracted three times with ethyl acetate (200 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:10 to 1:4) to give compound 25 (2.97 g, 89%) as a yellow oil.1H NMR (400 MHz, CDCl3) δ 7.37 - 7.21 (m, 10H), 4.54 (d, J = 1.2 Hz, 2H), 4.49 - 4.37 (m, 2H), 4.05 - 4.00 (m, 1H), 3.50 (br, 1H), 3.26 (dd, J = 9.4, 6.0 Hz, 1H), 3.18 (dd, J = 9.4, 7.3 Hz, 1H), 2.48 (m, 2H), 2.12 (m, 1H), 2.05 - 1.95 (m, 2H), 0.93 (ddd, J = 10.2, 5.9, 4.5 Hz, 1H), 0.68 (ddd, J = 10.0, 5.8, 4.5 Hz, 1H), 0.52 (ddd, J = 9.4, 6.3, 4.5 Hz, 1H), 0.37 (ddd, J = 9.8, 6.2, 4.5 Hz, 1H).; 13C NMR (101 MHz, CDCl3) δ 128.46, 127.76, 127.67, 127.63, 127.58, 83.19, 80.40, 73.19, 70.79, 70.28, 48.06, 39.97, 30.10, 8.78, 7.53. HRMS (FAB) m / z: [M + H]+ calcd for C。 22 H 27 O3, 339.1955; found, 339.1978.

[0207] [Example 44]

[0208]

[0209] 9-((4S,6S,7R)-6-(benzyloxy)-7-((benzyloxy)methyl)spiro[2.4]heptan-4-yl)-6-chloro-9H-purine (26). Under a nitrogen atmosphere, 6-chloropurine (553 mg, 3.58 mmol, 2.5 eq.) and DEAD (1.64 mL, 3.58 mmol) were added to a solution of triphenylphosphine (939 mg, 3.58 mmol) in anhydrous tetrahydrofuran (8 mL) at −20°C with stirring, and the mixture was stirred at the same temperature for 10 min. Compound 25 (485 mg, 1.43 mmol) diluted with anhydrous tetrahydrofuran (6.3 mL) was added, and the mixture was stirred at room temperature for 5 h. Saturated aqueous sodium bicarbonate (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:10 to 3:5) to give compound 26 (203 mg, 30%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.68 (s,1H), 8.64 (s,1H), 7.41 - 7.23 (m, 10H), 5.01 (t, J = 6.3 Hz, 1H), 4.58 - 4.44 (m, 4H), 4.29 (q, J = 5.8 Hz, 1H), 3.55 (dd, J = 9.8, 3.8 Hz, 1H), 3.35 (dd, J = 9.7, 4.1 Hz, 1H), 2.55 - 2.39 (m, 2H), 2.25 (q, J = 4.3 Hz, 1H), 0.93 - 0.85 (m, 1H), 0.85 - 0.70 (m, 2H), 0.06 (t, J = 4.8 Hz, 1H).; 13C NMR (101 MHz, CDCl3) δ 151.97, 151.71, 150.88, 145.60, 138.23, 137.66, 128.67, 128.54, 128.16, 128.13, 127.86, 127.71, 80.39, 73.58, 71.64, 67.86, 61.12, 50.61, 38.99, 25.52, 17.32, 8.87. HRMS (FAB) m / z: [M + H]+ calcd for C 27 H 28 ClN4O2, 475.1895; found, 475.1901.

[0210] [Example 45]

[0211]

[0212] 9-((4S,6S,7R)-6-(benzyloxy)-7-((benzyloxy)methyl)spiro[2.4]heptan-4-yl)-9H-purin-6-amine (27). Under a nitrogen atmosphere, compound 26 (200 mg, 0.420 mmol) was added to 7 M ammonia-methanol solution (10 mL) and stirred in a pressure-resistant vial at 110 °C for 18 hours. The reaction solution was returned to room temperature and then concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:3 → dichloromethane / methanol = 15:1) afforded compound 27 (162 mg, 85%) as a white foamy solid. 11H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 8.17 (s, 1H), 7.40 - 7.22 (m, 10H), 6.02 (s, 2H), 4.96 (dd, J = 7.2, 5.7 Hz, 1H), 4.58 - 4.42 (m, 4H), 4.28 (q, J = 5.8 Hz, 1H), 3.56 (dd, J = 9.7, 4.3 Hz, 1H), 3.28 (dd, J = 9.6, 4.4 Hz, 1H), 2.54 - 2.40 (m, 2H), 2.29 (q, J = 4.7 Hz, 1H), 0.87 (ddd, J = 9.4, 4.9, 2.9 Hz, 1H), 0.79 - 0.70 (m, 2H), 0.12 - 0.06 (m, 1H).; 13 13C NMR (101 MHz, CDCl3) δ 155.60, 152.80, 150.24, 140.55, 138.41, 137.96, 128.62, 128.50, 128.01, 127.75, 127.70, 119.46, 80.33, 73.53, 71.49, 68.40, 60.05, 50.46, 38.75, 25.73, 16.37, 8.49. HRMS (FAB) m / z: [M + H]+ calcd for C 27 1 30 H

