Double-stranded rnas containing carbamoyl ethyl modifications

Abstract

The present application aims to provide a chemically modified siRNA which further improves metabolic stability while maintaining activity. The present application provides a double-stranded RNA capable of suppressing expression of a target RNA, the double-stranded RNA comprising a sense strand and an antisense strand, the antisense strand having 14 to 40 nucleotides and having sufficient complementarity to the target RNA to mediate RNA interference, the sense strand having 14 to 40 nucleotides and having complementarity to the antisense strand, the double-stranded RNA comprising at least one 2'-O-XCE nucleotide.

Description

Technical Field

[0001] This invention relates to the following double-stranded RNA, pharmaceutical compositions comprising the thereof, and methods for regulating the expression of target RNA using the thereof, wherein the double-stranded RNA comprises carbamoyl ethyl modification (2'-O-XCE nucleotide) capable of inhibiting the expression of target RNA. Background Technology

[0002] One method for inhibiting the expression of target genes in cells, tissues, or individuals is the introduction of double-stranded RNA called small interfering RNA (siRNA) into such cells, tissues, or individuals. By introducing the double-stranded RNA, the target RNA, which is homologous to its sequence, is broken down, and the expression of the target gene is inhibited. This effect is called "RNA interference" or "RNAi." The siRNA is complementary to the target sequence on the transcript and, upon entering the cell, is loaded by the RNA-induced silencing complex (RISC). During this loading process, the sense strand is unloaded, and the antisense strand remains within the RISC; therefore, the antisense strand binds to its complementary site on the target RNA. The bound RNA is then cleaved by the nuclease activity of the RISC and can then be further broken down by cellular nucleases (see, for example, Non-Patent Literature 1).

[0003] siRNA, composed of natural RNA nucleotides, is rapidly degraded even when administered to organisms and does not easily reach target organs. Therefore, chemically modified siRNA is required for therapeutic applications. Typically, for siRNA, 2'-fluoro(2'-F) nucleotides and 2'-O-methyl(2'-O-Me) nucleotides are used as chemically modified nucleic acid units (modified nucleosides or modified nucleotides as their phosphate adducts) to improve metabolic stability (see, for example, Non-Patent Literature 2, 3).

[0004] As a modification of the oxygen atom at the 2' position of the sugar portion of ribonucleotides, methylcarbamoylethylated nucleotides (2'-O-MCE nucleotides) have been reported (see, for example, Patent Document 1 and Non-Patent Document 4). Furthermore, it has been reported that by introducing an amino or heterocyclic group into the nitrogen atom of the carbamoyl ethyl group via an alkyl group (2'-OR-ECE nucleotides), higher nuclease resistance can be obtained (see, for example, Patent Document 2 and Non-Patent Document 5). These nucleotides, such as 2'-O-MCE nucleotides and 2'-OR-ECE nucleotides, obtained by modifying the oxygen atom at the 2' position of the sugar portion with carbamoyl ethyl, are collectively referred to as 2'-O-XCE nucleotides.

[0005] Existing technical documents Patent documents Patent Document 1: International Publication No. 2007 / 102581 Patent Document 2: International Publication No. 2017 / 142054 Non-patent literature Non-patent literature 1: Nature, 1998, 391, pp 806-811 Non-patent literature 2: Nature Reviews Drug Discovery volume, 2019, 18, pp 421-446 Non-patent literature 3: Molecular Therapy, 2018, 26, pp 708-717 Non-patent literature 4: The Journal of Organic Chemistry, 2011, 76, pp 3042-3053 Non-patent literature 5: Organic & Biomolecular Chemistry, 2019, 17, pp 4835-4842 Summary of the Invention

[0006] The problem that the invention aims to solve In chemically modified siRNAs, new technologies are needed to further improve metabolic stability, activity, or sustained improvement while maintaining activity.

[0007] The purpose of this invention is to provide chemically modified siRNAs that further enhance metabolic stability, activity, or sustained improvement while maintaining activity.

[0008] Methods for solving problems The inventors of this application discovered that by partially replacing 2'-O-XCE nucleotides in chemically modified siRNA, metabolic stability, or activity and persistence, can be further improved while maintaining activity, thus completing the present invention. That is, the present invention comprises the following methods. 1. Double-stranded RNA is a type of double-stranded RNA that can repress the expression of its target RNA. The aforementioned double-stranded RNA contains a sense strand and an antisense strand. The aforementioned antisense strand has 14-40 nucleotides and is sufficiently complementary to the aforementioned target RNA for mediating RNA interference. The aforementioned sense strand has 14-40 nucleotides and is complementary to the aforementioned antisense strand. The aforementioned double-stranded RNA contains at least one 2'-O-XCE nucleotide. 2. As described in 1, the double-stranded RNA, wherein the aforementioned antisense strand contains at least one 2'-O-XCE nucleotide, or the aforementioned sense strand contains at least one 2'-O-XCE nucleotide.

[0011] 2-2. As described in 1, the double-stranded RNA, wherein the aforementioned antisense strand contains at least one 2'-O-XCE nucleotide.

[0012] 2-3. As described in 1, the double-stranded RNA, wherein the sense strand contains at least one 2'-O-XCE nucleotide.

[0013] 2-4. As described in 1, the double-stranded RNA, wherein the aforementioned antisense strand and sense strand each independently contain at least one 2'-O-XCE nucleotide. 3. The double-stranded RNA as described in any one of 1 to 2-4, wherein the aforementioned double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one 2'-fluoronucleotide. 4. The double-stranded RNA as described in any one of 1 to 3, wherein the aforementioned double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one 2'-O-Me nucleotide.

[0016] 4-2. The double-stranded RNA as described in any one of 1 to 4, wherein the nucleotides constituting the aforementioned double-stranded RNA are selected from 2'-O-XCE nucleotides, 2'-O-Me nucleotides, and 2'-fluoronucleotides. 5. The double-stranded RNA as described in any one of 1 to 4-2, wherein the aforementioned double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one deoxyribonucleotide.

[0018] 5-2. The double-stranded RNA as described in any one of 1 to 5, wherein the nucleotides constituting the aforementioned double-stranded RNA are selected from 2'-O-XCE nucleotides, 2'-O-Me nucleotides, 2'-fluoronucleotides and deoxyribonucleotides. 6. The double-stranded RNA as described in any one of 1 to 5-2, wherein at least one of positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the antisense strand hybridization region contains a 2'-O-XCE nucleotide.

[0020] 6-2. The double-stranded RNA as described in any one of 1 to 6, wherein the antisense strand comprises 2'-fluoronucleotides or deoxyribonucleotides at the 5' end of the hybridization portion of the antisense strand.

[0021] 6-3. The double-stranded RNA as described in any one of 1 to 6-2, wherein the aforementioned antisense strand contains 1 to 10 (preferably 1 to 9, more preferably 1, 2, 3 or 4) 2'-O-XCE nucleotides.

[0022] 6-4. The double-stranded RNA as described in any one of 1 to 6-3, wherein at least one of positions 1, 3, 4, 5, 6, 7, 8, 9 and 10 of the antisense strand hybridization portion, counted from the 5' end, contains a 2'-O-XCE nucleotide.

[0023] 6-5. The double-stranded RNA as described in any one of 1 to 6-4, wherein the antisense strand contains a 2'-O-XCE nucleotide at position 1, counted from the 5' end, of the antisense strand hybridization portion.

[0024] 6-6. The double-stranded RNA as described in any one of 1 to 6-5, wherein the antisense strand contains 2'-O-XCE nucleotides at the 3 positions counted from the 5' end of the antisense strand hybridization portion.

[0025] 6-7. The double-stranded RNA as described in any one of 1 to 6-6, wherein the aforementioned antisense strand contains 2'-O-XCE nucleotides at the 4 positions counted from the 5' end of the aforementioned antisense strand hybridization portion.

[0026] 6-8. The double-stranded RNA as described in any one of 1 to 6-7, wherein the antisense strand contains 2'-O-XCE nucleotides at the 5th position counting from the 5' end of the antisense strand hybridization portion.

[0027] 6-9. The double-stranded RNA as described in any one of 1 to 6-8, wherein the antisense strand contains 2'-O-XCE nucleotides at the 6 positions counted from the 5' end of the antisense strand hybridization portion.

[0028] 6-10. The double-stranded RNA as described in any one of 1 to 6-9, wherein the antisense strand contains 2'-O-XCE nucleotides at the 7 positions counted from the 5' end of the antisense strand hybridization portion.

[0029] 6-11. The double-stranded RNA as described in any one of 1 to 6-10, wherein the aforementioned antisense strand contains 2'-O-XCE nucleotides at the 8 positions counted from the 5' end of the aforementioned antisense strand hybridization portion.

[0030] 6-12. The double-stranded RNA as described in any one of 1 to 6-11, wherein the aforementioned antisense strand contains 2'-O-XCE nucleotides at the 9 positions counted from the 5' end of the aforementioned antisense strand hybridization portion.

[0031] 6-13. The double-stranded RNA as described in any one of 1 to 6-12, wherein the antisense strand contains 2'-O-XCE nucleotides at the 10 positions counted from the 5' end of the antisense strand hybridization portion. 7. The double-stranded RNA as described in any one of 1 to 6-13, wherein at least one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the sense strand, counted from the 3' end, contains a 2'-O-XCE nucleotide.

[0033] 7-2. The double-stranded RNA as described in any one of 1 to 7, wherein the sense strand comprises, independently, 2'-O-Me nucleotides, 2'-fluoronucleotides, or deoxyribonucleotides at positions 11, 12, and 13, counting from the 3' end, of the sense strand hybridization portion.

[0034] 7-3. The double-stranded RNA as described in any one of 1 to 7-2, wherein the sense strand comprises 2 to 10 (preferably 2 to 4, more preferably 2 or 4) 2'-O-XCE nucleotides.

[0035] 7-4. The double-stranded RNA as described in any one of 1 to 7-3, wherein at least one of positions 1, 2, 20 and 21 of the aforementioned sense strand at the aforementioned sense strand hybridization portion, counted from the 3' end, contains a 2'-O-XCE nucleotide.

[0036] 7-5. The double-stranded RNA as described in any one of 1 to 7-4, wherein the sense strand contains a 2'-O-XCE nucleotide at position 1, counted from the 3' end, of the sense strand hybridization portion.

[0037] 7-6. The double-stranded RNA as described in any one of 1 to 7-5, wherein the sense strand contains 2'-O-XCE nucleotides at position 21, counting from the 3' end, of the sense strand hybridization portion.

[0038] 7-7. The double-stranded RNA as described in any one of 1 to 7-6, wherein the sense strand contains 2'-O-XCE nucleotides at the 2 positions counted from the 3' end of the sense strand hybridization portion.

[0039] 7-8. The double-stranded RNA as described in any one of 1 to 7-7, wherein the sense strand contains 2'-O-XCE nucleotides at the 20th position counted from the 3' end of the sense strand hybridization portion.

[0040] 7-9. The double-stranded RNA as described in any one of 1 to 7-8, wherein the sense strand contains a 2'-O-XCE nucleotide at position 1, counted from the 5' end, of the sense strand hybridization portion.

[0041] 7-10. The double-stranded RNA as described in any one of 1 to 7-9, wherein the sense strand contains 2'-O-XCE nucleotides at the 2 positions counted from the 5' end of the sense strand hybridization portion.

[0042] 7-11. The double-stranded RNA as described in any one of 1 to 7-10, wherein the sense strand contains a 2'-O-XCE nucleotide at position 1, counted from the 5' end of the sense strand.

[0043] 7-12. The double-stranded RNA as described in any one of 1 to 7-11, wherein the sense strand contains 2'-O-XCE nucleotides at the 2 positions counted from the 5' end of the sense strand.

[0044] 7-13. The double-stranded RNA as described in any one of 1 to 7-12, wherein the sense strand contains a 2'-O-XCE nucleotide at position 1, counted from the 3' end of the sense strand.

[0045] 7-14. The double-stranded RNA as described in any one of 1 to 7-13, wherein the sense strand contains 2'-O-XCE nucleotides at the 2 positions counted from the 3' end of the sense strand. 8. The double-stranded RNA as described in any one of 1 to 7-13, wherein the aforementioned 2'-O-XCE nucleotide is a nucleotide comprising the local structure represented by the following formula (I): [Chemical Formula 1] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (Ia) represents the group: [Chemical Formula 2] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. 9. As described in 8, the double-stranded RNA, wherein the aforementioned X is a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups and cyano groups). 10. Double-stranded RNA as described in 8 or 9, wherein the aforementioned X is a methyl group. 11. As described in 8., the double-stranded RNA, wherein the aforementioned X is a group represented by the following formula (Ia): [Chemical Formula 3] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. 12. As described in 11, the double-stranded RNA, wherein the aforementioned R 1 and R 2 It is a hydrogen atom. 13. As described in 11 or 12, the double-stranded RNA, wherein the aforementioned Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form a 4-8 membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene rings, or... Y is a C2-9 aromatic heterocyclic group. 14. The double-stranded RNA as described in any one of 11 to 13, wherein the aforementioned Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine. 15. The double-stranded RNA as described in any one of 11 to 13, wherein the aforementioned Y is pyridyl, imidazole or benzimidazole. 16. The double-stranded RNA as described in any one of 11 to 15, wherein the aforementioned n is 2. 17. The double-stranded RNA as described in any one of 1 to 16, wherein the aforementioned antisense strand comprises 1, 2, 3, 4, 5 or 6 2'-fluoronucleotides.

[0056] 17-2. The double-stranded RNA as described in any one of 1 to 17, wherein the aforementioned antisense strand contains at least one of positions 2, 6, 8, 9, 14 and 16, counted from the 5' end, of the antisense strand hybridization region.

[0057] 17-3. The double-stranded RNA as described in any one of 1 to 17-2, wherein the aforementioned antisense strand contains 2'-fluoronucleotides at the 2 positions counted from the 5' end of the antisense strand hybridization portion.

[0058] 17-4. The double-stranded RNA as described in any one of 1 to 17-3, wherein the aforementioned antisense strand contains 2'-fluoronucleotides at the 14th position counted from the 5' end of the antisense strand hybridization portion.

[0059] 17-5. The double-stranded RNA as described in any one of 1 to 17-4, wherein the aforementioned antisense strand contains 2'-fluoronucleotides at the 16th position counted from the 5' end of the antisense strand hybridization portion.

[0060] 17-6. The double-stranded RNA as described in any one of 1 to 17-5, wherein the aforementioned antisense strand contains 2'-fluoronucleotides at the 6 positions counted from the 5' end of the antisense strand hybridization portion. 18. The double-stranded RNA as described in any one of 1 to 17-6, wherein the sense strand comprises 1, 2, 3, 4 or 5 2'-fluoronucleotides.

[0062] 18-2. The double-stranded RNA as described in any one of 1 to 18, wherein the aforementioned sense strand contains at least one of positions 11, 12, 13 and 15, counted from the 3' end, of the sense strand hybridization region, comprising a 2'-fluoronucleotide.

[0063] 18-3. The double-stranded RNA as described in any one of 1 to 18-2, wherein the sense strand comprises 2'-fluoronucleotides at the 15th position counting from the 3' end of the sense strand hybridization portion.

[0064] 18-4. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand comprises 2'-fluoronucleotides at positions 11, 12, and 13, counting from the 3' end, of the sense strand hybridization region.

[0065] 18-5. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand contains a deoxyribonucleotide at position 11, counting from the 3' end, and a 2'-fluoronucleotide at positions 12 and 13.

[0066] 18-6. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand contains deoxyribonucleotides at positions 12 from the 3' end of the sense strand hybridization region, and 2'-fluoronucleotides at positions 11 and 13.

[0067] 18-7. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand contains a deoxyribonucleotide at position 13, counting from the 3' end, and a 2'-fluoronucleotide at positions 11 and 12.

[0068] 18-8. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand contains 2'-O-Me nucleotides at positions 11 from the 3' end of the sense strand hybridization region, and 2'-fluoronucleotides at positions 12 and 13.

[0069] 18-9. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand contains 2'-O-Me nucleotides at positions 12 from the 3' end of the sense strand hybridization region, and 2'-fluoronucleotides at positions 11 and 13.

[0070] 18-10. The double-stranded RNA as described in any one of 1 to 18-3, wherein the sense strand contains a 2'-O-Me nucleotide at position 13, counting from the 3' end, and a 2'-fluoro nucleotide at positions 11 and 12. 19. The double-stranded RNA as described in any one of 1 to 18-10, wherein the sense strand comprises 7 to 18 2'-O-Me nucleotides.

[0072] 19-2. The double-stranded RNA as described in any one of 1 to 19, wherein the sense strand comprises 13, 14, 15, 16, 17 or 18 2'-O-Me nucleotides. 20. The double-stranded RNA as described in any one of 1 to 19-2, wherein the aforementioned antisense strand contains 13 to 20 2'-O-Me nucleotides.

[0074] 20-2. The double-stranded RNA as described in any one of 1 to 20, wherein the aforementioned antisense strand comprises 13 to 18 2'-O-Me nucleotides. twenty one. The double-stranded RNA as described in any one of 1 to 20-2, wherein the sense strand comprises 0 to 6 deoxyribonucleotides.

[0076] 21-2. The double-stranded RNA as described in any one of 1 to 21, wherein the sense strand comprises 0 or 1 deoxyribonucleotide. twenty two. The double-stranded RNA as described in any one of 1 to 21-2, wherein the aforementioned antisense strand comprises 0 to 6 deoxyribonucleotides.

[0078] 22-2. The double-stranded RNA as described in any one of 1 to 22, wherein the aforementioned antisense strand contains 1 to 6 (preferably 2 to 6, 3 to 5) deoxyribonucleotides at positions 2, 5, 7, 12, 14 and 16, selected from the 5' end of the antisense strand hybridization region.

[0079] 22-3. The double-stranded RNA as described in any one of 1 to 22-2, wherein the aforementioned antisense strand contains 1 to 4 (preferably 2, 3 or 4) deoxyribonucleotides at positions 2, 5, 7 and 12, counted from the 5' end of the antisense strand hybridization region.

[0080] 22-4. The double-stranded RNA as described in any one of 1 to 22-3, wherein the aforementioned antisense strand contains deoxyribonucleotides in one or two of the 5th and 7th positions selected from the antisense strand hybridization portion, counting from the 5' end.

[0081] 22-5. The double-stranded RNA as described in any one of 1 to 22, wherein the aforementioned antisense strand does not contain deoxyribonucleotides. twenty three. The double-stranded RNA as described in any one of 1 to 22-5, wherein the aforementioned double-stranded RNA contains at least one phosphate thioester bond.

[0083] 23-2. The double-stranded RNA as described in any one of 1 to 23, wherein the bonds linking the nucleotides constituting the aforementioned double-stranded RNA are each independently a phosphodiester bond or a modified phosphodiester bond (preferably a thiophosphate bond). twenty four. The double-stranded RNA as described in any one of 1 to 23-2, wherein the sense strand comprises 1, 2, 3 or 4 phosphate thioester bonds. 25. The double-stranded RNA as described in any one of 1 to 24, wherein the sense strand contains a phosphate thioester bond in at least one of the following groups: between positions 1 and 2, between positions 2 and 3, and between positions 1 and 2, and between positions 2 and 3, counted from the 5' end of the sense strand.

[0086] 25-2. The double-stranded RNA as described in any one of 1 to 25, wherein the sense strand contains a phosphate thioester bond between positions 1 and 2, between positions 2 and 3, and between positions 1 and 2, and between positions 2 and 3, counting from the 5' end of the sense strand. 26. The double-stranded RNA as described in any one of 1 to 25-2, wherein the aforementioned antisense strand contains 1, 2, 3 or 4 phosphate thioester bonds. 27. The double-stranded RNA as described in any one of 1 to 26, wherein the aforementioned antisense strand contains a phosphate thioester bond between positions 1 and 2, between positions 2 and 3, and between positions 1 and 2, and between positions 2 and 3, counting from the 5' end of the aforementioned antisense strand. 28. The double-stranded RNA as described in any one of 1 to 27, wherein the aforementioned antisense strand contains a 5'-phosphonate group at its 5' end. 29. As described in 28, the 5'-phosphonate group is a 5'-vinylphosphonate (VP) group. 30. Double-stranded RNA as described in 28 or 29, wherein the aforementioned antisense strand contains a 5'-vinylphosphonate (VP)-substituted 2'-O-Me nucleotide at position 1, counted from the 5' end of the aforementioned antisense strand. 31. Double-stranded RNA as described in 28 or 29, wherein the aforementioned antisense strand contains a 5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotide at position 1, counted from the 5' end of the aforementioned antisense strand. 32. As described in 28, the 5'-phosphonate group is a 5'-cyclopropanephosphonate (CPP) group. 33. Double-stranded RNA as described in 28 or 32, wherein the aforementioned antisense strand contains a 5'-cyclopropanephosphonate (CPP)-substituted 2'-O-Me nucleotide at position 1, counted from the 5' end of the aforementioned antisense strand. 34. Double-stranded RNA as described in 28 or 32, wherein the aforementioned antisense strand contains a 5'-cyclopropanephosphonate (CPP)-substituted 2'-O-XCE nucleotide at position 1, counted from the 5' end of the aforementioned antisense strand. 35. As described in 28, the 5'-phosphonate group is a 5'-ethylphosphonate (EP) group. 36. Double-stranded RNA as described in 28 or 35, wherein the aforementioned antisense strand contains 5'-ethylphosphonate (EP)-substituted 2'-O-Me nucleotides at position 1, counted from the 5' end of the aforementioned antisense strand. 37. Double-stranded RNA as described in 28 or 35, wherein the aforementioned antisense strand contains 5'-ethylphosphonate (EP)-substituted 2'-O-XCE nucleotides at position 1, counted from the 5' end of the aforementioned antisense strand. 38. The double-stranded RNA as described in any one of 1 to 37, wherein the length of the sense strand and the antisense strand is independently 19 to 25 nucleotides (preferably 19 to 23 nucleotides).

[0100] 38-2. The double-stranded RNA as described in any one of 1 to 38, wherein the length of the sense strand is 19 to 21 nucleotides and the length of the antisense strand is 21 to 23 nucleotides. 39. The double-stranded RNA as described in any one of 1 to 38-2, wherein the sense strand is 21 nucleotides long. 40. The double-stranded RNA as described in any one of 1 to 39, wherein the aforementioned antisense strand has a nucleotide length of 23. 41. The double-stranded RNA as described in any one of 1 to 40, wherein the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides. 42. The double-stranded RNA as described in any one of 1 to 41 includes an overhang at the 3' end of the aforementioned antisense strand.

[0105] 42-2. As described in 42, the double-stranded RNA, wherein the overhang at the 3' end of the aforementioned antisense strand is 2 nucleotides long. 43. The double-stranded RNA as described in any one of 1 to 42-2, wherein the 5' end of the aforementioned antisense strand comprises a blunt end. 44. The double-stranded RNA as described in any one of 1 to 43 further comprises a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target site. 45. As described in 44, the double-stranded RNA, wherein the aforementioned functional molecules are selected from the group consisting of sugars, lipids, peptides, proteins, and their derivatives. 46. The double-stranded RNA as described in 44 or 45, wherein the aforementioned functional molecules are selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, C5-30 unsaturated fatty acids, C5-30 alkyl groups and C5-30 alkenyl groups.

[0110] 46-2. As described in 46, the double-stranded RNA, wherein the aforementioned functional molecule is a lipid selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids and C5-30 unsaturated fatty acids.

[0111] 46-3. As described in 46, the double-stranded RNA wherein the aforementioned group derived from the functional molecule is a C5-30 alkyl group (preferably a C14-24 alkyl group, a C16-22 alkyl group, or a hexadecyl group). 47. Double-stranded RNA as described in 44 or 45, wherein the aforementioned functional molecule is a peptide or protein selected from the group consisting of receptor ligands and antibodies. 48. Double-stranded RNA as described in 44 or 45, wherein the aforementioned functional molecule is a sugar derivative capable of interacting with desialyl glycoprotein receptors.

[0114] 48-2. As described in 48, the double-stranded RNA, wherein the aforementioned group derived from the functional molecule is a group of a ligand represented by the following formula: [Chemical Formula 4] (In the formula, Rd is a hydroxyl group or a mercapto group (preferably a mercapto group)).

[0115] 48-3. As described in 48, the double-stranded RNA, wherein the aforementioned group derived from the functional molecule is a group of a ligand represented by the following formula: [Chemical Formula 5] (In the formula, Re is independently a hydroxyl group or a mercapto group (preferably a mercapto group)).

[0116] 48-4. The double-stranded RNA as described in any one of 44 to 48-3, wherein the aforementioned functional molecule is capable of binding to the 5' or 3' end of the sense or antisense strand.

[0117] 48-5. The double-stranded RNA as described in any one of 44 to 48-3, wherein the aforementioned functional molecule is capable of binding to the 3' end of the sense strand.

[0118] 48-6. The double-stranded RNA as described in any one of 44 to 48-3, wherein the aforementioned functional molecule is capable of binding to the inner nucleotide of the sense strand (preferably the oxygen atom at the 2' position of the inner nucleotide).

[0119] 48-7. The double-stranded RNA as described in any one of 48. to 48-3., wherein the aforementioned functional molecule can bind to the inner nucleotide of the antisense strand (preferably the oxygen atom at the 2' position of the inner nucleotide). 49. A pharmaceutical composition comprising a pharmacologically permissible carrier and any one of the following: 1 to 48-7. of double-stranded RNA. 50. A method for regulating the function of a target RNA, comprising the step of contacting a cell with the double-stranded RNA as described in any one of 1 to 49. 51. A method for regulating the function of target RNA in a mammal, comprising the step of administering the pharmaceutical composition described in 49 to the mammal. 52. Method for producing double-stranded RNA using any one of 1 to 48-7 using 2'-O-XCE nucleotides. 53. The following compounds or salts represented by formula (II): [Chemical Formula 6] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IIa) represents the group: [Chemical Formula 7] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T1 For the group represented by the following formula (IIb): [Chemical Formula 8] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano)] Rb represents either an oxygen atom or a sulfur atom. A 1 The group is selected from the following formula (IIc): [Chemical Formula 9] [Q1 and Q2 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy, and cyano groups)]}. 54. The compound or its salt as described in 53, wherein X is a methyl group and Q1 and Q2 are hydrogen atoms. 55. The compound or salt thereof as described in 53 or 54, wherein the aforementioned R 1 and R 2 For hydrogen atoms, Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine, where Q1 and Q2 are hydrogen atoms. 56. The compound or salt thereof as described in any one of 53 to 55, wherein the aforementioned phosphorus-containing group is cyanoethoxy(diisopropylamino)phosphinyl or hydroxyphosphinyl. 57. The following compounds or salts represented by formula (III): [Chemical Formula 10] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IIIa) represents the group: [Chemical Formula 11] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T1 The group represented by the following formula (IIIb): [Chemical Formula 12] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. B 1 The group represented by the following formula (IIIc): [Chemical Formula 13] [Q3 and Q4 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkyl, a C1-6 alkoxy, and a cyano group)]}. 58. The compound or salt thereof as described in 57, wherein X is a methyl group and Q3 and Q4 are hydrogen atoms. 59. The compound or salt thereof as described in 57 or 58, wherein the aforementioned R 1 and R 2 For hydrogen atoms, Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine, where Q3 and Q4 are hydrogen atoms. 60. The compound or salt thereof as described in any one of 57 to 59, wherein the aforementioned phosphorus-containing group is cyanoethoxy(diisopropylamino)phosphin or hydroxyphosphin. 61. The following compounds or salts of formula (IV) are shown: [Chemical Formula 14] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IVa) represents the group: [Chemical Formula 15] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T1 For groups represented by the following formula (IVb): [Chemical Formula 16] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. E 1 For groups represented by the following formula (IVc): [Chemical Formula 17] [Q5~Q8 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy, and cyano groups)]}. 62. The compound or its salt as described in 61, wherein X is a methyl group and Q5 to Q8 are hydrogen atoms. 63. The compound or salt thereof as described in 61 or 62, wherein the aforementioned R 1 and R 2 For hydrogen atoms, Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine, where Q5 to Q8 are hydrogen atoms. 64. The compound or salt thereof as described in any one of 61 to 63, wherein the aforementioned phosphorus-containing group is cyanoethoxy(diisopropylamino)phosphin or hydroxyphosphin.

[0136] Invention Effects According to the present invention, chemically modified siRNAs can be provided that further enhance metabolic stability, or activity and sustained enhancement while maintaining activity.

[0137] The double-stranded RNA of this invention can effectively regulate the expression of target RNA and is useful as a nucleic acid pharmaceutical. Attached Figure Description

[0138] [ Figure 1 The image shows the single-crystal X-ray analysis ORTEP plot of isomer A of compound 13.

