Antisense oligonucleotide having reduced toxicity

Abstract

Provided is an antisense oligonucleotide having reduced toxicity.　The antisense oligonucleotide is a gapmer antisense oligonucleotide having a central region, a 5'-side region, and a 3'-side region, and is characterized by having a 2'-4'-crosslinked nucleotide in the 5'-side region and the 3'-side region, and a 2'-O-R-ECE nucleotide in at least one of the 5'-side region and the 3'-side region.

Description

Antisense oligonucleotides with reduced toxicity

[0001] The present invention relates to antisense oligonucleotides with reduced toxicity.

[0002] Nucleic acid drugs are pharmaceuticals consisting of nucleic acids (oligonucleotides) that form complementary base pairs with target DNA or RNA, and are expected to be novel pharmaceuticals. Various artificial nucleic acid units (artificial nucleosides or artificial nucleotides, which are phosphate adducts thereof) have been developed as nucleic acid units for use in nucleic acid drugs. For example, it is known that methoxyethylation (MOE) of the oxygen atom at the 2'-position of the sugar moiety of ribonucleotides improves affinity for target nucleic acids and resistance to nucleases (see, for example, Patent Document 1). Furthermore, 2',4'-BNA and 2',4'-LNA are compounds in which the 2'- and 4'-positions of the sugar moiety of the nucleic acid unit are crosslinked, and are known to have high affinity for target nucleic acids (see, for example, Patent Documents 2 to 5). Furthermore, methylcarbamoylethyl (MCE)-modified nucleotides have been reported as modified forms of the oxygen atom at the 2'-position of the sugar moiety of ribonucleotides (see, for example, Patent Document 6 and Non-Patent Document 1). Furthermore, it has been reported that high nuclease resistance can be obtained by introducing an amino group or a heterocyclic group via an alkyl group into the nitrogen atom of a carbamoylethyl group (R-ECE-modified nucleotide) (see, for example, Patent Document 7 and Non-Patent Document 2).

[0003] Progress has been made in the development of gapmer-type antisense nucleic acids, in which artificial nucleic acid units are introduced into both ends (5' and 3' regions) of a single-stranded oligodeoxyribonucleotide. It is known that gapmer-type antisense nucleic acids form a double-stranded complex with a target RNA, and intracellular RNase H recognizes the double-stranded portion between the deoxyribonucleotide moiety and the target RNA and cleaves the RNA strand. In particular, gapmer-type antisense nucleic acids with 2'-4' bridged nucleotides, such as 2',4'-BNA and 2',4'-LNA, introduced into both ends are known to have potent target RNA knockdown effects. However, recent reports have shown that gapmer-type antisense nucleic acids with 2'-4' bridged nucleotides introduced into both ends can induce cytotoxicity due to their action on non-target RNA or non-specific protein binding within cells (see, for example, Non-Patent Document 3).

[0004] Japanese Patent Application Laid-Open No. 7-2889 WO 98 / 39352 WO 2009 / 006478 WO 2011 / 052436 WO 2015 / 125783 WO 2007 / 102581 WO 2017 / 142054

[0005] The Journal of Organic Chemistry, 2011, 76, pp 3042-3053Organic & Biomolecular Chemistry, 2019, 17, pp 4835-4842Nature biotechnology, 2019, 37, pp 640-650

[0006] There is a need for a new technique for reducing the toxicity of gapmer-type antisense nucleic acids in which 2'-4' bridged nucleotides are introduced at both ends while maintaining their activity.

[0007] An object of the present invention is to provide antisense oligonucleotides with reduced toxicity.

[0008] The present inventors have discovered that a gapmer-type antisense nucleic acid having, in the central region and / or the 5'-side region and / or the 3'-side region, a nucleotide in which an amino group or a heterocyclic group has been introduced via an alkyl group to the nitrogen atom of a 2'-position modified carbamoylethyl group (2'-O-R-ECE), can reduce toxicity, and have completed the present invention. That is, the present invention encompasses the following aspects.

[0009] 1. An antisense oligonucleotide having a central region, a 5' region, and a 3' region, wherein: - the central region consists of at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and the 3' and 5' ends thereof are each independently selected from deoxyribonucleotides and 5'-modified nucleotides, and the central region includes at least one oligonucleotide strand consisting of at least four consecutive nucleotides selected from deoxyribonucleotides and 5'-modified nucleotides; - the 5' region consists of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and includes at least one 2'-4' bridged nucleotide, and the 3' end thereof is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the 3' end nucleotide is linked to the central region, and does not include an oligonucleotide strand consisting of at least four consecutive nucleotides selected from deoxyribonucleotides and 5'-modified nucleotides; - the 3' region An antisense oligonucleotide consisting of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, containing at least one 2'-4' bridged nucleotide, the 5' end of which is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the 5' end nucleotide is linked to a central region, and does not contain an oligonucleotide chain consisting of at least four consecutive nucleotides selected from deoxyribonucleotides and 5'-modified nucleotides; and containing at least one 2'-O-R-ECE nucleotide in at least one of the central region, the 5' side region, and the 3' side region.

[0010] 2. The 2'-O-R-ECE nucleotide is represented by the following formula (I): (wherein Bx is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group), and R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ５ R ６ (The R ５ and R ６each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy 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 a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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 the R ５ and R ６are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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 a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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), and n is an integer from 1 to 3, provided that when n is 2 or 3, then 2 or 3 R ３ and R ４ 1. The antisense oligonucleotide according to 1., wherein the nucleotide contains a partial structure represented by the following formula:

[0011] 3. R １ , R ２ , R ３ and R ４ 3. The antisense oligonucleotide according to 2., wherein is a hydrogen atom.

[0012] 4. Y is NR ５ R ６ and the R５ and R ６ are taken together with the nitrogen atom to which they are attached to form a 4-8 membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene groups, or Y is a C2-9 aromatic heterocyclic group.

[0013] 5. Y is NR ５ R ６ and the R ５ and R ６ and , taken together with the nitrogen atom to which they are attached, form a morpholine.

[0014] 6. The antisense oligonucleotide according to any one of 2. to 4., wherein Y is a pyridyl group, an imidazolyl group, or a benzimidazolyl group.

[0015] 7. The antisense oligonucleotide according to any one of 2. to 6., wherein n is 2.

[0016] 8. The antisense oligonucleotide according to any one of 1. to 7., wherein the central region consists of 5 to 15 nucleotides, and the 5'-side region and the 3'-side region each independently consist of 2 to 7 nucleotides.

[0017] 9. The antisense oligonucleotide according to any one of 1. to 8., wherein the central region consists of 8 to 12 nucleotides, and the 5' region and the 3' region each independently consist of 2 to 5 nucleotides.

[0018] 10. The 2'-4' bridged nucleotide is LNA, cEt-BNA, ENA, BNA ＮＣ 10. The antisense oligonucleotide according to any one of 1. to 9., which is at least one selected from the group consisting of AmNA, scpBNA and GuNA.

[0019] 11. The antisense oligonucleotide according to any one of 1. to 9., wherein the 2'-modified nucleotide is at least one selected from the group consisting of a 2'-O-R-ECE nucleotide, a 2'-O-MCE nucleotide, a 2'-O-MOE nucleotide, a 2'-O-NMA nucleotide, and a 2'-O-Me nucleotide.

[0020] 12. The antisense oligonucleotide according to any one of 1. to 11., wherein the 5'-modified nucleotide is a 5'-CP nucleotide.

[0021] 13. The antisense oligonucleotide of any one of 1. to 12., wherein the antisense oligonucleotide comprises phosphorothioate linkages.

[0022] 14. The antisense oligonucleotide according to any one of 1. to 13., wherein each nucleotide constituting the antisense oligonucleotide is independently linked by a phosphodiester bond or a phosphorothioate bond.

[0023] 15. The antisense oligonucleotide according to any one of 1 to 14, 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.

[0024] 16. The antisense oligonucleotide according to 15., wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof.

[0025] 17. The antisense oligonucleotide according to 15. or 16., wherein the functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol, and tocotrienol.

[0026] 18. The antisense oligonucleotide according to 15. or 16., wherein the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor.

[0027] 19. The antisense oligonucleotide according to 15. or 16., wherein the functional molecule is a peptide or protein selected from the group consisting of receptor ligands and antibodies.

[0028] 20. A prodrug of the antisense oligonucleotide according to any one of 1. to 19.

[0029] 21. A double-stranded oligonucleotide complex comprising: (i) the antisense oligonucleotide according to any one of 1. to 19.; and (ii) an oligonucleotide comprising a region that hybridizes to the antisense oligonucleotide.

[0030] 22. An oligonucleotide comprising: (i) a group derived from the antisense oligonucleotide according to any one of 1. to 19.; and (ii) a group derived from an oligonucleotide comprising a region that hybridizes to the antisense oligonucleotide, wherein the group derived from the antisense oligonucleotide (i) and the group derived from the oligonucleotide (ii) are linked together.

[0031] 23. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22., and a pharmacologically acceptable carrier.

[0032] 24. A method for regulating the function of a target RNA, comprising the step of contacting a cell with the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22.

[0033] 25. A method for regulating the function of a target RNA in a mammal, comprising the step of administering to the mammal the pharmaceutical composition according to 23.

[0034] 26. A method for controlling expression of a target gene, comprising the step of contacting a cell with the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22.

[0035] 27. A method for regulating expression of a target gene in a mammal, comprising the step of administering to the mammal the pharmaceutical composition according to 23.

[0036] 28. A method for producing the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22., using a 2'-O-R-ECE nucleotide.

[0037] The present invention provides antisense oligonucleotides with reduced toxicity.

[0038] The terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Each term used in this specification is explained below. In this specification, each term has the same meaning whether used alone or together with other terms, unless otherwise specified.

[0039] "n-" means normal, "i-" means iso, "s-" means secondary, "t-" means tertiary, "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 dimethoxytrityl.

[0040] "Halogen atom" means a fluorine atom, chlorine atom, bromine atom or iodine atom.

[0041] The term "C1-6 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms. Examples of the C1-6 alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl. Similarly, the term "C1-3 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 1 to 3 carbon atoms.

[0042] The term "halo C1-6 alkyl group" refers to the above "C1-6 alkyl group" in which at least one hydrogen atom at any position is substituted with the above "halogen atom".

[0043] The term "C2-6 alkenyl group" refers to a monovalent linear or branched unsaturated aliphatic hydrocarbon group 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.

[0044] The term "C1-6 alkoxy group" refers to a group in which the above-mentioned "C1-6 alkyl group" is bonded to an oxy (-O-) group. Examples of C1-6 alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, isobutoxy, s-butoxy, n-pentyloxy, isopentyloxy, and n-hexyloxy.

[0045] The term "C2-6 alkenyloxy group" refers to a group in which the above "C2-6 alkenyl group" is bonded to an oxy (-O-) group. Examples of the C2-6 alkenyloxy group include a vinyloxy group, an allyloxy group, a propenyloxy group, an isopropenyloxy group, a butenyloxy group, an isobutenyloxy group, a butadienyloxy group, a 3-methyl-2-butenyloxy group, a pentenyloxy group, an isopentenyloxy group, a pentadienyloxy group, a hexenyloxy group, an isohexenyloxy group, and a hexadienyloxy group.

[0046] The term "C1-6 alkylcarbonyl group" refers to a group in which the above-mentioned "C1-6 alkyl group" is bonded to a carbonyl (-C(=O)-) group.

[0047] The term "C1-6 alkoxycarbonyl group" refers to a group in which the "C1-6 alkoxy group" is bonded to a carbonyl (-C(=O)-) group, and the term "C2-6 alkenyloxycarbonyl group" refers to a group in which the "C2-6 alkenyloxy group" is bonded to a carbonyl (-C(=O)-) group.

[0048] The term "C2-20 alkylene group" refers to a divalent group of a linear or branched saturated aliphatic hydrocarbon having from 2 to 20 carbon atoms. The term "C8-12 alkylene group" refers to a divalent group of a linear or branched saturated aliphatic hydrocarbon having from 8 to 12 carbon atoms, among the aforementioned "C2-20 alkylene groups". The term "C2-6 alkylene group" refers to a divalent group of a linear or branched saturated aliphatic hydrocarbon having from 2 to 6 carbon atoms, among the aforementioned "C2-20 alkylene groups", and examples thereof include an ethylene (ethanediyl) group, a propylene group, a propane-1,3-diyl (trimethylene) group, a propane-2,2-diyl (isopropylidene) group, a 2,2-dimethyl-propane-1,3-diyl group, a hexane-1,6-diyl (hexamethylene) group, and a 3-methylbutane-1,2-diyl group.

[0049] The term "C2-20 alkenylene group" refers to a divalent linear or branched unsaturated aliphatic hydrocarbon group containing 2 to 20 carbon atoms and at least one carbon-carbon double bond.

[0050] The term "C1-6 alkylamino group" includes mono-C1-6 alkylamino groups and di-C1-6 alkylamino groups. The term "mono-C1-6 alkylamino group" refers to a group in which one of the above-mentioned "C1-6 alkyl groups" is bonded to an amino group. Examples of mono-C1-6 alkylamino groups include methylamino group, ethylamino group, n-propylamino group, isopropylamino group, n-butylamino group, n-pentylamino group, and n-hexylamino group. The term "di-C1-6 alkylamino group" refers to a group in which two of the above-mentioned "C1-6 alkyl groups" are bonded to an amino group. The two alkyl groups may be the same or different. Examples of the di-C alkylamino group include a dimethylamino group, a diethylamino group, an N,N-diisopropylamino group, an N-methyl-N-ethylamino group, an N-isopropyl-N-methylamino group, an N-n-butyl-N-methylamino group, an N-tert-butyl-N-methylamino group, an N-methyl-N-pentylamino group, an N-n-hexyl-N-methylamino group, and an N-isopropyl-N-ethylamino group.

[0051] The term "C1-6 alkylaminocarbonyl group" means a group in which the above "C1-6 alkylamino group" is bonded to a carbonyl group.

[0052] The term "C1-6 alkylcarbonyloxy group" means a group in which the above "C1-6 alkylcarbonyl group" is bonded to an oxy group.

[0053] The term "C1-6 alkylcarbonylamino group" means a group in which one "C1-6 alkylcarbonyl group" as defined above is bound to an amino group.

[0054] The term "C1-6 alkoxycarbonylamino group" means a group in which one "C1-6 alkoxycarbonyl group" as defined above is bonded to an amino group.

[0055] The term "aryl group" refers to a monovalent group formed by removing one hydrogen atom at any position from a monocyclic or bicyclic aromatic hydrocarbon ring in which all atoms constituting the ring are carbon atoms, and specific examples include "C aryl groups" such as a phenyl group and a naphthyl group.

[0056] The term "aralkyl group" refers to a monovalent group in which a hydrogen atom at any position of the "C1-6 alkyl group" is replaced by the "C6-10 aryl group".

[0057] The term "3- to 11-membered nitrogen-containing non-aromatic heterocycle" refers to a monocyclic, fused polycyclic (in such a fused polycyclic, a non-aromatic ring may be fused to a non-aromatic ring or an aromatic ring), bridged ring, or spirocyclic non-aromatic heterocyclic compound containing at least one nitrogen atom and having 3 to 11 atoms constituting the ring, and examples thereof include azetidine, pyrrolidine, 2-oxopyrrolidine, piperidine, 3-oxopiperidine, piperazine, morpholine, thiomorpholine, homomorpholine, and homopiperazine.

[0058] The term "C2-9 aromatic heterocyclic group" refers to a monovalent group formed by removing one hydrogen atom at any position from an aromatic monocyclic or fused ring compound having 2 to 9 carbon atoms in the ring and one or more identical or different heteroatoms selected from oxygen atoms, sulfur atoms, and nitrogen atoms in the ring. Examples of C2-9 aromatic heterocyclic groups include purinyl, pyrimidinyl, thienyl, furyl, isobenzofuranyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, pteridinyl, benzofuranyl, benzothienyl, and benzimidazolyl groups.

[0059] The term "5- to 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 having 5 to 10 atoms constituting the ring and containing 1 to 5 heteroatoms (the heteroatoms refer to nitrogen atoms, oxygen atoms, or sulfur atoms, and when there are two or more heteroatoms, they may be the same or different) among the atoms constituting the ring.

[0060] Examples of the monocyclic "5- to 10-membered heterocyclic group" include a 2-thienyl group, a 3-thienyl group, a 2-furyl group, a 3-furyl group, a 2-pyranyl group, a 3-pyranyl group, a 4-pyranyl group, a 1-pyrrolyl group, a 2-pyrrolyl group, a 3-pyrrolyl group, a 1-imidazolyl group, a 2-imidazolyl group, a 4-imidazolyl group, a 1-pyrazolyl group, a 3-pyrazolyl group, a 4-pyrazolyl group, a 5-pyrazolyl group, 2-thiazolyl group, 4-thiazolyl group, 5-thiazolyl group, 3-isothiazolyl group, 4-isothiazolyl group, 5-isothiazolyl group, 1,2,4-triazol-1-yl group, 1,2,4-triazol-3-yl group, 1,2,4-triazol-5-yl group, 1,2,3-triazol-1-yl group, 1,2,3-triazol-4-yl group, 1,2,3-triazolyl group Examples thereof include an oxazol-5-yl group, a 2-oxazolyl group, a 4-oxazolyl group, a 5-oxazolyl group, a 3-isoxazolyl group, a 4-isoxazolyl group, a 5-isoxazolyl group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-pyrazinyl group, a 2-pyrimidinyl group, a 4-pyrimidinyl group, a 5-pyrimidinyl group, a 3-pyridazinyl group, a 4-pyridazinyl group, a 1,3,4-oxadiazol-2-yl group, a 1,3,4-thiadiazol-2-yl group, a 1,2,4-oxadiazol-3-yl group, a 1,2,4-oxadiazol-5-yl group, a 1,2,4-thiadiazol-3-yl group, a 1,2,4-thiadiazol-5-yl group, a 1,2,5-oxadiazol-3-yl group, and a 1,2,5-thiadiazol-3-yl group.

[0061] Examples of the fused polycyclic "5- to 10-membered heterocyclic group" include a 2-benzofuranyl group, a 3-benzofuranyl group, a 4-benzofuranyl group, a 5-benzofuranyl group, a 6-benzofuranyl group, a 7-benzofuranyl group, a 1-isobenzofuranyl group, a 4-isobenzofuranyl group, a 5-isobenzofuranyl group, a 2-benzothienyl group, a 3-benzothienyl group, a 4-benzothienyl group, a 5-benzothienyl group, a 6-benzothienyl group, a 7-benzothienyl group, a 1-isobenzothienyl group, a 4-isobenzothienyl group, a 5-isobenzothienyl group, a 2-benzothiazolyl group, a 4- ... azothiazolyl group, a 5-benzothiazolyl group, a 6-benzothiazolyl group, a 7-benzothiazolyl group, a 2-chromenyl group, a 3-chromenyl group, a 4-chromenyl group, a 5-chromenyl group, a 6-chromenyl group, a 7-chromenyl group, an 8-chromenyl group, a 1-indolizinyl group, a 2-indolizinyl group, a 3-indolizinyl group, a 5-indolizinyl group, a 6-indolizinyl group, a 7-indolizinyl group, an 8-indolizinyl group, a 1-isoindolyl group, a 2-isoindolyl group, a 4-isoindolyl group, a 5-isoindolyl group, a 1-indolyl group, a 2-indolyl group, a 3- Indolyl group, 4-indolyl group, 5-indolyl group, 6-indolyl group, 7-indolyl group, 1-indazolyl group, 2-indazolyl group, 4-indazolyl group, 5-indazolyl group, 6-indazolyl group, 7-indazolyl group, 2-purinyl group, 6-purinyl group, 7-purinyl group, 8-purinyl group, 2-quinolyl group, 3-quinolyl group, 4-quinolyl group, 5-quinolyl group, 6-quinolyl group, 7-quinolyl group, 8-quinolyl group, 1-isoquinolyl group, 3-isoquinolyl group, 4-isoquinolyl group, 5-isoquinolyl group, 6-isoquinolyl group, 7-isoquinolyl group a quinolyl group, an 8-isoquinolyl group, a 1-phthalazinyl group, a 5-phthalazinyl group, a 6-phthalazinyl group, a 2,7-naphthyridin-1-yl group, a 2,7-naphthyridin-3-yl group, a 2,7-naphthyridin-4-yl group, a 2,6-naphthyridin-1-yl group, a 2,6-naphthyridin-3-yl group, a 2,6-naphthyridin-4-yl group, a 1,8-naphthyridin-2-yl group, a 1,8-naphthyridin-3-yl group, a 1,8-naphthyridin-4-yl group, a 1,7-naphthyridin-2-yl group, a 1,7-naphthyridin-3-yl group, a 1,7-naphthyridin-4-yl group,7-naphthyridin-5-yl group, 1,7-naphthyridin-6-yl group, 1,7-naphthyridin-8-yl group, 1,6-naphthyridin-2-yl group, 1,6-naphthyridin-3-yl group, 1,6-naphthyridin-4-yl group, 1,6-naphthyridin-5-yl group, 1,6-naphthyridin-7-ynyl group, 1,6-naphthyridin-8-yl group, 1,5-naphthyridin-2-yl group, 1,5-naphthyridin-3-yl group, 1,5-naphthyridin-4-yl group , 2-quinoxalinyl group, 5-quinoxalinyl group, 6-quinoxalinyl group, 2-quinazolinyl group, 4-quinazolinyl group, 5-quinazolinyl group, 6-quinazolinyl group, 7-quinazolinyl group, 8-quinazolinyl group, 3-cinnolinyl group, 4-cinnolinyl group, 5-cinnolinyl group, 6-cinnolinyl group, 7-cinnolinyl group, 8-cinnolinyl group, 2-pteridinyl group, 4-pteridinyl group, 6-pteridinyl group and 7-pteridinyl group.

