Lipid-binding oligonucleotide or complex thereof

Abstract

The present invention addresses the problem of providing a lipid-binding oligonucleotide or a complex thereof that improves pharmacological effects for the liver and even for organs other than the liver.　The present invention provides a lipid-binding oligonucleotide represented by general formula (I) or a complex thereof, or provides a pharmaceutical composition including the same. (In the formula, W, L, R1, and R2 have the same meanings as in the specification and the claims.)

Description

Lipid-bound oligonucleotides or complexes thereof

[0001] The present invention relates to lipid-linked oligonucleotides or complexes thereof.

[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. Representative nucleic acid drugs include antisense nucleic acids (ASOs) and siRNAs, but the development of methods for efficiently delivering these to target organs, especially to organs other than the liver, has long been recognized as a challenge.

[0003] It has been reported that conjugating ASO with lipids such as cholesterol, tocopherol, or palmitic acid as a delivery vehicle improves its antisense effect in muscle and heart (see, for example, Non-Patent Document 1). However, the effect is limited. Furthermore, cholesterol conjugates have been shown to cause toxicity.

[0004] Furthermore, it has been reported that a complex consisting of a first oligonucleotide containing ASO and a second oligonucleotide bound to a lipid such as cholesterol improves the antisense effect on organs other than the liver (see, for example, Patent Document 1).

[0005] Thus, it has been shown that conjugating lipids to oligonucleotides can exert effects on organs other than the liver, but the effects are often limited, and there is still a need to develop lipids as more effective delivery means.

[0006] International Publication No. 2017 / 053999

[0007] Nucleic Acids Research, 2019, 47, 12, pp 6045-6058

[0008] An objective of the present invention is to provide a lipid-linked oligonucleotide or a complex thereof that improves pharmacological effects on the liver and organs other than the liver.

[0009] In order to solve the above problems, the present inventors have conducted extensive research and found that a novel oligonucleotide or a complex thereof bound to a dialkyl lipid improves its pharmacological effect on the liver and organs other than the liver. Based on these findings, the present inventors have completed the present invention. That is, the present invention includes the following aspects.

[0010] [1] A lipid-linked oligonucleotide or a complex thereof represented by the following general formula (I): wherein W is a group derived from an oligonucleotide compound or oligonucleotide conjugate; L is a substituted or unsubstituted C １－１０ Alkylene group or substituted or unsubstituted polyC １－１０ is a divalent group derived from alkylene glycol, R １ and R ２ are each independently a substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ may be bonded to each other to form a ring. １ and R ２ each independently represents an unsubstituted C ５－３２ alkyl group or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ [3] The lipid-linked oligonucleotide or complex thereof according to [1], wherein R １ and R ２ each independently represents an unsubstituted C ５－３２ alkyl group or unsubstituted C ５－３２ [4] The lipid-linked oligonucleotide or complex thereof according to [1] or [2], wherein R is an alkenyl group. １ and R ２ each independently represents an unsubstituted C １０－２０[5] The lipid-linked oligonucleotide or complex thereof according to any one of [1] to [3], wherein R is an alkyl group. １ and R ２ is unsubstituted C １４ The lipid-linked oligonucleotide or complex thereof according to any one of [1] to [4], wherein the lipid-linked oligonucleotide is an alkyl group.

[0011] [6] The L is unsubstituted C １－１０ [7] The lipid-linked oligonucleotide or the complex thereof according to any one of [1] to [5], wherein L is an alkylene group. ６

[10] The lipid-linked oligonucleotide or a conjugate thereof according to any one of [1] to [6], wherein the lipid-linked oligonucleotide is an alkylene group. [8] The lipid-linked oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide compound is a gapmer antisense oligonucleotide. [9] The lipid-linked oligonucleotide according to [8], wherein the gapmer antisense oligonucleotide comprises at least one selected from the group consisting of a 2'-modified nucleoside and a 2'-4'-bridged nucleoside.

[10] The lipid-linked oligonucleotide according to [8] or [9], wherein the gapmer antisense oligonucleotide comprises at least four consecutive deoxyribonucleosides.

[0012]

[11] The lipid-linked oligonucleotide according to any one of [8] to

[10] , wherein the gapmer antisense oligonucleotide consists of 13 to 25 nucleosides.

[12] The lipid-linked oligonucleotide according to any one of [8] to

[11] , wherein L is bound to the 5'-end of the gapmer antisense oligonucleotide.

[13] The lipid-linked oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide compound is a mixmer antisense oligonucleotide.

[14] The lipid-linked oligonucleotide according to

[13] , wherein the mixmer antisense oligonucleotide comprises at least one selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

[15] The lipid-linked oligonucleotide according to

[13] or

[14] , wherein the mixmer-type antisense oligonucleotide is an oligonucleotide in which nucleosides or oligonucleotides consisting of 1 to 20 sugar-modified nucleosides and nucleosides or oligonucleotides consisting of 1 to 3 deoxyribonucleosides are alternately linked, or an oligonucleotide composed only of sugar-modified nucleosides as nucleosides.

[0013]

[16] The oligonucleotide compound according to any one of

[13] to

[15] , wherein the mixmer antisense oligonucleotide consists of 13 to 25 nucleosides.

[17] The lipid-linked oligonucleotide compound according to any one of

[13] to

[16] , wherein L is bound to the 5'-terminus of the mixmer antisense oligonucleotide.

[18] The oligonucleotide conjugate according to any one of [1] to [7], wherein the oligonucleotide conjugate is a double-stranded oligonucleotide conjugate comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is a gapmer antisense oligonucleotide or a mixmer antisense oligonucleotide consisting of 7 to 100 nucleosides, and the second oligonucleotide comprises a sequence capable of hybridizing with at least a portion of the first oligonucleotide and consists of 4 to 100 nucleosides independently selected from deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, and the first oligonucleotide or the second oligonucleotide is bound to L, and the first oligonucleotide and the second oligonucleotide hybridize.

[19] The conjugate according to

[18] , wherein the second oligonucleotide comprises at least four consecutive ribonucleosides.

[20] The conjugate according to

[18] or

[19] , wherein L is bonded to the 5' end of the second oligonucleotide.

[0014]

[21] The lipid-linked oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide compound comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides, and the second oligonucleotide comprises a sequence capable of hybridizing with at least a portion of the first oligonucleotide and consists of 4 to 100 nucleosides independently selected from deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, and wherein the first oligonucleotide or the second oligonucleotide is bound to L, and the first oligonucleotide and the second oligonucleotide are linked, and the first oligonucleotide and the second oligonucleotide hybridize.

[22] The lipid-linked oligonucleotide according to

[21] , wherein the second oligonucleotide comprises at least four consecutive ribonucleosides.

[23] The lipid-linked oligonucleotide according to

[21] or

[22] , wherein L is bound to the 5'-end of the second oligonucleotide.

[24] The lipid-linked oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide compound or oligonucleotide complex is selected from siRNA, an aptamer, and a ribozyme.

[25] The lipid-linked oligonucleotide according to any one of [1] to [7] and

[24] , wherein the oligonucleotide compound or oligonucleotide complex is siRNA.

[26] A pharmaceutical composition comprising the lipid-linked oligonucleotide or complex thereof according to any one of [1] to

[25] and a pharmacologically acceptable carrier.