[0139] [ Figure 2 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001 to si-0014) in quantitative real-time PCR as measured in Evaluation Example 1.

[0140] [ Figure 3 The rat Sod1 gene expression levels of each siRNA (si-0001, si-0015 to si-0027) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0141] [ Figure 4 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0028 to si-0040) in quantitative real-time PCR as measured in Evaluation Example 1.

[0142] [ Figure 5 The rat Sod1 gene expression levels of each siRNA (si-0001, si-0041 to si-0053) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0143] [ Figure 6 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0054 to si-0066) in quantitative real-time PCR as measured in Evaluation Example 1.

[0144] [ Figure 7 The rat Sod1 gene expression levels of each siRNA (si-0067 to si-0080) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0145] [ Figure 8 The rat Sod1 gene expression levels of each siRNA (si-0067, si-0081 to si-0093) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0146] [ Figure 9 The table shows the rat Sod1 gene expression levels of each siRNA (si-0067, si-0094 to si-0106) in quantitative real-time PCR as measured in Evaluation Example 1.

[0147] [ Figure 10The rat Sod1 gene expression levels of each siRNA (si-0067, si-0107 to si-0119) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0148] [ Figure 11 The rat Sod1 gene expression levels of each siRNA (si-0067, si-0120 to si-0132) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0149] [ Figure 12 The rat Sod1 gene expression levels of each siRNA (s si-0001, i-0133 to si-0138) in quantitative real-time PCR, as measured in Evaluation Example 1, are shown.

[0150] [ Figure 13 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0139 to si-0145) in quantitative real-time PCR as measured in Evaluation Example 1.

[0151] [ Figure 14 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0146 to si-0157) in quantitative real-time PCR as measured in Evaluation Example 2.

[0152] [ Figure 15 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0158 to si-0161) in quantitative real-time PCR as measured in Evaluation Example 2.

[0153] [ Figure 16 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0162 to si-0169) in quantitative real-time PCR as measured in Evaluation Example 2.

[0154] [ Figure 17 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0170 to si-0182) in quantitative real-time PCR as measured in Evaluation Example 2.

[0155] [ Figure 18 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0183 to si-0190) in quantitative real-time PCR as measured in Evaluation Example 2.

[0156] [ Figure 19The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0191 to si-0197) in quantitative real-time PCR as measured in Evaluation Example 2.

[0157] [ Figure 20 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0198 to si-0208) in quantitative real-time PCR as measured in Evaluation Example 2.

[0158] [ Figure 21 The rat Sod1 gene expression levels of each siRNA (si-0001, si-0209 to si-0211) in quantitative real-time PCR, as measured in Evaluation Example 3, are shown.

[0159] [ Figure 22 The table shows the rat Sod1 gene expression levels of each siRNA (si-0001, si-0212 to si-0223) in quantitative real-time PCR as measured in Evaluation Example 3.

[0160] [ Figure 23 The text shows the expression levels of the mouse Sod1 gene in the lungs of each siRNA (si-0224 to si-0227) as measured by quantitative real-time PCR in Evaluation Example 6.

[0161] [ Figure 24 The text shows the expression levels of the mouse Sod1 gene in the lungs of each siRNA (si-0224 to si-0225) as measured by quantitative real-time PCR in Evaluation Example 7.

[0162] [ Figure 25 The mouse Sod1 gene expression levels in the brain of each siRNA (si-0224 to si-0226 and si-0228) measured by quantitative real-time PCR in Evaluation Example 8 are shown.

[0163] [ Figure 26 The text shows the expression levels of the mouse Sod1 gene in the hippocampus of each siRNA (si-0224 to si-0226 and si-0228) as measured by quantitative real-time PCR in Evaluation Example 8.

[0164] [ Figure 27 The image shows the expression levels of the mouse Sod1 gene in the cerebellum of each siRNA (si-0224 to si-0226 and si-0228) measured by quantitative real-time PCR in Evaluation Example 8.

[0165] [ Figure 28The image shows the mouse Sod1 gene expression levels in the medulla oblongata in Evaluation Example 8, measured by quantitative real-time PCR for each siRNA (si-0224 to si-0226 and si-0228).

[0166] [ Figure 29 The mouse Ttr gene expression levels of each siRNA (si-0229 to si-0241) in quantitative real-time PCR, as measured in Evaluation Example 9, are shown.

[0167] [ Figure 30 The mouse Ttr gene expression levels of each siRNA (si-0229, si-0242 to si-0250) in quantitative real-time PCR, as measured in Evaluation Example 9, are shown.

[0168] [ Figure 31 The mouse Ttr gene expression levels of each siRNA (si-0229, si-0251 to si-0259) in quantitative real-time PCR, as measured in Evaluation Example 9, are shown.

[0169] [ Figure 32 The mouse FXII gene expression levels of each siRNA (si-0260 to si-0270) in quantitative real-time PCR, as measured in Evaluation Example 10, are shown.

[0170] [ Figure 33 The mouse FXII gene expression levels of each siRNA (si-0260, si-0271 to si-0281) in quantitative real-time PCR, as measured in Evaluation Example 10, are shown.

[0171] [ Figure 34 The mouse FXII gene expression levels of each siRNA (si-0260, si-0282 to si-0289) in quantitative real-time PCR, as measured in Evaluation Example 10, are shown.

[0172] [ Figure 35 The table shows the TTR expression levels in mouse serum of each siRNA (si-0290 to si-0297) as measured by ELISA in Evaluation Example 11.

[0173] [ Figure 36 The mouse Ttr gene expression levels of each siRNA (si-0298 to si-0305) in quantitative real-time PCR, as measured in Evaluation Example 12, are shown.

[0174] [ Figure 37 The mouse Ttr gene expression levels of each siRNA (si-0306 to si-0312) in quantitative real-time PCR, as measured in Evaluation Example 12, are shown.

[0175] [ Figure 38 The mouse Ttr gene expression levels of each siRNA (si-0313 to si-0321) in quantitative real-time PCR, as measured in Evaluation Example 13, are shown.

[0176] [ Figure 39 The mouse Ttr gene expression levels of each siRNA (si-0322 to si-0329) in quantitative real-time PCR, as measured in Evaluation Example 13, are shown.

[0177] [ Figure 40 The mouse Ttr gene expression levels of each siRNA (si-0330 to si-0343) in quantitative real-time PCR, as measured in Evaluation Example 14, are shown.

[0178] [ Figure 41 The mouse Ttr gene expression levels of each siRNA (si-0298, si-0344 to si-0349) in quantitative real-time PCR, as measured in Evaluation Example 15, are shown.

[0179] [ Figure 42 The table shows the TTR expression levels in mouse serum of each siRNA (si-0350 to si-0351) as measured by ELISA in Evaluation Example 16.

[0180] [ Figure 43 The rat Sod1 gene expression levels of each siRNA (si-0209, si-0216, si-0352 to si-0362) in quantitative real-time PCR, as measured in Evaluation Example 17, are shown.

[0181] [ Figure 44 The rat Sod1 gene expression levels of each siRNA (si-0209, si-0216, si-0363 to si-0373) in quantitative real-time PCR, as measured in Evaluation Example 17, are shown.

[0182] [ Figure 45 The rat Sod1 gene expression levels of each siRNA (si-0209, si-0216, si-0374 to si-0383) in quantitative real-time PCR, as measured in Evaluation Example 18, are shown.

[0183] [ Figure 46 The rat Sod1 gene expression levels of each siRNA (si-0209, si-0216, si-0384 to si-0394) in quantitative real-time PCR, as measured in Evaluation Example 18, are shown.

[0184] [ Figure 47The mouse Ttr gene expression levels of each siRNA (si-0251, si-0253, si-0395 to si-0403) in quantitative real-time PCR, as measured in Evaluation Example 19, are shown.

[0185] [ Figure 48 The mouse Ttr gene expression levels of each siRNA (si-0251, si-0253, si-0404 to si-0416) in quantitative real-time PCR, as measured in Evaluation Example 19, are shown.

[0186] [ Figure 49 The mouse Ttr gene expression levels of each siRNA (si-0251, si-0411 to si-0412) in quantitative real-time PCR, as measured in Evaluation Example 19, are shown.

[0187] [ Figure 50 The mouse FXII gene expression levels of each siRNA (si-0282, si-0417 to si-0419) in quantitative real-time PCR, as measured in Evaluation Example 20, are shown.

[0188] [ Figure 51 The mouse FXII gene expression levels of each siRNA (si-0282, si-0284, si-0420 to si-0425) in quantitative real-time PCR, as measured in Evaluation Example 20, are shown.

[0189] [ Figure 52 The mouse Ttr gene expression levels of each siRNA (si-0298, si-0347, si-0426 to si-0429) in quantitative real-time PCR, as measured in Evaluation Example 21, are shown.

[0190] [ Figure 53 The mouse Ttr gene expression levels of each siRNA (si-0298, si-0430 to si-0436) in quantitative real-time PCR, as measured in Evaluation Example 21, are shown.

[0191] [ Figure 54 The mouse Ttr gene expression levels of each siRNA (si-0251, si-0437 to si-0448) in quantitative real-time PCR, as measured in Evaluation Example 22, are shown.

[0192] [ Figure 55 The mouse Ttr gene expression levels of each siRNA (si-0251, si-0449 to si-0453) in quantitative real-time PCR, as measured in Evaluation Example 23, are shown.

[0193] [ Figure 56The mouse Ttr gene expression levels of each siRNA (si-0251, si-0454 to si-0460) in quantitative real-time PCR, as measured in Evaluation Example 24, are shown.

[0194] [ Figure 57 The mouse Ttr gene expression levels of each siRNA (si-0251, si-0461 to si-0467) in quantitative real-time PCR, as measured in Evaluation Example 25, are shown.

[0195] [ Figure 58 The mouse Ttr gene expression levels of each siRNA (si-0298, si-0468 to si-0470) in quantitative real-time PCR, as measured in Evaluation Example 26, are shown.

[0196] [ Figure 59 The rat Sod1 gene expression levels of each siRNA (si-0209, si-0216, si-0220, si-0471 to si-0480) measured in quantitative real-time PCR in Evaluation Example 27 are shown.

[0197] [ Figure 60 The rat Sod1 gene expression levels of each siRNA (si-0209, si-0481 to si-0486) in quantitative real-time PCR, as measured in Evaluation Example 29, are shown.

[0198] [ Figure 61 The table shows the TTR expression levels in mouse serum for each siRNA (si-0488, si-00489, si-0497 to si-0499) as measured by ELISA in Evaluation Example 30.

[0199] [ Figure 62 The table shows the TTR expression levels in mouse serum for each siRNA (si-0488, si-00489, si-0493 to si-0495, si-0500) detected by ELISA in Evaluation Example 30.

[0200] [ Figure 63 The table shows the TTR expression levels in mouse serum for each siRNA (si-0488, si-00489, si-0490 to si-0492, si-0496) detected by ELISA in Evaluation Example 30.

[0201] [ Figure 64 The image shows the nuclease resistance of the oligonucleotides measured in Evaluation Example 31.

[0202] [ Figure 65 The image shows the nuclease resistance of the oligonucleotides measured in Evaluation Example 32. Detailed Implementation

[0203] Unless otherwise specified, the terms used in this specification are used in the sense they are commonly understood in the art. The terms used in this specification are explained below. It should be noted that, unless otherwise specified, each term in this specification has the same meaning when used alone or in combination with other terms. The foregoing summary and the following detailed description are merely illustrative and should not be construed as limiting the scope of the claimed invention. In this specification, unless otherwise specified, the singular form includes the plural form.

[0204] “n-” means normal, “i-” means iso, “s-” means secondary, “t-” means tert-, “o-” means ortho, “m-” means meta, and “p-” means para. “Ph” means phenyl, “Me” means methyl, “Bu” means butyl, “Ac” means acetyl, and “DMTr” means dimethoxytriphenylmethyl.

[0205] The term "halogen atom" refers to fluorine, chlorine, bromine, or iodine atoms.

[0206] The term "C1-6 alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 6 carbon atoms. Examples of C1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl. Similarly, "C1-4 alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 4 carbon atoms. Similarly, "C1-3 alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 3 carbon atoms. Similarly, "C5-30 alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon having 5 to 30 carbon atoms. Examples of C5-30 alkyl groups include n-pentyl, n-hexyl, isohexyl, n-heptyl, decyl, tetradecyl, hexadecyl, octadecyl, eicosyl, dodecyl, tetradecyl, and triacontyl. Similarly, "C14-24 alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon with 14 to 24 carbon atoms. Likewise, "C16-22 alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon with 16 to 22 carbon atoms.

[0207] The term "halogenated C1-6 alkyl" refers to a group obtained by replacing at least one hydrogen atom at any position of the aforementioned "C1-6 alkyl" with the aforementioned "halogen atom".

[0208] The term "C2-6 alkenyl" refers to a monovalent group in a straight-chain or branched unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond and having 2 to 6 carbon atoms. Examples of C2-6 alkenyl groups include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, butadienyl, 3-methyl-2-butenyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, and hexadienyl. Similarly, the term "C5-30 alkenyl" refers to a monovalent group in a straight-chain or branched unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond and having 5 to 30 carbon atoms. Examples of C5-30 alkenyl groups include pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, decenyl, tetradecenyl, hexadecenyl, octadecenyl, eicoseneyl, dodecenyl, tetradecenyl, triadecenyl, etc.

[0209] The term "C1-6 alkoxy" refers to a group formed by the bonding of a "C1-6 alkyl" group with an oxygen group (-O-). Examples of C1-6 alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, sec-butoxy, n-pentyloxy, isopentyloxy, and n-hexyloxy.

[0210] The term "C2-6 alkenyloxy group" refers to a group formed by bonding the aforementioned "C2-6 alkenyl group" with an oxygen group (-O-). Examples of C2-6 alkenyloxy groups include vinyloxy, allyloxy, propenyloxy, isopropenyloxy, butenyloxy, isobutenyloxy, butadienyloxy, 3-methyl-2-butenyloxy, pentenyloxy, isopentenyloxy, pentamenyloxy, hexenyloxy, isohexenyloxy, and hexadienyloxy.

[0211] "C1-6 alkyl carbonyl" refers to the group obtained by bonding the aforementioned "C1-6 alkyl" with a carbonyl group (-C(=O)-).

[0212] "C1-6 alkoxy carbonyl" refers to the group obtained by bonding the aforementioned "C1-6 alkoxy" with a carbonyl group (-C(=O)-), and "C2-6 alkenyloxy carbonyl" refers to the group obtained by bonding the aforementioned "C2-6 alkenyloxy" with a carbonyl group (-C(=O)-).

[0213] The term "C2-20 alkylene" refers to a divalent group in a straight-chain or branched saturated aliphatic hydrocarbon with 2 to 20 carbon atoms.

[0214] The term "C8-12 alkylene" refers to the divalent groups of straight-chain or branched saturated aliphatic hydrocarbons with 8 to 12 carbon atoms among the aforementioned "C2-20 alkylene".

[0215] The term "C2-6 alkylene" refers to the divalent group of a straight-chain or branched saturated aliphatic hydrocarbon with 2 to 6 carbon atoms among the aforementioned "C2-20 alkylene". Examples include ethylene (ethanediyl), propylene, propane-1,3-diyl (trimethylene), propane-2,2-diyl (isopropylidene), 2,2-dimethyl-propane-1,3-diyl, hexane-1,6-diyl (hexamethylene), and 3-methylbutane-1,2-diyl.

[0216] The term "C2-20 alkenyl group" refers to a divalent group of a straight-chain or branched unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond, with 2 to 20 carbon atoms.

[0217] "C1-6 alkylamino" includes mono-C1-6 alkylamino and di-C1-6 alkylamino. A "mono-C1-6 alkylamino" refers to a group formed by bonding one of the aforementioned "C1-6 alkyl" groups to an amino group. Examples of mono-C1-6 alkylamino groups include methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, n-pentylamino, and n-hexylamino. A "di-C1-6 alkylamino" refers to a group formed by bonding two of the aforementioned "C1-6 alkyl" groups to an amino group. The two alkyl groups can be the same or different. Examples of diC1-6 alkylamino groups include, for example, dimethylamino, diethylamino, N,N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-methylamino, N-n-butyl-N-methylamino, N-tert-butyl-N-methylamino, N-methyl-N-n-pentylamino, N-n-hexyl-N-methylamino, and N-isopropyl-N-ethylamino.

[0218] "C1-6 alkylamino carbonyl" refers to the group obtained by bonding the aforementioned "C1-6 alkylamino" with a carbonyl group.

[0219] "C1-6 alkyl carbonyloxy group" refers to the group obtained by bonding the aforementioned "C1-6 alkyl carbonyl" with an oxygen group.

[0220] "C1-6 alkyl carbonyl amino" refers to a group obtained by bonding one of the aforementioned "C1-6 alkyl carbonyl" groups with an amino group.

[0221] "C1-6 alkoxycarbonylamino" refers to a group obtained by bonding the aforementioned "C1-6 alkoxycarbonyl" group with an amino group.

[0222] The term "aryl" refers to a monovalent group obtained by removing one hydrogen atom at any position from an aromatic hydrocarbon that is a monocyclic or bicyclic hydrocarbon whose rings are all composed of carbon atoms. Specific examples include "C6-10 aryl" groups such as phenyl and naphthyl.

[0223] The term "aralkyl" refers to a monovalent group obtained by replacing a hydrogen atom at any position of the aforementioned "C1-6 alkyl" with a "C6-10 aryl" group. The term "C7-10 aralkyl" refers to a monovalent group obtained by replacing a hydrogen atom at any position of the aforementioned "C1-4 alkyl" with a phenyl group.

[0224] The term "3-11 member nitrogen-containing non-aromatic heterocycles" refers to non-aromatic heterocyclic compounds containing at least one nitrogen atom, with 3 to 11 atoms constituting the ring. These compounds can be monocyclic, fused polycyclic (in which the non-aromatic ring can be fused with other non-aromatic or aromatic rings), bridged, or spirocyclic. Examples include nitrogen-containing heterocyclic butanes, pyrrolidines, 2-oxopyrrolidines, piperidines, 3-oxopyridines, piperazines, morpholine, thiomorpholine, homomorpholine, and homopiperazines.

[0225] The term "C2-9 aromatic heterocyclic group" refers to a monovalent group obtained by removing one hydrogen atom at an arbitrary position from an aromatic monocyclic or fused-ring compound having one or more heteroatoms (selected arbitrarily from oxygen, sulfur, and nitrogen atoms) that are the same or different. Examples of C2-9 aromatic heterocycles include purine, pyrimidinyl, thiophene, furanyl, isobenzofuranyl, pyrroleyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, pyridinyl, pyrazinyl, indoleazinyl, indoleyl, isoindoleyl, isoquinolinyl, quinolinyl, naphthidyl, quinoxolinyl, pteridinyl, benzofuranyl, benzothiophene, and benzimidazolyl.

[0226] The term "5-10 membered heterocyclic group" refers to a monovalent group obtained by removing one hydrogen atom at any position from a monocyclic or fused polycyclic aromatic heterocyclic compound that has 5 to 10 atoms constituting the ring and contains 1 to 5 heteroatoms (the heteroatoms can be nitrogen, oxygen, or sulfur; if there are more than 5 heteroatoms, they can be the same or different).

[0227] Examples of monocyclic "5-10 membered heterocyclic groups" include 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyranyl, 3-pyranyl, 4-pyranyl, 1-pyrroleyl, 2-pyrroleyl, 3-pyrroleyl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 1,2,4-triazol-1-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-5-yl, 1,2,3-triazol-1-yl, 1,2,3-triazol- 4-yl, 1,2,3-triazol-5-yl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-isooxazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 1,3,4-oxadiazole-2-yl, 1,3,4-thiadiazole-2-yl, 1,2,4-oxadiazole-3-yl, 1,2,4-oxadiazole-5-yl, 1,2,4-thiadiazole-3-yl, 1,2,4-thiadiazole-5-yl, 1,2,5-oxadiazole-3-yl, and 1,2,5-thiadiazole-3-yl, etc.

[0228] Examples of fused polycyclic 5-10 membered heterocyclic groups include 2-benzofuranyl, 3-benzofuranyl, 4-benzofuranyl, 5-benzofuranyl, 6-benzofuranyl, 7-benzofuranyl, 1-isobenzofuranyl, 4-isobenzofuranyl, 5-isobenzofuranyl, 2-benzothiophenyl, 3-benzothiophenyl, 4-benzothiophenyl, 5-benzothiophenyl, 6-benzothiophenyl, 7-benzothiophenyl, 1-isobenzothiophenyl, 4-isobenzothiophenyl, 5-isobenzothiophenyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl, 2-benzopyranyl, 3-benzopyranyl, 4-benzopyranyl. 5-Benzopyranyl, 6-Benzopyranyl, 7-Benzopyranyl, 8-Benzopyranyl, 1-Indoleazinyl, 2-Indoleazinyl, 3-Indoleazinyl, 5-Indoleazinyl, 6-Indoleazinyl, 7-Indoleazinyl, 8-Indoleazinyl, 1-Isoindolyl, 2-Isoindolyl, 4-Isoindolyl, 5-Isoindolyl, 1-Indolyl, 2-Indolyl, 3-Indolyl, 4-Indolyl, 5-Indolyl, 6-Indolyl, 7-Indolyl, 1-Indazole, 2-Indazole, 4-Indazole, 5-Indazole, 6-Indazole, 7-Indazole, 2-Puryl, 6-Puryl, 7-Puryl, 8-Puryl, 2-Quinolinyl, 3-Quinolinyl, 4-Quinolinyl, 5-Quinolinyl, 6 -quinolinyl, 7-quinolinyl, 8-quinolinyl, 1-isoquinolinyl, 3-isoquinolinyl, 4-isoquinolinyl, 5-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl, 8-isoquinolinyl, 1-phthalazinyl, 5-phthalazinyl, 6-phthalazinyl, 2,7-naphthyl-1-yl, 2,7-naphthyl-3-yl, 2,7-naphthyl-4-yl, 2,6-naphthyl-1-yl, 2,6-naphthyl-3-yl, 2,6-naphthyl-4-yl, 1,8-naphthyl-2-yl, 1,8-naphthyl-3-yl, 1,8-naphthyl-4-yl, 1,7-naphthyl-2-yl, 1,7-naphthyl-3-yl, 1,7-naphthyl-4-yl, 1,7-naphthyl-5-yl, 1,7-naphthyl-6-yl The compounds include 1,7-naphthyl-8-yl, 1,6-naphthyl-2-yl, 1,6-naphthyl-3-yl, 1,6-naphthyl-4-yl, 1,6-naphthyl-5-yl, 1,6-naphthyl-7-ynyl, 1,6-naphthyl-8-yl, 1,5-naphthyl-2-yl, 1,5-naphthyl-3-yl, 1,5-naphthyl-4-yl, 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl, 2-quinazolinyl, 4-quinazolinyl, 5-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl, 8-quinazolinyl, 3-pterinyl, 4-pterinyl, 5-pterinyl, 6-pterinyl, 7-pterinyl, 8-pterinyl, 2-pterinyl, 4-pterinyl, 6-pterinyl, and 7-pterinyl, etc.

[0229] The term "oxo" refers to a group in which an oxygen atom is substituted through a double bond (=O). When an oxo group substitutes for a carbon atom, it forms a carbonyl group together with that carbon atom.

[0230] The term "thio" refers to a group in which a sulfur atom is substituted via a double bond (=S). When a carbon atom is substituted, it forms a thiocarbonyl group together with that carbon atom.

[0231] The terms "protected hydroxyl group," "protected amino group," and "protected thioalkyl group" refer to hydroxyl groups protected by hydroxyl protecting groups, amino groups protected by amino protecting groups, and thioalkyl groups protected by thioalkyl protecting groups, respectively. The hydroxyl protecting group, amino protecting group, and thioalkyl protecting group are only required to be stable during the synthesis of oligonucleotides, and therefore there are no special limitations. For example, the protecting groups described in Protective Groups in Organic Synthesis 4th Edition, TW Greene, PGM Wuts, John Wiley & Sons Inc. (2006), etc., which are well known to those skilled in the art, can be cited.

[0232] For example, examples of "hydroxyl protecting groups" include C1-6 alkyl groups (e.g., methyl, ethyl, tert-butyl, etc.), triarylmethyl groups (e.g., triphenylmethyl (triphenylmethyl), monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (DMTr), trimethoxytriphenylmethyl, etc.), ether-based protecting groups; methoxymethyl, methyl thio, methoxyethyl, benzyloxymethyl, 2-tetrahydropyranyl, ethoxyethyl, etc., acetal-based protecting groups; acyl groups (e.g., formyl, acetyl, pivaloyl, benzoyl, etc.), tri(C1-6 alkyl)silyl groups (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, dimethylsilyl, etc.), etc. Silyl groups such as isopropylsilyl, (C1-6 alkyl)diarylsilyl (e.g., tert-butyldiphenylsilyl, diphenylmethylsilyl, etc.), triarylsilyl (e.g., triphenylsilyl), tribenzylsilyl, [(triisopropylsilyl)oxy]methyl (Tomyl) and other silyl groups; 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpepyl), 9-phenylxanthone-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthone-9-yl (MOXyl), acyloxymethyl (e.g., methyl groups substituted with C1-6 alkyl carbonyloxy, specifically tert-pentyloxymethyl, acetyloxymethyl, etc.), etc.

[0233] For example, examples of "amino protecting groups" include acyl groups (e.g., formyl, acetyl, propionyl, p-pentanoyl (Pv), tigloyl, etc.), haloacyl groups (e.g., fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, etc.), aryl carbonyl groups (e.g., benzoyl, p-bromobenzoyl, p-nitrobenzoyl, 2,4-dinitrobenzoyl, etc.), and amide protecting groups; C1-6 alkoxy carbonyl groups (e.g., methoxy carbonyl, etc.). The following are examples of carbonyl groups: ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl (Boc), tert-pentyloxycarbonyl, etc. (preferably Boc), C2-6 alkenyloxycarbonyl (e.g., vinyloxycarbonyl (Voc), allyloxycarbonyl (Alloc), etc.), tris(C1-3)silylethoxycarbonyl (e.g., 2-(trimethylsilyl)ethoxycarbonyl (Teoc), etc.), halogenated C1-6 alkoxycarbonyl (e.g., ...). Examples of urethane protecting groups include 2,2,2-trichloroethoxycarbonyl (Troc), aryloxycarbonyl (e.g., benzyloxycarbonyl (Z or Cbz), p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl (Moz)), and alkylsulfonyl (e.g., methanesulfonyl (Ms), ethanesulfonyl, etc.) and arylsulfonyl (e.g., benzenesulfonyl, p-toluenesulfonyl (Ts), p-chlorobenzenesulfonyl, p-methoxybenzenesulfonyl (MBS), m-nitrobenzenesulfonyl, o-nitrobenzenesulfonyl). Sulfonamide protecting groups include p-nitrobenzenesulfonyl, 2,4-nitrobenzenesulfonyl, 2,6-dimethoxy-4-methylbenzenesulfonyl (iMds), 2,6-dimethyl-4-methoxybenzenesulfonyl (Mds), 2,4,6-trimethoxybenzenesulfonyl (Mtb), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonyl (Mte), 2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr), 2,4,6-trimethylbenzenesulfonyl (Mts), pentamethylbenzenesulfonyl (Pme), etc.

[0234] For example, examples of "thioalkyl protecting groups" include ether-based protecting groups such as benzyl, p-methoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-pyridinemethyl, 9-fluorenylmethyl, and di or triarylmethyl (e.g., diphenylmethyl, triphenylmethyl (triphenylmethyl), bis(4-methoxyphenyl)methyl, etc.); thioacetal-based protecting groups such as methoxymethyl, isobutoxymethyl, benzylmethylthio, and 2-tetrahydropyranyl; and thioester-based protecting groups such as acyl (e.g., acetyl, benzoyl, etc.). It should be noted that thioalkyl is sometimes referred to as mercapto in this specification.

[0235] For information on the protection and deprotection of the “hydroxyl protecting group,” “amino protecting group,” and “thioalkyl protecting group” in this invention, please also refer to Protective Groups in Organic Synthesis, 4th Edition, by TW Greene and PGM Wuts, John Wiley & Sons Inc. (2006), etc.

[0236] A phosphorus-containing group refers to a group containing a phosphorus atom that can be used to form a nucleoside internucleotide bond containing a phosphate diester structure or a thiophosphate structure. Phosphorus-containing groups known in the field can be used as phosphorus-containing groups, for example, groups derived from phosphorous amides, groups derived from H-phosphonates, groups derived from phosphate diesters, groups derived from phosphate triesters, etc.

