BBB-crossing lipid ligands of heterogeneous nucleic acids

Abstract

To provide a nucleic acid agent that can be efficiently delivered to the nervous system, particularly the central nervous system to which drug delivery is inhibited by the BBB mechanism, and can produce an antisense effect on a target transcriptional product at the delivered site, and a composition comprising the same.SOLUTION: Provided is a double-stranded nucleic acid complex formed by annealing a first nucleic acid strand which is capable of hybridizing with part of a target transcriptional product and having an antisense effect on the target transcriptional product, to a second nucleic acid strand which comprises a base sequence complementary to the first nucleic acid strand and is bound to tocopherol or an analog thereof, cholesterol or an analog thereof, or a substituted or unsubstituted C1-30 alkyl group, a substituted or unsubstituted C2-30 alkenyl group, or a substituted or unsubstituted C1-30 alkoxy group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a nucleic acid complex or a salt thereof capable of producing an antisense effect in the nervous system, particularly the central nervous system, and a composition comprising the same. [Background technology]

[0002] In recent years, oligonucleotides have attracted attention in the ongoing development of pharmaceuticals called nucleic acid drugs, and in particular, the development of nucleic acid drugs using the antisense method is being actively promoted in view of the high selectivity and low toxicity of the target gene. The antisense method is a method that involves selectively modifying or inhibiting the expression of a protein coded by a target gene or the activity of miRNA by introducing a complementary oligonucleotide (antisense oligonucleotide: often referred to as "ASO (AntiSense Oligonucleotide)" in this specification) into a cell using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand.

[0003] Patent Document 1 discloses a double-stranded nucleic acid molecule that is composed of a first oligomeric compound and a second oligomeric compound containing a conjugated group such as cholesterol and that can regulate the amount or activity of a target nucleic acid in extrahepatic tissues or extrahepatic cells, or in hepatic tissues or hepatic cells, and an antisense compound composed of the double-stranded nucleic acid molecule.

[0004] In order to exert an antisense effect in the central nervous system, including the brain, it is necessary to deliver nucleic acid agents such as ASO to the central nervous system. However, the brain has a mechanism called the blood-brain barrier (hereinafter often referred to as "BBB") that selects and restricts substances that are transferred to the brain via blood. While this BBB mechanism protects the brain from harmful substances, it also acts as a barrier to drug delivery to the brain. Therefore, a method for delivering nucleic acid agents such as ASO to the central nervous system, including the brain, is required. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 053999 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a nucleic acid agent that can be efficiently delivered to the nervous system, particularly the central nervous system where drug delivery is prevented by the BBB mechanism, and that exerts an antisense effect on a target transcript at the delivery site, and a composition containing the same. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors have conducted extensive research and found that a nucleic acid complex annealed with an ASO and a complementary strand of an ASO bound to tocopherol or cholesterol or an analog thereof is efficiently delivered to the central nervous system and exhibits a high antisense effect there. Based on these findings, the present inventors have completed the present invention. That is, the present invention includes the following aspects.

[0008] [1] A nucleic acid complex or a salt thereof comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of a target transcript and has an antisense effect on the target transcript; The second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand, and (1) Tocopherol or its analogues, (2) Cholesterol or its analogues, or (3) Substituted or unsubstituted C 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of1 ~ 30 Alkoxy group It is combined with The nucleic acid complex or a salt thereof, wherein the first nucleic acid strand is annealed to the second nucleic acid strand. [2] (1) Tocopherol or its analogues, (2) Cholesterol or its analogues, or (3) Substituted or unsubstituted C 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of 1 ~ 30 Alkoxy group wherein the nucleic acid strand or a salt thereof is bound to a target transcription product, the nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of a target transcription product, and has an antisense effect on the target transcription product. [3] The tocopherol or its analogue has the following formula (I): [ka] (In the formula, R 1 is a substituted or unsubstituted C 1 ~ 30 or a substituted or unsubstituted C 2 ~ 30 (represents an alkenyl group) The nucleic acid complex, nucleic acid strand, or salt thereof according to [1] or [2], [4] The cholesterol or its analogue is represented by the following formulae (II), (V), (VI), and (VII): [ka] (In the formula, Ring A represents a substituted or unsubstituted cyclohexane or a substituted or unsubstituted benzene; Ring B represents substituted or unsubstituted cyclohexene, substituted or unsubstituted cyclohexane, or substituted or unsubstituted cyclohexadiene; Ring C represents a substituted or unsubstituted cyclohexene or a substituted or unsubstituted cyclohexane; R 2 is a hydrogen atom, a substituted or unsubstituted C 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 Alkenyl groups of the formula: 1 ~ 6 Alkyl-carbonyl groups, substituted or unsubstituted C 1 ~ 6 represents an alkyl-carbonyl-oxy group or an oxo group; R 3 represents a hydrogen atom, R 2 and R 3 may be taken together to form a substituted or unsubstituted 1,6-dioxaspiro[4.5]decane ring; L 1 is -O-, -NH-, [ka] or [ka] ), [ka] (In the formula, R 4 is a substituted or unsubstituted C 1 ~ 30 or a substituted or unsubstituted C 2 ~ 30 represents an alkenyl group; [ka] (In the formula, R 5 and R 6 each independently represents a substituted or unsubstituted C 1 ~ 30 or a substituted or unsubstituted C 2 ~ 30 represents an alkenyl group, and

[0009] [ka] (In the formula, R 7 is a hydroxy group, a substituted or unsubstituted C 1 ~ 30 Alkyl-carbonyl-oxy group, or substituted or unsubstituted C 1 ~ 30 alkenyl-carbonyl-oxy group) The nucleic acid complex, the nucleic acid chain, or a salt thereof according to any one of [1] to [3], represented by a formula selected from the group consisting of: [5] The cholesterol or its analogue has the following formula (IIa): [ka] (In the formula, Ring A represents a substituted or unsubstituted cyclohexene, a substituted or unsubstituted cyclohexane, or a substituted or unsubstituted cyclohexadiene; R 1 is a substituted or unsubstituted C 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 Alkenyl groups of the formula: 1 ~ 6 Alkyl-carbonyl groups, substituted or unsubstituted C 1 ~6 represents an alkyl-carbonyl-oxy group or an oxo group; L 1 is -O-, -NH-, [ka] or [ka] (represents The nucleic acid complex, nucleic acid strand, or salt thereof according to [1] or [2], [6] The cholesterol or its analogue is represented by the following formulae (IIa-1), (IIa-2), (IIa-3), and (IIa-4): [ka] (IIa-1), TIFF0007669602000012.tif3265 (IIa-2), TIFF0007669602000013.tif3265 (IIa-3), TIFF0007669602000014.tif3865 (IIa-4) (In the formula, L 1 is -O-, -NH-, [ka] or [ka] ), The nucleic acid complex, nucleic acid strand, or salt thereof according to [1] or [2], represented by a formula selected from the group consisting of: [7] The second nucleic acid strand has the following formula (VIII): [ka] (In the formula, L 2 is a substituted or unsubstituted C 1 ~ 12 an alkylene group of the formula: 3 ~ 8 Cycloalkylene group, -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 - or CH(CH 2 -OH)-CH 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 - represents L 3 represents -NH- or a bond, L 4 is a substituted or unsubstituted C 1 ~ 12 an alkylene group of the formula: 3 ~ 8 a cycloalkylene group, -(CH 2 ) 2 -[O-(CH 2 ) 2 ] m - or a bond, where m is an integer of 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond. Through a linker represented by (1) Tocopherol or its analogues, (2) Cholesterol or its analogues, or (3) Substituted or unsubstituted C 1 ~ 30Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of 1 ~ 30 Alkoxy group The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of [1] to [6], wherein the nucleic acid complex, nucleic acid strand, or salt thereof is bound to: [8] The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of [1] to [7], wherein the first nucleic acid strand contains at least four consecutive deoxyribonucleosides. [9] The nucleic acid complex, nucleic acid strand, or a salt thereof according to any one of [1] to [8], wherein the first nucleic acid strand is a gapmer.

[10] The nucleic acid complex, nucleic acid strand, or salt thereof according to [8] or [9], wherein the second nucleic acid strand contains at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the first nucleic acid strand.

[11] The nucleic acid complex, nucleic acid strand, or a salt thereof according to any one of [1] to [7], wherein the first nucleic acid strand is a mixmer.

[12] The nucleic acid complex, nucleic acid strand, or salt thereof according to any one of [1] to

[11] , wherein the first nucleic acid strand has a length of 13 to 20 bases.

[13] The nucleic acid complex, the nucleic acid strand, or the salt thereof according to any one of [1] to

[12] , wherein the second nucleic acid strand does not contain a natural ribonucleoside.

[14] The nucleic acid complex, nucleic acid chain, or salt thereof according to any one of [1] to

[13] , wherein the nucleic acid portion of the second nucleic acid strand is composed of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside bonds.

[15] A composition for regulating expression or editing of a target transcript in the central nervous system of a subject, comprising the nucleic acid complex, nucleic acid strand, or a salt thereof according to any one of [1] to

[14] .

[16] The composition described in

[15] , which is for treating a central nervous system disease in a subject.

[17] The composition described in

[16] , wherein the central nervous system disease is an immune-mediated central nervous system disease.

[18] A composition for delivering a drug to the central nervous system of a subject, comprising the nucleic acid complex, the nucleic acid strand, or a salt thereof according to any one of [1] to

[14] .

[19] The composition according to any one of

[15] to

[18] , wherein the central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brainstem, the cerebellum, and the spinal cord.

[20] The composition according to any of

[15] to

[18] , wherein the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

[21] The composition according to any one of

[15] to

[20] , which is for intravenous or subcutaneous administration.

[22] The composition according to any one of

[15] to

[21] , comprising 5 mg / kg or more of the nucleic acid complex, nucleic acid strand, or a salt thereof in a single dose.

[23] The composition according to any one of

[15] to

[22] , wherein the nucleic acid complex, nucleic acid strand, or salt thereof passes through the blood-brain barrier (BBB).

[24] The composition described in

[17] , wherein the immune-mediated central nervous system disease is a microglia-associated disease.

[25] The composition described in

[24] , wherein the microglia-related disease is Alzheimer's disease, multiple sclerosis, ALS, or neuropathic pain.

[26] The composition described in any of

[15] to

[25] for regulating expression or editing of a target transcript in microglia. This specification includes the disclosure of Japanese Patent Application No. 2018-055372, which is the priority basis of this application. Effect of the Invention

[0010] INDUSTRIAL APPLICABILITY The present invention makes it possible to provide a nucleic acid agent that can be efficiently delivered to the central nervous system and that exerts an antisense effect at the delivery site, and a composition containing the same. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 is a schematic diagram showing an example of a specific embodiment of a nucleic acid complex used in the present invention. In this diagram, four patterns are shown according to the binding position of tocopherol or cholesterol in the second nucleic acid strand. FIG. 1a shows a nucleic acid complex in which tocopherol is bound to the 5' end of the second nucleic acid strand. FIG. 1b shows a nucleic acid complex in which cholesterol is bound to the 5' end of the second nucleic acid strand. FIG. 1c shows a nucleic acid complex in which tocopherol is bound to the 3' end of the second nucleic acid strand. FIG. 1d shows a nucleic acid complex in which cholesterol is bound to the 3' end of the second nucleic acid strand. [Diagram 2] Figure 2 shows an example of the general mechanism of the antisense method. In the figure, "X" indicates the site of suppression or inhibition in the process from gene expression to translation. The figure within the dashed line is a schematic diagram showing how the heteroduplex portion is recognized by RNase H and the mRNA of the target gene is degraded. [Diagram 3] FIG. 3 shows the structures of various bridged nucleic acids. [Figure 4] FIG. 4 shows the structures of various natural and non-natural nucleotides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A first aspect of the present invention is a nucleic acid complex, more preferably a blood-brain barrier penetrating nucleic acid complex. The nucleic acid complex comprises a first nucleic acid strand and a second nucleic acid strand. The second nucleic acid strand is a nucleotide strand comprising a base sequence complementary to that of the first nucleic acid strand. In the nucleic acid complex, the first nucleic acid strand is annealed to the second nucleic acid strand. In one embodiment, the second nucleic acid strand is selected from the group consisting of (1) tocopherol or an analog thereof, (2) cholesterol or an analog thereof, or (3) substituted or unsubstituted C. 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of 1 ~ 30(In this specification, the above (1) to (3) are often collectively referred to as "tocopherol or cholesterol or an analog thereof, etc.").

[0013] A representative schematic diagram of the nucleic acid complex is shown in FIG. 1. FIG. 1a shows a nucleic acid complex in which tocopherol is bound to the 5'-end of the second nucleic acid strand. FIG. 1b shows a nucleic acid complex in which cholesterol is bound to the 5'-end of the second nucleic acid strand. FIG. 1c shows a nucleic acid complex in which tocopherol is bound to the 3'-end of the second nucleic acid strand. FIG. 1d shows a nucleic acid complex in which cholesterol is bound to the 3'-end of the second nucleic acid strand. However, as described later, tocopherol or cholesterol or an analog thereof may be bound to the 5'-end, 3'-end or both ends of the second nucleic acid strand, or may be bound to an internal nucleotide of the second nucleic acid strand.

[0014] In one embodiment, the first nucleic acid strand is a nucleotide strand that comprises a base sequence capable of hybridizing to at least a part of a target transcript. In a specific embodiment, the first nucleic acid strand is a nucleotide strand that has an antisense effect on the transcript of a target gene or the target transcript.

[0015] In one embodiment, the present invention relates to a method for the preparation of a medicament ... 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of 1 ~ 30 The present invention relates to a single-stranded nucleic acid chain bonded to an alkoxy group of the above. The nucleic acid chain corresponds to the first nucleic acid chain of the nucleic acid complex, and is a nucleotide chain containing a base sequence capable of hybridizing to at least a part of a target transcription product. In a specific embodiment, the nucleic acid chain is a nucleotide chain having an antisense effect on a transcription product of a target gene or a target transcription product.

[0016] (Definition of terms) As used herein, the term "target transcript" refers to any RNA that can be the target of the nucleic acid complex of the present invention and is synthesized by DNA-dependent RNA polymerase. In general, the term refers to a transcript of a target gene. Specifically, the term may include mRNA (including mature mRNA, mRNA precursor, mRNA not modified by base, etc.) transcribed from a target gene, and non-coding RNA (ncRNA) such as miRNA.

[0017] As used herein, the term "target gene" is not particularly limited, but may be, for example, a gene derived from an organism into which the nucleic acid complex of the present invention is introduced, such as a gene whose expression increases in various diseases. The target transcription product includes mRNA transcribed from genomic DNA encoding the target gene, as well as unmodified mRNA and unprocessed mRNA precursors. The "target transcription product" may include not only mRNA but also non-coding RNA (ncRNA) such as miRNA. More generally, the "transcription product" may be any RNA synthesized by a DNA-dependent RNA polymerase. In one embodiment, the "target transcript" may be, for example, scavenger receptor B1 (often referred to herein as "SR-B1 mRNA") or metastasis associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1") non-coding RNA. SEQ ID NO: 3 shows the nucleotide sequence of mouse Malat1 non-coding RNA, and SEQ ID NO: 4 shows the nucleotide sequence of human Malat1 non-coding RNA. SEQ ID NO: 5 shows the nucleotide sequence of mouse SR-B1 mRNA, and SEQ ID NO: 6 shows the nucleotide sequence of human SR-B1 mRNA. SEQ ID NO: 7 shows the nucleotide sequence of mouse DMPK mRNA, and SEQ ID NO: 8 shows the nucleotide sequence of human DMPK mRNA. In addition, in SEQ ID NOs: 1 to 8, the nucleotide sequence of mRNA is replaced with the nucleotide sequence of DNA. The nucleotide sequence information of these genes and transcripts can be obtained from a known database such as the NCBI (National Center for Biotechnology Information) database.

[0018] As used herein, "antisense oligonucleotide (ASO)" or "antisense nucleic acid" refers to a single-stranded oligonucleotide that contains a complementary base sequence capable of hybridizing to at least a portion of a target transcript, for example, any target region, and can suppress and control the expression of the transcript of the target gene or the level of the target transcript by the antisense effect. In the nucleic acid complex of the present invention, the first nucleic acid strand functions as an ASO, and the target region may include a 3'UTR, a 5'UTR, an exon, an intron, a coding region, a translation initiation region, a translation termination region, or any other nucleic acid region. The target region of the target transcript can be at least 8 bases long, for example, 10 to 35 bases long, 12 to 25 bases long, 13 to 20 bases long, 14 to 19 bases long, or 15 to 18 bases long.

[0019] "Antisense effect" refers to the effect of regulating the expression or editing of a target transcript by hybridizing an ASO to the target transcript (e.g., RNA sense strand). "Regulating expression or editing of a target transcript" refers to suppression or reduction of the expression of a target gene or the expression level of a target transcript (herein, "expression level of a target transcript" is often referred to as "level of a target transcript"), inhibition of translation, splicing function modification effect (e.g., exon skipping, etc.), or degradation of a transcript. For example, as shown in Figure 2, in the inhibition of translation, when an oligonucleotide (e.g., RNA) is introduced into a cell as an ASO, the ASO binds to the mRNA, etc., which is the transcript of the target gene, to form a partial duplex. This partial duplex serves as a cover to prevent translation by ribosomes, thereby inhibiting the expression of the protein encoded by the target gene at the translation level (Figure 2, dashed line, x mark). On the other hand, when an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in the degradation of the mRNA of the target gene and the inhibition of the expression of the protein encoded by the target gene at the expression level (inside the dashed line in FIG. 2). This is referred to as the "RNase H-dependent pathway". Furthermore, in certain instances, the antisense effect can be achieved by targeting an intron of a pre-mRNA. The antisense effect can also be achieved by targeting an miRNA, in which case the function of the miRNA is inhibited and the expression of the gene whose expression the miRNA normally controls can be increased. In one embodiment, the modulation of the expression of the target transcript can be a reduction in the amount of the target transcript.

[0020] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a monomeric nucleoside or nucleotide, an oligomer, an oligonucleotide, or a polymer, a polynucleotide.