[0213] [Example 46]

[0214] N-(9-((4S,6S,7R)-6-(benzyloxy)-7-((benzyloxy)methyl)spiro[2.4]heptan-4-yl)-9H-purin-6-yl)benzamide (28). Under a nitrogen atmosphere, N,N-diisopropylethylamine (76 μL, 0.446 mmol) was added to a solution of compound 27 (135 mg, 0.296 mmol) and 4-dimethylaminopyridine (3.6 mg, 0.03 mmol) in anhydrous N,N-dimethylformamide (3.0 mL). Benzoic anhydride (134 mg, 0.594 mmol) was then added, and the mixture was stirred at 100 °C for 2 h. A 2M ammonia-methanol solution (3 mL) was added under ice cooling, and after stirring for 10 min, saturated aqueous sodium bicarbonate (10 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (10 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:1) to give compound 28 (71.5 mg, 43%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 9.11 (br, 1H), 8.74 (s, 1H), 8.46 (s, 1H), 8.06 - 7.99 (m, 2H), 7.83 - 7.77 (m, 1H), 7.64 - 7.55 (m, 1H), 7.55 - 7.50 (m, 3H), 7.47 - 7.40 (m, 1H), 7.40 - 7.23 (m, 10H), 5.03 (dd, J = 7.2, 5.4 Hz, 1H), 4.58 - 4.46 (m, 4H), 4.31 (q, J = 5.9 Hz, 1H), 3.56 (dd, J = 9.7, 4.0 Hz, 1H), 3.38 (dd, J = 9.7, 4.0 Hz, 1H), 2.57 - 2.39 (m, 2H), 2.28 (q, J = 4.5 Hz, 1H), 0.90 (ddd, J = 9.4, 4.7, 2.7 Hz, 1H), 0.83 - 0.69 (m, 2H), 0.13 - 0.04 (m, 1H).; 13C NMR (101 MHz, CDCl3) δ 164.78, 152.36, 152.28, 149.35, 143.25, 138.31, 137.84, 133.82, 132.81, 132.12, 128.93, 128.72, 128.63, 128.52, 128.08, 128.02, 127.98, 127.81, 127.72, 127.43, 122.92, 80.34, 73.54, 71.59, 68.06, 60.61, 50.56, 38.86, 25.57, 16.97, 8.77. HRMS (FAB) m / z: [M + H]+ calcd for C 34 H 34 N5O3, 560.2656; found, 560.2662.

[0215] [Example 47]

[0216]

[0217] N-(9-((4S,6S,7R)-6-hydroxy-7-(hydroxymethyl)spiro[2.4]heptan-4-yl)-9H-purin-6-yl)benzamide (29). Under a nitrogen atmosphere, 1 M boron trichloride (6.3 mL, 6.3 mmol) was added dropwise to a solution of compound 28 (351 mg, 0.627 mmol) in anhydrous dichloromethane (6.3 mL) at −78 °C with stirring. After stirring at the same temperature for 20 min, methanol (10 mL) was added to the reaction solution and stirred for 10 min. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 20:1 to 10:1) to give compound 29 (136 mg, 57%) as a white foamy solid. 11H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H), 8.67 (s, 1H), 8.06 (d, J = 7.5 Hz, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.4 Hz, 2H), 5.07 (t, J = 6.1 Hz, 1H), 4.50 (p, J = 6.1 Hz, 1H), 3.75 (dd, J = 11.3, 4.2 Hz, 1H), 3.61 (dd, J = 11.2, 4.6 Hz, 1H), 2.54 (dt, J = 11.1, 5.1 Hz, 1H), 2.37 (dt, J = 13.8, 6.9 Hz, 1H), 1.98 (p, J = 4.6 Hz, 1H), 0.92 (t, J = 6.8 Hz, 2H), 0.89 - 0.73 (m, 1H), 0.03 (t, J = 6.4 Hz, 1H).; 13 13C NMR (101 MHz, CD3OD) δ 132.72, 128.46, 128.17, 72.53, 61.21, 59.70, 54.22, 48.32, 48.11, 47.89, 47.68, 47.47, 47.25, 47.04, 40.56, 25.38, 16.15, 7.73. HRMS (FAB) m / z: [M + H] + calcd for C 20 H 22 N5O3, 380.1717; found, 380.1723.