[0237] Specifically, the following formula (Z) can be cited as an example. 3 -1) to formula (Z) 3 The group represented by any of the formulas in -3) (wherein the phosphorus atom is bonded to the sugar portion of the nucleotide by an oxygen atom).

[0238] [Chemical Formula 18] In the formula, R X1 and R X2 Each is independently a C1-6 alkyl group, which is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, cyano groups, C6-10 aryl groups and C2-9 aromatic heterocyclic groups.

[0239] [Chemical Formula 19] In the formula, R X3 R is an oxygen atom or a sulfur atom. X4 Each of the following is independently a hydrogen atom, a hydroxyl protecting group, a C1-6 alkyl group, or a C6-10 aryl group, wherein the alkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, a cyano group, a C6-10 aryl group, and a C2-9 aromatic heterocyclic group, and the aryl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, a C1-6 alkyl group, and a cyano group.

[0240] [Chemical Formula 20] In the formula, R X5 It consists of an oxygen atom or a sulfur atom. R X6It can be a hydrogen atom, a hydroxyl protecting group, or a C6-10 aryl group.

[0241] The preferred phosphorus-containing group is of formula (Z). 3 -4) to formula (Z) 3 Any of the groups shown in -6).

[0242] [Chemical Formula 21] In the formula, R X1 It is a C1-6 alkyl or a C1-6 alkyl substituted with a cyano group, R X2 It is a C1-6 alkyl group.

[0243] [Chemical Formula 22] The phosphorus-containing group is more preferably cyanoethoxy(diisopropylamino)phosphin (the group shown by formula: -P(OC2H4CN)(N(i-Pr)2)) or hydroxyphosphin (the group shown by formula: -P(=O)H(OH)).

[0244] The term "phosphonate group" refers to a monovalent group containing one phosphorus-carbon single bond, one phosphorus-oxygen double bond, and two phosphorus-oxygen single bonds.

[0245] The term "5'-phosphonate group" refers to the aforementioned phosphonate group located at the 5' position of an oligonucleotide. Preferably, the 5'-phosphonate group is located at the 5' end of the oligonucleotide. As a bonding agent between the nucleotide and the phosphonate group, the oxygen atom at the 5' position of the nucleotide is preferably replaced by a carbon atom, which is bonded to a phosphorus atom. In this specification, the aforementioned phosphonate group is included in the modification of the phosphate group.

[0246] "RNA interference" refers to the process by which the introduction of double-stranded RNA disrupts the expression of target RNA that is homologous to its sequence.

[0247] The term "mediated RNA interference" can be understood as having the ability to break down target RNA and inhibit its expression in a sequence-specific manner by introducing double-stranded RNA.

[0248] "siRNA" is a double-stranded RNA that generates the aforementioned RNA interference by hybridizing its antisense and sense strands with the target RNA. The antisense and sense strands are, for example, 14-40 nucleotides long, 14-20 nucleotides long, and preferably 19-23 nucleotides long. The antisense strand can be understood as a strand complementary to the target RNA, primarily composed of unmodified RNA, and the sense strand can be understood as a strand partially or entirely complementary to the antisense strand, primarily composed of unmodified RNA. The mechanism of RNA interference is not yet clear, but the following can be considered: After entering the cell, the antisense strand is loaded by the RNA-induced silencing complex (RISC). During this loading process, the sense strand is unloaded, and the antisense strand remains within the RISC. Therefore, the antisense strand binds to its complementary site on the target RNA. The bound RNA is then cleaved by the nuclease activity of the RISC and can then be further broken down by cellular nucleases.

[0249] "Target RNA" refers to mRNA, mRNA precursor, or ncRNA, including mRNA transcribed from genomic DNA encoding the target gene, unmodified mRNA, unspliced ​​mRNA precursor, and ncRNA. There are no particular limitations on the "target RNA" that is used to suppress expression through RNA interference; examples include RNAs associated with genes overexpressed in various diseases. The "target RNA" can be any RNA synthesized by DNA-dependent RNA polymerase, preferably mRNA or mRNA precursor. More preferably, it is mammalian mRNA, and even more preferably, human mRNA.

[0250] "Hybridization" refers to the act of oligonucleotides containing complementary sequences or groups derived from oligonucleotides forming double strands, and the phenomenon of oligonucleotides containing complementary sequences or groups derived from oligonucleotides forming double strands. The aforementioned antisense strand and the aforementioned sense strand can hybridize.

[0251] A "sequence portion" refers to a local structure within an oligonucleotide chain. For example, a sequence portion containing a nucleotide is the local structure of the region within an oligonucleotide chain that contains that nucleotide.

[0252] The term "hybridization region" refers to the complementary sequence portion that forms the aforementioned double strand. "Sense strand hybridization region" refers to a region of the sense strand that can hybridize with the antisense strand. "Antisense strand hybridization region" refers to a region of the antisense strand that can hybridize with the sense strand. The double-stranded RNA of this invention contains a sense strand hybridization region in its sense strand and an antisense strand hybridization region in its antisense strand. The sense strand hybridization region and the antisense strand hybridization region are complementary to each other, forming a double strand.

[0253] The term "complementary" or "having complementarity" refers to the ability of two nucleic acid bases to form Watson-Crick type base pairs (natural base pairs) or non-Watson-Crick type base pairs (Hoogsteen type base pairs, etc.) via hydrogen bonds. Two oligonucleotides or groups derived from oligonucleotides can "hybridize" if their sequences are complementary. For two oligonucleotides or groups derived from oligonucleotides to hybridize, they do not necessarily need to be perfectly complementary; the complementarity used to hybridize two oligonucleotides or groups derived from oligonucleotides is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%). Sequence complementarity can be determined using a computer program capable of automatically identifying portions of the oligonucleotide sequence. For example, OligoAnalyzer is one such software, provided by Integrated DNA Technologies. This program is also available online. Those skilled in the art can readily determine the conditions (temperature, salt concentration, etc.) under which two oligonucleotides or groups derived from oligonucleotides can hybridize. Furthermore, those skilled in the art can readily design antisense strands complementary to the target RNA, for example, based on information about the nucleotide sequence of the target RNA, using methods such as the BLAST procedure. For information on the BLAST procedure, see Proceedings of the National Academy of Sciences of the United States of America, 1990, 87, pp 2264-2268, ibid., 1993, 90, pp 5873-5877, and Journal of Molecular Biology, 1990, 215, pp 403-410, etc.

[0254] "Sufficient complementarity with the target RNA for mediating RNA interference" is not particularly limited as long as it is sufficient complementarity with the target RNA for mediating RNA interference. For example, it is preferred to be 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%).

[0255] A "nucleotide" is a molecule that can be a building block of nucleic acids (oligonucleotides), typically having a base and / or a sugar as its constituent elements. Nucleotides are composed, for example, of a sugar, a base, and a phosphate group. The sugar, base, and phosphate group can also be modified. Nucleotides include ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, as described below. Additionally, nucleotides include molecules that lack a base but are composed of a sugar and a phosphate group, and their modifications.

[0256] "Nucleoside" refers to a molecule that can be a building block of nucleic acids (oligonucleotides), typically containing a base as a building block. A nucleoside represents, for example, a local structure composed of sugars and bases, and lacking the phosphate or modified phosphate groups found in the aforementioned "nucleotides." "Nucleoside structure" refers to a structural unit within an oligonucleotide linked to adjacent nucleosides by a phosphodiester bond or a modified phosphodiester bond. In this specification, nucleoside structure is used to describe local structures lacking phosphodiester bonds or modified phosphodiester bonds.

[0257] "Oligonucleotide" refers to a molecule having a structure composed of one or more of the aforementioned nucleotides. When an "oligonucleotide" consists of only one nucleotide, it can be called a "nucleotide".

[0258] The nucleotides contained in the double-stranded RNA of the present invention are each independently linked to each other by a phosphodiester bond, or a modified phosphodiester bond as described later. Regarding the 3' terminal nucleotide of each oligonucleotide strand contained in the double-stranded RNA of the present invention, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 3' position, more preferably a hydroxyl group, and generally a hydroxyl group. Regarding the 5' terminal nucleotide of each oligonucleotide strand contained in the double-stranded RNA, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 5' position.

[0259] A "nucleotide internucleotide bond" refers to a group or bond in an oligonucleotide that forms a covalent bond between adjacent nucleosides. Nucleotide internucleotide bonds include phosphodiester bonds or modified phosphodiester bonds (e.g., thiophosphate bonds). Nucleotide internucleotide bonds can also be called nucleoside internucleotide bonds.

[0260] "A group derived from an oligonucleotide" refers to a group obtained by removing a hydrogen atom, hydroxyl group, etc., from the hydroxyl group at least one of the 3' and 5' ends of the aforementioned oligonucleotide, and indirectly connected via covalent bonding by forming a phosphodiester bond or a modified phosphodiester bond with other groups (e.g., groups derived from other oligonucleotides). The aforementioned 3' or 5' hydroxyl group includes the hydroxyl group present in the phosphate group. For example, a group obtained by removing a hydrogen atom from the 3' hydroxyl group of an oligonucleotide, and a group obtained by removing a hydroxyl group from the 5' phosphate group of another oligonucleotide, form a phosphodiester bond or a modified phosphodiester bond.

[0261] Similarly, "a group derived from a molecule" refers to a group obtained by removing hydrogen atoms, hydroxyl groups, etc., from that molecule.

[0262] "Nucleotide sequence" refers to the base sequence of the nucleotides that make up an oligonucleotide.

[0263] In this specification, "flat ends" refers to the ends of double-stranded RNA where neither end protrudes and is paired.

[0264] In this specification, the term "protruding end" refers to a protrusion on one of the terminal strands of a double-stranded RNA. The protruding end region refers to the aforementioned protrusion. The protruding end region can be of any length, preferably 1 to 50 nucleotides long, more preferably 1 to 30 nucleotides long, further preferably 1 to 15 nucleotides long, and most preferably 2 to 6 nucleotides long. Since the aforementioned protrusion does not form a double strand as in the double-stranded RNA of this specification, the aforementioned protruding end is also referred to as a single-stranded protruding end.

[0265] "Deoxyribonucleotide" refers to a molecule whose sugar is 2-deoxyribose, whose base is bonded to the carbon atom at position 1 of the 2-deoxyribose, and which has a phosphate group at position 3 or 5. The deoxyribonucleotide in this invention can be a naturally occurring deoxyribonucleotide, or a deoxyribonucleotide formed by modifying the base portion or phosphodiester bond portion of a naturally occurring deoxyribonucleotide. Modifications to the base portion and the phosphodiester bond portion can be combined in multiple ways for a single deoxyribonucleotide. The aforementioned modified deoxyribonucleotides are described in publications such as Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, and RNA, 2023, 29, pp 423-433.

[0266] When the aforementioned "deoxyribonucleotides" constitute the oligonucleotides comprising the double-stranded RNA of the present invention, typically, the 3' position of the deoxyribonucleotide is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond), and the 5' position of the deoxyribonucleotide is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond). Regarding the deoxyribonucleotides at the 3' end of the oligonucleotides comprising the double-stranded RNA of the present invention, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. Regarding the deoxyribonucleotides at the 5' end of the oligonucleotides comprising the double-stranded RNA, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0267] "Ribosylnucleotide" refers to a molecule in which the sugar is ribose, the base is bonded to the carbon atom at position 1 of the ribose, and a phosphate group is present at positions 2, 3, or 5. The ribosylnucleotide in this invention can be a naturally occurring ribosylnucleotide, or a ribosylnucleotide in which the base portion or phosphodiester bond portion of a naturally occurring ribosylnucleotide has been modified. Modifications to the base portion and phosphodiester bond portion can be combined in multiple ways for a single ribosylnucleotide. The aforementioned modified ribosylnucleotides are described, for example, in *Journal of Medicinal Chemistry*, 2016, 59, pp 9645-9667; *Medicinal Chemistry Communication*, 2014, 5, pp 1454-1471; *Future Medicinal Chemistry*, 2011, 3, pp 339-365; and *RNA*, 2023, 29, pp 423-433.

[0268] When the aforementioned "ribonucleotides" constitute the oligonucleotides comprising the double-stranded RNA of the present invention, typically, the 3' position of the ribonucleotide is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond), and the 5' position of the ribonucleotide is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond). Regarding the 3' terminal ribonucleotide of each oligonucleotide comprising the double-stranded RNA of the present invention, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. Regarding the 5' terminal ribonucleotide of each oligonucleotide comprising the double-stranded RNA, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0269] "Modified sugars" include: (Z1) A molecule in which ribose or 2-deoxyribose is partially substituted by one or more substituents; (Z2) Pentose or hexose sugars that are different from ribose and 2-deoxyribose (e.g., hexitol, thioglycolate, etc.); and (Z3) Molecules obtained by replacing the ribose or 2-deoxyribose monolithically, or their tetrahydrofuran ring, with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.).

[0270] Modified sugars include “2-modified sugars”, “2-4-bridging sugars” and “5-modified sugars”, which will be discussed later.

[0271] Examples of sugars and sugar-modified nucleotides disclosed in Japanese Patent Application Publication No. 10-304889, International Publication No. 2005 / 021570, Japanese Patent Application Publication No. 10-195098, Japanese Patent Application Publication No. 2002-521310, International Publication No. 2007 / 143315, International Publication No. 2008 / 043753, International Publication No. 2008 / 029619, International Publication No. 2008 / 049085 and International Publication No. 2017 / 142054, etc. In addition, modified sugars and sugar-modified nucleotides have also been disclosed in the Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, RNA, 2023, 29, pp 423-433, and International Publication No. 2018 / 155450.

[0272] As an example of a modified sugar that is partially substituted by one substituent, one can cite ribose or 2-deoxyribose obtained by substituting any position of the sugar moiety by one or more (preferably one or two) substituents of (i) or (ii) below.

[0273] (i) C1-6 alkyl. Here, when there is substitution based on two or more C1-6 alkyl groups, the two or more C1-6 alkyl groups can form a 3- to 6-membered ring together.

[0274] (ii) A C1-6 alkyl group selected from the group consisting of a halogen atom, a C1-6 alkoxy group, a halogenated C1-6 alkoxy group, a mono- or di-C1-6 alkylamino group, a 5- to 10-membered heterocyclic group, a carboxyl group, a carbamoyl group, and an N-substituted carbamoyl group.

[0275] Here, as an example of the aforementioned N-substituted carbamoyl group, N-methyl-carbamoyl and N-ethyl-carbamoyl can be used. Here, the methyl and ethyl groups of N-methyl-carbamoyl and N-ethyl-carbamoyl can be 5-10 membered heterocyclic groups or single or double carbon groups. 1-6 Alkylamino substitution. Specific examples of N-substituted carbamoyl groups include N-methylcarbamoyl, N-ethylcarbamoyl, N-dimethylaminoethyl-carbamoyl, N-(morpholinylethyl)carbamoyl, N-(2-pyridylethyl)carbamoyl, N-((benzimidazol-1-yl)ethyl)carbamoyl, etc.

[0276] In the case of "sugar-modified nucleotides," in addition to molecules that have the aforementioned "modified sugar" in place of the sugar moiety of deoxyribonucleotides or ribonucleotides, it also includes molecules that have a non-cyclic structure in place of the sugar moiety. For example, sugar-modified nucleotides include "2'-modified nucleotides," "2'-4' bridging nucleotides," "5'-modified nucleotides," and "non-cyclic nucleotides" as described below. When the modified sugar is (Z3) as defined above, sugar-modified nucleotides also include molecules obtained by bonding the modified sugar to nucleic acid bases via a methylene chain or similar bond.

[0277] "2-modified sugars" refer to non-bridged sugars in which the oxygen or carbon atom at position 2 of the ribose is modified, including "2-O-Me", "2-O-MOE", "2-O-MCE", "2-O-NMA", "2-O-AP", "2-fluorine", "2-O-DMAECE", "2-O-MоrECE", "2-O-PyECE", and "2-O-BimECE". The fourth position of "2-modified sugars" is preferably unmodified.

[0278] A "2' modified nucleotide" refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of the aforementioned "2' modified sugar," and a phosphate group is present at position 3 or 5. Examples include "2'-O-Me nucleotide," "2'-O-MOE nucleotide," "2'-O-MCE nucleotide," "2'-O-NMA nucleotide," "2'-O-AP nucleotide," "2'-F nucleotide," "2'-O-DMAECE nucleotide," "2'-O-MоrECE nucleotide," "2'-O-PyECE nucleotide," and "2'-O-BimECE nucleotide." The 5' position of the 2' modified nucleotide may or may not be modified.

[0279] "2-O-Me" (also known as 2-O-methyl) refers to sugars obtained by replacing the 2-hydroxyl group at the ribose position with a methoxy group.

[0280] "2'-O-Me nucleotide" (also known as 2'-O-methyl nucleotide) refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of "2-O-Me", and has a phosphate group at position 3 or 5.

[0281] "2-O-MOE" (also known as 2-O-methoxyethyl) refers to a sugar obtained by replacing the hydroxyl group at the 2-position of ribose with a 2-methoxyethyloxy group.

[0282] "2'-O-MOE nucleotide" (also known as 2'-O-methoxyethyl nucleotide) refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of "2-O-MOE" and has a phosphate group at position 3 or 5.

[0283] "2-O-MCE" (also known as 2-O-methylcarbamoylethyl) refers to a sugar obtained by replacing the 2-hydroxyl group of ribose with a methylcarbamoylethyloxy group.

[0284] "2'-O-MCE nucleotide" (also known as 2'-O-methylcarbamoylethyl nucleotide) refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of "2-O-MCE" and has a phosphate group at position 3 or 5.

[0285] "2-O-NMA" refers to a sugar obtained by replacing the 2-hydroxyl group at the ribose position with [2-(methylamino)-2-oxoethyl]oxy.

[0286] "2'-O-NMA nucleotide" refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of "2-O-NMA" and has a phosphate group at position 3 or 5.

[0287] "2-O-AP" refers to a sugar obtained by replacing the hydroxyl group at the 2-position of ribose with a 3-aminopropyloxy group.

[0288] "2'-O-AP nucleotide" refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of "2-O-AP" and has a phosphate group at position 3 or 5.

[0289] "2-Fluorine" refers to sugar obtained by replacing the hydroxyl group at the 2-position of ribose with a fluorine atom.

[0290] "2'-Fluoronucleotide" refers to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of "2-fluorine", and has a phosphate group at position 3 or 5.

[0291] “2-O-DMAECE”, “2-O-MоrECE”, “2-O-PyECE”, and “2-O-BimECE” are sugars obtained by replacing the hydroxyl group at the 2-position of the ribose with the structures shown below as DMAECE, MorECE, PyECE, and BimECE, respectively. In the following structures, the wavy line indicates the bonding position of the carbon atom to which the hydroxyl group at the 2-position of the ribose is bonded.

[0292] [Chemical Formula 23] “2'-O-DMAECE nucleotide”, “2'-O-MоrECE nucleotide”, “2'-O-PyECE nucleotide” and “2'-O-BimECE nucleotide” each refer to a molecule in which the base is bonded to the carbon atom at position 1 (the position 1 of the ribose before modification) of “2-O-DMAECE”, “2-O-MоrECE”, “2-O-PyECE” and “2-O-BimECE”, respectively, and which has a phosphate group at position 3 or 5.

[0293] "2'-O-XCE nucleotide" refers to a nucleotide in which the 2' hydroxyl group of a ribonucleotide is modified with a carbamoyl ethyl group, either substituted or unsubstituted.

[0294] In some embodiments of the various methods disclosed in this specification, the 2'-O-XCE nucleotide is a nucleotide comprising a nucleoside structure represented by the following formula (I): [Chemical Formula 24] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (Ia) represents the group: [Chemical Formula 25] [In the formula, R] 1 and R 2Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different.

[0295] It should be noted that the wavy line in formula (I) can be understood as the bonding position of the nucleotide bond formed with and adjacent nucleotides, the bonding position with the functional molecule or its linker, or the bonding position with hydrogen atoms, hydroxyl protecting groups or phosphorus-containing groups, etc. The wavy line in formula (Ia) shows the bonding position with the nitrogen atom in formula (I).

[0296] In some embodiments, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group. Here, the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups.

[0297] X is preferably a hydrogen atom or a C1-6 alkyl group, more preferably a C1-3 alkyl group, and even more preferably a methyl group.

[0298] In some embodiments, X is a group represented by the following formula (Ia): [Chemical Formula 26] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different.

[0299] R 1 and R 2 Each of these independently represents a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group. Here, the C1-6 alkyl and C2-6 alkenyl groups are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups. Here, when n is 2 or 3, R... 1 They can be the same or different, R 2 They can be the same or different.

[0300] R 1 Preferably, it is a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom. R 2 Preferably, it is a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.

[0301] Y is NR 3 R 4 At that time, R 3 and R 4 Each of these groups independently represents a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group, or a C7-10 aralkyl group. Here, the C1-6 alkyl and C2-6 alkenyl groups are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino, and C1-6 alkoxycarbonylamino. The C7-10 aralkyl group... It is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino.

[0302] R 3 Preferably, it contains hydrogen atoms or C1-3 alkyl groups, and more preferably, it contains methyl groups.

[0303] R 4 Preferably, it contains hydrogen atoms or C1-3 alkyl groups, and more preferably, it contains methyl groups.

[0304] Here, NR 3 R 4 It can also be expressed as N(R) 3 R4 , refers to R 3 and R 4 A group formed by bonding to the nitrogen atom.

[0305] As another way, R 3 and R 4 They can form 3-11 member nitrogen-containing non-aromatic heterocycles together with the nitrogen atoms they are bonded to. Here, the 3-11 member nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino, and C1-6 alkoxycarbonylamino.

[0306] R 3 and R 4 The 3-11 membered nitrogen-containing non-aromatic heterocycles formed together with the nitrogen atoms they are bonded to are preferably 4-8 membered nitrogen-containing non-aromatic heterocycles containing 4 to 6 methylene groups, such as piperidine, pyrrolidine, morpholine, thiomorpholine, homopiperidine, and homomorpholine. More preferably, they are rings that also contain oxygen or sulfur atoms in the constituent atoms, such as morpholine, thiomorpholine, and homomorpholine. Morpholine is particularly preferred. Furthermore, the 4-8 membered nitrogen-containing non-aromatic heterocycle is preferably unsubstituted.

[0307] Alternatively, Y may be a C2-9 aromatic heterocyclic group. It is preferably pyridyl, imidazole or benzimidazole, more preferably 2-pyridyl, imidazole-1-yl or (benzimidazole)-1-yl, and particularly preferably 2-pyridyl or (benzimidazole)-1-yl.

[0308] n is the number of repeating unit structures, and is an integer from 1 to 3, preferably 2.

[0309] Regarding the 2'-O-XCE nucleotide, the oligonucleotide may contain the nucleoside structure shown in formula (I) at either the 3' or 5' end. In the case where the 3' end contains the 2'-O-XCE nucleotide, for example, it becomes the structure shown in formula (I-1): [Chemical Formula 27] (The definitions of X and Base in equation (I-1) are the same as those in equation (I). Z) 2 It is a hydrogen atom, a hydroxyl protecting group, or a phosphorus-containing group, preferably a hydrogen atom.

[0310] The wavy lines in formula (I-1) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups or phosphorus-containing groups, etc.

[0311] Additionally, when the 5' end contains a 2'-O-XCE nucleotide, it results in, for example, the structure shown in formula (I-2): [Chemical Formula 28] (The definitions of X and Base in equation (I-2) are the same as those in equation (I), Z) 1 (Hydroxyl, phosphate group or modified phosphate group).

[0312] The wavy lines in formula (I-2) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups.

[0313] Exemplary phosphate groups or modified phosphate groups include groups suitable for RISC-mediated gene silencing. Examples of preferred phosphate groups or modified phosphate groups include monophosphate ((HO)₂(O)PO₄⁻), diphosphate ((HO)₂(O)POP(HO)(O)-O⁻), triphosphate ((HO)₂(O)PO₄⁻(HO)(O)POP(HO)(O)-O⁻), monothiophosphate (phosphorothioate: (HO)₂(S)PO₄⁻), monodithiophosphate (dithiophosphate: (HO)(HS)(S)PO₄⁻), phosphorothiolate ((HO)₂(O)PS⁻), any further combinations of substituted oxygen / sulfur monophosphates, diphosphates, and triphosphates (e.g., α-thiotriphosphate, γ-thiotriphosphate, etc.), and aminophosphate ((HO)₂(O)P⁻). NH-, (HO)(NH2)(O)PO-), 5'-alkylphosphonates (alkylene phosphates (e.g., (HO)2(O)P-CH2-5'(methyl phosphate), (HO)2(O)P-CH2CH2-5'-(ethylene phosphate), R′P(OH)(O)-O-5'-(R′ = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-cycloalkylphosphonates ((HO)2(O)P-C3H4-5'-(cyclopropyl phosphate)), 5'-alkenylphosphonates (as alkenyl, there are, for example, vinyl ((OH)2(O)P-CH=CH-5'-), substituted vinyl), 5'-alkoxyalkylphosphonates (R″P(OH)(O)-O-5'-(R″ = alkoxyalkyl, e.g., methoxymethyl, ethoxymethyl, etc.)), etc.

[0314] In one implementation, Z 1 Preferably, it is a hydroxyl group. In a further embodiment, Z 1 The preferred form is monophosphate ((HO)2(O)PO-).

[0315] In one embodiment, when the 5' end contains a 2'-O-XCE nucleotide, it becomes the structure shown in formula (II-1): [Chemical Formula 29] [X and Base in Equation (II-1) are defined in the same way as X and Base in Equation (II).] T 1 The group represented by the following formula (IIb): [Chemical Formula 30] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. A 1 The groups are selected from the following formula (IIc): [Chemical Formula 31] [Q1 and Q2 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl and an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano groups)].

[0316] The wavy lines in formula (II-1) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups, etc. The wavy lines in formula (IIb) show the A in formula (II-1). 1 The bonding positions, the wavy lines in equation (IIc) respectively show the T in equation (II-1). 1The bonding positions of the atoms and their bonds with sugar carbon atoms.

[0317] In some embodiments, Ra and Rc are preferably hydroxyl groups, and Rb is preferably an oxygen atom.

[0318] In another embodiment of the invention, Q1 and Q2 are preferably hydrogen atoms.

[0319] In some embodiments, when the 5' end of the oligonucleotide contains the nucleoside structure shown in formula (I), the structure shown in formula (II-2) is preferred (5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotide): [Chemical Formula 32] (X and Base in Equation (II-2) are defined in the same way as X and Base in Equation (II).)

[0320] The wavy line in formula (II-2) can be understood as the bonding position of the nucleotide bond formed with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with the hydrogen atom, hydroxyl protecting group or phosphorus-containing group, etc.

[0321] In one embodiment, when the 5' end contains a 2'-O-XCE nucleotide, it becomes the structure shown in formula (III-1): [Chemical Formula 33] {T in equation (III-1)} 1 X and Base and T in equation (III) 1 X and Base have the same definition. B 1 The group represented by the following formula (IIIc): [Chemical Formula 34] [Q3 and Q4 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkyl, a C1-6 alkoxy, and a cyano group)]}.

[0322] The wavy lines in formula (III-1) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups, etc. The wavy lines in formula (IIIc) respectively indicate the T values ​​of formula (III-1). 1 The bonding positions of the atoms and their bonds with sugar carbon atoms.

[0323] In another embodiment, Q3 and Q4 are preferably hydrogen atoms.

[0324] In some embodiments, when the 5' end of the oligonucleotide contains the nucleoside structure shown in formula (I), the structure shown in formula (III-2) is preferred (5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide): [Chemical Formula 35] (X and Base in Equation (III-2) are defined in the same way as X and Base in Equation (III).)

[0325] The wavy lines in formula (III-2) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups, etc. The wavy lines in formula (IIIc) respectively indicate the T values ​​in formula (III-2). 1 The bonding positions of the atoms and their bonds with sugar carbon atoms.

[0326] In one embodiment, when the 5' end contains a 2'-O-XCE nucleotide, it becomes the structure shown in formula (IV-1): [Chemical Formula 36] {T in equation (IV-1)} 1 X and Base and T in equation (IV) 1 X and Base have the same definition. E 1 For groups represented by the following formula (IVc): [Chemical Formula 37] [Q5~Q8 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy, and cyano groups)]}.

[0327] The wavy lines in formula (IV-1) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups, etc. The wavy lines in formula (IIIc) respectively indicate the T values ​​of formula (IV-1). 1 The bonding positions of the atoms and their bonds with sugar carbon atoms.

