Oligonucleotides containing carbon ring nucleosides

Abstract

In the present application, as one embodiment of the invention, an oligonucleotide or a salt thereof is disclosed, characterized in that, in the sequence of the oligonucleotide, at least one carbocyclic nucleoside derivative residue of a divalent group represented by the following formula (B) (in the formula, each symbol is consistent with the description record.) is contained, the oligonucleotide or the salt thereof shows excellent effects on a target RNA or the like while maintaining high safety.

Description

Technical Field

[0001] This invention provides oligonucleotides containing carbocyclic nucleosides as embodiments thereof. More specifically, it provides an oligonucleotide containing a carbocyclic nucleoside, which, by introducing the oligonucleotide, can increase the maximum tolerated dose of the oligonucleotide in an organism or improve the therapeutic index, for example, being effective in the pharmaceutical field. Background Technology

[0002] In recent years, the research and development of nucleic acid drugs has flourished. Nucleic acid drugs are drugs with natural or chemically modified nucleotides as their basic backbone. These chemically synthesized nucleic acids can directly act on organisms. Besides their high specificity based on base pairing, nucleic acid drugs can target molecules that were previously inaccessible to other drugs, such as mRNA and non-coding RNA. They also have the advantage of being easily standardized in a relatively short time once the platform is built. Therefore, nucleic acid drugs are expected to become the next generation of drugs after small molecule drugs and antibody drugs.

[0003] Based on their mechanisms of action, these nucleic acid drugs exist in various types, including antisense nucleic acids (RNase H-mediated, sterically hindered, splice-regulated, RNA-editing, etc.), siRNA, aptamers, and antigenic nucleic acids. For example, antisense nucleic acids and siRNA act on RNA in the body, exerting their effects by inhibiting or regulating its function. Aptamers exert their effects by utilizing their higher-order structures and binding to targets such as proteins. In addition, antigenic nucleic acids are expected to exert their effects by acting on genomic DNA. Due to these diverse mechanisms of action, applications are being developed for many genetic and intractable diseases, such as neurodegenerative diseases, metabolic diseases, cancer, and infectious diseases (see Non-Patent Literature 1-3).

[0004] Existing technical documents Non-patent literature Non-patent literature 1: Stanley T. Crooke, Xue-Hai Liang, Brenda F. Baker,Rosanne M. Crooke, Antisense technology: A review, Journal of BiologicalChemistry, Volume 296, 2021, 100416, https: / / doi.org / 10.1016 / j.jbc.2021.100416 Non-patent document 2: Guillermo Aquino-Jarquin, Novel Engineered ProgrammableSystems 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 Zhong Non-patent literature 3: The growth of siRNA-based therapeutics: Updated clinicalstudies, Biochemical Pharmacology, Volume 189, 2021, 114432 Non-patent document 4: Guideline 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.html Non-patent document 5: Terada C, Kawamoto S, Yamayoshi A, Yamamoto T. Chemistryof Therapeutic Oligonucleotides That Drives Interactions with Biomolecules.Pharmaceutics. 2022 Nov 29;14(12):2647. Non-patent literature 6: 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. Summary of the Invention

[0005] The technical problem that the invention aims to solve However, as mentioned above, conventional oligonucleotide therapeutics have safety issues such as side effects and toxicity caused by non-specific interactions (off-target effects) with proteins or RNA that are not therapeutic targets in the body (see Non-Patent Literature 4-6). The technical problem of the present invention is to solve the problems of the prior art and provide an oligonucleotide therapeutic that increases the maximum tolerated dose or improves the therapeutic index.

[0006] Solution to the technical problem This invention relates to a DNA analogue, such as entecavir, known to have antiviral activity, having a carbon ring 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 in which the oxygen atom is replaced by a spirocyclic structure, i.e., the DNA analogue has a structure in which the oxygen atom at the 4' position is replaced by an sp2 carbon or a spirocyclic carbon. In the case of introducing entecavir or its derivatives into oligonucleotide therapeutics, the removal of the 4' oxygen atom, which is crucial for nucleic acid recognition typically induced by enzymes, and the introduction of a large exo-olefin structure that can sterically hinder interactions, can reduce interactions with non-specific proteins (hybridization-independent interactions), which are known as the main side effect mechanism of oligonucleotide therapeutics. Simultaneously, it may reduce hybridization-dependent toxicity by affecting the activity of key enzymes such as RNase H and RISC (RNA-Induced Silencing Complex) and ADAR (RNA-specific adenosine deaminase). Under this novel concept, the inventors of this application conducted in-depth research to solve the aforementioned technical problems and discovered that by introducing the carbocyclic nucleoside derivative (A) represented by formula (A) (hereinafter also referred to as "nucleoside (A)") into the nucleotide sequence constituting the oligonucleotide, the toxicity of the oligonucleotide (e.g., antisense oligonucleotide) is reduced compared to before introduction, thus completing this invention.

[0007]

[0008] In the formula, each group and each part of the structure have the same meaning as the corresponding group and part of the structure defined for the carbon-cyclic nucleoside derivative residues represented by formula (B) in [1] below.

[0009] The following specific embodiments are provided to illustrate the present invention. However, the present invention is not limited to these embodiments.

[0010] [1] An oligonucleotide (hereinafter also referred to as "oligonucleotide (I)") or a salt thereof, characterized in that, The oligonucleotide sequence contains at least one carbocyclic nucleoside derivative residue (B) as a divalent group represented by the following formula (B) (hereinafter also referred to as "nucleoside residue (B)").

[0011] [In the formula, Base represents either purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl, each optionally having one or more substituents selected from substituent group (a). The substituent group (a) includes hydroxyl, hydroxyl protected by a nucleic acid synthesis protecting group, oxo group, straight-chain alkyl with 1 to 6 carbon atoms, straight-chain alkoxy with 1 to 6 carbon atoms, mercapto, mercapto protected by a nucleic acid synthesis protecting group, straight-chain alkylthio with 1 to 6 carbon atoms, amino, straight-chain alkylamino with 1 to 6 carbon atoms, amino protected by a nucleic acid synthesis protecting group, and halogen atom (here, when purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl has an oxo group as a substituent selected from substituent group (a), the bond between the carbon atom bonded by the oxo group and the adjacent atom is a single bond). R3 and R4 represent hydrogen atoms, respectively; R5 represents a hydrogen atom; The groups shown in partial structural formula (i) below represent the groups shown in partial structural formula (i-1) or (i-2) below.

[0012]

[0013] (In the formula, R6 and R7 each independently represent a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms, or R6 and R7 combine with each other to form a carbon ring with 3 to 6 carbon atoms. R8, R9, R 10 and R 11 Each of the following can be independently represented as a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms. * indicates a binding site on an adjacent oligonucleotide moiety, or R1 indicates when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence: (Where R1 represents a hydrogen atom, a protecting group of a hydroxyl group from nucleic acid synthesis, an alkyl group optionally forming a branched chain or ring with 1 to 7 carbon atoms, an alkenyl group optionally forming a branched chain or ring with 2 to 7 carbon atoms, an aryl group optionally having one or more substituents selected from substituent group (a) and optionally containing heteroatoms with 3 to 10 carbon atoms, an aralkyl group optionally having one or more substituents selected from substituent group (a) and optionally containing heteroatoms with 3 to 12 carbon atoms, an acyl group optionally having one or more substituents selected from substituent group (a), a silyl group optionally having one or more substituents selected from substituent group (a), a phosphate group optionally having one or more substituents selected from substituent group (a), a phosphate group protected by a protecting group from nucleic acid synthesis, -P(R) 12 R 13 (where R) 12 and R 13 Each of the following groups independently represents a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, a thiol group, a thiol group protected by a nucleic acid synthesis protecting group, 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, ** indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, R2 indicates: (Here, R2 has the same meaning as R1 above).

[0014] [2] The oligonucleotide or its salt as described in [1] above, wherein, In nucleoside residue (B), The group shown in partial structural formula (i) is the same group shown in the following partial structural formula (i-1).

[0015]

[0016] (In the formula, R6 and R7 have the same meaning as described above).

[0017] The specific structure of the nucleoside residue (B) in this section is shown below.

[0018]

[0019] [3] The oligonucleotide or its salt as described in [1] above, wherein, In nucleoside residue (B), The group shown in partial structural formula (i) is the same group shown in the following partial structural formula (i-2).

[0020]

[0021] (In the formula, R8, R9, R 10 and R 11 (These have the same meaning as the above).

[0022] The specific structure of the nucleoside residue (B) in this section is shown below.

[0023]

[0024] [4] An oligonucleotide or a salt thereof as described in any one of [1] to [3] above, wherein, This oligonucleotide sequence contains 1 to 10 nucleoside residues (B).

[0025] [5] An oligonucleotide or a salt thereof as described in any one of [1] to [4] above, wherein, Oligonucleotides are 7 to 30 bases in length.

[0026] [6] An oligonucleotide or a salt thereof as described in any one of [1] to [5] above, wherein, Oligonucleotides are 10 to 25 bases in length.

[0027] [7] An oligonucleotide or a salt thereof as described in any one of [1] to [6] above, wherein, The toxicity (e.g., hepatotoxicity and / or weight loss) was reduced compared to before the introduction of nucleoside residues (B).

[0028] [8] An oligonucleotide or a salt thereof as described in any one of [1] to [7] above, wherein, Oligonucleotides are gapmers consisting of a 2-14 base length interstitial region, a 2-5 base length 5' wing region, and a 2-5 base length 3' wing region. The gap region is located between the 5' wing region and the 3' wing region.

[0029] [9] The oligonucleotide or its salt as described in [8] above, wherein, The interstitial region contains at least one nucleoside residue (B).

[0030]

[10] An oligonucleotide or a salt thereof as described in [8] or [9] above, wherein, The 5' wing region and / or the 3' wing region contain at least one nucleoside residue (B).

[0031]

[11] An oligonucleotide or a salt thereof as described in any one of [1] to

[10] above, wherein, At least one of the nucleotide linkages in an oligonucleotide is a phosphate thioester linkage.

[0032]

[12] An oligonucleotide or a salt thereof as described in any one of [1] to

[11] above, wherein, In oligonucleotides, all nucleotide linkages are phosphate thioester linkages.

[0033]

[13] An oligonucleotide or a salt thereof as described in any one of [1] to

[12] above, wherein, In nucleoside residue (B), Base represents either purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl, each of which may be selected to have 1 to 3 substituents selected from substituent group (a). The substituent group (a) includes a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an oxo group, a straight-chain alkyl group with 1 to 6 carbon atoms, a straight-chain alkoxy group with 1 to 6 carbon atoms, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, a straight-chain alkyl thio group with 1 to 6 carbon atoms, an amino group, a straight-chain alkyl amino group with 1 to 6 carbon atoms, an amino group protected by a nucleic acid synthesis protecting group, and a halogen atom.

[0034]

[14] An oligonucleotide or a salt thereof as described in any one of [1], [2], [4] to

[13] above, wherein, In nucleoside residue (B), R6 and R7 represent hydrogen atoms; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, it indicates a hydrogen atom; and, ** indicates a binding site on an adjacent oligonucleotide component, or a hydrogen atom when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence.

[0035]

[15] An oligonucleotide or a salt thereof as described in any one of [1], [3] to

[13] above, wherein, In nucleoside residue (B), R8, R9, R 10 and R 11 They represent hydrogen atoms respectively; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, it indicates a hydrogen atom; and, ** indicates a binding site on an adjacent oligonucleotide component, or a hydrogen atom when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence.

[0036]

[16] The use of any one of the oligonucleotides or their salts described in any one of [1] to

[15] above as antisense oligonucleotides or oligonucleotides constituting siRNA.

[0037]

[17] A method for reducing the toxicity of an oligonucleotide or its salt, characterized in that, In this oligonucleotide sequence, at least one carbocyclic nucleoside derivative residue (B) representing a divalent group as shown in formula (B) (“nucleoside residue (B)”) is introduced.

[0038] (In the formula, each group and each part of the structure have the same meaning as the corresponding group defined in the above [1] regarding "nucleoside residue (B)").

[0039]

[18] Use of a carbocyclic nucleoside derivative (“nucleoside (A)”) or a salt thereof, represented by formula (A), in reducing oligonucleotide toxicity.

[0040] (In the formula, each group and each part of the structure have the same meaning as the corresponding group defined in the above [1] regarding "nucleoside residue (B)").

[0041]

[19] As described in

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

[0042]

[20] A drug containing, as an active ingredient, any one of [1] to

[16] above-mentioned oligonucleotides or their salts.

[0043] Invention Effects As one embodiment, the present invention provides an oligonucleotide that exhibits excellent effects on target RNA while maintaining high safety. Attached Figure Description

[0044] Figure 1 The evaluation results of the target gene expression inhibition effect in Example 5 below are shown.

[0045] Figure 2 The results of the cytotoxicity (cell viability) evaluation in Example 6 below are shown.

[0046] Figure 3 The results of the evaluation of cytotoxicity (Caspase 3 / 7 activity) in Example 7 below are shown.

[0047] Figure 4 The evaluation results of the target gene expression inhibition effect in Example 9 below are shown.

[0048] Figure 5 The evaluation results of weight change in Example 10 below are shown.

[0049] Figure 6 The evaluation results of ALT in Example 10 below are shown.

[0050] Figure 7 The evaluation results of total bilirubin in Example 10 below are shown.

[0051] Figure 8 The evaluation results of direct bilirubin in Example 10 below are shown.

[0052] Figure 9 The evaluation results of indirect bilirubin in Example 10 below are shown.

[0053] Figure 10 The evaluation results of the target gene expression inhibition effect in Example 12 below are shown.

[0054] Figure 11 The evaluation results of ALT in Example 13 below are shown.

[0055] Figure 12 The evaluation results of total bilirubin in Example 13 below are shown.

[0056] Figure 13 The evaluation results of direct bilirubin in Example 13 below are shown.

[0057] Figure 14 The evaluation results of indirect bilirubin in Example 13 below are shown.

[0058] Figure 15 The evaluation results of the target gene expression inhibition effect in Example 15 below are shown.

[0059] Figure 16 The results of the cytotoxicity (cell viability) evaluation in Example 16 below are shown.

[0060] Figure 17 shows the evaluation results of cytotoxicity (cell viability) in Example 17 below.

[0061] Figure 18 The results of the cytotoxicity (cell viability) evaluation in Example 19 below are shown.

[0062] Figure 19 The evaluation results of the target gene expression inhibition effect in Example 20 below are shown.

[0063] Figure 20 The evaluation results of weight change in Example 21 below are shown.

[0064] Figure 21 The evaluation results of ALT in Example 21 below are shown.

[0065] Figure 22 The evaluation results of AST in Example 21 below are shown.

[0066] Figure 23 The evaluation results of total bilirubin in Example 21 below are shown.

[0067] Figure 24 The results show the evaluation of the double-strand forming ability of siRNA with an entecavir derivative introduced into the antisense strand in Example 41 below.

[0068] Figure 25 The results show the evaluation of the in vitro target gene expression inhibition effect of siRNA with entecavir derivative introduced into the AS chain in Example 42 below.

[0069] Figure 26 The results of the stability evaluation in Example 56 below are shown.

[0070] Figure 27 The evaluation results of the in vitro target gene expression inhibition effect of the oligonucleotide drug in Example 57 below are shown.

[0071] Figure 28 The results of the evaluation of the cytotoxicity (cell viability) of the oligonucleotide drug in Example 58 below are shown. Detailed Implementation

[0072] The invention will be described in detail below. Unless otherwise stated herein, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Any methods and materials identical or equivalent to those described herein may be used in the practice or testing of this invention, with preferred methods and materials described below. All publications and patents referenced in this specification, such as those describing and disclosing constructs and methodologies described in related publications applicable to the invention, are incorporated herein by reference.