[0062] "Oxo" refers to a group (=O) in which an oxygen atom is substituted via a double bond. When oxo is substituted on a carbon atom, it combines with said carbon atom to form a carbonyl. "Thioxo" refers to a group (=S) in which a sulfur atom is substituted via a double bond. When thioxo is substituted on a carbon atom, it combines with said carbon atom to form a thiocarbonyl.

[0063] The terms "protected hydroxy group," "protected amino group," and "protected sulfanyl group" refer to a hydroxy group protected with a hydroxy-protecting group, an amino group protected with an amino-protecting group, and a sulfanyl group protected with a sulfanyl-protecting group, respectively. The hydroxy-protecting group, amino-protecting group, and sulfanyl-protecting group are not particularly limited as long as they are stable during the synthesis of antisense oligonucleotides, and examples thereof include protecting groups well known to those skilled in the art, such as those described in Protective Groups in Organic Synthesis, 4th Edition, by T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (2006).

[0064] For example, examples of the "hydroxy-protecting group" include ether-based protecting groups such as C1-6 alkyl (e.g., methyl, t-butyl, etc.), triarylmethyl (e.g., triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, etc.); acetal-based protecting groups such as methoxymethyl, methylthiomethyl, methoxyethyl, benzyloxymethyl, 2-tetrahydropyranyl, ethoxyethyl, etc.; acyl-based protecting groups such as acyl (e.g., formyl, acetyl, pivaloyl, benzoyl, etc.); tri(C1-6 alkyl)silyl (e.g., trimethylsilyl, triethylsilyl, etc.); , triisopropylsilyl, t-butyldimethylsilyl, dimethylisopropylsilyl, etc.), (C1-6 alkyl)diarylsilyl (for example, t-butyldiphenylsilyl, diphenylmethylsilyl, etc.), triarylsilyl (for example, triphenylsilyl, etc.), tribenzylsilyl, [(triisopropylsilyl)oxy]methyl (Tom group) and other silyl protecting groups; 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep group), 9-phenylxanthen-9-yl (Pixyl group), 9-(p-methoxyphenyl)xanthen-9-yl (MOX group), etc.

[0065] For example, the "amino-protecting group" may be an amide-based protecting group such as acyl (for example, formyl, acetyl, propionyl, pivaloyl (Pv), tigloyl, etc.), haloacyl (for example, fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, etc.), arylcarbonyl (for example, benzoyl, p-bromobenzoyl, p-nitrobenzoyl, 2,4-dinitrobenzoyl, etc.); C alkoxycarbonyl (for example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, i-propoxycarbonyl, n-butoxycarbonyl, i-butoxycarbonyl, t-butoxycarbonyl (Boc), t- amyloxycarbonyl, and the like, preferably Boc, and the like), C2-6 alkenyloxycarbonyl (for example, vinyloxycarbonyl (Voc), allyloxycarbonyl (Alloc), and the like), tri(C1-3 alkyl)silylethoxycarbonyl (for example, 2-(trimethylsilyl)ethoxycarbonyl (Teoc), and the like), haloC1-6 alkoxycarbonyl (for example, 2,2,2-trichloroethoxycarbonyl (Troc), and the like), aryloxycarbonyl (for example, benzyloxycarbonyl (Z or Cbz), p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl (Moz), and the like); carbamate-based protecting groups such as C2-6 alkenyloxycarbonyl (for example, vinyloxycarbonyl (Voc), allyloxycarbonyl (Alloc), and the like), tri(C1-3 alkyl)silylethoxycarbonyl (for example, 2-(trimethylsilyl)ethoxycarbonyl (Teoc)), haloC1-6 alkoxycarbonyl (for example, 2,2,2-trichloroethoxycarbonyl (Troc)), and aryloxycarbonyl (for example, benzyloxycarbonyl (Z or Cbz), p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl (Moz), and the like);and sulfonamide-based protecting groups such as alkylsulfonyl (for example, methanesulfonyl (Ms), ethanesulfonyl, etc.), arylsulfonyl (for example, benzenesulfonyl, p-toluenesulfonyl (Ts), p-chlorobenzenesulfonyl, p-methoxybenzenesulfonyl (MBS), m-nitrobenzenesulfonyl, o-nitrobenzenesulfonyl, 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.);

[0066] For example, examples of the "sulfanyl protecting group" include ether-based protecting groups such as benzyl, p-methoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 9-fluorenylmethyl, di- or triarylmethyl (for example, diphenylmethyl, triphenylmethyl (trityl), bis(4-methoxyphenyl)methyl, etc.); thioacetal-based protecting groups such as methoxymethyl, isobutoxymethyl, benzylthiomethyl, 2-tetrahydropyranyl, etc.; and thioester-based protecting groups such as acyl (for example, acetyl, benzoyl, etc.).

[0067] For the protection and deprotection of the "hydroxy-protecting group," "amino-protecting group," and "sulfanyl-protecting group" in the present invention, reference can also be made to Protective Groups in Organic Synthesis, 4th Edition, T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (2006), etc.

[0068] The "antisense effect" means that the function of a target RNA is controlled by hybridizing a target RNA selected corresponding to a target gene with, for example, an oligonucleotide having a sequence complementary to a partial sequence of the target RNA. For example, when the target RNA is mRNA, this means that hybridization inhibits translation of the target RNA, has a splicing function conversion effect such as exon skipping, or degrades the target RNA by recognizing the hybridized portion.

[0069] An "antisense oligonucleotide" is an oligonucleotide that produces the antisense effect. Examples include, but are not limited to, DNA and oligodeoxyribonucleotides, and may also include RNA, oligoribonucleotides, or oligonucleotides designed to normally produce an antisense effect. The same applies to antisense nucleic acids.

[0070] "Target RNA" refers to mRNA, mRNA precursor, or ncRNA, and includes mRNA transcribed from genomic DNA encoding the target gene, mRNA without base modifications, unspliced ​​mRNA precursor, and ncRNA. The "target RNA" whose function is controlled by the antisense effect is not particularly limited, and examples include RNA associated with genes whose expression is increased in various diseases. "Target RNA" may be any RNA synthesized by DNA-dependent RNA polymerase, and is preferably mRNA or mRNA precursor. More preferably, it is mammalian mRNA or mRNA precursor, even more preferably human mRNA or mRNA precursor, and particularly preferably human mRNA.

[0071] "Hybridize" refers to the act of forming a double strand between oligonucleotides or groups derived from oligonucleotides that contain complementary sequences, and the phenomenon in which oligonucleotides or groups derived from oligonucleotides that contain complementary sequences form a double strand.

[0072] "Complementary" means that two nucleobases can form Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (such as Hoogsteen base pairs) through hydrogen bonds. Two oligonucleotides or oligonucleotide-derived groups can "hybridize" if their sequences are complementary. Two oligonucleotides or oligonucleotide-derived groups do not need to be completely complementary to hybridize, but the degree of complementarity for two oligonucleotides or oligonucleotide-derived groups to hybridize is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more). Sequence complementarity can be determined using a computer program that automatically identifies subsequences of oligonucleotides. For example, OligoAnalyzer is one such software program provided by Integrated DNA Technologies. This program is also available on the website. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two oligonucleotides or oligonucleotide-derived groups can hybridize. Furthermore, those skilled in the art can easily design antisense oligonucleotides complementary to a target RNA, for example, by using a BLAST program based on information about the nucleotide sequence of the target RNA. For details of the BLAST program, 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.

[0073] "Nucleotide" refers to a molecule that can be a building block of a nucleic acid (oligonucleotide), and typically has a base as a constituent element. A nucleotide is composed of, for example, a sugar, a base, and a phosphate. Nucleotides include ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, which are described below.

[0074] "Oligonucleotide" refers to a molecule having a structure in which one or more of the above-mentioned nucleotides are polymerized. When an "oligonucleotide" is composed of one nucleotide, the oligonucleotide can be referred to as a "nucleotide." The nucleotides contained in the "antisense oligonucleotide" molecule of the present invention are each independently linked to each other by a phosphodiester bond, a modified phosphodiester bond described below, or a linking group containing a non-nucleotide structure described below. The 3'-terminal nucleotide of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group, more preferably a hydroxy group, and usually a hydroxy group, at its 3'-position. The 5'-terminal nucleotide of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group, more preferably a hydroxy group, and usually a hydroxy group, at its 5'-position.

[0075] The term "group derived from an oligonucleotide" refers to a group obtained by removing a hydrogen atom, a hydroxy group, or the like from a hydroxy group at at least one of the 3'- and 5'-ends of the oligonucleotide, and is indirectly covalently linked to another group (e.g., a group derived from another oligonucleotide) by forming a phosphodiester bond or a modified phosphodiester bond. The hydroxy group at the 3'- or 5'-end includes a hydroxy group contained in a phosphate group. For example, a group obtained by removing a hydrogen atom from the hydroxy group at the 3'-end of an oligonucleotide and a group obtained by removing a hydroxy group from the phosphate group at the 5'-end of another oligonucleotide form a phosphodiester bond or a modified phosphodiester bond.

[0076] "Nucleotide sequence" means the base sequence of the nucleotides that make up an oligonucleotide.

[0077] As used herein, the term "sequence portion" refers to a partial structure of an oligonucleotide chain. For example, a sequence portion containing a nucleotide is a partial structure of a region of an oligonucleotide chain that contains the nucleotide.

[0078] "Deoxyribonucleotide" refers to a molecule in which the sugar is 2-deoxyribose, a base is bound to the carbon atom at position 1 of the 2-deoxyribose, and a phosphate group is located at position 3 or 5. The deoxyribonucleotide of the present invention may be a naturally occurring deoxyribonucleotide, or a naturally occurring deoxyribonucleotide in which the base moiety or the phosphodiester bond moiety has been modified. A single deoxyribonucleotide may be modified in combination with multiple types of modifications to the base moiety or the phosphodiester bond moiety. The modified deoxyribonucleotides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; and Future Medicinal Chemistry, 2011, 3, pp. 339-365.

[0079] When the "deoxyribonucleotide" constitutes the antisense oligonucleotide molecule of the present invention, the 3' position of the deoxyribonucleotide is usually linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the deoxyribonucleotide is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The deoxyribonucleotide at the 3' end of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at its 3' position, and the 5' position is as described above. The deoxyribonucleotide at the 5' end of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group at its 5' position, and the 3' position is as described above.

[0080] The term "oligodeoxyribonucleotide" refers to an oligonucleotide composed of the above-mentioned deoxyribonucleotides. The deoxyribonucleotides constituting the oligodeoxyribonucleotide may be the same or different.

[0081] "DNA" refers to an oligonucleotide composed of natural deoxyribonucleotides. The natural deoxyribonucleotides that make up DNA may be identical or different.

[0082] "Ribonucleotide" refers to a molecule in which the sugar is ribose, a base is bound to the carbon atom at position 1 of the ribose, and a phosphate group is located at position 2, 3, or 5. The ribonucleotide of the present invention may be a naturally occurring ribonucleotide, or a naturally occurring ribonucleotide in which the base moiety or the phosphodiester bond moiety has been modified. A single ribonucleotide may be modified in combination with multiple types of modifications to the base moiety or the phosphodiester bond moiety. The modified ribonucleotides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; and Future Medicinal Chemistry, 2011, 3, pp. 339-365.

[0083] When the "ribonucleotide" constitutes the antisense oligonucleotide molecule of the present invention, the 3' position of the ribonucleotide is typically linked to another nucleotide via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the ribonucleotide is linked to another nucleotide, etc. via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The 3'-terminal ribonucleotide of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at its 3' position, and the 5'-position is as described above. The 5'-terminal ribonucleotide of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group at its 5' position, and the 3'-position is as described above.

[0084] The term "oligoribonucleotide" refers to an oligonucleotide composed of the above-mentioned ribonucleotides. The ribonucleotides constituting the oligoribonucleotide may be the same or different.

[0085] "RNA" refers to an oligonucleotide composed of natural ribonucleotides. The natural ribonucleotides that make up the RNA may be identical or different.

[0086] "Modified sugar" means: (Z1) a molecule in which ribose or 2-deoxyribose is partially replaced with one or more substituents; (Z2) a penta- or hexa-saccharide other than ribose and 2-deoxyribose (e.g., hexitol, threose, etc.); (Z3) a molecule in which the entire ribose or 2-deoxyribose, or the tetrahydrofuran ring thereof, is replaced with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.) or a partial structure capable of forming a 5- to 7-membered ring by hydrogen bonding (e.g., a peptide structure); or (Z4) a molecule in which ribose or 2-deoxyribose is replaced with a C2-6 alkylene glycol (e.g., ethylene glycol, propylene glycol, etc.). Modified sugars include "2-modified sugars," "2-4-linked sugars," and "5-modified sugars," which are described below. Examples of modified sugars and sugar-modified nucleotides described below include sugars and sugar-modified nucleotides disclosed as being suitable for use in antisense methods in JP-A-10-304889, WO 2005 / 021570, JP-A-10-195098, JP-T-2002-521310, WO 2007 / 143315, WO 2008 / 043753, WO 2008 / 029619, WO 2008 / 049085, and WO 2017 / 142054 (hereinafter, these documents are referred to as "documents related to antisense methods"), etc. Modified sugars and sugar-modified nucleotides are also 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 WO 2018 / 155450.

[0087] Examples of modified sugars that are partially substituted with one substituent include ribose or 2-deoxyribose substituted at any position of the sugar moiety with one or more (preferably one or two) of the following substituents (i) or (ii): (i) a C1-6 alkyl group. When substitutions with two or more C1-6 alkyl groups are contained, the two or more C1-6 alkyl groups may be joined together to form a 3- to 6-membered ring. (ii) a C1-6 alkyl group substituted with at least one selected from the group consisting of a halogen atom, a C1-6 alkoxy group, a haloC1-6 alkoxy group, a mono- or di-C1-6 alkylamino group, a 5- to 10-membered heterocyclic group, a carboxy group, a carbamoyl group, and an N-substituted carbamoyl group. Here, examples of the N-substituted carbamoyl group include an N-methyl-carbamoyl group and an N-ethyl-carbamoyl group, and the methyl group and the ethyl group of the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group are 5- to 10-membered heterocyclic groups or mono- or di-C １－６ It may be substituted with an alkylamino group. Specific examples of the N-substituted carbamoyl group include an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N-dimethylaminoethyl-carbamoyl group, an N-(morpholinoethyl)carbamoyl group, an N-(2-pyridylethyl)carbamoyl group, and an N-((benzimidazol-1-yl)ethyl)carbamoyl group.

[0088] A "sugar-modified nucleotide" refers to a molecule having the above-mentioned "modified sugar" in place of the sugar moiety of a deoxyribonucleotide or ribonucleotide. For example, it includes the "2'-modified nucleotide" and "2'-4' bridged nucleotide" described below, as well as a "5'-modified nucleotide." When the modified sugar is (Z3) as defined above, the sugar-modified nucleotide also includes a molecule in which the modified sugar and the nucleobase are linked via a methylene chain or the like.

[0089] "Dimodified sugar" means a non-bridged sugar in which the oxygen atom or carbon atom at position 2 of ribose is modified, and includes "2-O-Me," "2-O-MOE," "2-O-MCE," "2-O-NMA," "2-O-AP," "2-F," "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE." Positions 4 and 5 of a "dimodified sugar" are preferably unmodified. A "2'-modified nucleotide" refers to a molecule having a base bound to the carbon atom at position 1 (position 1 of the ribose before modification) of the "dimodified sugar" and a phosphate group at position 3 or 5, and examples thereof 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'-DMAECE nucleotide," "2'-MorECE nucleotide," "2'-PyECE nucleotide," and "2'-BimECE nucleotide."

[0090] "2-O-Me" (also called 2-O-methyl) refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a methoxy group. "2'-O-Me nucleotide" (also called 2'-O-methyl nucleotide) refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-Me" (position 1 of unmodified ribose) and which has a phosphate group at position 3 or 5.

[0091] "2-O-MOE" (also referred to as 2-O-methoxyethyl) refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a 2-methoxyethyloxy group. "2'-O-MOE nucleotide" (also referred to as 2'-O-methoxyethyl nucleotide) refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-MOE" (position 1 of the ribose before modification) and which has a phosphate group at position 3 or 5.

[0092] "2-O-MCE" (also called 2-O-methylcarbamoylethyl) refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a methylcarbamoylethyloxy group. "2'-O-MCE nucleotide" (also called 2'-O-methylcarbamoylethyl nucleotide) refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-MCE" (position 1 of the ribose before modification) and which has a phosphate group at position 3 or 5.

[0093] "2-O-NMA" refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a [2-(methylamino)-2-oxoethyl]oxy group. "2'-O-NMA nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-NMA" (position 1 of the unmodified ribose) and which has a phosphate group at position 3 or 5.

[0094] "2-O-AP" refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a 3-aminopropyloxy group. "2'-O-AP nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-AP" (position 1 of the unmodified ribose) and which has a phosphate group at position 3 or 5.

[0095] "2-F" refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a fluorine atom. "2'-F nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-F" (position 1 of unmodified ribose) and a phosphate group is at position 3 or 5.

[0096] "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE" are sugars in which the hydroxy group at position 2 of ribose has been replaced with the structures shown below as DMAECE, MorECE, PyECE, and BimECE, respectively. In the structures below, the wavy line indicates the bond position with the carbon atom to which the hydroxy group at position 2 of ribose is bonded.

[0097] "2'-DMAECE nucleotide," "2'-MorECE nucleotide," "2'-PyECE nucleotide," and "2'-BimECE nucleotide" refer to molecules in which a base is bound to the carbon atom at position 1 (position 1 of unmodified ribose) of "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE," respectively, and which have a phosphate group at position 3 or 5.

[0098] The "2'-O-R-ECE nucleotide" has the following formula (I):

[0099] (wherein Bx is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group), and R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ５ R ６ (The R ５ and R ６each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy 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 a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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 the R ５ and R ６are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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 a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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), and n is an integer from 1 to 3, provided that when n is 2 or 3, then 2 or 3 R ３ and R ４ may be the same or different. The 5'-end and 3'-end in formula (I) are hydrogen atoms.

[0100] R １ and R ２R each independently represents a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group, wherein the C1-6 alkyl group and the C2-6 alkenyl group are unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group. １ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom. ２ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.

[0101] R ３ and R ４ each 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 group and the C2-6 alkenyl group are unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group. Here, when n is 2 or 3, R ３ may be the same or different, R ４ may be the same or different. ３ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom. ４ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.

[0102] Y is NR ５ R ６ When R ５ and R ６each independently represents a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group, or a C7-10 aralkyl group, wherein the C1-6 alkyl group and C2-6 alkenyl group are unsubstituted or substituted by one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy 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, and The R group is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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. ５ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a methyl group. ６ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a methyl group.

[0103] In other embodiments, R ５ and R ６may form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle together with the nitrogen atom to which they are bonded. Here, the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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. R ５ and R ６ The 3-11-membered nitrogen-containing non-aromatic heterocycle formed by these together with the nitrogen atom to which they are bonded is preferably a 4-8-membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene groups, such as piperidine, pyrrolidine, morpholine, thiomorpholine, homopiperidine, and homomorpholine. More preferably, it is a ring further containing an oxygen atom or a sulfur atom as a ring-constituting atom, such as morpholine, thiomorpholine, and homomorpholine. Morpholine is particularly preferred. Furthermore, it is preferable that the 4-8-membered nitrogen-containing non-aromatic heterocycle is unsubstituted.

[0104] In another embodiment, Y may be a C2-9 aromatic heterocyclic group, preferably a pyridyl group, an imidazolyl group, or a benzimidazolyl group, more preferably 2-pyridyl, imidazol-1-yl, or (benzimidazol)-1-yl, and particularly preferably 2-pyridyl or (benzimidazol)-1-yl.