[27] A method for regulating the function of a target RNA, comprising the step of contacting a cell with the lipid-linked oligonucleotide or complex thereof according to any one of [1] to

[25] .

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

[26] to the mammal.

[29] A method for regulating the expression of a target gene, comprising the step of contacting a cell with the lipid-linked oligonucleotide or complex thereof according to any one of [1] to

[25] .

[30] 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

[26] .

[31] A compound represented by the following formula (II): wherein L is a substituted or unsubstituted C １－１０ Alkylene group or substituted or unsubstituted polyC １－１０ is a divalent group derived from alkylene glycol, R １ and R ２ are each independently a substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ may be bonded to each other to form a ring, R ３ are each independently C １－１０ alkyl group or R ３ The two alkyl groups may be bonded to each other to form a ring, ４ is a 2-cyanoethyl group.

[0015] According to the present invention, it is possible to provide a lipid-linked oligonucleotide or a complex thereof with enhanced pharmacological effects on the liver and organs other than the liver.

[0016] 1 is a graph showing the results of Malat1 expression levels 10 days after antisense oligonucleotide administration in mice in Evaluation Example 3, for antisense suppression of Malat1. 2 is a graph showing the results of Malat1 expression levels 5 days after antisense oligonucleotide administration in mice in Evaluation Example 3, for antisense suppression of Malat1. 3 is a graph showing the results of Hprt1 expression levels in the liver of mice in Evaluation Example 4, where 2.9 μmol / kg of antisense oligonucleotide was administered, for antisense suppression of Hprt1. 4 is a graph showing the results of Hprt1 expression levels in the liver of mice in Evaluation Example 4, where 2.9 μmol / kg of antisense oligonucleotide was administered, for antisense suppression of Hprt1. 5 is a graph showing the results of Hprt1 expression levels in the heart of mice in Evaluation Example 4, where 2.9 μmol / kg of antisense oligonucleotide was administered, for antisense suppression of Hprt1. 6 is a graph showing the results of Hprt1 expression levels in the heart of mice in Evaluation Example 4, where 2.9 μmol / kg of antisense oligonucleotide was administered, for antisense suppression of Hprt1. 1 is a graph showing the results of Hprt1 expression levels in the lungs of mice administered 2.9 μmol / kg of antisense oligonucleotide in Evaluation Example 4, where antisense suppression of Hprt1 was performed. 2 is a graph showing the results of Hprt1 expression levels in the muscle of mice administered 2.9 μmol / kg of antisense oligonucleotide in Evaluation Example 4, where antisense suppression of Hprt1 was performed. 3 is a graph showing the results of Hprt1 expression levels in the muscle 10 days after antisense oligonucleotide administration in mice administered 2.9 μmol / kg in Evaluation Example 4, where antisense suppression of Hprt1 was performed. 4 is a graph showing the results of Hprt1 expression levels in the heart of mice administered 2.9 μmol / kg of antisense oligonucleotide in Evaluation Example 6, where antisense suppression of Hprt1 was performed. 5 is a graph showing the results of Hprt1 expression levels in the liver of mice administered 2.9 μmol / kg of antisense oligonucleotide in Evaluation Example 6, where antisense suppression of Hprt1 was performed.1 is a graph showing the results of Hprt1 expression levels in muscle following administration of 2.9 μmol / kg of antisense oligonucleotide in antisense suppression of Hprt1 in mice in Evaluation Example 6. 2 is a graph showing the results of Sod1 expression levels in the cerebrum following administration of siRNA in RNA interference of Sod1 in mice in Evaluation Example 8. 3 is a graph showing the results of Sod1 expression levels in the hippocampus following administration of siRNA in RNA interference of Sod1 in mice in Evaluation Example 8.

[0017] <Terminology> The terms used in the present invention have the following definitions unless otherwise specified.

[0018] "Nucleoside" is a term well known to those skilled in the art and is generally understood to mean a molecule in which a sugar and a nucleic acid base are bound, and which can be a unit constituting a nucleic acid. In this specification, nucleoside is a broader concept and includes deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, which will be described later. The nucleic acid base may be modified.

[0019] "Deoxyribonucleoside" refers to a molecule having a nucleobase at the carbon atom 1 of 2-deoxyribose. The deoxyribonucleoside of the present invention may be a naturally occurring deoxyribonucleoside or a deoxyribonucleoside in which the nucleobase moiety of a naturally occurring deoxyribonucleoside has been modified. A naturally occurring deoxyribonucleoside is a deoxyribonucleoside having a naturally occurring nucleobase. A single deoxyribonucleoside may be modified by a combination of multiple types. The modified deoxyribonucleosides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and WO 2018 / 155450.

[0020] "Ribonucleoside" refers to a molecule having a nucleobase at the carbon atom 1 of ribose. The ribonucleoside of the present invention may be a naturally occurring ribonucleoside or a ribonucleoside in which the nucleobase moiety of a naturally occurring ribonucleoside has been modified. A naturally occurring ribonucleoside is a ribonucleoside having a naturally occurring nucleobase. A single ribonucleoside may be modified in combination with multiple types of modifications. The modified ribonucleosides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and WO 2018 / 155450.

[0021] "Modified sugar" means: (Z1) a molecule in which ribose or 2-deoxyribose is partially replaced by one or more substituents; (Z2) a molecule in which ribose or 2-deoxyribose is replaced by a penta- or hexa-saccharide different from 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 by 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 by a C2-6 alkylene glycol (e.g., ethylene glycol, propylene glycol, etc.). Modified sugars include "2-modified sugars" and "2-4 linked sugars" described below. Examples of modified sugars and sugar-modified nucleosides described below include sugars and sugar-modified nucleosides disclosed as being suitable for use in antisense methods in, for example, JP 10-304889 A, WO 2005 / 021570 A, JP 10-195098 A, JP 2002-521310 A, WO 2007 / 143315 A, WO 2008 / 043753 A, WO 2008 / 029619 A, WO 2008 / 049085 A, and WO 2017 / 142054 A (hereinafter, these documents are referred to as "documents related to antisense methods"), etc. Modified sugars and sugar-modified nucleosides 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.

[0022] 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 the following (i) or (ii): (i) C １－６alkyl group, wherein 2 or more C １－６ When substitution with an alkyl group is contained, 2 or more C １－６ The alkyl groups may be joined together to form a 3- to 6-membered ring. (ii) A halogen atom, C １－６ Alkoxy group, halo C １－６ Alkoxy group, mono- or di-C １－６ C substituted with at least one selected from the group consisting of an alkylamino group, a 5- to 10-membered heterocyclic group, a carboxy group, a carbamoyl group, and an N-substituted carbamoyl group １－６ Examples of the N-substituted carbamoyl group include an N-methyl-carbamoyl group and an N-ethyl-carbamoyl group, and the methyl and ethyl groups 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-morpholinoethylcarbamoyl group, an N-(2-pyridylethyl)carbamoyl group, and an N-((benzimidazol-1-yl)ethyl)carbamoyl group.