[0328] In another embodiment of the invention, Q5 to Q8 are preferably hydrogen atoms.

[0329] In some embodiments, when the 5' end of the oligonucleotide contains the nucleoside structure shown in formula (I), the structure shown in formula (IV-2) is preferred (5'-ethylphosphonate (EP) 2'-O-XCE nucleotide): [Chemical Formula 38] (X and Base in Equation (IV-2) are defined in the same way as X and Base in Equation (IV).)

[0330] The wavy lines in formula (IV-2) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups.

[0331] In some embodiments, T in the aforementioned formulas (II-1), (III-1), and (IV-1) 1 In this context, Rb is preferably an oxygen atom, and Ra and Rc are each independently preferably hydroxyl or protected hydroxyl, more preferably protected hydroxyl, further preferably acyloxymethyl (e.g., methyl substituted with C1-6 alkyl carbonyloxy) or alkyl (e.g., C1-6 alkyl), particularly preferably pivaloyloxymethyl or ethyl.

[0332] The preferred structure of X in the aforementioned formulas (II-1), (III-1) and (IV-1) is the same as the preferred corresponding structure contained in the double-stranded RNA agent.

[0333] Z in the aforementioned equations (II-1), (III-1), and (IV-1)3 The hydroxyl protecting group is preferably silyl, and particularly preferably tert-butyldimethylsilyl.

[0334] Sometimes isomers are present in the compounds shown in formulas (I) to (IV-2). In this case, the compounds of this embodiment are not limited to specific isomers, but include all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereomers, optical isomers, rotational isomers, etc.), racemates, and mixtures thereof.

[0335] "2'-Modified nucleotides" contain "2'-O-XCE nucleotides", "2'-OR-XCE nucleotides" contain "2'-O-MCE nucleotides", "2'-O-DMAECE nucleotides", "2'-O-MоrECE nucleotides", "2'-O-PyECE nucleotides" and "2'-O-BimECE nucleotides".

[0336] "2-4 bridging sugars" refer to sugars in which the bridging unit is substituted at positions 2 and 4 of the ribose. Examples of bridging units include C2-6 alkylene groups (where the alkylene group is unsubstituted, or substituted by one or more substituents selected from the group consisting of halogen atoms, oxo groups, and thio groups, and one or two methylene groups in the alkylene group are not substituted, or are independently substituted by groups selected from the group consisting of -O-, -NR1- (R1 represents a hydrogen atom, a C1-6 alkyl group, or a halo-C1-6 alkyl group), and -S-). Position 5 of the "2-4 bridging sugar" may be modified or left unmodified, but is preferably left unmodified.

[0337] A "2'-4'-bridged nucleotide" (2',4'-BNA) is a molecule in which a base is bonded to a carbon atom at position 1 (the position 1 of the unmodified ribose or 2-deoxyribose) of the aforementioned "2-4-bridged sugar," and a phosphate group is present at position 3 or 5. Examples include β-D-methyleneoxy(4'-CH2-O-2')BNA or α-L-methyleneoxy(4'-CH2-O-2')BNA, also known as LNA (Locked Nucleic Acid; registered trademark); ethyleneoxy(4'-(CH2)2-O-2')BNA, also known as ENA; (4β-D-thio'-CH2-S-2')BNA; and aminooxy(4'-CH2-ON(R)). 11 )-2')BNA(R 11 (Hydrogen atom or methyl group), also known as 2',4'-BNANC oxyamino group (4'-CH2-N(R) 12 )-O-2')BNA(R 12(Hydrogen atom or methyl group), 2',4'-BNACOC, 3'-amino-2',4'-BNA, 5'-methylBNA, also known as cEt (4'-CH(CH3)-O-2')BNA, also known as cMOE-BNA (4'-CH(CH2OCH3)-O-2')BNA, also known as AmNA amide type BNA (4'-C(=O)-N(R) 13 )-2')BNA(R 13 (4'-C(spiropropyl)-O-2')BNA, also known as scpBNA, and (4'-CH2-N(R)BNA, also known as GuNA) 14 )-2')BNA(R 14 For C (=NHR) 15 +) NHR 16 R 15 R 16 Each of these atoms is independently composed of a hydrogen atom, methyl, ethyl, isopropyl, or tert-butyl, and is also known as amino-LNA (4'-CH2-N(R)). 17 )-2')BNA(R 17 (e.g., hydrogen atom or methyl group), other BNAs known to those skilled in the art.

[0338] "5-modified sugars" refer to non-bridging sugars in which the oxygen or carbon atom at the 5-position of the ribose backbone is modified, including "5-CP", "5-methyl", and "5-aminopropyl". Preferably, the 2- and 4-positions of "5-modified sugars" are unmodified.

[0339] "5'-Modified nucleotide" refers to a molecule in which a base is bonded to a carbon atom at position 1 (the position 1 of the unmodified 2-deoxyribose) of the aforementioned "5'-modified sugar," and a phosphate group is present at positions 2, 3, or 5. Examples include "5'-CP nucleotide," "5'-methyl nucleotide," and "5'-aminopropyl nucleotide." The 2' position of the 5'-modified nucleotide may or may not be modified. The 2' and 4' positions of the 5'-modified nucleotide may or may not be bridged; preferably, they are not bridged.

[0340] "5-CP" refers to a sugar in which the 5th position of the ribose backbone is replaced by two methyl groups, and these two methyl groups together form a cyclopropane.

[0341] A "5'-CP nucleotide" is a molecule with the sugar being the aforementioned "5-CP", a base bonded to a carbon atom at position 1 (forming the position 1 of the basic 2-deoxyribose), and a phosphate group at position 3 or 5. It can be represented by the following structural formula. In the formula, Base represents the nucleic acid base. The wavy lines can be interpreted as the bonding positions of nucleotides with adjacent nucleotides, with functional molecules or their linkers, or with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups, etc.

[0342] [Chemical Formula 39] "5-Methyl" is a sugar obtained by replacing the 5-position of the ribose backbone with a methyl group.

[0343] "5-Aminopropyl" is a sugar obtained by replacing the 5-position of the ribose backbone with a 3-aminopropyl group.

[0344] "5'-methyl nucleotide" and "5'-aminopropyl nucleotide" refer to molecules in which the base is bonded to the carbon atom at position 1 (the position 1 of the basic 2-deoxyribose) of "5-methyl" and "5-aminopropyl", respectively, and which have a phosphate group at position 3 or 5.

[0345] "5-vinyl" refers to the sugar obtained by replacing the methylene group at the 5-position of ribose with ethylene-1,2-diyl (vinyl).

[0346] "5-CP" refers to the sugar obtained by replacing the methylene group at the 5-position of the ribose with cyclopropane-1,1-diyl (cyclopropyl).

[0347] Vinylphosphonate (VP) nucleotides, cyclopropanephosphonate (CPP) nucleotides, and ethylphosphonate (EP) nucleotides, described later, are also included in 5'-modified nucleotides.

[0348] Among the aforementioned "deoxyribonucleotides," "ribonucleotides," "2'-modified nucleotides," "2'-4'-bridged nucleotides," and "5'-modified nucleotides," the bonds formed between the carbon atom at the 1' position and the base can be α-glycosidic bonds or β-glycosidic bonds, but are usually β-glycosidic bonds. Therefore, β-D-methyleneoxy BNA is typically used as an LNA.

[0349] When the aforementioned "sugar-modified nucleotide" constitutes each strand of oligonucleotide contained in the double-stranded RNA of the present invention, for example, the 3' position of the sugar-modified nucleotide is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond), and the 5' position of the sugar-modified nucleotide is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond). Regarding the sugar-modified nucleotide at the 3' end of each strand of oligonucleotide contained in the double-stranded RNA of the present invention, for example, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. Regarding the sugar-modified nucleotide at the 5' end of each strand of oligonucleotide contained in the double-stranded RNA, for example, it preferably has a hydroxyl group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0350] Examples of modifications to the phosphodiester bond portion of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides include thiophosphorylation, methylphosphonic acid esterification (including chiral methylphosphonic acid esterification), methylthiophosphonic acid esterification, dithiophosphorylation, aminophosphorylation, diaminophosphorylation, thiophosphoramide esterification, borane phosphorylation, phosphorylguanidineation, or mesylate phosphodiesterylation. In addition, examples of modification of the phosphodiester bond portion of nucleotides are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, and RNA, 2023, 29, pp 423-433. These can also be used for the phosphodiester bond portion of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.

[0351] A "bridged nucleotide" is a nucleotide modified by substituting the bridging unit at position 2 of the sugar moiety. Examples include 2'-4' bridged nucleotides.

[0352] "Acyclic nucleotide" refers to any nucleotide that has a non-cyclic structure in place of a sugar in a ribonucleotide or deoxyribonucleotide. For example, in acyclic nucleotides, any of the bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, and / or at least one of the ribose carbon atoms or oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination in the nucleotide. In some embodiments, acyclic nucleotides are represented by the following formula (V): [Chemical Formula 40] (The Base in equation (V) is defined the same as the Base in equation (I), R) 36 and R 37 Independently, it is a hydrogen atom, halogen, hydroxyl group, C1-6 alkoxy group, or C1-6 alkyl group.

[0353] "UNA" is a non-locked acyclic nucleic acid, one of the acyclic nucleotides described in this specification. In UNA, either the sugar bond of the ribonucleotide or deoxyribonucleotide has been removed, forming a non-locked "sugar" residue. In one example, UNA contains monomers in which the C1' to C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, UNA contains monomers in which the C2' to C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) can be removed (see Tetrahedron Letters, 1985, 26(17), p. 2059 and Mol. Biosyst., 2009, 10, p. 1039) (the entirety of which is incorporated herein by reference). The acyclic structure imparts flexibility to the backbone without affecting Watson-Crick pairing. UNA can be connected via 2'-5' or 3'-5' keys.

[0354] “GNA” is generally referred to as ethylene glycol nucleic acid, and is one of the non-cyclic nucleotides described in this specification. GNA consists of repeating units of glycerol linked by phosphodiester bonds or modified phosphodiester bonds. Although the composition of the “backbone” differs, it is similar to DNA or RNA (deoxyribonucleotides or ribonucleotides). Its representative structure is represented by the following formula (VI): [Chemical Formula 41] (The Base in Equation (VI) is defined the same as the Base in Equation (I).)

[0355] Sugar-modified nucleotides are not limited to those illustrated herein. Many sugar-modified nucleotides are known in the art, and embodiments of the present invention may be derived from, for example, those described in U.S. Patent No. 8,299,039 to Tachas et al. (particularly columns 17 to 22), or in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365.

[0356] The term "nucleic acid base" generally refers to the base components that make up nucleic acids. Natural nucleic acid bases include purine bases: adenine (A) and guanine (G), and pyrimidine bases: thymine (T), cytosine (C), and uracil (U). The base portions of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides used in this specification may use natural nucleic acid bases and their modified forms. Modified nucleic acid bases can form base pairs (i.e., hydrogen bonds) with any nucleic acid base (preferably a base complementary to the unmodified nucleic acid base). Typically, modified nucleic acid bases include: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and... Thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil, and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, 3-deadenine and 3-deadenine. The further modified nucleic acid bases include tricyclic pyrimidines such as phenoxazincytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenthiazincytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), substituted phenoxazincytidine, and G-clamps such as 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one, carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and pyridinocytidine (H-pyridino[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). In addition, modified nucleic acid bases may also include nucleic acid bases obtained by replacing purine or pyrimidine bases with other heterocycles, such as 7-deadenine, 7-deadenine, 2-aminopyridine, and 2-pyridone.Furthermore, examples of modifications to the base moiety of nucleotides are disclosed in *Journal of Medicinal Chemistry*, 2016, 59, pp 9645-9667; *Medicinal Chemistry Communication*, 2014, 5, pp 1454-1471; *Future Medicinal Chemistry*, 2011, 3, pp 339-365; and International Publication No. 2007 / 090071. These modifications can be applied to the base moiety of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. The amino and hydroxyl groups of the base moiety may be left unprotected independently.

[0357] The base portion of the deoxyribonucleotide, ribonucleotide, and sugar-modified nucleotide is preferably selected from at least one of the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), and 5-methylcytosine (5-me-C).

[0358] The term "thermally unstable modification" refers to modifications of double-stranded RNA that result in an overall lower melting temperature (Tm) compared to double-stranded RNA without one or more of these modifications (preferably 1, 2, 3 to 4 degrees lower Tm). There are no limitations on what constitutes a "thermally unstable modification," and examples include debasement modifications, mismatches between opposite nucleotides in the antiparallel strand, 2'-deoxy modifications, and non-circular modifications (e.g., non-locked nucleic acids (UNA) or glycol-based nucleic acids (GNA)).

[0359] "Thermal unstable sugar modification" refers to the use of sugars in thermal unstable modifications to modify nucleotides, which may include the aforementioned 2'-deoxy modification, non-cyclic modification, sugar modification, etc.

[0360] A "debaseted nucleotide" is a nucleotide that does not have nucleic acid bases.

[0361] Debase modification can be achieved by using debase nucleotides.

[0362] As an example of a nucleoside structure used in a debasement modified form, without limitation, the following formula (VII) can be cited: [Chemical Formula 42] (R in equation (VII)) 31 R is a hydrogen atom, methyl, ethyl, or methoxy group. 32 R is a hydrogen atom, methyl, ethyl, or methoxy group. 33 It can be a hydrogen atom, methyl, ethyl, or methoxy group. (This indicates that the center of the solid is either R or S, or a racemic mixture).

[0363] As an example of a nucleoside structure used in the form of a heat-labile sugar modification, without limitation, the following formula (VIII) can be cited: [Chemical Formula 43] (The Base in equation (VIII) is defined the same as the Base in equation (I).) R 34 It is fluorine or methoxy. (This indicates that the center of the solid is either R or S, or a racemic mixture).

[0364] Other nucleoside structures used in the form of heat-labile sugar modifications are not limited, and examples include the following formula (IX): [Chemical Formula 44] (The Base in equation (IX) is defined the same as the Base in equation (I).) R 35 (represented by hydrogen atom, hydroxyl group, or C1-6 alkoxy group).

[0365] In one embodiment, the nucleoside structure used in the form of a heat-labile sugar modification is selected from the group consisting of the following formulas (X): [Chemical Formula 45] (The Base in equation (X) has the same definition as the Base in equation (I)) (This indicates that the center of the solid is either R or S, or a racemic mixture).

[0366] It should be noted that the wavy lines in formulas (V) to (X) can be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups, etc.

[0367] Thermally unstable modifications to double-stranded RNA molecules can be mismatches (i.e., non-complementary base pairs) between thermally unstable nucleotides within the dsRNA double strand and their antiparallel counterparts. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatched base pairings known in this art are also suitable for this invention. Mismatches can occur between any nucleotides in naturally occurring or modified nucleotides; that is, mismatched base pairings are independent of modifications to the ribose sugar of the nucleotide and can occur between nucleic acid bases from individual nucleotides.

[0368] In one embodiment, the thermally unstable modification in the seed region of the antisense strand comprises a nucleotide exhibiting a Watson-Crick binding barrier to complementary bases on the target RNA. Exemplary nucleotides for which Watson-Crick binding to complementary bases on the target RNA is impaired are not limited, but include nucleotides comprising nucleic acid bases independently selected from the following formula (XI): [Chemical Formula 46] .

[0369] Other examples of debased nucleotides, noncyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in International Publication No. 2011 / 133876 (which is incorporated herein by reference in its entirety).

[0370] Thermally unstable modifications can also include modifications to common nucleic acid bases and phosphodiester bonds that reduce or eliminate the ability to form hydrogen bonds with the opposite base.

[0371] In some embodiments, the thermally unstable modification is not limited and includes nucleotides with non-standard bases, such as nucleic acid base modifications that impede or completely eliminate the ability to form hydrogen bonds with antiparallel bases. These nucleic acid base modifications are described in International Publication No. 2010 / 0011895 (entirely incorporated herein by reference), which evaluates the instability of the central region of the dsRNA double helix. An exemplary nucleic acid base modification can be exemplified by the following formula (XII): [Chemical Formula 47] .

[0372] Thus, a portion of the thermally unstable modification can be contained within the modified nucleic acid bases.

[0373] It should be noted that the wavy lines in equations (XI) to (XII) can each be interpreted as the bonding positions with the sugar carbon atoms of the nucleotide, etc.

[0374] In some implementations, one or more of the following thermally unstable modifications of formula (XIII) are included: [Chemical Formula 48] (where R) 38 It can be a hydrogen atom, hydroxyl group, C1-6 alkoxy group, fluorine, amino group, N-methylamino group, or N,N'-dimethylamino group.

[0375] The wavy line in formula (XIII) can be interpreted as the bonding position of the nucleotide bond formed with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with the hydrogen atom, hydroxyl protecting group or phosphorus-containing group, etc.

[0376] As an example of a phosphodiester bond modification that is known to reduce the thermal stability of the dsRNA double strand compared to the natural phosphodiester bond, there are no limitations, and examples can be given by the following formula (XIV): [Chemical Formula 49] (where R) 39 It is a C1-6 alkyl group. Examples of C1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0377] It should be noted that the wavy lines in formula (XIV) can be interpreted as the bonding positions with the sugar carbon atoms of the nucleotide, etc.

[0378] Thus, a portion of the thermally unstable modification can be contained within the modified phosphodiester bond.

[0379] In some embodiments, the thermally unstable modification is selected from the following formula (XV): [Chemical Formula 50] (The Base in equation (XV) is defined the same as the Base in equation (I), R) 34 With R in equation (VIII) 34 (Same definition)

[0380] The wavy line in formula (XV) can be interpreted as the bonding position of the nucleotide bond formed with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with the hydrogen atom, hydroxyl protecting group or phosphorus-containing group, etc.

[0381] When the aforementioned "thermally unstable modification" constitutes the oligonucleotides of each strand contained in the double-stranded RNA of the present invention, for example, the 3' position of the thermally unstable modification is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond), and the 5' position of the thermally unstable modification is linked to other nucleotides via a phosphodiester bond or a modified phosphodiester bond (e.g., a thiophosphate bond). Regarding the thermally unstable modification at the 3' end of each strand of the oligonucleotide contained in the double-stranded RNA of the present invention, for example, a hydroxyl group, a phosphate group, or a modified phosphate group is preferably present at the 3' position, and the 5' position is as described above. Regarding the thermally unstable modification at the 5' end of each strand of the oligonucleotide contained in the double-stranded RNA, for example, a hydroxyl group, a phosphate group, or a modified phosphate group is preferably present at the 5' position, and the 3' position is as described above.

[0382] A portion of the “thermally unstable modification” can be included in the aforementioned “sugar-modified nucleotides”.

[0383] Next, the double-stranded RNA of the present invention will be described.

[0384] In the following, double-stranded RNA will sometimes be referred to as "dsRNA".

[0385] In one embodiment, the present invention provides a dsRNA capable of inhibiting the expression of a target RNA, the dsRNA comprising a sense strand (also known as a lagging strand) and an antisense strand (also known as a guide strand), wherein the antisense strand is sufficiently complementary to the target sequence for mediating RNA interference, and the dsRNA comprises at least one 2'-O-XCE nucleotide.

[0386] Each strand of the dsRNA independently has a length of 14 to 40 nucleotides. For example, each strand can independently have a length of 14 to 40, 17 to 37, 25 to 37, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, or 21 to 23 nucleotides. There is no limitation, and the lengths of the sense and antisense strands can be the same or different. In some embodiments, the antisense strand is longer than the sense strand, for example, by only 1, 2, 3, 4, or 5 nucleotides.

[0387] In one embodiment, the antisense strand is 18-35 nucleotides long. In another embodiment, the antisense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides long. In a particular embodiment, the antisense strand is 23 nucleotides long. In a particular embodiment, the antisense strand is 22 nucleotides long. In yet another particular embodiment, the antisense strand is 21 nucleotides long.

[0388] Similar to the antisense strand, the sense strand, in some embodiments, has a length of 18 to 35 nucleotides. In some embodiments, the sense strand has a length of 21 to 25, 19 to 25, 19 to 21, or 21 to 23 nucleotides. In a particular embodiment, the sense strand is 21 nucleotides long. In a particular embodiment, the sense strand is 20 nucleotides long. In a particular embodiment, the sense strand is 19 nucleotides long.

[0389] In one specific embodiment, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long. In another specific embodiment, the sense strand is 20 nucleotides long and the antisense strand is 23 nucleotides long. In another specific embodiment, the sense strand is 19 nucleotides long and the antisense strand is 23 nucleotides long. In another specific embodiment, the sense strand is 20 nucleotides long and the antisense strand is 22 nucleotides long. In another specific embodiment, the sense strand is 21 nucleotides long and the antisense strand is 21 nucleotides long. In another specific embodiment, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.

[0390] Double-stranded RNA has a hybridization region. Generally, the hybridization region is 12 to 40 nucleotide base pairs long. For example, dsRNA has a hybridization region of 12 to 25 nucleotide base pairs long. In one embodiment, the dsRNA has a hybridization region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs long. In a specific embodiment, the dsRNA has a hybridization region of 19, 20, 21, or 22 nucleotide base pairs long. In another specific embodiment, the dsRNA has a hybridization region of 21 nucleotide base pairs long.

[0391] The 2'-O-XCE nucleotide can be present at any position within the dsRNA. For example, the 2'-O-XCE nucleotide can be present on the sense strand of the dsRNA. Generally, the 2'-O-XCE nucleotide can be present at any position within the antisense strand. For example, the 2'-O-XCE nucleotide can be located at the 5' end of the sense strand, the 3' end of the sense strand, and / or within the sense strand.

[0392] In one embodiment, the 2'-O-XCE nucleotide is located at the 5' end of the sense strand. In another embodiment, the 2'-O-XCE nucleotide is located at the 3' end of the sense strand.

[0393] In some embodiments, the 2'-O-XCE nucleotide may be located within the sense strand. For example, the 2'-O-XCE nucleotide may be located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the sense strand, counting from the 5' end. In some embodiments, the 2'-O-XCE nucleotide may be located within the 5' region (i.e., positions 2 to 8 of the sense strand, counting from the 5' end). In some embodiments, the 2'-O-XCE nucleotide may be located in the central region of the sense strand.

[0394] In one embodiment, the 2'-O-XCE nucleotide may be present in the sense strand at a position opposite to the seed region of the antisense strand (i.e., positions 2 to 8 from the 5' end of the antisense strand hybridization region).

[0395] In one embodiment, the sense strand comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more 2'-O-XCE nucleotides. In another embodiment, the sense strand comprises at least one 2'-O-XCE nucleotide at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, counting from the 3' end of the sense strand hybridization region. In one embodiment, the sense strand preferably contains at least one 2'-O-XCE nucleotide at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3' end of the sense strand hybridization region. In another embodiment, the sense strand contains 1 to 10 (more preferably 2 to 10, further preferably 2 to 4, and even more preferably 2 or 4) 2'-O-XCE nucleotides. In another embodiment, the sense strand preferably contains at least one 2'-O-XCE nucleotide at positions 1, 2, 20, and 21, counting from the 3' end of the sense strand hybridization region. In another embodiment, the sense strand preferably contains at least one 2'-O-XCE nucleotide at positions 1 and 21, counting from the 3' end of the sense strand hybridization region. In a third embodiment, the sense strand contains at least two 2'-O-XCE nucleotides. In one embodiment, the sense strand preferably contains at least two 2'-O-XCE nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3' end of the sense strand hybridization region. In another embodiment, the sense strand preferably contains 2'-O-XCE nucleotides at positions 1, 2, 20, and 21, counting from the 3' end of the sense strand hybridization region. In yet another embodiment, the sense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 21, counting from the 3' end of the sense strand hybridization region. In a further embodiment, the sense strand contains at least three 2'-O-XCE nucleotides. In one embodiment, the sense strand preferably contains at least three 2'-O-XCE nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3' end. In another embodiment, the sense strand preferably contains at least three 2'-O-XCE nucleotides at positions 1, 2, 20, and 21, counting from the 3' end. In a third embodiment, the sense strand contains at least four 2'-O-XCE nucleotides.In one embodiment, the sense strand preferably contains 2'-O-XCE nucleotides at at least four of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3' end. In another embodiment, the sense strand preferably contains 2'-O-XCE nucleotides at positions 1, 2, 20, and 21, counting from the 3' end.

[0396] In one embodiment, the sense strand preferably contains a 2'-O-XCE nucleotide in at least one of the first and second positions counting from the 5' end of the sense strand, and more preferably, it contains a 2'-O-XCE nucleotide in the first and second positions counting from the 5' end of the sense strand. In another embodiment, the sense strand preferably contains a 2'-O-XCE nucleotide in at least one of the first and second positions counting from the 3' end of the sense strand, and more preferably, it contains a 2'-O-XCE nucleotide in the first and second positions counting from the 3' end of the sense strand. In another embodiment, the sense strand preferably contains a 2'-O-XCE nucleotide in at least one of the first and second positions counting from the 5' end of the sense strand and in at least one of the first and second positions counting from the 3' end of the sense strand, and more preferably, it contains a 2'-O-XCE nucleotide in all of the first and second positions counting from the 5' end of the sense strand and in all of the first and second positions counting from the 3' end of the sense strand.

[0397] In one embodiment, the sense strand preferably contains a 2'-O-XCE nucleotide at position 1, counting from the 5' end. In another embodiment, the sense strand preferably contains a 2'-O-XCE nucleotide at position 1, counting from the 3' end. In yet another embodiment, the sense strand preferably contains a 2'-O-XCE nucleotide at both positions 1, counting from the 5' end and 1, counting from the 3' end.

[0398] In one embodiment, the sense strand comprises at least two or more 2'-O-XCE nucleotides. When two or more 2'-O-XCE nucleotides are present, they can be adjacent to each other. Therefore, in one embodiment, the sense strand comprises at least two, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-O-XCE nucleotides that are adjacent to each other.

[0399] In one embodiment, the sense strand comprises three adjacent 2'-O-XCE nucleotides. In another embodiment, the 2'-O-XCE nucleotides are present in the antisense strand. Generally, the 2'-O-XCE nucleotides can be present at any position within the antisense strand. For example, the 2'-O-XCE nucleotides can be located at the 5' end, the 3' end, and / or an internal position of the antisense strand. In one embodiment, the 2'-O-XCE nucleotides are present at the 5' end of the antisense strand. In another embodiment, the 2'-O-XCE nucleotides are present at the 3' end of the antisense strand.

[0400] In one embodiment, the 2'-O-XCE nucleotide may be present in an internal position of the antisense strand. For example, the 2'-O-XCE nucleotide may be present at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the 2'-O-XCE nucleotide may be present within the seed region (i.e., positions 2 to 8, counting from the 5' end of the antisense strand hybridization region). Positions 2 to 8, counting from the 5' end of the antisense strand hybridization region, are referred to as the seed region. The seed region is the first to recognize the target RNA. Introducing a thermolabile modification into the seed region can suppress off-target effects. For example, at least one of positions 3 to 8, counting from the 5' end of the antisense strand hybridization region, contains the 2'-O-XCE nucleotide. In one embodiment, the 2'-O-XCE nucleotide may be present in the central region of the antisense strand.

[0401] In one embodiment, the antisense strand comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more, 2'-O-XCE nucleotides. In another embodiment, the antisense strand comprises at least one 2'-O-XCE nucleotide. In yet another embodiment, the antisense strand comprises 1 to 10 (preferably 1 to 9, more preferably 1, 2, 3 or 4) 2'-O-XCE nucleotides. In a further embodiment, the antisense strand preferably comprises at least one 2'-O-XCE nucleotide from positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5' end of the antisense hybridization portion. In one embodiment, the antisense strand preferably contains at least one of positions 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains at least one of positions 1, 5, and 10, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the antisense strand contains at least two 2'-O-XCE nucleotides. In yet another embodiment, the antisense strand preferably contains at least two of positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5' end, of the antisense strand hybridization region. In one embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at at least two of positions 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at at least two of positions 1, 5, and 10, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 3, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 4, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 5, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 6, counting from the 5' end of the antisense strand hybridization region. In one embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 7, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 8, counting from the 5' end, of the antisense strand hybridization region.In one embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 9, counting from the 5' end, of the antisense hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 1 and 10, counting from the 5' end, of the antisense hybridization region. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at positions 5 and 10, counting from the 5' end, of the antisense hybridization region. In another embodiment, the antisense strand contains at least three 2'-O-XCE nucleotides. In another embodiment, the antisense strand preferably contains 2'-O-XCE nucleotides at at least three of positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5' end, of the antisense hybridization region. In one embodiment, the antisense strand further preferably contains 2'-O-XCE nucleotides at at least three of positions 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand particularly preferably contains 2'-O-XCE nucleotides at positions 1, 5, and 10, counting from the 5' end of the antisense strand hybridization region.