[0021] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is usually, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is usually a heterocyclic base moiety, including, but not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0022] "Nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides that contain a pentofuranosyl sugar, the phosphate group is usually linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0023] "Oligonucleotide" refers to a linear oligomer formed by covalently linking several to several tens of hydroxyl groups and phosphate groups of the sugar moieties between adjacent nucleotides. "Polynucleotide" refers to a linear polymer formed by linking several tens or more, preferably several hundreds or more, of nucleotides more numerous than an oligonucleotide by the covalent bond. Within an oligonucleotide or polynucleotide structure, the phosphate group is generally considered to form an internucleoside bond.

[0024] The term "nucleic acid strand" or simply "strand" as used herein refers to an oligonucleotide or polynucleotide. A nucleic acid strand can be made full length or partial by chemical synthesis, for example using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand can include natural and / or non-natural nucleotides.

[0025] In this specification, the term "natural nucleoside" refers to a nucleoside that exists in nature. For example, ribonucleosides consisting of ribose and bases such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and bases such as adenine, cytosine, guanine, or thymine, can be mentioned. Ribonucleosides found in RNA and deoxyribonucleosides found in DNA are sometimes referred to as "DNA nucleosides" and "RNA nucleosides", respectively. Similarly, "natural nucleotide" refers to a nucleotide that exists in nature, and a molecule in which a phosphate group is covalently bonded to the sugar portion of the natural nucleoside. For example, ribonucleotides, which are known as building blocks of RNA and in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, which are known as building blocks of DNA and in which a phosphate group is bonded to a deoxyribonucleoside, can be mentioned.

[0026] As used herein, "unnatural nucleoside" refers to any nucleoside other than natural nucleosides. For example, it includes modified nucleosides and nucleoside mimetics. As used herein, "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase. Nucleic acid chains, including unnatural oligonucleotides, are often preferred over natural forms due to desirable properties such as, for example, enhanced cellular uptake, enhanced affinity to nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0027] As used herein, a "mimetic" refers to a functional group that replaces the sugar, nucleobase, and / or internucleoside linkage. In general, a mimetic is used in place of a sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. A "nucleoside mimetic" includes structures that are used to replace the sugar, or to replace the sugar and base, or to replace the linkage between the monomeric subunits that make up an oligomeric compound, at one or more positions of the oligomeric compound. An "oligomeric compound" refers to a polymer of linked monomeric subunits that is at least hybridizable to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, such as nucleoside mimetics having non-furanose sugar units.

[0028] As used herein, the term "bicyclic nucleoside" refers to a modified nucleoside that contains a bicyclic sugar moiety. Nucleic acids that contain a bicyclic sugar moiety are generally referred to as bridged nucleic acids (BNAs). Nucleosides that contain a bicyclic sugar moiety may also be referred to as "bridged nucleosides" herein. Some examples of bridged nucleic acids are shown in Figure 3.

[0029] A bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those of skill in the art. One subgroup of nucleic acids (BNAs) that contain bicyclic sugars are 4'-(CH 2 ) p -O-2',4'-(CH 2 ) p -CH 2 -2',4'-(CH 2 ) p -S-2',4'-(CH 2 ) p -O CH 2 O-2',4'-(CH 2 ) n -N(R 3 )-O-(CH 2 ) mIn the formula, p, m, and n each represent an integer of 1 to 4, an integer of 0 to 2, and an integer of 1 to 3, respectively; 3 can be described as having the 2'- and 4'-carbon atoms bridged by a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, and a unit substituent (fluorescent or chemiluminescent labeling molecule, a functional group having nucleic acid cleavage activity, an intracellular or intranuclear localization signal peptide, etc.). Furthermore, with respect to BNAs according to certain embodiments, an OR on the 3'-carbon atom can be described as having the 2'- and 4'-carbon atoms bridged by a 2 Substituents and OR on the 5' carbon atom 1 In the substituent, R 1 and R 2 are typically hydrogen atoms, but may be the same or different from each other, and may further be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R 4 )R 5 [Where, R 4 and R 5 may be the same or different and each represent a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms. Non-limiting examples of such BNAs include methyleneoxy(4'-CH 2 -O-2')BNA (LNA (Locked Nucleic Acid®, also known as 2',4'-BNA), e.g., α-L-methyleneoxy (4'-CH 2 -O-2')BNA or β-D-methyleneoxy(4'-CH 2 -O-2')BNA, ethyleneoxy (4'-(CH 2 ) 2-O-2')BNA (also known as ENA), β-D-thio(4'-CH 2 -S-2')BNA, aminooxy (4'-CH 2 -ON(R 3 )-2')BNA, oxyamino(4'-CH 2 -N(R 3 )-O-2')BNA(2',4'-BNA NC Also known as 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH 3 )-O-2')BNA (also known as cEt BNA), (4'-CH(CH 2 OCH 3 )-O-2') BNAs (also known as cMOE BNAs), amide BNAs (4'-C(O)-N(R)-2') BNAs (R = H, Me) (also known as AmNAs), 2'-O,4'-C-spirocyclopropylene bridged nucleic acids (also known as scpBNAs) and other BNAs known to those of skill in the art. 2 Bicyclic nucleosides having an -O-2') bridge are sometimes referred to as "LNA nucleosides."

[0030] As used herein, the term "non-natural nucleotide" refers to any nucleotide other than a naturally occurring nucleotide, including modified nucleotides and nucleotide mimetics. As used herein, the term "modified nucleotide" refers to a nucleotide having any one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.

[0031] "Nucleotide mimics" include structures used to replace nucleosides and linkages at one or more positions of an oligomeric compound. Nucleotide mimics include, for example, peptide nucleic acids or morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide Nucleic Acids (PNA) are nucleotide mimics with a backbone in which N-(2-aminoethyl)glycine is linked by amide bonds instead of sugars. An example of the structure of a morpholino nucleic acid is shown in FIG. 4. Nucleic acid strands, including non-natural oligonucleotides herein, often have desirable properties, such as enhanced cellular uptake, enhanced affinity to nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Thus, they are preferred over natural nucleotides.

[0032] As used herein, the term "modified internucleoside bond" refers to an internucleoside bond having a substitution or any change from a naturally occurring internucleoside bond (i.e., a phosphodiester bond). Modified internucleoside bond includes phosphorus-containing internucleoside bond containing a phosphorus atom and non-phosphorus-containing internucleoside bond not containing a phosphorus atom. Representative phosphorus-containing internucleoside bond includes, but is not limited to, phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphotriester bond, alkylphosphonate bond, alkylthiophosphonate bond, boranophosphate bond, and phosphoroamidate bond. A phosphorothioate bond is an internucleoside bond in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing bond are well known. The modified internucleoside bond is preferably a bond having a higher nuclease resistance than a naturally occurring internucleoside bond.

[0033] As used herein, "modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleobases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8-bromoguanine. A preferred modified nucleobase is 5-methylcytosine. "Unmodified nucleobase" or "unmodified base" refers to the same as natural nucleobases, and refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0034] As used herein, "modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)). As used herein, a nucleic acid strand may contain one or more modified nucleosides, optionally including modified sugars. Sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the nucleic acid strand. A nucleoside may contain a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), and the replacement of ribosyl ring oxygen atoms with S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently selected from H, C, H ... 1 -C 12 As used herein, examples of nucleosides having modified sugar moieties include, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F (2'-fluoro), 2'-OCH 3 (2'-OMe group or 2'-O-methyl group), and 2'-O(CH 2 ) 2 OCH 3Nucleosides containing a substituent are also included. The substituent at the 2' position can also be allyl, amino, azido, thio, -O-allyl, -OC 1 -C 10 Alkyl, -OCF 3 , -O(CH 2 ) 2 SCH 3 , -O(CH 2 ) 2 -ON(Rm)(Rn), and O-CH 2 -C(=O)-N(Rm)(Rn), each Rm and Rn can be independently selected from H or substituted or unsubstituted C 1 -C 10 By "2'-modified sugar" herein is meant a furanosyl sugar modified at the 2' position.

[0035] Methods for the preparation of modified sugars are well known to those of skill in the art. In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) may be maintained for hybridization with an appropriate nucleic acid target.

[0036] Generally, modifications can be made such that nucleotides in the same chain can be independently modified differently. The same nucleotides can also have modified internucleoside linkages (e.g., phosphorothioate linkages) and modified sugars (e.g., 2'-O-methyl modified sugars or bicyclic sugars) to confer resistance to enzymatic cleavage. The same nucleotides can also have modified nucleobases (e.g., 5-methylcytosine) and modified sugars (e.g., 2'-O-methyl modified sugars or bicyclic sugars).

[0037] The number, type and position of non-natural nucleotides in the nucleic acid chain may affect the antisense effect and the like provided by the nucleic acid complex of the present invention. The selection of the modification may vary depending on the sequence of the target gene and the like, but a person skilled in the art can determine a suitable embodiment by referring to the descriptions in the literature related to the antisense method (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). Furthermore, when the antisense effect of the modified nucleic acid complex is measured, if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification), the related modification can be evaluated.

[0038] The term "complementary" as used herein means a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds. In the present invention, the first nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the target transcription product (e.g., the transcription product of the target gene), and it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. Similarly, the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the first nucleic acid strand, and it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.

[0039] As used herein, the term "blood-brain barrier (BBB)" is, as described above, a mechanism that selects and restricts substances that pass into the brain, and plays a role in protecting the brain from harmful substances.

[0040] The term "central nervous system" as used herein refers to tissues consisting of the brain and spinal cord, which together with the peripheral nervous system constitute the nervous system. The brain includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei), and brainstem (midbrain, substantia nigra, pons, medulla oblongata). The spinal cord includes the cervical spinal cord, thoracic spinal cord, lumbar spinal cord, sacral spinal cord, and caudal spinal cord. The central nervous system in this specification may be any of these regions, but is preferably the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical spinal cord, thoracic spinal cord, or lumbar spinal cord.

[0041] As used herein, "salts thereof" refers to salts of the nucleic acid complex of the present invention that are physiologically and pharma- ceutically acceptable salts of the nucleic acid complex of the present invention, i.e., salts that retain the desired biological activity of the nucleic acid complex and do not impart undesired toxicological effects. Examples of such salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic salts such as ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylammonium salts, tris(hydroxymethyl)aminomethane salts, diolamine salts, and meglumine salts. hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, phosphate; lower alkane sulfonate such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate; arylsulfonate such as benzenesulfonate, p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate; and amino acid salts such as glycine, lysine, arginine, ornithine, glutamate, aspartate.

[0042] In certain embodiments, the nucleic acid complex of the present invention includes any pharma- ceutically acceptable salt of the nucleic acid complex, an ester of the nucleic acid complex, or a salt of the ester. Suitable pharma- ceutically acceptable salts include, but are not limited to, sodium, potassium, and meglumine salts.

[0043] (Configuration of the first and second nucleic acid strands) In one aspect, the present invention relates to a nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand.

[0044] The first nucleic acid strand is a single-stranded oligonucleotide strand that contains a base sequence capable of hybridizing to at least a portion of a target transcript and exerts an antisense effect on the target transcript.

[0045] The second nucleic acid strand is a single-stranded oligonucleotide strand containing a base sequence complementary to the first nucleic acid strand. The second nucleic acid strand is bound to tocopherol or cholesterol or an analog thereof. In the nucleic acid complex, the second nucleic acid strand is annealed to the first nucleic acid strand via hydrogen bonds of complementary base pairs.

[0046] In one embodiment, the present invention relates to a method for the preparation of a medicament ... 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of 1 ~ 30 The structure of the single-stranded nucleic acid is the same as that of the first nucleic acid strand, except that the single-stranded nucleic acid is linked to tocopherol, cholesterol, or an analog thereof. The structure of tocopherol, cholesterol, or an analog thereof, and the form of linkage with the nucleic acid strand are as described in the nucleic acid complex in this specification.

[0047] The base length of the first and second nucleic acid strands may be, but is not limited to, at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base length of the first and second nucleic acid strands may be 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The first and second nucleic acid strands may be about 100 bases long, or 10 to 35 bases long, 12 to 25 bases long, 13 to 20 bases long, 14 to 19 bases long, or 15 to 18 bases long. The first and second nucleic acid strands may be the same length or different lengths (e.g., one of them may be 1-3 bases shorter or longer). The double-stranded structure formed by the first and second nucleic acid strands may include a bulge. The length selection may be determined by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost, synthesis yield, etc.

[0048] The internucleoside bond in the first and second nucleic acid strands may be naturally occurring internucleoside bond and / or modified internucleoside bond. It is preferred, without limitation, that at least one, at least two, or at least three internucleoside bond from the end (5' end, 3' end, or both ends) of the first and / or second nucleic acid strand are modified internucleoside bond. Here, for example, the two internucleoside bond from the end of the nucleic acid strand means the internucleoside bond closest to the end of the nucleic acid strand and the internucleoside bond adjacent thereto and located on the opposite side to the end. Modified internucleoside bond in the terminal region of the nucleic acid strand is preferred because it can suppress or inhibit undesired degradation of the nucleic acid strand. In one embodiment, all internucleoside bond in the first and / or second nucleic acid strand may be modified internucleoside bond. The modified internucleoside bond may be a phosphorothioate bond.

[0049] At least one (e.g., three) internucleoside bond from the 3'-end of the second nucleic acid strand may be a modified internucleoside bond such as a phosphorothioate bond, which has high RNase resistance. When the 3'-end of the second nucleic acid strand contains a modified internucleoside bond such as a phosphorothioate modification, the gene suppression activity of the double-stranded nucleic acid complex is improved, which is preferable.

[0050] At the 5'-end and 3'-end of the second nucleic acid strand, the internucleoside bonds of 2 to 6 bases at the ends not bound to tocopherol or cholesterol or an analogue thereof may be modified internucleoside bonds (eg, phosphorothioate bonds).

[0051] At least one (e.g., three) nucleosides from the 3'-terminus of the second nucleic acid strand may be, for example, a modified nucleoside with high RNase resistance, such as 2'F-RNA, 2'-OMe, etc. When the 3'-terminus of the second nucleic acid strand contains a modified nucleoside, such as 2'F-RNA, 2'-OMe, etc., it is preferable because the gene suppression activity of the double-stranded nucleic acid complex is enhanced.

[0052] At the 5'-end and 3'-end of the second nucleic acid strand, the 1 to 5 nucleosides at the ends not bound to tocopherol or cholesterol or an analogue thereof may be modified nucleosides such as 2'F-RNA having high RNase resistance.

[0053] The nucleosides in the first and second nucleic acid strands can be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and / or non-natural nucleosides.

[0054] In the present specification, the base sequence of the first nucleic acid strand is complementary to at least a part of the base sequence of the target transcript, and therefore can hybridize (or anneal) to the target transcript. The complementarity of the base sequence can be determined by using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to the target transcript, for example, based on information on the base sequence of the target gene.

[0055] The hybridization conditions may be various stringent conditions, such as low stringent conditions and high stringent conditions. Low stringent conditions may be conditions of relatively low temperature and high salt concentration, such as 30°C, 2xSSC, 0.1% SDS. High stringent conditions may be conditions of relatively high temperature and low salt concentration, such as 65°C, 0.1xSSC, 0.1% SDS. The stringency of hybridization can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0056] The first nucleic acid strand may contain at least 4, at least 5, at least 6, or at least 7 consecutive nucleosides that are recognized by RNase H when hybridized to the target transcript. Generally, the region may contain 4 to 20, 5 to 16, or 6 to 12 consecutive nucleosides. As the nucleoside recognized by RNase H, for example, a natural deoxyribonucleoside may be used. Modified deoxyribonucleosides and suitable nucleosides containing other bases are well known in the art. It is also known that nucleosides having a hydroxy group at the 2' position, such as ribonucleosides, are unsuitable as the nucleoside. The suitability of the nucleoside for use in this region containing "at least 4 consecutive nucleosides" can be easily determined. In one embodiment, the first nucleic acid strand may contain at least 4 consecutive deoxyribonucleosides.

[0057] In one embodiment, the entire length of the first nucleic acid strand is not composed of only natural ribonucleosides. Preferably, the first nucleic acid strand does not contain any natural ribonucleosides, or the half or less of the entire length of the first nucleic acid strand is natural ribonucleosides.

[0058] In one embodiment, the second nucleic acid strand may contain at least four consecutive ribonucleosides complementary to the at least four consecutive nucleosides (e.g., deoxyribonucleosides) in the first nucleic acid strand, such that the second nucleic acid strand forms a partial DNA-RNA heteroduplex with the first nucleic acid strand and is recognized and cleaved by RNase H. The at least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring internucleoside bonds, i.e., phosphodiester bonds.

[0059] All nucleosides in the second nucleic acid strand may be composed of ribonucleosides and / or modified nucleosides. All nucleosides in the second nucleic acid strand may be composed of deoxyribonucleosides and / or modified nucleosides, or may not contain ribonucleosides.

[0060] The first and / or second nucleic acid strands constituting the nucleic acid complex of the present invention may be gapmers. As used herein, the term "gapmer" refers to a single-stranded nucleic acid consisting of a central region (DNA gap region) and 5' and 3' wing regions arranged on both sides of the 5' and 3' ends of the central region. The central region in a gapmer contains at least four consecutive deoxyribonucleosides, and the 5' and 3' wing regions contain non-natural nucleosides. When the non-natural nucleosides constituting the 5' and 3' wing regions contain or consist of bridged nucleosides, the gapmer is particularly referred to as a "BNA / DNA gapmer". When the non-natural nucleosides constituting the 5' and 3' wing regions contain or consist of peptide nucleic acids, the gapmer is particularly referred to as a "peptide nucleic acid gapmer". When the non-natural nucleosides constituting the 5' wing region and the 3' wing region contain or consist of peptide nucleic acid, the gapmer is particularly referred to as a "morpholino nucleic acid gapmer". The number of bridged nucleosides contained in the 5' wing region and the 3' wing region may be 2 or 3. The bridged nucleosides contained in the 5' wing region and the 3' wing region may be contiguous or discontinuous in the 5' wing region and the 3' wing region. The bridged nucleoside may further contain a modified nucleic acid base (e.g., 5-methylcytosine). When the bridged nucleoside is an LNA nucleoside, the gapmer is referred to as an "LNA / DNA gapmer". The base length of the 5' wing region and the 3' wing region may be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, or 3 to 5 bases long, independently of each other. The 5' wing region and the 3' wing region may contain at least one unnatural nucleoside and may further contain natural nucleosides.