[0105] n is the number of repeating unit structures and is an integer of 1 to 3, preferably 2. A wavy line indicates bonding with adjacent nucleotides.

[0106] The compound represented by formula (I) may have isomers. In such cases, the compound of the present embodiment is not limited to a specific isomer, and includes all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereoisomers, optical isomers, rotational isomers, etc.), racemates, and mixtures thereof.

[0107] The nucleoside structure represented by formula (I) may be contained at the 3'-end or 5'-end of the oligonucleotide. When it is contained at the 3'-end, it has a structure represented by the following formula (II), for example.

[0108] R in formula (II) １ ~R ４ , Bx, Y and n are R in formula (I). １ ~R ４ , Bx, Y and n are synonymous. ２ is a hydrogen atom or a phosphate group, preferably a hydrogen atom. When it is contained at the 5'-end, the structure is, for example, represented by the following formula (III).

[0109] R in formula (III) １ ~R ４ , Bx, Y and n are R in formula (I). １ ~R ４ , Bx, Y and n are synonymous. １ is a hydrogen atom or a phosphate group, preferably a hydrogen atom.

[0110] The "2'-modified nucleotide" includes a "2'-O-R-ECE nucleotide", and the "2'-O-R-ECE nucleotide" includes a "2'-DMAECE nucleotide", a "2'-MorECE nucleotide", a "2'-PyECE nucleotide", and a "2'-BimECE nucleotide".

[0111] "2-4 bridged sugar" refers to a sugar in which the bridge unit is substituted by two substitutions at the 2- and 4-positions of ribose. Examples of the bridge unit include a C2-6 alkylene group (the alkylene group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are unsubstituted or independently substituted with a group selected from the group consisting of -O-, -NR'- (R' represents a hydrogen atom, a C1-6 alkyl group, or a haloC1-6 alkyl group), and -S-). By combining the above substitutions, the bridge unit can be -C(=O)-O-, -O-C(=O)-NR ２１ - (R ２１ represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), —C(═O)—NR ２１ - (R ２１ represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -C(=S)-NR ２１ - (R ２１ represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl). The 5-position of the "2-4-linked sugar" is preferably not modified.

[0112] The term "2',4' bridged nucleotide" (2',4'-BNA) refers to a molecule in which a base is bound to the carbon atom at position 1 (position 1 of unmodified ribose or 2-deoxyribose) of the "2,4-bridged sugar" and a nucleic acid base is attached to a phosphate group at position 3 or 5. For example, β-D-methyleneoxy (4'-CH), also known as LNA (Locked Nucleic Acid (registered trademark)), is used. ２ -O-2')BNA or α-L-methyleneoxy(4'-CH ２ -O-2')BNA, also known as ENA, ２ ) ２ -O-2')BNA, β-D-thio(4'-CH ２ -S-2')BNA, aminooxy (4'-CH ２ -O-N(R １１ )-2')BNA(R １１is a hydrogen atom or methyl), BNA ＮＣ Also called oxyamino (4'-CH ２ -N(R １２ )-O-2')BNA(R １２ is a hydrogen atom or methyl), 2',4'-BNA ＣＯＣ , 3'-amino-2',4'-BNA, 5'-methyl BNA, cEt-BNA (4'-CH(CH ３ )-O-2')BNA, also called cMOE-BNA (4'-CH(CH ２ OCH ３ )-O-2') BNA, amide type BNA (also called AmNA) (4'-C(=O)-N(R １３ )-2')BNA(R １３ is a hydrogen atom or methyl), also called scpBNA (4'-C(spiro-cyclopropyl)-O-2')BNA, also called GuNA (4'-CH ２ -N(R １４ )-2')BNA(R １４ is C(=NHR １５＋ ) NHR １６ and R １５ , R １６ are each independently a hydrogen atom, methyl, ethyl, isopropyl, or t-butyl), also known as amino LNA (4'-CH ２ -N(R １７ )-2')BNA(R １７ is a hydrogen atom or methyl), and other BNAs known to those skilled in the art. AmNA[Me] is a １３ AmNA is AmNA where R is methyl. １５ is t-butyl, and R １６ is H and is GuNA.

[0113] A "5-modified sugar" refers to a non-bridged sugar modified at the 5-position of the 2-deoxyribose, and includes "5-CP," "5-methyl," and "5-aminopropyl." Positions 2 and 4 of a "5-modified sugar" are preferably unmodified. A "5'-modified nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of the "5-modified sugar" (position 1 of the 2-deoxyribose before modification) and has a nucleic acid base attached to a phosphate group at position 2, 3, or 5, and includes, for example, a "5'-CP nucleotide," a "5'-methyl nucleotide," and a "5'-aminopropyl nucleotide."

[0114] "5-CP" is a sugar in which the 5-position of 2-deoxyribose is substituted with two methyl groups, and these two methyl groups together form a cyclopropane. A "5'-CP nucleotide" is a molecule in which the sugar is the above-mentioned "5-CP," a base is bound to the carbon atom at position 1 (position 1 of the base 2-deoxyribose), and a phosphate group is at position 3 or 5, and can be represented by the following structural formula. In the formula, Base is a nucleic acid base. The wavy line is understood to represent a bond to an adjacent nucleotide, linker, etc., or a hydrogen atom or a phosphate group. "5-methyl" is a sugar in which the 5-position of 2-deoxyribose is substituted with a methyl group. "5-aminopropyl" is a sugar in which the 5-position of 2-deoxyribose is substituted with a 3-aminopropyl group. "5'-methyl nucleotide" and "5'-aminopropyl nucleotide" refer to molecules in which a base is bound to the carbon atom at position 1 of "5-methyl" and "5-aminopropyl" (position 1 of the parent 2-deoxyribose), respectively, and a nucleic acid base is attached to a phosphate group at position 3 or 5.

[0115] In the "deoxyribonucleotide," "ribonucleotide," "2'-modified nucleotide," "2'-4' bridged nucleotide," and "5'-modified nucleotide," the bond between the carbon atom at the 1' position and the base can be an α-glycosidic bond or a β-glycosidic bond, but is usually a β-glycosidic bond. Therefore, β-D-methyleneoxy BNA is usually used as LNA.

[0116] When the "sugar-modified nucleotide" constitutes an antisense oligonucleotide molecule of the present invention, for example, the 3' position of the sugar-modified nucleotide is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the sugar-modified nucleotide is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The sugar-modified nucleotide at the 3' end of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at its 3' position, and the 5' position is as described above. The sugar-modified nucleotide at the 5' end of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group at its 5' position, and the 3' position is as described above.

[0117] Examples of modifications of the phosphodiester bond in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides include phosphorothioation, methylphosphonation (including chiral-methylphosphonation), methylthiophosphonation, phosphorodithioation, phosphoramidate, phosphorodiamidate, phosphoramidothioate, boranophosphate, etc. Examples of modifications of the phosphodiester bond in 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, etc., and these can be used for the phosphodiester bond in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.

[0118] A "bridged nucleotide" is a sugar-modified nucleotide in which the bridge unit is substituted by two substitutions in the sugar moiety, and examples thereof include 2'-4' bridged nucleotides.

[0119] Sugar-modified nucleotides are not limited to those exemplified herein. Many sugar-modified nucleotides are known in the art, and sugar-modified nucleotides described in, for example, U.S. Patent No. 8,299,039 to Tachas et al. (especially columns 17 to 22), 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, etc., can also be used in embodiments of the present invention.

[0120] Those skilled in the art can select and use an appropriate sugar-modified nucleotide from among such sugar-modified nucleotides, taking into consideration aspects such as antisense effect, affinity for a partial sequence of the target RNA, and resistance to nucleases.

[0121] "Nucleobase" generally refers to a base component constituting a nucleic acid, and natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (u). Natural nucleobases and modified nucleobases thereof can be used in the base moiety of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides used herein. Modified nucleobases can form base pairs (i.e., hydrogen bonds) with any nucleobase (preferably a base complementary to the nucleobase before modification). Typically, modified nucleobases 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-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, the pyrimidine base 5-propynyl (-C≡C-CH ３) uracil and cytosine and other alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils, and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is substituted with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Examples of modifications of the base moiety in 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. WO 2007 / 090071, among others. These modifications can be used for the base moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. The amino and hydroxy groups in the base moiety may each be independently protected.

[0122] The base moiety in the deoxyribonucleotide, ribonucleotide, and sugar-modified nucleotide is preferably at least one selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), and 5-methylcytosine (5-me-C).

[0123] "RNase H" is generally known as a ribonuclease that recognizes a double-stranded structure formed by hybridizing DNA and RNA and cleaves the RNA to produce single-stranded DNA. RNase H is not limited to double-stranded structures formed by hybridizing DNA and RNA, but can also recognize double-stranded structures in which at least one of the base moiety, phosphodiester bond moiety, and sugar moiety of at least one of the DNA and RNA is modified. For example, it can also recognize double-stranded structures formed by hybridizing oligodeoxyribonucleotides and oligoribonucleotides. Thus, DNA can be recognized by RNase H when hybridized with RNA. The same applies when at least one of the base moiety, phosphodiester bond moiety, and sugar moiety of at least one of the DNA and RNA is modified. For example, a typical example is an oligonucleotide in which the phosphodiester bond moiety of DNA is modified to phosphorothioate. RNA can be cleaved by RNase H when hybridized with DNA. The same applies to modifications of at least one of the base moiety, phosphodiester bond moiety, and sugar moiety in at least one of DNA and RNA. Examples of modifications of DNA and / or RNA that can be recognized by RNase H are described in, for example, Nucleic Acids Research, 2014, 42, pp. 5378-5389, Bioorganic & Medicinal Chemistry Letters, 2008, 18, pp. 2296-2300 (Non-Patent Document 1, mentioned above), Molecular BioSystems, 2009, 5, pp. 838-843, Nucleic Acid Therapeutics, 2015, 25, pp. 266-274, The Journal of Biological Chemistry, 2004, 279, pp. 36317-36326 (Non-Patent Document 2, mentioned above), etc. The RNase H used in the present invention is preferably mammalian RNase H, more preferably human RNase H, and particularly preferably human RNase H1.

[0124] The "gap region" is a region containing "at least four consecutive nucleotides recognized by RNase H," and is not particularly limited as long as it contains four or more consecutive nucleotides and is recognized by RNase H, but the consecutive nucleotides are preferably independently selected from deoxyribonucleotides and 5'-modified nucleotides.

[0125] The "5' wing region" is a region that is linked to the 5' side of the gap region and contains "at least one nucleotide" but does not contain the "at least four consecutive nucleotides recognized by RNase H," wherein the sugar moiety of the 3'-terminal nucleotide of the 5' wing region is different from the sugar moiety of the 5'-terminal nucleotide of the gap region. The difference in sugar moiety identifies the boundary between the 5' wing region and the gap region. The 3'-terminal nucleotide of the 5' wing region is generally a 2'-modified nucleotide or a 2'-4' bridged nucleotide. The 5' wing region is not particularly limited as long as it satisfies the above definition, but preferably is independently selected from deoxyribonucleotides and sugar-modified nucleotides and contains at least one 2'-modified nucleotide or 2'-4' bridged nucleotide.

[0126] The "3' wing region" is a region that is linked to the 3' side of the gap region and contains "at least one nucleotide" but does not contain the "at least four consecutive nucleotides recognized by RNase H," wherein the sugar moiety of the 5'-terminal nucleotide of the 3' wing region is different from the sugar moiety of the 3'-terminal nucleotide of the gap region. The difference in sugar moiety identifies the boundary between the 3' wing region and the gap region. The 5'-terminal nucleotide of the 3' wing region is generally a 2'-modified nucleotide or a 2'-4' bridged nucleotide. The 3' wing region is not particularly limited as long as it satisfies the above definition, but preferably is independently selected from deoxyribonucleotides and sugar-modified nucleotides and contains at least one 2'-modified nucleotide or 2'-4' bridged nucleotide.

[0127] An antisense oligonucleotide having a gap region, a 5' wing region and a 3' wing region is called a gapmer-type antisense oligonucleotide.

[0128] The "central region" is the central region in an oligonucleotide. The "5' region" is a region linked to the 5' side of the "central region" and comprising at least one nucleotide. The "3' region" is a region linked to the 3' side of the "central region" and comprising at least one nucleotide.

[0129] The sugar moiety of the 5'-terminal nucleotide in the 3' region is different from the sugar moiety of the 3'-terminal nucleotide in the central region. The difference in sugar moiety identifies the boundary between the 3' region and the central region. The sugar moiety of the 3'-terminal nucleotide in the 5' region is different from the sugar moiety of the 5'-terminal nucleotide in the central region. The difference in sugar moiety identifies the boundary between the 5' region and the central region.

[0130] "At least four consecutive nucleotides recognized by RNase H" includes four or more consecutive nucleotides and is not particularly limited as long as it is recognized by RNase H, but examples include "at least four consecutive deoxyribonucleotides" and "at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides." The number of nucleotides is, for example, 5 to 30, preferably 5 to 15, and more preferably 8 to 12. Those skilled in the art can determine whether a certain set of at least four consecutive nucleotides is "at least four consecutive nucleotides recognized by RNase H" based on the structure of the sugar moieties of the consecutive nucleotides.

[0131] Next, the antisense oligonucleotide of the present invention will be described. The antisense oligonucleotide of the present invention does not need to hybridize with the entire target RNA, but only needs to hybridize with at least a portion of the target RNA, and usually hybridizes with at least a portion of the target RNA. For example, the expression of the target gene is controlled by hybridizing at least a portion of the target RNA with an oligonucleotide (such as DNA, oligodeoxyribonucleotide, or an oligonucleotide designed to produce an antisense effect) having an antisense sequence complementary to a partial sequence of the target RNA. In addition, the entire antisense oligonucleotide does not need to hybridize, and only a portion may not hybridize. Although the entire antisense sequence portion may not hybridize, it is preferable that it hybridizes.

[0132] The term "antisense sequence" refers to the base sequence of nucleotides constituting an oligonucleotide that enables hybridization with a target RNA, and the term "antisense sequence portion" refers to the partial structure of the region of an oligonucleotide chain that has the antisense sequence.

[0133] The complementarity between the antisense sequence portion of the antisense oligonucleotide and the partial sequence of the target RNA is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, 99% or more). In order for the antisense sequence portion of the antisense oligonucleotide and at least a part of the target RNA to hybridize, their sequences do not need to be completely complementary, but completely complementary is even more preferable.

[0134] Those skilled in the art can easily determine a base sequence that matches an antisense sequence that is "capable of hybridizing with the target RNA" by using a BLAST program or the like.

[0135] The antisense oligonucleotide of the present invention has a central region, a 5' region, and a 3' region. The central region is preferably a gap region, the 5' region is preferably a 5' wing region, and the 3' region is a 3' wing region. The central region is composed of at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and the 3' end and 5' end are each independently selected from deoxyribonucleotides and 5'-modified nucleotides, and the central region includes at least one oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides.

[0136] The number of nucleotides contained in the central region is 5 to 30, preferably 5 to 15, more preferably 8 to 12, and particularly preferably 9 or 10. The number of nucleotides contained in the central region is usually selected depending on other factors such as the strength of the antisense effect on the target RNA, low toxicity, cost, and synthesis yield.

[0137] The central region may contain both deoxyribonucleotides and 5'-modified nucleotides. The number (total number) of 5'-modified nucleotides contained in the central region is 1 to 30, preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and particularly preferably 1. The number of 5'-modified nucleotides contained in the central region is usually selected depending on other factors such as the strength of the antisense effect on the target RNA, low toxicity, cost, and synthesis yield. The 5'-modified nucleotides contained in the central region can be contained at any position in the central region, but are preferably contained between the third nucleotide counting from the 5' end and the 5' end of the central region. The position where the 5'-modified nucleotide is contained is usually selected depending on other factors such as the strength of the antisense effect on the target RNA and low toxicity. All of the nucleotides contained in the central region may be deoxyribonucleotides.

[0138] Of the nucleotides contained in the central region, it is preferred that at least one nucleotide is phosphorothioated, more preferred that 80% of the nucleotides are phosphorothioated, even more preferred that 90% of the nucleotides are phosphorothioated, and especially preferred that all are phosphorothioated.

[0139] The 5' region is composed of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and includes at least one 2'-4' bridged nucleotide, and its 3'-terminus is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the sugar-modified nucleotide at the 3'-terminus is attached to the central region, and does not include an oligonucleotide strand composed of at least four consecutive nucleotides independently selected from the group consisting of at least four consecutive deoxyribonucleotides and 5'-modified nucleotides. The 3' region is composed of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and includes at least one 2'-4' bridged nucleotide, and its 5'-terminus is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the sugar-modified nucleotide at the 5'-terminus is attached to the central region, and does not include an oligonucleotide strand composed of at least four consecutive nucleotides independently selected from the group consisting of at least four consecutive deoxyribonucleotides and 5'-modified nucleotides.

[0140] At least one 2'-O-R-ECE nucleotide is contained in at least one of the central region, 5' region, and 3' region. Preferably, at least one 2'-O-R-ECE nucleotide is contained in at least one of the 5' region and 3' region. More preferably, at least one 2'-O-R-ECE nucleotide is contained in the 5' region.

[0141] The number of nucleotides contained in the 5' region is 2 to 15, preferably 2 to 7, more preferably 2 to 5, and particularly preferably 3. The number of nucleotides contained in the 5' region is usually selected depending on other factors such as the strength of the antisense effect on the target RNA, low toxicity, cost, and synthesis yield. The 3' region is the same as the 5' region.

[0142] The sugar-modified nucleotide contained in the 5' region is preferably a nucleotide whose affinity for a partial sequence of a target RNA has been increased by substitution or whose resistance to nucleases has been increased, and more preferably, is independently selected from 2'-modified nucleotides and 2',4'-BNAs. The 2'-modified nucleotide is preferably independently selected from the group consisting of 2'-O-methylated nucleotides, 2'-O-methoxyethyl (MOE)-modified nucleotides, 2'-O-aminopropyl (AP)-modified nucleotides, 2'-fluorinated nucleotides, 2'-O-(N-methylacetamido) (NMA)-modified nucleotides, 2'-O-methylcarbamoylethyl (MCE)-modified nucleotides, and 2'-O-R-ECE nucleotides, more preferably independently selected from 2'-O-methoxyethyl (MOE)-modified nucleotides, 2'-O-methylcarbamoylethyl (MCE)-modified nucleotides, and 2'-O-R-ECE nucleotides, even more preferably 2'-O-R-ECE nucleotides, and particularly preferably independently selected from 2'-DMAECE nucleotides, 2'-MorECE nucleotides, 2'-PyECE nucleotides, and 2'-BimECE nucleotides. 2',4'-BNA is preferably LNA, cEt-BNA, ENA, BNA ＮＣ , AmNA and scpBNA, more preferably LNA. The 3' region is the same as the 5' region.

[0143] The type, number, and position of sugar-modified nucleotides, deoxyribonucleotides, and ribonucleotides in the 5' region can affect the antisense effect, etc., of the antisense oligonucleotides disclosed herein. The type, number, and position of these nucleotides vary depending on the sequence of the target RNA, etc., and therefore cannot be generalized. However, those skilled in the art can determine a preferred embodiment by taking into consideration the descriptions in literature related to the antisense method. Furthermore, the antisense effect of an oligonucleotide after modification of the base moiety, sugar moiety, or phosphodiester linkage moiety can be measured, and if the measured value is not significantly lower than that of the oligonucleotide before modification (e.g., if the measured value of the modified oligonucleotide is 30% or more of the measured value of the oligonucleotide before modification), the modification can be evaluated as a preferred embodiment. The antisense effect can be measured, for example, as shown in the Examples below, by introducing a test oligonucleotide into cells, etc., and measuring the expression level of the target RNA, the expression level of cDNA associated with the target RNA, the amount of protein associated with the target RNA, etc., controlled by the antisense effect of the test oligonucleotide, appropriately using known techniques such as Northern blotting, quantitative PCR, and Western blotting. The 3' region is similar to the 5' region.