[0402] In one embodiment, the antisense strand comprises at least two or more 2'-O-XCE nucleotides. When two or more 2'-O-XCE nucleotides are present, they can be adjacent to each other. Therefore, in one embodiment, the antisense strand comprises at least two, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-O-XCE nucleotides that are adjacent to each other.

[0403] In one implementation, the antisense strand comprises three 2'-O-XCE nucleotides that are adjacent to each other.

[0404] In one embodiment, the 2'-O-XCE nucleotide is 2'-O-MCE.

[0405] In one embodiment, the 2'-O-XCE nucleotide is 2'-O-DMAECE.

[0406] In one embodiment, the 2'-O-XCE nucleotide is 2'-O-MORECE.

[0407] In one embodiment, the 2'-O-XCE nucleotide is 2'-O-PyECE.

[0408] In one embodiment, the 2'-O-XCE nucleotide is 2'-O-BimECE.

[0409] When dsRNA contains at least two 2'-O-XCE nucleotides, the 2'-O-XCE nucleotides can be the same or different.

[0410] The double-stranded RNA described in this specification may contain one or more sugar-modified nucleotides. Note that the double-stranded RNA may contain one or more 2'-O-XCE nucleotides and one or more sugar-modified nucleotides.

[0411] In some embodiments, the dsRNA may contain 2'-fluoronucleotides. For example, the dsRNA may contain at least one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more 2'-fluoronucleotides. There is no limitation; all 2'-fluoronucleotides may be present on a single strand. In some embodiments, both the sense strand and the antisense strand contain at least two 2'-fluoronucleotides. 2'-fluoronucleotides may appear on any nucleotide of the sense strand or the antisense strand. For example, 2'-fluoronucleotides may appear on all nucleotides of the sense strand and / or the antisense strand; each 2'-fluoronucleotide may appear in an alternating pattern on the sense strand or the antisense strand; or the sense strand and the antisense strand may contain 2'-fluoronucleotides in an alternating pattern. The alternation pattern of 2'-fluoronucleotides on the sense strand may be the same as or different from that on the antisense strand, and the alternation pattern of 2'-fluoronucleotides on the sense strand may be shifted relative to the alternation pattern of 2'-fluoronucleotides on the antisense strand. The antisense strand of the dsRNA may contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotide. In one embodiment, the antisense strand contains 1, 2, 3, 4, 5 or 6 2'-fluoronucleotides. There is no limitation; the 2'-fluoronucleotides of the antisense strand can be present at any position. In one embodiment, the antisense strand contains at least one 2'-fluoronucleotide. For example, the antisense strand contains a 2'-fluoronucleotide at at least one of positions 2, 6, 8, 9, 14, and 16, counted from the 5' end, of the antisense strand hybridization region. Preferably, the antisense strand contains a 2'-fluoronucleotide at at least one of positions 2 or 14, counted from the 5' end, of the antisense strand hybridization region. In one embodiment, the antisense strand contains at least two 2'-fluoronucleotides. For example, the antisense strand contains 2'-fluoronucleotides at least two of the positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense hybridization region. Preferably, the antisense strand contains 2'-fluoronucleotides at positions 2 and 14, counting from the 5' end of the antisense hybridization region. In one embodiment, the antisense strand contains at least three 2'-fluoronucleotides. For example, the antisense strand contains 2'-fluoronucleotides at at least three of the positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense hybridization region. Preferably, the antisense strand contains 2'-fluoronucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense hybridization region. In some other embodiments, the antisense strand contains at least four 2'-fluoronucleotides. For example, the antisense strand contains 2'-fluoronucleotides at at least four of the positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense hybridization region. Preferably, the antisense strand contains 2'-fluoronucleotides at positions 2, 6, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In some other embodiments, the antisense strand contains at least five 2'-fluoronucleotides.For example, the antisense strand contains at least five 2'-fluoronucleotides selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region. Preferably, the antisense strand contains 2'-fluoronucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region. In some other embodiments, the antisense strand contains at least six 2'-fluoronucleotides. For example, the antisense strand contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region.

[0412] The sense strand of the dsRNA may contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotide. In one embodiment, the sense strand contains 1, 2, 3, 4 or 5 2'-fluoronucleotides. There is no limitation; the 2'-fluoronucleotides of the sense strand can be present at any position. In one embodiment, the sense strand contains at least one 2'-fluoronucleotide. For example, the sense strand contains a 2'-fluoronucleotide in one of the 11th, 12th, 13th or 15th positions selected from the 3' end of the sense strand hybridization region. In one embodiment, the sense strand contains at least two 2'-fluoronucleotides. For example, the sense strand contains a 2'-fluoronucleotide in two of the 11th, 12th, 13th or 15th positions selected from the 3' end of the sense strand hybridization region. In one embodiment, the sense strand contains at least three 2'-fluoronucleotides. For example, the sense strand contains 2'-fluoronucleotides at three of the positions selected from positions 11, 12, 13, or 15, counting from the 3' end of the sense strand hybridization region. In some other embodiments, the sense strand contains at least four 2'-fluoronucleotides. For example, the sense strand contains 2'-fluoronucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridization region.

[0413] In one embodiment, the sense strand comprises at least one of the 11th, 12th, or 13th positions, opposite or complementary to the antisense strand, counting from the 5' end of the hybridization region. In another embodiment, the sense strand comprises at least one of the 11th, 12th, or 15th positions, opposite or complementary to the antisense strand, counting from the 5' end of the hybridization region. In yet another embodiment, the sense strand comprises at least one of the 11th, 12th, 13th, or 15th positions, opposite or complementary to the antisense strand, counting from the 5' end of the hybridization region.

[0414] In one embodiment, the sense strand contains 2'-fluoronucleotides at positions 11 and 12, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the sense strand contains 2'-fluoronucleotides at positions 11 and 13, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14, counting from the 5' end, of the antisense strand hybridization region. In yet another embodiment, the sense strand contains 2'-fluoronucleotides at positions 11 and 13, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14, counting from the 5' end, of the antisense strand hybridization region. In one embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 12, and 13, counting from the 3' end, of the sense hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2 and 14, counting from the 5' end, of the antisense hybridization region. In another embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 13, and 15, counting from the 3' end, of the sense hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 14, and 16, counting from the 5' end, of the antisense hybridization region. In yet another embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 13, and 15, counting from the 3' end, of the sense hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 6, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region. In another embodiment, the sense strand contains at least 2'-fluoronucleotides at positions 11, 13, and 15 starting from the 3' end of the sense strand hybridization region, and the antisense strand contains at least 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 starting from the 5' end of the antisense strand hybridization region. In another embodiment, the sense strand contains 2'-fluoronucleotides at positions 11, 12, and 15 starting from the 3' end of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14 starting from the 5' end of the antisense strand hybridization region. In yet another embodiment, the sense strand contains 2'-fluoronucleotides at positions 11, 13, and 15 starting from the 3' end of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14 starting from the 5' end of the antisense strand hybridization region. In one embodiment, the sense strand contains 2'-fluoronucleotides at positions 11, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the sense strand contains 2'-fluoronucleotides at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2 and 14, counting from the 5' end, of the antisense strand hybridization region.In one embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 12, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In yet another embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In one embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In yet another embodiment, the sense strand contains a 2'-fluoronucleotide at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 6, 9, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the sense strand contains 2'-fluoronucleotides at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end, of the antisense strand hybridization region.

[0415] In one embodiment, the antisense strand does not contain 2'-fluoronucleotides at positions 3 to 9, counting from the 5' end, of the antisense strand hybridization region.

[0416] The dsRNA may contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-O-Me nucleotides. There is no limitation; all 2'-O-Me nucleotides may be present on one strand. In one embodiment, both the sense strand and the antisense strand contain at least one 2'-O-Me nucleotide. The 2'-O-Me nucleotide may appear on any nucleotide of the sense strand or the antisense strand. For example, the 2'-O-Me nucleotide may appear on all nucleotides of the sense strand and / or the antisense strand; the 2'-O-Me nucleotide may appear in an alternating pattern on the sense strand or the antisense strand; or the sense strand and the antisense strand may contain 2'-O-Me nucleotides in an alternating pattern. The alternation pattern of 2'-O-Me nucleotides on the sense strand can be the same as or different from that on the antisense strand, and the alternation pattern of 2'-O-Me nucleotides on the sense strand can be shifted relative to the alternation pattern of 2'-O-Me nucleotides on the antisense strand. The antisense strand of the dsRNA can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more 2'-O-Me nucleotides. There is no limitation; the 2'-O-Me nucleotides of the antisense strand can be present at any position. In one embodiment, the antisense strand contains at least three 2'-O-Me nucleotides. In another embodiment, the antisense strand does not contain 2'-O-Me nucleotides at positions 2 and 14, counting from the 5' end, of the antisense strand hybridization region. For example, the antisense strand does not contain 2'-O-Me nucleotides at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In some other embodiments, the antisense strand does not contain a 2'-O-Me nucleotide at positions 2, 6, 14, and 16, counting from the 5' end, of the antisense hybridization region. In some other embodiments, the antisense strand does not contain a 2'-O-Me nucleotide at positions 2, 6, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region. In yet another embodiment, the antisense strand does not contain a 2'-O-Me nucleotide at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region.

[0417] The sense strand of the dsRNA may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more 2'-O-Me nucleotides. There is no limitation; the 2'-O-Me nucleotides of the sense strand can be present at any position. In one embodiment, the sense strand does not contain 2'-O-Me nucleotides at positions 11, 12, and 13, counting from the 3' end, of the sense strand hybridization region. In another embodiment, the sense strand does not contain 2'-O-Me nucleotides at positions 11, 12, and 15, counting from the 3' end, of the sense strand hybridization region. In yet another embodiment, the sense strand does not contain 2'-O-Me nucleotides at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region. In one embodiment, the sense strand contains a 2'-O-Me nucleotide at at least one of positions 11, 12, and 13, selected from the 3' end of the sense strand hybridization region. In another embodiment, the sense strand contains a 2'-O-Me nucleotide at position 11, selected from the 3' end of the sense strand hybridization region. In another embodiment, the sense strand contains a 2'-O-Me nucleotide at position 12, selected from the 3' end of the sense strand hybridization region. In another embodiment, the sense strand contains a 2'-O-Me nucleotide at position 13, selected from the 3' end of the sense strand hybridization region.

[0418] The dsRNA may additionally include 2'-modified nucleotides other than 2'-O-XCE nucleotides, 2'-fluoronucleotides, and 2'-O-Me nucleotides. For example, the dsRNA may include at least one, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more 2'-modified nucleotides. There is no limitation that all 2'-modified nucleotides may be present on one strand. In one embodiment, both the sense strand and the antisense strand contain at least 2'-modified nucleotides. 2'-modified nucleotides may appear on any nucleotide of the sense strand or the antisense strand. For example, 2'-modified nucleotides may appear on all nucleotides of the sense strand and / or the antisense strand; each 2'-modified nucleotide may appear in an alternating pattern on the sense strand or the antisense strand; or the sense strand and the antisense strand may contain 2'-modified nucleotides in an alternating pattern. The alternation pattern of 2' modifying nucleotides on the sense strand can be the same as or different from that on the antisense strand, and the alternation pattern of 2' modifying nucleotides on the sense strand can be shifted relative to the alternation pattern of 2' modifying nucleotides on the antisense strand. The antisense strand of dsRNA can contain at least one, for example, two, three, four, five, six, seven, eight, nine, ten or more 2' modifying nucleotides. There are no restrictions; the 2' modifying nucleotides on the antisense strand can be present at any position.

[0419] The sense strand of the dsRNA may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2' modified nucleotides. There is no limitation; the 2' modified nucleotides of the sense strand can be present at any position. In one embodiment, the sense strand contains at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2' modified nucleotides, and the antisense strand does not contain 2'-fluorinated nucleotides at positions 3 to 9, counting from the 5' end, of the antisense hybridization region.

[0420] The dsRNA may contain 2'-4' bridging nucleotides. For example, the dsRNA may contain at least one, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, 2'-4' bridging nucleotides. There is no limitation; all 2'-4' bridging nucleotides may be present on one strand. In one embodiment, both the sense strand and the antisense strand contain at least 2'-4' bridging nucleotides. The 2'-4' bridging nucleotides may appear on any nucleotide of the sense strand or the antisense strand. For example, the 2'-4' bridging nucleotides may appear on all nucleotides of the sense strand and / or the antisense strand; each 2'-4' bridging nucleotide may appear in an alternating pattern on the sense strand or the antisense strand; or the sense strand and the antisense strand may contain 2'-4' bridging nucleotides in an alternating pattern. The alternation pattern of 2'-4' bridging nucleotides on the sense strand can be the same as or different from that on the antisense strand, and the alternation pattern of 2'-4' bridging nucleotides on the sense strand can be shifted relative to the alternation pattern of 2'-4' bridging nucleotides on the antisense strand. The antisense strand of dsRNA can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-4' bridging nucleotides. There are no restrictions; the 2'-4' bridging nucleotides on the antisense strand can be present at any position.

[0421] The sense strand of the dsRNA may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-4' bridging nucleotides. There is no limitation; the 2'-4' bridging nucleotides of the sense strand can be present at any position. In one embodiment, the sense strand contains at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-4' bridging nucleotides, and the antisense strand does not contain a 2'-fluorinated nucleotide at positions 3 to 9, counting from the 5' end, of the antisense hybridization region.

[0422] In some embodiments, the dsRNA may comprise deoxyribonucleotides. For example, the dsRNA may comprise at least one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deoxyribonucleotides. There is no limitation; all deoxyribonucleotides may be present on a single strand. In some embodiments, both the sense strand and the antisense strand comprise at least two deoxyribonucleotides. Deoxyribonucleotides may appear on any nucleotide of the sense strand or the antisense strand. For example, deoxyribonucleotides may appear on all nucleotides of the sense strand and / or the antisense strand; each deoxyribonucleotide may appear in an alternating pattern on the sense strand or the antisense strand; or the sense strand and the antisense strand may contain deoxyribonucleotides in an alternating pattern. The alternation pattern of deoxyribonucleotides on the sense strand may be the same as or different from that on the antisense strand, and the alternation pattern of deoxyribonucleotides on the sense strand may be shifted relative to the alternation pattern of deoxyribonucleotides on the antisense strand.

[0423] The antisense strand of the dsRNA may contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) deoxyribonucleotide. In one embodiment, the antisense strand contains 1, 2, 3, 4, 5 or 6 deoxyribonucleotides. There is no limitation; the deoxyribonucleotides of the antisense strand can be present at any position. In one embodiment, the antisense strand contains at least one deoxyribonucleotide. For example, the antisense strand contains a deoxyribonucleotide in at least one (e.g., 1 to 6, preferably 2 to 6, 3 to 5) of positions 2, 5, 6, 7, 8, 9, 12, 14 and 16, counting from the 5' end of the antisense strand hybridization region. Preferably, the antisense strand contains a deoxyribonucleotide in at least one (e.g., 1 to 6, preferably 2 to 6, 3 to 5) of positions 2, 5, 7, 12, 14 and 16, counting from the 5' end of the antisense strand hybridization region. In one embodiment, the antisense strand comprises at least two deoxyribonucleotides. For example, the antisense strand contains deoxyribonucleotides at at least two of the positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. Preferably, the antisense strand contains deoxyribonucleotides at at least two of the positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. In another embodiment, the antisense strand comprises at least three deoxyribonucleotides. For example, the antisense strand contains deoxyribonucleotides at at least three of the positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. Preferably, the antisense strand contains deoxyribonucleotides at at least three of the positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. In some other embodiments, the antisense strand comprises at least four deoxyribonucleotides. For example, the antisense strand contains deoxyribonucleotides at least four of the positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. Preferably, the antisense strand contains deoxyribonucleotides at least four of the positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. In some other embodiments, the antisense strand comprises at least five deoxyribonucleotides. For example, the antisense strand contains deoxyribonucleotides at least five of the positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. Preferably, the antisense strand contains deoxyribonucleotides at least five of the positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand hybridization region. In some other embodiments, the antisense strand contains at least six deoxyribonucleotides.For example, the antisense strand contains deoxyribonucleotides at at least six of the positions selected from the 2nd, 5th, 6th, 7th, 8th, 9th, 12th, 14th, and 16th positions of the antisense strand hybridization region, counting from the 5' end. Preferably, the antisense strand contains deoxyribonucleotides at the 2nd, 5th, 7th, 12th, 14th, and 16th positions of the antisense strand hybridization region, counting from the 5' end.

[0424] It should be noted that, as part of other embodiments, deoxyribonucleotides are included in 1 to 4 (preferably 2, 3, or 4) of positions selected from 2, 5, 7, and 12. As part of other embodiments, deoxyribonucleotides are included in 1 or 2 of positions selected from 5 and 7. As part of other embodiments, the antisense strand may not contain deoxyribonucleotides.

[0425] The sense strand of dsRNA may contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) deoxyribonucleotide. In one embodiment, the sense strand contains 1, 2, 3, 4 or 5 deoxyribonucleotides. There is no limitation; the deoxyribonucleotides in the sense strand can be present at any position. In one embodiment, the sense strand contains at least one deoxyribonucleotide. For example, the sense strand contains a deoxyribonucleotide at one of positions selected from positions 11, 12, 13 or 15, counting from the 3' end, of the sense strand hybridization region. In one embodiment, the sense strand contains a deoxyribonucleotide at position 11, counting from the 3' end, of the sense strand hybridization region. In one embodiment, the sense strand contains a deoxyribonucleotide at position 12, counting from the 3' end, of the sense strand hybridization region. In one embodiment, the sense strand contains a deoxyribonucleotide at position 13, counting from the 3' end, of the sense strand hybridization region. In one embodiment, the sense strand comprises at least two deoxyribonucleotides. For example, the sense strand contains deoxyribonucleotides at two of the positions selected from positions 11, 12, 13, or 15, counting from the 3' end of the sense strand hybridization region. In another embodiment, the antisense strand comprises at least three deoxyribonucleotides. For example, the sense strand contains deoxyribonucleotides at three of the positions selected from positions 11, 12, 13, or 15, counting from the 3' end of the sense strand hybridization region. In yet another embodiment, the sense strand comprises at least four deoxyribonucleotides. For example, the sense strand contains deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridization region.

[0426] It should be noted that, as another approach, the sense strand may not contain deoxyribonucleotides.

[0427] In one embodiment, at least one of the opposite or complementary positions (positions 11, 12, or 13, counting from the 5' end) of the sense strand in the hybridization region with the antisense strand contains a deoxyribonucleotide. In another embodiment, at least one of the opposite or complementary positions (positions 11, 12, or 15, counting from the 5' end) of the antisense strand in the hybridization region with the sense strand contains a deoxyribonucleotide. In yet another embodiment, at least one of the opposite or complementary positions (positions 11, 12, 13, or 15, counting from the 5' end) of the sense strand in the hybridization region with the antisense strand contains a deoxyribonucleotide.

[0428] In one embodiment, the sense strand contains deoxyribonucleotides at positions 11, 12, and 13, counting from the 3' end, of the sense hybridization region, and the antisense strand contains deoxyribonucleotides at positions 2 and 14, counting from the 5' end, of the antisense hybridization region. In another embodiment, the sense strand contains deoxyribonucleotides at positions 11, 13, and 15, counting from the 3' end, of the sense hybridization region, and the antisense strand contains deoxyribonucleotides at positions 2, 14, and 16, counting from the 5' end, of the antisense hybridization region. In yet another embodiment, the sense strand contains deoxyribonucleotides at positions 11, 13, and 15, counting from the 3' end, of the sense hybridization region, and the antisense strand contains deoxyribonucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end, of the antisense hybridization region. In another embodiment, the sense strand contains at least deoxyribonucleotides at positions 11, 13, and 15 starting from the 3' end of the sense strand hybridization region, and the antisense strand contains at least deoxyribonucleotides at positions 2, 6, 8, 9, 14, and 16 starting from the 5' end of the antisense strand hybridization region.

[0429] In one embodiment, the sense strand contains deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains deoxyribonucleotides at positions 2, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In another embodiment, the sense strand contains deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains deoxyribonucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end, of the antisense strand hybridization region. In yet another embodiment, the sense strand contains deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end, of the sense strand hybridization region, and the antisense strand contains deoxyribonucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end, of the antisense strand hybridization region.

[0430] In one embodiment, the antisense strand does not contain deoxyribonucleotides at positions 3 to 9, counting from the 5' end, of the antisense strand hybridization region.

[0431] In some embodiments, the dsRNA includes one or more overhanging regions (i.e., single-stranded regions) and / or capping groups at the 3' end, 5' end, or both ends of the strand. There is no limitation; the overhangs can be 1-10 nucleotides long, 1-6 nucleotides long, 1-5 nucleotides long, 1-4 nucleotides long, 1-3 nucleotides long, 2-6 nucleotides long, 2-5 nucleotides long, 2-4 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhang can result from one strand being longer than the other, or from two strands of the same length being misaligned. The overhang can form a mismatch with the target sequence, or it can be a sequence complementary to the target sequence or other sequences. The sense strand and antisense strand are joined, for example, by forming a hairpin based on the addition of an additional nucleotide, or by other non-nucleotide linkers. There is no limitation; the overhang can be located at the 3' end of the sense strand, antisense strand, or both strands.

[0432] In one embodiment, the dsRNA includes a single overhang. For example, the dsRNA has a single overhang that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In one embodiment, the overhang is located at the 3' end of the antisense strand. In a particular embodiment, the dsRNA includes a 2-nucleotide overhang at the 3' end of the antisense strand. In a particular embodiment, the dsRNA includes a 3-nucleotide overhang at the 3' end of the antisense strand. In a particular embodiment, the dsRNA includes a 4-nucleotide overhang at the 3' end of the antisense strand.

[0433] dsRNA can also have blunt ends. For example, one end of the dsRNA is blunt, and the other end has a blunt end. There is no limitation; the blunt end can be located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3' end and a smooth 5' end. For example, dsRNA with an asymmetric blunt end at the 5' end of the antisense strand and an overhang at the 3' end of the antisense strand is advantageous for loading the guide strand into RISC. In one embodiment, the dsRNA has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In another embodiment, the dsRNA has a 3-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In yet another embodiment, the dsRNA has a 4-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0434] In some other embodiments, the dsRNA has two blunt ends, that is, blunt ends at both ends of the dsRNA.

[0435] Regarding the nucleotides in the overhang region of the dsRNA, examples can be sugar-modified nucleotides, deoxyribonucleotides, ribonucleotides, and any combination thereof, and can be modified or unmodified nucleotides, not limited to these. In some other embodiments, examples include 2'-O-XCE nucleotides, 2'-fluoronucleotides, 2'-O-methylnucleotides, deoxyribonucleotides, ribonucleotides, 2'-O-methoxyethyl nucleotides, and any combination thereof. For example, as a base sequence, TT (or UU) can be an overhang sequence targeting any end of any strand. The 5'- or 3'-overhangs of the sense strand, antisense strand, or both strands of the dsRNA can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphate thioester bond between them; these two nucleotides in the overhang region can be the same or different.

[0436] The dsRNA may contain at least one, for example, two, three, four, five, six, seven, eight, nine, ten or more modified phosphodiester bonds. In some other embodiments, thiophosphate bonds may be included. Modified phosphodiester bonds may be generated on any nucleotide at any position on the sense strand or antisense strand, or on either strand. For example, modified phosphodiester bonds may be generated on all nucleotides of the sense strand and / or antisense strand, or each modified phosphodiester bond may be generated in an alternating pattern on the sense strand or antisense strand, or both the sense strand and antisense strand may contain modified phosphodiester bonds in an alternating pattern. The alternation pattern of modified phosphodiester bonds on the sense strand may be the same as or different from that on the antisense strand, and the alternation pattern of modified phosphodiester bonds on the sense strand may vary relative to the alternation pattern of modified phosphodiester bonds on the antisense strand.

[0437] In some embodiments, the dsRNA may or may not contain modified phosphodiester bonds in the overhang region, but preferably it does. In some other embodiments, the dsRNA may contain phosphate-thioester bonds in the overhang region. For example, the overhang region contains two nucleotides with a modified phosphodiester bond between them. Modified phosphodiester bonds may also be provided to link the overhang nucleotides to a pairing nucleotide at the end of the hybridization portion. For example, at least two, three, four, or all of the overhang nucleotides may be linked by modified phosphodiester bonds, and optionally, additional modified phosphodiester bonds may be present to link the overhang nucleotides to a pairing nucleotide adjacent to the overhang nucleotide. For example, at least two modified phosphodiester bonds may be present between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is a pairing nucleotide adjacent to the overhang nucleotide. Preferably, these three terminal nucleotides may be present at the 3' end of the antisense strand.

[0438] In some embodiments, the dsRNA contains one or more modified phosphodiester bonds within positions 1 to 10 of the terminal position of the sense and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides contain modified phosphodiester bonds at one or both ends of the sense and / or antisense strand.

[0439] In one embodiment, the dsRNA contains 1 to 5 modified phosphodiester bonds within positions 1 to 5 from the 5' end of the sense strand, 1 to 5 modified phosphodiester bonds within positions 1 to 5 from the 3' end of the sense strand, and 1 to 5 modified phosphodiester bonds within positions 1 to 5 from the 5' end of the antisense strand, and 1 to 5 modified phosphodiester bonds within positions 1 to 5 from the 3' end of the antisense strand.

[0440] In one embodiment, the dsRNA contains 1 to 5 phosphate thioester bonds within positions 1 to 5 from the 5' end of the sense strand, 1 to 5 modified phosphate thioester bonds within positions 1 to 5 from the 3' end of the sense strand, and 1 to 5 phosphate thioester bonds within positions 1 to 5 from the 5' end of the antisense strand, and 1 to 5 phosphate thioester bonds within positions 1 to 5 from the 3' end of the antisense strand.

[0441] In one embodiment, the dsRNA contains two phosphate thioester bonds within positions 1 to 5 from the 5' end of the sense strand, one modified phosphate thioester bond within positions 1 to 5 from the 3' end of the sense strand, and one phosphate thioester bond within positions 1 to 5 from the 5' end of the antisense strand, and two phosphate thioester bonds within positions 1 to 5 from the 3' end of the antisense strand.

[0442] In one embodiment, the dsRNA contains two phosphate thioester bonds within positions 1 to 5 from the 5' end of the sense strand, two modified phosphate thioester bonds within positions 1 to 5 from the 3' end of the sense strand, and one phosphate thioester bond within positions 1 to 5 from the 5' end of the antisense strand, and two phosphate thioester bonds within positions 1 to 5 from the 3' end of the antisense strand.

[0443] In one embodiment, the dsRNA contains two phosphate thioester bonds within positions 1 to 5 from the 5' end of the sense strand, two modified phosphate thioester bonds within positions 1 to 5 from the 3' end of the sense strand, and one phosphate thioester bond within positions 1 to 5 from the 5' end of the antisense strand, and one phosphate thioester bond within positions 1 to 5 from the 3' end of the antisense strand.

[0444] In one embodiment, the dsRNA contains one phosphate thioester bond within positions 1 to 5 from the 5' end of the sense strand, one modified phosphate thioester bond within positions 1 to 5 from the 3' end of the sense strand, and two phosphate thioester bonds within positions 1 to 5 from the 5' end of the antisense strand, and two phosphate thioester bonds within positions 1 to 5 from the 3' end of the antisense strand.

[0445] In one embodiment, the dsRNA contains two phosphate thioester bonds within positions 1 to 5 from the 5' end of the sense strand, two modified phosphate thioester bonds within positions 1 to 5 from the 3' end of the sense strand, and two phosphate thioester bonds within positions 1 to 5 from the 5' end of the antisense strand, and two phosphate thioester bonds within positions 1 to 5 from the 3' end of the antisense strand.

[0446] In one embodiment, the dsRNA contains one phosphate thioester bond within positions 1 to 5 counting from the 5' end of the sense strand, two phosphate thioester bonds within positions 1 and 2 counting from the 5' end of the antisense strand, and one phosphate thioester bond within positions 1 to 5 counting from the 3' end of the antisense strand.

[0447] In one embodiment, the dsRNA contains two phosphate thioester bonds within positions 1 to 5 counting from the 5' end of the sense strand, two phosphate thioester bonds within positions 1 and 2 counting from the 5' end of the antisense strand, and two phosphate thioester bonds within positions 1 to 5 counting from the 3' end of the antisense strand.