[0061] The first nucleic acid strand and / or the second nucleic acid strand constituting the gapmer may be composed of, in order from the 5'-terminus, a bridged nucleoside having a length of 2 to 7 bases or 3 to 5 bases, a ribonucleoside or deoxyribonucleoside having a length of 4 to 15 bases or 8 to 12 bases, and a bridged nucleoside having a length of 2 to 7 bases or 3 to 5 bases.

[0062] The first and / or second nucleic acid strands constituting the nucleic acid complex of the present invention may be mixmers. As used herein, the term "mixmer" refers to a nucleic acid strand that contains alternating natural and non-natural nucleosides of periodic or random segment lengths, and does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. In a mixmer, a mixmer in which the non-natural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is particularly referred to as a "BNA / DNA mixmer." In a mixmer, a mixmer in which the non-natural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is particularly referred to as a "peptide nucleic acid / DNA mixmer." In the mixmer, the non-natural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside, and the mixmer is specifically referred to as a "morpholino nucleic acid / DNA mixmer". The mixmer is not limited to only contain two kinds of nucleosides. The mixmer can contain any number of kinds of nucleosides, whether natural or modified nucleosides or nucleoside mimics. For example, it may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside). The bridged nucleoside may further contain a modified nucleobase (e.g., 5-methylcytosine).

[0063] At least one, at least two, at least three, or at least four nucleosides from the termini (5'-terminus, 3'-terminus, or both termini) of the second nucleic acid strand may be modified nucleosides. The modified nucleosides may include modified sugars and / or modified nucleobases. The modified sugars may be 2'-modified sugars (e.g., sugars including a 2'-O-methyl group). The modified nucleobases may also be 5-methylcytosines.

[0064] The second nucleic acid strand may be composed of, in order from the 5'-terminus, a 2- to 7- or 3- to 5-base long modified nucleoside (e.g., a modified nucleoside containing a 2'-modified sugar), a 4- to 15- or 8- to 12-base long ribonucleoside or deoxyribonucleoside (optionally linked via a modified internucleoside bond), and a 2- to 7- or 3- to 5-base long modified nucleoside (e.g., a modified nucleoside containing a 2'-modified sugar). In this case, the first nucleic acid strand may be a gapmer.

[0065] The first and second nucleic acid strands may comprise, in whole or in part, nucleoside or nucleotide mimetics. The nucleotide mimetics may be peptide nucleic acids and / or morpholino nucleic acids. The first nucleic acid strand may comprise at least one modified nucleoside. The modified nucleoside may comprise a 2'-modified sugar. The 2'-modified sugar may be a sugar comprising a 2'-O-methyl group.

[0066] The first and second nucleic acid strands may contain any combination of the modified internucleoside linkages and modified nucleosides described above.

[0067] The second nucleic acid strand is selected from the group consisting of (1) tocopherol or an analog thereof, (2) cholesterol or an analog thereof, and (3) substituted or unsubstituted C 1 ~ 30 Alkyl groups of the formula: 2 ~ 30 or a substituted or unsubstituted alkenyl group of 1 ~ 30Tocopherol or cholesterol or analogues thereof can be produced by a method known per se by those skilled in the art.

[0068] As used herein, the term "analog" refers to a compound having similar structure and properties that has the same or a similar basic skeleton. Analogs include, for example, biosynthetic intermediates, metabolic products, etc. Whether a compound is an analog of another compound can be determined by one of ordinary skill in the art.

[0069] The tocopherol may be selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol. Analogs of tocopherol include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, etc. Preferably, the tocopherol is α-tocopherol.

[0070] Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol backbone, including, but not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0071] The second nucleic acid strand bound to tocopherol or an analogue thereof may have a group represented by the following general formula (I):

[0072] [ka] [In the formula, R 1 is a substituted or unsubstituted C 1 ~ 30 (e.g., methyl, 4,8,12-trimethyltridecyl) or substituted or unsubstituted C 2 ~ 30alkenyl group (e.g., 4,8,12-trimethyl-3,7,11-tridecatrien-1-yl) (wherein the substitution is preferably by a halogen atom).

[0073] The second nucleic acid strand bound to cholesterol or an analogue thereof may have a group selected from the group consisting of the following general formulas (II), (V), (VI), and (VII): [ka] (In the formula, Ring A represents substituted or unsubstituted cyclohexane or substituted or unsubstituted benzene; ring B represents substituted or unsubstituted cyclohexene, substituted or unsubstituted cyclohexane, or substituted or unsubstituted cyclohexadiene; ring C represents substituted or unsubstituted cyclohexene or substituted or unsubstituted cyclohexane; R 2 is a hydrogen atom, a substituted or unsubstituted C 1 ~ 30 alkyl groups (e.g., ethyl, isopropyl, 1-methylpropyl, 1,5-dimethylhexyl, 4-ethyl-1,5-dimethylhexyl), substituted or unsubstituted C 2 ~ 30 Alkenyl groups (e.g., 1,5-dimethyl-4-hexen-1-yl, 1,4,5-trimethyl-2-hexen-1-yl, 4-ethyl-1,5-dimethyl-2-hexen-1-yl), substituted or unsubstituted C 1 ~ 6 Alkyl-carbonyl groups (e.g., 1-oxoethyl), substituted or unsubstituted C 1 ~ 6 R represents an alkyl-carbonyl-oxy group (e.g., (1-oxoheptyl)oxy) or an oxo group; 3 represents a hydrogen atom, R 2 and R 3may be taken together to form a substituted or unsubstituted 1,6-dioxaspiro[4.5]decane ring.

[0074] In the formula, L 1 is -O-, -NH-, [ka] or [ka] ), [ka] (In the formula, R 4 is a substituted or unsubstituted C 1 ~ 30 (e.g., 1,5-dimethylhexyl), or a substituted or unsubstituted C 2 ~ 30 represents an alkenyl group; [ka] (In the formula, R 5 and R 6 each independently represents a substituted or unsubstituted C 1 ~ 30 (e.g., butyl, 1,5-dimethylhexyl), or substituted or unsubstituted C 2 ~ 30 represents an alkenyl group, and [ka] (In the formula, R 7 is a hydroxy group, a substituted or unsubstituted C 1 ~ 30 Alkyl-carbonyl-oxy groups (e.g., (1-oxododecyl)oxy), or substituted or unsubstituted C 1~ 30 In the group selected from the group consisting of (II), (V), (VI), and (VII), any of the substitutions is preferably a halogen atom (e.g., a fluorine atom), C 1 ~ 3 Alkyl group (e.g., methyl), hydroxyl group, C 1 ~ 6 Alkyl-carbonyl group (e.g., 1-oxo-2-methylpropyl), 1-5 C substituted with 1-3 halogen atoms 1 ~ 3 Carbamoyl groups mono- or di-substituted with alkyl-substituted phenyl groups (e.g., [3,5-bis(trifluoromethyl)benzoyl]amino), or C 1 ~ 6 This can be done with an alkyl-carbonyl group (eg, 2-methyl-1-oxopropyl).

[0075] In one embodiment, the second nucleic acid strand comprises a substituted or unsubstituted C 1 ~ 30 Alkyl groups (e.g., heptadecyl, 16-methylheptadecyl, henicosyl), substituted or unsubstituted C 2 ~ 30 alkenyl groups (e.g., 2,6-dimethyl-1,3,5,7-octatetraen-1-yl, 8-heptadecen-1-yl, 8,11-heptadecadien-1-yl, 8,11,14-heptadecatrien-1-yl), or substituted or unsubstituted C 1 ~ 30 wherein the substituent is preferably a halogen atom or 1 to 5 C 1 ~ 3 This can be achieved by a cyclohexenyl group substituted with an alkyl group (eg, 2,6,6-trimethyl-1-cyclohexen-1-yl).

[0076] In one embodiment, the second nucleic acid strand bound to tocopherol or an analogue thereof may have a group represented by formula (IIa): [ka] (In the formula, Ring A represents a substituted or unsubstituted cyclohexene, a substituted or unsubstituted cyclohexane, or a substituted or unsubstituted cyclohexadiene; R 1 is a substituted or unsubstituted C 1 ~ 30 Alkyl groups (e.g., C 2 ~ 20、 C 4 ~ 16、 C 6 ~ 12 Or C 8 ~ 10 (alkyl group), substituted or unsubstituted C 2 ~ 30 Alkenyl groups (e.g., C 2 ~ 20、 C 4 ~ 16、 C 6 ~ 12 Or C 8 ~ 10 Alkenyl groups of the formula (I) and (II), substituted or unsubstituted C 1 ~ 6 Alkyl-carbonyl groups, substituted or unsubstituted C 1 ~ 6 represents an alkyl-carbonyl-oxy group or an oxo group; L 1 is -O-, -NH-, [ka] or [ka] (represents

[0077] In one embodiment, the second nucleic acid strand bound to tocopherol or an analogue thereof may have groups represented by the following formulae (IIa-1), (IIa-2), (IIa-3), and (IIa-4). [ka] (IIa-1) TIFF0007669602000029.tif3563 (IIa-2) TIFF0007669602000030.tif3263 (IIa-3) TIFF0007669602000031.tif3765 (IIa-4) (In the formula, L 1 is -O-, -NH-, [ka] or [ka] ), is selected from the group consisting of:

[0078] In one embodiment, the second nucleic acid strand bound to tocopherol or an analogue thereof does not have a group represented by the following general formula (IIa-3). [ka]

[0079] Tocopherol or cholesterol or an analog thereof may be linked to the 5'-end, 3'-end, or both ends of the second nucleic acid strand. Alternatively, tocopherol or cholesterol or an analog thereof may be linked to an internal nucleotide of the second nucleic acid strand. For example, cholesterol may be bound to the 5'-end of the second nucleic acid strand. In another embodiment, the second nucleic acid strand contains two or more tocopherols or cholesterol or an analog thereof, which may be linked to multiple positions of the second nucleic acid strand and / or may be linked as a group to one position of the second nucleic acid strand. Tocopherol or cholesterol or an analog thereof may be linked to the 5'-end and 3'-end of the second nucleic acid strand, one each. Cholesterol or an analog thereof may be linked to the 5'-end and 3'-end of the second nucleic acid strand, one each.

[0080] The bond between the second nucleic acid strand and tocopherol or cholesterol or its analogue may be direct or indirect. Direct bond means that two molecules are directly bonded. Indirect bond means that two molecules to be bonded are bonded via another substance.

[0081] In one embodiment, the bond between the second nucleic acid strand and tocopherol or cholesterol and / or its analogue or the like may be via a phosphate bond or a phosphorothioate bond to the 5' end, 3' end, or an internal nucleotide of the second nucleic acid strand.

[0082] In one embodiment, it is attached to the 5' end of the second nucleic acid strand via a phosphate ester or phosphorothioate bond.

[0083] In one embodiment, it is attached to the 5' end of the second nucleic acid strand via a phosphate bond.

[0084] When the second nucleic acid strand and tocopherol, cholesterol, or an analog thereof are indirectly bound to each other, they may be bound via a linking group (often referred to as a "linker" in this specification). The linker may be bound to the 5'-end, 3'-end, or an internal nucleotide of the second nucleic acid strand via a phosphate bond or a phosphorothioate bond.

[0085] In one embodiment, the linker is attached to the 5' end of the second nucleic acid strand via a phosphate ester or phosphorothioate bond.

[0086] In one embodiment, the linker is attached to the 5'-end of the second nucleic acid strand via a phosphate bond.

[0087] A specific example of the linker is a linker represented by the following general formula (VIII): [ka] (In the formula, L 2 is a substituted or unsubstituted C 1 ~ 12 Alkylene groups (e.g., propylene, hexylene, dodecylene), substituted or unsubstituted C 3 ~ 8 Cycloalkylene groups (e.g., cyclohexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 - or CH(CH 2 -OH)-CH 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 - represents L3 represents -NH- or a bond; L 4 is a substituted or unsubstituted C 1 ~ 12 Alkylene groups (e.g., ethylene, pentylene, heptylene, undecylene), substituted or unsubstituted C 3 ~ 8 Cycloalkylene groups (e.g., cyclohexylene), -(CH 2 ) 2 -[O-(CH 2 ) 2 ] m - or a bond, where m is an integer of 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond, wherein the substitution is preferably made by a halogen atom.

[0088] When the second nucleic acid strand and tocopherol or cholesterol or an analogue thereof are indirectly bound to each other, they may be bound to each other via a cleavable linker. The term "cleavable linker" refers to a linking group that can be cleaved under physiological conditions, for example, within a cell or an animal body (for example, within the human body). The cleavable linker may be selectively cleaved by endogenous enzymes such as nucleases and peptidases, under acidic conditions, under a reducing environment, etc. Specific examples of such linkers include amide bonds, ester bonds, phosphate ester bonds, ester bonds at one or both ends of phosphodiester bonds, carbamate bonds, and disulfide bonds, as well as nucleotide linkers such as natural DNA linkers.

[0089] On the other hand, when the second nucleic acid strand and tocopherol or cholesterol or an analogue thereof are indirectly bound to each other, they may be bound to each other via a non-cleavable linker. The term "non-cleavable linker" refers to a linking group that is not cleaved under physiological conditions. Examples of such non-cleavable linkers include a phosphorothioate bond, and a linker consisting of a modified or unmodified deoxyribonucleoside or a modified or unmodified ribonucleoside linked by a phosphorothioate bond.

[0090] The chain length of the cleavable linker or non-cleavable linker is not particularly limited in the case of a nucleic acid such as DNA or an oligonucleotide, but may usually be 1 to 20 bases long, 1 to 10 bases long, or 1 to 6 bases long.

[0091] In a specific embodiment, when the nucleic acid complex of the present invention contains optical isomers, stereoisomers, positional isomers, or rotational isomers, these are also contained in the nucleic acid complex of the present invention, and each can be obtained as a single product by a synthesis method or separation method known per se. For example, when an optical isomer is present in the nucleic acid complex of the present invention, the optical isomer separated from the compound is also included in the nucleic acid complex of the present invention.

[0092] In certain embodiments, the nucleic acid complex of the present invention includes a prodrug and a pharma- ceutically acceptable salt of the prodrug. The prodrug of the nucleic acid complex of the present invention and a pharma- ceutically acceptable salt of the prodrug refer to a compound that is converted to the nucleic acid complex of the present invention by a reaction with an enzyme, gastric acid, or the like under physiological conditions in a living body, i.e., a compound that is enzymatically oxidized, reduced, hydrolyzed, or the like to change to the nucleic acid complex of the present invention, or a compound that is hydrolyzed by gastric acid or the like to change to the nucleic acid complex of the present invention. In certain embodiments, the prodrug of the nucleic acid complex includes one or more tocopherol or cholesterol or analogs thereof bound to the first or second nucleic acid strand.

[0093] In the nucleic acid complex of the present invention, the antisense effect of the first nucleic acid strand on the target transcript can be measured by a method known in the art. For example, after introducing the nucleic acid complex into a cell, it can be measured by using known techniques such as Northern blotting, quantitative PCR, or Western blotting. Specifically, it is sufficient to verify that the expression level of the target gene or the level of the target transcript in the cell (for example, the amount of mRNA or the amount of RNA such as microRNA, the amount of cDNA, the amount of protein, etc.) is reduced by the antisense effect using the known techniques described above.

[0094] The antisense effect of the nucleic acid complex of the present invention in the central nervous system and the passage of the blood-brain barrier can also be measured by a method known in the art. For example, the nucleic acid complex of the present invention is administered to a subject (e.g., a mouse) and the expression level of the target gene or the level of the target transcript in the central nervous system is measured whether it is suppressed several days to several months later (e.g., 2 to 7 days or 1 month later), and the above-mentioned judgment can be made by measuring whether the expression level of the target gene or the level of the target transcript in the central nervous system is suppressed or not, without being limited thereto. The criterion for the above-mentioned judgment is that the nucleic acid complex of the present invention has passed the blood-brain barrier and brought about an antisense effect in the central nervous system when the measured value of the expression level of the target gene or the measured value of the target transcript is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 40% compared to the measured value of a negative control (e.g., vehicle administration). Furthermore, passage through the blood-brain barrier can be determined by administering the nucleic acid complex of the present invention to a subject (e.g., a mouse) and measuring the amount (concentration) of the nucleic acid complex of the present invention in the central nervous system several days to several months later (e.g., 2 to 7 days or 1 month later).

[0095] As described above, exemplary embodiments of the nucleic acid complex of the present invention have been described, but the nucleic acid complex of the present invention is not limited to the above exemplary embodiments. Furthermore, a person skilled in the art can produce the first and second nucleic acid strands constituting the nucleic acid complex according to various embodiments of the present invention by appropriately selecting a known method. For example, the nucleic acid molecule of the present invention can be produced by designing each nucleic acid molecule based on the base sequence information of the target transcription product (e.g., the base sequence of the target gene), synthesizing the nucleic acid using a commercially available automated nucleic acid synthesizer such as GE Healthcare, Thermo Fisher Scientific, or Beckman Coulter, and then purifying the obtained oligonucleotide using a reverse phase column or the like.

[0096] The second nucleic acid strand may further comprise at least one functional moiety attached to the polynucleotide. There are no particular limitations on the structure of the "functional moiety" according to certain embodiments, so long as the functional moiety confers a desired function to the nucleic acid complex and / or the strand to which the functional moiety is attached. The desired functions include labeling and purification functions. Examples of moieties that confer a labeling function include compounds such as fluorescent proteins and luciferase. Examples of moieties that confer a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide. The binding position and type of binding of the functional moiety in the second nucleic acid strand are as described above for the binding of tocopherol or cholesterol or their analogs, etc., to the second nucleic acid strand.

[0097] In one embodiment, the nucleic acid complex to which the functional moiety is bound can be produced by carrying out the above synthesis, purification and annealing using a nucleic acid species to which a functional moiety is previously bound. For example, the second nucleic acid strand can be produced by carrying out the above synthesis and purification using a nucleic acid species to which tocopherol or cholesterol or an analogue thereof is previously bound.