[0218] [Example 48]

[0219]

[0220] N-(9-((4S,6S,7R)-7-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-hydroxyspiro[2.4]heptan-4-yl)-9H-purin-6-yl)benzamide (30). Compound 29 (32.2 mg, 0.0849 mmol) was added to anhydrous pyridine solution (1.2 mL) under a nitrogen atmosphere, followed by the addition of 4,4'-dimethoxytrityl chloride (40.7 mg, 0.127 mmol) and stirring at room temperature for 4.5 hours. Saturated aqueous sodium bicarbonate solution (40 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (40 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane=2:1 to dichloromethane / methanol=20:1) to obtain compound 30 (30 mg, 55%) as a yellow foamy solid. 1 H NMR (400 MHz, CD3OD) δ 8.55 (s, 1H), 8.27 (s, 1H), 8.09 - 8.05 (m, 2H), 7.67 - 7.58 (m, 1H), 7.58 - 7.53 (m, 2H), 7.49 - 7.43 (m, 2H), 7.37 - 7.26 (m, 6H), 7.23 - 7.11 (m, 1H), 6.88 - 6.79 (m, 4H), 5.08 - 5.04 (m, 1H), 4.57 (q, J = 5.7 Hz, 1H), 3.74 (s, 6H), 3.24 (d, J = 6.0 Hz, 2H), 2.53 (dt, J = 13.5, 5.8 Hz, 1H), 2.34 (ddd, J = 13.7, 8.0, 6.1 Hz, 1H), 2.23 (q, J = 5.9 Hz, 1H), 0.83 - 0.73 (m, 2H), 0.54 (dt, J = 9.7, 6.0 Hz, 1H), -0.31 (dt, J = 9.9, 5.9 Hz, 1H).; 13C NMR (101 MHz, CD3OD) δ 166.79, 158.78, 152.22, 151.53, 149.59, 145.08, 143.79, 136.01, 135.92, 133.68, 132.56, 129.98, 129.94, 128.42, 128.08, 128.00, 127.49, 126.49, 123.42, 112.77, 86.47, 72.85, 62.42, 60.67, 54.38, 52.64, 40.01, 26.49, 14.78, 6.86. HRMS (FAB) m / z: [M+ H] + calcd for C 41 H 40 N5O5, 682.3024; found, 682.3028.

[0221] [Example 49]

[0222]

[0223] (4R,5S,7S)-7-(6-benzamido-9H-purin-9-yl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)spiro[2.4]heptan-5-yl(2-cyanoethyl) diisopropylphosphoramidite (31) Compound 30 (53.0 mg, 0.078 mmol) and 1H-tetrazole (8.2 mg, 0.012 mmol) were added to a heated reaction vessel, and anhydrous dichloromethane solution (0.78 mL) and DIPEA (20 μL, 0.012 mmol) were added under a nitrogen atmosphere. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (37 μL, 0.012 mmol) was added and the mixture was stirred at room temperature for 1 hour. After confirming the consumption of the starting material by TLC, saturated aqueous sodium bicarbonate (20 mL) was added and the mixture was extracted three times with dichloromethane (20 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:1 → 4 / 1, 1% triethylamine added) to obtain compound 31 (45 mg, 66%) as a white foamy solid. 1H NMR (400 MHz, CDCl3) δ 9.20 (br, 1H), 8.68 - 8.67 (m, 1H), 8.04 - 7.97 (m, 2H), 7.88 - 7.83 (m, 1H), 7.62 - 7.58 (m, 1H), 7.53 - 7.49 (m, 2H), 7.45 - 7.41 (m, 2H), 7.36 - 7.27 (m, 6H), 7.24 - 7.20 (m, 1H), 6.82 (ddd, J = 8.9, 4.3, 1.4 Hz, 4H), 5.06 (dt, J = 8.9, 7.4 Hz, 1H), 4.64 - 4.51 (m, 1H), 3.87 - 3.68 (m, 8H), 3.67 - 3.56 (m, 2H), 3.53 - 3.10 (m, 2H), 2.66 - 2.39 (m, 5H), 1.23 - 1.14 (m, 12H), 0.92 - 0.74 (m, 2H), 0.60 - 0.50 (m, 1H), -0.25 -0.36 (m, 1H).; 31 P NMR (162 MHz, CDCl3) δ 148.15, 147.89. HRMS(FAB) m / z: [M + H] + calcd for C 50 H 57 N7O6P, 882.4102; found, 882.4109.

[0224] [Production of Nucleoside (A)] The synthesis of a phosphoramidite of an entecavir derivative (5-methyl cytosine) by a different route, its introduction into an oligonucleotide, and its functional evaluation will be described in detail below. First, the synthesis scheme of the phosphoramidite is shown below.