[0023] "Sugar-modified nucleoside" refers to a molecule having the above-mentioned "modified sugar" in place of the sugar moiety of a deoxyribonucleoside or ribonucleoside. Examples include "2'-modified nucleosides" and "2'-4'-bridged nucleosides" described below. When the modified sugar is (Z3) as defined above, the sugar-modified nucleoside also includes molecules in which the modified sugar and the nucleobase are linked via a methylene chain or the like.

[0024] "Dimodified sugar" means a non-bridged sugar in which the oxygen atom or carbon atom at the 2-position 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." "2'-modified nucleoside" means a molecule having a nucleobase at the 1-position of the di-modified sugar, and examples include "2'-O-Me nucleoside," "2'-O-MOE nucleoside," "2'-O-MCE nucleoside," "2'-O-NMA nucleoside," "2'-O-AP nucleoside," "2'-F nucleoside," "2'-DMAECE nucleoside," "2'-MorECE nucleoside," "2'-PyECE nucleoside," and "2'-BimECE nucleoside."

[0025] "2-O-Me" (also referred to as 2-O-methyl) refers to a sugar in which the hydroxy group at position 2 of the ribose has been replaced with a methoxy group. "2'-O-Me nucleoside" (also referred to as 2'-O-methyl nucleoside) refers to a molecule having a nucleobase at position 1 of "2-O-Me".

[0026] "2-O-MOE" (also referred to as 2-O-methoxyethyl) refers to a sugar in which the hydroxy group at position 2 of ribose is replaced with a 2-methoxyethyloxy group. "2'-O-MOE nucleoside" (also referred to as 2'-O-methoxyethyl nucleoside) refers to a molecule having a nucleobase at position 1 of "2-O-MOE".

[0027] "2-O-MCE" (also referred to as 2-O-methylcarbamoylethyl) refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a methylcarbamoylethyloxy group. "2'-O-MCE nucleoside" (also referred to as 2'-O-methylcarbamoylethyl nucleoside) refers to a molecule having a nucleobase at position 1 of "2-O-MCE."

[0028] "2-O-NMA" refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a 2-[(methylamino)-2-oxoethyl]oxy group. "2'-O-NMA nucleoside" refers to a molecule having a nucleobase at position 1 of "2-O-NMA."

[0029] "2-O-AP" refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a 3-aminopropyloxy group. "2'-O-AP nucleoside" refers to a molecule having a nucleobase at position 1 of "2-O-AP".

[0030] "2-F" refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a fluorine atom. "2'-F nucleoside" refers to a molecule having a nucleobase at position 1 of "2-F".

[0031] "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE" are modified sugars in which the hydroxy group at position 2 of ribose has been replaced with 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.

[0032] "2'-DMAECE nucleoside," "2'-MorECE nucleoside," "2'-PyECE nucleoside," and "2'-BimECE nucleoside" refer to molecules having a nucleobase at position 1 of "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE," respectively.

[0033] The term "2-4 linked sugar" refers to a sugar in which a linking unit is formed by two substitutions at the 2- and 4-positions of ribose. Examples of the linking unit include C ２－６ an 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 —O—, —NR １０ - (R １０ is a hydrogen atom, C１－６ Alkyl group or halo C １－６ and -S-, which is substituted with a group selected from the group consisting of -S-, -S-, and -S-.

[0034] The term "2',4'-bridged nucleoside" (2',4'-BNA) refers to a molecule having a nucleic acid base at the 1-position of the 2,4-bridged sugar. For example, β-D-methyleneoxy (4'-CH), also known as LNA (Locked Nucleic Acid (registered trademark)), which will be described later, 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 １１ is H or CH ３ ), 2',4'-BNA ＮＣ Also called oxyamino (4'-CH ２ -N(R １２ )-O-2')BNA(R １２ is H or CH ３ ), 2',4'-BNA ＣＯＣ , 3'-amino-2',4'-BNA, 5'-methyl BNA, cEt (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 H or CH ３ (4'-C(spiro-cyclopropyl)-O-2')BNA, 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(=NH ２ + ) NHR１５ and R １５ is H or CH ３ ), and other BNAs known to those skilled in the art.

[0035] A "5-modified sugar" refers to an unbridged sugar in which the oxygen or carbon atom at the 5-position of the ribose backbone has been modified, and includes "5-CP," "5-methyl," and "5-aminopropyl." The 2- and 4-positions of a "5-modified sugar" are preferably unmodified. A "5'-modified nucleoside" refers to a molecule having a nucleobase at the 1-position carbon atom of the "5-modified sugar" (the 1-position of the 2-deoxyribose before modification), and includes, for example, a "5'-CP nucleoside," a "5'-methyl nucleoside," and a "5'-aminopropyl nucleoside." The 2'-position of a 5'-modified nucleoside may or may not be bridged. The 2'- and 4'-positions of a 5'-modified nucleoside may or may not be bridged, but are preferably unbridged.

[0036] "5-CP" is a sugar in which the 5-position of the ribose skeleton is substituted with two methyl groups, and these two methyl groups together form a cyclopropane. A "5'-CP nucleoside" is a molecule in which the sugar is the above-mentioned "5-CP" and has a nucleobase at the carbon atom at position 1 (position 1 of the parent 2-deoxyribose), and can be represented by the following structural formula. In the formula, Base is the nucleobase. The wavy line is understood to represent a bond to an adjacent nucleoside, a linker, etc., or a hydrogen atom or a phosphate group. "5-Methyl" is a sugar in which the 5-position of the ribose backbone is substituted with a methyl group. "5-Aminopropyl" is a sugar in which the 5-position of the ribose backbone is substituted with a 3-aminopropyl group. "5'-Methylnucleoside" and "5'-aminopropylnucleoside" refer to molecules having a nucleobase at the 1-position (1-position of the parent 2-deoxyribose) carbon atom of "5-methyl" and "5-aminopropyl," respectively.

[0037] "5-vinyl" is a sugar in which the methylene group at the 5-position of ribose is replaced with an ethene-1,2-diyl group (vinyl group).

[0038] Vinylphosphonate (VP) nucleosides and cyclopropanephosphonate (CPP) nucleosides, which will be described later, are also included in the 5'-modified nucleosides.

[0039] In the "deoxyribonucleosides," "ribonucleosides," "2'-modified nucleosides," "2'-4'-bridged nucleosides," and "5'-modified nucleosides," the bond between the 1' carbon atom and the nucleic acid 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.

[0040] "n-" means normal, "s-" means secondary, and "t-" means tertiary.

[0041] The term "halogen atom" or "halo" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0042] "C １－６ The term "alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, and an isohexyl group (including various isomers).

[0043] "C １－１０ The term "alkyl group" means a straight-chain or branched alkyl group having 1 to 10 carbon atoms, and examples thereof include the above-mentioned "C １－６ In addition to examples of the "alkyl group," there may be mentioned, for example, a heptyl group, an octyl group, a nonyl group, a decyl group (including various isomers), and the like.

[0044] "C ５－３２The term "alkyl group" refers to a linear or branched alkyl group having 5 to 32 carbon atoms, and examples thereof include pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, docosyl, tetracosyl, hexacosyl, octacosyl, triacontyl, hentriacontyl, and dotriacontyl groups (including various isomers).

[0045] "C ８－２８ The term "alkyl group" means a linear or branched alkyl group having 8 to 28 carbon atoms, and examples thereof include an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a docosyl group, a tetracosyl group, a hexacosyl group, and an octacosyl group (including various isomers).