[0098] In one embodiment, tocopherol or cholesterol or an analog thereof can be bound to the second nucleic acid strand produced by carrying out the above synthesis and purification by a method known per se. Methods for linking functional moieties to nucleic acids are well known in the art. The nucleic acid produced by this method is mixed in an appropriate buffer solution, denatured at about 90°C to 98°C for several minutes (e.g., 5 minutes), and then annealed at about 30°C to 70°C for about 1 to 8 hours to produce one of the nucleic acid complexes of the present invention. The nucleic acid strand can also be ordered and obtained from various manufacturers (e.g., Gene Design Co., Ltd.) by specifying the base sequence and the modification site and type. The annealing step can be carried out by leaving the mixture at room temperature (about 10°C to about 35°C) for about 5 to 60 minutes.

[0099] The nucleic acid complex of some embodiments of the present invention may be prepared by dissolving the first nucleic acid strand and the second nucleic acid strand in a buffer solution (e.g., phosphate buffered saline) or water at about 70° C. to 98° C., mixing the two obtained solutions, and holding the mixture at about 70° C. to 98° C. for several minutes (e.g., 5 minutes), and then holding the mixture at about 30° C. to 70° C. (or 30° C. to 50° C.) for about 1 to 8 hours. The first nucleic acid strand and the second nucleic acid strand can also be dissolved in a buffer solution (e.g., phosphate buffered saline) or water at room temperature (about 10° C. to about 35° C.).

[0100] However, the annealing conditions (time and temperature) in preparing the nucleic acid complex are not limited to the above conditions, and suitable conditions for promoting annealing of nucleic acid strands are well known in the art.

[0101] (Effect of nucleic acid complex) The nucleic acid complex of the present invention can inhibit the effect of target miRNA in the central nervous system of a subject.Specifically, for example, the first nucleic acid strand includes a base sequence capable of hybridizing with at least a part of the target miRNA and has an antisense effect on the target miRNA, and the second nucleic acid strand includes a base sequence complementary to the first nucleic acid strand and is bound to tocopherol or cholesterol or its analogue, and the first nucleic acid strand and the second nucleic acid strand are annealed to each other.By inhibiting the effect of the target miRNA with this nucleic acid complex, the expression of the gene that the target miRNA normally downregulates can be upregulated.

[0102] The nucleic acid complex of the present invention can regulate the expression or editing of a target RNA in the central nervous system of a subject. For example, specifically, the first nucleic acid strand includes a base sequence capable of hybridizing to at least a part of the target RNA and has an antisense effect on the target RNA, the second nucleic acid strand includes a base sequence complementary to the first nucleic acid strand and is bound to tocopherol or cholesterol or an analog thereof, and the first nucleic acid strand and the second nucleic acid strand are annealed to each other. Here, "regulation of expression of target RNA" includes, for example, upregulation and downregulation of the expression level. In addition, "regulation of editing of target RNA" includes regulation of splicing by RNA editing, for example, exon skipping and exon inclusion. The target RNA may be viral or bacterial RNA, or toxic RNA.

[0103] The nucleic acid complex of the present invention can inhibit the translation of target mRNA in the central nervous system of a subject. For example, specifically, the first nucleic acid strand includes a base sequence capable of hybridizing with at least a part of the target mRNA and has an antisense effect on the target mRNA, and the second nucleic acid strand includes a base sequence complementary to the first nucleic acid strand and is bound to tocopherol or cholesterol or an analog thereof, and the first nucleic acid strand and the second nucleic acid strand are annealed to each other. The first nucleic acid strand binds to the target mRNA by this nucleic acid complex, resulting in a steric block, which inhibits the translation of mRNA.

[0104] The nucleic acid complex of the present invention can be effectively used in regulating the expression or editing of target transcripts in microglia (microglial cells) of the central nervous system, although this is not intended to be limiting.

[0105] <Composition> The second aspect of the present invention is a composition. The composition of the present invention includes the nucleic acid complex or single-stranded nucleic acid strand of the first aspect as an active ingredient and / or as a drug delivery molecule. The nucleic acid complex or single-stranded nucleic acid strand of the first aspect can pass through the BBB and regulate the expression level of a target transcript in the central nervous system by the antisense effect (e.g., reduce the expression level). Therefore, the composition of the present invention may be a composition that delivers the nucleic acid complex and treats the subject by administering it to the subject, or may be a pharmaceutical composition. The composition of the present invention can be effectively used in regulating the expression or editing of a target transcript in microglia (microglial cells) of the central nervous system, although it is not limited thereto.

[0106] Furthermore, one embodiment of the present invention relates to a method for treating each central nervous system disease by administering a composition containing the nucleic acid complex.

[0107] (Formulation) In the present specification, the composition can be prepared by a method known per se.For example, the composition can be used orally or parenterally in the form of capsules, tablets, pills, liquids, powders, granules, microgranules, film coatings, pellets, lozenges, sublinguals, peptizers, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, injections and suppositories.

[0108] For the formulation of these preparations, a pharma- ceutically acceptable carrier or solvent or a carrier or solvent acceptable for food and beverage products may be appropriately incorporated.Specific examples of such carriers or solvents include sterilized water, physiological saline, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavorings, dissolution aids, and other additives.

[0109] (Dosage form and dosage) In the present specification, there is no particular limitation on the preferred administration form of the composition. For example, it may be administered orally or parenterally. Specific examples of parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, tracheal / bronchial administration, rectal administration, and intramuscular administration, as well as administration by blood transfusion. It may also be administered by intramuscular injection, intravenous drip administration, or implanted continuous subcutaneous administration. Subcutaneous administration is preferred because it allows the patient to self-inject. In addition, in the case of intravenous administration, the amount of nucleic acid complex contained in one dose of the composition, i.e., a single dose of the nucleic acid complex, can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 00.25 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more. For example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.

[0110] (Subject / Applicable Subject) As used herein, the term "subject" refers to a subject to which the composition of the present invention is applied. Subjects include individuals, as well as organs, tissues, and cells. When the subject is an individual, the composition of the present invention can be applied to any animal, including humans. Subjects other than humans may include, for example, various livestock, poultry, pets, laboratory animals, etc. The subject may be an individual in need of reducing the expression level of a target transcript in the central nervous system, or an individual in need of treatment for a central nervous system disease.

[0111] The composition of the present invention can reduce the expression level of a target transcript in the central nervous system due to the BBB penetrating effect and antisense effect of the nucleic acid complex or single-stranded nucleic acid strand of the first aspect that it comprises.

[0112] When the composition of the present invention is applied for treating central nervous system diseases, the target disease is preferably a central nervous system disease associated with an increase or decrease in gene expression, particularly a disease associated with an increase in expression of a target transcript or target gene (such as a tumor), including, but not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, and the like.

[0113] In one embodiment, the composition of the present invention is applied for the treatment of immune central nervous system diseases, such as microglia-associated diseases, including Alzheimer's disease, multiple sclerosis, ALS, and neuropathic pain.

[0114] The delivery site of the composition of the present invention, more specifically, the delivery site of the active ingredient contained in the composition, is not particularly limited, but more effective results can be obtained by delivering to an appropriate site according to each disease. For example, in the treatment of Alzheimer's disease, drug delivery to the hippocampus and / or parietal lobe can be effective. In addition, in the treatment of frontotemporal dementia (FTD) (including frontotemporal lobar degeneration (FTLD), semantic dementia (SD), progressive non-fluent aphasia (PNFA), etc.) and Pick's disease, drug delivery to the frontal lobe, temporal lobe and / or substantia nigra can be effective. In addition, in the treatment of Parkinson's disease dementia, drug delivery to the occipital lobe, substantia nigra and / or striatum can be effective. In the treatment of Parkinson's disease, drug delivery to the substantia nigra and / or striatum can be effective. In the treatment of corticobasal degeneration (CBD), drug delivery to the frontal lobe, parietal lobe, basal ganglia and / or substantia nigra can be effective. In the treatment of progressive supranuclear palsy (PSP), drug delivery to the frontal lobe, basal ganglia, and / or substantia nigra may be effective. In the treatment of amyotrophic lateral sclerosis, drug delivery to the frontal lobe, parietal lobe, basal ganglia, and / or substantia nigra may be effective. In the treatment of spinocerebellar degeneration (SCD) SCA1 to SCA34, drug delivery to the brainstem and / or cerebellum may be effective. In the treatment of dentatorubral-pallidoluysian degeneration (DRPLA), drug delivery to the basal ganglia, brainstem, and / or cerebellum may be effective. In the treatment of spinal-bulbar atrophy (SBMA), drug delivery to the brainstem and / or spinal cord may be effective. In the treatment of Friedreich's ataxia (FA), drug delivery to the brainstem and / or cerebellum may be effective. In the treatment of Huntington's disease, drug delivery to the striatum, frontal lobe, parietal lobe, and / or basal ganglia may be effective. In the treatment of prion diseases (including mad cow disease and GSS), drug delivery to the cerebral cortex, cerebral white matter, basal ganglia and / or substantia nigra may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of encephalitis (including viral, bacterial, fungal, and tuberculous) and meningitis (including viral, bacterial, fungal, and tuberculous), drug delivery to the entire brain may be effective. In the treatment of metabolic encephalopathy, toxic encephalopathy, and nutritional encephalopathy, drug delivery to the entire brain may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective.In the treatment of cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, moyamoya disease, and anoxic encephalopathy, drug delivery to the entire brain may be effective. In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective. In the treatment of diffuse axonal injury, drug delivery to the cerebral white matter may be effective. In the treatment of head trauma, drug delivery to the entire brain may be effective. In the treatment of multiple sclerosis (MS) and neuromyelitis optica (NMO), drug delivery to the cerebral white matter, cerebral cortex, optic nerve, and / or spinal cord may be effective. In the treatment of myotonic dystrophy (DM1, DM2), drug delivery to skeletal muscle, cardiac muscle, cerebral cortex, and / or cerebral white matter may be effective. In the treatment of familial spastic paraplegia (HSP), drug delivery to the parietal lobe and / or spinal cord may be effective. In the treatment of Fukuyama muscular dystrophy, drug delivery to skeletal muscle, cerebral cortex and / or cerebral white matter may be effective. In the treatment of dementia with Lewy bodies (DLB), drug delivery to the substantia nigra, striatum, occipital lobe, frontal lobe and / or parietal lobe may be effective. In the treatment of multiple system atrophy (MSA), drug delivery to the striatum, basal ganglia, cerebellum, substantia nigra, frontal lobe and / or temporal lobe may be effective. In the treatment of Alexander disease, drug delivery to cerebral white matter may be effective. In the treatment of CADASIL and CARASIL, drug delivery to cerebral white matter may be effective.

[0115] When the composition is administered or ingested, the dosage or intake may be appropriately selected according to the subject's age (including months and weeks), body weight, symptoms and health condition, type of composition (medicine, food, beverage, etc.), etc. The effective intake amount of the composition of the present invention to a subject may be, for example, 0.00001 mg / kg / day to 10000 mg / kg / day or 0.001 mg / kg / day to 100 mg / kg / day of the nucleic acid complex contained therein. The composition may be administered once or multiple times. In the case of multiple administration, it may be administered every day or at appropriate time intervals (for example, at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times, etc. The dosage of the nucleic acid complex per administration can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 00.25 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, 15 ... g / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and for example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.

[0116] The nucleic acid complex of the present invention may be administered at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg) twice a week for four times. Alternatively, the nucleic acid complex may be administered at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg) once or twice a week for two to four times, e.g., twice a week for two times. By adopting such an administration regimen (divided administration), toxicity can be reduced and the burden on the subject can be reduced compared to a single administration of a higher dose.

[0117] Although there is a limit (upper limit) to the amount of nucleic acid complex that can pass through the BBB and BNB by a single administration, it is believed that the inhibitory effect works additively in cells even with repeated administration. In other words, at a high dose (e.g., 25 mg / kg or more) that exceeds the limit of BBB and BNB passage, the enhancement of efficacy is reduced by increasing the single dose, but it is believed that efficacy can be improved by repeated administration with a certain interval between administrations (e.g., half a day or more).

[0118] (Drug Delivery) The composition of the present invention can deliver a specific drug to the nervous system, particularly the central nervous system, by binding the drug to the first nucleic acid strand and / or the second nucleic acid strand, utilizing the fact that the nucleic acid complex or single-stranded nucleic acid strand of the first aspect contained as an active ingredient can pass through the BBB and be efficiently delivered to the central nervous system. The drug delivered to the nervous system can be, but is not limited to, a peptide, a protein or a nucleic acid drug, or other organic compounds, such as antitumor drugs, hormonal drugs, antibiotics, antiviral agents, and anti-inflammatory drugs. The drug is preferably a small molecule drug. The term "small molecule drug" is well understood in the art. A small molecule drug typically refers to a drug having a molecular weight of less than 1,000 daltons. The drug may be a lipophilic drug. The nucleic acid drug can be, but is not limited to, an ASO, an antagomir, a splice switching oligonucleotide, an aptamer, a single-stranded siRNA, a microRNA, a pre-microRNA, and the like. The binding position and type of bond of the drug in the second nucleic acid strand are as described above for the binding of tocopherol or cholesterol or an analog thereof to the second nucleic acid strand.

[0119] As disclosed in the following examples, the composition of the present invention can be delivered to the central nervous system with high efficiency and effectively modify or suppress the expression of a target gene or the level of a target transcript. Therefore, a method for reducing the expression level of a target transcript in the central nervous system of a subject is provided, comprising administering to the subject a composition comprising the above-mentioned nucleic acid complex. The method may be a method for treating a central nervous system disease in a subject. Also provided is a drug delivery method for delivering a drug to the central nervous system of a subject, comprising administering to the subject a composition comprising the above-mentioned nucleic acid complex. EXAMPLES

[0120] The present invention will be further explained in detail by the following Reference Examples and Examples, but these are not intended to limit the present invention and may be modified without departing from the scope of the present invention.

[0121] In the following examples, "room temperature" generally refers to about 10° C. to about 35° C. Ratios shown in mixed solvents are volume ratios unless otherwise specified. % refers to weight % unless otherwise specified.

[0122] In the examples, elution in column chromatography was performed under observation by TLC (Thin Layer Chromatography) unless otherwise specified. In the TLC observation, a 60 F TLC plate manufactured by Merck was used. 254 The developing solvent was the same as the elution solvent used in column chromatography. A UV detector was used for detection. In preparative HPLC (high performance liquid chromatography), when C18 is used, octadecyl-bonded silica gel was used. The ratio of elution solvents is by volume unless otherwise specified. 1 H NMR analysis was performed using ACD / SpecManager (trade name) software, etc. Peaks with very gentle proton peaks such as hydroxyl and amino groups may not be recorded.

[0123] The meanings of the abbreviations used in the examples are shown below. M: Molar concentration N: Normality CDCl 3 : deuterated chloroform DMSO-d 6 : Deuterated dimethyl sulfoxide 1 H NMR: Proton nuclear magnetic resonance LC / MS: Liquid chromatograph mass spectrometer DIPEA: N,N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate NMP: N-methyl-2-pyrrolidone PBS: Phosphate-buffered saline TEA: Triethylamine TEAA: Triethylamine acetate THF: tetrahydrofuran

[0124] The structures of the oligonucleotides used in the following examples are summarized in Table 1. Among the oligonucleotides used in the examples, ASO(Malat1), Y61-cRNA(Malat1), Y62-cRNA(Malat1), Y63-cRNA(Malat1), Y64-cRNA(Malat1), Y59-cRNA(Malat1), Y60-cRNA(Malat1) and Chol-cRNA(Malat1) were synthesized by GeneDesign Inc. (Osaka, Japan).

[0125] [Table 1] TIFF0007669602000037.tif209152 TIFF0007669602000038.tif209153 TIFF0007669602000039.tif209154 TIFF0007669602000040.tif228124

[0126] The 5'-terminal structure of the oligonucleotide Y61-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0127] [ka]

[0128] The 5'-terminal structure of the oligonucleotide Y62-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0129] [ka]

[0130] The 5'-terminal structure of the oligonucleotide Y63-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0131] [ka]

[0132] The 5'-terminal structure of the oligonucleotide Y64-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0133] [ka]

[0134] The 5'-terminal structure of oligonucleotide Y1-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0135] [ka]

[0136] The 5'-terminal structure of oligonucleotide Y3-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0137] [ka]

[0138] The 5'-terminal structure of oligonucleotide Y4-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0139] [ka]

[0140] The 5'-terminal structure of oligonucleotide Y5-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0141] [ka]

[0142] The 5'-terminal structure of the oligonucleotide Y6-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0143] [ka]

[0144] The 5'-terminal structure of the oligonucleotide Y7-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0145] [ka]

[0146] The 5'-terminal structure of the oligonucleotide Y8-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0147] [ka]

[0148] The 5'-terminal structure of the oligonucleotide Y9-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0149] [ka]

[0150] The 5'-terminal structure of the oligonucleotide Y10-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0151] [ka]

[0152] The 5'-terminal structure of the oligonucleotide Y11-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0153] [ka]

[0154] The 5'-terminal structure of the oligonucleotide Y12-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0155] [ka]

[0156] The 5'-terminal structure of the oligonucleotide Y13-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0157] [ka]

[0158] The 5'-terminal structure of the oligonucleotide Y14-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0159] [ka]

[0160] The 5'-terminal structure of the oligonucleotide Y15-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0161] [ka]

[0162] The 5'-terminal structure of the oligonucleotide Y16-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0163] [ka]

[0164] The 5'-terminal structure of the oligonucleotide Y17-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0165] [ka]

[0166] The 5'-terminal structure of oligonucleotide Y18-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0167] [ka]

[0168] The 5'-terminal structure of the oligonucleotide Y19-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0169] [ka]

[0170] The 5'-terminal structure of the oligonucleotide Y20-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0171] [ka]

[0172] The 5'-terminal structure of the oligonucleotide Y59-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0173] [ka]

[0174] The 5'-terminal structure of the oligonucleotide Y60-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0175] [ka]

[0176] The 5'-terminal structure of the oligonucleotide Y21-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0177] [ka]

[0178] The 5'-terminal structure of the oligonucleotide Y22-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0179] [ka]

[0180] The 5'-terminal structure of the oligonucleotide Y23-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0181] [ka]

[0182] The 5'-terminal structure of the oligonucleotide Y24-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0183] [ka]