[0225]

[0226] [Example 50]

[0227]

[0228] N-(1-((1S,2S,3S,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-hydroxycyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (32). Compound 7 (56.0 mg, 0.181 mmol) was added to anhydrous N,N-dimethylformamide (1.8 mL) under a nitrogen atmosphere, followed by the addition of lithium chloride (15.3 mg, 0.362 mmol) and DBU (54.0 μL, 0.362 mmol). Subsequently, N-benzoyl-5-methylcytosine (83 mg, 0.362 mmol) was added, and the mixture was stirred at 140 °C for 7 h. Saturated aqueous sodium bicarbonate (20 mL) was added, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:2) to give compound 32 (42.4 mg, 43%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 8.30 (dd, J = 8.2, 1.5 Hz, 2H), 7.54 - 7.48 (m, 1H), 7.46 - 7.40 (m, 2H), 7.31 (qd, J = 8.9, 4.4 Hz, 16H), 7.25 (d, J = 0.9 Hz, 4H), 7.17 (d, J = 1.4 Hz, 1H), 4.61 - 4.46 (m, 7H), 4.42 (d, J = 11.8 Hz, 1H), 4.32 (t, J = 8.4 Hz, 1H), 4.18 (s, 1H), 3.93 - 3.87 (m, 1H), 3.78 (dd, J = 9.0, 4.7 Hz, 1H), 3.61 (dd, J = 9.0, 7.4 Hz, 1H), 2.34 - 2.21 (m, 3H), 2.05 (q, J = 1.0 Hz, 3H), 1.28 - 1.22 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 159.94, 149.01, 140.37, 138.03, 137.89, 137.28, 132.51, 129.97, 128.66, 128.58, 128.51, 128.23, HRMS(FAB) m / z: [M + H] + calcd for C 32 H 34 N3O5, 540.2493; found, 540.2498.

[0229] [Example 51]

[0230]

[0231] N-(1-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (33). Compound 32 (56.0 mg, 0.104 mmol) was added to anhydrous dichloromethane (1.0 mL) under a nitrogen atmosphere, and Dess-Martin periodinane (57.3 mg, 0.135 mmol) was added with stirring at 0°C, followed by stirring at room temperature for 3 hours. Aqueous sodium thiosulfate solution (15 mL) and saturated sodium bicarbonate (5 mL) were added with stirring at 0°C, followed by stirring at the same temperature for 20 minutes. The reaction solution was extracted three times with dichloromethane (20 mL). The organic layer was washed with saturated brine, dehydrated over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in anhydrous tetrahydrofuran (1.0 mL) under a nitrogen atmosphere. 0.5 M Tebbe reagent (0.18 mL, 0.091 mmol) was added at 0 °C with stirring, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was quenched by adding 0.1 M aqueous NaOH (20 mL) and stirring for 30 minutes at 0 °C. H2O (20 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (20 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:12 to 1:3) to obtain compound 33 (23.4 mg, 42% over two steps) as a yellow oil. 11H NMR (500 MHz, CDCl3) δ 8.36 - 8.25 (m, 2H), 7.54 - 7.48 (m, 1H), 7.46 - 7.40 (m, 3H), 7.39 - 7.27 (m, 15H), 5.76 (dd, J = 10.4, 7.9 Hz, 1H), 5.30 (t, J = 2.4 Hz, 1H), 5.02 (t, J = 2.5 Hz, 1H), 4.70 (s, 1H), 4.60 - 4.46 (m, 5H), 4.13 (tt, J = 5.5, 2.8 Hz, 1H), 3.82 - 3.72 (m, 2H), 2.93 (s, 1H), 2.40 (ddt, J = 13.2, 8.0, 1.9 Hz, 1H), 2.11 - 2.05 (m, 1H), 1.73 (d, J = 1.1 Hz, 3H).; 13 13C NMR (126 MHz, CDCl3) δ 179.49, 159.81, 149.18, 148.99, 139.85, 138.06, 137.75, 137.31, 132.30, 129.83, 128.59, 128.57, 128.44, 128.10, 128.07, 127.96, 127.68, 127.60, 127.56, 127.53, 126.99, 111.94, 111.60, 80.22, 77.22, 73.64, 72.90, 70.70, 65.41, 58.11, 49.58, 36.77, 13.15. HRMS(FAB) m / z: [M + H] + calculated for C 33 H 34 N3O4, 536.2544; found, 536.2550.