[0073] [About Oligonucleotides (I)] According to one embodiment of the present invention, it provides "[1] An oligonucleotide or a salt thereof, characterized in that, The oligonucleotide sequence contains at least one carbocyclic nucleoside derivative residue (B) as a divalent group represented by the following formula (B) (“nucleoside residue (B)”).

[0074] (In the formula, each group and each part of the structure have the same meaning as the corresponding group and part of the structure defined in item [1] of the above [Solution to the technical problem]). The oligonucleotide (I) will be described in detail below.

[0075] As described above, the oligonucleotide (I) consists of (1) nucleoside residues (B) and (2) nucleosides (or nucleoside derivatives) selected according to the target oligonucleotide sequence.

[0076] (1) Regarding nucleoside residues (B) First, a detailed description of "nucleoside residue (B)" will be provided. The definitions of each group of nucleoside residue (B) are as described above, and their preferred manner is as follows.

[0077] In this specification, when the expressions “Ca-b” (e.g., C1-6) or “Ca-Cb” (e.g., C1-C6) are used, it indicates that the number of carbon atoms constituting the group is a~b (e.g., 1~6).

[0078] In this specification, "a straight-chain alkyl group having 1 to 6 carbon atoms" can be listed as a straight-chain alkyl group having 1 to 6 carbon atoms, specifically, examples include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. On the other hand, "an alkyl group having 1 to 6 carbon atoms" can be listed as a straight-chain, branched, or cyclic alkyl group having 1 to 6 carbon atoms, specifically, in addition to the above, examples include branched alkyl groups such as isopropyl, isobutyl, tert-butyl, and isopentyl, and any cyclic alkyl group having 3 to 6 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0079] In this specification, "a straight-chain alkoxy group having 1 to 6 carbon atoms" can be listed as a straight-chain alkoxy group having 1 to 6 carbon atoms, specifically, examples include methoxy, ethoxy, and n-propoxy. On the other hand, "an alkoxy group having 1 to 6 carbon atoms" can be listed as any straight-chain, branched, or cyclic alkoxy group having 1 to 6 carbon atoms, in addition to the above, examples include branched alkoxy groups such as isopropoxy, isobutoxy, tert-butoxy, and isopentoxy, and any cyclic alkoxy group having 3 to 6 carbon atoms such as cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy.

[0080] Furthermore, "a straight-chain alkoxy group having 1 to 6 carbon atoms that is optionally substituted with a straight-chain alkoxy group having 1 to 6 carbon atoms" refers to the aforementioned "straight-chain alkoxy group having 1 to 6 carbon atoms" and alkoxy groups in which one or more hydrogen atoms constituting the "straight-chain alkoxy group having 1 to 6 carbon atoms" are substituted with other "straight-chain alkoxy groups having 1 to 6 carbon atoms" of the same or different nature. Examples of such "straight-chain alkoxy groups having 1 to 6 carbon atoms that is optionally substituted with a straight-chain alkoxy group having 1 to 6 carbon atoms" include, for example, methoxy, ethoxy, n-propoxy, methoxymethoxy, ethoxymethoxy, n-propoxymethoxy, methoxyethoxy (e.g., 2-methoxyethoxy), ethoxyethoxy (e.g., 2-ethoxyethoxy), and n-propoxyethoxy.

[0081] In this specification, "cyanoalkoxy group having 1 to 6 carbon atoms" can be exemplified as a group in which at least one hydrogen atom of any straight-chain, branched or cyclic alkoxy group having 1 to 6 carbon atoms is replaced by a cyano group.

[0082] In this specification, "a straight-chain alkyl thio group having 1 to 6 carbon atoms" can be exemplified as an alkyl thio group having any straight-chain alkyl group having 1 to 6 carbon atoms. Examples include methyl thio, ethyl thio, and n-propyl thio. On the other hand, "an alkyl thio group having 1 to 6 carbon atoms" can be exemplified as any straight-chain, branched, or cyclic alkyl thio group having 1 to 6 carbon atoms.

[0083] In this specification, "a straight-chain alkylamino group having 1 to 6 carbon atoms" includes amino groups having one or two straight-chain alkyl groups having 1 to 6 carbon atoms. Examples include methylamino, dimethylamino, ethylamino, and diethylamino.

[0084] In this specification, the term "alkyl group having 1 to 7 carbon atoms that optionally form branches or rings" can include any straight-chain 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. It is sometimes simply referred to as "C1-7 alkyl group". Examples of straight-chain alkyl groups having 1 to 7 carbon atoms include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and n-heptyl; examples of branched alkyl groups having 3 to 7 carbon atoms include isopropyl, isobutyl, tert-butyl, and isopentyl; and examples of cyclic alkyl groups having 3 to 7 carbon atoms include cyclobutyl, cyclopentyl, and cyclohexyl.

[0085] In this specification, "alkenyl groups having 2 to 7 carbon atoms that optionally form branches or rings" includes any straight-chain 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 is sometimes simply referred to as "C1-7 alkenyl group". Examples of straight-chain alkenyl groups with 2 to 7 carbon atoms include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, and 1-hexenyl. Examples of branched alkenyl groups with 3 to 7 carbon atoms include isopropenyl, 1-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, and 1-methyl-2-butenyl. Examples of cyclic alkenyl groups with 3 to 7 carbon atoms include cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0086] In this specification, cyclopropane, cyclobutane, cyclopentane, and cyclohexane may be listed as examples of "carbon rings with 3 to 6 carbon atoms".

[0087] In this specification, "aryl groups with 3 to 10 carbon atoms that optionally contain heteroatoms" can be categorized as any aryl group consisting solely of hydrocarbons with 6 to 10 carbon atoms, and any heteroaryl group with 3 to 12 carbon atoms in which at least one carbon atom constituting the aryl ring structure is replaced by a heteroatom (e.g., nitrogen, oxygen, and sulfur atoms, or combinations thereof). Examples of aryl groups with 6 to 10 carbon atoms include phenyl, naphthyl, indole, and azulel, and examples of heteroaryl groups with 3 to 12 carbon atoms include pyridyl, pyrroleyl, quinolinyl, indoleyl, imidazolyl, furanyl, and thiopheneyl.

[0088] In this specification, examples of "aralkyl groups having an aryl moiety having an optional heteroatom containing 3 to 12 carbon atoms" include benzyl, phenethyl, naphthylmethyl, 3-phenylpropyl, 2-phenylpropyl, 4-phenylbutyl, 2-phenylbutyl, pyridylmethyl, indolylmethyl, furanylmethyl, thienylmethyl, pyrroleylmethyl, 2-pyridylethyl, 1-pyridylethyl, 3-thienylpropyl, etc.

[0089] In this specification, aliphatic acyl groups and aromatic acyl groups may be listed as "acyl groups".

[0090] Specifically, examples of aliphatic acyl groups include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3... Alkyl carbonyl groups such as 7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecananoyl, 14-methylpentadecananoyl, 13,13-dimethyltetradecanoyl, heptadecananoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecananoyl, nonadecananoyl, eicosanoyl, and dodecanoyl; carboxylated alkyl carbonyl groups such as succinoyl, glutaryl, and adipyl; halogenated C1-6 alkyl carbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl, and trifluoroacetyl; C1-6 alkoxy-C1-6 alkyl carbonyl groups such as methoxyacetyl; and unsaturated hydrocarbon carbonyl groups such as (E)-2-methyl-2-butenoyl.

[0091] In addition, examples of aromatic acyl groups include aryl carbonyl groups such as benzoyl, α-naphthoyl, and β-naphthoyl; haloaryl carbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl; C1-6 alkylated aryl carbonyl groups such as 2,4,6-trimethylbenzoyl and 4-toluyl; C1-6 alkoxylated aryl carbonyl groups such as 4-anisanoyl; carboxylated aryl carbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, and 4-carboxybenzoyl; nitrated aryl carbonyl groups such as 4-nitrobenzoyl and 2-nitrobenzoyl; C1-6 alkoxylated aryl carbonyl groups such as 2-(methoxycarbonyl)benzoyl; and aryllated aryl carbonyl groups such as 4-phenylbenzoyl. The preferred groups are formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, neovaleryl, and benzoyl.

[0092] In this specification, the term "silyl" may include trimethylsilyl, triethylsilyl, isopropyl dimethylsilyl, tert-butyl dimethylsilyl, methyl diisopropylsilyl, methyl ditert-butylsilyl, triisopropylsilyl, and triC1-6 alkylsilyl groups such as trimethylsilyl, triethylsilyl, isopropyl dimethylsilyl, tert-butyl dimethylsilyl, methyl ditert-butylsilyl, and triisopropylsilyl; and triC1-6 alkylsilyl groups substituted with 1 to 2 aryl groups such as diphenylmethylsilyl, butyl diphenyl butylsilyl, diphenyl isopropylsilyl, and phenyl diisopropylsilyl. Trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyl dimethylsilyl, and tert-butyl diphenylsilyl are preferred, and trimethylsilyl is more preferred.

[0093] In this specification, "halogen atom" may be listed as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. Fluorine atom or chlorine atom is preferred.

[0094] In this specification, the term "protecting group" in phrases such as "protecting group of amino group in nucleic acid synthesis," "protecting group of hydroxyl group in nucleic acid synthesis," "hydroxyl group protected by protecting group in nucleic acid synthesis," "phosphate group protected by protecting group in nucleic acid synthesis," and "thiol group protected by protecting group in nucleic acid synthesis" is not particularly limited as long as it is a group capable of stably protecting amino, hydroxyl, phosphate, or thiol groups 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 C1-6 alkyl, C1-6 alkenyl, acyl, tetrahydropyranyl or tetrahydrothiaranyl, tetrahydrofuranyl or tetrahydrothiofuranyl, silyl, C1-6 alkoxymethyl, C1-6 alkoxylated C1-6 alkoxymethyl, halo-C1-6 alkoxymethyl, C1-6 alkoxylated ethyl, halo-ethyl, methyl substituted with 1 to 3 aryl groups, "methyl substituted with 1 to 3 aryl groups, wherein the aryl ring of the aryl group is substituted with a C1-6 alkyl, C1-6 alkoxy, halogen atom or cyano", C1-6 alkoxycarbonyl, "aryl substituted with a halogen atom, C1-6 alkoxy or nitro", "C1-6 alkoxycarbonyl substituted with a halogen atom or tri-C1-6 alkylsilyl", alkenyloxycarbonyl, "aralkyloxycarbonyl with optional aryl ring substituted with C1-6 alkoxy or nitro", etc.

[0095] More specifically, examples of tetrahydropyranyl or tetrahydrothiaranyl groups include tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiaran-4-yl, and 4-methoxytetrahydrothiaran-4-yl. Examples of tetrahydrofuranyl or tetrahydrothiofuranyl groups include tetrahydrofuran-2-yl and tetrahydrothiofuran-2-yl. Examples of C1-6 alkoxymethyl groups include methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, and tert-butoxymethyl. Examples of C1-6 alkoxylated C1-6 alkoxymethyl groups include 2-methoxyethoxymethyl. Examples of halosubstituted C1-6 alkoxymethyl groups include 2,2,2-trichloroethoxymethyl and bis(2-chloroethoxy)methyl. Examples of C1-6 alkoxylated ethyl groups include 1-ethoxyethyl and 1-(isopropoxy)ethyl. Examples of haloethyl groups include 2,2,2-trichloroethyl. Examples of methyl groups substituted with 1 to 3 aryl groups include benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, and 9-anthraylmethyl. Examples of methyl groups substituted with 1 to 3 aryl groups, wherein the aryl ring of the aryl group is substituted with a C1-6 alkyl group, a C1-6 alkoxy group, a halogen atom, or a cyano group, include 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, and 4-cyanobenzyl. Examples of C1-6 alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, and isobutoxycarbonyl. Examples of aryl groups substituted with a halogen atom, C1-6 alkoxy group, or nitro group include 4-chlorophenyl, 2-chlorophenyl, 4-methoxyphenyl, 4-nitrophenyl, and 2,4-dinitrophenyl. Examples of C1-6 alkoxycarbonyl groups substituted with a halogen atom or a tri-C1-6 alkylsilyl group include 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl. Examples of alkenyloxycarbonyl groups include vinyloxycarbonyl and aryloxycarbonyl. Examples of "arylalkyloxycarbonyl groups in which the aryl ring is optionally substituted by a C1-6 alkoxy or nitro group" include benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl.

[0096] In one embodiment, examples of "protecting groups for hydroxyl groups in nucleic acid synthesis" include aliphatic acyl groups, aromatic acyl groups, methyl groups substituted with 1 to 3 aryl groups, "methyl groups substituted with 1 to 3 aryl groups, wherein the aryl ring of the aryl group is substituted with a C1-6 alkyl group, a C1-6 alkoxy group, a halogen group, or a cyano group", and silyl groups. Alternatively, in other embodiments, examples of "protecting groups for the hydroxyl groups of nucleic acid synthesis" include acetyl, benzoyl, benzyl, p-methoxybenzoyl, dimethoxytriphenylmethyl, monomethoxytriphenylmethyl, tert-butyldiphenylsilyl, tert-butyldimethylsilyl (TBDMS), [(triisopropylsilyl)oxy]methyl (TOM), [(2-nitrobenzyl)oxy]methyl (NBOM), bis(acetoxyethoxy)methyl ether (ACE), tetrahydro-4-methoxy-2H-pyran-2-yl (Mthp), 1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM), tert-butyldithiomethyl (DTM), 2-(4-toluenesulfonyl)ethoxymethyl (TEM), and 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl (4-MABOM).

[0097] In one embodiment, examples of protecting groups that are "hydroxyl groups protected by a protecting group of nucleic acid synthesis" include aliphatic acyl groups, aromatic acyl groups, "methyl groups substituted with 1 to 3 aryl groups", "aryl groups substituted with halogen atoms, C1-6 alkoxy groups, or nitro groups", C1-6 alkyl groups, and C1-6 alkenyl groups. Alternatively, in other embodiments, examples of protecting groups that are "hydroxyl groups protected by a protecting group of nucleic acid synthesis" include benzoyl, benzyl, 2-chlorophenyl, 4-chlorophenyl, and 2-propenyl groups.

[0098] In one embodiment, an acyl group, preferably a benzoyl group, can be used as a "protecting group for the amino group of nucleic acid synthesis".

[0099] In one embodiment, the "protecting group" as "the phosphate group protected by the protecting group of nucleic acid synthesis" can include, for example, C1-6 alkyl, C1-6 alkyl substituted with cyano, aralkyl, "aralkyl ring substituted with nitro or halogen atom", and "aryl group substituted with C1-6 alkyl, halogen atom or nitro". Alternatively, in one embodiment, the "protecting group" as "the phosphate group protected by the protecting group of nucleic acid synthesis" can include, for example, 2-cyanoethyl, 2,2,2-trichloroethyl, benzyl, 2-chlorophenyl and 4-chlorophenyl.

[0100] In one embodiment, the "protecting group" as "the thiol group protected by the protecting group of nucleic acid synthesis" can be exemplified by, for example, aliphatic acyl groups and aromatic acyl groups, preferably benzoyl groups.

[0101] In this specification, -P(R)12 R 13 [In the formula, R] 12 and R 13 Each of the following groups independently represents a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, a thiol group, a thiol group protected by a nucleic acid synthesis protecting group, 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 For R 12a And R 13 For NR 13a The group refers to the "phosphamide group" (here, R...). 12a For example, cyanoalkoxy groups having 1 to 6 carbon atoms, and R 13a For example, an alkyl group having 1 to 6 carbon atoms. As a phosphoramidyl group, preferably a group represented by the formula -P(OC2H4CN)(N(iPr)2) or a group represented by the formula -P(OCH3)(N(iPr)2). Here, iPr represents isopropyl.