[0184] The 5'-terminal structure of the oligonucleotide Y25-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0185] [ka]

[0186] The 5'-terminal structure of the oligonucleotide Y26-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0187] [ka]

[0188] The 5'-terminal structure of the oligonucleotide Y27-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0189] [ka]

[0190] The 5'-terminal structure of the oligonucleotide Y28-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0191] [ka]

[0192] The 5'-terminal structure of the oligonucleotide Y29-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0193] [ka]

[0194] The 5'-terminal structure of the oligonucleotide Y30-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0195] [ka]

[0196] The 5'-terminal structure of the oligonucleotide Y31-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0197] [ka]

[0198] The 5'-terminal structure of the oligonucleotide Y32-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0199] [ka]

[0200] The 5'-terminal structure of the oligonucleotide Y33-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0201] [ka]

[0202] The 5'-terminal structure of the oligonucleotide Y34-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0203] [ka]

[0204] The 5'-terminal structure of the oligonucleotide Y35-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0205] [ka]

[0206] The 5'-terminal structure of the oligonucleotide Y36-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0207] [ka]

[0208] The 5'-terminal structure of the oligonucleotide Y37-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0209] [ka]

[0210] The 5'-terminal structure of the oligonucleotide Y38-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0211] [ka]

[0212] The 5'-terminal structure of the oligonucleotide Y39-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0213] [ka]

[0214] The 5'-terminal structure of the oligonucleotide Y40-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0215] [ka]

[0216] The 5'-terminal structure of the oligonucleotide Y41-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0217] [ka]

[0218] The 5'-terminal structure of the oligonucleotide Y42-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0219] [ka]

[0220] The 5'-terminal structure of the oligonucleotide Y43-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0221] [ka]

[0222] The 5'-terminal structure of the oligonucleotide Y44-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0223] [ka]

[0224] The 5'-terminal structure of the oligonucleotide Y45-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0225] [ka]

[0226] The 5'-terminal structure of the oligonucleotide Y46-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0227] [ka]

[0228] The 5'-terminal structure of the oligonucleotide Y47-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0229] [ka]

[0230] The 5'-terminal structure of the oligonucleotide Y48-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0231] [ka]

[0232] The 5'-terminal structure of the oligonucleotide Y49-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0233] [ka]

[0234] The 5'-terminal structure of the oligonucleotide Y50-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0235] [ka]

[0236] The 5'-terminal structure of the oligonucleotide Y51-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0237] [ka]

[0238] The 5'-terminal structure of the oligonucleotide Y52-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0239] [ka]

[0240] The 5'-terminal structure of the oligonucleotide Y53-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0241] [ka]

[0242] The 5'-terminal structure of the oligonucleotide Y54-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0243] [ka]

[0244] The 5'-terminal structure of the oligonucleotide Y55-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0245] [ka]

[0246] The 5'-terminal structure of the oligonucleotide Y57-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, "oligo" refers to an oligonucleotide.

[0247] [ka]

[0248] The 5'-terminal structure of the oligonucleotide Chol-cRNA (Malat1) shown in Table 1 is shown below. In the following chemical formula, the term "oligo" refers to an oligonucleotide.

[0249] [ka]

[0250] Reference example 1 Synthesis of IY1 Trimethylsilyl chloride (569 μL) was added to a pyridine solution (5567 μL) of 6-aminohexanoic acid (146 mg) at 0° C. The reaction mixture was stirred at room temperature for 30 minutes under a nitrogen atmosphere. Cholesterol chloroformate (500 mg) was added at 0° C., and the reaction mixture was stirred at room temperature for 30 minutes under a nitrogen atmosphere. The reaction mixture was neutralized by adding 1N aqueous hydrochloric acid at 0° C., and extracted with ethyl acetate. The organic layer was separated, washed with water and then with saturated saline, dried over magnesium sulfate, and concentrated to dryness. The obtained solid was washed with diisopropyl ether to give 200 mg of the title compound.

[0251] In the same manner as in Reference Example 1, the compounds of Reference Examples 7, 8, 14 and 15 were prepared.

[0252] Table 2 below shows the chemical structures and NMR data of the compounds of Reference Examples 1, 7, 8, 14 and 15.

[0253] [Table 2] TIFF0007669602000104.tif24872

[0254] Reference example 9 Synthesis of IY9 Nitrophenyl carbonochloridate (210 mg) was added to a solution (5 mL) of estradiol enanthate (200 mg) and pyridine (535 μL) in THF at 0° C. The reaction mixture was reacted at room temperature for 3 hours. The precipitate was removed by filtration through Celite, and the filtrate was washed with diisopropyl ether. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 80 mg of the title compound.

[0255] In the same manner as in Reference Example 9, the compounds of Reference Examples 10, 36, 39 and 40 were prepared. Table 3 below shows the chemical structures and NMR data of the compounds of Reference Examples 9, 10, 36, 39 and 40.

[0256] [Table 3]

[0257] Reference example 11 Synthesis of IY11 A) Synthesis of IY11-1 The title compound was obtained using cholesterol chloroformate (300 mg) and 3-(2-aminoethoxy)propanoic acid (89 mg) in the same manner as in Reference Example 1. This compound was used in the next step without purification.

[0258] B) Synthesis of IY11 Bis(pentafluorophenyl) carbonate (528 mg) was added to a tetrahydrofuran solution (6700 μL) of IY11-1 obtained in step A, and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. The reaction mixture was added to water at room temperature and extracted with ethyl acetate. The extract was washed with water and saturated saline, and then dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 127 mg of the title compound.

[0259] IY16-1 of Reference Example 16 and IY17-1 of Reference Example 17 were produced in the same manner as in step A) of Reference Example 11. IY16 of Reference Example 16 and IY17 of Reference Example 17 were produced in the same manner as in step B) of Reference Example 11.

[0260] Table 4 below shows the chemical structures and NMR data of IY11-1, IY11, IY16-1, IY16, IY17-1 and IY17.

[0261] [Table 4] TIFF0007669602000107.tif248126

[0262] Reference example 18 Synthesis of IY18

[0263] [ka]

[0264] Aqueous sodium hydroxide solution (0.1N, 2.4 mL) of IY18-1 (138 mg) was added to a solution of cholesterol chloroformate (49.1 mg) in THF (3 mL) under ice cooling, and the mixture was stirred at room temperature for 5 hours. The reaction solution was acidified with 1N hydrochloric acid, and the reaction mixture was extracted with THF-ethyl acetate (1:1). The organic layer was dried over magnesium sulfate and then concentrated to dryness to obtain 151 mg of the title compound.

[0265] The NMR results of the obtained compound are as follows: 1H NMR (300 MHz, CDCl 3 ) δ 0.68 (3H, s), 0.80-1.69 (37H, m), 1.73-2.07 (6H, m), 2.18-2.71 (11H, m), 3.27-3.46 (3H, m), 3.47-3.73 (80H, m), 3.78 (2H, t, J = 6.1 Hz), 4.39-4.66 (1H, m), 5.24 (1H, brs), 5.37 (1H, d, J = 5.4 Hz), 6.98 (1H, s).

[0266] Reference example 19 Synthesis of IY19 A mixture of diosgenin (445 mg), bis(2,5-dioxopyrrolidin-1-yl) carbonate (1650 mg), triethylamine (2.244 mL) and acetonitrile (10 mL) was reacted under ultrasonic conditions for 3 hours. The reaction mixture was diluted with saturated sodium bicarbonate solution and extracted with a mixed solvent of ethyl acetate and diethyl ether. The extract was washed with water and saturated saline, and then dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 55 mg of the title compound.

[0267] The compounds of Reference Examples 20, 21, 22, 24, 25, 28, 29, 32, 33, 34, 35, 43, 44, 48, 51 and 52 were prepared in the same manner as in Reference Example 19.

[0268] Table 5 below shows the chemical structures and NMR data of compounds of Reference Examples 19, 20, 21, 22, 24, 25, 28, 29, 32, 33, 34, 35, 43, 44, 48, 51 and 52.

[0269] [Table 5] TIFF0007669602000110.tif201135 TIFF0007669602000111.tif202150 TIFF0007669602000112.tif199146 TIFF0007669602000113.tif203147

[0270] Reference example 23 Synthesis of IY-23 [ka] A) Synthesis of IY23-2 Imidazole (152 mg) and DMAP (91 mg) were added to a dichloromethane solution (10 mL) of IY23-1 (300 mg) at room temperature and stirred for 15 minutes. tert-Butyldimethylsilyl trifluoromethanesulfonate (0.34 mL) was added and stirred at room temperature for 16 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 150 mg of the title compound.

[0271] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ -0.01 (6H, s), 0.67 (3H, s), 0.66 - 0.96 (15H, m), 0.91 - 1.14 (9H, m), 1.11 - 1.40 (8H, m), 1.42 (2H, dd, J = 8.0, 4.6 Hz), 1.44 - 1.53 (6H, m), 1.77 - 1.89 (3H, m), 1.91 - 2.05 (2H, m), 2.17 - 2.33 (2H, m), 3.34 (1H, dd, J = 9.7, 6.8 Hz), 3.42 (1H, dd, J = 9.6, 6.0 Hz), 3.47 - 3.57 (1H, m), 5.34 (1H, d, J = 4.8 Hz).

[0272] B) Synthesis of IY23 Using IY23-1 obtained in Step A, the title compound was obtained in the same manner as in Reference Example 19. The NMR results of the obtained compound are as follows: 1H NMR (300 MHz, CDCl 3 ) δ 0.04 (s, 6 H) 0.68 (s, 3 H) 0.77 - 1.63 (m, 38 H) 1.67 - 2.18 (m, 6 H) 2.48 (d, J = 7.54 Hz, 2 H) 2.83 (s, 4 H) 3.28 - 3.51 (m, 2 H) 4.51 - 4.70 (m, 1 H) 5.42 (d, J = 4.14 Hz, 1 H)

[0273] Reference example 27 Synthesis of IY27 [ka] A) Synthesis of IY27-2 Dicyclohexylcarbodiimide (226 mg), IY27-1 (250 mg), and DMAP (26.7 mg) were added to a dichloromethane solution (15 mL) of lauric acid (148 mg), and the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with dichloromethane (50 mL), and the organic layer was washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 230 mg of the title compound.

[0274] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3) δ0.68 (3H, s), 0.87 (3H, t, J = 5.5 Hz), 0.95 (3H, d, J = 6.1 Hz), 1.01(3H, s), 1.05 - 1.18 (3H, m), 1.25 (16H, s), 1.36-1.65 (14H, m), 1.80 - 1.89 (4H, m), 1.91 - 2.04 (2H, m), 2.21 - 2.33 (4H, m), 2.34 - 2.45 (1H, m), 2.46 - 2.59 (1H, m), 4.55 - 4.65 (1H, m), 5.27 (2H, s), 5.36 (1H, d, J = 2.8 Hz), 7.62 (2H, d, J = 8.4 Hz), 7.77 (2H, d, J = 8.5 Hz).

[0275] B) Synthesis of IY27 Acetic acid (10 mL) and zinc powder (113 mg) were added to a dichloromethane solution (10 mL) of IY27-2 (180 mg) obtained in step A, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 100 mg of the title compound.

[0276] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 0.67 (3H, s), 0.86 (3H, d, J = 6.9 Hz), 0.93 (3H, d, J = 6.3 Hz), 1.01(3H, s), 1.06 - 1.19 (3H, m), 1.24 (14H, s), 1.36 - 1.65 (14H, m), 1.80 - 1.88 (5H, m), 1.91 - 2.02 (2H, m),2.27 (7H, dt, J = 15.1, 7.3 Hz), 4.53 - 4.67 (1H, m), 5.36 (1H, d, J = 5.8 Hz).

[0277] Reference example 37 Synthesis of IY37 [ka] A) Synthesis of IY37-2 Imidazole (460 mg) and DMAP (275 mg) were added to a DMF solution (10 mL) of IY37-1 (1.0 g) at room temperature and stirred for 15 minutes. tert-Butyldimethylsilyl trifluoromethanesulfonate (1.03 mL) was added and stirred at room temperature for 16 hours. Water was added to the reaction mixture, and it was extracted with ethyl acetate. The organic layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 1 g of the title compound.

[0278] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 0.04 (6H, d, J = 4.6 Hz), δ 0.66 (3H, s), 0.81 - 0.93 (17H, m), 0.95 - 1.17 (12H, m), 1.22 - 1.39 (6H, m), 1.39 - 1.54 (4H, m), 1.71 - 1.91 (5H, m), 1.94 - 2.06 (4H, m), 2.31 (2H, q, J = 7.5 Hz), 3.93 (1H, d, J = 7.9 Hz), 4.54 - 4.66 (1H, m), 5.29 (1H, s).

[0279] B) Synthesis of IY37-3 To a solution of IY37-2 (1 g) obtained in step A in ethanol (4 mL) and dichloromethane (2 mL), an aqueous sodium hydroxide solution (10%, 2.86 mL) was added and stirred at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and ethyl acetate was added to the residue, which was washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain 800 mg of the title compound.

[0280] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ -0.04 - 0.09 (6H, m), 0.66 (3H, s), 0.82 - 0.93 (18H, m), 0.94 - 1.16 (12H, m), 1.24 (1H, d, J = 7.3 Hz), 1.28 - 1.41 (4H, m), 1.52 (6H, d, J = 32.7 Hz), 1.73 - 1.86 (4H, m), 1.95 - 2.05 (1H, m), 2.24 (2H, d, J = 10.9 Hz), 3.47 - 3.58 (1H, m), 3.93 (1H, d, J = 7.6 Hz), 5.26 (1H, s).

[0281] C) Synthesis of IY37-4 To a solution of IY37-3 (800 mg) obtained in step B in acetonitrile (4 mL) and dichloromethane (4 mL), triethylamine (3.26 mL) and bis(2,5-dioxopyrrolidin-1-yl) carbonate (2.38 g) were added and stirred at 45 °C for 16 hours. The reaction mixture was filtered through Celite, 5% citric acid solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 800 mg of the title compound.

[0282] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3) δ 0.05 (6H, d, J = 4.6 Hz), 0.66 (3H, s), 0.84 - 0.92 (17H, m), 0.95 - 1.16 (12H, m), 1.21 - 1.37 (6H, m), 1.40 - 1.55 (3H, m), 1.70 - 1.82 (3H, m), 1.89 (1H, d, J = 13.8 Hz), 1.95 - 2.06 (3H, m), 2.47 (2H, d, J = 7.8 Hz), 2.82 (4H, s), 3.92 (1H, d, J = 7.5 Hz), 4.53 - 4.67 (1H, m), 5.31 (1H, d, J = 15.7 Hz).

[0283] D) Synthesis of IY37 Triethylamine trihydrofluoride (2.64 mL) was added to a solution of IY37-4 (800 mg) obtained in step C in THF (8 mL) at 0°C, and the mixture was stirred at room temperature for 16 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 110 mg of the title compound.

[0284] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, CDCl 3 ) δ 0.68 (3H, s), 0.86 (6H, d, J = 5.0 Hz), 0.91 (3H, d, J = 6.2 Hz), 0.98 - 1.17 (11H, m), 1.22 - 1.43 (8H, m), 1.41 - 1.53 (3H, m), 1.70 - 1.83 (2H, m), 1.84 - 1.96 (2H, m), 2.04 (2H, s), 2.51 (2H, t, J = 9.1 Hz), 2.83 (4H, s), 3.85 (1H, s), 4.55 - 4.69 (1H, m), 5.35 (1H, s).

[0285] Reference example 38 Synthesis of IY38 [ka] The title compound was synthesized in the same manner as in Step C of Reference Example 37 using 5α-cholestane-3β,5α,6β-triol.

[0286] The NMR results of the obtained compound are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 0.63 (3H, s), 0.86 (8H, dd, J = 16.0, 5.6 Hz), 0.94 - 1.38 (21H, m), 1.44 - 1.69 (6H, m), 1.71 - 1.81 (1H, m), 1.82 - 1.97 (2H, m), 2.15 (1H, t, J = 12.0 Hz), 2.80 (4H, s), 3.38 (1H, s), 4.13 (1H, s), 4.64 (1H, d, J = 4.0 Hz), 5.03 - 5.14 (1H, m)

[0287] Reference example 41 Synthesis of IY41 [ka]

[0288] A) Synthesis of IY41-2 8N sodium hydroxide solution (2.92 mL) and hydrogen peroxide solution (14.3 mL) were added to a mixed solution of IY41-1 (1.8 g) in methanol (3.12 mL)-hexane (6.24 mL). The reaction mixture was stirred at room temperature overnight. Hydrogen peroxide solution (1.43 mL) was added. Urea-hydrogen peroxide (1.32 g) was added and stirred for 1 hour. The reaction mixture was neutralized with 6N hydrochloric acid solution, sodium thiosulfate solution was added, and extracted with ethyl acetate. The organic layer was separated, washed with 1N hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 700 mg of the title compound as a stereoisomeric mixture (4α,5α epoxy and 4β,5β epoxy).

[0289] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.64-0.73 (3H, m), 0.80-0.93 (9H, m), 0.94-2.47 (31H, m), 2.98 (1H, s, 4-H beta), 3.03 (1H, 2, 4-H alpha).

[0290] B) Synthesis of IY41-3 To a mixed solution of IY41-2 (700 mg) obtained in step A in acetic acid (10 mL) and THF (10 mL), p-toluenesulfonyl hydrazide (342 mg) was added in small portions, and the reaction mixture was stirred at room temperature for 4 hours. Saturated saline (30 mL) was added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate, washed with a saturated aqueous solution of sodium bicarbonate, dried over sodium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 560 mg of the title compound.

[0291] The NMR results of the obtained compound are as follows: 1H NMR (300 MHz, CDCl 3 ) δ 0.73 (3H, s), 0.82-0.95 (9H, m), 0.96-1.08 (3H, m), 1.09 (3H, s), 1.10-2.39 (25H, m), 2.44-2.62 (1H, m).

[0292] C) Synthesis of IY41-4 Sodium borohydride (110 mg) was added to a methanol solution (10 mL) of IY41-3 (560 mg) obtained in step B at room temperature. The reaction mixture was stirred at room temperature for 30 minutes and then diluted with ethyl acetate. The organic layer was separated, washed with water and saturated saline, dried over sodium sulfate, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 560 mg of the title compound as a stereoisomer mixture (5α and 5β hydroxyl).