[0144] The 5' region preferably consists of 2 to 5 nucleotides independently selected from the group consisting of 2'-modified nucleotides, 2',4'-BNAs, and deoxyribonucleotides, and includes at least one 2'-O-R-ECE nucleotide and at least one 2',4'-BNA. More preferably, it consists of 2 to 5 nucleotides independently selected from the group consisting of 2'-O-R-ECE nucleotides and 2',4'-BNAs, and even more preferably, it consists of 3 to 5 nucleotides independently selected from the group consisting of 2'-O-R-ECE nucleotides and 2',4'-BNAs. Even more preferably, it consists of 4 to 5 nucleotides independently selected from LNAs and 2'-O-R-ECE nucleotides. Even more preferably, it consists of 1 to 3 LNAs and 1 to 3 nucleotides selected from 2'-DMAECE nucleotides, 2'-MorECE nucleotides, 2'-PyECE nucleotides, and 2'-BimECE nucleotides. Even more preferably, it consists of 2 to 3 LNAs and 1 to 2 nucleotides selected from 2'-DMAECE nucleotides, 2'-MorECE nucleotides, 2'-PyECE nucleotides, and 2'-BimECE nucleotides. Particularly preferably, it consists of 3 LNAs and 1 to 2 nucleotides selected from 2'-DMAECE nucleotides, 2'-MorECE nucleotides, 2'-PyECE nucleotides, and 2'-BimECE nucleotides. The 3' region is the same as the 5' region. In another embodiment, the 3' region preferably consists of 2 to 5 2',4'-BNAs, and particularly preferably consists of 3 LNAs.

[0145] Of the nucleotides contained in the 5' region, at least one nucleotide is preferably phosphorothioated, more preferably 50% of the nucleotides are phosphorothioated, even more preferably 80% of the nucleotides are phosphorothioated, and particularly preferably all are phosphorothioated. In another embodiment, it is preferred that all of the nucleotides contained in the 5' region are linked by phosphodiester bonds. The 3' region is the same as the 5' region.

[0146] In the antisense oligonucleotide of the present invention, the 3'-end of the 5'-side region and the 5'-end of the central region are linked via a phosphodiester bond or a modified phosphodiester bond, and the 5'-end of the 3'-side region and the 3'-end of the central region are linked via a phosphodiester bond or a modified phosphodiester bond. Preferably, the 3'-end of the 5'-side region and the 5'-end of the central region are linked via a modified phosphodiester bond, and the 5'-end of the 3'-side region and the 3'-end of the central region are linked via a modified phosphodiester bond. More preferably, the 3'-end of the 5'-side region and the 5'-end of the central region are linked via a phosphorothioate bond, and the 5'-end of the 3'-side region and the 3'-end of the central region are linked via a phosphorothioate bond. The nucleotide at the 3'-end of the 5'-side region is preferably an LNA or a 2'-O-R-ECE nucleotide, more preferably a 2'-O-R-ECE nucleotide. The 5'-terminal nucleotide of the 3'-region is preferably an LNA or 2'-O-R-ECE nucleotide, more preferably an LNA.

[0147] The antisense oligonucleotide of the present invention may be directly or indirectly bound to a functional molecule.The binding between the functional molecule and the antisense oligonucleotide may be direct or indirect via other substances, but it is preferred that the oligonucleotide and the functional molecule are bound by a covalent bond, an ionic bond or a hydrogen bond.From the viewpoint of high stability of the binding, it is more preferred that they are bound by a covalent bond directly or by a covalent bond via a linker (linking group).

[0148] When the functional molecule is covalently bound to the antisense oligonucleotide, it is preferred that the functional molecule be directly or indirectly bound to the 3'-end or 5'-end of the antisense oligonucleotide. The bond between the linker or functional molecule and the terminal nucleotide of the antisense oligonucleotide is selected depending on the functional molecule. The linker or functional molecule and the terminal nucleotide of the antisense oligonucleotide are preferably linked via a phosphodiester bond or a modified phosphodiester bond, more preferably via a phosphodiester bond. The linker or functional molecule may be directly linked to the 3'-position oxygen atom of the 3'-terminal nucleotide or the 5'-position oxygen atom of the 5'-terminal nucleotide of the antisense oligonucleotide.

[0149] The structure of the "functional molecule" is not particularly limited, and its binding confers a desired function to the antisense oligonucleotide. Desired functions include labeling, purification, and delivery to a target site. Examples of molecules that impart a labeling function include compounds such as fluorescent proteins and luciferase. Examples of molecules that impart a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.

[0150] Furthermore, from the viewpoint of highly specific and efficient delivery of antisense oligonucleotides to target sites (e.g., target cells) and highly effective control of target gene expression by the antisense oligonucleotides, it is preferable that the functional molecule be a molecule having the function of delivering the antisense oligonucleotide to the target site. For molecules having such delivery functions, see, for example, European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp. 321-340; Advanced Drug Delivery Reviews, 2016, 104, pp. 78-92; Expert Opinion on Drug Delivery, 2014, 11, pp. 791-822, etc.

[0151] Molecules that impart a delivery function to target RNA include, for example, lipids and sugars, which can deliver antisense oligonucleotides to the liver and other tissues 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; and derivatives thereof. Among these, cholesterol and vitamin E (tocopherols, tocotrienols) are preferred from the viewpoint of greater safety. Among these, tocopherols are more preferred, tocopherol is even more preferred, and α-tocopherol is particularly preferred. Examples of sugars include sugar derivatives that interact with the asialoglycoprotein receptor.

[0152] The "asialoglycoprotein receptor" is present on the surface of liver cells and recognizes the galactose residue of asialoglycoprotein, taking up the molecule into the cell and degrading it. The "sugar derivative that interacts with the asialoglycoprotein receptor" is preferably a compound that has a structure similar to that of a galactose residue and is taken up into the cell through interaction with the asialoglycoprotein receptor, such as GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives. From the perspective of being able to deliver the antisense oligonucleotide of the present invention to the brain with high specificity and efficiency, the "functional molecule" may be a sugar (e.g., glucose, sucrose, etc.). From the perspective of being able to deliver the antisense oligonucleotide to each organ with high specificity and efficiency by interacting with various proteins on the cell surface of the organ, the "functional molecule" may be a receptor ligand, an antibody, or a peptide or protein fragment thereof.

[0153] The linker used to bond the functional molecule to the antisense oligonucleotide is not particularly limited as long as it can exhibit the function of the functional molecule as an antisense oligonucleotide molecule, and can stably bond the functional molecule to the oligonucleotide. Examples of such linkers include groups derived from oligonucleotides containing 1 to 20 nucleotides, groups derived from polypeptides containing 2 to 20 amino acids, alkylenes containing 2 to 20 carbon atoms, and alkenylenes containing 2 to 20 carbon atoms. The group derived from an oligonucleotide containing 2 to 20 nucleotides is a group obtained by removing a hydroxyl, hydrogen atom, or the like from an oligonucleotide containing 2 to 20 nucleotides. For examples of groups derived from oligonucleotides containing 1 to 20 nucleotides, see International Publication No. 2017 / 053995. International Publication No. 2017 / 053995 describes, for example, a three-base linker having a TCA motif and a one- to five-base linker without a TCA motif. The group derived from a polypeptide having 2 to 20 amino acids is a group obtained by removing a hydroxyl, hydrogen atom, amino, or the like from a polypeptide having 2 to 20 amino acids.

[0154] The linker is preferably a C2-20 alkylene or a C2-20 alkenylene (methylenes contained in the alkylene and alkenylene are each independently unsubstituted or substituted with one or two substituents selected from the group consisting of a halogen atom, hydroxy, protected hydroxy, oxo, and thioxo. In addition, methylenes in the alkylene and alkenylene are each independently unsubstituted or substituted with —O—, —NR Ｂ - (R Ｂ is a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -S-, -S(=O)- or -S(=O) ２ Here, by combining the above substitutions, the linker may contain a group represented by -C(=O)-O-, -O-C(=O)-NR"- (R" represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -C(=O)-NR"- (R" represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -C(=S)-NR"- (R" represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -NR"-C(=O)-NR"- (R" each independently represent a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), or the like. For example, a linker structure of -[C2-6 alkylene]-[C(=O)-NR"-(R" represents a hydrogen atom or a C1-6 alkyl)]-[C2-6 alkylene]-[C(=O)]- can be used. Here, preferably, the left-side [C2-6 alkylene] binds to the functional molecule, and the right-side [C(=O)] binds to the oligonucleotide.

[0155] The linker is more preferably a C2-20 alkylene (wherein each methylene of the alkylene is independently unsubstituted or replaced by —O—; each unsubstituted methylene is independently unsubstituted or substituted by hydroxy or protected hydroxy), and even more preferably a C8-12 alkylene (wherein each methylene of the alkylene is independently unsubstituted or replaced by —O—; each unsubstituted methylene is independently unsubstituted or substituted by hydroxy).

[0156] In another embodiment, the linker is more preferably a C2-20 alkylene (each methylene of the alkylene is independently unsubstituted or -O- or -NR Ｂ - (R Ｂ is a hydrogen atom or a C1-6 alkyl). Each unsubstituted methylene is independently unsubstituted or substituted by oxo), and more preferably C8-12 alkylene (each methylene of the alkylene is independently unsubstituted or substituted by —O— or —NR Ｂ - (R Ｂ is replaced by a hydrogen atom or a C1-6 alkyl. Each unsubstituted methylene is independently unsubstituted or substituted by oxo.

[0157] One to five (preferably one to three, particularly preferably three) 3- to 11-membered nitrogen-containing non-aromatic heterocycle-diyldimethanol structures may be linked to the 5'- or 3'-end of the antisense oligonucleotide via phosphodiester or phosphorothioate bonds, with the linker being linked to the 3- to 11-membered nitrogen-containing non-aromatic heterocycle. In this case, the number of functional molecules linked via the linker is also preferably one to five, more preferably one to three, and particularly preferably three. The 3- to 11-membered nitrogen-containing non-aromatic heterocycle is preferably pyrrolidine or piperidine. The 3- to 11-membered nitrogen-containing non-aromatic heterocycle-diyldimethanol is particularly preferably piperidine-4,4-diylmethanol.

[0158] The protecting group for the "protected hydroxy" is not particularly limited as long as it is stable when the functional molecule and the oligonucleotide are linked together. The linker is not particularly limited, and examples thereof include any protecting group described in Protective Groups in Organic Synthesis, 4th Edition, by T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (2006). Specifically, as described in the explanation of terms, preferred protecting groups are benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, triphenylmethyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthen-9-yl, and 9-(p-methoxyphenyl)xanthen-9-yl, more preferably monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl, and even more preferably dimethoxytrityl.

[0159] The present invention also includes prodrugs of the antisense oligonucleotides. A prodrug is a derivative of a pharmaceutical compound having a chemically or metabolically decomposable group, which is converted into a pharmacologically active pharmaceutical compound by solvolysis or in vivo decomposition under physiological conditions. Methods for selecting and preparing suitable prodrug derivatives are described, for example, in Design of Prodrugs (Elsevier, Amsterdam, 1985). In the case of the present invention, when a compound has a hydroxy group, examples of prodrugs include acyloxy derivatives prepared by reacting the compound with a suitable acyl halide, a suitable acid anhydride, or a suitable alkyloxycarbonyl halide compound. A particularly preferred structure for a prodrug is -O-C(=O)C ２ H ５ , -OC(=O)(t-Bu), -OC(=O)C １５ H ３１ , -OC(=O)-(m-CO ２ Na-Ph), -OC(=O)CH ２ CH ２ CO ２ Na-OC(=O)CH(NH ２ ) CH ３ , -OC(=O)CH ２ N (CH ３ ) ２ or -O-CH ２ OC(=O)CH ３ When the antisense oligonucleotide of the present invention has an amino group, examples of the prodrug include those prepared by reacting a compound having an amino group with an appropriate acid halide, an appropriate mixed acid anhydride, or an appropriate alkyloxycarbonyl halide compound. A particularly preferred structure for the prodrug is -NH-C(=O)-(CH ２ ) ２０ OCH ３ , -NH-C(=O)CH(NH ２ )CH ３ , —NH—CH ２ OC(=O)CH ３ etc.

[0160] Other preferred structures of prodrugs included in the present invention include double-stranded oligonucleotides containing ribonucleotides (e.g., oligoribonucleotides, RNA), peptide nucleic acids (PNAs), or deoxyribonucleotides (e.g., oligodeoxyribonucleotides, DNA) that are complementary to the antisense oligonucleotide (e.g., as described in WO 2013 / 089283, WO 2017 / 068791, WO 2017 / 068790, or WO 2018 / 003739), and single-stranded oligonucleotides in which an RNA oligonucleotide complementary to the antisense oligonucleotide is linked via a linker (e.g., as described in WO 2017 / 131124). The linker is not limited to those described in WO 2017 / 131124 and may, for example, contain a non-nucleotide structure. Examples also include single-stranded oligonucleotides in which an RNA oligonucleotide complementary to the antisense oligonucleotide is directly linked.

[0161] More specific examples of the prodrugs of the present invention are given below.

[0162] (A) an oligonucleotide complex comprising: (i) the antisense oligonucleotide; and (ii) an oligonucleotide comprising at least one ribonucleotide and comprising a region that hybridizes to the (i) antisense oligonucleotide.

[0163] (B) An oligonucleotide comprising: (i) a group derived from the antisense oligonucleotide; and (ii) a group derived from an oligonucleotide that contains at least one ribonucleotide and includes a region that hybridizes to the antisense oligonucleotide (i), wherein the group derived from the antisense oligonucleotide (i) and the group derived from the oligonucleotide (ii) are linked together.

[0164] In (B), (i) the group derived from an antisense oligonucleotide and (ii) the group derived from an oligonucleotide may be linked via a group derived from an oligonucleotide that is degraded under physiological conditions, or may be linked via a linking group containing a non-nucleotide structure, or may be linked directly.

[0165] (C) An oligonucleotide complex comprising: (iii) an oligonucleotide having an oligonucleotide chain containing at least one ribonucleotide linked to a group derived from the antisense oligonucleotide; and (iv) an oligonucleotide having an oligonucleotide chain containing at least four consecutive nucleotides that are recognized by RNase H, wherein the oligonucleotide chain containing at least one ribonucleotide of (iii) and the oligonucleotide chain containing at least four consecutive nucleotides that are recognized by RNase H of (iv) hybridize.

[0166] (D) An oligonucleotide comprising: (iii) a group derived from an oligonucleotide in which an oligonucleotide chain containing at least one ribonucleotide is linked to the group derived from the antisense oligonucleotide; and (iv) a group derived from an oligonucleotide comprising an oligonucleotide chain containing at least four consecutive nucleotides that are recognized by RNase H, wherein the group derived from the oligonucleotide (iii) is linked to the group derived from the oligonucleotide (iv), and the oligonucleotide chain containing at least one ribonucleotide of (iii) hybridizes with the oligonucleotide chain containing at least four consecutive nucleotides that are recognized by RNase H of (iv).

[0167] In (D), (iii) the group derived from an oligonucleotide and (iv) the group derived from an oligonucleotide may be linked via a group derived from an oligonucleotide that is degraded under physiological conditions, or may be linked via a linking group containing a non-nucleotide structure, or may be linked directly.

[0168] In (C) and (D), the group derived from an antisense oligonucleotide and the oligonucleotide chain containing at least one ribonucleotide may be linked by a group derived from an oligonucleotide that is degraded under physiological conditions, by a linking group containing a non-nucleotide structure, or directly.

[0169] The "oligonucleotide degradable under physiological conditions" may be any oligonucleotide that is degraded under physiological conditions by enzymes such as various DNases (deoxyribonucleases) and RNases (ribonucleases), and the nucleotides constituting the oligonucleotide may or may not be chemically modified in the base, sugar, or phosphate bond in part or all of them. The "oligonucleotide degradable under physiological conditions" is, for example, an oligonucleotide containing at least one phosphodiester bond, preferably linked by a phosphodiester bond, more preferably an oligodeoxyribonucleotide or oligoribonucleotide, even more preferably DNA or RNA, and even more preferably RNA. The oligonucleotide degradable under physiological conditions may or may not contain a partially complementary sequence within the oligonucleotide degradable under physiological conditions, but preferably does not contain a partially complementary sequence. An example of such an oligonucleotide is (N) linked by a phosphodiester bond. ｋ’ (Each N is independently adenosine, uridine, cytidine, guanosine, 2'-deoxyadenosine, thymidine, 2'-deoxycytidine, or 2'-deoxyguanosine, and k is an integer (number of repetitions) of 1 to 40. Among these, k' is preferably 3 to 20, more preferably 4 to 10, even more preferably 4 to 7, still more preferably 4 or 5, and particularly preferably 4.

[0170] The oligonucleotide (ii) in (A) above, the group derived from the oligonucleotide (ii) in (B) above, and the oligonucleotide (iii) in the oligonucleotide complexes shown in (C) and (D) above, which contain at least one ribonucleotide (the portion that hybridizes to an oligonucleotide chain containing at least four consecutive nucleotides recognized by RNase H), will be described below. The type, number, and position of sugar-modified nucleotides, deoxyribonucleotides, and ribonucleotides can affect the antisense effect, etc., of the antisense oligonucleotide prodrug disclosed herein. The type, number, and position of these nucleotides vary depending on the sequence of the target RNA, etc., and therefore cannot be generalized. However, those skilled in the art can determine preferred embodiments by referring to the descriptions in literature related to the antisense method. Furthermore, the antisense effect of a prodrug of an antisense oligonucleotide after modification of the base moiety, sugar moiety, or phosphodiester bond moiety can be measured, and if the measured value obtained is not significantly lower than that of the prodrug of the antisense oligonucleotide before modification (for example, if the measured value of the prodrug of the antisense oligonucleotide after modification is 30% or more of the measured value of the prodrug before modification), the modification can be evaluated as a preferable embodiment. Measurement of the antisense effect can be carried out, for example, as shown in the Examples described below, by introducing a test oligonucleotide into cells or the like, and appropriately utilizing known techniques such as Northern blotting, quantitative PCR, and Western blotting to determine the expression level of the target RNA, the expression level of cDNA associated with the target RNA, and the amount of protein associated with the target RNA, which are controlled by the antisense effect of the test oligonucleotide.

[0171] The oligonucleotide (ii) in the oligonucleotide conjugate shown in (A) or the group derived from the oligonucleotide (ii) in the oligonucleotide shown in (B) is independently selected from ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, and is preferably selected from ribonucleotides. When the oligonucleotide (ii) or the group derived from the oligonucleotide is selected from ribonucleotides, the ribonucleotides are preferably linked to each other via a phosphodiester bond. In another embodiment, the oligonucleotide (ii) or the group derived from the oligonucleotide is selected from ribonucleotides and sugar-modified nucleotides, and the sugar-modified nucleotide is selected from sugar-modified nucleotides excluding 5'-modified nucleotides. In this case, it is preferable that the terminal of the oligonucleotide has at least one sugar-modified nucleotide. This sugar-modified nucleotide is preferably a 2'-O-methylated nucleotide, and is preferably linked to an adjacent nucleotide via a phosphorothioate bond. In the oligonucleotide complex shown in (C) or the oligonucleotide shown in (D), the oligonucleotide (iii) contains at least one ribonucleotide (the same applies to the portion that hybridizes with an oligonucleotide chain containing at least four consecutive nucleotides recognized by RNase H).

[0172] In the case of (A), the 3'- and 5'-terminal nucleotides of the oligonucleotide (ii) are preferably sugar-modified nucleotides. In the case of (B), the terminal nucleotides of the 3'- and 5'-terminals of the group derived from the oligonucleotide (ii) that are not bound to the group derived from the antisense oligonucleotide (i) are preferably sugar-modified nucleotides. In the cases of (C) and (D), the terminal nucleotides of the 3'- and 5'-terminals of the group derived from the oligonucleotide (iii) that are not bound to the group derived from the antisense oligonucleotide are preferably sugar-modified nucleotides.

[0173] The number of bases of the oligonucleotide (ii) or the group derived from the oligonucleotide is not particularly limited, and may be the same or different from the number of bases of the antisense oligonucleotide (i) (or the group derived from it).In (A), the number of bases of the oligonucleotides (i) and (ii) is preferably the same, and it is preferred that the oligonucleotides (i) and (ii) are hybridized as a whole.In (B), the same applies when the group derived from the oligonucleotides (i) and (ii) is linked by a group derived from the oligonucleotide that is degraded under physiological conditions or a linking group that includes a non-nucleotide structure.

[0174] The oligonucleotide (iv) in the oligonucleotide conjugate shown in (C) and the group derived from the oligonucleotide (iv) in the oligonucleotide in (D) are independently selected from ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, and are preferably selected from deoxyribonucleotides and sugar-modified nucleotides. The sugar-modified nucleotide contained in the oligonucleotide (iv) or the group derived from the oligonucleotide is preferably selected from sugar-modified nucleotides excluding 5'-modified nucleotides. In this case, the 5'-terminus and 3'-terminus of the oligonucleotide or the group derived from the oligonucleotide preferably contain at least one sugar-modified nucleotide. The at least one sugar-modified nucleotide is preferably at least one selected from 2'-modified nucleotides and 2',4'-BNA, and more preferably 2'-O-methylated nucleotides, 2'-O-methoxyethyl (MOE)-modified nucleotides, 2'-O-aminopropyl (AP)-modified nucleotides, 2'-fluorinated nucleotides, 2'-O-(N-methylacetamido) (NMA)-modified nucleotides, 2'-O-methylcarbamoylethyl (MCE)-modified nucleotides, LNA, cEt-BNA, ENA, BNA ＮＣ (iv) or at least one selected from the group consisting of AmNA and scpBNA. The nucleotides contained in the oligonucleotide of the group derived from the oligonucleotide are preferably linked to each other via phosphorothioate bonds.