[0102] (2) Regarding the nucleoside (or nucleoside derivative) selected based on the target oligonucleotide sequence. In this specification, the terms "nucleoside" and "nucleoside analogue" are not specifically defined, but refer to substances commonly used in this technical field. They refer to non-natural nucleosides formed by the combination of purine or pyrimidine bases with sugars, as well as substances formed by the combination of purine or pyrimidine bases with sugars using aromatic heterocycles and aromatic hydrocarbon rings other than purine and pyrimidine.

[0103] In embodiments of the present invention, those skilled in the art can select appropriate oligonucleotides (I) based on their sequence.

[0104] In this specification, the term "oligonucleotide (I)" further includes "artificial oligonucleotide" and "oligonucleotide analog." Here, "artificial oligonucleotide" and "oligonucleotide analog" refer to non-natural derivatives of "oligonucleotides" formed by the linkage of the same or different "nucleosides" or "nucleoside analogs" (e.g., 2 to 50) through phosphodiester bonds. As such analogs, preferred examples include sugar derivatives with modified sugar moiety; thioate derivatives with thioate-modified phosphodiester moiety; ester bodies with esterified terminal phosphate moiety; and amide bodies with amidated amino groups on purine bases. Sugar derivatives with modified sugar moiety are even more preferred examples.

[0105] In this specification, the salts of oligonucleotides (I) may include, for example, alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such as aluminum, iron, zinc, copper, nickel, and cobalt salts; inorganic salts such as ammonium salts; tert-octylamine salts; dibenzylamine salts; morpholine salts; glucosamine salts; alkyl phenylglycine ester salts; ethylenediamine salts; N-methylglucosamine salts; guanidine salts; diethylamine salts; triethylamine salts; dicyclohexylamine salts; and N,N Amino salts of organic salts such as ′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenylethylamine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt; inorganic acid salts such as hydrohalic acid salts, hydrochloride salts, hydrobromide salts, and hydroiodide salts; nitrates, perchlorates, sulfates, and phosphates; organic acid salts such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; aryl sulfonates such as benzenesulfonates and p-toluenesulfonates; acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and salts of amino acids such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, and aspartate salts.

[0106] The preferred embodiments of the nucleoside residues (B) in the oligonucleotide (I) are described in detail below.

[0107] Nucleoside residue (B) (I) A nucleoside residue (B), in which, Base represents either purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl, each optionally having 1 to 3 substituents selected from group α. Among them, 1) the α group includes hydroxyl, hydroxyl protected by a protecting group of nucleic acid synthesis, straight-chain alkyl with 1 to 6 carbon atoms, straight-chain alkoxy with 1 to 6 carbon atoms, mercapto, mercapto protected by a protecting group of nucleic acid synthesis, straight-chain alkyl thio with 1 to 6 carbon atoms, amino, straight-chain alkyl amino with 1 to 6 carbon atoms, amino protected by a protecting group of nucleic acid synthesis, and halogen atoms; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, R1 indicates: (Where R1 represents a hydrogen atom, a protecting group of the hydroxyl group in nucleic acid synthesis, an alkyl group with 1 to 7 carbon atoms that optionally form a branch or ring, or an alkenyl group with 2 to 7 carbon atoms that optionally form a branch or ring); and, ** indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, R2 indicates: (Here, R2 has the same meaning as R1 above).

[0108] Nucleoside residue (B)(II) The above nucleoside residue (B) (I), wherein the part of the structure represented by formula (i) in nucleoside residue (B) is the group shown in the following partial structural formula (i-1),

[0109] (In the formula, R6 and R7 each independently represent a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms, or R6 and R7 combine with each other to form a carbon ring with 3 to 6 carbon atoms together with the adjacent carbon atoms.

[0110] More preferably, the above-mentioned nucleoside residue (B) (I), wherein, in the above formula (i-1), R6 and R7 represent hydrogen atoms; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, indicates a hydrogen atom; and, ** indicates a binding site on an adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, it indicates a hydrogen atom.

[0111] Nucleoside residue (B)(III) The above nucleoside residue (B) (I), wherein the part of the structure represented by formula (i) in nucleoside residue (B) is the group shown in the following partial structural formula (i-2),

[0112] (In the formula, R8, R9, R 10 and R 11 Each can be independently represented by a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms.

[0113] More preferably, the above-mentioned nucleoside residue (B) (I), wherein, in the above formula (i-2), R8, R9, R 10 and R 11 Each atom represents a hydrogen atom independently; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, indicates a hydrogen atom; and, ** indicates a binding site on an adjacent oligonucleotide component, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, it indicates a hydrogen atom.

[0114] The above describes the implementation of oligonucleotides (I) primarily from the perspective of "nucleoside residues (B)" that constitute their nucleic acid monomers. The following describes implementations from other perspectives.

[0115] There is no particular limitation on the number of nucleoside residues (B) contained in the oligonucleotide (I). However, as an embodiment of the present invention, the sequence of the oligonucleotide may include, for example, oligonucleotide (I) containing 1 to 25 nucleoside residues (B) and oligonucleotide (I) containing 1 to 10 nucleoside residues (B) or their salts.

[0116] However, as mentioned above, there is no limit to the number of introduced nucleoside residues (B), which can be appropriately determined according to the intended use of the oligonucleotide (I). For example, the entire sequence of the oligonucleotide (I) can be composed of nucleoside residues (B).

[0117] By introducing at least one (e.g., 1–10) nucleoside residue (B), the toxicity of oligonucleotides (hereinafter also referred to as "parent oligonucleotides") having a base sequence determined in a sequence-dependent manner with the target RNA can be effectively reduced. Examples of toxicity reductions associated with parent oligonucleotides include hepatotoxicity and weight loss.

[0118] The "method for reducing the toxicity of oligonucleotides or their salts" is another embodiment of the present invention.

[0119] Those skilled in the art can appropriately determine the preferred number of nucleoside residues (B) introduced into the sequence based on the sequence of the parent oligonucleotide, the type and severity of toxicity. Examples include 1 to 30 residues, with 1 to 10 and 1 to 6 residues being more preferred.

[0120] The insertion position of nucleoside residue (B) in oligonucleotide (I) is not particularly limited, and those skilled in the art may determine it appropriately as needed.

[0121] There is no particular limitation on the total base length of the oligonucleotide (I), however, in one embodiment of the invention, the oligonucleotide is an oligonucleotide (I) or a salt thereof having a length of 7 to 30 bases.

[0122] Those skilled in the art can appropriately determine the base length of the oligonucleotide (I) according to its intended use, etc., to design the oligonucleotide (I). When using the oligonucleotide (I) or its salt, for example as an antisense oligonucleotide, a preferred embodiment may be a length of 7 to 30 bases. Alternatively, as a more preferred embodiment, a length of 10 to 25 or 10 to 20 bases may be used.

[0123] In one embodiment of the present invention, the oligonucleotide (I) can be a gapmer comprising a gap region of 2 to 10 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, wherein the gap region is located between the 5' wing region and the 3' wing region.

[0124] In one embodiment, a modified nucleic acid that has a strong binding affinity to RNA may be configured in the wing region.

[0125] In this embodiment, there are no particular limitations on the number and position of the nucleoside residues (B) introduced into the oligonucleotide (I). For example, the interstitial region may contain at least one nucleoside residue (B), and the 5' wing region and / or 3' wing region may contain at least one nucleoside residue (B). In addition, the interstitial region, as well as the 5' wing region and / or 3' wing region, may each contain at least one nucleoside residue (B).

[0126] Those skilled in the art can appropriately determine the number and location of nucleoside residues (B) introduced into oligonucleotides (I) based on, for example, the sequence of the target RNA or the sequence of the parent oligonucleotide.

[0127] As one embodiment of the present invention, the oligonucleotide (I) may be an oligonucleotide constituting the antisense strand (guide strand) and / or sense strand (passenger strand) of siRNA composed of a double-stranded RNA of about 21 bases in length.

[0128] In this embodiment, the base sequences of the antisense and sense strands in the oligonucleotide (I) can be designed based on the sequence of the target RNA, and 2'-O-methyl modification (2'-OMe) and 2'-F-RNA (2'-F) are introduced to reduce off-target effects, control in vivo dynamics, and immune responses.

[0129] Furthermore, in this embodiment, there are no particular limitations on the number and location of nucleoside residues (B) introduced into the oligonucleotide (I). For example, nucleoside modifications can be introduced into the overhang region, seed region, or cleavage region. Those skilled in the art can appropriately determine the number and location of modifications to the oligonucleotide (I) based on the sequence of the target RNA, the sequence of the parent oligonucleotide, etc.

[0130] In one embodiment of the present invention, in the oligonucleotide (I), at least one of the nucleotide linkages in the nucleic acid sequence can be a phosphate thioester linkage. Alternatively, all nucleotide linkages can be phosphate thioester linkages.

[0131] In oligonucleotide (I), from the viewpoint of improving the oligonucleotide's resistance to nucleases, phosphate thioester linkages can be used instead of phosphate diester linkages as needed. Those skilled in the art can appropriately determine the number and position of phosphate thioester linkages; however, as an example of a preferred embodiment, all internucleotide linkages are phosphate thioester linkages.

[0132] In one embodiment of the invention, the oligonucleotide (I) can be used as a single-stranded oligonucleotide (e.g., an antisense oligonucleotide) or as an oligonucleotide constituting a double-stranded oligonucleotide (e.g., siRNA). In this case, the oligonucleotide (I) can constitute one strand of the double-stranded oligonucleotide or both strands thereof.

[0133] [Regarding the method for manufacturing oligonucleotide (I)] Oligonucleotides (I) or their salts can be manufactured by using said “nucleoside A” as one of the nucleic acid monomers constituting the oligonucleotide and introducing it together with other nucleic acid monomers into a suitable oligonucleotide sequence. One or more nucleoside A molecules can be introduced according to the sequence of the target oligonucleotide, not only into the sequence but also into target positions containing a 3' or 5' end. When introducing more than two, they can be introduced sequentially into the sequence or in a manner that separates them from one or more other nucleic acid monomers.

[0134] The specific oligonucleotide (I) can be produced using methods common in this technical field, such as solid-phase synthesis, which can be performed in the following sequence. However, it is not limited to this.

[0135] In the chemical synthesis of oligonucleotides, solid-phase synthesis using the phosphoramidite method is widely used. In the initial stage of the synthesis cycle, the 3' end nucleoside of the oligonucleotide (nucleic acid) sequence to be synthesized is immobilized on a solid support. It should be noted that if the phosphoramidite for the nucleic acid monomer is commercially available, it can be purchased and used. Alternatively, those skilled in the art can appropriately synthesize phosphoramidite including nucleoside (A) using, for example, the method described in Example 1 below, or based on that method.

[0136] In recent years, universal linkers that can be coupled with any nucleoside have been widely used in solid-phase synthesis because they can arbitrarily introduce nucleosides into oligonucleotides and efficiently synthesize oligonucleotides with arbitrary sequences without being limited by the nucleoside at the 3' end.

[0137] More specifically, universal linkers are pre-loaded on a solid support via cleavable linkers (spacer arms) such as succinyl groups and coupled with any 3'-terminal nucleoside. Then, an oligonucleotide extension reaction, typically consisting of the following steps, is carried out in a reaction column according to the synthesis program of an automated nucleic acid synthesis device: (1) The step of deprotecting the 5'-OH group of the protected nucleoside by using an acid such as trichloroacetic acid / dichloromethane solution; (2) The step of coupling nucleoside phosphoramide (also referred to as “nucleic acid monomer”) to the deprotected 5'-OH group in the presence of an activator (tetrazole, etc.); (3) The step of capping the unreacted 5'-OH group with acetic anhydride, etc.; and, (4) The step of oxidizing the phosphite by iodine water or the step of sulfiding the phosphite by 3-((N,N-dimethylaminomethylene)amino)-3H-1,2,4-dithiazole-5-thione or the like.

[0138] The above synthesis cycle is repeated according to the target sequence, and the oligonucleotide extension reaction is carried out from the 3' end to the 5' end to synthesize an oligonucleotide (I) with the target sequence.

[0139] Finally, oligonucleotide (I) can be obtained by hydrolyzing the cleavable linker with ammonia or methylamine solution and by cleaving the synthesized oligonucleotide from the solid support and the universal linker.

[0140] (Regarding nucleoside (A)) The meanings of each group and part of the structure of nucleoside (A) are the same as those of the corresponding groups and parts of the structure defined in the item [1] of the above-mentioned [Solution to the technical problem].

[0141] Therefore, the preferred configurations for each group and part of the structure, as well as the preferred configuration for nucleoside (A), can be found in the corresponding descriptions detailed regarding "nucleoside residue (B)".

[0142] Here, a preferred embodiment of nucleoside (A) is shown by its structural formula, as detailed below. In the following text, nucleosides having this structure are also referred to as nucleoside (Ai-1) and nucleoside (Ai-2), respectively.

[0143]

[0144] Nucleosides (A) can be used to reduce the toxicity of oligonucleotides. More specifically, by introducing at least one nucleoside (A) into the sequence of the target oligonucleotide, the toxicity of the oligonucleotide can be reduced.

[0145] The use of nucleoside (A) in this method is another embodiment of the present invention.

[0146] (Regarding the method for manufacturing nucleoside derivative (A)) Those skilled in the art can appropriately manufacture nucleoside derivatives (A) using known methods, starting from known compounds; for example, they can be manufactured using the methods specifically described in the examples below. The starting material compounds can also be appropriately obtained by those skilled in the art from known compounds.

[0147] [Regarding the use of oligonucleotides (I)] As demonstrated in the examples below, the oligonucleotide (I) or its salts (hereinafter collectively referred to as "this oligonucleotide") exhibit excellent effects in enhancing or maintaining efficacy while reducing toxicity by introducing a nucleoside derivative (A) into the original antisense oligonucleotide sequence. Furthermore, this oligonucleotide itself can, for example, be used as a novel antisense oligonucleotide. Therefore, this oligonucleotide is effective as a drug for the prevention or treatment of diseases, as an antisense oligonucleotide, for example.

[0148] The use of this oligonucleotide as a drug is another embodiment of the present invention.

[0149] The following is a detailed description of how this oligonucleotide is used as a drug.

[0150] In this instruction manual, "prevention" includes preventing the onset of a disease (the condition as a whole, or one or more conditions) and delaying the onset of the disease. "Effective dose for prevention" refers to the amount of this oligonucleotide sufficient to achieve such an objective.

[0151] In this instruction manual, "treatment" includes curing a disease (the overall condition, or one or more conditions), improving the disease, and inhibiting the progression of the disease's severity. "Therapeutic effective amount" refers to the amount of this oligonucleotide sufficient to achieve such an effect.

[0152] In carrying out this invention, the oligonucleotide may be used either in its standalone form or in the form of a pharmaceutical composition containing the oligonucleotide as an active ingredient and a pharmaceutically acceptable carrier.

[0153] Examples of pharmaceutical compositions include, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, lozenges, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), syrups, liquid preparations, 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 patches), injections (e.g., subcutaneous injections, intravenous injections (e.g., boluses), intramuscular injections, intraperitoneal injections, intramedullary injections, intraventricular injections), drops, transdermal absorption preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories), microcapsules, nasal preparations, inhaled preparations, eye drops, etc.

[0154] In this specification, various carriers commonly used in the field of formulation technology may be used as “pharmaceutically acceptable carriers”.