[0448] In one embodiment, the dsRNA contains two phosphate-thioester bonds within positions 1 to 5 from the 5' end of the sense strand, one phosphate-thioester bond within positions 1 to 5 from the 3' end of the sense strand, two phosphate-thioester bonds within positions 1 and 2 from the 5' end of the antisense strand, and one phosphate-thioester bond within positions 1 to 5 from the 3' end of the antisense strand.

[0449] In one embodiment, the dsRNA contains two phosphate-thioester bonds within positions 1 to 5 counting from the 5' end of the sense strand, one phosphate-thioester bond within positions 1 to 5 counting from the 3' end of the sense strand, two phosphate-thioester bonds within positions 1 and 2 counting from the 5' end of the antisense strand, and two phosphate-thioester bonds within positions 1 to 5 counting from the 3' end of the antisense strand.

[0450] In one embodiment, the dsRNA contains two phosphate thioester bonds at positions 1 and 2 counting from the 5' end of the sense strand, two phosphate thioester bonds at positions 1 and 2 counting from the 3' end of the sense strand, one phosphate thioester bond at position 1 counting from the 5' end of the antisense strand, and one phosphate thioester bond at position 1 counting from the 3' end of the antisense strand.

[0451] In one embodiment, the dsRNA contains one phosphate thioester bond at position 1 counting from the 5' end of the sense strand, one phosphate thioester bond at position 1 counting from the 3' end of the sense strand, two phosphate thioester bonds at positions 1 and 2 counting from the 5' end of the antisense strand, and two phosphate thioester bonds at positions 1 and 2 counting from the 3' end of the antisense strand.

[0452] In a subset of exemplary dsRNAs, the sense strand may contain 0, 1, 2, 3, or 4 phosphate-thioester bonds. For example, the sense strand may contain phosphate-thioester bonds between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the sense strand.

[0453] In some exemplary dsRNAs, the antisense strand may contain 1, 2, 3, or 4 phosphate-thioester bonds. For example, the sense strand contains phosphate-thioester bonds between positions 1 and 2, and between positions 2 and 3, counting from the 3' end of the sense strand. In another example, the antisense strand contains phosphate-thioester bonds between positions 1 and 2, and between positions 2 and 3, counting from the 5' end of the antisense strand, between positions 1 and 2, and between positions 2 and 3, counting from the 3' end of the antisense strand.

[0454] In some embodiments, the sense strand contains a phosphate thioester bond between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 5' end of the sense strand, and the antisense strand contains a phosphate thioester bond between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 3' end of the antisense strand. For example, the sense strand contains a phosphate thioester bond between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 5' end of the sense strand, and the antisense strand contains a phosphate thioester bond between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, counting from the 5' end of the antisense strand, and between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 3' end of the antisense strand.

[0455] In some embodiments, the sense strand and / or antisense strand of the dsRNA may be 5' phosphorylated, or a phosphate modifier may be included at the 5' end. Exemplary phosphate groups or modified phosphate groups contain groups suitable for RISC-mediated gene silencing. For example, preferred phosphate groups or modified phosphate groups include 5'-monophosphate ((HO)₂(O)PO-5'), 5'-diphosphate ((HO)₂(O)POP(HO)(O)-O-5'), 5'-triphosphate ((HO)₂(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-monothiophosphate (thiophosphate ester; (HO)₂(S)PO-5'), and 5'-monodithiophosphate (dithiophosphate ester). Acids, (HO)(HS)(S)PO-5'), 5'-thiophosphates ((HO)2(O)PS-5'), any further combinations of substituted oxygen / sulfur monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-aminophosphates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (alkylene phosphates (e.g.) (HO)2(O)P-CH2-5' (methyl phosphate), (HO)2(O)P-CH2CH2-5' (ethylene phosphate), R′P(OH)(O)-O-5'- (R′ = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-cycloalkylphosphonates ((HO)2(O)P-C3H4-5' (cyclopropylene phosphate)), 5'-alkenylphosphonates (as alkenyl groups, for example, vinyl ((OH)2(O) P-CH=CH-5'-), substituted vinyl groups), and 5'-alkoxyalkyl phosphonates (R″P(OH)(O)-O-5'- (R″=alkoxyalkyl, e.g., methoxymethyl, ethoxymethyl, etc.)). Modifications can be configured within the antisense strand of the dsRNA. For example, the antisense strand may contain a 5' phosphonate group at the 5' end, and may contain 5'-vinylphosphonate nucleotides, 5'-ethylphosphonate nucleotides, or 5'-cyclopropanephosphonate nucleotides.

[0456] In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises a 5'-E-vinylphosphonate. For example, the structure shown in formula (II-3) below (5'-vinylphosphonate (VP)-modified 2'-O-Me nucleotide) is preferred as the 5'-E-vinylphosphonate: [Chemical Formula 51] (The Base in Equation (II-3) is defined the same as X and Base in Equation (II).)

[0457] In some additional embodiments, the preferred structure is that shown in formula (II-2) (5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotide): [Chemical Formula 52] (X and Base in Equation (II-2) are defined in the same way as X and Base in Equation (II).)

[0458] In some embodiments, the preferred structure is the one shown in formula (III-3) (5'-cyclopropanephosphonate (CPP)-substituted 2'-O-Me nucleotide): [Chemical Formula 53] (The Base in Equation (III-3) is defined the same as X and Base in Equation (III).)

[0459] In another embodiment of the invention, the preferred structure is the one shown in formula (III-2) (5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide): [Chemical Formula 54] (X and Base in Equation (III-2) are defined in the same way as X and Base in Equation (III).)

[0460] In another embodiment of the invention, the preferred structure is the one shown in formula (IV-3) (5'-ethylphosphonate (EP)-substituted 2'-O-Me nucleotide): [Chemical Formula 55] (The Base in Equation (IV-3) is defined the same as X and Base in Equation (IV).)

[0461] In another embodiment of the invention, the preferred structure is the one shown in formula (IV-2) (5'-ethylphosphonate (EP)-substituted 2'-O-XCE nucleotide): [Chemical Formula 56] (X and Base in Equation (IV-2) are defined in the same way as X and Base in Equation (IV).)

[0462] The wavy lines in formulas (II-2) to (IV-3) can each be interpreted as the bonding positions of nucleotide bonds formed with adjacent nucleotides, bonding positions with functional molecules or their linkers, or bonding positions with hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups.

[0463] In some embodiments, the sense strand includes a 5'-morpholino group, a 5'-dimethylamino group, a 5'-deoxy group, an inverted debasement group, or an inverted debasement group locked nucleic acid group at the 5' end.

[0464] As an example of a nucleoside structure used in the form of 5'-morpholinoyl modification, without limitation, the following formula (XVI) can be cited: [Chemical Formula 57] (The Base in equation (XVI) is defined the same as the Base in equation (I).) R 40 The atoms are hydrogen, hydroxyl, C1-6 alkoxy, fluorine, amino, N-methylamino, N,N'-dimethylamino, 2-methoxyethoxy (O-MOE), 2-(N-methylcarbamoyl)ethoxy (O-MCE), DMAECE, MorECE, PyECE, and BimECE.

[0465] As an example, a nucleoside structure modified with 5'-dimethylamino is used, without limitation, and the following formula (XVII) can be cited: [Chemical Formula 58] (The Base in equation (XVII) is defined the same as the Base in equation (I).) R 40 With R in equation (XVI) 40 (Same definition)

[0466] As an example of a nucleoside structure used in the form of 5'-deoxy modification, without limitation, the following formula (XVIII) can be cited: [Chemical Formula 59] (The Base in equation (XVIII) is defined the same as the Base in equation (I).) R 40 With R in equation (XVI) 40 (Same definition)

[0467] As an example of a nucleoside structure used in the form of inverted debasement modification, without limitation, the following formula (XIX) can be cited: [Chemical Formula 60] (In the formula, the wavy line indicates the bonding position that can bond with the 5' position of the adjacent nucleotide).

[0468] As an example, a nucleoside structure used in the form of an inverted debasement locked nucleic acid modification is not limited, and the following formula (XX) can be cited: [Chemical Formula 61] (In the formula, the wavy line indicates the bonding position that can bond with the 5' position of the adjacent nucleotide).

[0469] Generally, dsRNA has a melting temperature (Tm) ranging from about 40°C to about 80°C. For example, dsRNA has a melting temperature at the lower end of the range of about 40°C, 45°C, 50°C, 55°C, 60°C, or more than 65°C, and at the upper end of the range of about 70°C, 75°C, or more than 80°C. In some embodiments, dsRNA has a melting temperature ranging from about 55°C to about 70°C or from about 60°C to about 75°C. In some embodiments, dsRNA has a melting temperature ranging from about 57°C to about 67°C. In some specific embodiments, dsRNA has a melting temperature ranging from about 60°C to about 67°C. In some other embodiments, dsRNA has a melting temperature ranging from about 62°C to about 66°C.

[0470] For example, dsRNA with a melting temperature of at least 60°C has improved efficacy in vivo or in vitro. Therefore, in some embodiments, dsRNA has a melting temperature of at least 60°C.

[0471] For example, thermally unstable modifications at positions 2 to 9, counting from the 5' end, of the antisense hybridization region can reduce or inhibit off-target gene silencing. Therefore, in one embodiment, the antisense strand includes at least one (e.g., 1, 2, 3, 4, 5, or more) thermally unstable modification of the double strand within a range of 9 nucleotide positions counting from the 5' end of the antisense hybridization region. The term "one or more thermally unstable modifications" includes one or more modifications of dsRNA that should result in an overall lower melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower) compared to dsRNA without such one or more modifications.

[0472] In some embodiments, the thermally unstable modification is located at positions 2, 3, 4, 5, 6, 7, 8, or 9, counting from the 5' end of the antisense hybridization region, or preferably at positions 4, 5, 6, 7, or 8. In some embodiments, the thermally unstable modification is located at positions 2, 3, 4, 5, or 9, counting from the 5' end of the antisense hybridization region. In some other embodiments, the thermally unstable modification is located at positions 6, 7, or 8, counting from the 5' end of the antisense hybridization region. In a particular embodiment, the thermally unstable modification is located at position 7, counting from the 5' end of the antisense hybridization region.

[0473] In some embodiments, the antisense strand includes at least one 2' modifying nucleotide or 2'-4' bridging nucleotide adjacent to the thermally unstable modification. For example, the 2' modifying nucleotide or 2'-4' bridging nucleotide may be a nucleotide located at the 5' or 3' end of the thermally unstable modification, i.e., at position -1 or +1 from the thermally unstable modification position. In some embodiments, the antisense strand includes a 2' modifying nucleotide or 2'-4' bridging nucleotide at both the 5' and 3' ends of the thermally unstable modification, i.e., at positions -1 and +1 from the thermally unstable modification position.

[0474] In one embodiment, the antisense strand contains at least two modifying nucleotides, each independently selected from 2' modifying nucleotides or 2'-4' bridging nucleotides, at the 3' end of the thermally unstable modification, i.e., at the position +1 and +2 of the self-thermally unstable modification.

[0475] In one embodiment, the sense strand does not contain a 2' modified nucleotide or a 2'-4' bridging nucleotide at a position opposite to or complementary to the thermally unstable modification of the double strand located on the antisense strand.

[0476] In one embodiment, the antisense strand contains a 2'-fluoronucleotide in at least one of the positions adjacent to the thermally unstable modification. For example, the 2'-fluoronucleotide may be a nucleotide located at the 5' or 3' end of the thermally unstable modification, i.e., at position -1 or +1 of the self-instability modification position. In another embodiment, the antisense strand contains a 2'-fluoronucleotide at both the 5' and 3' ends of the thermally unstable modification, i.e., at positions -1 and +1 of the self-instability modification position.

[0477] In one embodiment, the antisense strand contains at least two 2'-fluoronucleotides at the 3' end of the thermally unstable modification, i.e., at positions +1 and +2 of the self-thermally unstable modification.

[0478] In one embodiment, the sense strand does not contain 2'-fluoronucleotides at positions opposite to or complementary to the thermally unstable modification of the double strand located on the antisense strand.

[0479] In some embodiments, specific bases may be included at the overhang, or modified nucleotides or nucleotide substitutes may be included at the single-stranded overhang, such as the 5' or 3' overhang, or both. For example, it is sometimes desirable to include purine nucleotides at the overhang. In some embodiments, all or part of the bases in the 3' or 5' overhang may be modified, for example, by modifications described herein. Modifications may include, for example, the use of a 2' modified nucleotide, such as a 2'-O-XCE nucleotide, a 2'-fluoronucleotide, or a 2'-O-Me nucleotide, and a modified phosphate group, such as the use of a thiophosphate bond. The overhang need not be identical to the target sequence.

[0480] In some embodiments, each residue of the sense and antisense strands is independently modified by 2' modification (2'-O-MOE, 2'-O-Me, or 2'-fluorine, etc.), 2'-4' bridging modification (LNA, etc.), or 2'-deoxy. Each strand may contain more than two modifications. In some embodiments, each residue of the sense and antisense strands is independently modified by 2'-O-Me or 2'-fluorine. It should be understood that in dsRNA, in addition to one or more 2'-O-XCE nucleotides, the above-mentioned modifications are also present.

[0481] In some embodiments, in addition to one or more 2'-O-XCE nucleotides, at least one (preferably two) different modifying nucleotides may be present on the sense strand and / or antisense strand. The respective modifications can be deoxyribonucleotides, 2'-O-Me nucleotides, 2'-fluoronucleotides, acyclic nucleotides, etc. In some embodiments, the sense strand and antisense strand each contain one or two different modifying nucleotides selected from 2'-O-Me nucleotides, 2'-fluoronucleotides, and / or 2'-deoxyribonucleotides. In some embodiments, the sense strand and antisense strand are independently modified by 2'-O-Me nucleotides, deoxyribonucleotides, 2'-fluoronucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides (also known as 2'-fluoroarabinonucleotides, 2'-F-ANA). In this case, it should still be understood that the above-mentioned modifications exist in the dsRNA in addition to one or more 2'-O-XCE nucleotides.

[0482] In some embodiments, the dsRNA may contain alternating patterns of modification. The term "alternating pattern," as used in this specification, refers to a modification pattern in which a strand has repeating units containing one or more modifications, with the repeating number being two or more. Nucleotides appearing in the alternating pattern can alternate between one and three. For example, where A, B, and C each represent a modification of one type of nucleotide, examples of alternating patterns with two repeats include "ABAB," "AABBAABB," "AABAAB," "AAABAAAB," "AAABBBAAABBB," or "ABCABC," etc. The alternating patterns within each strand can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0483] The types of modifications included in the alternation pattern can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, that is, the modifications of nucleotides that are separated by one nucleotide, can be the same, and the sense strand or antisense strand can each be selected from several possibilities of modifications within the range of alternation patterns such as "ABAB...", "ACAC...", "BDBD...", or "CDCD...".

[0484] In one embodiment, the dsRNA includes a shift of alternation patterns on the sense strand relative to alternation patterns on the antisense strand. This shift can be a corresponding or opposite shift of a modified set of nucleotides on the sense strand to a different modified set of nucleotides on the antisense strand. For example, when the sense and antisense strands are paired in the dsRNA double strand, the alternation pattern on the sense strand within the hybridization region can begin with "ABABAB" along the 5'-3' of the strand, and the alternation pattern on the antisense strand can begin with "BABABA" along the 3'-5' of the strand. As another example, the alternation pattern on the sense strand within the hybridization region can begin with "AABBAABB" along the 5'-3' of the strand, and the alternation pattern on the antisense strand can begin with "BBAABBAA" along the 3'-5' of the strand, thus resulting in a complete or partial shift of the modification pattern between the sense and antisense strands.

[0485] In some embodiments, the dsRNA contains one or more mismatches with the target RNA, within the range of the double strand, or combinations thereof. Mismatches may occur in overhanging regions or hybridization portions. Base pairs may be ordered based on a tendency to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular pair; the simplest method is to investigate the pairs on each base pair, but adjacent base analysis or the same analysis can also be used). Regarding promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; and I:C (I = inosine) is preferred over G:C. Mismatches, such as non-standard or non-standard pairings (as described in other parts of this specification), are preferred over standard (A:T, A:U, G:C) pairings; and pairings containing universal bases are preferred over standard pairings.

[0486] In one embodiment, the dsRNA comprises at least one of the first 1, 2, 3, 4, or 5 base pairs (which may be independently selected from the group A:U, G:U, I:C) of the antisense hybridization portion, counted from the 5' end, and mispairing, such as non-standard or non-standard pairings, or pairings containing universal bases, thereby promoting the dissociation of the antisense strand at the 5' end of the double strand.

[0487] In one embodiment, the nucleotide at position 1, counted from the 5' end, of the antisense hybridization region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs, counted from the 5' end, of the antisense hybridization region is an AU base pair. For example, the first base pair, counted from the 5' end, of the antisense hybridization region is an AU base pair.

[0488] For example, if 4'-modified and / or 5'-modified nucleotides are introduced into the 3' end of the phosphodiester bond, thiophosphate bond, and / or dithiophosphate bond of a dinucleotide located at any position in a single-stranded or double-stranded oligonucleotide, a steric effect is produced on the internucleotide bond, thus protecting it from nucleases or stabilizing it.

[0489] In one embodiment, the 5'-modified nucleotide is introduced into the 3' end of a dinucleotide located at any position on the dsRNA. For example, a 5'-alkylated nucleotide may be introduced into the 3' end of a dinucleotide located at any position on the dsRNA.

[0490] In one embodiment, a 4'-modified nucleotide is introduced to the 3' end of a dinucleotide located at any position on the dsRNA. For example, a 4'-alkylated nucleotide may be introduced to the 3' end of a dinucleotide located at any position on the dsRNA. Alternatively, a 4'-O-alkylated nucleotide may be introduced to the 3' end of a dinucleotide located at any position on a single-stranded or double-stranded siRNA. An exemplary 4'-O-alkylated nucleotide is a 4'-O-methyl nucleotide. The 4'-O-methyl group may be a racemic mixture or a chiral pure R or S isomer.

[0491] In one embodiment, a 5'-alkylated nucleotide is introduced at any position on the sense or antisense strand of the dsRNA, a modification that maintains or improves the efficacy of the dsRNA. The 5'-alkyl can be either a racemic mixture or a chiral pure R or S isomer. An exemplary 5'-alkylated nucleotide is a 5'-methyl nucleotide. The 5'-methyl can be either a racemic mixture or a chiral pure R or S isomer. Another exemplary 5'-alkylated nucleotide is a 5'-CP nucleotide.

[0492] In one embodiment, a 4'-alkylated nucleotide is introduced at any position on the sense or antisense strand of the dsRNA, a modification that maintains or improves the efficacy of the dsRNA. The 4'-alkyl can be either a racemic mixture or a chiral pure R or S isomer. An exemplary 4'-alkylated nucleotide is a 4'-methyl nucleotide. The 4'-methyl can be either a racemic mixture or a chiral pure R or S isomer.

[0493] In one embodiment, a 4'-O-alkylated nucleotide is introduced at any position on the sense or antisense strand of the dsRNA, which maintains or improves the efficacy of the dsRNA. The 5'-alkyl can be either a racemic mixture or a chiral pure R or S isomer. An exemplary 4'-O-alkylated nucleotide is a 4'-O-methyl nucleotide. The 4'-O-methyl can be either a racemic mixture or a chiral pure R or S isomer.

[0494] In some embodiments, the dsRNA may contain a 2'-5' bond (containing 2'-H, 2'-OH, and 2'-O-Me and containing P=O or P=S). For example, 2'-5' bond modification can be used to promote nuclease resistance or inhibit the binding of the sense and antisense strands, or it can be used at the 5' end of the sense strand to prevent RISC-based sense strand activation. In some embodiments, the sense strand contains a 2'-5' bond between the nucleotides at positions 1 and 2, counting from the 5' end.

[0495] In some embodiments, a functional molecule may be directly or indirectly bound to the dsRNA. The binding of the functional molecule to the sense strand and / or antisense strand of the dsRNA can be direct or indirect via other substances, but oligonucleotides are preferably bound to the functional molecule via covalent bonds, ionic bonds, or hydrogen bonds. From the viewpoint of high stability of this binding, direct binding via covalent bonds or binding via covalent bonds through a linker (connecting group) is more preferred.

[0496] When the aforementioned functional molecule binds to the sense strand and / or antisense strand of dsRNA via covalent bonds, the functional molecule preferably binds directly or indirectly to the 3' or 5' end of the sense strand and / or antisense strand of dsRNA. In some embodiments, it preferably binds directly or indirectly to internal nucleotides (e.g., sugar portions, base portions, or internucleotide bonds) of the sense strand and / or antisense strand of dsRNA. The binding of the aforementioned linker or functional molecule to the terminal nucleotides or internal nucleotides of the sense strand and / or antisense strand of dsRNA can be selected based on the functional molecule.

[0497] The aforementioned linker or functional molecule, and the terminal nucleotides or internal nucleotides of the sense strand and / or antisense strand of the dsRNA, are preferably linked via phosphodiester bonds or modified phosphodiester bonds, more preferably via phosphodiester bonds. In one embodiment, the aforementioned linker or functional molecule, and the terminal nucleotides of the sense strand and / or antisense strand of the dsRNA, are preferably linked via modified phosphodiester bonds, more preferably via thiophosphate bonds. The aforementioned linker or functional molecule can be directly linked to the 3' oxygen atom of the 3' terminal nucleotide or the 5' oxygen atom of the 5' terminal nucleotide of the sense strand and / or antisense strand of the dsRNA. In one embodiment, the aforementioned linker or functional molecule can be directly linked to the 2' oxygen atom of the terminal nucleotide or internal nucleotide of the sense strand and / or antisense strand of the dsRNA.

[0498] The structure of a "functional molecule" is not particularly restricted, and it can confer a desired function to dsRNA through binding. Desired functions include labeling, purification, and delivery to the target site. Examples of molecules that confer labeling functions include fluorescent proteins and luciferases. Examples of molecules that confer purification functions include biotin, avidin, His-tagged peptides, GST-tagged peptides, and FLAG-tagged peptides.

[0499] Furthermore, from the viewpoint of delivering dsRNA to the target site (e.g., target cells) with high specificity and efficiency, and of effectively regulating the expression of the target RNA through this dsRNA, it is preferable to have a molecule that enables the delivery of dsRNA to the target site as a functional molecule. Molecules with this delivery function can be found, for example, in *European Journal of Pharmaceutics and Biopharmaceutics*, 2016, 107, pp 321-340; *Advanced Drug Delivery Reviews*, 2016, 104, pp 78-92; and *Expert Opinion on Drug Delivery*, 2014, 11, pp 791-822.

[0500] As molecules that confer delivery to target sites, lipids and sugars can be cited as examples, considering the ability to deliver dsRNA to the liver, central nervous system, lungs, etc., with high specificity and efficiency. Examples of such lipids include cholesterol; fatty acids; fat-soluble vitamins such as vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D, and vitamin K; intermediate metabolites such as acylcarnitine and acyl-CoA; glycolipids; glycerides; long-chain hydrocarbons; and their derivatives. Among these, cholesterol and vitamin E (tocopherols, tocotrienols) are preferred from the viewpoint of higher safety. Tocopherols are preferred, more preferably tocopherol, and particularly preferably α-tocopherol. Examples of sugars include sugar derivatives that interact with desialyl glycoprotein receptors.

[0501] In another embodiment of the dsRNA, preferably in the sense strand and / or antisense strand, and more preferably in the sense strand, a lipid-containing structure as shown in formula (XXI) is included: [Chemical Formula 62] (The Base in equation (XXI) is defined the same as the Base in equation (I), R) 50 (C5-30 alkyl or C5-30 alkenyl). In another embodiment of the part, it is preferably included at least one of the 14 to 21 positions selected from the sense hybridization moiety counting from the 3' end (XXI), more preferably included at the 16 position (XXI). In another embodiment of the part, R 50 Preferably C14-24 alkyl, R 50 Further preferred are C16-22 alkyl groups.

[0502] The "desialyl glycoprotein receptor" is located on the surface of liver cells and has the function of recognizing the galactose residues of desialyl glycoprotein, taking the molecule into the cell, and breaking it down. The "glycoprotein derivative that can interact with the desialyl glycoprotein receptor" is preferably a compound having a structure similar to the galactose residues and being taken into the cell through interaction with the desialyl glycoprotein receptor, such as GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives.

[0503] In another embodiment of the part, as a "glyco derivative capable of interacting with the desialylate glycoprotein receptor", it is preferably contained in the sense strand and / or antisense strand of the dsRNA, and more preferably in the sense strand, the GalNAc structure shown in formula (i) below: [Chemical Formula 63] (In the formula, Rd is a hydroxyl group or a mercapto group, preferably a mercapto group).

[0504] In another embodiment of the invention, it is preferred to bind directly to the 3' or 5' end of the sense strand of the dsRNA, and more preferably to the 5' end of the sense strand.

[0505] In another embodiment of the part, as a "glyco derivative capable of interacting with the desialylate glycoprotein receptor", it is preferably contained in the sense strand and / or antisense strand of the dsRNA, and more preferably in the sense strand, the GalNAc structure shown in formula (ii) below: [Chemical Formula 64] (In the formula, Re is independently a hydroxyl group or a thiol group, preferably a thiol group).

[0506] In another embodiment of the invention, it is preferred to bind directly to the 3' or 5' end of the sense strand of the dsRNA, and more preferably to the 3' end of the sense strand.

[0507] Furthermore, from the viewpoint of being able to efficiently deliver the dsRNA of the present invention into the brain with high specificity, sugars (e.g., glucose, sucrose, etc.) can be cited as "functional molecules." Additionally, from the viewpoint of being able to efficiently deliver the dsRNA into organs with high specificity by interacting with various proteins present on the cell surface of organs, receptor ligands, antibodies, peptides or proteins as fragments of these proteins can be cited as "functional molecules."

[0508] Regarding adapters that mediate the binding of functional molecules to the sense strand and / or antisense strand of dsRNA, any adapter that can perform the function of the functional molecule in the form of dsRNA is acceptable. Therefore, there are no particular limitations as long as the adapter stably binds the functional molecule to the oligonucleotide. Examples of such adapters include groups derived from oligonucleotides with 1 to 20 nucleotides, groups derived from polypeptides with 2 to 20 amino acids, alkylene groups with 2 to 20 carbon atoms, and alkenyl groups with 2 to 20 carbon atoms. The aforementioned groups derived from oligonucleotides with 2 to 20 nucleotides are groups obtained by removing hydroxyl groups, hydrogen atoms, etc., from oligonucleotides with 2 to 20 nucleotides. For example, International Publication No. 2017 / 053995 describes adapters with, for example, a 3-base TCA motif and adapters with 1 to 5 bases without a TCA motif. The aforementioned groups derived from polypeptides with 2 to 20 amino acids are groups obtained by removing hydroxyl, hydrogen, and amino groups from polypeptides with 2 to 20 amino acids.

[0509] The connector is preferably a C2-20 alkylene or C2-20 alkenylene group (where the methylene group in the alkylene and alkenylene groups is independently unsubstituted or substituted by one or two substituents selected from the group consisting of halogen atoms, hydroxyl groups, protected hydroxyl groups, oxo groups, and thio groups). Additionally, the methylene groups in the alkylene and alkenylene groups are independently unsubstituted or substituted with -O- or -NR-. B -(R) B This indicates substitution of hydrogen atoms, C1-6 alkyl or halo-C1-6 alkyl atoms, -S-, -S(=O)-, or -S(=O)2-. Here, combining the aforementioned substitutions, the connector may contain elements of -C(=O)-O-, -OC(=O)-NR, etc. 23 -(R) 23 Represents hydrogen atom, C1-6 alkyl or halo-C1-6 alkyl, -C (=O)-NR 23 -(R) 23 Represents hydrogen atom, C1-6 alkyl or halo-C1-6 alkyl, -C (=S)-NR 23 -(R) 23 (representing hydrogen atom, C1-6 alkyl or halo-C1-6 alkyl), -NR 23 -C (=O) -NR 23 -(R) 23 Each group independently represents a hydrogen atom, a C1-6 alkyl group, or a halo-C1-6 alkyl group.

[0510] For example, it can be used as -[C2-6 alkylene]-[C(=O)-NR 23 -(R) 23 This represents a connector structure consisting of a hydrogen atom or a C1-6 alkyl group (C1-6 alkylene group) and a C2-6 alkylene group (C=O group). Preferably, the C2-6 alkylene group on the left is bound to the aforementioned functional molecule, and the C(O) group on the right is bound to an oligonucleotide.