[0175] In (A), it is believed that the hybridized portion between the (i) antisense oligonucleotide and the (ii) region containing at least one ribonucleotide and hybridizing to the (i) antisense oligonucleotide is recognized by RNase H, and the (ii) region containing at least one ribonucleotide and hybridizing to the (i) antisense oligonucleotide is cleaved. As a result, the antisense oligonucleotide of the present invention is produced in target cells, etc., and the prodrug of (A) is believed to exert a therapeutic effect, etc. The same applies to (B). In (C), it is believed that the hybridized portion between the (iii) oligonucleotide chain containing at least one ribonucleotide and the (iv) oligonucleotide chain containing at least four consecutive nucleotides recognized by RNase H is recognized by RNase H, and the (iii) oligonucleotide chain containing at least one ribonucleotide is cleaved. As a result, the antisense oligonucleotide of the present invention is produced in the target cells etc., and the prodrug of (C) is thought to exert a therapeutic effect etc. The same applies to the above (D).

[0176] When the oligonucleotide complex shown in (A) has a functional molecule, it is preferable that the oligonucleotide (ii) contains a functional molecule, and it is preferable that the functional molecule is bound to the end of the oligonucleotide (ii). The same applies to the oligonucleotide shown in (B). When the oligonucleotide complex shown in (C) has a functional molecule, it is preferable that the oligonucleotide (iv) contains a functional molecule, and it is preferable that the functional molecule is bound to the end of the oligonucleotide (iv). The same applies to the oligonucleotide shown in (D). The preferred embodiments of the functional molecule and its bond are as described above.

[0177] Of the (A) and (B), the oligonucleotide or oligonucleotide-derived group (ii) may further have a group derived from an antisense oligonucleotide. The group derived from the antisense oligonucleotide (ii) may be the same as or different from the group derived from the antisense oligonucleotide or oligonucleotide (i). Furthermore, it may or may not be a group derived from the antisense oligonucleotide of the present invention. It is preferable that the group derived from the antisense oligonucleotide (ii) does not hybridize with the antisense oligonucleotide or antisense oligonucleotide (i). Of the (C) and (D), the oligonucleotide or oligonucleotide-derived group (iv) may be an antisense oligonucleotide or antisense oligonucleotide-derived group. The group derived from the antisense oligonucleotide or antisense oligonucleotide (iv) may be the same as or different from the group derived from the antisense oligonucleotide contained in the oligonucleotide (iii). It may or may not be a group derived from the antisense oligonucleotide of the present invention. It is preferable that the group derived from the antisense oligonucleotide or antisense oligonucleotide (iv) does not hybridize with the group derived from the antisense oligonucleotide or antisense oligonucleotide (iii). Examples of antisense oligonucleotides that are not the antisense oligonucleotides of the present invention include the following antisense oligonucleotides:

[0178] (1) An antisense oligonucleotide having a central region, a 5' region, and a 3' region, wherein: - the central region consists of at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, wherein the sugar-modified nucleotides are selected from sugar-modified nucleotides excluding 5'-modified nucleotides, and the 3' and 5' ends of the central region are each independently a deoxyribonucleotide or a ribonucleotide, and the antisense oligonucleotide comprises at least one oligonucleotide chain composed of at least four consecutive nucleotides independently selected from deoxyribonucleotides and 5'-modified nucleotides; - the 5' region consists of at least one nucleotide independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and the 3' end is a sugar-modified nucleotide, wherein the sugar-modified nucleotide at the 3' end is linked to the central region and is selected from sugar-modified nucleotides excluding 5'-modified nucleotides, and the antisense oligonucleotide does not comprise an oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides; an antisense oligonucleotide, wherein the 3' region is composed of at least one nucleotide independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and the 5' end of the 3' region is a sugar-modified nucleotide, wherein the sugar-modified nucleotide at the 5' end is linked to the central region and is selected from sugar-modified nucleotides excluding 5'-modified nucleotides; and the antisense oligonucleotide does not contain an oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides.

[0179] Among these, the following antisense oligonucleotide (2) can be mentioned: (2) An antisense oligonucleotide having a central region, a 5' region, and a 3' region, wherein: - the central region consists of at least five nucleotides independently selected from deoxyribonucleotides, - the 5' region consists of at least one nucleotide independently selected from the group consisting of deoxyribonucleotides and sugar-modified nucleotides, the 3' terminus of which is a sugar-modified nucleotide, wherein the sugar-modified nucleotide at the 3' terminus is linked to the central region and selected from sugar-modified nucleotides excluding 5'-modified nucleotides, and does not include an oligonucleotide chain consisting of at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides; - the 3' region consists of at least one nucleotide independently selected from the group consisting of deoxyribonucleotides and sugar-modified nucleotides, the 5' terminus of which is a sugar-modified nucleotide, wherein the sugar-modified nucleotide at the 5' terminus is linked to the central region and selected from sugar-modified nucleotides excluding 5'-modified nucleotides, and An antisense oligonucleotide, which does not contain an oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides.

[0180] In the above (1) and (2), the central region is preferably a gap region, the 5'-side region is preferably a 5'-wing region, and the 3'-side region is preferably a 3'-wing region. Preferred aspects of the 5'-side region and the 3'-side region are the same as those of the 5'-side region and the 3'-side region in the antisense oligonucleotide of the present invention. Preferred aspects of the central region are the same as those of the central region in the antisense oligonucleotide of the present invention.

[0181] Other examples include the following antisense oligonucleotide (so-called mixmer) (3): (3) An antisense oligonucleotide consisting of at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and comprising alternating oligonucleotide strands consisting of 1 to 3 nucleotides independently selected from deoxyribonucleotides and 5'-modified nucleotides and oligonucleotide strands consisting of 1 to 3 nucleotides independently selected from 2'-modified nucleotides and 2',4'-BNAs.

[0182] A linking group containing a non-nucleotide structure can be bonded to an oligonucleotide by the general amidite or H-phosphonate method. For example, after protecting one of the hydroxy groups of a compound having two hydroxy groups, the compound can be converted into an amidite form using an amidite reagent (e.g., 2-cyanoethyl chloro(diisopropylamino)phosphinite, 2-cyanoethyl bis(diisopropylamino)phosphinite, etc.) or into an H-phosphonate form using an H-phosphonate reagent (e.g., diphenyl phosphite, phosphorous acid, etc.), and then bonded to an oligonucleotide. The protected hydroxy group can be deprotected, and further nucleotides can be elongated using a commercially available automated nucleic acid synthesizer. The compound having two hydroxy groups can be synthesized from raw materials such as amino acids, carboxylic acids, and diol compounds by combining protection and deprotection reactions known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, 4th Edition), oxidation reactions, reduction reactions, and condensation reactions (see, for example, Comprehensive Organic Transformations, 2nd Edition, R.C. Larock, Wiley-VCH (1999) for details of oxidation, reduction, and condensation reactions). When the linking group containing a non-nucleotide structure has a functional group other than the two hydroxy groups (e.g., an amino group, a hydroxy group, or a thiol group), it can be efficiently elongated by protecting it with a protecting group known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, 4th Edition). Furthermore, reference can be made to WO2012 / 017919, WO2013 / 103146, WO2013 / 133221, WO2015 / 099187, WO2016 / 104775, etc., for the synthesis of oligonucleotides having linking groups containing non-nucleotide structures.

[0183] Alternatively, two oligonucleotides can be synthesized separately and then linked to a linking group containing a non-nucleotide structure. An example of a synthesis method is shown below. A partial structure having a functional group such as an amino group is linked to the 5'-end of one oligonucleotide by a method known to those skilled in the art (e.g., using 6-(trifluoroacetylamino)hexyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, etc.), and a partial structure having a functional group such as an amino group is linked to the 3'-end of another oligonucleotide by a method known to those skilled in the art (e.g., using 2-((4,4'-dimethoxytrityl)oxymethyl)-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-long-chain alkylamino-CPG (Glen Research, Product No. 20-2958), etc.). The two functional groups of the linking group containing a non-nucleotide structure can be converted to desired functional groups that react with the amino group, etc., to link the two oligonucleotides. For example, the two functional groups of the linking group containing a non-nucleotide structure can be converted to a carboxylic acid, ester, activated ester (e.g., N-hydroxysuccinimide), acid chloride, activated carbonate diester (e.g., 4-nitrophenyl carbonate diester), isocyanate, or the like, followed by reaction under known N-carbonylation conditions to link the two functional groups. The N-carbonylation conditions can be found in, for example, Comprehensive Organic Transformations Second Edition, 1999, John Wiley & Sons, INC. Those skilled in the art can protect one of the two functional groups as necessary, bind one oligonucleotide to the linking group containing a non-nucleotide structure, deprotect the functional group, and then similarly bind another oligonucleotide to the linking group containing a non-nucleotide structure.

[0184] Antisense oligonucleotides or prodrugs thereof may exist via their tautomers and geometric isomers, as well as mixtures thereof or mixtures of each isomer. Furthermore, if an asymmetric center exists or an asymmetric center is generated as a result of isomerization, the present invention also includes compounds that exist as individual optical isomers and mixtures of each optical isomer in any ratio. Furthermore, in the case of compounds with two or more asymmetric centers, diastereomers based on each optical isomer also exist. The present invention also includes compounds that contain all of these forms in any ratio. Furthermore, optically active forms can be obtained by methods well known for this purpose.

[0185] For example, when the antisense oligonucleotide or prodrug thereof of the present invention contains a modified phosphodiester bond (e.g., a phosphorothioate bond) and the phosphorus atom is an asymmetric atom, both oligonucleotides in which the phosphorus atom is stereoregulated and oligonucleotides in which the phosphorus atom is not stereoregulated are included within the scope of the present invention.

[0186] The antisense oligonucleotides, prodrugs thereof, or pharmaceutically acceptable salts thereof of the present invention may exist in any crystalline form or any hydrate depending on the manufacturing conditions, and these crystalline forms, hydrates, and mixtures thereof are also included within the scope of the present invention. They may also exist as solvates containing organic solvents such as acetone, ethanol, 1-propanol, and 2-propanol, and all of these forms are included within the scope of the present invention.

[0187] The antisense oligonucleotides of the present invention or their prodrugs can be converted into pharmaceutically acceptable salts as needed, or can be released from the resulting salts. Examples of pharmaceutically acceptable salts of antisense oligonucleotides or their prodrugs include salts 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.). In particular, when the partial structure represented by -P(=O)(OH)- is -P(=O)(O － The partial structure represented by -P(=O)(SH)-, which forms a phosphorothioate bond, may be converted to an anionic partial structure represented by -P(=O)(SH)-, and form a salt with an alkali metal (lithium, sodium, potassium, etc.), an alkaline earth metal (magnesium, calcium, etc.), ammonium, etc. In addition, the partial structure represented by -P(=O)(SH)-, which forms a phosphorothioate bond, may be converted to an anionic partial structure represented by -P(=O)(S － )-, and similarly form a salt with an alkali metal, alkaline earth metal, ammonium, etc. The same applies to other modified phosphodiester bonds.

[0188] Those skilled in the art can prepare the antisense oligonucleotide or prodrug thereof of the present invention by appropriately selecting a known method. For example, those skilled in the art can design the nucleotide sequence of the antisense oligonucleotide based on the nucleotide sequence information of the target RNA and synthesize it using a commercially available automated nucleic acid synthesizer (Applied Biosystems, Beckman, GeneDesign, etc.). Synthesis can also be performed by an enzymatic reaction. Examples of the enzyme include, but are not limited to, polymerase, ligase, and restriction enzyme. That is, the method for producing the antisense oligonucleotide or prodrug thereof according to this embodiment can include a step of extending the nucleotide chain at the 3' or 5' end.

[0189] Many methods for binding a functional molecule to the oligonucleotide are well known in the art, and reference can be made to, for example, European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp. 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp. 78-92, Expert Opinion on Drug Delivery, 2014, 11, pp. 791-822, etc. For example, the functional molecule and linker may be bound by a known method, and then the resulting linker may be converted into an amidite form using an amiditizing reagent or into an H-phosphonate form using an H-phosphonate reagent, and then bound to the oligonucleotide.

[0190] The resulting oligonucleotide can be purified by reverse phase column chromatography or the like to prepare an antisense oligonucleotide or a prodrug thereof.

[0191] The antisense oligonucleotides of the present invention or their prodrugs can effectively control the expression of target genes. Therefore, the present invention can provide compositions containing the antisense oligonucleotides of the present invention as an active ingredient, for example, compositions for controlling the expression of target genes by antisense effects. In particular, the antisense oligonucleotides of the present invention or their prodrugs can achieve high efficacy when administered at low concentrations, and in some embodiments, pharmaceutical compositions for treating, preventing, or ameliorating diseases associated with increased expression of target genes, such as metabolic diseases, tumors, and infectious diseases, can also be provided.

[0192] The composition containing the antisense oligonucleotide of the present invention or a prodrug thereof can be formulated by known pharmaceutical methods, and can be used enterally (orally, etc.) or parenterally as, for example, capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc.

[0193] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a carrier that is pharmacologically or acceptable as a food or beverage, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0194] The administration form of the composition containing the antisense oligonucleotide of the present invention or a prodrug thereof is not particularly limited, and includes enteral (oral, etc.) or parenteral administration. More preferred examples include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, intranasal administration, intravitreal administration, etc., and administration by infusion.

[0195] Diseases that can be treated, prevented, or ameliorated by the nucleic acid pharmaceuticals using the antisense oligonucleotides or prodrugs thereof of the present invention are not particularly limited, and examples include diseases caused by gene expression, such as metabolic diseases, cardiovascular diseases, tumors, infectious diseases, eye diseases, inflammatory diseases, autoimmune diseases, and rare genetic diseases. More specifically, hypercholesterolemia, hypertriglyceridemia, spinal muscular atrophy, muscular dystrophies (Duchenne muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy (Fukuyama congenital muscular dystrophy, Ullrich congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, integrin deficiency, Walker-Warburg syndrome, etc.), Becker muscular dystrophy, limb-girdle muscular dystrophy, Miyoshi muscular dystrophy, facioscapulohumeral muscular dystrophy, etc.), Huntington's disease, and rheumatoid arthritis. Examples of the diseases that can be targeted include Ralzheimer's disease, transthyretin amyloidosis, familial amyloidotic cardiomyopathy, multiple sclerosis, Crohn's disease, inflammatory bowel disease, acromegaly, type 2 diabetes, chronic nephropathy, respiratory syncytial 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, colon cancer, renal cell carcinoma, bile duct cancer, cervical cancer, liver cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, atopic dermatitis, glaucoma, and age-related macular degeneration. Depending on the type of disease, a gene that causes the disease is set as the target gene, and the expression control sequence (e.g., an antisense sequence) can be appropriately set depending on the sequence of the target gene.

[0196] In addition to primates such as humans, a variety of other mammalian diseases can be treated, prevented, or ameliorated with compositions containing the antisense oligonucleotides of the present invention or their prodrugs. For example, diseases in mammalian species, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, or other bovine, ovine, equine, canine, feline, and rodent species such as mice, can be treated. Compositions containing antisense oligonucleotides can also be used in other species, such as birds (e.g., chickens).

[0197] When a composition containing the antisense oligonucleotide of the present invention or a prodrug thereof is administered to or ingested by an animal, including a human, the dosage or intake is appropriately selected depending on the age, body weight, symptoms, health condition, type of composition (drug, food or drink, etc.) of the subject, etc., but the dosage or intake is preferably 0.0001 mg / kg / day to 100 mg / kg / day in terms of antisense oligonucleotide.

[0198] The antisense oligonucleotides or prodrugs thereof of the present invention can effectively control the expression of target genes while exhibiting reduced toxicity compared to conventional antisense oligonucleotides. Therefore, a method can be provided in which the antisense oligonucleotides or prodrugs thereof of the present invention are administered to animals, including humans, to more safely control the expression of target genes through the antisense effect. Furthermore, a method can be provided for treating, preventing, or ameliorating various diseases accompanied by increased expression of target genes, which comprises administering a composition containing the antisense oligonucleotides or prodrugs thereof of the present invention to animals, including humans.

[0199] Preferred methods for using the antisense oligonucleotide of the present invention include the following: A method for regulating the function of a target RNA, comprising the step of contacting a cell with the antisense oligonucleotide of the present invention or a prodrug thereof. A method for regulating the function of a target RNA in a mammal, comprising the step of administering to the mammal a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or a prodrug thereof. Use of the antisense oligonucleotide of the present invention or a prodrug thereof for regulating the function of a target RNA in a mammal. Use of the antisense oligonucleotide of the present invention or a prodrug thereof for producing a medicament for regulating the function of a target RNA in a mammal. A method for regulating the expression of a target gene, comprising the step of contacting a cell with the antisense oligonucleotide of the present invention or a prodrug thereof. A method for regulating the expression of a target gene in a mammal, comprising the step of administering to the mammal a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or a prodrug thereof. Use of the antisense oligonucleotide of the present invention or a prodrug thereof for regulating the expression of a target gene in a mammal. Use of the antisense oligonucleotide of the present invention or a prodrug thereof for producing a medicament for regulating the expression of a target gene in a mammal.

[0200] In the present invention, control of the function of a target RNA means that the antisense sequence portion coats a part of the target RNA through hybridization, thereby regulating or converting a splicing function such as translation inhibition or exon skipping, or that the function of the target RNA is suppressed through degradation of the target RNA, which can occur when the part where the antisense sequence portion hybridizes with a part of the target RNA is recognized.

[0201] The mammal is preferably a human. The route of administration is preferably enteral. In another embodiment, the route of administration is parenteral.

[0202] The 2'-O-R-ECE nucleotide according to the embodiment of the present invention can be produced by referring to WO2017 / 142054 and the like.

[0203] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples. In the examples, "NMR" means nuclear magnetic resonance, and "(v / v)" means (volume / volume). １ H-NMR data, when provided, were measured at 300 MHz (JNM-ECX300; JEOL Ltd.) or 400 MHz (JNM-ECZ400; JEOL Ltd.) and represent the chemical shift δ (unit: ppm) (splitting pattern, integral value) of the signal using tetramethylsilane as an internal standard. "s" stands for singlet, "d" for doublet, "t" for triplet, "q" for quartet, "dd" for doublet of doublets, "td" for triplet of doublets, "m" for multiplet, "brs" for broad singlet, "CDCl3" for deuterated chloroform, "DMSO-d6" for deuterated dimethyl sulfoxide, and "METHANOL-d4" for deuterated methanol. ３１ When P-NMR data is given, it is measured at 162 MHz (JNM-ECZ400; manufactured by JEOL Ltd.) and the chemical shift δ (unit: ppm) of the signal is shown using phosphoric acid as an internal standard.

[0204] Unless otherwise specified, MS was measured using the ESI (electrospray ionization) method under any of the following analytical conditions 1 to 5. ＋ " means ESI positive ion mode, "ESI － " means ESI negative ion mode.

[0205] Analysis condition 1: Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient conditions: Flow rate: 0.6 mL / min, after starting measurement with a mixing ratio of solution A to solution B of 90 / 10, the mixing ratio of solution A to solution B was changed linearly to 10 / 90 over 3 minutes. Detection wavelength: PDA (210-400 nm)

[0206] Analysis condition 2: Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 0.026% v / v ammonium bicarbonate aqueous solution Solution B: 10% water-acetonitrile solution (0.026% v / v ammonium bicarbonate) Gradient conditions: Flow rate 0.6 mL / min, after starting measurement with a mixing ratio of solution A to solution B of 91 / 9, the mixing ratio of solution A to solution B was changed linearly to 19 / 81 over 3 minutes. Detection wavelength: PDA (210-400 nm)

[0207] Analysis condition 3: Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 0.026% v / v ammonium bicarbonate aqueous solution Solution B: 10% water-acetonitrile solution (0.026% v / v ammonium bicarbonate) Gradient conditions: Flow rate 0.6 mL / min, after starting measurement with a mixing ratio of solution A to solution B of 82 / 18, the mixing ratio of solution A to solution B was changed linearly to 10 / 90 over 2.5 minutes. Detection wavelength: PDA (210-400 nm)

[0208] Analysis condition 4: Apparatus: Waters ACQUITY UPLC H CLASS / QDa Detector Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient conditions: Flow rate: 0.6 mL / min, after starting measurement with a mixing ratio of solution A and solution B of 90 / 10, the mixing ratio of solution A and solution B was changed linearly to 10 / 90 over 3 minutes. Detection wavelength: PDA (210-400 nm)

[0209] Analysis condition 5: Apparatus: Waters ACQUITY UPLC H CLASS / QDa Detector Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40 ° C Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient conditions: Flow rate 0.6 mL / min, after starting measurement with a mixture ratio of solution A and solution B of 80 / 20, the mixture ratio of solution A and solution B was changed linearly to 0 / 100 over 2.5 minutes. Detection wavelength: PDA (210-400 nm)

[0210] Unless otherwise specified, purification by silica gel column chromatography was performed using Purif-Pack (registered trademark)-EX (SI-50 μm) manufactured by SHOKO SCIENCE or Presep (registered trademark) (Luer Lock) NH manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. ２ (HC) was used.