[0155] As specific examples of "pharmaceutically acceptable carriers", excipients (e.g., lactose, white sugar, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silica, etc.), lubricants (e.g., magnesium stearate, talc, colloidal silica, etc.), binders (e.g., crystalline cellulose, white sugar, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethyl cellulose, etc.) and disintegrants (e.g., starch, calcium carboxymethyl cellulose, sodium carboxymethyl starch, L-hydroxypropyl cellulose, etc.) can be used in solid dosage forms.

[0156] Liquid formulations may use solvents (e.g., water for injection, isotonic saline, ethanol, propylene glycol, polyethylene glycol, sesame oil, etc.), solubilizers (e.g., polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, etc.), suspending agents (e.g., stearyltriethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, glyceryl monostearate, etc. surfactants; e.g., polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, etc. hydrophilic polymers, etc.), isotonic agents (e.g., glucose, D-sorbitol, sodium chloride, glycerol, D-mannitol, etc.), buffers (e.g., phosphate, citrate, etc. buffers, etc.), and analgesics (e.g., benzyl alcohol, etc.).

[0157] Depending on the needs, preservatives (such as parabens, chlorobutanol, benzyl alcohol, sorbic acid, etc.), antioxidants (such as sulfites, ascorbic acid, α-tocopherol, etc.), colorants, sweeteners, and other pharmaceutical additives may also be added.

[0158] Although the dosage form, administration method, and carrier may differ, the oligonucleotides of the present invention can be manufactured by adding them relative to the total amount of the formulation at a typically 0.01–99% (w / w), preferably 0.1–85% (w / w) ratio. The pharmaceutical composition can be manufactured according to its morphology using methods conventional in the field of pharmaceutical formulation technology. The pharmaceutical composition of the present invention can be formulated into a sustained-release formulation containing the active ingredient.

[0159] (Regarding the target population for drug administration) This oligonucleotide is expected to have low toxicity and few side effects, and also possesses excellent properties as a drug. Therefore, this oligonucleotide can be safely administered to mammals (especially humans).

[0160] (Regarding the route of administration) In carrying out the present invention, the oligonucleotide may be administered orally or non-orally, alone or as a pharmaceutical composition (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, ophthalmic, intracerebral (intraventricular), intramedullary, rectal, vaginal, intraperitoneal, and lesion-specific administration).

[0161] (Regarding dosage) The dosage of the pharmaceutical composition of the present invention varies depending on the purpose of administration, method of administration, type and severity of the target disease, and the condition of the target patient (sex, age, weight, etc.), and is not particularly limited. For example, when the complex of the present invention is administered systemically to an adult, a single dose is generally expected to be 0.01 mg / kg or more and 1000 mg / kg or less; when administered locally, a dose is generally expected to be 0.001 mg / body or more and 100 mg / body or less. It is desirable to administer this dosage 1 to 10 times, preferably 5 to 10 times.

[0162] The pharmaceutical compositions of the present invention can be used in combination with, for example, commercially available therapeutic agents for diseases. These combined agents can be formulated with the pharmaceutical compositions of the present invention as a single-ingredient formulation for administration, or formulated separately from the pharmaceutical compositions of the present invention and administered simultaneously or at different times via the same or different routes as the pharmaceutical compositions of the present invention. Furthermore, the dosage of these combined agents can be the usual dosage used when administered alone, or a reduced dosage compared to the usual dosage.

[0163]

Example

[0164] The following examples illustrate various instances of using entecavir as a nucleoside (A) to synthesize oligonucleotides (I) and the results of pharmacological evaluation (efficacy / toxicity) of these oligonucleotides.

[0165] [Example 1] Synthesis of Amidite The phosphoramidoid of entecavir was synthesized according to the following synthetic scheme (already reported).

[0166]

[0167] Under a nitrogen atmosphere, anhydrous MeOH (17 mL) was added to compound 1 (entecavir) (500 mg, 1.80 mmol), followed by the addition of N,N-dimethylformamide dimethyl acetal (905 μL, 6.77 mmol). The mixture was stirred at room temperature for 12 hours. After confirming the consumption of the starting material by TLC, the solvent was concentrated under reduced pressure to obtain target compound 2 (581 mg, 98%).

[0168] Under a nitrogen atmosphere, anhydrous pyridine (19 mL) and anhydrous DMF (5 mL) were added to compound 2 (658 mg, 1.98 mmol), followed by the addition of 4,4-dimethoxytrityl chloride (470 mg, 1.39 mmol), and the mixture was stirred at room temperature for 1 hour. Further addition of 4,4-dimethoxytrityl chloride (470 mg, 1.39 mmol) and stirring at room temperature for 3 hours was performed. After TLC confirmation of feed consumption, the solvent was concentrated under reduced pressure. A saturated NaHCO3 solution (100 mL) was added to the residue, and the mixture was extracted three times with EtOAc (100 mL). The organic layer was then washed with brine and dried over Na2SO4. The solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: DCM / MeOH = 98:2 + 0.5% TEA) to give target compound 3 (1.01 g, 81%).

[0169] Compound 3 (563 mg, 0.89 mmol) and 1H-tetrazole (93.5 mg, 1.34 mmol) were added to a heated and dried reaction vessel, and anhydrous 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 hours. 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 and dried with Na2SO4. The solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: DCM / MeOH = 100:0 to 95:5 + 1% TEA) to give target compound 4 (514 mg, 0.616 mmol, 69%).

[0170] [Example 2] Objective: To synthesize oligonucleotides Methods and Results: (1) Synthesis scheme The oligonucleotides were synthesized by preparing a 0.1 M anhydrous acetonitrile solution of commercially available dA(Bz), dG(iBu), dC(Bz), dT, LNA-A(Bz), LNA-G(DMF), LNA-mC(Bz), LNA-T, and entecavir synthesized in Example 1 using phosphorus amide, and then using a DNA / RNA synthesis apparatus (NTS M-2-TRS, Nihon Techno Service Co., LTD) according to the conventional phosphorus amide method.

[0171] The synthesis was performed at a 1 µmol scale under triel-on conditions. 5-Benzylthio-1H-tetrazole (0.25 M anhydrous acetonitrile) was used as the activator, with condensation times of 3 min for the LNA amide block and 30 s for the native amide block. After synthesis, the sample was treated with 28% ammonia at 55 °C for 13 h, cleaved from the column support, and deprotected of the base and phosphate diester portions. The sample was then transferred to a simple reverse-phase column (Glen-Pak). TM DNA was purified using a Glen Research DNA purification kit, followed by reversed-phase HPLC. The HPLC determination conditions are shown below.

[0172] (Eluent) Solution A: 100mM hexafluoro-2-propanol + 8.6mM triethylamine (pH 8.36) Solution B: Methanol (gradient) Solution B concentration: 1) 5-30% (30 min) (purification) 2) 5-40% (20 min) (purity confirmation) (Chromatographic column) 1) Nacalai 5C 18 -MS-II (10×250mm) (purified) 2) Nacalai 5C 18 -MS-II (4.6×50mm) (Purity confirmed) 3) Column temperature 60℃ (Flow rate) 1) 2.0 mL / min (purification) 2) 0.5 mL / min (purity confirmation) (Detection) UV (260nm) (2) The synthesized oligonucleotides According to the above synthetic scheme, oligonucleotides with the sequences shown in Table 1 were obtained (nucleotide linkages: all are phosphate thioester linkages). In the table, E represents entecavir.

[0173] It should be noted that in Table 1 and Tables 2-9, 12-14 and 16 below, unless otherwise stated, nucleosides represented by uppercase letters represent LNA, and nucleosides represented by lowercase letters represent DNA. Additionally, entecavir is sometimes indicated by the marginal note "E". In hApo1-cRNA and hApo1-cDNA, uppercase letters represent RNA and DNA, respectively.

[0174] Table 1

[0175] Research: Based on the above, it can be shown that nucleoside (A) can be introduced into oligonucleotide sequences according to the prescribed method, and multiple nucleosides can be introduced consecutively within a single sequence.

[0176] [Example 3] Objective: To determine the double-strand melting temperature (T) for complementary RNA. m ) Methods and Results: A sample solution (150 μL) was prepared with a final concentration of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM EDTA, and 4 μM of each oligonucleotide and complementary strand RNA (hApo1-cRNA, Table 1) listed in Table 2. The solution was heated to 95 °C for 3 minutes, then gradually cooled to 20 °C at a rate of 1 °C per minute for annealing before measurement. The absorbance at 260 nm was plotted at 0.5 °C per minute increments up to 95 °C. m All values ​​were calculated using the midpoint method.

[0177] The test results are shown in Table 2 below. In the table, E represents entecavir.

[0178] Table 2

[0179] Research: Based on the above, it is shown that nucleoside (A) is a unique non-natural nucleotide that does not impair the thermodynamic stability of ASO to target RNA.

[0180] [Example 4] Objective: To determine the double-strand melting temperature (T) for complementary DNA. m ) Methods and Results: A sample solution (150 μL) was prepared to a final concentration of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM EDTA, and 4 μM of each oligonucleotide and complementary strand DNA (hApo1-cDNA, Table 1). After heating to 95 °C for 3 minutes, it was gradually cooled to 20 °C at a rate of 1 °C per minute for annealing, followed by measurement. The absorbance was plotted at 260 nm at 0.5 °C per minute increments, increasing to 95 °C. m All values ​​were calculated using the midpoint method.

[0181] The test results are shown in Table 3 below. In the table, E represents entecavir.

[0182] Table 3

[0183] Based on the above, it is shown that nucleoside (A) is a unique non-natural nucleotide that does not compromise thermodynamic stability even for complementary strand DNA.

[0184] [Example 5] Objective: To evaluate the in vitro inhibitory effect of oligonucleotide drugs with nucleoside (A) inserted into the chain on the expression of target genes. Methods and Results: ASO was diluted to a final concentration of 2 μM using cell culture medium (with 9 mM CaCl2 added) and added to 96-well plates. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and incubated for 24 hours. cDNA was then prepared from cell lysates using the SuperPrep (trademarked) II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's instructions. ApoB mRNA expression levels were analyzed using the CFX Real-Time PCR System (BIO RAD). In the analysis of human ApoB, hApoB-F: 5'-TTCTCAAGAGTTACAGCAGATCCA-3' (Sequence No. 1) and hApoB-R: 5'-TGGAAGTCCTTAAGAGCAACTAACA-3' (Sequence No. 2) were used. In the analysis of human Gapdh as a housekeeping gene, primer sets hGAPDH-F: 5'-GCACCGTCAAGGCTGAGAAC-3' (Sequence No. 3) and hGAPDH-R: 5'-TGGTGAAGACGCCAGTGGA-3' (Sequence No. 4) were used to calculate KD activity based on relative expression levels. The relative expression level was obtained by converting the difference in Ct values ​​to the difference in expression levels.

[0185] The evaluation results are shown in Figure 1 .

[0186] Research: according to Figure 1 The activities of gap2, 2mod, and 3mod were slightly reduced, while gap3 showed activity comparable to or slightly higher than that of hApo1 (parental strand), indicating that oligonucleotides (I) (oligonucleotide drugs) with nucleoside (A) introduced into the strand have gene-inhibiting activity.

[0187] [Example 6] Objective: To evaluate the cytotoxicity (cell viability) of oligonucleotide drugs into which nucleoside (A) has been introduced into the chain. Methods and Results: ASO was diluted to a final concentration of 1 or 2 μM using cell culture medium (with 9 mM CaCl2 added) and added to 96-well plates. Huh-7 cells diluted in the above medium were seeded at 10,000 cells per well and incubated for 72 hours. Afterward, Cell Counting Kit-8 (Tongren Chemical) was added, and the plates were incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0188] The evaluation results are shown in Figure 2 .

[0189] Research: according to Figure 2 Cell death inhibition was confirmed in gap2, gap3, 2mod, and 3mod, and cytotoxicity was suppressed. Oligonucleotides (I) (oligonucleotide drugs) introduced into the positions shown in Table 3 exhibited low cytotoxicity.

[0190] [Example 7] Objective: To evaluate the cytotoxicity (Caspase 3 / 7 activity) of oligonucleotide drugs with nucleoside (A) inserted into the chain. Methods and Results: Dilute ASO to a final concentration of 1 or 2 μM using cell culture medium (with 9 mM CaCl2 added) and add to 96-well plates. Seed Huh-7 cells diluted with the above medium at 10,000 cells per well and incubate for 72 hours. Activation of Caspase 3 / 7 was then determined using Caspase-Glo (registered trademark) 3 / 7 Assay Systems, Caspase-Glo (registered trademark) (Promega), and according to the manufacturer's instructions.

[0191] The measurement results are shown in Figure 3 .

[0192] Research: according to Figure 3 In gaps 2, 2mod, and 3mod, the increase in Caspase 3 / 7 was significantly inhibited. This demonstrates that the detoxicization of oligonucleotide drugs is possible by introducing nucleosides (A) into the chain at the positions shown in Table 3.

[0193] [Example 8] Objective: To synthesize an oligonucleotide drug containing nucleoside (A) carrying a targeting ligand. Methods and Results: (1) Synthesis scheme The oligonucleotides were synthesized by preparing commercially available dA(Bz), dG(iBu), dC(Bz), dT, LNA-A(Bz), LNA-G(DMF), LNA-mC(Bz), LNA-T, and the entecavir phosphorusamide synthesized in Example 1 into 0.1 M anhydrous acetonitrile solutions, and using a DNA / RNA synthesis apparatus (NTS M-2-TRS, Nihon Techno Service Co., LTD) according to the conventional phosphorusamide method. The synthesis scale was set at 1 µmol and carried out under triel-on conditions. 5-Benzylthio-1H-tetrazole (0.25 M anhydrous acetonitrile) was used as an activator, and the condensation time was 3 minutes for the amide segment of LNA and 30 seconds for the native amide segment.

[0194] After synthesis, the solid support was transferred to a 1.0 mL airtight syringe, and the ligand moiety was extended manually. The ligand moiety was extended by using GalNAc amide segments (0.1 M anhydrous acetonitrile) and 5-ethylthio-1H-tetrazole (0.5 M anhydrous acetonitrile) as activators and acting them according to the usual phosphoramidite method. It should be noted that the GalNAc amide segment was prepared according to the report "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, it was treated with 28% ammonia at 55°C for 13 hours, excised from the column support, and deprotected from the base and phosphodiester portions. Then it was processed using a simple reverse-phase column (Glen-Pak). TM DNA was purified using a Glen Research DNA purification kit, followed by reversed-phase HPLC. The HPLC determination conditions are shown below.

[0195] (Eluent) Solution A: 100mM hexafluoro-2-propanol + 8.6mM triethylamine (pH 8.36) Solution B: Methanol (gradient) Concentration of solution B: 1) 5-30% (30 min) (purification) 2) 5-40% (20 min) (purity confirmation) (Chromatographic column) 1) Nacalai 5C 18 -MS-II (10×250mm) (purified) 2) Nacalai 5C 18 -MS-II (4.6×50mm) (Purity confirmed) 3) Column temperature 60℃ (Flow rate) 1) 2.0 mL / min (purification) 2) 0.5 mL / min (purity confirmation) (Detection) UV (260nm) (2) The synthesized oligonucleotides According to the above synthetic scheme, oligonucleotides with the sequences shown in Table 4 were obtained (nucleotide linkages: the two linkages from the 5' end are phosphodiester linkages, and the remaining linkages are thiophosphate linkages). In the table, E represents entecavir, and X represents GalNAc. APD .

[0196] Table 4

[0197] Research: Based on the above, it can be shown that oligonucleotide drugs (oligonucleotides (I)) containing nucleosides (A) carrying target ligands can be constructed using conventional methods.