[0046] "C １０－２０ The term "alkyl group" refers to a straight-chain or branched alkyl group having 10 to 20 carbon atoms, and examples thereof include decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and icosyl groups (including various isomers). １２－１４ The term "alkyl group" refers to a linear or branched alkyl group having 12 to 14 carbon atoms, and examples thereof include a dodecyl group, a tridecyl group, and a tetradecyl group (including various isomers). １２ The term "alkyl group" means a linear or branched alkyl group having 12 carbon atoms, and examples thereof include a 1-dodecyl group, a 6-dodecyl group, a 1-methylundecyl group, and a 6-methylundecyl group. １４ The term "alkyl group" means a straight-chain or branched alkyl group having 14 carbon atoms, and examples thereof include a 1-tetradecyl group, a 7-tetradecyl group, a 1-methyltridecyl group, and a 12-methyltridecyl group.

[0047] "C ５－３２The term "alkenyl group" refers to a linear or branched unsaturated hydrocarbon group containing one or more carbon-carbon double bonds and having 5 to 32 carbon atoms, and examples thereof include pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, docosenyl, tetracosenyl, hexacosenyl, octacosenyl, triacontenyl, hentriacontenyl, and dotriacontenyl groups (including various isomers). ８－２８ The term "alkenyl group" means a straight-chain or branched unsaturated hydrocarbon group containing one or more carbon-carbon double bonds and having 8 to 28 carbon atoms, and examples thereof include octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, docosenyl, tetracosenyl, hexacosenyl, and octacosenyl groups (including various isomers). １０－２０ The term "alkenyl group" refers to a linear or branched unsaturated hydrocarbon group having 10 to 20 carbon atoms and containing one or more carbon-carbon double bonds, and examples thereof include a decenyl group, an undecenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, and an icocenyl group (including various isomers). １４ The term "alkenyl group" means a straight-chain or branched alkenyl group having 14 carbon atoms, and examples thereof include a tetradec-2-enyl group, a tetradec-7-enyl group, a 1-methyltridec-2-enyl group, and a 12-methyltridec-2-enyl group.

[0048] "Haro C １－６ The "C alkyl group" is defined as １－６ The term "alkyl group" refers to a group in which a hydrogen atom at any position of the "alkyl group" is substituted with one or more of the above-mentioned "halogen atoms."

[0049] "C ２－６The term "alkylene group" refers to a divalent group (alkanediyl group) obtained by removing two hydrogen atoms at any position from a linear or branched saturated hydrocarbon group having 2 to 6 carbon atoms, and examples thereof include an ethylene (ethanediyl) group, a propane-1,3-diyl (trimethylene) group, a propane-2,2-diyl 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.

[0050] "C １－１０ The term "alkylene group" means a linear or branched alkylene group having 1 to 10 carbon atoms, and examples thereof include the above-mentioned "C ２－６ Examples of "alkylene groups" include methylene, propylene, tetramethylene, 1-methylpropylene, 2-methylpropylene, dimethylethylene, ethylethylene, pentamethylene, 1-methyl-tetramethylene, 2-methyl-tetramethylene, 1,1-dimethyl-trimethylene, 1,2-dimethyl-trimethylene, 1-ethyl-trimethylene, octamethylene, and decamethylene.

[0051] "C ２－８ The term "alkylene group" means a linear or branched alkylene group having 2 to 8 carbon atoms, and examples thereof include the above-mentioned "C ２－６ Examples of "alkylene groups" include methylene, propylene, tetramethylene, 1-methylpropylene, 2-methylpropylene, dimethylethylene, ethylethylene, pentamethylene, 1-methyl-tetramethylene, 2-methyl-tetramethylene, 1,1-dimethyl-trimethylene, 1,2-dimethyl-trimethylene, 1-ethyl-trimethylene, and octamethylene.

[0052] "C ３－６The term "alkylene group" means a straight-chain or branched alkylene group having 3 to 6 carbon atoms, and examples thereof include a propylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 1-ethyl-trimethylene group, a hexamethylene group, and the like.

[0053] "C ６ The term "alkylene group" means a straight-chain or branched alkylene group having 6 carbon atoms, and examples thereof include a hexamethylene group, a 1-methyl-pentamethylene group, a 2-methyl-pentamethylene group, and a 1,1-dimethyl-tetramethylene group.

[0054] "C １－６ The "alkoxy group" is the same as the "C １－６ "Alkyl" refers to a group in which an "alkyl" is bonded to an oxy group.

[0055] "Haro C １－６ The "alkoxy group" is the same as the "C １－６ The term "alkoxy group" refers to a group in which a hydrogen atom at any position of the "alkoxy group" is substituted with one or more of the above-mentioned "halogen atoms."

[0056] "Mono- or di-C １－６ An "alkylamino group" is a group in which one hydrogen atom of an amino group is substituted with one "C １－６ A group in which two hydrogen atoms of an amino group are replaced by "C alkyl groups" or two hydrogen atoms of an amino group are replaced by the same or different "C １－６ It refers to a group substituted with an "alkyl group," and examples thereof include a methylamino group, an ethylamino group, a propylamino group, an isopropylamino group, a butylamino group, a dimethylamino group, a diethylamino group, a dipropylamino group, a dibutylamino group, and an N-ethyl-N-methylamino group.

[0057] The term "3- to 6-membered ring" means a monocyclic saturated or unsaturated hydrocarbon ring having 3 to 6 carbon atoms, and examples thereof include cyclopropane, cyclobutane, and cyclohexane.

[0058] The term "5- to 10-membered heterocyclic group" refers to a 5- to 10-membered monocyclic or fused polycyclic aromatic or non-aromatic heterocyclic group containing, as ring-constituting atoms other than carbon atoms, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom. Preferable examples of the "5- to 10-membered heterocyclic group" include a thienyl group, a furyl group, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a triazolyl group, a tetrazolyl group, a triazinyl group, a benzothiophenyl group, a benzofuranyl group, a benzimidazolyl group, a benzoxazolyl group, a benzisoxazolyl group, a benzothiazolyl group, a benzisothiazolyl group, a benzotriazolyl group, an imidazopyridinyl group, a thienopyridinyl group, a pyrrolopyridinyl group, a pyrazolopyridinyl group, an oxazolopyridinyl group, a thiazolopyridinyl group, an imidazopyrazinyl group, an imidazopyrimidinyl group, an aziridinyl group, Examples of such groups include an oxiranyl group, an azetidinyl group, an oxetanyl group, a thietanyl group, a tetrahydrothienyl group, a tetrahydrofuranyl group, a pyrrolinyl group, a pyrrolidinyl group, an oxopyrrolidinyl group, an imidazolinyl group, an oxoimidazolinyl group, an imidazolidinyl group, an oxazolinyl group, an oxazolidinyl group, a pyrazolinyl group, a pyrazolidinyl group, a thiazolinyl group, a thiazolidinyl group, a tetrahydroisothiazolyl group, a tetrahydrooxazolyl group, a tetrahydroisoxazolyl group, a piperidinyl group, a piperazinyl group, a tetrahydropyridinyl group, a dihydropyridinyl group, a tetrahydropyridazinyl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a morpholinyl group, and a thiomorpholinyl group.