[0211] [Reference production example 1]

[0212] Synthesis of nucleoside analogue: diisopropylphosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-5-(6-acetamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(3-((2-morpholinoethyl)amino)-3-oxopropoxy)tetrahydrofuran-3-yl (Compound 6)

[0213] 4-(2-aminoethyl)morpholine (31.1 mL, 239 mmol) was added to compound 1 (synthesized according to the method described in The Journal of Organic Chemistry, 2011, 76, p. 3042) (9.5 g, 15.9 mmol), and the mixture was stirred at 120°C overnight. After the reaction, water was added, and the mixture was extracted with dichloromethane. The organic layer was recovered, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain a crude product containing the target compound 2. The resulting crude product (8.2 g) was dissolved in dichloromethane (40 mL). To the solution, pyridine (2.5 mL) and acetyl chloride (1.3 mL, 17.7 mmol) were added, followed by further addition of acetyl chloride (0.84 mL, 11.8 mmol), and the mixture was stirred at room temperature overnight. After the reaction, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with dichloromethane. The organic layer was recovered, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 60 / 40 → 0 / 100). The resulting crude product containing Compound 3 was then purified by silica gel column chromatography (ethyl acetate / methanol = 100 / 0 → 50 / 50) to obtain the target Compound 3 (3.7 g, two-stage yield 31%) as a solid. MASS (ESI) ＋ ) (Condition 5): [M + H] ＋ 736 1 H-NMR (400 MHz, CDCl3) δ 0.91-1.15 (m, 24H), 1.62-1.70 (m, 4H), 2.30-2.53 (m, 8H), 2.62 (s, 3H), 2.65-2.74 (m, 1H), 3.34-3.42 (m, 1H), 3.44-3.60 (m, 6H), 3.92-4.06 (m, 2H), 4.25-4.33 (m, 1H), 4.60 (dd, 1H), 6.23 (s, 1H), 8.35 (s, 1H), 8.60 (s, 1H), 8.69 (s, 1H).

[0214] Compound 3 (3.0 g, 4.1 mmol) was dissolved in tetrahydrofuran (15 mL). To this solution, triethylamine (2.8 mL, 20 mmol) and triethylamine trihydrofluoride (2.0 mL, 12 mmol) were added, and the mixture was stirred at room temperature for 2 hours and 30 minutes. After the reaction, methoxytrimethylsilane (3.4 mL, 25 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 50 / 50 → 5 / 90) to obtain a crude product containing the target compound 4. The resulting crude product (2.0 g) was dissolved in pyridine (20 mL). To this solution, 4,4'-dimethoxytrityl chloride (2.44 g, 7.2 mmol) was added, followed by further addition of 4,4'-dimethoxytrityl chloride (0.40 g, 1.2 mmol), and the mixture was stirred at room temperature for 45 minutes. After the reaction, methanol and saturated aqueous sodium bicarbonate solution were added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / methanol / triethylamine = 95 / 5 / 0.5 → 50 / 50 / 0.5). The resulting crude product containing Compound 5 was then further purified by silica gel column chromatography (chloroform / methanol / triethylamine = 100 / 0 / 0.5 → 80 / 20 / 0.5) to obtain the target Compound 5 (2.7 g, two-stage yield 82%) as a solid. MASS (ESI) ＋ ) (Condition 5): [M + H] ＋ 796 1 H NMR (400 MHz, DMSO-d6) δ 2.21-2.26 (5H, m), 2.26-2.32 (6H, m), 3.10 (q, 2H), 3.18-3.28 (m, 2H), 3.51 (t, 4H), 3.64-3.88 (m, 8H), 4.07 (q, 1H), 4.48 (q, 1H), 4.67 (t, 1H), 5.36 (d, 1H), 6.09 (d, 1H), 6.79-6.87 (m, 4H), 7.11-7.29 (m, 7H), 7.33-7.36 (m, 2H), 7.87 (t, 1H), 8.56-8.57 (m, 2H), 10.73 (s, 1H)

[0215] Dichloromethane (20 mL) was added to compound 5 (2.7 g, 3.3 mmol). To the solution, N,N-diisopropylethylamine (1.7 mL, 10.0 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.9 mL, 8.3 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was purified by silica gel column chromatography (chloroform / methanol / triethylamine = 100 / 0 / 0.5 → 80 / 20 / 0.5) to obtain the target compound 6 (2.5 g, yield 70%) as a solid. MASS (ESI ＋ ) (Condition 3): [M + H] ＋ 997 1 H-NMR (400 MHz, DMSO-d6) δ 1.02 (d, J = 6.8 Hz, 3H), 1.09-1.15 (m, 9H), 2.10-2.36 (m, 11H), 2.61 (t, 1H), 2.79-2.82 (m, 1H), 3.01-3.13 (m, 2H), 3.22-3.38 (m, 2H), 3.50-3.90 (m, 16H), 4.18-4.26 (m, 1H), 4.67-4.75 (m, 1H), 4.85-4.93 (m, 1H), 6.09-6.20 (m, 1H), 6.75-6.88 (m, 4H), 7.13-7.29 (m, 7H), 7.33-7.46 (m, 2H), 7.69-7.77 (m, 1H), 8.52-8.70 (m, 2H), 10.71-10.79 (m, 1H). 31 P-NMR (162 MHz, DMSO-d6) δ 149.8, 149.6

[0216]

[0217] Synthesis of nucleoside analogue: diisopropylphosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-5-(4-acetamido-5-methyl-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(3-((2-morpholinoethyl)amino)-3-oxopropoxy)tetrahydrofuran-3-yl (Compound 13)

[0218] Compound 7 (synthesized according to the method described in Organic and Biomolecular Chemistry, Vol. 12, p. 6457 (2014)) (11 g, 14.5 mmol) was dissolved in methanol (11 mL). 4-(2-aminoethyl)morpholine (19 mL, 144 mmol) was added to the resulting solution, and the mixture was stirred at 80°C for 30 minutes. After the reaction, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / ethyl acetate / methanol = 60 / 40 / 0 → 0 / 100 / 0 → 95 / 0 / 5 → 80 / 0 / 20) to obtain a crude product containing the desired compound 8.

[0219] The crude product (8.3 g) obtained as described above was dissolved in acetonitrile (120 mL). Triethylamine (16.8 mL, 121 mmol) and 4-dimethylaminopyridine (5.2 g, 42.2 mmol) were added to the solution, which was then cooled to 0°C. p-toluenesulfonyl chloride (6.9 g, 36.4 mmol) was added, and the mixture was stirred at room temperature for 1 hour and 30 minutes. 28% by mass aqueous ammonia (87 mL) was added to the resulting mixture. After the reaction, 1 M hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The organic layer was recovered, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 70 / 30 → 0 / 100) and then silica gel column chromatography (chloroform / methanol = 90 / 10 → 70 / 30) to obtain a crude product containing the target compound 9.

[0220] The crude product (7.5 g) obtained as described above was dissolved in pyridine (35 mL). The solution was cooled to 0°C, and acetyl chloride (0.94 mL, 13.1 mmol) was added, followed by stirring at room temperature overnight. After the reaction, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 80 / 20) to obtain the target compound 10 (5.5 g, three-stage yield 31%) as a solid. MASS (ESI) ＋ ) (Condition 5): [M + H] ＋ 727 1 H-NMR (400 MHz, CDCl3) δ 0.99-1.07 (m, 24H), 1.11-1.15 (m, 4H), 2.05 (s, 3H), 2.35 (s, 3H), 2.38-2.52 (m, 8H), 3.45-3.62 (m, 8H), 3.62-3.68 (brs, 1H), 3.91-4.01 (m, 2H), 4.14-4.19 (m, 2H), 4.24-4.29 (m, 1H), 5.89 (s, 1H), 7.30 (s, 1H), 7.99 (brs, 1H).

[0221] Compound 10 (5.5 g, 7.5 mmol) was dissolved in tetrahydrofuran (55 mL). Triethylamine (3.1 mL, 22.6 mmol) and triethylamine trihydrofluoride (2.5 mL, 15.1 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour. After the reaction, methoxytrimethylsilane (4.3 mL, 30.1 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 20 / 80) to obtain a crude product containing the target compound 11. The resulting crude product (3.0 g) was azeotroped with toluene, and pyridine (30 mL) was added. 4,4'-Dimethoxytrityl chloride (3.5 g, 10.6 mmol) was added to the solution, and the mixture was stirred overnight. After the reaction, methanol and saturated aqueous sodium bicarbonate were added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol / triethylamine = 100 / 0 / 0.5 → 80 / 20 / 0.5), and the crude product containing Compound 12 was further purified by silica gel column chromatography (ethyl acetate / methanol / triethylamine = 100 / 0 / 0.5 → 50 / 50 / 0.5) to obtain the target Compound 12 (2.9 g, two-stage yield: 49%) as a solid. MASS (ESI) ＋ ) (Condition 3): [M + H] ＋ 787 1 H-NMR (400 MHz, CDCl3) δ 1.35 (s, 3H), 2.36 (s, 3H), 2.39-2.56 (m, 6H), 2.69 (s, 2H), 3.29-3.74 (m, 9H), 3.75-3.83 (m, 6H), 3.94-4.22 (m, 4H), 4.52-4.57 (m, 1H), 5.93-6.02 (m, 1H), 6.80-6.86 (m, 4H), 7.13-7.35 (m, 9H), 7.38-7.44 (m, 2H), 8.15 (brs, 1H)

[0222] Compound 12 (2.5 g, 3.2 mmol) was dissolved in dichloromethane (25 mL). To the solution, N,N-diisopropylethylamine (1.7 mL, 9.6 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.8 mL, 8.0 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was purified by silica gel column chromatography (chloroform / methanol / triethylamine = 95 / 5 / 0.5 → 80 / 20 / 0.5) to obtain the target compound 13 (2.4 g, yield 76%) as a solid. MASS (ESI) ＋ ) (Condition 3): [M + H] ＋ 987 1 H-NMR (400 MHz, DMSO-d6) δ 0.86-0.97 (m, 3H), 1.02-1.14 (m, 9H), 1.40-1.50 (m, 3H), 2.20-2.25 (m, 2H), 2.27-2.37 (m, 7H), 2.52-2.63 (m, 1H), 2.75-2.81 (m, 1H), 3.08-3.19 (m, 2H), 3.3-3.34 (m, 7H), 3.46-3.61 (m, 6H), 3.72-3.73 (m, 6H), 3.90-4.03 (m, 1H), 4.08-4.20 (m, 2H), 4.34-4.47 (m, 1H), 5.82-5.92 (m, 1H), 6.82-6.95 (m, 4H), 7.11-7.20 (m, 1H), 7.22-7.36 (m, 6H), 7.37-7.45 (m, 2H), 7.73-7.81 (m, 1H), 7.89-7.97 (m, 1H), 9.71-9.84 (brs, 1H). 31 P-NMR (162 MHz, DMSO-d6) δ 149.9, 149.0.

[0223]

[0224] Synthesis of nucleoside analogue: (2-cyanoethyl)(2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-(3-((2-morpholinoethyl)amino)-3-oxopropoxy)tetrahydrofuran-3-yl diisopropylphosphoramidite (Compound 20)

[0225] Dioxane (6 mL) and water (6 mL) were added to compound 14 (synthesized according to the method described in PCT / JP2022 / 031705) (6.0 g, 8.8 mmol). Lithium hydroxide monohydrate (839 mg, 20.0 mmol) was added to the solution and stirred at room temperature for 1 hour. After the reaction, the mixture was cooled to 0°C, 1N hydrochloric acid was added, and the mixture was extracted with dichloromethane. The resulting organic layer was collected, and the solvent was evaporated under reduced pressure to obtain a crude product containing the target compound 15. The resulting crude product was dissolved in dimethylformamide (30 mL). To this solution, N,N-diisopropylethylamine (6.2 mL, 35.6 mmol), 4-(2-aminoethyl)morpholine (4.6 mL, 35.6 mmol), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (6.7 g, 17.8 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was recovered, and the solvent was evaporated under reduced pressure. Next, 4-(2-aminoethyl)morpholine (2.3 mL, 17.7 mmol) was added to the resulting residue at room temperature, and the mixture was stirred at 80°C for 1 hour. After the reaction, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 90 / 10 → 60 / 40) to obtain a crude product containing compound 16. The resulting crude product (6.4 g) was dissolved in tetrahydrofuran (60 mL). Triethylamine (3.7 mL, 26.7 mmol) and triethylamine trihydrofluoride (2.9 mL, 17.8 mmol) were added to the solution, and the mixture was stirred at room temperature for 2 hours. After the reaction, methoxytrimethylsilane (1.9 mL, 18.0 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 70 / 30 → 50 / 50) to obtain a crude product containing the target compound 17. The resulting crude product (4 g) was dissolved in methanol (20 mL).The solution was heated to 40°C, N,N-dimethylformamide dimethyl acetal (2.0 mL, 15.0 mmol) was added, and the mixture was stirred at 40°C for 1 hour. Further N,N-dimethylformamide dimethyl acetal (1.0 mL, 7.5 mmol) was added, and the mixture was stirred at 40°C for 1 hour. After the reaction, the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 70 / 30 → 30 / 70) to obtain the target compound 18 (2.0 g, four-step yield: 44%) as a solid. MASS (ESI). ＋ ) (Condition 5): [M + H] ＋ 523 1 H-NMR (400 MHz, METHANOL-d4) δ 2.37-2.51 (m, 8H), 3.11 (s, 3H), 3.21 (s, 3H), 3.24-3.36 (m, 3H), 3.61-3.76 (m, 6H), 3.78-3.88 (m, 2H), 3.89-3.97 (m, 1H), 4.05-4.10 (m, 1H), 4.40-4.53 (m, 2H), 6.00 (d, 1H), 8.10 (s, 1H), 8.65 (s, 1H).

[0226] Compound 18 (2.0 g, 3.8 mmol) was dissolved in pyridine (20 mL). 4,4'-Dimethoxytrityl chloride (2.2 g, 6.5 mmol) was added to the solution, and the mixture was stirred at room temperature for 50 minutes. After the reaction, methanol and saturated aqueous sodium bicarbonate solution were added, and the mixture was extracted with ethyl acetate. The organic layer was collected and evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / methanol / triethylamine = 90 / 10 / 0.5 → 80 / 20 / 0.5) to obtain the desired compound 19 (2.1 g, yield 66%) as a solid. MASS (ESI ＋ ) (Condition 5): [M + H] ＋ 825 1H-NMR (400 MHz, CDCl3) δ 2.40-2.65 (m, 8H), 3.04 (s, 3H), 3.09 (s, 3H), 3.32-3.50 (m, 4H), 3.68-3.72 (m, 4H), 3.76-3.88 (m, 7H), 3.94-4.02 (m, 1H), 4.23-4.27 (m, 1H), 4.42 (dd, 1H), 4.58 (dd, 1H), 6.05 (d, 1H), 6.60-6.68 (brs, 1H), 6.78-6.83 (m, 4H), 7.13-7.24 (m, 2H), 7.25-7.36 (m, 6H), 7.41-7.43 (m, 2H), 7.73 (s, 1H), 8.60 (s, 1H), 9.01-9.13 (brs, 1H).

[0227] Compound 19 (2.1 g, 2.54 mmol) was dissolved in dichloromethane (20 mL). To the solution, N,N-diisopropylethylamine (1.34 mL, 7.70 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.4 mL, 6.40 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was purified by silica gel column chromatography (chloroform / methanol / triethylamine = 100 / 0 / 0.5 → 70 / 30 / 0.5) to obtain the target compound 20 (1.5 g, yield 57%) as a solid. MASS (ESI) ＋ ) (Condition 3): [M + H] ＋ 1026 1H-NMR (300 MHz, DMSO-d6) δ 0.94 (d, 3H), 1.01-1.18 (m, 9H), 2.21-2.37 (m, 10H), 2.53-2.60 (m, 1H), 2.72-2.81 (m, 1H), 3.02 (s, 3H), 3.08-3.13 (m, 5H), 3.19-3.27 (m, 2H), 3.50-3.62 (m, 6H), 3.65-3.85 (m, 9H), 4.03-4.19 (m, 1H), 4.47-4.59 (m, 1H), 5.94-5.99 (m, 1H), 6.79-6.86 (m, 4H), 7.11-7.41 (m, 9H), 7.68-7.78 (m, 1H), 7.86-7.96 (m, 1H), 8.48-8.54 (m, 1H), 11.39 (brs, 1H). 31 P-NMR (162 MHz, DMSO-d6) δ 149.7, 149.9.

[0228]

[0229] Synthesis of nucleoside analog: diisopropylphosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-5-(6-acetamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propoxy)tetrahydrofuran-3-yl (Compound 27)

[0230] Compound 21 (synthesized according to the method described in The Journal of Organic Chemistry, 2011, 76, p. 3042) (12.0 g, 23.6 mmol) was dissolved in t-butanol (60 mL). 2,2,2-trifluoroethyl acrylate (44.2 mL, 353 mmol) was added to the solution, which was then cooled to 0°C. Potassium t-butoxide (23.6 mL, 1 M, 23.6 mmol, tetrahydrofuran solution) was added, and the mixture was stirred at 0°C for 1 hour. After the reaction, aqueous acetic acid was added at 0°C, and the mixture was extracted with methyl t-butyl ether. The resulting organic layer was washed with water, recovered, and the solvent was evaporated under reduced pressure to obtain a crude product containing the target compound 22. 2-(2-aminoethyl)pyridine (15.4 mL) was added to the resulting crude product (15.4 g), and the mixture was stirred at 75°C for 2 hours and 30 minutes. After the reaction, water was added, and the organic layer was extracted with ethyl acetate. The organic layer was collected, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel chromatography (chloroform / ethyl acetate = 90 / 10 → 100 / 0), and the resulting crude product containing compound 23 was further purified by silica gel chromatography (chloroform / methanol) to obtain the target compound 23 (7.2 g, two-step yield 44%) as a solid. MASS (ESI) ＋ ) (Condition 2): [M + H] ＋ 686 1 H-NMR (400 MHz, DMSO-d6) δ 0.88-1.06 (m, 28H), 2.22-2.38 (m, 2H), 2.72-2.83 (m, 2H), 3.34-3.43 (m, 1H), 3.60-3.66 (m, 1H), 3.71-3.80 (m, 1H), 3.85-3.89 (m, 1H), 3.94-4.00 (m, 2H), 4.42 (dd, 1H), 4.54 (t, 1H), 5.30 (d, 1H), 5.96 (d, 1H), 7.17-7.23 (m, 2H), 7.31-7.36 (brs, 2H), 7.62-7.69 (m, 1H), 8.04 (t, 1H), 8.11-8.12 (m, 1H), 8.25-8.27 (m, 1H), 8.44-8.47 (m, 1H).