[0198] [Example 9] Objective: To verify the in vivo inhibitory effect of an oligonucleotide drug containing nucleoside (A) carrying a targeting ligand on the expression of a target gene. Methods and Results: All animal experimental protocols were approved by the Animal Experimentation Committee of Nagasaki University. For 7-week-old C57Bl / 6J (male, Japanese SLC), ASO targeting ApoB (a common human-mouse sequence) was administered subcutaneously at a single dose of 200 nmol / kg. 72 hours after administration, whole blood was collected under isoflurane inhalation anesthesia, followed by liver collection. Liver samples were stored overnight in RNAlater™ solution at 4°C and then at -20°C until analysis. Total RNA was extracted from the liver using the QuickGene RNAtissue kit SII (Fujifilm Corporation) according to the accompanying instruction manual. Using the extracted total RNA as a template, reverse transcription was performed using the High Capacity RNA-to-cDNA™ kit (Thermo Fisher Scientific), and ApoB mRNA expression levels were analyzed using the CFX Real-Time PCR System (BIO RAD). In the analysis of mouse ApoB, the primer sets mApoB-F: 5'-TCCTCGGTGAGTTCAATGACTTTC-3' (Sequence No. 5) and mApoB-R: 5'-TGGACCTGCTGTAGCTTGTAGGA-3' (Sequence No. 6) were used. In the analysis of mouse Gapdh, which is a housekeeping gene, the primer sets mGAPDH-F: 5'-TGTGTCCGTCGTGGATCTGA-3' (Sequence No. 7) and mGAPDH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3' (Sequence No. 8) were used to calculate KD activity based on relative expression levels. The relative expression level is obtained by converting the difference in Ct values ​​to the difference in expression levels.

[0199] The evaluation results are shown in Figure 4 .

[0200] Research: according to Figure 4 The results showed that the in vivo knockdown activity was consistent with the in vitro evaluation results, with gap2 still showing a slightly reduced activity but a high gene expression repression effect. On the other hand, gap1 and gap3 showed high activity comparable to hApo1 (parental strand). This indicates that the differences caused by different introduction sites have been confirmed; however, oligonucleotides (I) generally exhibit high activity.

[0201] [Example 10] Objective: To verify the effect of nucleoside (A) on the toxicity of oligonucleotide drugs using mice.

[0202] Methods and Results: All animal experimental protocols were approved by the Animal Experimentation Committee of Nagasaki University. For 7-week-old C57Bl / 6J (male, Japanese SLC), the ASO targeting ApoB obtained in Example 8 was administered subcutaneously at a dose of 200 nmol / kg. Whole blood was collected 72 hours after administration under isoflurane inhalation anesthesia, followed by liver collection. Serum samples collected from the inferior vena cava were used to evaluate changes in body weight. ALT, total bilirubin, direct bilirubin, and indirect bilirubin were determined by Oriental Yeast Industry Co., Ltd.

[0203] The evaluation results are shown in Figures 5-9 .

[0204] Research: By introducing nucleoside (A), weight loss caused by the side effects of antisense nucleic acid drugs was eliminated in all oligonucleotides (I) of gap1, gap2, and gap3. Figure 5 ), hepatotoxicity ( Figure 6 ) and jaundice symptoms ( Figure 7 , 8 (9). In particular, gap2, which also showed effectiveness in cell experiments, significantly improved the abnormalities in ALT and bilirubin values ​​found in hApo1.

[0205] This indicates that, in the oligonucleotide (I) of the present invention, the toxicity of the oligonucleotide is improved by introducing nucleoside (A) compared with that before introduction, and oligonucleotide drugs with excellent activity and safety can also be obtained according to the selection of its introduction position and amount.

[0206] [Example 11] Objective: To determine the double-strand melting temperature (T) for complementary RNA. m ) Methods and Results: (1) Synthesis of oligonucleotides The same method described in Example 2 was used to synthesize various oligonucleotides (nucleotide linkages: all phosphate thioester linkages) with mPCS2 as the parent sequence as described in Table 5 below, and the results were evaluated as follows.

[0207] (2) Determine the double-strand melting temperature (T) for complementary RNA. m ) A sample solution (150 μL) was prepared with a final concentration of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM EDTA, and 4 μM of each oligonucleotide and complementary strand RNA. After heating to 95 °C for 3 minutes, it was gradually cooled to 20 °C at a rate of 1 °C per minute for annealing, and then the measurement was started. The temperature was increased to 95 °C at a rate of 0.5 °C per minute, and the absorbance at 260 nm was plotted at 1 °C intervals. m All values ​​were calculated using the midpoint method.

[0208] The test results are shown in Table 5 below. In the table, E represents entecavir.

[0209] Table 5

[0210] Research: Based on the above, it is shown that even in different ASO sequences (mPCS2), the nucleoside (A) is a unique non-natural nucleotide that does not impair the thermodynamic stability of the complementary strand RNA.

[0211] [Example 12] Objective: To verify the in vivo inhibitory effect of an oligonucleotide drug containing nucleoside (A) carrying a targeting ligand on the expression of a target gene. Methods and Results: (1) Synthesize oligonucleotide drugs containing nucleoside (A), which carries a targeting ligand. As described in Example 8, oligonucleotide drugs containing nucleoside (A) as described in Table 6 below were synthesized and evaluated as follows.

[0212] Table 6

[0213] (2) Verify the in vivo inhibitory effect of an oligonucleotide drug containing nucleoside (A) on the expression of the target gene, wherein the nucleoside (A) carries a targeting ligand. All animal experimental protocols were approved by the Animal Experimentation Committee of Nagasaki University. For 7-week-old C57Bl / 6J (male, Japanese SLC), the PCSK9-targeting ASO listed in Table 6 was administered subcutaneously at a single dose of 200 nmol / kg. 72 hours after administration, whole blood was collected under isoflurane inhalation anesthesia, followed by liver collection. The liver was stored overnight in RNAlater™ solution at 4°C and then at -20°C until analysis. Total RNA was extracted from the liver using the QuickGene RNA tissue kit SII (Fujifilm Corporation) according to the accompanying instruction manual. Using the extracted total RNA as a template, reverse transcription was performed using the HighCapacity RNA-to-cDNA™ kit (Thermo Fisher Scientific), and PCSK9 mRNA expression was analyzed using the CFX Real-Time PCR System (BIO RAD).

[0214] In the analysis of mouse PCSK9, mPSCK9-F: 5'-TCAGTTCTGCACACCTCCAG-3' (Sequence No. 9) and mPCSK9-R: 5'-GGGTAAGGTGCGGTAAGTCC-3' (Sequence No. 10) were used. In the analysis of mouse Gapdh, which is a housekeeping gene, primer sets mGAPDH-F: 5'-TGTGTCCGTCGTGGATCTGA-3' (Sequence No. 7) and mGAPDH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3' (Sequence No. 8) were used to calculate KD activity based on relative expression levels. The relative expression level is obtained by converting the difference in Ct values ​​to the difference in expression levels.

[0215] The evaluation results are shown in Figure 10 .

[0216] Research: This indicates that the introduction of nucleoside (A) can achieve the same or greater activity compared to the parental sequence (mPCS2).

[0217] [Example 13] Objective: To verify the effect of nucleoside (A) on the toxicity of oligonucleotide drugs using mice.

[0218] Methods and Results: The experiments were conducted using oligonucleotides carrying various target ligands synthesized in Example 12, identical to those described in Example 10. Serum collected from the inferior vena cava was used to evaluate changes in body weight. ALT, total bilirubin, direct bilirubin, and indirect bilirubin were determined by Oriental Yeast Industry Co., Ltd.

[0219] The evaluation results are shown in Figures 11-14 .

[0220] Research: This indicates that the toxicity found when using the parental sequence (mPCS2) can be improved by adjusting the insertion site of nucleoside (A).

[0221] [Example 14] Objective: To determine the double-strand melting temperature (T) for complementary RNA. m ) Methods and Results: (1) Synthesis of oligonucleotides The same method described in Example 2 was used to synthesize various oligonucleotides (nucleotide linkages: all phosphate thioester linkages) with mPCS2 as the parent sequence as described in Table 7 below, and the results were evaluated as follows.

[0222] Here, in addition to the entecavir derivative (E), a thymidine derivative of carbon-cyclic DNA (refer to compound 17 (E) in Example 32 below) was used as nucleoside (A). T To synthesize and evaluate oligonucleotides.

[0223] (2) Determine the double-strand melting temperature (T) for complementary RNA. m ) A sample solution (150 μL) was prepared with a final concentration of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM EDTA, and 4 μM of each oligonucleotide and complementary strand RNA. After heating to 95 °C for 3 minutes, it was gradually cooled to 20 °C at a rate of 1 °C per minute for annealing, and then the assay was performed. The temperature was increased to 95 °C at a rate of 0.5 °C per minute, and absorbance was plotted at 260 nm at 1 °C intervals. m All values ​​were calculated using the midpoint method after three measurements.

[0224] The test results are shown in Table 7 below. In the table, E represents entecavir, E T Thymidine derivatives representing carbon-cyclic DNA.

[0225] Table 7

[0226] Research: Regarding the thermodynamic stability of the ASO-target RNA complex, it is the same as that of the guanosine derivative (E: entecavir) and the carbocyclic DNA thymidine derivative (E... T The introduction into ASO did not produce a significant effect. This shows that the same thermodynamic effect can be obtained regardless of the type of base used.

[0227] [Example 15] Objective: To evaluate the in vitro inhibitory effect of oligonucleotide drugs with nucleoside (A) inserted into the chain on the expression of target genes. Methods and Results: ASO (mPCS2, mPCS2-gap1, mPCS2-gap2, mPCS2-gap3, mPCS2-gapT1, and mPCS2-gapT2 as described in Table 7 above) was diluted to a final concentration of 1 μM using cell culture medium (with 9 mM CaCl2 added) and added to 96-well plates. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and incubated for 24 hours. cDNA was then prepared from the cell lysates using the SuperPrep (trademarked) II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's instructions. The expression level of Pcsk9 mRNA was analyzed using the CFX Real-Time PCR System (BIO RAD). In the analysis of human PCSK9, hPCSK9-F: 5'-AAGGGAAGGGCACGGTTAG-3' (SEQ ID NO. 11) and hPCSK9-R: 5'-GAGTAGAGGCAGGCATCGTC-3' (SEQ ID NO. 12) were used. In the analysis of human Gapdh as a housekeeping gene, primer sets hGAPDH-F: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO. 3) and hGAPDH-R: 5'-TGGTGAAGACGCCAGTGGA-3' (SEQ ID NO. 4) were used. KD activity was calculated based on relative expression levels, which were obtained by converting the difference in Ct values ​​to the difference in expression levels. Significance was tested using Dunnett's multiple comparison test after one-way ANOVA. **p<0.01, *p<0.05. No significant difference in "ns" (p>0.05).

[0228] The evaluation results are shown in Figure 15 .

[0229] Research: Even when a thymidine derivative of a carbon-cyclic DNA is introduced into the strand, the same knockdown activity as the parental sequence (mPCS2) can still be achieved. This indicates that the same efficacy can be obtained regardless of the type of nucleoside (A) base.

[0230] [Example 16] Objective: To evaluate the cytotoxicity (cytotoxicity rate) of oligonucleotide drugs with nucleoside (A) inserted into the chain (LDH assay). Methods and Results: (Cellular Experiment) Dilute ASO to a final concentration of 2 μM using cell culture medium (with 9 mM CaCl2 added) and add to 96-well plates. Seed Huh-7 cells diluted with the above medium at 10,000 cells per well and incubate for 72 hours.

[0231] (LDH detection) Add 10 μL of lysis solution to untreated cells and incubate at 37 °C for 30 min. Transfer 80 μL of supernatant from each well to another 96-well plate and add 80 μL of working solution to each well. After incubation at room temperature for 30 min, add 40 μL of stop solution to each well. Measure the absorbance at 490 nm using a microplate reader and subtract the background value from the control or detection value. Calculate the cytotoxicity rate by ((experimental sample) – (low control)) / ((high control) – (low control)) × 100.

[0232] Significance was determined by Dunnett's multiple comparison test after one-way ANOVA. **p<0.01, *p<0.05. No significant difference was found in "ns" (p>0.05).

[0233] The evaluation results are shown in Figure 16 .

[0234] Research: Compared to the parental strand mPCS2, a carbon-cyclic DNA guanosine derivative (E) and a thymidine derivative (E) were introduced. T The ASO of the parent strand reduces or eliminates cytotoxicity with a high probability. This suggests the existence of introduction sites that do not affect the toxicity of the parent strand.

[0235] Based on the above, it is shown that regardless of the target gene or base sequence, nucleic acid drugs with reduced toxicity can be obtained by introducing nucleosides (A) into the ASO chain.

[0236] [Example 17] Objective: To evaluate the cytotoxicity (cell viability) of oligonucleotide drugs into which nucleoside (A) has been introduced (PI staining / dead cell staining). Methods and Results: (Cellular Experiment) Dilute ASO to a final concentration of 2 μM using cell culture medium (with 9 mM CaCl2 added) and add it to a 96-well plate. Seed Huh-7 cells diluted with the above medium at 10,000 cells per well and incubate for 96 hours.

[0237] (PI staining / dead cell staining) Replace the culture medium with cell culture medium (add 9mM CaCl2, 100μL), and add PI solution (Dongren Chemical) diluted to a final concentration of 1.0 μg / mL to each well. Incubate at 37°C for 15 minutes and observe with a fluorescence microscope.

[0238] The evaluation results are shown in Figure 17. In each figure, from left to right, bright field, PI, and merge (bright field / PI) are shown sequentially. Stained dead cells appear white fluorescent 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 in gap 3, which contained a guanosine derivative (E) with parental mPCS2 and carbocyclic DNA, and cell death was observed. On the other hand, gap 1 and gap 2, which contained a guanosine derivative (E) with carbocyclic DNA, and gap 3, which contained a thymidine derivative (E) with carbocyclic DNA, showed significant PI-positive cells. T In the gapT1 and gapT2 of ASO, the number of PI-positive cells was significantly reduced.

[0239] Research: The same tendency was also confirmed in the PI staining experiment of this Example 17, based on the LDH test results in Example 16.

[0240] [Example 18] Objective: To determine the double-strand melting temperature (T) for complementary RNA. m ) Methods and Results: (1) Synthesis of oligonucleotides The same method described in Example 2 was used to synthesize various oligonucleotides (nucleotide linkages: all thiophosphate linkages) with hApoC3 as the parental sequence as described in Table 8 below, and the results were evaluated as follows.

[0241] (2) Determine the double-strand melting temperature (T) for complementary RNA. m ) (chain decomposition temperature (T) m Determination (evaluation of double-strand forming ability) A sample solution (150 μL) was prepared to a final concentration of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM EDTA, and 4 μM of each oligonucleotide and complementary strand RNA. After heating to 95 °C for 3 minutes, it was gradually cooled to 20 °C at a rate of 1 °C per minute for annealing, and then the assay was performed. The temperature was increased to 95 °C at a rate of 0.5 °C per minute, and absorbance was plotted at 260 nm at 1 °C intervals. m All values ​​were calculated using the midpoint method.

[0242] The test results are shown in Table 8 below. In the table, E represents entecavir.

[0243] Table 8

[0244] Research: The introduction of nucleoside (A) into the ASO complex did not significantly affect the thermodynamic stability of the ASO-target RNA complex (SD = 0.52 °C / mod). Moreover, considering the positive correlation between thermodynamic stability and toxicity (see Reference 1 below), this derivative can be considered an excellent artificial nucleic acid that can modulate toxicity parameters without altering the target binding affinity of ASO.