[0293] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.65 (9H, d, J = 3.4 Hz), 0.74-2.36 (35H, m), 3.43-3.56 (1H, m, 5-H alpha), (1H, d, J = 2.4 Hz, 5-H alpha).

[0294] D) Synthesis of IY41-5 Palladium / carbon (7.4 mg) was added to an ethanol solution (7 mL) of IY41-4 (540 mg) obtained in step C, and the mixture was stirred overnight under a hydrogen atmosphere. The reaction mixture was filtered through an amino silica pad, and the solvent was distilled off to obtain 540 mg of the title compound as a stereoisomer mixture (5α and 5β hydroxy forms).

[0295] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3) δ 0.61-0.69 (3H, m), 0.80 (3H, s), 0.81-1.91 (42H, m), 1.92-2.03 (1H, m), 3.43-3.55 (1H, m, 5-H alpha), 3.63 (1H, brs, 5-H beta).

[0296] E) Synthesis of IY41 The title compound was obtained as a single isomer by the same method as in Reference Example 9 using IY41-5 obtained in Step D. The α-hydroxy form of IY41-5 that remained unreacted under these reaction conditions was removed during the purification process.

[0297] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.66 (4H, s), 0.73-2.07 (47H, m), 4.72 (1H, dd, J = 11.2, 4.2 Hz), 7.36 (2H, d, J = 8.9 Hz), 8.28 (2H, d, J = 8.3 Hz).

[0298] Reference example 42 Synthesis of IY42 [ka] A) Synthesis of IY42-2 To a pyridine solution (30 mL) of IY42-1 (3.03 g), p-toluenesulfonyl chloride (2.40 g) was added at room temperature. The reaction mixture was stirred for 3 hours, and then p-toluenesulfonyl chloride (2.40 g) was added and stirred overnight. 1N aqueous hydrochloric acid was added to the reaction mixture at room temperature, and the mixture was extracted with ethyl acetate. The organic layer was separated, washed with saturated saline, and dried over magnesium sulfate, and the solvent was removed by distillation. The residue was washed with diisopropyl ether to obtain 1.45 g of the title compound. The crude product was used in the next step without purification.

[0299] B) Synthesis of IY42-3 Sodium azide (238 mg) was added to DMF (3 mL) of IY42-2 (377 mg) obtained in step A at room temperature, and the mixture was stirred at 70°C for 4 hours. The reaction mixture was added to water at room temperature, and extracted with ethyl acetate. The organic layer was separated, washed with saturated saline, dried over magnesium sulfate, and then the solvent was distilled off. The residue was washed with diisopropyl ether to obtain 310 mg of the title compound. The crude product was used in the next step without purification.

[0300] C) Synthesis of IY42-5 Polymer-supported triphenylphosphine (3 mmol / g) (1.32 g) was added to a mixed solvent of IY42-3 (493 mg) obtained in step B in THF (10 mL) and water (1 mL), and the mixture was stirred at 70° C. for 3 hours. The insoluble solid was filtered and washed with ethyl acetate. Saturated saline was added to the filtrate, the organic layer was separated, dried over magnesium sulfate, and the solvent was distilled off. The residue was dissolved in pyridine (10 mL), and 3,5-bis(trifluoromethyl)-benzoyl chloride (0.241 mL) was added at room temperature. The reaction mixture was stirred overnight, and then 1N aqueous hydrochloric acid was added and extracted with ethyl acetate. The organic layer was separated, washed with water and then saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 370 mg of the title compound.

[0301] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.81 (3H, s), 0.94-1.74 (22H, m), 1.74-2.12 (5H, m), 2.33 (2H, d, J = 7.3 Hz), 4.20-4.38 (1H, m), 4.62 (1H, brs), 5.39 (1H, brs), 5.96 (1H, d, J = 8.9 Hz), 8.00 (1H, s), 8.16 (2H, s).

[0302] D) Synthesis of IY42-6 IY49-5 (370 mg) obtained in step C was dissolved in methanol (3 mL)-THF (3 mL), potassium carbonate (256 mg) was added, and the mixture was stirred at room temperature for 2 hours. The solvent of the reaction mixture was distilled off, and the residue was added with 1N aqueous hydrochloric acid, and extracted with ethyl acetate. The aqueous layer was neutralized with saturated aqueous sodium bicarbonate at 0°C, and extracted with ethyl acetate. The organic layer was separated, washed with water and then with saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 183 mg of the title compound.

[0303] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.80 (3H, s), 0.90-2.12 (25H, m), 2.16-2.65 (2H, m), 3.53 (1H, brs), 4.25 (1H, d, J = 6.8 Hz), 5.36 (1H, brs), 6.08 (1H, d, J =8.7 Hz), 7.99 (1H, s), 8.16 (2H, s).

[0304] E) Synthesis of IY42 The title compound was synthesized in the same manner as in Reference Example 19 using IY42-6 obtained in Step D.

[0305] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3) δ 0.81 (3H, s), 0.90-1.24 (7H, m), 1.32 (3H, d, J = 6.4 Hz), 1.41-2.16 (14H, m), 2.49 (2H, d, J = 7.0 Hz), 2.84 (4H, s), 4.27 (1H, s),4.62 (1H, s), 5.42 (1H, d, J = 4.7 Hz), 5.92 (1H, d, J = 9.4 Hz), 8.00 (1H, s), 8.15 (2H, s).

[0306] Reference example 46 Synthesis of IY46 [ka]

[0307] A mixture of IY42-3 (830 mg) obtained in step B of Reference Example 42, triphenylphosphine (577 mg), THF (10 mL), and water (1 mL) was stirred at 70° C. for 1 hour. The reaction mixture was added to 1N aqueous hydrochloric acid, and the insoluble solid was filtered off. The organic layer was separated, washed with saturated saline, dried over magnesium sulfate, and the solvent was distilled off. THF (20 mL), triethylamine (0.562 mL), and triphosgene (598 mg) were added to the residue at 0° C., and the mixture was stirred overnight at room temperature. A saturated aqueous sodium bicarbonate solution was added to the reaction mixture at 0° C. to neutralize it, and the mixture was extracted with a mixed solvent of ethyl acetate and THF. The organic layer was separated, washed with water and then saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 485 mg of the title compound.

[0308] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) δ 0.67 (3H, s), 0.78-2.12 (38H, m), 2.35 (2H, d, J = 8.3 Hz), 3.24 (1H, d, J = 9.0 Hz), 5.37 (1H, d, J = 5.1 Hz).

[0309] Reference example 47 Synthesis of IY47 [ka]

[0310] Sodium ascorbate (1920 mg) was added to a mixture of IY42-3 (570 mg) obtained in step B of Reference Example 42, hepta-6-ynoic acid (0.226 mL), copper (II) sulfate pentahydrate (346 mg), ethanol (5 mL), water (5 mL) and THF (10 mL) at room temperature, and the mixture was stirred overnight. Insoluble solids were filtered, and the solvent of the filtrate was distilled off under reduced pressure. A 1N aqueous hydrochloric acid solution was added to the residue, and ethyl acetate-THF (4:1) was added. The organic layer was separated, washed with water and then with saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 188 mg of the title compound.

[0311] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.69 (3H, s), 0.79-2.19 (43H, m), 2.27-2.89 (6H, m), 4.34 (1H, brs), 5.45 (1H, brs), 7.32 (1H, s).

[0312] Reference example 49 Synthesis of IY49 [ka]

[0313] A mixture of IY42-3 (200 mg) obtained in Step B of Reference Example 42, (1R,9S)-bicyclo[6.1.0]nona-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate (142 mg) and THF (15 mL) was stirred at room temperature for 2 hours. The reaction mixture was added to water and extracted with ethyl acetate. The organic layer was separated, washed with saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to obtain 134 mg of the title compound.

[0314] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl3) - 0.70 (3H, s), 0.80-1.66 (36H, m), 1.70-2.12 (6H, m), 2.17-2.51 (4H, m), 2.68 (1H, ddt, J = 16.0, 10.0, 3.2 Hz), 2.85 (4H, s),2.89-3.27 (4H, m), 4.01 (1H, tt, J = 12.2, 3.9 Hz), 4.29-4.44 (1H, m), 4.47-4.61 (1H, m), 5.42 (1H, d, J = 1.9 Hz).

[0315] Reference example 50 Synthesis of IY50 [ka]

[0316] A) Synthesis of IY50-2 Using IY50-1 (1.02 g) as a starting material, 825 mg of the title compound was obtained in the same manner as in Step A of Reference Example 42.

[0317] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3) δ 0.64 (3H, s), 0.81-2.39 (33H, m), 2.45 (3H, s), 3.50 (2H, brs), 3.67-4.06 (3H, m), 4.71 (1H, brs), 5.33 (1H, brs), 7.34 (2H, d, J = 7.5 Hz), 7.78 (2H, d, J = 7.9 Hz).

[0318] B) Synthesis of IY50-3 Using IY50-2 (411 mg) obtained in Step A as a starting material, 258 mg of the title compound was obtained in the same manner as in Step B of Reference Example 42.

[0319] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ ppm 0.69 (3H, s), 0.82-2.43 (33H, m), 3.04 (1H, dd, J = 11.8, 7.4 Hz), 3.28-3.68 (3H, m), 3.81-4.03 (1H, m), 4.72 (1H, brs), 5.35(1H, brs).

[0320] C) Synthesis of IY50-5 Using IY50-3 (258 mg) obtained in Step B as a starting material, 190 mg of the title compound was obtained in the same manner as in Step C of Reference Example 42.

[0321] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.73 (3H, s), 0.87-1.43 (22H, m), 1.63-2.14 (8H, m), 2.37 (1H, s), 3.03-4.03 (7H, m), 4.72 (1H, brs), 5.36 (1H, s), 6.13 (1H, s), 8.01 (1H, s), 8.20 (2H, s).

[0322] D) Synthesis of IY50-6 A mixture of IY50-5 (190 mg) obtained in step C, pyridinium p-toluenesulfonate (101 mg) and methanol (5 mL) was stirred under heating and reflux for 1 hour. The solvent was removed under reduced pressure, and the residue was added to water and extracted with ethyl acetate. The organic layer was separated, washed with saturated saline, dried over magnesium sulfate, and then the solvent was removed to obtain 170 mg of the title compound. The crude product was used in the next step without purification.

[0323] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.74 (3H, s), 0.86-2.45 (28H, m), 3.10-3.33 (1H, m), 3.41-3.69 (2H, m), 5.35 (1H, brs), 6.15 (1H, brs), 8.01 (1H, s), 8.20 (2H, s).

[0324] E) Synthesis of IY50 Using IY53-6 ​​obtained in Step D, the title compound was obtained in the same manner as in Reference Example 19.

[0325] The NMR results of the obtained compound are as follows: 1 H NMR (300 MHz, CDCl 3 ) δ 0.74 (3H, s), 0.88-2.21 (25H, m), 2.49 (2H, d, J = 7.9 Hz), 2.84 (4H, s), 3.09-3.32 (1H, m), 3.59 (1H, d, J = 12.8 Hz), 4.60 (1H, brs), 5.43 (1H, brs), 6.15 (1H, brs), 8.01 (1H, s), 8.20 (2H, s).

[0326] Other compounds used in the examples are shown in the table below. Commercially available products may be used as they are, or they may be prepared by known methods or methods similar thereto.

[0327] [Table 6] TIFF0007669602000125.tif28163

[0328] Example 1 A) Synthesis of cRNA with Y1-linked to the 5' end (Y1-cRNA(Malat1)) IY1 in 50 mM NMP solution (800 μL), HATU in 75 mM NMP solution (800 μL), and DIPEA in 150 mM NMP solution (800 μL) were mixed in an Eppendorf tube, stirred, precipitated, and then reacted at room temperature for 30 minutes. An aqueous solution (2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1, NMP (2100 μL), distilled water (153 μL), and DIPEA (63 μL) were added thereto, stirred, precipitated, and then reacted at room temperature for 1 hour. The reaction solution was purified using an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). The resulting solution was mixed with 10 times the amount of 1 M meglumine acetate, stirred thoroughly, and left to stand for 5 minutes to carry out ion exchange. After that, desalting was carried out by ultrafiltration (Amicon ultra 3 kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter, and 690 nmol of the title compound was obtained after lyophilization. B) Synthesis of double-stranded nucleic acid agent Y1-HDO A 16-mer single-stranded LNA / DNA gapmer (ASO(Malat1)) targeting the metastasis-associated lung adenocarcinoma transcript (Malat1) non-coding RNA contains three LNA nucleosides at the 5' end and three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them. This LNA / DNA gapmer has a base sequence complementary to 1316-1331 of mouse Malat1 non-coding RNA (GenBank accession number NR_002847, SEQ ID NO: 3). ASO(Malat1) (first strand) and Y1-cRNA(Malat1) (second strand) obtained in the previous step A were mixed in equimolar amounts, and the mixture was heated at 70°C for 5 minutes. The mixture was then gradually cooled to room temperature to prepare the above-mentioned double-stranded nucleic acid agent, Y1-conjugated heteroduplex oligonucleotide (Y1-HDO).

[0329] Example 3 A) Synthesis of cRNA with Y3-linked at the 5' end (Y3-cRNA(Malat1)) A 50 mM DMSO solution of IY3 (240 μL), a 75 mM NMP solution of HATU (240 μL), and a 150 mM NMP solution of DIPEA (240 μL) were mixed in an Eppendorf flask, stirred, and allowed to settle, and then reacted at room temperature for 15 minutes. An aqueous solution of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 (253 μL, 1200 nmol), DMSO (1980 μL), distilled water (47.4 μL), and DIPEA (38 μL) were added thereto, stirred, allowed to settle, and then reacted at room temperature for 1 hour. The reaction solution was diluted with distilled water to an NMP concentration of 10 v / v%, extracted with ethyl acetate, and the aqueous layer was purified by ultrafiltration (Amicon ultra 3 kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and freeze-dried to quantitatively obtain the title compound. B) Synthesis of double-stranded nucleic acid agent Y3-HDO Using ASO (Malat1) (first strand) and Y3-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y3-conjugated heteroduplex oligonucleotide (Y3-HDO), was prepared in the same manner as in step B of Example 1.

[0330] Example 4 A) Synthesis of cRNA with Y4-linked at the 5' end (Y4-cRNA(Malat1)) Using an aqueous solution (2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY4, 590 nmol of the title compound was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y4-HDO Using ASO (Malat1) (first strand) and Y4-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y4-conjugated heteroduplex oligonucleotide (Y4-HDO), was prepared in the same manner as in step B of Example 1.

[0331] Example 5 A) Synthesis of cRNA with Y5-linked at the 5' end (Y5-cRNA(Malat1)) Using an aqueous solution (1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY5, 776 nmol of the title compound was obtained in the same manner as in Step A of Example 3. B) Synthesis of double-stranded nucleic acid agent Y5-HDO Using ASO (Malat1) (first strand) and Y5-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y5-conjugated heteroduplex oligonucleotide (Y5-HDO), was prepared in the same manner as in step B of Example 1.

[0332] Example 6 A) Synthesis of cRNA with Y6-linked at the 5' end (Y6-cRNA(Malat1)) Using an aqueous solution (1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY6, 880 nmol of the title compound was obtained in the same manner as in Step A of Example 3. B) Synthesis of double-stranded nucleic acid agent Y6-HDO Using ASO (Malat1) (first strand) and Y6-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y6-conjugated heteroduplex oligonucleotide (Y6-HDO), was prepared in the same manner as in step B of Example 1.

[0333] Example 7 A) Synthesis of cRNA with Y7-linked at the 5' end (Y7-cRNA(Malat1)) IY7 in 50 mM NMP solution (1200 μL), HATU in 75 mM NMP solution (1200 μL), and DIPEA in 150 mM NMP solution (1200 μL) were mixed in an Eppendorf tube, stirred, precipitated, and then reacted at room temperature for 30 minutes. An aqueous solution (548 μL, 3000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1, NMP (3150 μL), distilled water (202 μL), and DIPEA (94 μL) were added thereto, stirred, precipitated, and then reacted at room temperature for 1 hour. The reaction solution was purified using an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). The resulting solution was freeze-dried and then dissolved in distilled water to obtain 780 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y7-HDO A 16-mer single-stranded LNA / DNA gapmer (ASO(Malat1)) targeting the metastasis-associated lung adenocarcinoma transcript (Malat1) non-coding RNA contains three LNA nucleosides at the 5' end and three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them. This LNA / DNA gapmer has a base sequence complementary to 1316-1331 of mouse Malat1 non-coding RNA (GenBank accession number NR_002847, SEQ ID NO: 3). ASO(Malat1) (first strand) and Y1-cRNA(Malat1) (second strand) obtained in the previous step A were mixed in equimolar amounts, and the mixture was heated at 70°C for 5 minutes. The mixture was then gradually cooled to room temperature. The resulting mixture was then ion-exchanged to a sodium salt using BT AG 50W-X8 Resin, after which it was lyophilized and distilled water was added to prepare a double-stranded nucleic acid agent, Y1-conjugated heteroduplex oligonucleotide (Y7-HDO).

[0334] Example 8 A) Synthesis of cRNA with Y8-linked at the 5' end (Y8-cRNA(Malat1)) Using an aqueous solution (3,000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY8, 630 nmol of the title compound was obtained in the same manner as in Step A of Example 7. B) Synthesis of double-stranded nucleic acid agent Y8-HDO Using ASO (Malat1) (first strand) and Y8-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y8-conjugated heteroduplex oligonucleotide (Y8-HDO), was prepared in the same manner as in step B of Example 7.

[0335] Example 9 A) Synthesis of cRNA with Y9-linked at the 5' end (Y9-cRNA(Malat1)). An aqueous solution (260 μL, 1500 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf tube with a 50 mM NMP solution (900 μL), distilled water (190 μL), NMP (2100 μL), and borate buffer, pH 8.5+ / -0.2, 5X concentrated (375 μL), in that order, and then stirred and precipitated, and reacted at room temperature for 2 hours. The reaction solution was purified with an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3kDa, manufactured by Merck Millipore, distilled water). The resulting solution was lyophilized to obtain 578 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y9-HDO Using ASO (Malat1) (first strand) and Y9-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y9-conjugated heteroduplex oligonucleotide (Y9-HDO), was prepared in the same manner as in step B of Example 7.