[0231] Compound 23 (6.5 g, 9.5 mmol) was dissolved in pyridine (13 mL). The solution was heated to 100°C, and acetic anhydride (1.3 mL, 14.2 mmol) was added at 100°C. After stirring at 100°C for 3 hours, acetic anhydride (89.8 μl, 0.95 mmol) was added and stirred at 100°C for 1 hour. After the reaction, water (0.14 mL) was added, and the mixture was stirred at 100°C for 1 hour, followed by extraction with ethyl acetate. The organic layer was recovered, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel chromatography (hexane / ethyl acetate = 50 / 50 → 0 / 100), and the resulting crude product containing compound 24 was further purified by silica gel chromatography (ethyl acetate / methanol = 100 / 0 → 70 / 30) to obtain a crude product containing the desired compound 24. The resulting crude product containing compound 24 (4.5 g) was dissolved in tetrahydrofuran (45 mL). To this solution, triethylamine (4.1 mL, 29.4 mmol) and triethylamine trihydrofluoride (3.0 mL, 18.5 mmol) were added and stirred at room temperature for 1 hour. After the reaction, methoxytrimethylsilane (4.3 mL, 31 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 70 / 30 → 40 / 60) to obtain a crude product containing the target compound 25. The resulting crude product containing compound 25 (3.4 g) was dissolved in pyridine (34 mL). 4,4'-Dimethoxytrityl chloride (4.8 g, 12.6 mmol) was added to the solution, and the mixture was stirred overnight at room temperature. After the reaction, methanol and water were added, followed by extraction with ethyl acetate, and the organic layer was washed with saturated aqueous sodium bicarbonate. The organic layer was collected and evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / ethyl acetate / triethylamine=50 / 50 / 0.5→0 / 100 / 0.5), and the resulting crude product containing compound 26 was further purified by silica gel column chromatography (chloroform / methanol / triethylamine=100 / 0 / 0.5→50 / 50 / 0.5) and then by silica gel column chromatography (ethyl acetate / methanol / triethylamine=100 / 0 / 0.5→40 / 60 / 0.5) to give a crude product containing the target compound 26.The resulting crude product (4.0 g) containing compound 26 was dissolved in dichloromethane (40 mL). To the solution, N,N-diisopropylethylamine (2.7 mL, 15.3 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (2.8 mL, 12.9 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was purified by silica gel column chromatography (chloroform / methanol / triethylamine) to obtain the target compound 27 (3.2 g, four-step yield: 34%) as a solid. MASS (ESI). ＋ ) (Condition 3): [M + H] ＋ 989 1 H-NMR (400 MHz, CDCl3) δ 1.05 (d, 3H), 1.12-1.18 (m, 9H), 2.32-2.44 (m, 5H), 2.57-2.61 (m, 2H), 2.86-2.96 (m, 2H), 3.33-3.38 (m, 1H), 3.51-3.74 (m, 6H), 3.75-3.79 (m, 7H), 3.83-3.90 (m, 2H), 3.98-4.10 (m, 1H), 4.30-4.37 (m, 1H), 4.59-4.63 (m, 1H), 6.08-6.12 (m, 1H), 6.78-6.83 (m, 4H), 7.03-7.34 (m, 9H), 7.38-7.44 (m, 2H), 7.51-7.59 (m, 1H), 8.23-8.26 (m, 1H), 8.40-8.45 (m, 1H), 8.54-8.59 (m, 1H), 8.74 (brs, 1H). 31 P-NMR (162 MHz, CDCl3) δ 150.7, 150.8.

[0232]

[0233] Synthesis of nucleoside analog: diisopropylphosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-(3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propoxy)tetrahydrofuran-3-yl (Compound 32)

[0234] Compound 15 (3.1 g, 4.7 mmol) was dissolved in dimethylformamide (15 mL). To the solution, N,N-diisopropylethylamine (3.3 mL, 18.8 mmol), 2-(2-aminoethyl)pyridine (2.3 mL, 18.8 mmol), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (3.5 g, 9.4 mmol) were added and stirred for 2 hours. Then, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (1.8 g, 4.7 mmol) and 2-(2-aminoethyl)pyridine (1.1 mL, 9.4 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was evaporated under reduced pressure. Next, 2-(2-aminoethyl)pyridine (1.1 mL, 9.4 mmol) was added to the resulting residue, and the mixture was stirred at room temperature for 2 hours. After the reaction, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was evaporated under reduced pressure. Ammonia methanol solution (30 mL) was added to the resulting residue, and the mixture was stirred for 2 hours. After that, ammonia methanol solution (30 mL) was added, and the mixture was stirred for 2 hours. After the reaction, the solvent was evaporated under reduced pressure. Ammonia methanol solution (30 mL) was added to the resulting residue, and the mixture was stirred overnight at 30°C. After the reaction, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol) to obtain a crude product containing compound 28. The resulting crude product (3.2 g) was dissolved in tetrahydrofuran (32 mL). To the solution, triethylamine (1.9 mL, 14 mmol) and triethylamine trihydrofluoride (1.5 mL, 9.0 mmol) were added and stirred at room temperature for 2 hours. After the reaction, methoxytrimethylsilane (2.4 mL, 18 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 90 / 10 → 50 / 50) to obtain a crude product containing compound 29. The resulting crude product (820 mg) was dissolved in methanol (4 mL).To this solution, N,N-dimethylformamide dimethyl acetal (720 μL, 5.35 mmol) was added at 40°C and stirred for 1 hour. Then, N,N-dimethylformamide dimethyl acetal (300 μL, 2.2 mmol) was added at room temperature and stirred at 40°C for 20 minutes. After the reaction, the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 90 / 10 → 60 / 40) to obtain the target compound 30 (820 mg, three-step yield: 34%) as a solid. MASS (ESI). ＋ ) (Condition 4): [M + H] ＋ 515 1 H-NMR (400 MHz, DMSO-d6) δ 2.32 (t, 2H), 2.81 (t, 2H), 3.01 (s, 3H), 3.13 (s, 3H), 3.34-3.39 (m, 2H), 3.51-3.78 (m, 3H), 3.88-3.92 (m, 1H), 4.02-4.12 (m, 1H), 4.31-4.39 (m, 2H), 5.08 (t, 1H), 5.25 (d, 1H), 5.85 (d, 1H), 7.17-7.21 (m, 2H), 7.67 (td, 1H), 8.01-8.04 (m, 1H), 8.07 (s, 1H), 8.43-8.49 (m, 1H), 8.54 (s, 1H), 11.34 (brs, 1H).

[0235] Compound 30 (830 mg, 1.61 mmol) was dissolved in pyridine (8.3 mL). 4,4'-Dimethoxytrityl chloride (929 mg, 2.74 mmol) was added to the solution, and the mixture was stirred at room temperature for 50 minutes. After the reaction, methanol and saturated aqueous sodium bicarbonate solution were added, and the mixture was extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol / triethylamine) to obtain the desired compound 31 (1.14 g, yield 87%) as a solid. MASS (ESI ＋ ) (Condition 3): [M + H] ＋ 817 1H-NMR (400 MHz, DMSO-d6) δ 2.34 (t, 2H), 2.81 (t, 2H), 3.01 (s, 3H), 3.09 (s, 3H), 3.12-3.26 (m, 2H), 3.34-3.39 (m, 3H), 3.72-3.82 (m, 7H), 3.96-4.02 (m, 1H), 4.29-4.36 (m, 1H), 4.40 (t, 1H), 5.29-5.30 (m, 1H), 5.90 (d, 1H), 6.81-6.85 (m, 4H), 7.12-7.27 (m, 9H), 7.34-7.36 (m, 2H), 7.65 (td, 1H), 7.91 (s, 1H), 8.01-8.04 (m, 1H), 8.45-8.47 (m, 1H), 8.52 (s, 1H), 11.37 (brs, 1H).

[0236] Compound 31 (950 mg, 1.16 mmol) was dissolved in toluene, and the solution was evaporated under reduced pressure. The solution was then dissolved in dichloromethane (11 mL). N,N-diisopropylethylamine (2.0 mL, 11.3 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.3 mL, 5.8 mmol) were added to the resulting solution, and the mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was purified by silica gel column chromatography (chloroform / acetone / triethylamine = 90 / 10 / 0.5 → 5 / 95 / 0.5). The resulting crude product containing compound 32 was further purified by silica gel column chromatography (chloroform / methanol / triethylamine = 90 / 10 / 0.5 → 70 / 30 / 0.5) to obtain the desired compound 32 (720 mg, yield 61%) as a solid. MASS (ESI) ＋ ) (Condition 3): [M + H] ＋ 1018 1H-NMR (400 MHz, DMSO-d6) δ 0.93 (d, 3H), 1.07-1.13 (m, 9H), 2.23-2.34 (m, 2 H), 2.54 (t, 1H), 2.72-2.82 (m, 3H), 3.00 (d, 3H), 3.07 (d, 3H), 3.18-3.39 (m, 5H), 3.46-3.64 (m, 3H), 3.68-3.74 (m, 7H), 3.78 (t, 2H), 4.08-4.17 (m, 1H), 4.48-4.59 (m, 1H), 5.96 (d, 1H), 6.79-6.85 (m, 4H), 7.13-7.27 (m, 9H), 7.32-7.36 (m, 2H), 7.62-7.67 (m, 1H), 7.85-7.91 (m, 1H), 7.91-7.96 (m, 1H), 8.43-8.47 (m, 1H), 8.48-8.53 (m, 1H), 11.38 (brs, 1H). 31 P-NMR (162 MHz, DMSO-d6) δ 149.7, 149.9.

[0237]

[0238] Synthesis of nucleoside analog: diisopropylphosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-4-(3-((2-(1H-benzo(d)imidazol-1-yl)ethyl)amino)-3-oxopropoxy)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (Compound 38)

[0239] Compound 1 (6.3 g, 10.7 mmol) (synthesized according to the method described in The Journal of Organic Chemistry, 2011, 76, p. 3042) was dissolved in water (12 mL) and dioxane (30 mL). Lithium hydroxide monohydrate (1.8 g, 42.8 mmol) was added to the solution, and the mixture was stirred for 4 hours. After the reaction, the solvent was evaporated under reduced pressure to obtain a crude product containing compound 33. 2-(1H-benzimidazol-1-yl)ethylamine (3.7 g, 22.7 mmol) was added to the resulting crude product containing compound 33 (3.4 g). The resulting mixture was dissolved in dimethylformamide (34 mL), N,N-diisopropylethylamine (2.9 mL, 17.0 mmol) was added, and the mixture was cooled to 0°C. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (6.5 g, 17.0 mmol) was added, and the mixture was stirred at room temperature for 3 hours. After the reaction, the mixture was cooled to 0°C, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The resulting organic layer was collected, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel chromatography (chloroform / methanol = 100 / 0 → 70 / 30) to obtain the desired compound 34 (3.4 g, two-step yield 81%). MASS (ESI ＋ ) (Condition 1): [M + H ２ O + H] ＋ 743 1 H-NMR (400 MHz, DMSO-d6) δ 0.88-1.04 (m, 24H), 2.25-2.33 (m, 4H), 3.39-3.46 (m, 2H), 3.51-3.62 (m, 2H), 3.71-3.77 (m, 1H), 3.85-3.89 (m, 1H), 3.95-4.01 (m, 2H), 4.26 (t, 2H), 4.38-4.43 (m, 1H), 4.53 (t, 1H), 5.30 (d, 1H), 5.98 (d, 1H), 7.12-7.30 (m, 5H), 7.54-7.58 (m, 1H), 7.61-7.64 (m, 1H), 8.10 (m 2H), 8.27 (s, 1H).

[0240] Compound 34 (3.4 g, 4.6 mmol) was dissolved in tetrahydrofuran (34 mL). Triethylamine (2.6 mL, 18.4 mmol) and triethylamine trihydrofluoride (2.3 mL, 13.8 mmol) were added to the solution, and the mixture was stirred at room temperature for 3 hours. After the reaction, methoxytrimethylsilane (3.2 mL, 23.0 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 / 0.5 → 50 / 50 / 0.5) to obtain a crude product (1.1 g) containing compound 35. The resulting crude product (960 mg) containing compound 35 was dissolved in pyridine (34 mL). The solution was cooled to 0 °C, trimethylsilyl chloride (1.26 mL, 9.9 mmol) was added, and the mixture was stirred at 0 °C for 30 minutes. To the resulting reaction solution, benzoyl chloride (1.1 mL, 9.9 mmol) was added and stirred at room temperature for 2 hours. After the reaction, the reaction solution was cooled to 0°C, and aqueous ammonia (13.4 mL, 14%, w / w) was added. The mixture was stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate / chloroform / methanol) to obtain a crude product (1.1 g) containing compound 36. The resulting crude product (1.1 g) containing compound 36 was dissolved in pyridine (11 mL). 4,4'-Dimethoxytrityl chloride (1.1 g, 3.4 mmol) was added to the solution, and the mixture was stirred at room temperature for 45 minutes. Further 4,4'-Dimethoxytrityl chloride (1.1 g, 3.4 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After the reaction, methanol and saturated aqueous sodium bicarbonate were added, and the mixture was extracted with ethyl acetate. The organic layer was recovered, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol / triethylamine), and the resulting crude product containing Compound 37 was further purified by silica gel column chromatography (ethyl acetate / methanol / triethylamine) to obtain the target Compound 37 (1.4 g, three-stage yield: 34%) as a solid. － ) (Condition 4): [M-H] － 887 1H-NMR (400 MHz, CDCl3) δ 2.39-2.54 (m, 2H), 3.39-3.66 (m, 3H), 3.70-3.82 (m, 9H), 3.93 (td, 1H), 4.28-4.46 (m, 2H), 4.54-4.63 (m, 3H), 6.12 (d, 1H), 6.78-6.81 (m, 4H), 7.14-7.34 (m, 10H), 7.37-7.45 (m, 3H), 7.50-7.54 (m, 2H), 7.59-7.67 (m, 2H), 8.01-8.03 (m, 2H), 8.24 (s, 1H), 8.66 (s, 1H), 9.10 (s, 1H).

[0241] Compound 37 (1.4 g, 1.6 mmol) was dissolved in dichloromethane (14 mL). To the solution, N,N-diisopropylethylamine (820 μL, 4.7 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (880 μL, 3.9 mmol) were added and stirred at room temperature for 30 minutes. After the reaction, the crude product containing compound 38 was purified by silica gel column chromatography (chloroform / methanol / triethylamine). The crude product was further purified by silica gel column chromatography (hexane / ethyl acetate / methanol) using Chromatorex (registered trademark) DIOL MB100-40 / 75 (Fuji Silysia Corporation), yielding the target compound 38 (634 mg, yield 37%) as a solid. MASS (ESI － ) (Condition 1): [M-H] － 1088 1H-NMR (400 MHz, DMSO-d6) δ 1.01 (d, 3H), 1.08-1.24 (m, 9H), 2.21-2.27 (m, 2H), 2.59 (t, 1H), 2.75-2.80 (m, 1H), 2.88 (t, 1H), 3.21-3.26 (m, 1H), 3.52-3.66 (m, 4H), 3.68-3.70 (m, 8H), 3.73-3.88 (m, 2H), 4.17-4.22 (m, 3H), 4.66-4.74 (m, 1H), 4.88-4.92 (m, 1H), 6.15-6.17 (m, 1H), 6.78-6.83 (m, 4H), 7.12-7.26 (m, 9H), 7.33-7.35 (m, 2H), 7.48-7.59 (m, 3H), 7.59-7.66 (m, 2H), 7.91-8.03 (m, 3H), 8.08 (s, 1H), 8.61-8.63 (m, 2H), 11.18-11.24 (brs, 1H). 31 P-NMR (162 MHz, DMSO-d6) δ 149.7, 149.8.

[0242]

[0243] Synthesis of nucleoside analog: diisopropyl (2-cyanoethyl) phosphoramidate (2R,3R,4R,5R)-4-(3-((2-(1H-benzo(d)imidazol-1-yl)ethyl)amino)-3-oxopropoxy)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (Compound 43)

[0244] Compound 14 (4.1 g, 6.0 mmol) (synthesized according to the method described in PCT / JP2022 / 031705) was dissolved in a mixed solvent of dioxane (41 mL) and water (21 mL). Lithium hydroxide monohydrate (785 mg, 18.7 mmol) was added to the solution and stirred at room temperature for 1 hour. After the reaction, 1 M hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product containing compound 15. 2-(1H-benzimidazol-1-yl)ethylamine (4.8 g, 30 mmol) was added to the resulting crude product containing compound 15 (3.7 g), and the mixture was then dissolved in dimethylformamide (37 mL). To this solution, N,N-diisopropylethylamine (3.1 mL, 18 mmol) was added at room temperature, followed by the addition of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (6.8 g, 18 mmol) at 0°C, followed by stirring at room temperature for 2 hours and 30 minutes. After the reaction, the mixture was cooled to 0°C, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was recovered, and the solvent was distilled off under reduced pressure. Ammonia methanol solution was added to the resulting residue, and the mixture was stirred overnight. The solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 50 / 50), and the resulting crude product containing compound 39 was further purified by silica gel column chromatography (chloroform / methanol → 100 / 0 → 70 / 30) to obtain the desired compound 39 (1.7 g, two-step yield 37%) as a solid. MASS (ESI ＋ ) (Condition 1): [M + H ２ O + H] ＋ 759 1H-NMR (300 MHz, DMSO-d6) δ 0.78-1.02 (m, 28H), 2.22-2.29 (t, 2H), 3.38-3.96 (m, 6H), 4.22-4.35 (m, 5H), 5.78 (d, 1H), 6.51-6.62 (brs, 2H), 7.15-7.25 (m, 3H), 7.54-7.64 (m, 2H), 7.83 (s, 1H), 8.08-8.18 (m, 2H).

[0245] Compound 39 (1.7 g, 2.2 mmol) was dissolved in tetrahydrofuran (17 mL). Triethylamine (1.2 mL, 8.8 mmol) and triethylamine trihydrofluoride (1.1 mL, 6.6 mmol) were added to the solution, and the mixture was stirred at room temperature overnight. After the reaction, methoxytrimethylsilane (1.5 mL, 11 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 90 / 10 → 60 / 40) to obtain a crude product containing compound 40. The resulting crude product (1.0 g) containing compound 40 was dissolved in methanol (15 mL). To this solution, N,N-dimethylformamide dimethyl acetal (820 μL, 6.1 mmol) was added dropwise at 40°C, and the mixture was stirred at 40°C for 1 hour. Then, N,N-dimethylformamide dimethyl acetal (410 μL, 3.1 mmol) was added dropwise at 40°C, and the mixture was stirred at 40°C for 1 hour. After the reaction, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol = 90 / 10 → 20 / 80) to obtain the target compound 41 (1.1 g, two-stage yield 90%) as a solid. MASS (ESI ＋ ) (Condition 1): [M + H] ＋ 554 1H-NMR (400 MHz, DMSO-d6) δ 2.23-2.35 (m, 2H), 3.29-3.46 (m, 8H), 3.51-3.62 (m, 2H), 3.70-3.75 (m, 1H), 3.91-3.93 (m, 1H), 4.26-4.38 (m, 3H), 5.10 (t, 1H), 5.20 (d, 1H), 5.78 (d, 1H), 6.40-6.65 (brs, 2H), 7.14-7.27 (m, 3H), 7.57 (d, 1H), 7.64-7.66 (m, 1H), 7.95 (s, 1H), 8.11-8.18 (m, 2H), 10.59-10.65 (brs, 1H).

[0246] Compound 41 (1.1 g, 2.0 mmol) was dissolved in pyridine, and the solution was evaporated under reduced pressure. The solution was then dissolved in pyridine (10 mL). 4,4'-Dimethoxytrityl chloride (1.2 g, 3.6 mmol) was added to the solution and stirred at room temperature for 2 hours. 4,4'-Dimethoxytrityl chloride (1.2 g, 3.6 mmol) was then added and stirred overnight. After the reaction, methanol and saturated aqueous sodium bicarbonate solution were added, and the mixture was extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 70 / 30) to obtain the desired compound 42 (1.4 g, yield 79%) as a solid. MASS (ESI ＋ ) (Condition 3): [M + H] ＋ 856 1H-NMR (400 MHz, DMSO-d6) δ 2.29-2.34 (m, 4H), 3.21-3.46 (m, 8H), 3.64-3.85 (m, 8H), 4.03-4.12 (m, 1H), 4.22-4.29 (m, 2H), 4.46-4.52 (m, 1H), 4.70 (t, 1H), 5.37 (d, 1H), 6.14 (d, 1H), 6.80-6.88 (m, 4H), 7.11-7.26 (m, 5H), 7.30-7.43 (m, 2H), 7.52-7.68 (m, 5H), 8.03 (d, 2H), 8.09-8.15 (m, 1H), 8.59 (s, 1H), 8.65 (s, 1H),11.18-11.26 (d, 1H).

[0247] Compound 42 (1.4 g, 1.6 mmol) was dissolved in dichloromethane (14.0 mL). To the solution, N,N-diisopropylethylamine (808 μL, 4.7 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (872 μL, 3.9 mmol) were added and stirred at room temperature for 3 hours. Then, N,N-diisopropylethylamine (380 μL, 2.2 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (100 μL, 0.4 mmol) were added to the reaction solution and stirred for 2 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol / triethylamine = 100 / 0 / 0.5 → 70 / 30 / 0.5), and the resulting crude product containing compound 43 was further purified by silica gel column chromatography (chloroform / acetone / triethylamine = 50 / 50 / 0.5 → 0 / 100 / 0.5) and then by silica gel column chromatography (chloroform / methanol / triethylamine = 70 / 30 / 0.5) to obtain the target compound 43 (1.0 g, yield 60%) as a solid. MASS (ESI) ＋ ) (Condition 2): [M + H] ＋ 1057 1H-NMR (400 MHz, DMSO-d6) δ 0.93 (d, 3H), 1.01-1.15 (m, 9H), 2.20-2.31 (m, 2H), 2.52-2.57 (m, 1H), 2.72-2.81 (m, 1H), 2.93-3.06 (m, 6H), 3.15-3.60 (m, 7H), 3.67-3.86 (m, 9H), 4.11-4.33 (m, 3H), 4.49-4.58 (m, 2H), 5.98 (d, 1H), 6.77-6.84 (m, 4H), 7.11-7.27 (m, 9H), 7.33-7.39 (m, 2H), 7.53 (dd, 1H), 7.61 (dd, 1H), 7.94-8.10 (m, 3H), 8.42-8.46 (m, 1H), 11.37 (brs, 1H). 31 P-NMR (162 MHz, DMSO-d6) δ 149.7, 149.9.