[0245] 1) Watt AT, Swayze G, Swayze EE, Freier SM. Likelihood of NonspecificActivity of Gapmer Antisense Oligonucleotides Is Associated with RelativeHybridization Free Energy. Nucleic Acid Ther. 2020 Aug;30(4):215-228. doi:10.1089 / nat.2020.0847. [Example 19] Objective: To evaluate the cytotoxicity of oligonucleotide drugs into which nucleoside (A) has been introduced into the chain. Methods and Results: (Cellular Experiment) The ASO concentration listed in Table 8 above was diluted to a final concentration of 2 μM using cell culture medium (with 9 mM CaCl2 added), and then added to 96-well plates. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and incubated for 72 hours before various assays were performed.

[0246] (Cell viability assay) After adding 10 μL ( / well) of Cell Counting Kit-8 (Tongren Chemical) to the culture medium, incubate for 2 hours. Measure the absorbance at 450 nm using a microplate reader, and subtract the background value from the control or detection value. Significance was determined by Dunnett's multiple comparison test after one-way ANOVA. **p<0.01, *p<0.05. "ns" showed no significant difference (p>0.05).

[0247] The evaluation results are shown in Figure 18 .

[0248] Research: Compared to the parental strand hApoC3, gap1, gap2, and gap3 all demonstrated reduced or eliminated cytotoxicity. This indicates that, regardless of the target gene or base sequence, nucleic acid drugs with reduced cytotoxicity can be obtained by introducing nucleosides (A).

[0249] [Example 20] Objective: To evaluate the in vivo inhibitory effect of oligonucleotide drugs with nucleoside (A) inserted into the chain on the expression of target genes. Methods and Results: (Cellular Experiment) ASO was diluted to a final concentration of 1 μM using cell culture medium (with 9 mM CaCl2 added) and added to 96-well plates. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and incubated for 24 hours. cDNA was then prepared from the cell lysates using the SuperPrep (registered trademark) II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's instructions. ApoC3 mRNA expression levels were analyzed using the CFX Real-Time PCR System (BIO RAD). In the analysis of human ApoC3, hApoC3-F: 5'-CTGCTCCAGGAACAGAGGTG-3' (Sequence No. 13) and hApoC3-R: 5'-GTGGCGTGCTTCATGTAACC-3' (Sequence No. 14) were used. In the analysis of human Gapdh as a housekeeping gene, primer sets hGAPDH-F: 5'-GCACCGTCAAGGCTGAGAAC-3' (Sequence No. 3) and hGAPDH-R: 5'-TGGTGAAGACGCCAGTGGA-3' (Sequence No. 4) were used. KD activity was calculated based on relative expression levels, which were obtained by converting the difference in Ct values ​​to the difference in expression levels. Significance was tested using Dunnett's multiple comparison test after one-way ANOVA. **p<0.01, *p<0.05. No significant difference in "ns" (p>0.05).

[0250] The evaluation results are shown in Figure 19 .

[0251] Research: Compared to hApoC3 (the parental chain), the ASOs of gap1-3, which exhibited reduced cytotoxicity, confirmed knockdown activity. Gap1 and gap3, in particular, showed activity equivalent to the parental chain. This demonstrates that introducing nucleosides (A) can improve safety while maintaining efficacy.

[0252] [Example 21] Objective: To verify the effect of nucleosides (A) carrying target ligands on the toxicity of oligonucleotide drugs using mice. Methods and Results: (1) Synthesize oligonucleotide drugs containing nucleosides (A) carrying target ligands. As described in Example 8, oligonucleotides carrying various targeting ligands with hApoC3 as the parental sequence, as shown in Table 9 below, were synthesized (nucleotide linkages: the two linkers from the 5' end are phosphodiester linkages, and the remaining linkers are thiophosphate linkages), and evaluated as follows. In the table, E represents entecavir.

[0253] Table 9

[0254] (2) Determination of KD activity of ASO in mouse liver.

[0255] All animal experimental protocols were approved by the Animal Experimentation Committee of Nagasaki University before implementation. For 7-week-old C57Bl / 6J (male, Japanese SLC), ASO, targeting human ApoC3 (with no homology to mice), was administered subcutaneously at a single dose of 200 nmol / kg. While observing changes in body weight, whole blood was collected under isoflurane inhalation anesthesia 72 hours after administration. Subsequently, serum collected from the inferior vena cava was used, and ALT, AST, and total bilirubin were measured by Oriental Yeast Industry Co., Ltd. For statistical analysis, after the Smirnov-Grubbs test, significance was assessed using Dunnett's multiple comparison test after one-way ANOVA (AST, ALT, T-BIL) or two-way ANOVA (body weight change) (**p<0.01, *p<0.05. "ns" indicates no significant difference (p>0.05)).

[0256] The evaluation results are shown in Figures 20-23 .

[0257] (Research) A significant decrease in body weight following drug administration was confirmed only in hApoC3 (parental strand). No decrease in body weight was confirmed in gaps 1-3. Furthermore, hepatotoxicity was confirmed in hApoC3 (parental strand). Compared to the parental strand, ASOs incorporating nucleoside (A) inhibited the increase in hepatic excretion enzymes and bilirubin levels. The degree and trend were consistent with the cytotoxic trend. This indicates that introducing nucleoside (A) can maintain efficacy while improving safety (however, the ASOs used here are not homologous to the mouse ApoC3 gene; therefore, knockdown activity in mice was not confirmed). [Production of nucleoside (A)] Regarding nucleosides (A) other than those described in Example 1, their specific methods of preparation and manufacturing are shown below (Examples 22-38).

[0258] First, the synthetic scheme for compound 13, which serves as a common intermediate in the synthesis of various derivatives, is shown below. It should be noted that compounds 8 onwards are new compounds. Starting from compound 13, various nucleosides (A) can be synthesized.

[0259]

[0260] [Example 22]

[0261] (1S,2S,3S,5S)-3-(benzyloxy)-2-((benzyloxy)methyl)-5-(tert-butoxy)-cyclopentan-1-ol (8) Under a nitrogen atmosphere and with stirring at -20°C, tert-butanol (4.48 mL, 47.2 mmol, 4 eq.) dehydrated with MS4A was added to a 100 mL solution of anhydrous dichloromethane containing compound 7 (3.66 g, 11.8 mmol). At the same temperature, BF3·OEt2 (148 μL, 1.18 mmol, 0.1 eq.) diluted with anhydrous dichloromethane (30 mL) was added dropwise, and the mixture was stirred at room temperature for 23 hours. Saturated sodium bicarbonate (200 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, dehydrated with 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) yielded a colorless oily compound 8 (3.78 g, 83%). 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).; 13C 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 H 33 O4, 385.2373; found, 385.2379. [Example 23]

[0262] (2R,3S,5S)-3-(benzyloxy)-2-((benzyloxy)methyl)-5-(tert-butoxy)-cyclopentan-1-one (9) Under a nitrogen atmosphere and with stirring at 0°C, Dess-Martin periodinane (4.5 g, 10.63 mmol) was added to an anhydrous dichloromethane solution (88 mL) of compound 8 (3.40 g, 8.86 mmol), followed by stirring at room temperature for 1 hour. An aqueous solution of sodium thiosulfate (70 mL) and saturated sodium bicarbonate (10 mL) were added to the reaction solution with stirring at 0°C, and the mixture was stirred for 20 minutes at the same temperature. An aqueous solution of sodium thiosulfate to saturated sodium bicarbonate (7:1) (150 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (200 mL). After washing the organic layer with saturated brine, the mixture was dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure to give compound 9 (3.25 g, 96%) as a yellow oil. 1H 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).; 13 C 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. [Example 24]

[0263] ((1R,3S,5S)-5-(benzyloxy)-3-(tert-butoxy)-2-methylenecyclopentyl)methoxy)methyl)benzene (10) Under a nitrogen atmosphere and with stirring at 0°C, 0.5 M Tebbe reagent (19.7 mL, 9.87 mmol) was added dropwise to an anhydrous tetrahydrofuran solution (50 mL) of compound 9 (2.90 g, 7.59 mmol) and stirred at room temperature for 50 min. After quenching the reaction by adding 0.1 M NaOH aq (50 mL) and stirring for 30 min at 0°C, H₂O (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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. Purification by silica gel column chromatography (SiO₂, ethyl acetate / hexane = 1:3) yielded compound 10 (2.22 g, 77%) as a yellow oil. 1H NMR (400MHz, 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 C 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, 28.63. HRMS (FAB) m / z: [M + H]+ calcd forC 25 H 33 O3, 381.2424; found, 381.2428. [Example 25]

[0264] (1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentan-1-ol (11) Under a nitrogen atmosphere and with stirring at 0°C, 14 mL of trifluoroacetic acid was added to an anhydrous dichloromethane solution of compound 10 (2.14 g, 5.64 mmol) in 37 mL of dichloromethane, and the mixture was stirred at room temperature for 1 hour. After further addition of 5 mL of trifluoroacetic acid and stirring for 30 minutes, 200 mL of cold saturated sodium bicarbonate solution 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 with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Purification was performed by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:3). The compound with the secondary hydroxyl group trifluoroacetylated was recovered in the reaction and hydrolyzed with 10 mL of 50 mM potassium carbonate-methanol solution. 100 mL of saturated sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 100 mL of ethyl acetate. After washing the organic layer with saturated brine, the mixture was dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 11 (1.66 g, 90%) as a yellow oily substance. 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, 4.3Hz, 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).; 13 C 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. [Example 26]

[0265] (1R,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentylacetate (12).

[0266] DEAD (4.2 mL, 9.05 mmol) was added to an anhydrous tetrahydrofuran solution of triphenylphosphine (2.37 g, 9.05 mmol) in 22 mL under a nitrogen stream and stirring at -20 °C, and the mixture was stirred for 20 min at the same temperature. Compound 11 (1.42 g, 4.52 mmol) diluted with anhydrous tetrahydrofuran solution (20 mL) was added, and the mixture was stirred for 10 min at the same temperature. Acetic acid (679 μL, 11.3 mmol) was added, and the mixture was stirred for 20 h at room temperature. A saturated aqueous solution of 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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:4) to give compound 12 (1.20 g, 73%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ7.37 - 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) δ171.05, 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 27O4, 367.1904; found, 367.1908. [Example 27]

[0267] (1R,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentan-1-ol (13).

[0268] Compound 12 (1.06 g, 2.89 mmol) was added to a nitrogen atmosphere with 50 mM potassium carbonate-methanol (20 mL) and stirred at room temperature for 1 hour. After adding H₂O (5 mL), the mixture was concentrated under reduced pressure. The residue was then extracted with ethyl acetate (100 mL) and saturated sodium bicarbonate (100 mL). The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 13 (935 mg, quant) as a colorless oil. 1 H 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, 8.1Hz, 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). [Example 28]

[0269] 1-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentyl)-3-((benzyloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (14).

[0270] DEAD (514 μL, 1.13 mmol) was added to a solution of triphenylphosphine (297 mg, 1.13 mmol) in anhydrous acetonitrile (2 mL) under a nitrogen stream and stirring at 0 °C, and the mixture was stirred for 20 min at the same temperature. N3-(benzyloxymethyl)thymidine (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. A saturated aqueous solution of 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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product 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, CDCl3) 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, CDCl3) 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, HRMS (FAB) m / z: [M+H] + calcd for C 34 H 37N2O5,553.2697; found, 553.2702. [Example 29]

[0271] 1-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl)-5-methylpyrimidine-2,4(1H,3H)-dione (15).

[0272] Under a nitrogen atmosphere and with stirring at -40°C, 1 M BCl3-dichloromethane solution (3.43 mL, 3.43 mmol) was added to an anhydrous dichloromethane solution (1.0 mL) of compound 14 (189 mg, 0.343 mmol), and the mixture was stirred for 1.5 hours at the same temperature. Then, 2 M ammonia-methanol solution (2 mL) was added with stirring at -40°C, and the mixture was stirred for 1 hour at room temperature. After concentrating the reaction solution under reduced pressure, the solution was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 10:1) to give compound 15 (71 mg, 82%) as a colorless oil. 1 H 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 = 2.5Hz, 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 CNMR (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 forC 12 H 17 N2O4, 253.1183; found, 253.1191. [Example 30]

[0273] Anhydrous pyridine (2.8 mL) was added to compound 15 (71 mg, 0.282 mmol) under a nitrogen atmosphere, followed by the addition of 4,4'-dimethoxytriphenylmethyl chloride (142 mg, 0.42 mmol), and the mixture was stirred at room temperature for 20 hours. A saturated aqueous solution of 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, dehydrated with 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) to give compound 16 (122 mg, 78%) as a white foamy solid. 1 H NMR (400 MHz, CDCl3) 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 (101 MHz, CDCl3) δ163.93,158.73, 151.56, 148.84, 144.53, 138.09, 135.76, 135.48, 130.20, 128.23,128.08, 127.18, 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] + calcd forC 33 H 35N2O6, 555.2490; found, 555.2502. [Example 31]

[0274] Compound 16 (122 mg, 0.220 mmol) and 1H-tetrazole (23 mg, 0.33 mmol) were added to a heated and dried reaction vessel. 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 (rt) for 1.5 h. After confirming the consumption of the starting material by TLC, saturated sodium bicarbonate aqueous solution (30 mL) was added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with saturated brine, dehydrated with 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, with the addition of 1% triethylamine) to give 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).;31 PNMR (162 MHz, CDCl3) δ 147.97, 147.69. HRMS (FAB) m / z: [M + H] + calcd forC 42 H 52 N4O7P, 755.3574; found, 755.3574. [Example 32]

[0275] 1,2,4-triazole (123 mg, 1.78 mmol) was added to a heated and dried reaction vessel, followed by anhydrous acetonitrile (2 mL) under a nitrogen atmosphere. Phosphorus oxychloride (37.5 μL, 0.403 mmol) was added dropwise, and the mixture was stirred at room temperature for 10 minutes. Triethylamine (331 μL, 2.385 mmol) was added dropwise, and the mixture was stirred further at room temperature for 30 minutes. Next, compound 17 (40 mg, 0.053 mmol) dissolved in anhydrous acetonitrile (1 mL) was added dropwise, and the mixture was stirred further at room temperature for 1 hour. After confirming the consumption of the starting material by TLC, a saturated aqueous solution of 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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 18 (41 mg, 96%) as a white, foamy solid. 1H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.07 (d, J = 1.0Hz, 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 P 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. [Example 33]

[0276] 9-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentyl)-6-chloro-9H-purine (19) DEAD (140 μL, 0.308 mmol) was added to an anhydrous tetrahydrofuran solution (1 mL) of triphenylphosphine (81 mg, 0.308 mmol) under a nitrogen stream and stirring at 0 °C, and the mixture was stirred for 20 min at the same temperature. 6-Chloropurin (35.5 mg, 0.308 mmol) was added, and the mixture was stirred for another 10 min. Then, compound 13 (50 mg, 0.154 mmol) diluted with anhydrous tetrahydrofuran solution (500 μL) was added, and the mixture was stirred at room temperature for 24 h. A saturated aqueous sodium bicarbonate solution (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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:2) gave compound 19 (47 mg, 66%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ8.70 (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).; 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 26 H 26 ClN4O2, 461.1739; found, 461.1754. [Example 34]

[0277] 9-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylene-cyclopentyl)-9H-purine-6-amine (20) Under a nitrogen atmosphere, 3 mL of 7 M ammonia-methanol solution was added to compound 19 (180 mg, 0.392 mmol), and the mixture was stirred in a pressure-resistant flask at 110 °C for 2 hours. The reaction solution was returned to room temperature and concentrated under reduced pressure to obtain the crude product. Purification was performed by silica gel column chromatography (SiO2, ethyl acetate / hexane = 2:1 → dichloromethane / methanol = 10:1) to give 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).; 13 C 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 forC 26 H 28 N5O2, 442.2238; found, 442.2243. [Example 35]