[0511] The connector is more preferably a C2-20 alkylene group (each methylene group is independently unsubstituted or substituted with -O-. The unsubstituted methylene groups are independently unsubstituted or substituted with hydroxyl or protected hydroxyl groups), and even more preferably a C8-12 alkylene group (each methylene group is independently unsubstituted or substituted with -O-. The unsubstituted methylene groups are independently unsubstituted or substituted with hydroxyl groups).

[0512] Alternatively, the connector is more preferably a C2-20 alkylene group (where the methylene groups of the alkylene group are each independently unsubstituted, or are replaced by -O- or -NR). B -(R) BThe methylene group is replaced by a hydrogen atom or a C1-6 alkyl group. Each unsubstituted methylene group is independently unsubstituted or oxysubstituted, more preferably a C8-12 alkylene group (where each methylene group is independently unsubstituted or oxysubstituted). B -(R) B (replaced by hydrogen atoms or C1-6 alkyl groups). Unreplaced methylene groups are individually either unsubstituted or oxysubstituted.

[0513] One to five (preferably three, particularly preferably three) 3-11-membered nitrogen-containing non-aromatic heterocyclic dimethyldiethanol structures can be linked to the 5' or 3' ends of the sense strand and / or antisense strand of the dsRNA via phosphodiester bonds or thiophosphate bonds. The aforementioned linker can be attached to this 3-11-membered nitrogen-containing non-aromatic heterocycle. In this case, the number of functional molecules linked by the linker is also preferably one to five, more preferably one to three, and particularly preferably three. The aforementioned 3-11-membered nitrogen-containing non-aromatic heterocycle is preferably pyrrolidine or piperidine. The 3-11-membered nitrogen-containing non-aromatic heterocyclic dimethyldiethanol is particularly preferably piperidine-4,4-dimethylethanol.

[0514] The aforementioned "protected hydroxyl group" is not particularly limited as long as it is stable when the functional molecule is bound to the oligonucleotide. The linker is not particularly limited either; for example, any protecting group described in Protective Groups in Organic Synthesis, 4th Edition, TW Greene, PGM Wuts, John Wiley & Sons Inc. (2006), etc., can be cited. Specifically, as stated in the description of the terminology, benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 9-phenylxanth-9-yl or 9-(p-methoxyphenyl)xanth-9-yl are preferred, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl or trimethoxytriphenylmethyl are more preferred, and dimethoxytriphenylmethyl is even more preferred.

[0515] In dsRNA, the functional molecule can bind to one or both strands. In some embodiments, the dsRNA has a functional molecule that binds to the sense strand. In other embodiments, the dsRNA has a functional molecule that binds to the antisense strand.

[0516] In some embodiments, the functional molecule can bind to the nucleic acid bases, sugar moieties, or nucleoside bonds of the sense strand and / or antisense strand of dsRNA. Binding to purine nucleic acid bases or their derivatives can occur at any position, including intra- and extra-ring atoms. In some embodiments, binding occurs at positions 2, 6, 7, or 8 of purine nucleic acid bases. Binding to pyrimidine nucleic acid bases or their derivatives can also occur at any position. In some embodiments, binding occurs at positions 2, 5, and 6 of pyrimidine nucleic acid bases. Binding to the sugar moieties of nucleotides can occur at any carbon atom. Examples of carbon atoms in the sugar moieties that can be bound include 2', 3', and 5' carbon atoms. Binding can also occur at the 1' position, for example, at a debasement residue. Nucleoside bonds can also have coupling portions. In phosphorus-containing bonds (e.g., phosphodiesters, thiophosphates, dithiophosphates, aminophosphates, etc.), binding can occur directly to a phosphorus atom or to an O, N, or S atom bonded to a phosphorus atom. In nucleoside internucleotide bonds containing amines or amides (e.g., PNA), the nitrogen atom of the amine or amide or an adjacent carbon atom may be bonded.

[0517] In some embodiments, the functional molecule is bound to the sense strand. As described in this specification, the functional molecule may bind to the 3' end, 5' end, or internal position of the sense strand. In some embodiments, the functional molecule binds to the 3' end of the sense strand. Additionally, the functional molecule may bind to nucleic acid bases, sugar moieties, or intermolecular bonds of the sense strand.

[0518] dsRNAs also include substances existing via their tautomerism, geometric isomerism, and mixtures thereof or mixtures of isomers. Furthermore, in the case of the presence of a chiral center, or the formation of a chiral center due to isomerization, substances existing as optical isomers and mixtures thereof in any proportion are also included. In the case of compounds having two or more chiral centers, diastereomers arising from their respective optical isomerism are further present. The present invention also includes substances containing all these types in any proportion. Furthermore, optically active substances can be obtained by methods well known for achieving this purpose.

[0519] For example, in the case where the dsRNA of the present invention contains a modified phosphodiester bond (e.g., a thiophosphate bond) and the phosphorus atom is a chiral atom, either an oligonucleotide with stereocontrolled phosphorus atom or an oligonucleotide with uncontrolled phosphorus atom is included within the scope of the present invention.

[0520] The dsRNA of the present invention, or its pharmaceutically permissible salts, may exist in any crystalline form or in any hydrate form, depending on the manufacturing conditions. These crystalline forms, hydrates, and mixtures thereof are also included within the scope of the present invention. Additionally, it may sometimes exist as a solvate containing organic solvents such as acetone, ethanol, 1-propanol, and 2-propanol; these forms are also included within the scope of the present invention.

[0521] The dsRNA of the present invention can be converted into a pharmaceutically permissible salt as needed, or it can be freed from the generated salt. Examples of pharmaceutically permissible salts for dsRNA include salts formed with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, organic bases (triethylamine, trimethylamine, etc.), amino acids (glycine, lysine, glutamic acid, etc.), inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.), or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.).

[0522] Specifically, the local structure represented by -P(=O)(OH)- can be transformed into an anionic local structure represented by -P(=O)(O-)-, forming salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), or ammonium. Similarly, the local structure representing the formation of thiophosphate bonds, represented by -P(=O)(SH)-, can be transformed into an anionic local structure represented by -P(=O)(S-)-, similarly forming salts with alkali metals, alkaline earth metals, or ammonium. The same applies to other modified phosphodiester bonds.

[0523] For the dsRNA of the present invention, those skilled in the art can prepare it by appropriately selecting known methods. For example, those skilled in the art can design the nucleotide sequence of the dsRNA based on information about the nucleotide sequence of the target RNA and synthesize it using a commercially available automated nucleic acid synthesizer (manufactured by Applied Biosystems, Beckman, GeneDesign, etc.). Alternatively, it can be synthesized by using an enzymatic reaction. Examples of such enzymes include polymerases, ligases, and restriction endonucleases, but are not limited thereto. That is, the method for manufacturing the dsRNA according to this embodiment may include the step of extending the nucleotide chain at the 3' or 5' end of the sense strand and / or antisense strand.

[0524] Various methods for binding functional molecules to the sense and / or antisense strands of the dsRNA are known in the art, for example, see European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp 78-92, ExpertOpinion on Drug Delivery, 2014, 11, pp 791-822, etc. For example, after binding the functional molecule to the adapter using known methods, it can be derivatized into an amide body using an amidation reagent or into an H-phosphonate body using an H-phosphonate reagent, and then bound to an oligonucleotide.

[0525] The obtained oligonucleotides can be purified using reversed-phase column chromatography, thereby preparing the sense and antisense strands of dsRNA. dsRNA can be prepared by mixing equivalent amounts of the sense and antisense strands and then annealing.

[0526] The dsRNA of the present invention can effectively regulate the expression of target RNA. Therefore, the present invention can provide compositions containing the dsRNA of the present invention as an active ingredient for regulating the expression of target RNA, for example, through RNA interference. In particular, the dsRNA of the present invention can achieve high efficacy with low concentrations, and in some embodiments, pharmaceutical compositions can also be provided for treating, preventing, and improving diseases such as metabolic diseases, tumors, and infectious diseases accompanied by hyperexpression of target RNA.

[0527] Compositions containing the dsRNA of the present invention can be formulated using known pharmaceutical methods. For example, they can be used via the intestinal tract (orally, etc.) or non-intestinal tract in the form of capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated formulations, pellets, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, ointments, plasters, poultices, transdermal formulations, lotions, inhalers, aerosols, injections, suppositories, etc.

[0528] In the above formulation, it can be appropriately combined with pharmacologically permissible or food and beverage carriers (specifically sterile water, physiological saline, vegetable oil, solvent, matrix, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, aromatic agent, excipient, vehicle, preservative, binder, diluent, isotonic agent, analgesic agent, expander, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring and odor-correcting agent, solubilizer or other additives, etc.).

[0529] There are no particular limitations on the administration method of the dsRNA composition comprising the present invention, and examples include intestinal (oral, etc.) administration or non-intestinal administration. More preferably, examples include intravenous administration, intra-arterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intramedullary administration, intraventricular administration, nasal administration, and intravitreal administration, as well as infusion-based administration.

[0530] There are no particular limitations on the diseases that can be treated, prevented, or improved using the nucleic acid pharmaceuticals based on the dsRNA of the present invention. Examples include metabolic diseases, circulatory system diseases, tumors, infectious diseases, eye diseases, inflammatory diseases, autoimmune diseases, rare hereditary diseases, and other diseases caused by gene expression. More specifically, examples include hypercholesterolemia, hypertriglyceridemia, spinal muscular atrophy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy (Fukuyama type, Ullrich type, Merosin deficiency type, integrin deficiency, Walker-Warburg syndrome, etc.), Becker muscular dystrophy, limb-girdle muscular dystrophy, Miyoshi muscular dystrophy, facioscapulohumeral muscular dystrophy, etc.), Huntington's disease, Alzheimer's disease, and transthyretin amyloidosis. Amyloidosis, familial amyloid cardiomyopathy, multiple sclerosis, Crohn's disease, inflammatory bowel disease, acromegaly, type II diabetes, chronic kidney disease, RS virus infection, Ebola hemorrhagic fever, Marburg fever, HIV, influenza, hepatitis B, hepatitis C, cirrhosis, chronic heart failure, myocardial fibrosis, atrial fibrillation, prostate cancer, melanoma, breast cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, bile duct cancer, cervical cancer, liver cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, atopic dermatitis, glaucoma, age-related macular degeneration, etc. The genes that could be the cause of the aforementioned diseases can be designated as the target RNAs, and then, based on the sequence of the target RNAs, the expression regulatory sequences (e.g., antisense sequences) can be appropriately set.

[0531] Besides primates such as humans, various other mammalian diseases can also be treated, prevented, or improved using compositions containing the double-stranded RNA or its prodrug of the present invention. For example, while not limited to this, diseases can be treated in mammalian species including cows, sheep, goats, horses, dogs, cats, guinea pigs, or other bovine, ovine, equine, canine, feline, and rodent species such as mice. Furthermore, compositions containing double-stranded RNA can also be used in other species such as birds (e.g., chickens).

[0532] When the composition containing the dsRNA of the present invention is administered or ingested into animals, including humans, the dosage or intake may be appropriately selected according to the age, weight, symptoms, health status, and type of composition (pharmaceutical, food, etc.) of the subject, and the dosage or intake is preferably 0.0001 mg / kg / day to 100 mg / kg / day.

[0533] The dsRNA of the present invention effectively regulates the expression of target RNA while exhibiting higher metabolic stability than conventional siRNAs. Therefore, a method can be provided to administer the dsRNA of the present invention to animals, including humans, to regulate the expression of target RNA for a longer period through RNA interference. Furthermore, a method can be provided for treating, preventing, and improving various diseases accompanied by hyperexpression of target RNA, the method comprising administering a composition containing the dsRNA of the present invention to animals, including humans.

[0534] As a preferred method for using the dsRNA of the present invention, the following methods can be cited.

[0535] A method for regulating the function of target RNA, comprising the step of contacting the dsRNA of the present invention with a cell.

[0536] A method for regulating the function of target RNAs in mammals, comprising the step of administering a dsRNA pharmaceutical composition comprising the present invention to the mammal.

[0537] The present invention relates to the use of dsRNA for regulating the function of target RNA in mammals.

[0538] The use of the dsRNA of the present invention for manufacturing pharmaceuticals for regulating the function of target RNAs in mammals.

[0539] A method for regulating the expression of target RNA, comprising the step of contacting the dsRNA of the present invention with cells.

[0540] A method for regulating the expression of target RNA in a mammal, comprising the step of administering a pharmaceutical composition containing the dsRNA of the present invention to the mammal.

[0541] The present invention relates to the use of dsRNA for regulating the expression of target RNA in mammals.

[0542] The dsRNA of the present invention is used for manufacturing pharmaceuticals for regulating the expression of target RNA in mammals.

[0543] The preferred mammal among the aforementioned is human.

[0544] The preferred route of administration is transenteral (intestinal). Alternatively, administration may be non-transenteral.

[0545] The 2'-O-XCE nucleotide involved in the embodiments of the present invention can be manufactured with reference to International Publication No. 2007 / 102581, The Journal of Organic Chemistry, 2011, 76, pp 3042-3053, WO2017 / 142054, Organic & Biomolecular Chemistry, 2019, 17, pp 4835-4842, etc.

[0546] Some embodiments of the present invention comprise the following 5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotides, 5'-cyclopropanephosphonate (CPP)-substituted 2'-O-XCE nucleotides, and 5'-ethylphosphonate (EP)-substituted 2'-O-XCE nucleotides.

[0547] 1. The compound or its salt represented by the following formula (II): [Chemical Formula 65] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IIa) represents the group: [Chemical Formula 66] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R)3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2They can be the same or different. T 1 The group represented by the following formula (IIb): [Chemical Formula 67] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. A 1 The groups are selected from the following formula (IIc): [Chemical Formula 68] [Q1 and Q2 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy, and cyano groups)]}.

[0548] 2. The compound or its salt represented by formula (III) below: [Chemical Formula 69] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IIIa) represents the group: [Chemical Formula 70] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T 1 The group represented by the following formula (IIIb): [Chemical Formula 71] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. B 1 The group represented by the following formula (IIIc): [Chemical Formula 72] [Q3 and Q4 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkyl, a C1-6 alkoxy, and a cyano group)]}.

[0549] 3. The compound or its salt represented by the following formula (IV): [Chemical Formula 73] {In the formula, Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IVa) represents the group: [Chemical Formula 74] [In the formula, R] 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T1 For the group represented by the following formula (IVb): [Chemical Formula 75] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. E 1 For groups represented by the following formula (IVc): [Chemical Formula 76] [Q5~Q8 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl, and an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy, and cyano groups)]}.

[0550] The 5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotide, 5'-cyclopropanephosphonate (CPP)-substituted 2'-O-XCE nucleotide, and 5'-ethylphosphonate (EP)-substituted 2'-O-XCE nucleotide involved in this embodiment can be manufactured by the methods shown below. However, the following manufacturing methods show an example of conventional manufacturing methods and do not limit the manufacturing methods of the nucleotides involved in this embodiment.

[0551] 5'-Vinylphosphonate (VP)-modified 2'-O-XCE nucleotides (3'-amidized form) can be synthesized, for example, by a method based on that described in J. Med. Chem. 2018, 61, 734-744. Specifically, it can be produced by protecting the 3'-hydroxyl group of the 2'-O-XCE nucleotide (e.g., tert-butyldimethylsilyl, etc.), then converting the 5'-hydroxyl group to an aldehyde body by an oxidation reaction (e.g., Dys-Martin reagent, etc.), then converting it to an olefin body by the Horner-Wozworth-Emmons reaction of tetratetra[(pivaloyloxy)methyl]methylene diphosphonate, then deprotecting the 3'-hydroxyl protecting group, and finally amidating with an amidating agent.

[0552] 5'-Cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotides (3'-amidation) can be synthesized, for example, by a method based on that described in Chem. Commun., 2021, 57, 6808. Specifically, it can be manufactured by protecting the 3'-hydroxyl group of the 2'-O-XCE nucleotide (e.g., tert-butyldimethylsilyl, etc.), then converting the 5'-hydroxyl group to an aldehyde (e.g., Des Martin reagent, etc.) by oxidation, then converting it to an olefinic body by Wittig reaction of p-[(triphenylphosphine-1,2-methyl)methyl]O,O-thiophosphonate diethyl ester, then converting it to a cyclopropaneic body by Cory-Tchaikovsky reaction, etc., then deprotecting the 3'-hydroxyl protecting group, then converting the thiophosphonate to a phosphonate (e.g., Oxone, etc.) by oxidation, and finally amidation using an amidating agent.

[0553] 5'-Ethylphosphonate (EP)-modified 2'-O-XCE nucleotide (3'-amidide) can be manufactured by reducing an olefinic precursor, which is a synthetic precursor of 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide (3'-amidide), through a hydrogenation reaction or similar process, and then amidating it with an amidating agent.

[0554] Oligonucleotides comprising the 5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotide, 5'-cyclopropanephosphonate (CPP)-substituted 2'-O-XCE nucleotide, and 5'-ethylphosphonate (EP)-substituted 2'-O-XCE nucleotide of this embodiment at their 5' ends can be manufactured using commercially available phosphoramide reagents required for manufacturing each of the above-mentioned 3'-amidates and oligonucleotide analogs of the desired nucleotide sequence, through solid-phase synthesis using an automated nucleic acid synthesis apparatus (e.g., nS-8II (GeneDesign)).

[0555] Oligonucleotides containing 5'-vinylphosphonate (VP)-substituted 2'-O-XCE nucleotides and 5'-ethylphosphonate (EP)-substituted 2'-O-XCE nucleotides at the 5' end can be manufactured by treating the solid-phase support after the reaction using the above-mentioned automated nucleic acid synthesis apparatus with an amine (e.g., ammonia, methylamine, diethylamine, etc.).

[0556] Oligonucleotides containing 5'-cyclopropanephosphonate (CPP)-substituted 2'-O-XCE nucleotides at the 5' end can be manufactured by treating the solid support after the above-mentioned reaction using an automated nucleic acid synthesis apparatus with a mixture of, for example, trimethyliodosilane, pyridine and acetonitrile, followed by treatment with an amine (e.g., ammonia, methylamine, diethylamine, etc.).

[0557] Example The present invention will now be described in more detail based on embodiments, but these embodiments do not constitute any limitation on the scope of the present invention. It should be noted that "NMR" refers to Nuclear Magnetic Resonance, "LC / MS" refers to High Performance Liquid Chromatography / Mass Spectrometry, "(v / v)" refers to (volume / volume), and "(w / v)" refers to (mass / volume).

[0558] Records 1 For H NMR data, the chemical shift δ (unit: ppm) of the signal measured at 400 MHz (JNM-ECZ400; manufactured by JEOL Ltd.) or JNM-ECX300; manufactured by JEOL Ltd., with tetramethylsilane as an internal standard (fracture mode, integrated value) is indicated. “s” indicates a singlet, “d” indicates a doublet, “t” indicates a triplet, “dd” indicates a doublet, “m” indicates a multiplet, “br” indicates a broad peak, “brs” indicates a broad singlet, “J” indicates the coupling constant, “CDCl3” indicates deuterated chloroform, “DMSO-D6” indicates deuterated dimethyl sulfoxide, and “ACETONITRILE-D3” indicates deuterated acetonitrile.

[0559] Records 31 When using p-NMR data, the chemical shift δ (unit: ppm) of the signal measured using a 162 MHz (JNM-ECZ400; manufactured by JEOL Ltd.) with phosphoric acid as an internal standard is expressed.

[0560] Unless otherwise specified, LC / MS is performed using any of the following conditions 1 to 4, employing ESI (electrospray ionization). ＋ "" refers to ESI positive ion mode. - "" refers to ESI negative ion mode. It should be noted that the mixing ratio of the two solutions described in the following LC / MS conditions is a volume ratio, and the formic acid content is expressed as volume %

[0561] LC / MS condition 1: Device: Waters ACQUITY UPLC H CLASS / QDa Detector Column: Waters ACQUITY UPLC BEH C18 (1.7μm, 2.1×50mm) Column temperature: 40℃ Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient condition: With a flow rate of 0.6 mL / min and a mixing ratio of solution A to solution B set to 90 / 10, the mixing ratio of solution A to solution B was linearly changed to 10 / 90 after 3 minutes.

[0562] After 0.7 minutes, the mixing ratio of solution A to solution B was fixed at 10 / 90.

[0563] After 0.1 minutes, the mixing ratio of solution A to solution B was linearly changed to 90 / 10 and the flow rate was 0.8 mL / min.

[0564] After one minute, the mixing ratio of solution A to solution B was fixed at 90 / 10.

[0565] Detection wavelength: PDA (190-400nm) LC / MS condition 2: Device: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7μm, 2.1×50mm) Column temperature: 40℃ Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient condition: With a flow rate of 0.6 mL / min and a mixing ratio of solution A to solution B set to 90 / 10, the mixing ratio of solution A to solution B was linearly changed to 10 / 90 after 3 minutes.

[0566] After 0.7 minutes, the mixing ratio of solution A to solution B was fixed at 10 / 90.

[0567] After 0.1 minutes, the mixing ratio of solution A to solution B was linearly changed to 90 / 10 and the flow rate was 0.8 mL / min.

[0568] After one minute, the mixing ratio of solution A to solution B was fixed at 90 / 10.

[0569] Detection wavelength: PDA (190-400nm) LC / MS condition 3: Device: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7μm, 2.1×50mm) Column temperature: 40℃ Solvent: Solution A: 10 mmol / L ammonium bicarbonate aqueous solution Solution B: 10 mmol / L ammonium bicarbonate-acetonitrile solution (containing 10% water of the total solution volume) Gradient condition: With a flow rate of 0.6 mL / min and a mixing ratio of solution A to solution B set to 91 / 9, the mixing ratio of solution A to solution B was linearly changed to 19 / 81 after 3 minutes.

[0570] After 0.7 minutes, the mixing ratio of solution A to solution B was fixed at 19 / 81.

[0571] After 0.1 minutes, the mixing ratio of solution A to solution B was linearly changed to 91 / 9 and the flow rate was 0.8 mL / min.

[0572] After one minute, the mixing ratio of solution A to solution B was fixed at 91 / 9.

[0573] Detection wavelength: PDA (190-400nm) LC / MS condition 4: Device: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7μm, 2.1×50mm) Column temperature: 40℃ Solvent: Solution A: 10 mmol / L ammonium bicarbonate aqueous solution Solution B: 10 mmol / L ammonium bicarbonate-acetonitrile solution (containing 10% water of the total solution volume) Gradient condition: With a flow rate of 0.6 mL / min and a mixing ratio of solution A to solution B set to 82 / 18, the mixing ratio of solution A to solution B was linearly changed to 10 / 90 after 2.5 minutes.

[0574] After 1.2 minutes, the mixing ratio of solution A to solution B was fixed at 19 / 81.

[0575] After 0.1 minutes, the mixing ratio of solution A to solution B was linearly changed to 82 / 18 and the flow rate was 0.8 mL / min.

[0576] After one minute, the mixing ratio of solution A to solution B was fixed at 82 / 18.

[0577] Detection wavelength: PDA (190-400nm) Unless otherwise specified, purification by silica gel column chromatography was performed using SHOKO SCIENCE Purif-Pack (registered trademark)-EX (SI-50 μm).

[0578] Unless otherwise specified, purification using silica gel reversed-phase column chromatography employed either Xbridge PREPC18 5μm 19×100mm or Xselect CSH Prep C18 5μm 19×100mm.

[0579] The content and amount of formic acid and triethylamine in the solvent used in silica gel column chromatography are shown in volume % (%).

[0580] Purification using supercritical fluid chromatography was carried out under the following conditions.

[0581] Installation: Waters Prep 80q Column: DAICL CHIRALPAK IG (20 mm) (×250mm, 5μm) Column temperature: 40℃ Mobile phase: Carbon dioxide / methanol = 50 / 50 Flow rate: 15 mL / min Single-crystal X-ray structural analysis was performed using the XtaLAB Synergy R, DW system, Hypix (Rigoro Kabushiki Co., Ltd.), and measurements were taken in accordance with the instruction manual.

[0582] [Manufacturing Example 1] Synthesis of Compound 8 [Chemical Formula 77] Step (1): Synthesis of Compound 2 Imidazole (947.7 mg, 13.9 mmol) was added to a mixture of compound 1 (synthesized according to the method described in International Publication No. 2023 / 054708) (3.00 g, 4.64 mmol) and dehydrated N,N-dimethylformamide (9.30 mL), and dissolved by stirring at room temperature. Tert-butyldimethylchlorosilane (1.40 g, 9.28 mmol) was added to this mixture, and the mixture was stirred overnight (approximately 16–20 hours) at room temperature under an argon atmosphere. A saturated aqueous sodium bicarbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with water and a saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give target compound 2 (3.47 g, 98% yield) as a white solid.

[0583] LC / MS: Condition 1 LC / MS (ESI-) m / z; 758.1 [MH] - 1 H-NMR (CDCl3) δ: 8.20 (1H, br s), 7.78-7.77 (1H, m), 7.40-7.38 (2H,m), 7.32-7.28 (7H, m), 6.85-6.83 (4H, m), 6.35-6.34 (1H, m), 5.95 (1H, d, J =3.3 Hz), 4.34-4.32 (1H, m), 4.10-4.07 (1H, m), 3.95-3.89 (3H, m), 3.80 (6H,s), 3.66 (1H, dd, J = 11.0, 2.2 Hz), 3.26 (1H, dd, J = 11.0, 2.6 Hz), 2.78(3H, d, J = 4.8 Hz), 2.52-2.48 (2H, m), 1.37-1.37 (3H, m), 0.80 (9H, s), 0.04(3H, s), -0.07 (3H, s). Step (2): Synthesis of compound 3 To a mixture of compound 2 (3.47 g, 4.57 mmol) and dichloromethane (44.0 mL), 1-dodecyl mercaptan (2.17 mL, 9.13 mmol) was added, followed by trifluoroacetic acid (0.525 mL, 6.85 mmol). The mixture was stirred at room temperature for 15 minutes under an argon atmosphere. A saturated aqueous sodium bicarbonate solution was added to the reaction mixture until the pH changed to 8–9, followed by extraction with dichloromethane. The resulting organic layer was washed with a saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate → ethyl acetate / methanol) to give target compound 3 (1.88 g, 90%) as a white solid.

[0584] LC / MS: Condition 1 LC / MS (ESI+) m / z; 458.4 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.44 (1H, br s), 7.55-7.55 (1H, m), 6.22-6.20 (1H,m), 5.70 (1H, d, J = 4.0 Hz), 4.37 (1H, t, J = 5.1 Hz), 4.08-4.03 (2H, m),4.02-3.98 (1H, m), 3.92-3.90 (1H, m), 3.85-3.74 (2H, m), 2.78 (3H, d, J = 4.8Hz), 2.54-2.52 (1H, m), 2.48-2.46 (2H, m), 1.93 (3H, d, J = 1.1 Hz), 0.92(9H, s), 0.12 (6H, s). Step (3): Synthesis of Compound 6 To a mixture of compound 3 (500.0 mg, 1.1 mmol) and dichloromethane (25.0 mL), 1,1,1-triacetoxy-1,1-dihydro-1,2-benzoiodoxapram-3-(1H)-one (Dess-Martin periodinane) (648.8 mg, 1.5 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 1 hour, and then further stirred at room temperature for 1 hour. After the reaction, a mixture of 10% (w / v) aqueous sodium thiosulfate solution (10.8 mL) and saturated sodium bicarbonate solution (10.8 mL) was added at 0 °C, and the mixture was extracted with ethyl acetate. The resulting organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give a crude product containing target compound 4. Next, tetrahydrofuran (2.5 mL) was added to sodium hydride (55 wt% liquid paraffin, 182.1 mg, 3.8 mmol), and compound 5 was added dropwise at -78 °C under an argon atmosphere, with stirring at -78 °C for 15 minutes. Then, a tetrahydrofuran solution (6.9 mL) containing the crude product of compound 4 was added dropwise to the reaction mixture, and the mixture was stirred at -78 °C for 1 hour, then heated to 0 °C and stirred for 1 hour, and then further heated to room temperature and stirred for 4 hours. After the reaction, a saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with a saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate → ethyl acetate / methanol) to give target compound 6 (713.0 mg, 86% yield in two stages) as a colorless, transparent oil.