[0248] [Production Example 1] Synthesis and Purification of Antisense Oligonucleotides The antisense oligonucleotides listed in Table 1 were prepared using an automatic nucleic acid synthesizer according to the following procedure. In the sequence notations in Table 1, "(L)" means an LNA nucleotide, "(Mo)" means a 2'-MorECE nucleotide, "(Py)" means a 2'-PyECE nucleotide, "(Bz)" means a 2'-BimECE nucleotide, lowercase letters mean deoxyribonucleotides, "5(x)" means that the nucleobase of the deoxyribonucleotide is 5-methylcytosine, "^" means a phosphorothioate bond, "5" means that the base of the nucleotide is 5-methylcytosine, and "X" means a nucleotide represented by the following formula (4): (where * represents the bonding position to the oligonucleotide) is bonded to the oxygen atom of the 5'-terminal hydroxy group of the oligonucleotide via a group represented by -P(=O)(OH)-.

[0249] The columns in Table 1 are denoted as follows: "Compd. No.": Compound number. "Chemical Structure": Chemical structure. "Expected MW": Theoretical molecular weight (Da) of each compound. "Observed MW": The observed molecular weight (Da) of each compound. "Target RNA": The name of the mouse gene (transcript) targeted by each compound.

[0250]

[0251] The antisense oligonucleotides shown in Table 1 were synthesized using an automatic nucleic acid synthesizer nS-8II (Gene Design) or an automatic nucleic acid synthesizer M-8-SE (Nihon Techno Service). Solid-phase-supported oligonucleotide analogs, whose 5'-ends were protected with a DMTr group, were treated with saturated aqueous ammonia. These were then purified by reverse-phase HPLC (Shimadzu Corporation, using a Waters XBridge® BEH C18 OBD® Prep Column 130A, 5 μm (10 mm × 250 mm) as the preparative column).

[0252] The synthesized antisense oligonucleotides were subjected to mass analysis by ESI-MS under the following analytical condition 6, and each was identified as the target compound (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 1).

[0253] (Analysis Condition 6) Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: ACQUITY UPLC Oligonucleotide BEH C18 Column (130 Å, 1.7 μm, 2.1 × 50 mm) Column temperature: 60°C Solvent: Solution A: Water (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Solution B: 90% v / v aqueous methanol solution (0.75% v / v hexafluoroisopropanol; 0.038% v / v triethylamine; 5 μM ethylenediaminetetraacetic acid) Gradient conditions: Measurement was started at a flow rate of 0.36 mL / min with a fixed mixing ratio of Solution A to Solution B of 95 / 5 (v / v). 0.56 minutes later, the mixing ratio of Solution A to Solution B was linearly changed to 40 / 60 (v / v) over a period of 5.5 minutes. Detection wavelength: 260 nm. Deconvolution: Ion signals were analyzed using ProMass for MassLynx Software (Waters).

[0254] Evaluation Example 1 Antisense Inhibition of Mouse Cxcl12 in 3T3-L1 Cells (Mouse Fibroblasts) 3T3-L1 cells were seeded in a 96-well plate at a density of 10,000 cells / well, and after approximately 24 hours, P22790047, P23790187, P23790188, P23790189, P23790190, P23790191, and P23790192 were added to a final concentration of 0.3 to 30 nM in the presence of Lipofectamine® 3000 Reagent (Thermo Fisher Scientific) (Lipofection). After 24 hours, cell lysates containing RNA were prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio). Mouse Cxcl12 gene expression levels were measured by quantitative real-time PCR using One Step PrimeScript® III RT-qPCR Mix (Takara Bio) and TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA level of the housekeeping gene Gapdh was simultaneously quantified, and the Cxcl12 mRNA level relative to the Gapdh mRNA level was evaluated as the Cxcl12 expression level. Results are expressed as percent inhibition at 10 nM relative to untreated control cells and as 50% inhibitory concentrations (IC) calculated from percent expression of Cxcl12 using GraphPad PRISM 9.0 (Dotmatics). ５０ ) are shown in Table 2.

[0255] Evaluation Example 2 Inhibition of Increased Mouse Caspase 3 / 7 Activity in NMuLi Cells (Mouse Hepatocytes) Approximately 24 hours later, P22790047, P23790187, P23790188, P23790189, P23790190, P23790191, and P23790192 were added to NMuLi cells seeded at a density of 10,000 cells / well in a 96-well plate in the presence of Lipofectamine® 3000 Reagent (Thermo Fisher Scientific) to a final concentration of 10 nM (Lipofection). After 24 hours, caspase 3 / 7 activity was measured using the Caspase-Glo® 3 / 7 Assay System (Promega). The results are shown in Table 2 as the percentage of caspase 3 / 7 activity relative to untreated control cells and as the percentage of caspase 3 / 7 activity divided by the percentage of Cxcl12 expression inhibition in 3T3-L1 cells at 10 nM.

[0256]

[0257] As is clear from Table 2, compounds P23790187 to P23790192 were able to significantly reduce toxicity while maintaining the same or weaker mRNA inhibitory activity as P22790047 (an LNA gapmer without an R-ECE nucleotide).

[0258] Evaluation Example 3 Antisense Suppression of Mouse F11 in Hepa-1c1c7 Cells (Mouse Hepatocytes) Hepa-1c1c7 cells were seeded in a 96-well plate at a density of 5,000 cells / well, and after approximately 24 hours, P22790056, P22790062, and P22790063 were added to a final concentration of 0.1 to 30 nM in the presence of Lipofectamine® 3000 Reagent (Thermo Fisher Scientific) (Lipofection). After 24 hours, cell lysates containing RNA were prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio). Then, mouse F11 gene expression levels were measured by quantitative real-time PCR using One Step PrimeScript® III RT-qPCR Mix (Takara Bio) and TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA level of the housekeeping gene Gapdh was also quantified, and the F11 mRNA level relative to the Gapdh mRNA level was evaluated as the F11 expression level. Results are expressed as percent inhibition at 10 nM relative to untreated control cells and as 50% inhibitory concentrations (IC) calculated from percent expression of F11 using GraphPad PRISM 9.0 (Dotmatics). ５０ ) are shown in Table 3.

[0259] Evaluation Example 4 Inhibition of Increased Mouse Caspase 3 / 7 Activity in Hepa-1c1c7 Cells (Mouse Hepatocytes) Hepa-1c1c7 cells were seeded in a 96-well plate at a density of 5,000 cells / well. After approximately 24 hours, P22790056, P22790062, and P22790063 were added to a final concentration of 10 nM in the presence of Lipofectamine® 3000 Reagent (Thermo Fisher Scientific) (Lipofection). After 24 hours, caspase 3 / 7 activity was measured using the Caspase-Glo® 3 / 7 Assay System (Promega). The results are shown in Table 3 as the percentage of caspase 3 / 7 activity relative to untreated control cells and as the percentage of caspase 3 / 7 activity divided by the percentage of F11 expression inhibition at 10 nM in Hepa-1c1c7.

[0260]

[0261] As is clear from Table 3, compounds P22790062 and P22790063 were able to significantly reduce toxicity while maintaining the same or weaker mRNA inhibitory activity compared to P22790056 (an LNA gapmer without an R-ECE nucleotide).

[0262] Evaluation Example 5: Antisense inhibition of mouse Pten in 3T3-L1 cells Using the same evaluation method as in Evaluation Example 1, P23790177, P23790204, P23790205, P23790206, and P23790208 were added to a final concentration of 0.3 to 30 nM, and the amount of Pten mRNA relative to the amount of mouse Gapdh mRNA was evaluated as the Pten expression level. The results were reported as the percentage inhibition rate at 10 nM relative to untreated control cells, and the 50% inhibitory concentration (IC) calculated from the percentage expression of Pten. ５０ ) are shown in Table 4.

[0263] [Evaluation Example 6] Inhibition of Increased Mouse Caspase 3 / 7 Activity in NMuLi Cells Caspase 3 / 7 activity was measured using P23790177, P23790204, P23790205, P23790206, and P23790208 at a final concentration of 10 nM using the same evaluation method as in Evaluation Example 2. The results are shown in Table 4 as the percentage of caspase 3 / 7 activity relative to untreated control cells and the percentage of caspase 3 / 7 activity divided by the percentage of Pten expression inhibition in 3T3-L1 at 10 nM.

[0264]

[0265] As is clear from Table 4, compounds P23790204, P23790205, P23790206 and P23790208 were able to significantly reduce toxicity while maintaining the same or weaker mRNA inhibitory activity compared to P23790177 (an LNA gapmer without an R-ECE nucleotide).

[0266] Evaluation Example 7: Antisense inhibition of mouse Sod1 in 3T3-L1 cells Using the same evaluation method as in Evaluation Example 1, P23790178, P23790212, P23790213, P23790214, P23790215, P23790216, and P23790217 were used to give final concentrations of 0.3 to 30 nM, and the amount of Sod1 mRNA relative to the amount of mouse Gapdh mRNA was evaluated as the Sod1 expression level. The results were reported as the percentage inhibition rate at 10 nM relative to untreated control cells and the 50% inhibitory concentration (IC) calculated from the percentage expression of Sod1. ５０ ) are shown in Table 5.

[0267] [Evaluation Example 8] Inhibition of Increased Mouse Caspase 3 / 7 Activity in NMuLi Cells Using the same evaluation method as in Evaluation Example 2, P23790178, P23790212, P23790213, P23790214, P23790215, P23790216, and P23790217 were added to a final concentration of 10 nM, and caspase 3 / 7 activity was measured. The results are shown in Table 5 as the percentage of caspase 3 / 7 activity relative to untreated control cells and the percentage of caspase 3 / 7 activity divided by the percentage of Sod1 expression inhibition at 10 nM in 3T3-L1 cells.

[0268]

[0269] As is clear from Table 5, compounds P23790212 to P22790217 were able to significantly reduce toxicity while maintaining the same or weaker mRNA inhibitory activity as P23790178 (an LNA gapmer without an R-ECE nucleotide).

[0270] Evaluation Example 9: Antisense inhibition of mouse Dnm2 in 3T3-L1 cells Using the same evaluation method as in Evaluation Example 1, P23790181, P23790221, and P23790222 were used to give final concentrations of 0.3 to 30 nM, and the amount of Dnm2 mRNA relative to the amount of mouse Gapdh mRNA was evaluated as the Dnm2 expression level. The results were reported as the percentage inhibition rate at 10 nM relative to untreated control cells, and the 50% inhibitory concentration (IC) calculated from the percentage expression of Dnm2. ５０ ) are shown in Table 6.

[0271] [Evaluation Example 10] Inhibition of Increased Mouse Caspase 3 / 7 Activity in NMuLi Cells Caspase 3 / 7 activity was measured using P23790181, P23790221, and P23790222 at a final concentration of 10 nM using the same evaluation method as in Evaluation Example 2. The results are shown in Table 6 as the percentage of caspase 3 / 7 activity relative to untreated control cells and the percentage of caspase 3 / 7 activity divided by the percentage of Dnm2 expression inhibition in 3T3-L1 at 10 nM.

[0272]

[0273] As is clear from Table 6, compounds P23790221 and P22790222 were able to significantly reduce toxicity while maintaining the same or weaker mRNA inhibitory activity as P23790181 (an LNA gapmer without an R-ECE nucleotide).

[0274] Evaluation Example 11: Antisense inhibition of Cxcl12 in mice Balb / c mice (male, 6 weeks old, Jackson Laboratory Japan) were subcutaneously administered P23790268 and P23790270 dissolved in saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) to the back at doses of 0.04, 0.2, 1.0, and 5.0 mg / kg per mouse in terms of antisense oligonucleotide. As a control, saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) alone was administered. Three days after administration, livers were harvested under isoflurane anesthesia. After RNA isolation from liver using the MagMAX® mirVana® Total RNA Isolation Kit (Thermo Fisher Scientific), mouse Cxcl12 gene expression levels were measured by quantitative real-time PCR using One Step PrimeScript® III RT-qPCR Mix (Takara Bio) and TaqMan® Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA level of the housekeeping gene Gapdh was also quantified, and the Cxcl12 mRNA level relative to the Gapdh mRNA level was evaluated as the Cxcl12 expression level. The results were calculated as the 50% effective dose (EC ) using GraphPad PRISM 9.0 (Dotmatics) based on the percent expression of Cxcl12 relative to the untreated control group. ５０ ) were calculated and are shown in Table 7.

[0275] Evaluation Example 12: Inhibition of Hepatotoxicity in Mice Balb / c mice (male, 6 weeks old, Jackson Laboratory Japan) were subcutaneously administered P23790268 and P23790270 dissolved in saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) to the back at doses of 0.04, 0.2, 1.0, and 5.0 mg / kg of antisense oligonucleotide per mouse. As a control, saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) alone was administered. Three days after administration, venous blood was collected from the abdominal vena cava under isoflurane anesthesia. Plasma was obtained by centrifugation, and plasma AST and ALT levels were measured using Fuji DryChem Slide GOT / AST-PIII and Fuji DryChem Slide GPT / ALT-PIII. The results are shown in Table 7.

[0276]

[0277] As is clear from Table 7, compound P23790270 was able to significantly reduce toxicity while maintaining a weak level of mRNA inhibitory activity compared to P23790268 (an LNA gapmer without an R-ECE nucleotide).

[0278] The oligonucleotides of the present invention are believed to be able to suppress off-target effects and therefore reduce toxicity, and are useful as pharmaceutical compositions for treating and preventing diseases associated with increased function of target RNA and / or increased expression of target genes, such as metabolic diseases, tumors, and infectious diseases.

Claims

1. An antisense oligonucleotide having a central region, a 5' region and a 3' region, wherein: the central region is composed of at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, the 3' and 5' ends of which are each independently selected from deoxyribonucleotides and 5'-modified nucleotides, and comprises at least one oligonucleotide strand composed of at least four consecutive nucleotides selected from deoxyribonucleotides and 5'-modified nucleotides; the 5' region is composed of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, comprises at least one 2'-4' bridged nucleotide, and the 3' end of which is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the 3' end nucleotide is bound to the central region, and does not comprise an oligonucleotide strand composed of at least four consecutive nucleotides selected from deoxyribonucleotides and 5'-modified nucleotides; the 3' region is composed of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, comprises at least one 2'-4' bridged nucleotide, and comprises a 2'-4' bridged nucleotide or a 2'-modified nucleotide. An antisense oligonucleotide consisting of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, containing at least one 2'-4' bridged nucleotide, the 5' end of which is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the 5' end nucleotide is linked to a central region, and does not contain an oligonucleotide chain consisting of at least four consecutive nucleotides selected from deoxyribonucleotides and 5'-modified nucleotides; and containing at least one 2'-O-R-ECE nucleotide in at least one of the central region, the 5' side region and the 3' side region.

2. The 2'-O-R-ECE nucleotide has the following formula (I): (wherein Bx is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group and a protected sulfanyl group), R １ , R ２ , R ３ and R ４ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group); Y is NR ５ R ６ (The R ５ and R ６ each independently represents a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy 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 a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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 the R ５ and R ６ form a 3-11-membered nitrogen-containing non-aromatic heterocycle together with the nitrogen atom to which they are bonded (the 3-11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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 a C2-9 aromatic heterocyclic group (which is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl 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 haloalkyl 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), n is an integer from 1 to 3, provided that when n is 2 or 3, then 2 or 3 R ３ and R ４ The antisense oligonucleotide according to claim 1, which is a nucleotide containing a partial structure represented by the following formula (III):

3. R １ , R ２ , R ３ and R ４ The antisense oligonucleotide of claim 2, wherein is a hydrogen atom.

4. Y is NR ５ R ６ and the R ５ and R ６ are taken together with the nitrogen atom to which they are attached to form a 4-8 membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene groups, or Y is a C2-9 aromatic heterocyclic group.

5. Y is NR ５ R ６ and the R ５ and R ６ The antisense oligonucleotide of claim 2 , wherein, together with the nitrogen atom to which they are attached, form a morpholine.

6. The antisense oligonucleotide of any one of claims 2 to 4, wherein Y is a pyridyl group, an imidazolyl group, or a benzimidazolyl group.

7. The antisense oligonucleotide of any one of claims 2 to 6, wherein n is 2.

8. An antisense oligonucleotide according to any one of claims 1 to 7, wherein the central region consists of 5 to 15 nucleotides, and the 5' region and the 3' region each independently consist of 2 to 7 nucleotides.

9. An antisense oligonucleotide according to any one of claims 1 to 8, wherein the central region consists of 8 to 12 nucleotides, and the 5' region and the 3' region each independently consist of 2 to 5 nucleotides.

10. The 2'-4' bridged nucleotide is LNA, cEt-BNA, ENA, BNA ＮＣ 10. The antisense oligonucleotide of claim 1, which is at least one selected from the group consisting of AmNA, scpBNA and GuNA.

11. The antisense oligonucleotide of any one of claims 1 to 9, wherein the 2'-modified nucleotide is at least one selected from the group consisting of 2'-O-R-ECE nucleotides, 2'-O-MCE nucleotides, 2'-O-MOE nucleotides, 2'-O-NMA nucleotides, and 2'-O-Me nucleotides.

12. The antisense oligonucleotide of any one of claims 1 to 11, wherein the 5'-modified nucleotide is a 5'-CP nucleotide.

13. The antisense oligonucleotide of any one of claims 1 to 12, wherein the antisense oligonucleotide comprises phosphorothioate linkages.

14. The antisense oligonucleotide of any one of claims 1 to 13, wherein each nucleotide constituting the antisense oligonucleotide is independently linked by a phosphodiester bond or a phosphorothioate bond.

15. An antisense oligonucleotide described in any one of claims 1 to 14, 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.

16. The antisense oligonucleotide of claim 15, wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides and proteins, and derivatives thereof.

17. The antisense oligonucleotide of claim 15 or 16, wherein the functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol and tocotrienol.

18. The antisense oligonucleotide of claim 15 or 16, wherein the functional molecule is a sugar derivative that interacts with the asialoglycoprotein receptor.

19. The antisense oligonucleotide of claim 15 or 16, wherein the functional molecule is a peptide or protein selected from the group consisting of receptor ligands and antibodies.

20. A prodrug of the antisense oligonucleotide of any one of claims 1 to 19.

21. A double-stranded oligonucleotide complex comprising: (i) an antisense oligonucleotide according to any one of claims 1 to 19; and (ii) an oligonucleotide comprising a region that hybridizes to the antisense oligonucleotide.

22. An oligonucleotide comprising: (i) a group derived from an antisense oligonucleotide according to any one of claims 1 to 19; and (ii) a group derived from an oligonucleotide comprising a region that hybridizes to the antisense oligonucleotide, wherein the group derived from the antisense oligonucleotide (i) and the group derived from the oligonucleotide (ii) are linked together.

23. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 19, a prodrug according to claim 20, an oligonucleotide conjugate according to claim 21, or an oligonucleotide according to claim 22, and a pharmacologically acceptable carrier.

24. A method for controlling the function of a target RNA, comprising the step of contacting a cell with an antisense oligonucleotide described in any one of claims 1 to 19, a prodrug described in claim 20, an oligonucleotide conjugate described in claim 21, or an oligonucleotide described in claim 22.

25. A method for regulating the function of a target RNA in a mammal, comprising the step of administering to the mammal the pharmaceutical composition according to claim 23.

26. A method for controlling expression of a target gene, comprising the step of contacting a cell with an antisense oligonucleotide described in any one of claims 1 to 19, a prodrug described in claim 20, an oligonucleotide conjugate described in claim 21, or an oligonucleotide described in claim 22.

27. A method for regulating expression of a target gene in a mammal, comprising the step of administering to the mammal the pharmaceutical composition according to claim 23.

28. A method for producing an antisense oligonucleotide according to any one of claims 1 to 19, a prodrug according to claim 20, an oligonucleotide conjugate according to claim 21, or an oligonucleotide according to claim 22, using a 2'-O-R-ECE nucleotide.

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