[0278] N-(9-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylene-cyclopentyl)-9H-purine-6-yl)benzamide (21) Under a nitrogen atmosphere, N,N-diisopropylethylamine (76 μL, 0.446 mmol) was added to an anhydrous N,N-dimethylformamide solution (2.9 mL) containing compound 20 (131 mg, 0.297 mmol) and 4-dimethylaminopyridine (3.6 mg, 0.030 mmol). Benzoic anhydride (134 mg, 0.594 mmol) was then added, and the mixture was stirred at 100 °C for 7.5 h. Under ice bath cooling, 2 M ammonia-methanol solution (1 mL) was added, and the mixture was stirred for 10 min. A saturated sodium bicarbonate aqueous solution (50 mL) was then added to the reaction solution, followed by extraction three times with ethyl acetate (50 mL). The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:1) yielded compound 21 (109 mg, 67%) as a white foamy solid. 1 H NMR (400 MHz, CDCl3) δ9.09 (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).; 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. [Example 36]

[0279] N-(9-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl)-9H-purine-6-yl)benzamide (22) Under a nitrogen atmosphere and with stirring at -40°C, 1M boron trichloride (1.98 mL, 1.98 mL) was added dropwise to an anhydrous dichloromethane solution (500 μL) of compound 21 (108 mg, 0.198 mmol). After stirring at the same temperature for 20 min, triethylamine (1 mL) and methanol (1 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 30 min. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (SiO2, dichloromethane / methanol = 20:1). After concentration under reduced pressure, a saturated ammonium chloride aqueous solution (40 mL) was added to the residue, and the mixture was extracted five times with ethyl acetate (40 mL). After dehydration of the organic layer with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to give compound 22 (60 mg, 83%) as a white foamy solid. 1H 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).; 13 C 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. [Example 37]

[0280] N-(9-((1S,3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylenecyclopentyl)-9H-purine-6-yl)benzamide (23) Under a nitrogen atmosphere, anhydrous pyridine (1.6 mL) was added to compound 22 (60 mg, 0.164 mmol), followed by the addition of 4,4'-dimethoxytriphenylmethyl chloride (67 mg, 0.197 mmol), and the mixture was stirred at room temperature for 6 hours. A saturated aqueous solution of sodium bicarbonate (40 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (40 mL). The organic layer was washed with saturated brine, dehydrated with 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 give compound 23 (74 mg, 68%) as a white foamy solid. 1 H 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 C 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] +calcd for C 40 H 38 N5O5,668.2867; found,668.2873. [Example 38]

[0281] Compound 23 (74.1 mg, 0.111 mmol) and 1H-tetrazole (12 mg, 0.167 mmol) were added to a heated and dried reaction vessel. Anhydrous dichloromethane (1.1 mL) and DIPEA (28 μL, 0.167 mmol) were added under a nitrogen atmosphere. 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine (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 sodium bicarbonate aqueous solution (30 mL) was added, and the mixture was extracted three times with ethyl acetate (30 mL). The organic layer was washed with saturated brine, dehydrated with 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, with the addition of 1% triethylamine). The residue was dissolved in dichloromethane (1 mL), followed by the addition of hexane (49 mL), and centrifuged at 25 °C and 5000 g for 5 minutes. The supernatant was removed, and the process was repeated once for the precipitate. The precipitate was then dissolved again 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 = 7.2Hz, 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. [Example 39]

[0282] The phosphoramidoids of the two cytosine derivatives shown in the above structural formulas can be synthesized by those skilled in the art, for example, starting from compound 13 (Example 27), by referring to the methods described in Examples 28-32 and other methods known in the art.

[0283] [Example 40] Objective: Synthesis of siRNA Methods and Results: (1) Synthesis scheme The siRNA was synthesized by preparing a 0.1 M anhydrous acetonitrile solution of commercially available phosphoramides 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, and then synthesizing them according to the phosphoramide method.

[0284] (2) The sequence and structure of the synthesized siRNA Oligonucleotides with the sequences shown in Table 10 below were prepared based on the structure of Vutrisiran (dots indicate PS links, and the remaining links are phosphodiester links). In the table, "E" represents entecavir, "N" represents 2'-OMe RNA, and "Nf" represents 2'-F RNA.

[0285] Table 10

[0286] Research: Based on the above, it was shown that oligonucleotides containing entecavir derivatives can be synthesized using conventional methods, and siRNAs can be constructed.

[0287] [Example 41] Objective: To evaluate the double-strand forming ability of siRNA with an entecavir derivative introduced into the antisense strand. Methods and Results: The sense strand and the antisense strands (AS, AS@6, AS@5, AS@5, 6) synthesized in Example 40 were mixed in equal volumes in a sample solution (10 μL). The mixture 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. 10 pmol of each sample relative to 20 μM was mixed with Orange DNA Ladder Dye (6X) (Thermo Fisher Scientific) and 10x TBE. The mixture was loaded onto a 20% TBE gel and electrophoresed at 4°C and 200 V for 60 minutes. The electrophoresed gel was stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific) and photographed using FAS-V (Genetics, Japan). The evaluation results are shown below. Figure 24 .

[0288] The study revealed that introducing one or more entecavir derivatives into any AS strand can form a double strand identical to that of unmodified siRNA by mixing it with a sense strand.

[0289] [Example 42] Objective: To evaluate the in vitro inhibitory effect and IC50 of entecavir derivative-derived siRNA on target gene expression when introduced into the AS strand. 50 Methods and Results: A sample containing equal volumes of sense strands and various antisense strands (AS, AS@6, AS@5, AS@5,6) 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. The annealed siRNA was diluted to final concentrations of 0, 0.01, 0.1, 1, 10, and 100 nM in cell culture medium (with CaCl2 added) and added to 96-well plates. Huh-7 cells diluted in the above medium were seeded at 10,000 cells per well and incubated for 24 hours. cDNA was then prepared from the cell lysate using the SuperPrep (trademarked) II Cell Lysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's instructions. TTR mRNA expression levels were analyzed using a QuantStudio3 (Thermo Fisher Scientific). In the analysis of human TTR, the TaqMan probe (Hs00174914_m1) was labeled with FAM, and in the analysis of human Gapdh as a housekeeping gene, the TaqMan probe (Hs02758991_g1) was labeled with VIC. KD activity was calculated based on relative expression levels, which were obtained by converting the difference in Ct values ​​to the difference in expression levels. The 50% inhibitory concentration (IC50) was calculated based on the dose-response curve. 50 The evaluation results are illustrated in [the following text is incomplete and likely refers to a separate topic:] Figure 25 In addition, each IC 50 As shown in Table 11.

[0290] Table 11

[0291] Study: When one or more entecavir derivatives were introduced into the AS strand of siRNA in cultured cells, it showed high knockdown activity comparable to that of Vutrisiran.

[0292] [Production of nucleoside (A)] The following section presents representative compounds of nucleosides (Ai-2) and their synthetic schemes for phosphoramidoids.

[0293]

[0294] The following section details each synthesis step in the above synthesis scheme.

[0295] [Example 43]

[0296] 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 and with stirring at 0°C, 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 an anhydrous diethyl ether solution (98.4 mL) of compound 13 (3.189 g, 9.84 mmol). 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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:10 to 1:4) yielded 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. [Example 44]

[0297] 9-((4S,6S,7R)-6-(benzyloxy)-7-((benzyloxy)methyl)spiro[2.4]heptane-4-yl)-6-chloro-9H-purine (26).

[0298] Under a nitrogen atmosphere and with stirring at -20°C, 6-chloropurine (553 mg, 3.58 mmol, 2.5 eq.) and DEAD (1.64 mL, 3.58 mmol) were added to an anhydrous tetrahydrofuran solution (8 mL) of triphenylphosphine (939 mg, 3.58 mmol) and stirred for 10 minutes. Compound 25 (485 mg, 1.43 mmol) diluted with anhydrous tetrahydrofuran solution (6.3 mL) was added and stirred at room temperature for 5 hours. A saturated aqueous solution of 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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product 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). 13 C 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 forC 27H 28 ClN4O2, 475.1895; found, 475.1901. [Example 45]

[0299] 9-((4S,6S,7R)-6-(benzyloxy)-7-((benzyloxy)methyl)spiro[2.4]heptane-4-yl)-9H-purine-6-amine (27).

[0300] Under a nitrogen atmosphere, 10 mL of 7 M ammonia-methanol solution was added to compound 26 (200 mg, 0.420 mmol), and the mixture was stirred in a pressure-resistant flask at 110 °C for 18 hours. The reaction solution was returned to room temperature and concentrated under reduced pressure to give the crude product. Purification was performed by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:3 → dichloromethane / methanol = 15:1) to give compound 27 (162 mg, 85%) as a white foamy solid. 1 H 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, 5.7Hz, 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 C 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 H30 N5O, 456.2394; found, 456.2400. [Example 46]

[0301] N-(9-((4S,6S,7R)-6-(benzyloxy)-7-((benzyloxy)methyl)spiro[2.4]heptane-4-yl)-9H-purine-6-yl)benzamide (28) Under a nitrogen atmosphere, N,N-diisopropylethylamine (76 μL, 0.446 mmol) was added to an anhydrous N,N-dimethylformamide solution (3.0 mL) containing compound 27 (135 mg, 0.296 mmol) and 4-dimethylaminopyridine (3.6 mg, 0.03 mmol), followed by the addition of benzoic anhydride (134 mg, 0.594 mmol), and the mixture was stirred at 100 °C for 2 h. Under ice bath cooling, 2 M ammonia-methanol solution (3 mL) was added, and the mixture was stirred for 10 min. A saturated sodium bicarbonate aqueous solution (10 mL) was then added to the reaction solution, followed by extraction three times with ethyl acetate (10 mL). The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (SiO2, ethyl acetate / hexane = 1:1) yielded 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).;13 C 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. [Example 47]

[0302] N-(9-((4S,6S,7R)-6-hydroxy-7-(hydroxymethyl)spiro[2.4]heptane-4-yl)-9H-purine-6-yl)benzamide (29) Under a nitrogen atmosphere and with stirring at -78°C, 1M boron trichloride (6.3 mL, 6.3 mmol) was added dropwise to an anhydrous dichloromethane solution of compound 28 (351 mg, 0.627 mmol). After stirring at the same temperature for 20 minutes, methanol (10 mL) was added to the reaction solution and stirred for 10 minutes. After concentration under reduced pressure, the solution 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. 1H 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 C 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] + calcdfor C 20 H 22 N5O3, 380.1717; found, 380.1723. [Example 48]

[0303] N-(9-((4S,6S,7R)-7-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-hydroxyspiro[2,4]heptane-4-yl)-9H-purine-6-yl)benzamide (30) Under a nitrogen atmosphere, 1.2 mL of anhydrous pyridine solution was added to compound 29 (32.2 mg, 0.0849 mmol), followed by the addition of 4,4'-dimethoxytriphenylmethyl chloride (40.7 mg, 0.127 mmol), and the mixture was stirred at room temperature for 4.5 hours. A saturated aqueous solution of sodium bicarbonate (40 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (40 mL). The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to give 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 give 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, 6.1Hz, 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) δ166.79, 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. [Example 49]

[0304] (4R,5S,7S)-7-(6-benzoylamino-9H-purine-9-yl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)spiro[2,4]heptane-5-yl(2-cyanoethyl)diisopropylphosphonamide (31) Compound 30 (53.0 mg, 0.078 mmol) and 1H-tetrazole (8.2 mg, 0.012 mmol) were added to a heated and dried reaction vessel. 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 sodium bicarbonate aqueous solution (20 mL) was added, and the mixture was extracted three times with dichloromethane (20 mL). The organic layer was washed with saturated brine, dehydrated with 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, with the addition of 1% triethylamine) to give 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. [Production of nucleoside (A)] The following details the synthesis of phosphoramids via other pathways using entecavir derivatives (5-methylcytosine), their introduction into oligonucleotides, and their functional evaluation.

[0305] First, the synthesis scheme of this phosphorous amide is shown below.

[0306]

[0307] [Example 50]

[0308] N-(1-((1S,2S,3S,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-hydroxycyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (32) Under a nitrogen atmosphere, 1.8 mL of anhydrous N,N-dimethylformamide solution was added to compound 7 (56.0 mg, 0.181 mmol), followed by the addition of lithium chloride (15.3 mg, 0.362 mmol) and DBU (54.0 μL, 0.362 mmol). Then, N-benzoyl-5-methylcytosine (83 mg, 0.362 mmol) was added, and the mixture was stirred at 140 °C for 7 hours. A saturated aqueous solution of 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, dehydrated with 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 = 1:2) 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).; 13CNMR (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, 128.03, 127.92, 127.82,111.86, 77.33, 76.64, 76.36, 73.67, 71.41, 70.85, 65.70, 51.93, 33.52, 13.58.HRMS(FAB) m / z: [M + H] + calcd for C 32 H 34 N3O5, 540.2493; found, 540.2498. [Example 51]

[0309] N-(1-((1S,3R,4S)-4-(benzyloxy)-3-((benzyloxy)methyl)-2-methylenecyclopentyl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (33) Under a nitrogen atmosphere, 1.0 mL of anhydrous dichloromethane solution was added to compound 32 (56.0 mg, 0.104 mmol), followed by the addition of Dys-Martin reagent (57.3 mg, 0.135 mmol) with stirring at 0 °C, and the mixture was stirred at room temperature for 3 hours. Then, 15 mL of aqueous sodium thiosulfate solution and 5 mL of saturated sodium bicarbonate were added with stirring at 0 °C, and the mixture was stirred for 20 minutes at the same temperature. The reaction solution was extracted three times with dichloromethane (20 mL). After washing the organic layer with saturated brine, the solution was dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure. Under a nitrogen atmosphere, the residue was dissolved in anhydrous tetrahydrofuran (1.0 mL), followed by the addition of 0.5 M Tebbe reagent (0.18 mL, 0.091 mmol) with stirring at 0 °C, and the mixture was stirred for another 1.5 hours at room temperature. The reaction was terminated by adding 0.1 M NaOH aq (20 mL) with stirring at 0 °C and stirring for 30 minutes. H₂O (20 mL) was then 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, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (SiO₂, ethyl acetate / hexane = 1:12 to 1:3) yielded compound 33 (23.4 mg, 42% in 2 steps) as a yellow oil. 1H 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 CNMR (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] + calcd for C 33 H 34 N3O4, 536.2544; found, 536.2550. [Example 52]

[0310] 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 and with stirring at -40°C, 1M boron trichloride (1.23 mL, 1.23 mmol) was added dropwise to an anhydrous dichloromethane solution of compound 33 (65.9 mg, 0.123 mmol). After stirring for 5 minutes at the same temperature, methanol (2 mL) was added to the reaction solution, and the mixture was stirred for 1 hour. After concentration under reduced pressure, the solution 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) δ149.56, 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. [Example 53]

[0311] 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) Under a nitrogen atmosphere, 1.1 mL of anhydrous pyridine solution was added to compound 34 (38.5 mg, 0.108 mmol), followed by the addition of 4,4'-dimethoxytriphenylmethyl chloride (54.9 mg, 0.162 mmol), and the mixture was stirred at room temperature for 1 hour. A saturated aqueous solution of sodium bicarbonate (10 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL). The organic layer was washed with saturated brine, dehydrated with 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 give 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. [Example 54]

[0312] (1S,2R,4S)-4-(4-benzoylamino-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-methylenecyclopentyl(2-cyanoethyl)diisopropylphosphonamide (36).