[0336] Example 10 A) Synthesis of cRNA with Y10-linked at the 5' end (Y10-cRNA(Malat1)). An aqueous solution (421 μL, 2000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf with 50 mM NMP solution (800 μL), distilled water (79 μL), NMP (3200 μL), and borate buffer, pH 8.5+ / -0.2, 5X concentrated (500 μL) in that order, stirred, precipitated, and reacted at room temperature for 1 hour. The reaction solution was diluted with distilled water to an NMP concentration of 10v / v%, and then concentrated and low molecular weight reagents such as NMP were removed by ultrafiltration (Amicon ultra 3kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 1185 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y10-HDO Using ASO (Malat1) (first strand) and Y10-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y10-conjugated heteroduplex oligonucleotide (Y10-HDO), was prepared in the same manner as in step B of Example 1.

[0337] Example 11 A) Synthesis of cRNA with Y11-linked at the 5' end (Y11-cRNA(Malat1)) An aqueous solution (173.5 μL, 1000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf with 50 mM NMP solution (400 μL), distilled water (76.5 μL), NMP (1600 μL), ×10 PBS (250 μL), and DIPEA (31 μL) in that order, stirred, precipitated, and reacted at 70°C for 1 hour. The reaction solution was purified with an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). 1 M meglumine acetate was added in a 10-fold amount to the obtained solution, stirred well, and left for 5 minutes to perform ion exchange. Thereafter, desalting was carried out by ultrafiltration (Amicon ultra 3 kDa, Merck Millipore, distilled water), and the final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 592 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y11-HDO Using ASO(Malat1) (first strand) and Y11-cRNA(Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y11-conjugated heteroduplex oligonucleotide (Y11-HDO), was prepared in the same manner as in step B of Example 1.

[0338] Example 12 A) Synthesis of cRNA with Y12-linked at the 5' end (Y12-cRNA(Malat1)). Using an aqueous solution (3000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY12, the same procedure as in Step A of Example 7 was followed to give 1770 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y12-HDO Using ASO(Malat1) (first strand) and Y12-cRNA(Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y12-conjugated heteroduplex oligonucleotide (Y12-HDO), was prepared in the same manner as in step B of Example 7.

[0339] Example 13 A) Synthesis of cRNA with Y13-linked at the 5' end (Y13-cRNA(Malat1)). Using an aqueous solution (3000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY13, 1230 nmol of the title compound was obtained in the same manner as in Step A of Example 7. B) Synthesis of double-stranded nucleic acid agent Y13-HDO Using ASO (Malat1) (first strand) and Y13-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y13-conjugated heteroduplex oligonucleotide (Y13-HDO), was prepared in the same manner as in step B of Example 7.

[0340] Example 14 A) Synthesis of cRNA with Y14-linked at the 5' end (Y14-cRNA(Malat1)) Using an aqueous solution (3000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY14, the same procedure as in Step A of Example 1 was followed to give 1650 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y14-HDO Using ASO (Malat1) (first strand) and Y14-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y14-conjugated heteroduplex oligonucleotide (Y14-HDO), was prepared in the same manner as in step B of Example 1.

[0341] Example 15 A) Synthesis of cRNA with Y15-linked at the 5' end (Y15-cRNA(Malat1)). Using aqueous solutions (210 μl nmol, 1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY15, 900 nmol of the title compound was obtained in the same manner as in Step A of Example 3. B) Synthesis of double-stranded nucleic acid agent Y15-HDO Using ASO (Malat1) (first strand) and Y15-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y15-conjugated heteroduplex oligonucleotide (Y15-HDO), was prepared in the same manner as in step B of Example 1.

[0342] Example 16 A) Synthesis of cRNA with Y16-linked at the 5' end (Y16-cRNA(Malat1)). Using an aqueous solution (176 μL, 1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY16, 532 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y16-HDO Using ASO (Malat1) (first strand) and Y16-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y16-conjugated heteroduplex oligonucleotide (Y16-HDO), was prepared in the same manner as in step B of Example 1.

[0343] Example 17 A) Synthesis of cRNA with Y17-linked at the 5' end (Y17-cRNA(Malat1)). Using an aqueous solution (176 μL, 1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY17, 686 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y17-HDO Using ASO (Malat1) (first strand) and Y17-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y17-conjugated heteroduplex oligonucleotide (Y17-HDO), was prepared in the same manner as in step B of Example 1.

[0344] Example 18 A) Synthesis of cRNA with Y18-linked at the 5' end (Y18-cRNA(Malat1)). Using an aqueous solution (274 μL, 1500 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY18, 1398 nmol of the title compound was obtained in the same manner as in Step A of Example 3. B) Synthesis of double-stranded nucleic acid agent Y18-HDO Using ASO (Malat1) (first strand) and Y18-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y18-conjugated heteroduplex oligonucleotide (Y18-HDO), was prepared in the same manner as in step B of Example 1.

[0345] Example 19 A) Synthesis of cRNA with Y19-linked at the 5' end (Y19-cRNA(Malat1)). An aqueous solution (210.5 μL, 1000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf flask with 50 mM NMP solution (400 μL), distilled water (39.5 μL), NMP (1600 μL), ×10 PBS (250 μL), and DIPEA (31 μL) in that order, stirred, precipitated, and reacted at room temperature for 1 hour. The reaction solution was diluted with distilled water to an NMP concentration of 10v / v%, and then concentrated and low molecular weight reagents such as NMP were removed by ultrafiltration (Amicon ultra 3kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 927 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y19-HDO Using ASO (Malat1) (first strand) and Y19-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y19-conjugated heteroduplex oligonucleotide (Y19-HDO), was prepared in the same manner as in step B of Example 1.

[0346] Example 20 A) Synthesis of cRNA with Y20-attached to the 5' end (Y20-cRNA(Malat1)). Using an aqueous solution (260 μL, 1500 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY19, 686 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y20-HDO Using ASO (Malat1) (first strand) and Y20-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y20-conjugated heteroduplex oligonucleotide (Y20-HDO), was prepared in the same manner as in step B of Example 1.

[0347] Example 21 A) Synthesis of cRNA with Y21-linked at the 5' end (Y21-cRNA(Malat1)). Aqueous solution (260 μL, 1500 nmol) of RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf with 50 mM NMP solution (300 μL), distilled water (115 μL), NMP (2400 μL), ×10 PBS (375 μL), and 50 mM DMAP (300 μL) in that order, stirred, precipitated, and reacted at 70 ° C. for 1 hour. The reaction solution was purified with an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3kDa, manufactured by Merck Millipore, distilled water). The final product was filtered with a 0.20 μm membrane filter, and 917 nmol of the title compound was obtained after lyophilization. B) Synthesis of double-stranded nucleic acid agent Y21-HDO Using ASO (Malat1) (first strand) and Y21-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y21-conjugated heteroduplex oligonucleotide (Y21-HDO), was prepared in the same manner as in step B of Example 1.

[0348] Example 22 A) Synthesis of cRNA with Y22-linked at the 5' end (Y22-cRNA(Malat1)). The title compound (1253 nmol) was obtained in the same manner as in Step A of Example 21 using an aqueous solution (365 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY22. B) Synthesis of double-stranded nucleic acid agent Y22-HDO Using ASO (Malat1) (first strand) and Y22-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y22-conjugated heteroduplex oligonucleotide (Y22-HDO), was prepared in the same manner as in step B of Example 1.

[0349] Example 23 A) Synthesis of cRNA with Y23-linked at the 5' end (Y23-cRNA(Malat1)). An aqueous solution (382 μL, 2200 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf flask with a 50 mM NMP solution (440 μL), distilled water (168 μL), NMP (3520 μL), ×10 PBS (550 μL), and 50 mM DMAP (440 μL) in that order, stirred, precipitated, and reacted at 70° C. for 1 hour. The reaction solution was purified using an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). The obtained solution was diluted with distilled water to 1000 μL, and then mixed in an Eppendorf with distilled water (200 μL), NMP (400 μL), methanol (4400 μL), and 50 mM hydrogen fluoride in DMSO (2000 μL) in that order, stirred, precipitated, and reacted at room temperature for 1 hour. A 300 mM trimethylethoxysilane in DMSO solution (2000 μL) was added to the reaction solution. The reaction mixture was purified with an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3kDa, manufactured by Merck Millipore, distilled water). The final product was filtered with a 0.20 μm membrane filter, and 1114 nmol of the title compound was obtained after lyophilization. B) Synthesis of double-stranded nucleic acid agent Y23-HDO Using ASO (Malat1) (first strand) and Y23-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y23-conjugated heteroduplex oligonucleotide (Y23-HDO), was prepared in the same manner as in step B of Example 7.

[0350] Example 24 A) Synthesis of cRNA with Y24-linked at the 5' end (Y24-cRNA(Malat1)). Using an aqueous solution (174 μL, 1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY24, 870 nmol of the title compound was obtained in the same manner as in Step A of Example 21. B) Synthesis of double-stranded nucleic acid agent Y24-HDO Using ASO (Malat1) (first strand) and Y24-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y24-conjugated heteroduplex oligonucleotide (Y24-HDO), was prepared in the same manner as in step B of Example 7.

[0351] Example 25 A) Synthesis of cRNA with Y25-linked at the 5' end (Y25-cRNA(Malat1)). Using an aqueous solution (274 μL, 1500 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY25, 760 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y25-HDO Using ASO(Malat1) (first strand) and Y25-cRNA(Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y25-conjugated heteroduplex oligonucleotide (Y25-HDO), was prepared in the same manner as in step B of Example 1.

[0352] Example 26 A) Synthesis of cRNA with Y26-linked at the 5' end (Y26-cRNA(Malat1)). A 50 mM THF solution of IY26 (400 μL), a 75 mM NMP solution of HATU (400 μL), and a 150 mM NMP solution of DIPEA (400 μL) were mixed in an Eppendorf flask, stirred, allowed to settle, and then reacted at room temperature for 30 minutes. An aqueous solution of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1 (315 μL, 2000 nmol), NMP (1450 μL), THF (1850 μL), distilled water (184 μL), and DIPEA (63 μL) were added, stirred, allowed to settle, and then reacted at room temperature for 1 hour. Further, 50 mM THF solution (400 μL) of IY26, 75 mM NMP solution (400 μL) of HATU, and 150 mM NMP solution (400 μL) of DIPEA were mixed in a Falcon tube and left to stand for 15 minutes, then combined with the reaction solution, DIPEA (125 μL) was added, and reacted at room temperature for 2 hours. The reaction solution was purified with an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). 10 times the amount of 1 M meglumine acetate was added to the obtained solution, stirred well, and left to stand for 5 minutes to perform ion exchange. Then, desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 237 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y26-HDO Using ASO (Malat1) (first strand) and Y26-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y26-conjugated heteroduplex oligonucleotide (Y26-HDO), was prepared in the same manner as in step B of Example 1.

[0353] Example 27 A) Synthesis of cRNA with Y27-linked at the 5' end (Y27-cRNA(Malat1)). Using an aqueous solution (312 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY27, 705 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y27-HDO Using ASO (Malat1) (first strand) and Y27-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y27-conjugated heteroduplex oligonucleotide (Y27-HDO), was prepared in the same manner as in step B of Example 1.

[0354] Example 28 A) Synthesis of cRNA with Y28-linked at the 5' end (Y28-cRNA(Malat1)). The title compound (1046 nmol) was obtained in the same manner as in Step A of Example 11 using an aqueous solution (346 μL, 2530 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY28. B) Synthesis of double-stranded nucleic acid agent Y28-HDO Using ASO (Malat1) (first strand) and Y28-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y28-conjugated heteroduplex oligonucleotide (Y28-HDO), was prepared in the same manner as in step B of Example 1.

[0355] Example 29 A) Synthesis of cRNA with Y29-linked at the 5' end (Y29-cRNA(Malat1)). The procedure was carried out in the same manner as in Step A of Example 21 using an aqueous solution (369 μL, 1500 nmol) of an RNA strand (Y62-cRNA(Malat1)) shown in Table 1 and IY29 to give 903 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y29-HDO Using ASO (Malat1) (first strand) and Y29-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y29-conjugated heteroduplex oligonucleotide (Y29-HDO), was prepared in the same manner as in step B of Example 7.

[0356] Example 30 A) Synthesis of cRNA with Y30-linked at the 5' end (Y30-cRNA(Malat1)). Using an aqueous solution (358 μL, 1500 nmol) of an RNA strand (Y63-cRNA(Malat1)) shown in Table 1 and cholesterol chloroformate, 1228 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y30-HDO Using ASO (Malat1) (first strand) and Y30-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y30-conjugated heteroduplex oligonucleotide (Y30-HDO), was prepared in the same manner as in step B of Example 1.

[0357] Example 31 A) Synthesis of cRNA with Y31-linked at the 5' end (Y31-cRNA(Malat1)). Using an aqueous solution (355 μL, 1400 nmol) of an RNA strand (Y64-cRNA(Malat1)) shown in Table 1 and IY29, 657 nmol of the title compound was obtained in the same manner as in Step A of Example 21. B) Synthesis of double-stranded nucleic acid agent Y31-HDO Using ASO(Malat1) (first strand) and Y31-cRNA(Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y31-conjugated heteroduplex oligonucleotide (Y31-HDO), was prepared in the same manner as in step B of Example 7.

[0358] Example 32 A) Synthesis of cRNA with Y32-linked at the 5' end (Y32-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY32, 864 nmol of the title compound was obtained in the same manner as in Step A of Example 11. B) Synthesis of double-stranded nucleic acid agent Y32-HDO Using ASO (Malat1) (first strand) and Y32-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y32-conjugated heteroduplex oligonucleotide (Y32-HDO), was prepared in the same manner as in step B of Example 1.

[0359] Example 33 A) Synthesis of cRNA with Y33-linked at the 5' end (Y33-cRNA(Malat1)). An aqueous solution (365 μL, 2000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf with a 50 mM NMP solution (400 μL), distilled water (135 μL), NMP (3200 μL), ×10 PBS (500 μL), and 50 mM DMAP (400 μL) in that order, stirred, precipitated, and reacted at 70° C. for 1 hour. The reaction solution was purified with an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile), and desalted by ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). 1 M meglumine acetate was added in a 10-fold amount to the obtained solution, stirred well, and left for 5 minutes to perform ion exchange. Then, desalting was performed by ultrafiltration (Amicon ultra 3 kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 963 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y33-HDO Using ASO (Malat1) (first strand) and Y33-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y33-conjugated heteroduplex oligonucleotide (Y33-HDO), was prepared in the same manner as in step B of Example 1.

[0360] Example 34 A) Synthesis of cRNA with Y34-linked at the 5' end (Y34-cRNA(Malat1)). Using an aqueous solution (365 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY34, 1083 nmol of the title compound was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y34-HDO Using ASO (Malat1) (first strand) and Y34-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y34-conjugated heteroduplex oligonucleotide (Y34-HDO), was prepared in the same manner as in step B of Example 1.

[0361] Example 35 A) Synthesis of cRNA with Y35-linked at the 5' end (Y35-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1, 830 nmol of the title compound was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y35-HDO Using ASO (Malat1) (first strand) and the Y35-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y35-conjugated heteroduplex oligonucleotide (Y35-HDO), was prepared in the same manner as in step B of Example 1.

[0362] Example 36 A) Synthesis of cRNA with Y36-linked at the 5' end (Y36-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY36, 830 nmol of the title compound was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y36-HDO Using ASO (Malat1) (first strand) and Y36-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y36-conjugated heteroduplex oligonucleotide (Y36-HDO), was prepared in the same manner as in step B of Example 1.

[0363] Example 37 A) Synthesis of cRNA with Y37-linked at the 5' end (Y37-cRNA(Malat1)). Using an aqueous solution (400 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY37, 1189 nmol of the title compound was obtained in the same manner as in Step A of Example 33. B) Synthesis of double-stranded nucleic acid agent Y37-HDO Using ASO (Malat1) (first strand) and Y37-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y37-conjugated heteroduplex oligonucleotide (Y37-HDO), was prepared in the same manner as in step B of Example 1.

[0364] Example 38 A) Synthesis of cRNA with Y38-linked at the 5' end (Y38-cRNA(Malat1)). The title compound (1032 nmol) was obtained in the same manner as in Step A of Example 33 using an aqueous solution (329 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY38. B) Synthesis of double-stranded nucleic acid agent Y38-HDO Using ASO (Malat1) (first strand) and the Y38-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y38-conjugated heteroduplex oligonucleotide (Y38-HDO), was prepared in the same manner as in step B of Example 1.

[0365] Example 39 A) Synthesis of cRNA with Y39-linked at the 5' end (Y39-cRNA(Malat1)). An aqueous solution (329 μL, 2000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf flask with 50 mM NMP solution of IY39 (800 μL), distilled water (270 μL), NMP (2800 μL), Borate Buffer, pH 8.5+ / -0.2, 5X Concentrate (500 μL), in that order, stirred, precipitated, and reacted at room temperature for 1 hour. Further, a 50 mM NMP solution of IY39 (400 μL) was added and reacted at room temperature for 1 hour. Further, a 50 mM NMP solution of IY39 (400 μL), Borate Buffer, pH 8.5+ / -0.2, 5X Concentrate (100 μL) was added and reacted at room temperature overnight. The reaction solution was purified using an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile) and desalted using ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). 10 times the amount of 1 M meglumine acetate was added to the resulting solution, which was then thoroughly stirred and allowed to stand for 5 minutes to carry out ion exchange. Thereafter, the solution was desalted using ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). The final product was filtered using a 0.20 μm membrane filter and lyophilized to obtain 1554 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y39-HDO Using ASO (Malat1) (first strand) and Y39-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y39-conjugated heteroduplex oligonucleotide (Y39-HDO), was prepared in the same manner as in step B of Example 1.

[0366] Example 40 A) Synthesis of cRNA with Y40-linked at the 5' end (Y40-cRNA(Malat1)). Using an aqueous solution (364 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY40, 607 nmol of the title compound was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y40-HDO Using ASO (Malat1) (first strand) and Y40-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y40-conjugated heteroduplex oligonucleotide (Y40-HDO), was prepared in the same manner as in step B of Example 1.