[0059] An "oxo group" refers to a group in which an oxygen atom is substituted via a double bond (=O). When an oxo group is substituted on a carbon atom, it combines with the carbon atom to form a carbonyl group. A "thioxo group" refers to a group in which a sulfur atom is substituted via a double bond (=S). When a thioxo group is substituted on a carbon atom, it combines with the carbon atom to form a thiocarbonyl group.

[0060] A "nucleobase" is a purine base or a pyrimidine base, and may be a naturally occurring nucleobase or a modified naturally occurring nucleobase. Naturally occurring nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The "nucleobase" includes naturally occurring nucleobases and the "modified nucleobases" described below.

[0061] Examples of modifications of nucleobases in "modified nucleobases" include halogenation, methylation, ethylation, n-propylation, isopropylation, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, cyclobutylation, hydroxylation, amination, thiolation, demethylation, etc. More specific examples include 5-methylation, 5-fluorolation, 5-bromination, 5-iodination, and N4-methylation of cytosine; 2-thiolation, 5-demethylation, 5-fluorolation, 5-bromination, and 5-iodination of thymine; 2-thiolation, 5-fluorolation, 5-bromination, and 5-iodination of uracil; N6-methylation and 8-bromination of adenine; and N2-methylation and 8-bromination of guanine. Furthermore, examples of modifications of the nucleic acid base moiety in a nucleoside 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, WO 2018 / 155450, and the like.

[0062] The nucleic acid base in the nucleoside is preferably at least one selected from the group consisting of adenine, guanine, thymine, cytosine, uracil and 5-methylcytosine.

[0063] "5-methylcytosine" means a cytosine with a methyl group at the 5-position.

[0064] "Nucleic acid base sequence" means the sequence of nucleic acid bases from the 5' to the 3' side of each nucleoside contained in an oligonucleotide.

[0065] The term "contiguous nucleobases" refers to a sequence of contiguous nucleobases from the 5' side to the 3' side in the "nucleobase sequence".

[0066] "Internucleoside linkage" means a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. "Internucleoside linkage" includes phosphodiester linkages and "modified internucleoside linkages," as defined below.

[0067] The term "modified internucleoside bond" refers to a modified phosphodiester bond, and examples thereof include a phosphorothioate bond, a methylphosphonate bond (including a chiral-methylphosphonate bond), a methylthiophosphonate bond, a phosphorodithioate bond, a phosphoramidate bond, a phosphorodiamidate bond, a phosphoramidothioate bond, and a boranophosphate bond. Examples of modified phosphodiester bonds 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 can be used for modified phosphodiester bonds.

[0068] "Modified nucleoside" means a nucleoside having a modified sugar and / or a modified nucleobase.

[0069] An "oligonucleotide" refers to a molecule having a structure in which two or more identical or different "nucleosides" are linked by the "internucleoside bond" (e.g., a phosphodiester bond or a modified phosphodiester bond) that is independently selected from each other. An "oligodeoxyribonucleotide" refers to a polynucleotide or oligonucleotide in which two or more identical or different "deoxyribonucleosides" are linked by the "internucleoside bond" that is independently selected from each other. An "oligoribonucleotide" refers to a polynucleotide or oligonucleotide in which two or more identical or different "ribonucleosides" are linked by the "internucleoside bond" that is independently selected from each other. When the 5' position of a nucleoside is linked to another nucleoside, the 5' position is linked to the 2', 3', or 5' position (preferably the 3' position) of the other nucleoside by an internucleoside bond. When the 3' position of a nucleoside is linked to another nucleoside, the 3' position is linked to the 2', 3', or 5' position (preferably the 5' position) of the other nucleoside via an internucleoside bond.

[0070] "DNA" refers to a naturally occurring polynucleotide or oligonucleotide in which two or more of the above-mentioned "deoxyribonucleosides" are linked by phosphodiester bonds. The naturally occurring deoxyribonucleosides that make up DNA may be the same or different from each other. "RNA" refers to a naturally occurring polynucleotide or oligonucleotide in which two or more of the above-mentioned "ribonucleosides" are linked by phosphodiester bonds. The naturally occurring ribonucleosides that make up RNA may be the same or different from each other.

[0071] The term "oligonucleotide complex" refers to a complex in which multiple "oligonucleotides" (molecules) that are not covalently linked to each other are combined together by intermolecular hybridization.

[0072] "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.

[0073] 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.

[0074] An "antisense oligonucleotide" (ASO) is an oligonucleotide that produces the antisense effect. Examples include, but are not limited to, DNA, oligodeoxyribonucleotide, gapmer-type antisense oligonucleotide (also simply referred to as "gapmer"), and mixmer-type antisense oligonucleotide (also simply referred to as "mixmer"), and may also be RNA, oligoribonucleotide, or an oligonucleotide designed to normally produce an antisense effect.

[0075] "Hybridize" refers to the act of forming a double strand between oligonucleotides or portions thereof containing complementary sequences, and the phenomenon in which oligonucleotides or portions thereof containing complementary sequences form a double strand.

[0076] The term "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 portions thereof, can "hybridize" if their sequences are complementary. Two oligonucleotides, or portions thereof, do not need to be completely complementary to hybridize; however, the degree of complementarity for two oligonucleotides, or portions thereof, to hybridize is preferably 70% or greater, more preferably 80% or greater, and even more preferably 90% or greater (e.g., 95%, 96%, 97%, 98%, or 99% or greater). Sequence complementarity is determined by 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. When two oligonucleotides have the above-described preferred complementarity, one skilled in the art can determine that the two oligonucleotides hybridize.

[0077] The term "gapmer" refers to an oligonucleotide that includes a "gap segment," a "5' wing segment," and a "3' wing segment," as described below.

[0078] A "gap segment" is a region containing "at least four consecutive nucleosides recognized by RNase H." It is not particularly limited as long as it contains four or more consecutive nucleosides and is recognized by RNase H, but the consecutive nucleosides are preferably independently selected from deoxyribonucleosides and sugar-modified nucleosides, and include at least two deoxyribonucleosides. The nucleosides at the 5' and 3' ends of the gap segment are deoxyribonucleosides or 5'-modified nucleosides, more preferably deoxyribonucleosides. In one embodiment, preferably, the 5' end of the gap segment is a 5'-modified nucleoside, and the 3' end of the gap segment is a deoxyribonucleoside.