[0232] [Example 52]

[0233]

[0234] N-(1-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (34). Under a nitrogen atmosphere, 1 M boron trichloride (1.23 mL, 1.23 mmol) was added dropwise to a solution of compound 33 (65.9 mg, 0.123 mmol) in anhydrous dichloromethane (1.2 mL) at −40°C with stirring. After stirring at the same temperature for 5 min, methanol (2 mL) was added to the reaction solution and stirred for 1 h. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 20:1 to 10:1) to give compound 34 (38.5 mg, 88%) as a white foamy solid. 1 H NMR (400 MHz, CD3OD) δ 8.26 (d, J = 7.6 Hz, 2H), 7.64 (s, 1H), 7.52 (s, 1H), 7.44 (d, J = 7.7 Hz, 2H), 5.67 (s, 1H), 5.31 (t, J = 2.5 Hz, 1H), 4.99 (s, 1H), 4.33 (d, J = 3.4 Hz, 1H), 3.78 (q, J = 6.7 Hz, 2H), 2.61 (s, 1H), 2.15 (d, J = 10.5 Hz, 2H), 2.06 (s, 3H). 13 C NMR (101 MHz, CD3OD) δ 149.56, 132.30, 130.12, 129.36, 127.93, 112.83, 111.64, 110.38, 71.90, 63.27, 58.90, 54.44, 53.82, 47.49, 47.27, 47.06, 46.42, 38.45, 12.29, 8.02. HRMS (FAB) m / z: [M + H] + calcd for C 19 H 22 N3O4, 356.1605; found, 356.1610.

[0235] [Example 53]

[0236]

[0237] N-(1-((1S,3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylenecyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (35). Compound 34 (38.5 mg, 0.108 mmol) was added to anhydrous pyridine solution (1.1 mL) under a nitrogen atmosphere, followed by the addition of 4,4'-dimethoxytrityl chloride (54.9 mg, 0.162 mmol) and stirring at room temperature for 1 hour. Saturated aqueous sodium bicarbonate solution (10 mL) was added to the reaction solution, which was then extracted three times with ethyl acetate (10 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane=2:1 to dichloromethane / methanol=20:1) to obtain compound 35 (28.7 mg, 40%) as a yellow foamy solid. 1 H NMR (400 MHz, CDCl3) δ 8.32 - 8.26 (m, 2H), 7.54 - 7.47 (m, 1H), 7.46 - 7.37 (m, 5H), 7.33 - 7.26 (m, 7H), 7.11 (d, J = 1.2 Hz, 1H), 6.84 (dd, J = 8.9, 1.4 Hz, 4H), 5.77 (t, J = 9.1 Hz, 1H), 4.97 (t, J = 2.7 Hz, 1H), 4.88 (t, J = 2.6 Hz, 1H), 4.44 (q, J = 4.0 Hz, 1H), 3.79 (s, 7H), 3.61 (dd, J = 9.4, 4.5 Hz, 2H), 3.32 - 3.21 (m, 3H), 2.70 (s, 1H), 2.29 (ddd, J = 12.8, 8.9, 3.6 Hz, 2H), 2.17 (ddd, J = 13.9, 9.1, 5.7 Hz, 1H), 1.96 (s, 3H).; 13C NMR (101 MHz, CDCl3) δ 159.82, 158.77, 149.13, 148.89, 144.52, 139.44, 137.30, 135.74, 135.45, 132.49, 132.14, 130.22, 129.95, 129.23, 128.74, 128.26, 128.20, 128.10, 127.46, 127.20, 113.34, 112.40, 111.50, 87.16, 77.33, 73.94, 65.25, 57.54, 55.37, 52.98, 51.69, 39.68, 34.59, 14.93, 13.15, 8.07. HRMS (FAB) m / z: [M+ H] + calcd for C 40 H 40 N3O6, 658.2912; found, 658.2916.

[0238] [Example 54]

[0239]

[0240] (1S,2R,4S)-4-(4-benzamido-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-methylenecyclopentyl (2-cyanoethyl) diisopropylphosphoramidite (36). Compound 35 (84.0 mg, 0.128 mmol) and 1H-tetrazole (13.5 mg, 0.192 mmol) were added to a heated and dried reaction vessel, and anhydrous dichloromethane solution (1.28 mL) and DIPEA (32.6 μL, 0.192 mmol) were added under a nitrogen atmosphere. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (60.9 μL, 0.192 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After confirming the consumption of the starting material by TLC, saturated aqueous sodium bicarbonate (10 mL) was added and the mixture was extracted twice with dichloromethane (10 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 2:5, 1% triethylamine added) to obtain compound 36 (66.3 mg, 60%) as a white foamy solid. 1H NMR (400 MHz, CDCl3) δ 8.28 (dq, J = 7.2, 1.4 Hz, 2H), 7.58 - 7.46 (m, 1H), 7.46 - 7.34 (m, 5H), 7.25 (s, 14H), 6.87 - 6.79 (m, 5H), 5.79 (t, J = 9.5 Hz, 1H), 5.05 - 4.85 (m, 2H), 4.56 (dd, J = 10.0, 4.6 Hz, 1H), 3.91 - 3.65 (m, 12H), 3.65 - 3.41 (m, 4H), 3.24 (ddd, J = 23.8, 9.3, 4.8 Hz, 1H), 2.86 (d, J = 23.5 Hz, 1H), 2.64 (q, J = 6.9 Hz, 2H), 2.51 (t, J = 6.4 Hz, 1H), 2.48 - 2.30 (m, 1H), 2.24 - 2.11 (m, 1H), 1.61 (d, J = 1.1 Hz, 3H), 1.26 - 1.09 (m, 18H).; 31 P NMR (162 MHz, CDCl3) δ 148.02, 147.75. HRMS(FAB) m / z: [M + H] + calcd for C 49 H 57 N5O7P, 858.3990; found, 858.3996.