[0367] Example 41 A) Synthesis of cRNA with Y41-linked at the 5' end (Y41-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY41, 1400 nmol of the title compound was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y41-HDO Using ASO (Malat1) (first strand) and Y41-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y41-conjugated heteroduplex oligonucleotide (Y41-HDO), was prepared in the same manner as in step B of Example 1.

[0368] Example 42 A) Synthesis of cRNA with Y42-linked at the 5' end (Y42-cRNA(Malat1)). The title compound (1061 nmol) was obtained in the same manner as in Step A of Example 39 using an aqueous solution (365 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY42. B) Synthesis of double-stranded nucleic acid agent Y42-HDO Using ASO (Malat1) (first strand) and Y42-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y42-conjugated heteroduplex oligonucleotide (Y42-HDO), was prepared in the same manner as in step B of Example 1.

[0369] Example 43 A) Synthesis of cRNA with Y43-linked at the 5' end (Y43-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY43, 820 nmol of the title compound was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y43-HDO Using ASO (Malat1) (first strand) and Y43-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y43-conjugated heteroduplex oligonucleotide (Y43-HDO), was prepared in the same manner as in step B of Example 1.

[0370] Example 44 A) Synthesis of cRNA with Y44-linked at the 5' end (Y44-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY44, 969 nmol of the title compound was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y44-HDO Using ASO (Malat1) (first strand) and Y44-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y44-conjugated heteroduplex oligonucleotide (Y44-HDO), was prepared in the same manner as in step B of Example 1.

[0371] Example 45 A) Synthesis of cRNA with Y45-linked at the 5' end (Y45-cRNA(Malat1)). Using an aqueous solution (164 μL, 1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY45, 653 nmol of the title compound was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y45-HDO Using ASO (Malat1) (first strand) and Y45-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y45-conjugated heteroduplex oligonucleotide (Y45-HDO), was prepared in the same manner as in step B of Example 1.

[0372] Example 46 A) Synthesis of cRNA with Y46-linked at the 5' end (Y46-cRNA(Malat1)). An aqueous solution (312 μL, 2000 nmol) of the RNA strand (Y61-cRNA (Malat1)) shown in Table 1 was mixed in an Eppendorf flask with 50 mM NMP solution (400 μL), distilled water (188 μL), NMP (3200 μL), ×10 PBS (500 μL), and 50 mM DMAP (400 μL) in that order, stirred, precipitated, and reacted at 70 °C for 1 hour. Furthermore, a 50 mM NMP solution (400 μL) of IY46 and 50 mM DMAP (400 μL) were added, and the mixture was reacted at 70 °C for 1 hour and at room temperature. Furthermore, a 50 mM NMP solution (400 μL) of IY46 and 150 mM DIPEA (400 μL) were added, and the mixture was reacted at 70 °C for 1 hour. The reaction solution was purified using an ODS column (column: Purif-Pack (registered trademark)-EX ODS-50 size 60, manufactured by Shoko Science, mobile phase: TEAA / acetonitrile) and desalted using ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). 10-fold amount of 1 M meglumine acetate was added to the obtained solution, stirred well, and allowed to stand for 5 minutes to carry out ion exchange. Then, desalted using ultrafiltration (Amicon ultra 3 kDa, manufactured by Merck Millipore, distilled water). The final product was filtered using a 0.20 μm membrane filter and lyophilized to obtain 980 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y46-HDO Using ASO (Malat1) (first strand) and Y46-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y46-conjugated heteroduplex oligonucleotide (Y46-HDO), was prepared in the same manner as in step B of Example 1.

[0373] Example 47 A) Synthesis of cRNA with Y47-linked at the 5' end (Y47-cRNA(Malat1)). The same procedure as in Step A of Example 1 was carried out using an aqueous solution (315 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY47 to obtain 569 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y47-HDO Using ASO (Malat1) (first strand) and Y47-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y47-conjugated heteroduplex oligonucleotide (Y47-HDO), was prepared in the same manner as in step B of Example 1.

[0374] Example 48 A) Synthesis of cRNA with Y48-linked at the 5' end (Y48-cRNA(Malat1)). IY48 in 50 mM NMP solution (800 μL), HATU in 75 mM NMP solution (800 μL), and DIPEA in 150 mM NMP solution (800 μL) were mixed in an Eppendorf tube, stirred, precipitated, and then reacted at room temperature for 30 minutes. An aqueous solution (2000 nmol) of the RNA strand (Y61-cRNA(Malat1)) shown in Table 1, NMP (2100 μL), distilled water (153 μL), and DIPEA (63 μL) were added thereto, stirred, precipitated, and then reacted at room temperature for 1 hour. The reaction solution was diluted with distilled water to an NMP concentration of 10v / v%, and then concentrated and low molecular weight reagents such as NMP were removed by ultrafiltration (Amicon ultra 3kDa, Merck Millipore, distilled water). The final product was filtered through a 0.20 μm membrane filter and lyophilized to obtain 1085 nmol of the title compound. B) Synthesis of double-stranded nucleic acid agent Y48-HDO Using ASO (Malat1) (first strand) and Y48-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y48-conjugated heteroduplex oligonucleotide (Y48-HDO), was prepared in the same manner as in step B of Example 1.

[0375] Example 49 A) Synthesis of cRNA with Y49-linked at the 5' end (Y49-cRNA(Malat1)). Using an aqueous solution (329 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY49, 1176 nmol of the title compound was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y49-HDO Using ASO (Malat1) (first strand) and the Y49-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y49-conjugated heteroduplex oligonucleotide (Y49-HDO), was prepared in the same manner as in step B of Example 1.

[0376] Example 50 A) Synthesis of cRNA with Y50-linked at the 5' end (Y50-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY50, 674 nmol of the title compound was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y50-HDO Using ASO (Malat1) (first strand) and Y50-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y50-conjugated heteroduplex oligonucleotide (Y50-HDO), was prepared in the same manner as in step B of Example 1.

[0377] Example 51 A) Synthesis of cRNA with Y51-linked at the 5' end (Y51-cRNA(Malat1)). Using an aqueous solution (315 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY51, 827 nmol of the title compound was obtained in the same manner as in Step A of Example 39. B) Synthesis of double-stranded nucleic acid agent Y51-HDO Using ASO(Malat1) (first strand) and Y51-cRNA(Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y51-conjugated heteroduplex oligonucleotide (Y51-HDO), was prepared in the same manner as in step B of Example 1.

[0378] Example 52 A) Synthesis of cRNA with Y52-linked at the 5' end (Y52-cRNA(Malat1)). Using an aqueous solution (164 μL, 1000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY52, 653 nmol of the title compound was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y52-HDO Using ASO (Malat1) (first strand) and Y52-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y52-conjugated heteroduplex oligonucleotide (Y52-HDO), was prepared in the same manner as in step B of Example 1.

[0379] Example 53 A) Synthesis of cRNA with Y53-linked at the 5' end (Y53-cRNA(Malat1)). Using an aqueous solution (313 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY53, 865 nmol of the title compound was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y53-HDO Using ASO (Malat1) (first strand) and the Y53-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y53-conjugated heteroduplex oligonucleotide (Y53-HDO), was prepared in the same manner as in step B of Example 1.

[0380] Example 54 A) Synthesis of cRNA with Y54-linked at the 5' end (Y54-cRNA(Malat1)). The title compound (1165 nmol) was obtained in the same manner as in Step A of Example 1 using an aqueous solution (313 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY54. B) Synthesis of double-stranded nucleic acid agent Y54-HDO Using ASO (Malat1) (first strand) and Y54-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y54-conjugated heteroduplex oligonucleotide (Y54-HDO), was prepared in the same manner as in step B of Example 1.

[0381] Example 55 A) Synthesis of cRNA with Y55-linked at the 5' end (Y55-cRNA(Malat1)). The title compound (1401 nmol) was obtained in the same manner as in Step A of Example 1 using an aqueous solution (313 μL, 2000 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY55. B) Synthesis of double-stranded nucleic acid agent Y55-HDO Using ASO (Malat1) (first strand) and the Y55-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y55-conjugated heteroduplex oligonucleotide (Y55-HDO), was prepared in the same manner as in step B of Example 1.

[0382] Example 57 A) Synthesis of cRNA with Y57-linked at the 5' end (Y57-cRNA(Malat1)). Using an aqueous solution (394 μL, 2500 nmol) of an RNA strand (Y61-cRNA(Malat1)) shown in Table 1 and IY57, 641 nmol of the title compound was obtained in the same manner as in Step A of Example 1. B) Synthesis of double-stranded nucleic acid agent Y57-HDO Using ASO (Malat1) (first strand) and Y57-cRNA (Malat1) (second strand) obtained in the previous step A, a double-stranded nucleic acid agent, Y57-conjugated heteroduplex oligonucleotide (Y57-HDO), was prepared in the same manner as in step B of Example 1.

[0383] Example 58 Synthesis of double-stranded nucleic acid agent Y59-HDO A double-stranded nucleic acid agent, Y59-conjugated heteroduplex oligonucleotide (Y59-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first strand) and Y59-cRNA (Malat1) (second strand) shown in Table 1.

[0384] Example 59 Synthesis of double-stranded nucleic acid agent Y60-HDO A double-stranded nucleic acid agent, Y60-conjugated heteroduplex oligonucleotide (Y60-HDO), was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first strand) and Y60-cRNA (Malat1) (second strand) shown in Table 1.

[0385] Example 60 Synthesis of double-stranded nucleic acid agent Chol-HDO A Chol-conjugated heteroduplex oligonucleotide (Chol-HDO), which is a double-stranded nucleic acid agent, was prepared in the same manner as in step B of Example 1 using ASO (Malat1) (first strand) and Chol-cRNA (Malat1) (second strand) shown in Table 1.

[0386] Example 61 (A) In vivo experiment The experimental animals used were 7-week-old male C57BL / 6J mice (Charles River Japan), with 2-4 mice per group. In the experimental group, a solution containing nucleic acid was administered intravenously once at a dose of 5mL / kg through the tail vein of the mice. In the comparison group, the solvent used to prepare the nucleic acid solution (5% glucose solution) was administered intravenously to the mice using the same procedure as in the experimental group.

[0387] (B) Expression analysis 72 hours after administration of the nucleic acid solution, the mice were anesthetized by intraperitoneal administration of 50 mg / kg pentobarbital, blood was collected, the mice were sacrificed, and the brains (cerebral cortex) were removed. Total RNA was extracted from the brain tissue using the RNA extraction reagent ISOGEN (Nippon Gene Co., Ltd.). The brain tissues removed in ISOGEN solution were crushed and then separated into RNA fractions using chloroform. This was then carried out using the nucleic acid isolation system QuickGene RNA tissue kit SII (Kurashiki Boseki Co., Ltd.). cDNA synthesis from total RNA was carried out using the ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd.), and quantitative PCR was carried out using THUNDERBIRD qPCR Mix (Toyobo Co., Ltd.). The quantitative PCR was carried out using the fluorescent probe method, and mouse Malat1 (Integrated DNA Technologies Co., Ltd.) and mouse Gapdh (Thermo Fisher Scientific Co., Ltd.) were used as fluorescent probes. The gene fragment amplification reaction conditions for quantitative PCR were in accordance with the protocol for THUNDERBIRD qPCR Mix (Toyobo Co., Ltd.). The expression levels of mouse Malat1 and Gapdh (internal standard gene) were calculated using a relative calibration curve, and the relative expression level was calculated as Malat1 / Gapdh. The average value of the relative expression level was calculated from the results of 2-4 mice per group. The ratio of the average value of the relative expression level of the experimental group to the average value of the relative expression level of the comparison group, which was set as 100%, was calculated as the relative Malat1 ncRNA expression level.

[0388] (C) Result The results of Example 61 are shown in Table 7. In the table, the dose indicates the amount of ASO (Malat1).

[0389] All of the duplexes Nos. 1, 3 to 55, 57, 59 and 60 suppressed the expression of Malat1 non-coding RNA in the cerebral cortex. This result demonstrated that the nucleic acid complex of the present invention can be delivered to the brain and exert an antisense effect there.

[0390] [Table 7] TIFF0007669602000127.tif7998

[0391] Example 62 (A) In vivo experiment The experimental animals used were 10-week-old male C57BL / 6J mice (Charles River Japan), with three mice per group. In the experimental group, a solution containing nucleic acid was administered intravenously once at a dose of 5mL / kg through the tail vein of the mice. In the comparison group, the solvent (saline) used to prepare the nucleic acid solution was administered intravenously to the mice using the same procedure as in the experimental group.

[0392] (B) Microglia isolation 72 hours after administration of the nucleic acid solution, the mouse was anesthetized with isoflurane, the diaphragm was incised to expose the heart, saline was injected from the left ventricle with a syringe, the right atrium was incised to allow blood to escape, and the brain was removed. The brain was treated with enzymes using Neural Tissue Dissociation Kits (Miltenyi Biotec) according to the protocol, debris was removed using Debris Removal Solution (Miltenyi Biotec), and brain cell fluid was prepared. Microglia were isolated from the brain cell fluid using CD11b (Microglia) Microbeads (Miltenyi Biotec) according to the protocol.

[0393] (C) Expression analysis Total RNA was extracted from isolated microglia using RNeasy Micro Kit (QIAGEN). cDNA synthesis from total RNA was performed using High Capacity cDNA RT Kit (Thermo Fisher Scientific), and quantitative PCR was performed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). Fluorescent probe method was adopted for quantitative PCR, and mouse Malat1 (Thermo Fisher Scientific) and mouse Gapdh (Thermo Fisher Scientific) were used as fluorescent probes. The gene fragment amplification reaction conditions for quantitative PCR were in accordance with the protocol for TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). The expression levels of mouse Malat1 and Gapdh (internal control genes) were calculated using the comparative Ct method, and the relative expression level was calculated as Malat1 / Gapdh. The average value of the relative expression level was calculated from the results of three mice per group. The ratio of the average value of the relative expression level of the experimental group to the average value of the relative expression level of the comparison group, which was set to 100%, was calculated as the relative Malat1 ncRNA expression level.

[0394] (D)Result The results of Example 62 are shown in Table 8. In the table, the dose indicates the amount of ASO (Malat1). The duplex No. 58 suppressed the expression of Malat1 non-coding RNA in microglia. This result demonstrated that the nucleic acid complex of the present invention can be delivered to the brain and exert an antisense effect in microglia.

[0395] [Table 8] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of a target transcript and has an antisense effect on the target transcript; the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is bound to a cholesterol analog; The cholesterol analogue is Below formula (VI): 【Chemistry 1】 (wherein R 5 and R 6 each independently represent a substituted or unsubstituted C 1-30 alkyl group, or a substituted or unsubstituted C 2-30 alkenyl group); or Below formula (VII): 【Chemistry 2】 (wherein R 7 represents a hydroxy group, a substituted or unsubstituted C 1-30 alkyl-carbonyl-oxy group, or a substituted or unsubstituted C 1-30 alkenyl-carbonyl-oxy group). It is expressed as The first nucleic acid strand is annealed to the second nucleic acid strand.

2. The second nucleic acid strand has the following formula (VIII): 【Chemistry 3】 (In the formula, L ２ is a substituted or unsubstituted C １ ~ １２ an alkylene group of the formula: ３ ~ ８ Cycloalkylene group, -(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ - or CH(CH ２ —OH)—CH ２ -O(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ - represents L ３ represents -NH- or a bond; L ４ is a substituted or unsubstituted C １ ~ １２ an alkylene group of the formula: ３ ~ ８ a cycloalkylene group of -(CH ２ ) ２ -[O-(CH ２ ) ２ ] ｍ - or a bond, where m is an integer from 1 to 25; L ５ represents -NH-(C=O)-, -(C=O)-, or a bond. The nucleic acid complex according to claim 1, which is bound to a cholesterol analogue via a linker represented by the following formula:

3. The nucleic acid complex of claim 1 or 2, wherein the first nucleic acid strand comprises at least four consecutive deoxyribonucleosides.

4. The nucleic acid complex according to any one of claims 1 to 3, wherein the first nucleic acid strand is a gapmer.

5. 5. The nucleic acid complex of claim 3 or 4, wherein the second nucleic acid strand comprises at least four consecutive ribonucleosides that are complementary to at least four consecutive deoxyribonucleosides in the first nucleic acid strand.

6. The nucleic acid complex according to claim 1 or 2, wherein the first nucleic acid strand is a mixmer.

7. The nucleic acid complex according to any one of claims 1 to 6, wherein the first nucleic acid strand is 13 to 20 bases in length.

8. The nucleic acid complex according to any one of claims 1 to 7, wherein the second nucleic acid strand does not contain natural ribonucleosides.

9. The nucleic acid complex according to any one of claims 1 to 8, wherein the nucleic acid portion of the second nucleic acid strand is composed of deoxyribonucleosides and / or sugar-modified nucleosides linked by modified or unmodified internucleoside bonds.

10. A composition comprising the nucleic acid complex according to any one of claims 1 to 9, for use in treating a central nervous system disease in a subject.

11. The composition of claim 10, wherein the central nervous system disease is an immune-mediated central nervous system disease.

12. 12. The composition of claim 10 or 11 for delivering a drug to the central nervous system of a subject.

13. The composition of any one of claims 10 to 12, wherein the central nervous system is selected from the group consisting of the cerebral cortex, the basal ganglia, the cerebral white matter, the diencephalon, the brainstem, the cerebellum, and the spinal cord.

14. The composition according to any one of claims 10 to 13, wherein the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigra, pons, medulla oblongata, cerebellar cortex, cerebellar nuclei, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord.

15. The composition according to any one of claims 10 to 14, which is for intravenous or subcutaneous administration.

16. The composition according to any one of claims 10 to 15, comprising 5 mg / kg or more of the nucleic acid complex in a single dose.

17. The composition of any one of claims 10 to 16, wherein the nucleic acid complex crosses the blood-brain barrier (BBB).

18. The composition according to claim 11, wherein the immune-mediated central nervous system disease is a microglia-associated disease.

19. 19. The composition of claim 18, wherein the microglia-related disease is Alzheimer's disease, multiple sclerosis, ALS, or neuropathic pain.

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