[0079] The "5' wing segment" is a region linked to the 5' side of the gap segment and containing "at least one nucleoside" without containing the "at least four consecutive nucleosides recognized by RNase H." The sugar moiety of the 3'-terminal nucleoside of the 5' wing segment is different from the sugar moiety of the 5'-terminal nucleoside of the gap segment. The boundary between the 5' wing segment and the gap segment is confirmed by the difference in sugar moiety. In one preferred embodiment of the present invention, the difference in sugar moiety is determined by the presence or absence of modification at the 2-position of the corresponding sugar. Note that, as defined above, a "dimodified sugar" is a non-bridged sugar in which the oxygen atom or carbon atom at the 2-position of ribose is modified, and a "2-4 bridged sugar" is a sugar in which the bridge unit is substituted by substitutions at two positions, the 2- and 4-positions of the ribose, as defined above, and therefore the 2-position is modified in both cases. (For example, the 5'-terminal nucleoside of the gap segment is a deoxyribonucleoside, and the 3'-terminal nucleoside of the 5'-wing segment is a sugar-modified nucleoside. This sugar-modified nucleoside is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside. Also, in one embodiment, for example, the 5'-terminal nucleoside of the gap segment is a 5'-modified nucleoside, and the 3'-terminal nucleoside of the 5'-wing segment is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside.) The 3'-terminal nucleoside of the 5'-wing segment is generally a sugar-modified nucleoside, and preferably a 2'-4'-bridged nucleoside or a 2'-modified nucleoside. The 5' wing segment is not particularly limited as long as it satisfies the above definition, but the at least one nucleoside is preferably independently selected from a deoxyribonucleoside and a sugar-modified nucleoside, and includes at least one sugar-modified nucleoside. The 5' wing segment is preferably independently selected from a sugar-modified nucleoside, and more preferably independently selected from a 2'-4'-bridged nucleoside and a 2'-modified nucleoside.

[0080] The "3' wing segment" is a region linked to the 3' side of the gap segment and containing "at least one nucleoside" without containing the "at least four consecutive nucleosides recognized by RNase H," in which the sugar moiety of the 5'-terminal nucleoside of the 3' wing segment is different from the sugar moiety of the 3'-terminal nucleoside of the gap segment. The difference in sugar moieties identifies the boundary between the 3' wing segment and the gap segment. In one preferred embodiment of the present invention, the difference in sugar moieties is determined by the presence or absence of modification at the 2-position of the corresponding sugar. (For example, the nucleoside at the 3' end of the gap segment is a deoxyribonucleoside, and the nucleoside at the 5' end of the 3' wing segment is a sugar-modified nucleoside. This sugar-modified nucleoside is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside. Also, in one embodiment, for example, the nucleoside at the 3' end of the gap segment is a 5'-modified nucleoside, and the nucleoside at the 5' end of the 3' wing segment is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside.) The 5'-terminal nucleoside of the 3' wing segment is generally a sugar-modified nucleoside, and preferably a 2'-4'-bridged nucleoside or a 2'-modified nucleoside. The 3' wing segment is not particularly limited as long as it satisfies the above definition, but the at least one nucleoside is preferably independently selected from a deoxyribonucleoside and a sugar-modified nucleoside, and includes at least one sugar-modified nucleoside. The 3' wing segment is preferably independently selected from a sugar-modified nucleoside, and more preferably independently selected from a 2'-4'-bridged nucleoside and a 2'-modified nucleoside.

[0081] In one typical example, the portion from the 5' end where 2'-modified nucleosides or 2'-4'-bridged nucleosides continue is the 5' wing segment, and the boundary where the 3'-terminal nucleoside of the 5' wing segment is connected to another nucleoside (deoxyribonucleoside, ribonucleoside, etc.) excluding the 2'-modified nucleoside or 2'-4'-bridged nucleoside is the boundary between the 5' wing segment and the gap segment. In one typical example, the portion from the 3' end where 2'-modified nucleosides or 2'-4'-bridged nucleosides continue is the 3' wing segment, and the boundary where the 5'-terminal nucleoside of the 3' wing segment is connected to another nucleoside (deoxyribonucleoside, ribonucleoside, etc.) excluding the 2'-modified nucleoside or 2'-4'-bridged nucleoside is the boundary between the 3' wing segment and the gap segment.

[0082] "RNase H" is generally known as a ribonuclease that recognizes a double-stranded structure formed by hybridizing DNA and RNA in vivo 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 nucleobase moiety, phosphodiester bond moiety, and sugar moiety of at least one of the DNA and RNA is modified. For example, it can recognize a double-stranded structure formed by hybridizing DNA modified with a phosphorothioate bond with RNA. It can also recognize a double-stranded structure formed by hybridizing an oligodeoxyribonucleotide with an oligoribonucleotide. Thus, DNA can be recognized by RNase H when hybridized with RNA. Furthermore, RNA can be cleaved by RNase H when hybridized with DNA. The same applies to structures in which at least one of the nucleobase moiety, phosphodiester bond moiety, and sugar moiety of at least one of the DNA and RNA is modified. Representative examples include oligonucleotides in which the phosphodiester bond of DNA is modified to phosphorothioate. 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, 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, etc. In the present invention, RNase H is preferably mammalian RNase H, more preferably human RNase H, and particularly preferably human RNase H1.

[0083] The term "at least four consecutive nucleosides recognized by RNase H" includes four or more consecutive nucleosides and is not particularly limited as long as it is recognized by RNase H, and examples thereof include "at least four consecutive deoxyribonucleosides." The number of nucleosides constituting "at least four consecutive nucleosides recognized by RNase H" is, for example, 5 to 30, preferably 5 to 15, more preferably 8 to 12, and particularly preferably 10. These nucleosides are independently preferably deoxyribonucleosides or 5'-modified nucleosides, and more preferably deoxyribonucleosides. Those skilled in the art can determine whether certain at least four consecutive nucleosides are "at least four consecutive nucleosides recognized by RNase H" based on the structure of the sugar moieties of the consecutive nucleosides.

[0084] The number of nucleosides constituting the 5' wing segment and the 3' wing segment is, independently, for example, 1 to 10, preferably 2 to 6, more preferably 3 to 5, and particularly preferably 3.

[0085] The number of nucleosides constituting a gapmer is preferably 7 to 50, more preferably 7 to 27, even more preferably 14 to 22, and particularly preferably 16.

[0086] A mixmer is an oligonucleotide that contains multiple sugar-modified nucleosides and produces an antisense effect without having the gap segment, and examples thereof include an oligonucleotide in which nucleosides or oligonucleotides consisting of 1 to 20 sugar-modified nucleosides and nucleosides or oligonucleotides consisting of 1 to 3 deoxyribonucleosides are alternately linked, an oligonucleotide composed only of sugar-modified nucleosides, etc. In one embodiment, the mixmer is preferably an oligonucleotide in which nucleosides or oligonucleotides consisting of 1 to 20 sugar-modified nucleosides independently selected from 2'-modified nucleosides and 2',4'-BNA are alternately linked with nucleosides or oligonucleotides consisting of 1 to 3 deoxyribonucleosides.

[0087] "SiRNA" (small interfering RNA) is a short double-stranded oligoribonucleotide. SiRNA is involved in a phenomenon called RNA interference (RNAi) and suppresses gene expression by destroying the target mRNA. SiRNA comprises an antisense strand and a sense strand. In some embodiments, the antisense and sense strands of siRNA comprise RNA. SiRNA typically consists of 15 to 30 base pairs, preferably 21 to 23 base pairs. The 3' ends of the two nucleic acid strands often have one to five (preferably two) overhanging deoxythymidines (commonly designated as tt, dTdT, etc.) (commonly referred to as overhangs). In some cases, only the 3' end of the sense strand has two overhanging deoxythymidines, and in other cases, only the 3' end of the antisense strand has two overhanging deoxythymidines.