[0241] [Example 55] Objective: To measure the double-strand melting temperature (T m ) Measurement Methods and Results: Oligonucleotide Synthesis Various oligonucleotides (internucleotide linkages: all phosphorothioate linkages) containing mPCS2 as the parent sequence, as shown in Table 12 below, were synthesized in the same manner as in Example 2, and were subjected to the following evaluations. A entecavir, E A3 indicates a 4'-spiro form. A3 are the compounds synthesized in Examples 43 to 49 above.

[0242]

[0243] (2) Double-strand melting temperature (Tm ) Measurement: A sample solution (150 μL) containing 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, and 4 μM of the oligonucleotides and complementary strand RNA listed in Table 12 was heated to 95°C for 3 minutes, then gradually cooled to 20°C at a rate of 1°C per minute to allow annealing, after which measurement was initiated. The temperature was raised to 95°C at a rate of 0.5°C per minute, and the absorbance at 260 nm was plotted at 1°C intervals. T m All values ​​were calculated using the midline method. The measurement results are shown in Table 13 below.

[0244]

[0245] Discussion: Based on the above, oligonucleotides incorporating the nucleoside analogs described in this invention could be synthesized using standard methods. Furthermore, although the thermodynamic stability of the complementary strand varied slightly depending on the analog, the binding strength was not significantly impaired. Previously reported unsubstituted carbocyclic nucleotides (Org. Lett. 2019, 21, 7, 1963-1967) have been shown to significantly impair the binding strength of the complementary RNA strand. Therefore, suppressing conformational fluctuations in the cyclopentane ring due to sp2 and spiro carbons is thought to contribute to maintaining high binding strength. Furthermore, it is suggested that thermodynamic stability can be finely tuned by the type of substituents extending out from the ring.

[0246] [Example 56] Objective: Evaluation of the stability of oligonucleotides containing an entecavir analogue introduced into the chain against 3'-exonuclease Methods and Results: Synthesis of oligonucleotides Various oligonucleotides (internucleotide linkages: all phosphodiester linkages) listed in Table 14 below were synthesized in the same manner as in Example 2 and subjected to the following evaluations. In the table, mC represents 2'-deoxy-5-methylcytidine, E mC indicates a 5-methylcytidine derivative of carbocyclic DNA. Evaluation of the stability of oligonucleotides against 3'-exonucleases Entecavir analogues (E mCAntisense oligonucleotides (2.7 nmol) containing the 3'-terminally modified nucleotide (Table 14) were mixed in a buffer solution [10 mM MgCl2, 50 mM Tris-HCl (pH 8.0)] with 0.003 units of Crotalus admanteus venom phosphodiesterase (CAVP) (Worthington Biochemical) and incubated at 37°C. Samples were taken at 5, 15, 30, 45, and 60 minutes of reaction time and heated to 90°C for 2 minutes. The time course of the full-length oligonucleotides was then analyzed by reverse-phase HPLC. The HPLC conditions are as follows: (Eluent) Solution A: 100 mM hexafluoro-2-propanol, 8.6 mM triethylamine (pH 8.36) Solution B: Methanol (Gradient) Solution B concentration: 0-30% (10 min) (Column) 1) YMC Accura Triart Bio C18, 5.0 μm (4.6 A x 50 mm) 2) Column temperature: 60°C (Flow rate): 1.0 mL / min (Detection): UV (260 nm) (Oligonucleotides evaluated) The sequences of the oligonucleotides evaluated are shown in Table 14 below.

[0247]

[0248] In the table, mC represents 5-methylcytosine DNA, E mC indicates an entecavir analogue with 5-methylcytosine as the base. (2) The area ratio (%) relative to the unreacted oligonucleotide is shown in the table and graph (Table 15, Figure 26).

[0249]

[0250] Discussion: From the figure, the parent chain 3'-dmC was completely decomposed within 5 minutes of nuclease treatment, whereas the nucleoside (E mC In the case of 3'-eneNA(mC) containing the DNA analog, over 40% remained unreacted even after 60 minutes of nuclease treatment, demonstrating high enzyme resistance. Oligonucleotides incorporating this DNA analog can confer metabolic stability to nucleic acids due to the effect of the substituents overhanging the outer ring.