[0585] LC / MS: Condition 1 LC / MS (ESI-) m / z; 760.4 [MH] - 1 H-NMR (400 MHz, CDCl3) δ: 0.10 (s, 6H), 0.88-0.94 (m, 9H), 1.21-1.23(m, 18H), 1.94-1.98 (m, 3H), 2.45-2.49 (m, 2H), 2.79-2.82 (m, 3H), 3.81-3.86(m, 1H), 3.89-3.95 (m, 2H), 4.06-4.10 (m, 1H), 4.44-4.48 (m, 1H), 5.63-5.72(m, 4H), 5.82 (d, J = 3.3 Hz, 1H), 6.07-6.09 (m, 1H), 6.81-6.92 (m, 1H),7.12-7.12 (m, 1H), 8.01 (s, 1H). 31 P-NMR (162 MHz, CDCl3) δ: 14.3, 16.9. Step (4): Synthesis of Compound 7 A formic acid / water mixture (35.7 mL, 1 / 1 (v / v)) was added to compound 6 (713.0 g, 0.94 mmol), and the mixture was stirred at room temperature for 8 hours. Then, another formic acid / water mixture (35.7 mL, 1 / 1 (v / v)) was added to the reaction mixture, and the mixture was stirred at room temperature for 13 hours. Methanol was added to the reaction mixture, and the mixture was stirred and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol) to give a crude product containing target compound 7. This crude product was purified by reversed-phase silica gel column chromatography (0.1% trifluoroacetic acid-acetonitrile / 0.1% trifluoroacetic acid aqueous solution) to give target compound 7 (400.1 mg, 66% yield) as a colorless, transparent oil.

[0586] LC / MS: Condition 4 LC / MS (ESI+) m / z; 648.7 [M+H] ＋ 1 H NMR (400 MHz, DMSO-D6) δ: 0.97-1.17 (m, 18H), 2.06 (s, 3H), 2.31-2.36 (m, 2H), 2.54 (d, 3H), 3.64-3.79 (m, 3H), 4.10-4.19 (m, 2H), 4.30-4.33(m, 1H), 5.58-5.62 (m, 4H), 5.78 (d, 1H), 6.02-6.12 (m, 1H), 6.77-6.89 (m,1H), 7.44-7.44 (m, 1H), 7.84-7.85 (m, 1H), 11.40 (s, 1H) 31 P-NMR (162 MHz, DMSO-D6) δ: 17.9 Step (5): Synthesis of Compound 8 To a mixture of compound 7 (400.1 mg, 0.62 mmol) and acetonitrile (8.0 mL), 4,5-dicyanimidazole (80.3 mg, 0.68 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (235.2 μL, 0.74 mmol) were added, and the mixture was stirred at room temperature for 2 hours. A 1.5% aqueous solution of potassium bicarbonate (8.0 mL) was added to the reaction mixture, and after stirring, hexane (2.0 mL) and ethyl acetate (2.0 mL) were added. After stirring for 10 minutes, the aqueous layer was removed. Next, a 1.5% aqueous solution of potassium bicarbonate (8.0 mL) was added to the organic layer, and after stirring for 10 minutes, the aqueous layer was removed. This step was repeated once more. Water and a saturated sodium chloride aqueous solution were added to the organic layer, and after stirring, the aqueous layer was removed. The organic layer was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate (with 0.15% triethylamine) / methanol) to give target compound 8 (197.7 mg, yield 38%) as a white solid.

[0587] LC / MS: Condition 3 LC / MS (ESI-) m / z; 846.3, 846.2 [MH] - 1 H-NMR (400 MHz, ACETONITRILE-D3) δ: 1.17-1.23 (m, 30H), 1.82-1.83 (m,2H), 2.07-2.10 (m, 3H), 2.28-2.36 (m, 2H), 2.62 (t, 3H), 2.66-2.76 (m, 2H),3.59-3.89 (m, 4H), 4.09-4.13 (m, 1H), 4.24-4.37 (m, 1H), 4.51-4.58 (m, 1H),5.57-5.65 (m, 4H), 5.82-5.84 (m, 1H), 6.04-6.14 (m, 1H), 6.32-6.35 (m, 1H), 6.78-7.00 (m, 1H), 7.16-7.17 (m, 1H). 31 P-NMR (162 MHz, ACETONITRILE-D3) δ: 150.4, 150.3, 17.3, 16.9. [Manufacturing Example 2] Synthesis of Compound 14 [Chemical Formula 78] Step (1): Synthesis of Compound 10 A mixture of compound 3 (1.06 g, 2.32 mmol) and dichloromethane (46.5 mL) was cooled to 0 °C. 1,1,1-triacetoxy-1,1-dihydro-1,2-benzoiodoxypentane-3-(1H)-one (Dies Martin oxidant) (1.18 g, 2.79 mmol) and saturated sodium bicarbonate (19.5 mg, 0.23 mmol) were added. The mixture was stirred at 0 °C for 1 hour under an argon atmosphere, followed by stirring at room temperature for 30 minutes. A mixture of saturated sodium bicarbonate aqueous solution and saturated sodium thiosulfate aqueous solution (40.0 mL, 1 / 1 (v / v)) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated sodium chloride aqueous solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product of target compound 4 (1.08 g) as a white solid. The crude product (1.07 mmol) was dissolved in dehydrated tetrahydrofuran (8.0 mL). Under an argon atmosphere, a tetrahydrofuran solution (6.0 mL) of compound 9 (synthesized according to the method described in Chem. Commun., 2021, 57, 6808) (1.19 g, 2.79 mmol) was added dropwise at 0 °C. The mixture was then heated to room temperature and stirred overnight (approximately 16–20 hours). The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate → ethyl acetate / methanol) to give target compound 10 (976.7 mg, 69% yield in two stages) as a white solid.

[0588] LC / MS: Condition 1 LC / MS (ESI+) m / z; 606.2 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.78 (1H, br s), 7.14-7.13 (1H, m), 6.86-6.78 (1H,m), 6.28-6.22 (2H, m), 5.82 (1H, d, J = 2.5 Hz), 4.55-4.49 (1H, m), 0.92 (9H, s), 0.12 (6H, d, J = 3.7Hz). 31 P-NMR (162 MHz, CDCl3) δ: 84.4 Step (2): Synthesis of compound 12 Under an argon atmosphere, sodium hydride (55% liquid paraffin, 112.9 mg, 4.7 mmol) was added to a mixture of trimethyl sulfoxide (1.04 g, 4.7 mmol) and dehydrated dimethyl sulfoxide (4.0 mL), and the mixture was stirred at room temperature for 30 minutes. This suspension was then added dropwise to a dimethyl sulfoxide solution (3 mL) of compound 10 (949.8 mg, 1.57 mmol). After the addition was complete, the mixture was stirred at room temperature for 90 minutes, then heated and stirred at 40 °C for 6 hours. The reaction mixture was cooled to 0 °C, ice water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and a saturated sodium chloride aqueous solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 11 as a white solid. Next, a formic acid / water mixture (10.08 mL, 1 / 1 (v / v)) was added to the crude compound 11 (937.1 mg), and the mixture was stirred overnight (approximately 16–20 hours) at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to obtain isomer A (455.0 mg, LC / MS (condition 1) RT = 1.69 min, white solid, yield 60%) and isomer B (204.5 mg, LC / MS (condition 1) RT = 1.74 min, white solid, yield 27%) of target compound 12.

[0589] Isomer A LC / MS: Condition 1 LC / MS (ESI+) m / z; 506.3 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.64 (1H, br s), 7.23-7.22 (1H, m), 5.88-5.86 (2H, m), 4.23-4.21 (1H, m), 4.19-4.03 (4H, m), 4.00-3.97 (2H, m), 1.66-1.51 (2H, m), 1.36-1.24 (7H, m), 1.23-1.15(1H, m), 1.11-1.00 (1H, m). 31P-NMR (162 MHz, CDCl3) δ 101.4 Isomer B LC / MS: Condition 1 LC / MS (ESI+) m / z; 506.3 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.74 (1H, s), 7.16 (1H, d, J = 1.2 Hz), 5.98-5.96(1H, m), 5.77 (1H, d, J = 3.7 Hz), 4.25-3.94 (7H, m), 3.88-3.82 (1H, m),3.54-3.47 (1H, m), 2.82 (3H, d, J = 4.9 Hz), 2.60-2.53 (1H, m), 2.45-2.40(1H, m), 1.96-1.96 (3H, m), 1.65-1.50 (1H, m), 1.36-1.29 (7H, m), 1.28-1.18(1H, m), 1.04–0.98 (1H, m). 31 P-NMR (162 MHz, CDCl3) δ: 101.0 Step (3): Synthesis of isomer A of compound 13 Compound 12 (isomer A: 455.0 mg, 0.9 mmol) was dissolved in a tetrahydrofuran / water mixture (6.5 mL, 1 / 1 (v / v)), and OXONE (registered trademark) and a monopersulfate compound (553.3 mg, 1.8 mmol) were added. The mixture was stirred at room temperature for 5 hours. Ethanol was added to the reaction mixture, and the mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel reversed-phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid aqueous solution) to give isomer A of target compound 13 as a white solid (102.25 mg, LC / MS (condition 1) RT = 1.14 min, yield 23%).

[0590] LC / MS: Condition 1 LC / MS (ESI+) m / z; 490.2 [M+H] ＋ 1H-NMR (CDCl3) δ: 9.43 (1H, br s), 7.22-7.22 (1H, m), 6.32-6.31 (1H,m), 5.82 (1H, d, J = 4.5 Hz), 4.70 (1H, br s), 4.24-4.22 (1H, m), 4.16-4.05(4H, m), 4.00-3.97 (2H, m), 3.85-3.79 (1H, m), 3.43-3.40 (1H, m), 2.80 (3H,d, J = 4.5 Hz), 2.62-2.39 (2H, m), 1.93 (3H, s), 1.71-1.57 (1H, m), 1.34-1.29(6H, m), 1.24-1.18 (1H, m), 1.09-0.92 (2H, m). 31 P-NMR (162 MHz, CDCl3) δ: 29.6 Step (3): Synthesis of isomer A of compound 13 (other methods) Another example of the preparation of isomer A of compound 13 is shown. Compound 12 (isomer A: 485.3 mg, 0.96 mmol) was dissolved in dichloromethane (33 mL), and 3-chloroperoxybenzoic acid (containing about 30% water by mass) (1.08 g, 1.63 mmol) was added at 0 °C, followed by heating to room temperature and stirring for 15 minutes. Then, a mixture of 30% (w / v) aqueous sodium thiosulfate and saturated sodium bicarbonate (1 / 1 (v / v)) was added to the reaction mixture, and the mixture was stirred at room temperature for 5 minutes. The mixture was concentrated under reduced pressure, and ethanol and methanol were added to the resulting residue. The precipitated solid was filtered, and the filtrate was collected in a round-bottom flask. Ethanol was added to the filtered solid, and the solid was filtered. The filtrates obtained from the first and second filtrations were combined and concentrated under reduced pressure. The residue was purified by silica gel reversed-phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid aqueous solution) to give isomer A of target compound 13 (241.2 mg, yield 51%) as a white solid.

[0591] 1 mg of isomer A of compound 13 obtained in step (3) above was diluted with 50 μL of acetone and allowed to stand at 25°C for 24 hours to obtain crystals.

[0592] To determine the structure of the crystal, single-crystal X-ray diffraction was performed. The obtained diffraction data were analyzed using CrysAlisPro software (Rigaku Corporation) for integration and absorption correction. The initial structure was obtained using SHELX T (Acta Crystallogr.Sect. A 2015, 71, 3-8), and refined using SHELX L (Acta Crystallogr. Sect. C2015, 71, 3-8). The results are shown in Table 1, and the ORTEP diagram is plotted below. Figure 1 .

[0593] [Table 1] Based on the above results, the structure of isomer A of compound 13 is determined to be as follows (13A).

[0594] [Chemical Formula 79] In addition, the structure of isomer B of compound 13 is determined as shown in the following formula (13B).

[0595] [Chemical Formula 80] Step (3'): Synthesis of isomer B of compound 13 Compound 12 (isomer B: 204.5 mg, 0.40 mmol) was dissolved in a tetrahydrofuran / water mixture (3.2 mL, 1 / 1 (v / v)), and OXONE (registered trademark) and a monopersulfate compound (248.1 mg, 0.81 mmol) were added. The mixture was stirred at room temperature for 5 hours. Ethanol was added to the reaction mixture, and the mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel reversed-phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid aqueous solution) to give isomer B of target compound 13 as a colorless, transparent oil (76.60 mg, LC / MS (condition 1) RT = 1.17 min, yield 39%).

[0596] LC / MS: Condition 1 LC / MS (ESI+) m / z; 490.3 [M+H] ＋ 1H-NMR (CDCl3) δ: 8.90 (1H, br s), 7.17-7.17 (1H, m), 6.07-6.06 (1H,m), 5.77 (1H, d, J = 3.7 Hz), 4.20-4.07 (5H, m), 4.02-3.97 (2H, m), 3.88-3.82(1H, m), 3.42 (1H, dd, J = 8.4, 5.9 Hz), 2.82 (3H, d, J = 4.9 Hz), 2.59-2.54(1H, m), 2.45-2.40 (1H, m), 1.95-1.95 (3H, m), 1.68-1.55 (1H, m), 1.35 (6H,td, J = 7.0, 2.0 Hz), 1.29-1.17 (1H, m), 1.08-0.92 (2H, m). 31 P-NMR (162 MHz, CDCl3) δ: 29.7 Step (3'): Synthesis of isomer B of compound 13 (other methods) Another example of the preparation of isomer B of compound 13 is shown. Compound 12 (isomer B: 1.83 g, 3.62 mmol) was dissolved in dichloromethane (124 mL), and 3-chloroperoxybenzoic acid (containing about 30% water by mass) (3.70 g, 5.61 mmol) was added at 0 °C, and the mixture was stirred for 15 minutes. Then, a mixture of 30% (w / v) aqueous sodium thiosulfate and saturated sodium bicarbonate (1 / 1 (v / v)) was added to the reaction mixture, and the mixture was stirred at room temperature for 5 minutes. The mixture was concentrated under reduced pressure, and ethanol was added to the resulting residue. The precipitated solid was filtered, and the filtrate was recovered into a round-bottom flask. Silica gel was added to the filtrate, and the mixture was concentrated under reduced pressure. After concentration, the filtrate was purified by silica gel column chromatography (ethyl acetate / methanol) to give isomer B of target compound 13 (1.99 g, crude purified) as a yellow oil.

[0597] Step (4): Synthesis of isomer A of compound 14 Under an argon atmosphere, a mixture of isomer A of compound 13 (112.0 mg, 0.23 mmol) and dehydrated dichloromethane (1.4 mL) was added to a dichloromethane solution (0.6 mL) of 4,5-dicyanimidazole (10.8 mg, 0.092 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.109 mL, 0.34 mmol), and stirred at room temperature for 5 hours. The reaction mixture was purified by silica gel column chromatography (chloroform (with 0.2% triethylamine) / methanol) to give isomer A of target compound 14 (81.2 mg) as a white solid. Subsequently, isomer A (50 mg, 0.10 mmol) of compound 13, recovered by column chromatography, was dissolved in dehydrated dichloromethane (0.6 mL), and a dichloromethane solution (0.32 mL) of 4,5-dicyanimidazole (7.2 mg, 0.061 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.0486 mL, 0.15 mmol) was added. The mixture was stirred at room temperature for 5 hours. The reaction mixture was purified by silica gel column chromatography (chloroform (with 0.2% triethylamine) / methanol) to give isomer A (50.8 mg) of target compound 14 as a white solid. (Total yield: 132.0 mg, 84% yield) LC / MS: Condition 3 LC / MS (ESI-) m / z; 688.2, 688.2 [MH] - 1 H-NMR (CDCl3) δ: 7.25-7.24 (1H, m), 6.43-6.39 (1H, m), 5.79 (1H, dd,J = 20.4, 2.9 Hz), 4.30-4.02 (7H, m), 4.00-3.81 (3H, m), 3.78-3.41 (4H, m), 2.78 (3H, d, J ...

Claims

1. Double-stranded RNA, which is a double-stranded RNA that can repress the expression of target RNA. The double-stranded RNA contains a sense strand and an antisense strand. The antisense strand has 14-40 nucleotides and is sufficiently complementary to the target RNA for mediating RNA interference. The sense strand has 14-40 nucleotides and is complementary to the antisense strand. The double-stranded RNA contains at least one 2'-O-XCE nucleotide.

2. The double-stranded RNA as described in claim 1, wherein, The antisense strand contains at least one 2'-O-XCE nucleotide, or the sense strand contains at least one 2'-O-XCE nucleotide.

3. The double-stranded RNA as described in claim 1 or 2, wherein, The double-stranded RNA contains at least one 2'-O-XCE nucleotide and at least one 2'-fluoronucleotide.

4. The double-stranded RNA according to any one of claims 1 to 3, wherein, The double-stranded RNA contains at least one 2'-O-XCE nucleotide and at least one 2'-O-Me nucleotide.

5. The double-stranded RNA according to any one of claims 1 to 4, wherein, The double-stranded RNA contains at least one 2'-O-XCE nucleotide and at least one deoxyribonucleotide.

6. The double-stranded RNA according to any one of claims 1 to 5, wherein, The antisense strand contains at least one of the following positions, counting from the 5' end: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, of the antisense strand hybridization region: 2'-O-XCE nucleotide.

7. The double-stranded RNA according to any one of claims 1 to 6, wherein, At least one of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the sense strand hybridization region, counting from the 3' end, contains a 2'-O-XCE nucleotide.

8. The double-stranded RNA according to any one of claims 1 to 7, wherein, The 2'-O-XCE nucleotide is a nucleotide containing a local structure represented by the following formula (I): [Chemical Formula 1] In formula (I), Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl and protected thioalkyl). X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (Ia) represents the group: [Chemical Formula 2] In formula (Ia), R 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different.

9. The double-stranded RNA as described in claim 8, wherein, X is a C1-6 alkyl or C2-6 alkenyl (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups and cyano groups).

10. The double-stranded RNA as described in claim 8 or 9, wherein, X is a methyl group.

11. The double-stranded RNA as described in claim 8, wherein, X is a group represented by the following formula (Ia): [Chemical Formula 3] In formula (Ia), R 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different.

12. The double-stranded RNA of claim 11, wherein, The R 1 and R 2 It is a hydrogen atom.

13. The double-stranded RNA as described in claim 11 or 12, wherein, The Y is NR 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form a 4-8 membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene rings, or... Y is a C2-9 aromatic heterocyclic group.

14. The double-stranded RNA according to any one of claims 11 to 13, wherein, The Y is NR 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine.

15. The double-stranded RNA according to any one of claims 11 to 13, wherein, The Y is pyridyl, imidazolyl, or benzimidazolyl.

16. The double-stranded RNA according to any one of claims 11 to 15, wherein, The value of n is 2.

17. The double-stranded RNA according to any one of claims 1 to 16, wherein, The antisense strand contains 1, 2, 3, 4, 5, or 6 2'-fluoronucleotides.

18. The double-stranded RNA according to any one of claims 1 to 17, wherein, The sense strand contains 1, 2, 3, 4 or 5 2'-fluoronucleotides.

19. The double-stranded RNA according to any one of claims 1 to 18, wherein, The sense strand contains 7 to 18 2'-O-Me nucleotides.

20. The double-stranded RNA according to any one of claims 1 to 19, wherein, The antisense strand contains 13 to 20 2'-O-Me nucleotides.

21. The double-stranded RNA according to any one of claims 1 to 20, wherein, The sense strand contains 0 to 6 deoxyribonucleotides.

22. The double-stranded RNA according to any one of claims 1 to 21, wherein, The antisense strand contains 0 to 6 deoxyribonucleotides.

23. The double-stranded RNA according to any one of claims 1 to 22, wherein, The double-stranded RNA contains at least one phosphate thioester bond.

24. The double-stranded RNA according to any one of claims 1 to 23, wherein, The sense chain contains 1, 2, 3 or 4 thiophosphate bonds.

25. The double-stranded RNA according to any one of claims 1 to 24, wherein, The sense strand contains a phosphate thioester bond in at least one of the following groups: between positions 1 and 2, between positions 2 and 3, and between positions 1 and 2, and between positions 2 and 3, counting from the 5' end of the sense strand.

26. The double-stranded RNA according to any one of claims 1 to 25, wherein, The antisense chain contains 1, 2, 3 or 4 thiophosphate bonds.

27. The double-stranded RNA according to any one of claims 1 to 26, wherein, The antisense strand contains a phosphate thioester bond between positions 1 and 2, 2 and 3, and between positions 1 and 2, and between positions 2 and 3, counting from the 5' end of the antisense strand.

28. The double-stranded RNA according to any one of claims 1 to 27, wherein, The antisense chain contains a 5'-phosphonate group at its 5' end.

29. The double-stranded RNA as claimed in claim 28, wherein, The 5'-phosphonate group is a 5'-vinylphosphonate group.

30. The double-stranded RNA as described in claim 28 or 29, wherein, The antisense strand contains a 5'-vinylphosphonate-treated 2'-O-Me nucleotide at position 1, counting from the 5' end.

31. The double-stranded RNA as described in claim 28 or 29, wherein, The antisense strand contains a 5'-vinylphosphonate-treated 2'-O-XCE nucleotide at position 1, counting from the 5' end.

32. The double-stranded RNA as described in claim 28, wherein, The 5'-phosphonate group is a 5'-cyclopropanephosphonate group.

33. The double-stranded RNA as described in claim 28 or 32, wherein, The antisense strand contains a 5'-cyclopropanephosphonate-treated 2'-O-Me nucleotide at position 1, counting from the 5' end.

34. The double-stranded RNA as described in claim 28 or 32, wherein, The antisense strand contains a 5'-cyclopropanephosphonate-treated 2'-O-XCE nucleotide at position 1, counting from the 5' end.

35. The double-stranded RNA as described in claim 28, wherein, The 5'-phosphonate group is a 5'-ethylphosphonate group.

36. The double-stranded RNA as described in claim 28 or 35, wherein, The antisense strand contains a 5'-ethylphosphonate-treated 2'-O-Me nucleotide at position 1, counting from the 5' end.

37. The double-stranded RNA as described in claim 28 or 35, wherein, The antisense strand contains a 5'-ethylphosphonate-treated 2'-O-XCE nucleotide at position 1, counting from the 5' end.

38. The double-stranded RNA according to any one of claims 1 to 37, wherein, The lengths of the sense strand and the antisense strand are independently 19 to 25 nucleotides.

39. The double-stranded RNA according to any one of claims 1 to 38, wherein, The sense strand has 21 nucleotides in length.

40. The double-stranded RNA according to any one of claims 1 to 39, wherein, The antisense strand has a nucleotide length of 23.

41. The double-stranded RNA according to any one of claims 1 to 40, wherein, The sense strand has a length of 21 nucleotides, and the antisense strand has a length of 23 nucleotides.

42. The double-stranded RNA according to any one of claims 1 to 41, wherein the 3' end of the antisense strand includes a protruding end.

43. The double-stranded RNA according to any one of claims 1 to 42, wherein the 5' end of the antisense strand comprises a blunt end.

44. The double-stranded RNA according to any one of claims 1 to 43, further comprising a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target site.

45. The double-stranded RNA as described in claim 44, wherein, The functional molecules are selected from the group consisting of sugars, lipids, peptides, proteins, and their derivatives.

46. ​​The double-stranded RNA as described in claim 44 or 45, wherein, The functional molecules are selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, C5-30 unsaturated fatty acids, C5-30 alkyl groups, and C5-30 alkenyl groups.

47. The double-stranded RNA as described in claim 44 or 45, wherein, The functional molecules are peptides or proteins selected from the group consisting of receptor ligands and antibodies.

48. The double-stranded RNA as described in claim 44 or 45, wherein, The functional molecule is a sugar derivative that can interact with the desialyl glycoprotein receptor.

49. A pharmaceutical composition comprising a pharmacologically permissible support and the double-stranded RNA of any one of claims 1 to 48.

50. A method for regulating the function of a target RNA, comprising the step of contacting the double-stranded RNA of any one of claims 1 to 48 with a cell.

51. A method for regulating the function of target RNA in a mammal, comprising the step of administering the pharmaceutical composition of claim 49 to the mammal.

52. A method for producing double-stranded RNA according to any one of claims 1 to 48 using 2'-O-XCE nucleotides.

53. The compound or its salt represented by the following formula (II): [Chemical Formula 4] In formula (II), Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IIa) represents the group: [Chemical Formula 5] In formula (IIa), R 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T 1 For the group represented by the following formula (IIb): [Chemical Formula 6] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. A 1 The group is selected from the following formula (IIc): [Chemical Formula 7] [Q1 and Q2 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano groups)].

54. The compound or a salt thereof as claimed in claim 53, wherein, X is a methyl group, and Q1 and Q2 are hydrogen atoms.

55. The compound or a salt thereof as described in claim 53 or 54, wherein, The R 1 and R 2 The atom is hydrogen, and Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine, where Q1 and Q2 are hydrogen atoms.

56. The compound or a salt thereof as claimed in any one of claims 53 to 55, wherein, The phosphorus-containing group is cyanoethoxy(diisopropylamino)phosphin or hydroxyphosphin.

57. The compound or its salt represented by the following formula (III): [Chemical Formula 8] In formula (III), Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl, and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl, and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IIIa) represents the group: [Chemical Formula 9] In formula (IIIa), R 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T 1 The group represented by the following formula (IIIb): [Chemical Formula 10] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. B 1 The group represented by the following formula (IIIc): [Chemical Formula 11] [Q3 and Q4 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano groups)].

58. The compound or a salt thereof as claimed in claim 57, wherein, X is a methyl group, and Q3 and Q4 are hydrogen atoms.

59. The compound or a salt thereof as claimed in claim 57 or 58, wherein, The R 1 and R 2 The atom is hydrogen, and Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine, where Q3 and Q4 are hydrogen atoms.

60. The compound or a salt thereof as claimed in any one of claims 57 to 59, wherein, The phosphorus-containing group is cyanoethoxy(diisopropylamino)phosphin or hydroxyphosphin.

61. The compound or its salt represented by the following formula (IV): [Chemical Formula 12] In formula (IV), Base is purine-9-yl, 2-oxo-pyrimidin-1-yl, or 2-thio-pyrimidin-1-yl (each of the purine-9-yl, 2-oxo-pyrimidin-1-yl and 2-thio-pyrimidin-1-yl is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, amino, protected amino, hydroxyl, protected hydroxyl, thioalkyl and protected thioalkyl). Z 3 It consists of hydrogen atoms, hydroxyl protecting groups, or phosphorus-containing groups. X is a hydrogen atom, a C1-6 alkyl or C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkoxy groups, and cyano groups), or The following formula (IVa) represents the group: [Chemical Formula 13] In formula (IVa), R 1 and R 2 Each is independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). Y is NR 3 R 4 (The R) 3 and R 4 Each is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (each of which is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxyl group, a carboxyl group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or C 7-10 aralkyl (the C7-10 aralkyl group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), or, the R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form 3-11 member nitrogen-containing non-aromatic heterocycles (these 3-11 member nitrogen-containing non-aromatic heterocycles are unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino) or C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atom, cyano, nitro, amino, hydroxyl, carboxyl, carbamoyl, C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C2-6 alkenyloxy, C1-6 alkoxycarbonyl, C2-6 alkenyloxycarbonyl, C1-6 alkylcarbonyl, C1-6 haloalkyl, C1-6 alkylamino, C1-6 alkylaminocarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylcarbonylamino and C1-6 alkoxycarbonylamino), When n is an integer from 1 to 3, and n is 2 or 3, there are 2 or 3 R. 1 and R 2 They can be the same or different. T 1 For groups represented by the following formula (IVb): [Chemical Formula 14] [Ra and Rc are each independently selected from hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, protected amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy (the hydroxyl, mercapto, amino, C1-6 alkyl, C2-6 alkenyl or C1-6 alkoxy are each independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano). Rb represents either an oxygen atom or a sulfur atom. E 1 The group represented by the following formula (IVc): [Chemical Formula 15] [Q5 to Q8 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl, a C1-6 alkoxy, a C2-6 alkenyl, a C2-6 alkynyl or an amino group (each of the C1-C6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl and amino groups is independently unsubstituted or substituted by one or more substituents selected individually or differently from the group consisting of halogen atoms, C1-6 alkyl, C1-6 alkoxy and cyano groups)].

62. The compound or a salt thereof as claimed in claim 61, wherein, X is a methyl group, and Q5 to Q8 are hydrogen atoms.

63. The compound or a salt thereof as claimed in claim 61 or 62, wherein, The R 1 and R 2 The atom is hydrogen, and Y is NR. 3 R 4 The R 3 and R 4 Together with the nitrogen atoms they are bonded to, they form morpholine, where Q5 to Q8 are hydrogen atoms.

64. The compound or a salt thereof as claimed in any one of claims 61 to 63, wherein, The phosphorus-containing group is cyanoethoxy(diisopropylamino)phosphin or hydroxyphosphin.

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