[0088] In some embodiments, neither the antisense strand nor the sense strand has an overhang. For example, an siRNA asymmetrically having a blunt end at the 5' end of the antisense strand and an overhang at the 3' end of the antisense strand favors the process of loading the guide strand into RISC. The siRNA may contain one or more sugar-modified nucleosides. For example, at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the nucleosides constituting the siRNA are sugar-modified nucleosides. The sugar-modified nucleoside used in the siRNA is, for example, at least one selected from 2'-modified nucleosides and 2'-4'-bridged nucleosides, preferably at least one selected from 2'-modified nucleosides, and particularly preferably 2'-O-Me nucleosides and 2'-F nucleosides. In one embodiment, the siRNA is composed of 2'-O-Me nucleosides and 2'-F nucleosides. The ratios are, for example, 10-30% 2'-F nucleosides and 90-70% 2'-O-Me nucleosides. Preferably, the sense strand contains 10-20% 2'-F nucleosides and 90-80% 2'-O-Me nucleosides, and the antisense strand contains 20-30% 2'-F nucleosides and 80-70% 2'-O-Me nucleosides.

[0089] Preferably, the antisense strand contains a 2'-fluoronucleoside at at least one position selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5' end. Particularly preferably, the antisense strand has 2'-fluoronucleosides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the hybridizing portion of the antisense strand. The other nucleosides are 2'-O-Me nucleosides.

[0090] Preferably, the sense strand contains a 2'-fluoronucleoside at at least one position selected from positions 7, 9, 10, and 11, counting from the 5'-terminus. Particularly preferably, the sense strand contains 2'-fluoronucleosides at positions 7, 9, 10, and 11, counting from the 5'-terminus. The other nucleosides are 2'-O-Me nucleosides.

[0091] The internucleoside linkages of the siRNA may be phosphodiester linkages or modified internucleoside linkages, but each internucleoside linkage is preferably independently a phosphodiester linkage or a phosphorothioate linkage. More preferably, 1 to 5 internucleoside linkages from the 5'-end and 3'-end of each of the antisense strand and the sense strand are phosphorothioate linkages, and the remaining internucleoside linkages are phosphodiester linkages. Even more preferably, 1 to 3 internucleoside linkages from the 5'-end and 3'-end of each of the antisense strand and the sense strand are phosphorothioate linkages, and the remaining internucleoside linkages are phosphodiester linkages. Even more preferably, 2 to 3 internucleoside linkages from the 5'-end and 3'-end of each of the antisense strand and the sense strand are phosphorothioate linkages, and the remaining internucleoside linkages are phosphodiester linkages. Particularly preferably, two internucleoside bonds at the 5'-end and 3'-end of each of the antisense and sense strands are phosphorothioate bonds, and the remaining internucleoside bonds are phosphodiester bonds.

[0092] In some embodiments, the sense strand and / or antisense strand of the siRNA may be 5' phosphorylated or may include a phosphate modification at the 5' end. Exemplary phosphate groups or modified phosphate groups include those compatible with RISC-mediated gene silencing. For example, suitable phosphate groups or phosphate modifications include 5'-monophosphate ((HO) ２ (O)P-O-5'), 5'-diphosphate ((HO) ２ (O)P-O-P(HO)(O)-O-5'), 5'-triphosphate ((HO) ２ (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate; (HO) ２ (S)P—O-5′), 5′-monodithiophosphate (dithiophosphate, (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO) ２(O)P-S-5'), any further combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidic acid ((HO) ２ (O)P-NH-5', (HO)(NH ２ ) (O)P-O-5'), methylene phosphate ((HO) ２ (O) P-CH ２ -5'), 5'-alkylphosphonate (R'P(OH)(O)-O-5'- (R'=alkyl, for example, methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkenylphosphonate (alkenyl is, for example, vinyl((OH) ２ (O)P-5'-CH=CH-), substituted vinyl), 5'-alkoxyalkylphosphonates (R"P(OH)(O)-O-5'- (R"=alkoxyalkyl, e.g., methoxymethyl, ethoxymethyl, etc.), 5'-cycloalkylphosphonates ((OH) ２ (O)P-5'-(cycloalkane-diyl)-, for example, (OH) ２ (O)P-5'-(cyclopropane-1,2-diyl)-). The modification may be located within the antisense strand of the siRNA. For example, the antisense strand may include a 5'-vinylphosphonate nucleoside (preferably a 5'-E-vinylphosphonate nucleoside) at the 5'-terminus. Also, for example, the antisense strand may include a 5'-cyclopropanephosphonate nucleoside (preferably a 5'-(trans)-cyclopropanephosphonate nucleoside) at the 5'-terminus.

[0093] In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises a 5'-E-vinyl phosphonate or a 5' cyclopropane phosphonate, for example, a nucleotide of the following formula (X): A structure (5'-vinylphosphonate (VP)-modified 2'-O-Me nucleoside) represented by the following formula (wherein Base in formula (X) has the same meaning as X and Base in formula (I)) is preferred as the 5'-E-vinylphosphonate.

[0094] In some embodiments, the compound of formula (XI): (Base in formula (XI) has the same meaning as X and Base in formula (I). The substituent on the cyclopropane is in a trans form) (5'-cyclopropanephosphonate (CPP)-modified 2'-O-Me nucleoside) is preferred.

[0095] Chemical modifications of siRNA are well known in the art, and reference can be made to, for example, Sig Transduct Target Ther 2020, 5, pp101, RNA Biol. 2022, 19(1) pp452-467, Trends Mol Med. 2024, 30(1) pp13-24, Nat Rev Drug Discov 2024, 23, pp341-364, etc.

[0096] An "aptamer" is an oligonucleotide that exhibits the ability to bind to a specific extracellular protein and inhibit its function.

[0097] A "ribozyme" is an RNA or oligoribonucleotide with catalytic activity.

[0098] "miRNA" (micro-RNA) is a non-coding RNA that is encoded on the genome but is not translated into protein, and has the effect of suppressing gene expression. It is typically an RNA of 21 to 25 bases.

[0099] "CpG oligo" is an oligonucleotide that contains deoxycytidine (C) and deoxyguanosine (G) and activates innate immunity by interacting with proteins such as Toll-like receptor 9 (TLR9). It is typically an oligodeoxyribonucleotide of 15 to 30 bases.

[0100] A "decoy nucleic acid" is a double-stranded oligodeoxyribonucleotide that binds to a transcription factor, among other proteins, to inhibit the transcription stage. The decoy nucleic acid can inhibit the gene expression of the transcription factor. The decoy nucleic acid typically consists of 15 to 30 base pairs.

[0101] <Lipid-linked oligonucleotide or complex thereof> The present invention provides a lipid-linked oligonucleotide or complex thereof. The lipid-linked oligonucleotide or complex thereof of the present invention is represented by the following general formula (I): wherein W is a group derived from an oligonucleotide compound or oligonucleotide conjugate; L is a substituted or unsubstituted C １－１０ Alkylene group or substituted or unsubstituted polyC １－１０ is a divalent group derived from alkylene glycol, R １ and R ２ are each independently a substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ may be bonded to each other to form a ring).

[0102] In the present invention, "substituted or unsubstituted C １－１０ An "alkylene group" is an (unsubstituted) C １－１０ C substituted with an alkylene group or one or more optional substituents １－１０ It means an alkylene group.

[0103] In the present invention, "substituted or unsubstituted" means that the group is substituted with a given substituent group, for example, a halogen atom, a hydroxy group, a carboxy group, a C １－６ It means that it is unsubstituted or substituted with at least one substituent selected from the group consisting of an alkoxy group and an aryl group.