[0251] Example 57 Objective: To evaluate the in vitro inhibitory effect of oligonucleotide drugs incorporating carbocyclic nucleosides with spiro ring structures in their chains. Methods and Results: The ASOs listed in Table 16 were diluted to a final concentration of 1 μM in cell culture medium (supplemented with 9 mM CaCl2) and added to a 96-well plate. A well without ASO was prepared as a negative control. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 24 hours. cDNA was prepared from the cell lysate using the SuperPrep® II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's protocol. Human PCSK9 mRNA expression was analyzed using a QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Human Pcsk9 was analyzed using TaqMan Gene Expression Assay (FAM) Assay ID: Hs00545399_m1 (Thermo Fisher Scientific). Human Gapdh, a housekeeping gene, was analyzed using TaqMan Gene Expression Assay (VIC primer-limited) Assay ID: Hs02758991_g1 (Thermo Fisher Scientific). KD activity was calculated based on relative expression levels, where the difference in Ct values ​​was converted to the difference in expression levels. Significance was determined by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. **p < 0.01, ***p < 0.001. The results are shown in Figure 27.

[0252]

[0253] Internucleotide linkage: All phosphorothioate linkages E in the table A3 indicates the 4'-spiro form of entecavir. Discussion: As can be seen from the figure, gapA3 significantly suppressed the expression of the target gene. These results indicate that nucleic acid drugs in which some monomers in the nucleic acid drug chain have been replaced with carbocyclic nucleosides with spiro ring structures, or which contain carbocyclic nucleosides with spiro ring structures in the chain, retain their activity.

[0254] Example 58 Objective: To evaluate the cytotoxicity (cell viability) of oligonucleotide drugs incorporating carbocyclic nucleosides with spiro ring structures. Methods and Results: The ASOs listed in Table 16 of Example 57 were diluted in cell culture medium (supplemented with 9 mM CaCl2) to final concentrations of 0.001 μM to 3 μM and added to a 96-well plate. A negative control well was prepared without ASO. HuH-7 cells diluted with the above medium were seeded at 10,000 cells per well and cultured in an incubator for 72 hours. The medium was then replaced with 110 μL of Cell Counting Kit-8 (Dojindo Laboratories) (10 μL / well) and incubated in the incubator for 2 hours. Absorbance was measured at 450 nm and at 650 nm (reference wavelength) using a microplate reader. After subtracting each absorbance value at 450 nm by the absorbance value at 650 nm, cell viability was calculated by subtracting the background value from the control or assay value. The IC50 value of each ASO was calculated using a four-parameter logistic model based on the obtained cell viability. The evaluation results are shown in Figure 28. Discussion: As can be seen from the figure, gapA3 exhibited significantly reduced cytotoxicity compared to the parent chain mPCS2, and its IC 50 The cytotoxicity value for the parent chain was 0.0391 nM, while for gapA3 it was 0.290 nM, a difference of approximately 7-fold. These results demonstrate that nucleic acid drugs containing carbocyclic nucleosides with spiro ring structures in the chain, or nucleic acid drugs obtained by replacing some monomers in the nucleic acid drug chain with carbocyclic nucleosides with spiro ring structures, are useful for obtaining nucleic acid drugs with reduced cytotoxicity.

[0255] According to one embodiment of the present invention, there is provided an oligonucleotide useful as a medicine, which exhibits excellent effects on target RNA etc. while maintaining high safety.

[0256] This application is based on patent application No. 2023-187759 filed in Japan (filing date: November 1, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. An oligonucleotide or a salt thereof, comprising, in the oligonucleotide sequence, a carbocyclic nucleoside derivative residue (B) ("nucleoside residue (B)"), which is a divalent group represented by the following formula (B): [wherein Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group, each of which may have one or more optional substituents selected from substituent group (a), wherein substituent group (a) consists of a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an oxo group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom (wherein, when the purin-9-yl group or the 2-oxo-1,2-dihydropyrimidin-1-yl group has an oxo group as a substituent selected from substituent group (a), the bond between the carbon atom to which the oxo group is bonded and the adjacent atom is a single bond); R ３ and R ４ each represents a hydrogen atom; ５ represents a hydrogen atom; The group represented by the following partial structural formula: (i-1) or (i-2): (In the formula, R ６ and R ７ each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms, or R ６ and R ７ are bonded to each other to form a carbon ring having 3 to 6 carbon atoms together with adjacent carbon atoms, R ８ , R ９ , R １０ and R １１ each independently represents a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms; * represents the position of attachment to the adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 5'-terminus of the oligonucleotide sequence, R １ : (where R １ is a hydrogen atom, a protecting group for a hydroxyl group in nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms which may form a branched or cyclic group, an alkenyl group having 2 to 7 carbon atoms which may form a branched or cyclic group, an aryl group having 3 to 10 carbon atoms which may have one or more optional substituents selected from the substituent group (a) and which may contain a heteroatom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may have one or more optional substituents selected from the substituent group (a) and which may contain a heteroatom, an acyl group which may have one or more optional substituents selected from the substituent group (a), a silyl group which may have one or more optional substituents selected from the substituent group (a), a phosphate group which may have one or more optional substituents selected from the substituent group (a), a phosphate group protected by a protecting group in nucleic acid synthesis, -P(R １２ ) R １３ (In the formula, R １２ and R １３ each independently represents a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a dialkylamino group having an alkyl group having 1 to 6 carbon atoms); and, ** represents the bonding position to the adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, R ２ : (where R ２ is the R １ and n is an integer of 1 to 5. The oligonucleotide or salt thereof, 2. The nucleoside residue (B) has the partial structural formula (i): is represented by the following partial structural formula: (i-1): (In the formula, R ６ and R ７ The oligonucleotide or salt thereof according to claim 1 , wherein the aryl group is a group represented by the following formula:

3. The nucleoside residue (B) has the partial structural formula (i): The group represented by the following partial structural formula: (i-2): (In the formula, R ８ , R ９ , R １０ and R １１ The oligonucleotide or salt thereof according to claim 1 , wherein R 1 is a group represented by the following formula (I):

4. The oligonucleotide or salt thereof according to any one of claims 1 to 3, which comprises 1 to 10 nucleoside residues (B) in the oligonucleotide sequence.

5. The oligonucleotide or a salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide has a length of 7 to 30 bases.

6. The oligonucleotide or a salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide has a length of 10 to 20 bases.

7. The oligonucleotide or salt thereof according to any one of claims 1 to 3, which has reduced toxicity compared to before introduction of the nucleoside residue (B).

8. The oligonucleotide or salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide is a gapmer consisting of a gap region of 2 to 14 bases in length, a 5' wing region of 2 to 5 bases in length, and a 3' wing region of 2 to 5 bases in length, and the gap region is located between the 5' wing region and the 3' wing region.

9. The oligonucleotide or salt thereof according to claim 8, wherein the gap region comprises at least one nucleoside residue (B).

10. The oligonucleotide or salt thereof according to claim 9, wherein the 5' wing region and / or the 3' wing region comprises at least one nucleoside residue (B).

11. The oligonucleotide or salt thereof according to any one of claims 1 to 3, wherein at least one of the internucleotide linkages in the oligonucleotide is a phosphorothioate linkage.

12. The oligonucleotide or salt thereof according to any one of claims 1 to 3, wherein all of the internucleotide linkages in the oligonucleotide are phosphorothioate linkages.

13. The oligonucleotide or salt thereof according to any one of claims 1 to 3, wherein in the nucleoside residue (B), Base represents a purine-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group, each of which may have 1 to 3 optional substituents selected from substituent group (a), wherein substituent group (a) consists of a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, an oxo group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected by a protecting group for nucleic acid synthesis, and a halogen atom; 14. In the nucleoside residue (B), R ６ and R ７ The oligonucleotide or salt thereof according to claim 13, wherein: * represents a bonding position to an adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, represents a hydrogen atom; and ** represents a bonding position to an adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, represents a hydrogen atom.

15. In the nucleoside residue (B), R ８ , R ９ , R １０ and R １１ The oligonucleotide or salt thereof according to claim 13, wherein each of the symbols represents a hydrogen atom; * represents a bonding position to an adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, represents a hydrogen atom; and ** represents a bonding position to an adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, represents a hydrogen atom.

16. Use of the oligonucleotide or a salt thereof according to any one of claims 1 to 3 as an antisense oligonucleotide or an oligonucleotide constituting siRNA.

17. A method for reducing the toxicity of an oligonucleotide or its salt, comprising: a carbocyclic nucleoside derivative residue (B) ("nucleoside residue (B)"), which is a divalent group represented by the following formula (B), in the oligonucleotide sequence: (wherein each group and partial structure has the same meaning as the corresponding group and partial structure defined for "nucleoside residue (B)" in claim 1), 18. Use of a carbocyclic nucleoside derivative represented by the following formula (A) ("nucleoside (A)") or a salt thereof for reducing the toxicity of an oligonucleotide: (In the formula, each group and partial structure has the same meaning as the corresponding group and partial structure defined for "nucleoside residue (B)" in claim 1, respectively.) 19. The use according to claim 18, wherein the reduction of the toxicity of an oligonucleotide comprises the introduction of at least one nucleoside (A) into the oligonucleotide sequence.

20. A medicine comprising, as an active ingredient, the oligonucleotide or a salt thereof according to any one of claims 1 to 3.

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