[0313] 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. 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'-tetraisopropylphosphonic diamine (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 sodium bicarbonate aqueous solution (10 mL) was added, and the mixture was extracted twice with dichloromethane (10 mL). The organic layer was washed with saturated brine, dehydrated with 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, with the addition of 1% triethylamine) to give compound 36 (66.3 mg, 60%) as a white foamy solid. 1H NMR (400MHz, 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 = 23.5Hz, 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 PNMR (162 MHz, CDCl3) δ 148.02, 147.75. HRMS(FAB) m / z: [M + H] + calcd forC 49 H 57 N5O7P, 858.3990; found, 858.3996. [Example 55] Objective: To determine the double-strand melting temperature (T) for complementary RNA. m ) Methods and Results: • Synthesis of oligonucleotides As described in Example 2, various oligonucleotides (nucleotide linkages: all phosphate thioester linkages) using mPCS2 as the parental sequence, as shown in Table 12 below, were synthesized and evaluated as follows. In the table, E... A Entecavir, E A3 Represents 4'-spirocyclic body. E A3 These are the compounds synthesized in Examples 43-49 above.

[0314] Table 12

[0315] (2) Determine the double-strand melting temperature (T) for complementary RNA.m ) A sample solution (150 μL) was prepared with a final concentration of 10 mM phosphate buffer (pH 7.0), 100 mM sodium chloride, 0.1 mM EDTA, and 4 μM oligonucleotides and complementary strand RNA as listed in Table 12. After heating to 95 °C for 3 minutes, it was gradually cooled to 20 °C at a rate of 1 °C per minute for annealing, and then the assay was performed. The absorbance at 260 nm was plotted at 0.5 °C per minute increments, increasing the temperature to 95 °C. m All values ​​were calculated using the midpoint method. The measurement results are shown in Table 13 below.

[0316] Table 13

[0317]

[0318] Research: Based on the above, the oligonucleotides incorporating nucleoside analogs shown in this invention can be synthesized using conventional methods. Furthermore, although the thermodynamic stability with the complementary strand varies depending on the analog, the binding affinity is not significantly impaired. Since previously reported unsubstituted carbon-cyclic nucleotides (Org. Lett. 2019, 21, 7, 1963-1967) showed significantly impaired binding affinity with complementary RNA, it is believed that inhibiting sp... 2 The conformational fluctuations of the cyclopentane ring caused by carbon and spirocyclic carbons are related to maintaining high binding strength. Furthermore, it suggests that the type of exocyclic substituents can fine-tune thermodynamic stability.

[0319] [Example 56] Objective: To evaluate the stability of oligonucleotides containing entecavir analogs to 3'-exonucleases. Methods and Results: • Synthesis of oligonucleotides The various oligonucleotides (nucleotide linkages: all phosphodiester linkages) listed in Table 14 below were synthesized using the same method as described in Example 2, and evaluated as follows. In the table, mC represents 2'-deoxy-5-methylcytidine, E... mC This represents a 5-methylcytidine derivative of carbon-cyclic DNA.

[0320] • Evaluate the stability of oligonucleotides to 3'-exonucleases To a substance containing an entecavir analogue (E) introduced at the 3' end mC0.003 units of rattlesnake venom phosphodiesterase (CAVP) (Worthington Biochemicals) were added to the buffer [10 mM MgCl2, 50 mM Tris-HCl (pH 8.0)] of the antisense nucleic acid (2.7 nmol) (Table 14) and mixed, and incubated at 37°C. Samples were taken at reaction times of 5, 15, 30, 45, and 60 minutes, and after heating at 90°C for 2 minutes, the changes of the full-length oligonucleotide over time were analyzed by reverse HPLC. The HPLC determination conditions are shown below.

[0321] (Eluent) Solution A: 100mM hexafluoro-2-propanol + 8.6mM triethylamine (pH 8.36) Solution B: Methanol (gradient) Solution B concentration: 0-30% (10 min) (Chromatographic column) 1) YMC Accura Triart Bio C18, 5.0 μm (4.6 A x 50 mm) 2) Column temperature 60℃ (Flow rate) 1.0 mL / min (Testing) UV (260nm) (The evaluated oligonucleotides) The sequences of the evaluated oligonucleotides are shown in Table 14 below.

[0322] Table 14

[0323] In the table, mC represents 5-methylcytosine DNA, and E... mC This refers to entecavir analogs with 5-methylcytosine as the base.

[0324] (2) The area ratio (%) relative to unreacted oligonucleotides is shown in the table and figure (Table 15, Figure 26 ) Table 15

[0325] Research: According to the figure, the 3'-dmC of the parental strand was completely decomposed within 5 minutes of nuclease treatment, introducing nucleoside (E). mCThe 3'-eneNA (mC) of the DNA analog remained unreacted for over 40 minutes after nuclease treatment, demonstrating high enzyme resistance. Oligonucleotides incorporating this DNA analog can confer metabolic stability to nucleic acids through the action of substituents protruding from the loop.

[0326] [Example 57] Objective: To evaluate the in vitro inhibitory effect of oligonucleotide drugs containing carbocyclic nucleosides with spirocyclic structures on target gene expression. Methods and Results: ASO (as described in Table 16) was diluted to a final concentration of 1 μM and added to 96-well plates using cell culture medium (with 9 mM CaCl2 added). Wells without ASO were prepared as negative controls. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and incubated for 24 hours. cDNA was then prepared from cell lysates using the SuperPrep (registered trademark) II CellLysis & RT Kit for qPCR (TOYOBO) according to the manufacturer's instructions. Human PCSK9 mRNA expression was analyzed using a QuantStudio® 3 real-time PCR system (Thermo Fisher Scientific). The TaqMan Gene Expression Assay (FAM) Assay ID: Hs00545399_m1 (Thermo Fisher Scientific) was used for human PCSK9 analysis, and the TaqMan Gene Expression Assay (VIC primer-limited) Assay was used for analysis of human Gapdh, a housekeeping gene. ID: Hs02758991_g1 (Thermo Fisher Scientific), calculated based on the relative expression level of KD activity, where the relative expression level is obtained by converting the difference in Ct values ​​to the difference in expression levels. Significance was tested using Tukey's multiple comparison test after one-way ANOVA. **p<0.01, ***p<0.001. Evaluation results are shown in... Figure 27 .

[0327] Table 16

[0328] Nucleotide linkages: all are phosphate thioester linkages.

[0329] In the table, E A3 This indicates the 4'-spirocyclic form of entecavir.

[0330] Research: As shown in the figure, gapA3 significantly inhibited the expression of the target gene. This indicates that drugs in which some monomers in the nucleic acid drug chain are replaced by carbocyclic nucleosides with a spirocyclic structure, or drugs containing carbocyclic nucleosides with a spirocyclic structure in their chain, can maintain their activity.

[0331] [Example 58] Objective: To evaluate the cytotoxicity (cell viability) of oligonucleotide drugs containing carbocyclic nucleosides with spirocyclic structures inserted into the chain. Methods and Results: The ASO described in Table 16 of Example 57 was diluted to a final concentration of 0.001 μM to 3 μM using cell culture medium (with 9 mM CaCl2 added) and added to 96-well plates. Wells without added ASO were prepared as negative controls. Huh-7 cells diluted with the above medium were seeded at 10,000 cells per well and incubated for 72 hours. Afterward, the medium was replaced with 110 μL of Cell Counting Kit-8 (Dongren Chemical) containing 10 μL per well, and incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the absorbance at 650 nm was measured as a reference. Cell viability was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm and then subtracting the background value from the control or detection value. The IC50 value of each ASO was calculated based on the obtained cell viability using a four-parameter logistic model.

[0332] The evaluation results are shown in Figure 28 .

[0333] Research: According to the figure, compared with the parental strand mPCS2, gapA3 showed a significant reduction in cytotoxicity, with an IC50 value of 0.0391 nM relative to the parental strand, compared to approximately a 7-fold difference of 0.290 nM in gapA3. This indicates that nucleic acid drugs containing carbocyclic nucleosides with spirocyclic structures, or nucleic acid drugs in which some monomers are replaced by carbocyclic nucleosides with spirocyclic structures, are effective in obtaining nucleic acid drugs with reduced cytotoxicity.

[0334] Industrial availability According to one embodiment of the present invention, an oligonucleotide is provided for use as a drug, which maintains high safety while exhibiting excellent effects on target RNA and the like.

[0335] This application is based on Japanese Patent Application No. 2023-187759 (filed on November 1, 2023), the entire contents of which are incorporated herein by reference.

Claims

1. An oligonucleotide or a salt thereof, characterized in that, The oligonucleotide sequence contains at least one carbocyclic nucleoside derivative residue (B) represented by the following formula (B), namely "nucleoside residue (B)". In formula (B), Base represents either purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl, each optionally having one or more substituents selected from substituent group (a). Wherein, substituent group (a) includes hydroxyl, hydroxyl protected by a nucleic acid synthesis protecting group, oxo group, straight-chain alkyl with 1 to 6 carbon atoms, straight-chain alkoxy with 1 to 6 carbon atoms, mercapto, mercapto protected by a nucleic acid synthesis protecting group, straight-chain alkylthio with 1 to 6 carbon atoms, amino, straight-chain alkylamino with 1 to 6 carbon atoms, amino protected by a nucleic acid synthesis protecting group, and halogen atom. Here, when purine-9-yl or 2-oxo-1,2-dihydropyrimidine-1-yl has an oxo group as a substituent selected from substituent group (a), the bond between the carbon atom bonded by the oxo group and the adjacent atom is a single bond; R3 and R4 represent hydrogen atoms, respectively; R5 represents a hydrogen atom; The groups shown in partial structural formula (i) below represent the groups shown in partial structural formula (i-1) or (i-2) below. In equation (i-1) or (i-2), R6 and R7 each independently represent a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms, or... R6 and R7 combine with each other to form a carbon ring with 3 to 6 carbon atoms. R8, R9, R 10 and R 11 Each can independently represent a hydrogen atom, a halogen atom, or an alkyl group having 1 to 6 carbon atoms; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, R1 is represented as follows: Wherein, R1 represents a hydrogen atom, a protecting group of a hydroxyl group synthesized from nucleic acid, an alkyl group optionally forming a branched chain or ring with 1 to 7 carbon atoms, an alkenyl group optionally forming a branched chain or ring with 2 to 7 carbon atoms, an aryl group optionally having one or more substituents selected from substituent group (a) and optionally containing heteroatoms with 3 to 10 carbon atoms, an aralkyl group optionally having one or more substituents selected from substituent group (a) and optionally containing heteroatoms with 3 to 12 carbon atoms, an acyl group optionally having one or more substituents selected from substituent group (a), a silyl group optionally having one or more substituents selected from substituent group (a), a phosphate group optionally having one or more substituents selected from substituent group (a), a phosphate group protected by a protecting group synthesized from nucleic acid, -P(R 12 R 13 Formula -P(R) 12 R 13 In the middle, R 12 and R 13 Each of these groups independently represents a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, 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, ** indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence, R2 is represented as follows: R2 has the same meaning as R1 mentioned above.

2. The oligonucleotide or its salt as described in claim 1, wherein, In nucleoside residue (B), The group shown in partial structural formula (i) is the same group shown in the following partial structural formula (i-1). In equation (i-1), R6 and R7 have the same meaning as described above.

3. The oligonucleotide or its salt as described in claim 1, wherein, In nucleoside residue (B), The group shown in partial structural formula (i) is the same group shown in the following partial structural formula (i-2). In equation (i-2), R8, R9, R 10 and R 11 They have the same meanings as those mentioned above.

4. The oligonucleotide or its salt as described in any one of claims 1 to 3, wherein, This oligonucleotide sequence contains 1 to 10 nucleoside residues (B).

5. The oligonucleotide or its salt as described in any one of claims 1 to 3, wherein, Oligonucleotides are 7 to 30 bases in length.

6. The oligonucleotide or its salt as described in any one of claims 1 to 3, wherein, Oligonucleotides are 10 to 20 bases in length.

7. The oligonucleotide or its salt as described in any one of claims 1 to 3, wherein, The toxicity was reduced compared to before the introduction of nucleoside residue (B).

8. The oligonucleotide or its salt as described in any one of claims 1 to 3, wherein, Oligonucleotides are interstitial structures containing interstitial regions of 2 to 14 bases in length, 5'-wing regions of 2 to 5 bases in length, and 3'-wing regions of 2 to 5 bases in length; The gap region is located between the 5' wing region and the 3' wing region.

9. The oligonucleotide or a salt thereof as described in claim 8, wherein, The interstitial region contains at least one nucleoside residue (B).

10. The oligonucleotide or a salt thereof as described in claim 9, wherein, The 5' wing region and / or the 3' wing region contain at least one nucleoside residue (B).

11. The oligonucleotide or a salt thereof as described in any one of claims 1 to 3, wherein, At least one of the nucleotide linkages in an oligonucleotide is a phosphate thioester linkage.

12. The oligonucleotide or a salt thereof as described in any one of claims 1 to 3, wherein, In oligonucleotides, all nucleotide linkages are phosphate thioester linkages.

13. The oligonucleotide or its salt as described in any one of claims 1 to 3, wherein, In nucleoside residue (B), Base represents either purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl, each of which may be selected to have 1 to 3 substituents selected from substituent group (a). The substituent group (a) includes a hydroxyl group, a hydroxyl group protected by a nucleic acid synthesis protecting group, an oxo group, a straight-chain alkyl group with 1 to 6 carbon atoms, a straight-chain alkoxy group with 1 to 6 carbon atoms, a mercapto group, a mercapto group protected by a nucleic acid synthesis protecting group, a straight-chain alkyl thio group with 1 to 6 carbon atoms, an amino group, a straight-chain alkyl amino group with 1 to 6 carbon atoms, an amino group protected by a nucleic acid synthesis protecting group, and a halogen atom.

14. The oligonucleotide or a salt thereof as described in claim 13, wherein, In nucleoside residue (B), R6 and R7 represent hydrogen atoms; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, it indicates a hydrogen atom; and, ** indicates a binding site on an adjacent oligonucleotide component, or a hydrogen atom when the nucleoside residue (B) is located at the 3' end of the oligonucleotide sequence.

15. The oligonucleotide or a salt thereof as described in claim 13, wherein, In nucleoside residue (B), R8, R9, R 10 and R 11 They represent hydrogen atoms respectively; * indicates a binding site on an adjacent oligonucleotide moiety, or, when the nucleoside residue (B) is located at the 5' end of the oligonucleotide sequence, it indicates a hydrogen atom; and, ** indicates a binding site on an adjacent oligonucleotide component, or 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 as described in 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, characterized in that, In this oligonucleotide sequence, at least one carbocyclic nucleoside derivative residue (B) representing a divalent group as shown in formula (B), namely "nucleoside residue (B)", is introduced. In formula (B), each group and each part of the structure have the same meaning as the corresponding group and part of the structure defined in claim 1 above regarding "nucleoside residue (B)".

18. The use of the carbocyclic nucleoside derivative represented by the following formula (A), namely "nucleoside (A)" or its salt, in reducing oligonucleotide toxicity, In the formula, each group and each part of the structure has the same meaning as the corresponding group and part of the structure defined in claim 1 regarding "nucleoside residue (B)".

19. The use as described in claim 18, wherein, Reducing oligonucleotide toxicity involves introducing at least one nucleoside (A) into the oligonucleotide sequence.

20. A drug, wherein, It contains any one of the oligonucleotides or their salts as active ingredients according to claims 1 to 3.

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