[0104] "Substituted C １－１０ The term "alkylene group" refers to a group in which one or more arbitrary hydrogen atoms of the alkylene group are replaced with a halogen atom, a hydroxy group, a carboxy group, a C １－６ It means one substituted with a substituent selected from the group consisting of an alkoxy group and an aryl group.

[0105] Poly C １－１０ The divalent group derived from alkylene glycol is, for example, represented by the formula: -(O) ｌ-(Alk-O-) ｍ -Alk-(O) ｎ wherein l and n are independently 0 or 1, m is an integer from 2 to 20, and Alk is C １－１０ an alkylene group, and the alkylene group is selected from the group consisting of a hydroxy group, a protected hydroxy group, a halogen atom, a hydroxy group, a carboxy group, and C １－６ The protected hydroxy group is not particularly limited as long as it is stable when bound to an oligonucleotide, and examples thereof include ether-based protecting groups such as triarylmethyl (e.g., triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, etc.); acetal-based protecting groups such as methoxymethyl, methylthiomethyl, methoxyethyl, benzyloxymethyl, 2-tetrahydropyranyl; acyl-based protecting groups such as acyl (e.g., formyl, acetyl, pivaloyl, benzoyl, etc.); tri(alkyl)silyl (e.g., trimethylsilyl, triethylsilyl, silyl-based protecting groups such as (triisopropylsilyl, t-butyldimethylsilyl, dimethylisopropylsilyl, etc.), (alkyl)diarylsilyl (for example, t-butyldiphenylsilyl, diphenylmethylsilyl, etc.), triarylsilyl (for example, triphenylsilyl, etc.), tribenzylsilyl, and [(triisopropylsilyl)oxy]methyl (Tom group); 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep group), 9-phenylxanthen-9-yl (Pixyl group), and 9-(p-methoxyphenyl)xanthen-9-yl (MOX group). The protecting group for the "protected hydroxy group" is preferably benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, triphenylmethyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl.

[0106] Among these, L in the general formula (I) is unsubstituted C １－１０ An alkylene group is preferred, and C ２－８ An alkylene group is more preferred, and C ３－６ An alkylene group is more preferred, and C ６ An alkylene group is particularly preferred. For example, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a hexamethylene group, or an octamethylene group (including various isomers) is more preferred, a trimethylene group, a tetramethylene group, or a hexamethylene group (including various isomers) is even more preferred, and a hexamethylene group (including various isomers) is particularly preferred. Furthermore, a 1,6-hexamethylene group (1,6-hexanediyl group) is particularly preferred.

[0107] In the present invention, "substituted or unsubstituted C ５－３２ "Alkyl group" refers to an (unsubstituted) C ５－３２ C substituted with an alkyl group or one or more optional substituents ５－３２ It means an alkyl group.

[0108] "Substituted C ５－３２ The term "alkyl group" refers to a group in which one or more arbitrary hydrogen atoms of the alkyl group are substituted with a halogen atom, a hydroxy group, a carboxy group, a C １－６ It means one substituted with a substituent selected from the group consisting of an alkoxy group and an aryl group.

[0109] Among these, R in general formula (I) １ and R ２ As for C, unsubstituted C ５－３２ Alkyl groups are preferred, and C ８－２８ Alkyl groups are more preferred, and C １０－２０ Alkyl groups are more preferred, and C １２－１４ Alkyl groups are even more preferred, C １４ Alkyl groups are particularly preferred. １２Alkyl groups are also particularly preferred. For example, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and icosyl groups (including various isomers) are more preferred, and tetradecyl groups (including various isomers) are particularly preferred. １ and R ２ The alkyl group is preferably a straight-chain alkyl group.

[0110] In the present invention, "substituted or unsubstituted C ５－３２ An "alkenyl group" is an (unsubstituted) C ５－３２ C substituted with an alkenyl group or one or more optional substituents ５－３２ It means an alkenyl group.

[0111] "Substituted C ５－３２ The term "alkenyl group" refers to a group in which one or more arbitrary hydrogen atoms of the alkenyl group are replaced with a halogen atom, a hydroxy group, a carboxy group, a C １－６ It means one substituted with a substituent selected from the group consisting of an alkoxy group and an aryl group.

[0112] Among these, R in general formula (I) １ and R ２ As for C, unsubstituted C ５－３２ Alkenyl groups are preferred, C ８－２８ An alkenyl group is more preferred, and C １０－２０ An alkenyl group is more preferred, and C １４ Alkenyl groups are particularly preferred, for example, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, and icocenyl groups (including various isomers) are more preferred, and tetradecenyl groups (including various isomers) are particularly preferred.

[0113] In the present invention, "may be bonded to each other to form a ring" means R １ and R ２ Substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２The alkenyl groups are bonded to each other at parts thereof, and the "-O-CH" in the general formula (I) to which they are bonded is ２ This means that a 10- to 64-membered ring may be formed, including the "-CH-O-" portion. The 10- to 64-membered ring is preferably a 10- to 40-membered ring, more preferably a 12- to 30-membered ring, even more preferably a 20- to 30-membered ring, and particularly preferably a 22-membered ring. The ring is more preferably unsubstituted.

[0114] The "group derived from an oligonucleotide compound or oligonucleotide conjugate" of the present invention refers to a partial structure of an oligonucleotide compound formed by removing a hydrogen atom, a hydroxy group, or the like from the 3'-end or 5'-end of at least one oligonucleotide compound constituting the oligonucleotide compound or oligonucleotide conjugate. Thus, one of the 3'-end or 5'-end of the oligonucleotide compound is covalently bound to L in general formula (I). One of the 3'-end or 5'-end of the oligonucleotide compound and L are preferably linked via a phosphodiester bond or a modified phosphodiester bond, more preferably via a phosphodiester bond. In one embodiment, they are preferably linked via a phosphorothioate bond.

[0115] The oligonucleotide compound or oligonucleotide conjugate of the present invention is not particularly limited as long as it is an oligonucleotide useful as a nucleic acid drug, and may be, for example, an antisense oligonucleotide, siRNA, aptamer, ribozyme, miRNA, CpG oligo, decoy nucleic acid, etc. Antisense oligonucleotides and siRNA can be designed to be complementary to target RNA. The complementarity 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).

[0116] The oligonucleotide compound of the present invention is preferably an antisense oligonucleotide. Examples of antisense oligonucleotides include, but are not limited to, DNA, oligodeoxyribonucleotides, gapmer-type antisense oligonucleotides (also simply referred to as "gapmers"), and mixmer-type antisense oligonucleotides (also simply referred to as "mixmers"). They may also include, but are not limited to, RNA, oligoribonucleotides, or oligonucleotides designed to typically produce an antisense effect. Alternatively, the "oligonucleotide complex" of the present invention may be an HDO (heteroduplex nucleic acid) comprising a single-stranded DNA antisense oligonucleotide (first oligonucleotide) and a complementary RNA oligonucleotide (second oligonucleotide). Alternatively, the first and second oligonucleotides may be single-stranded oligonucleotide compounds linked by a nucleic acid linker, single-stranded oligonucleotide compounds linked by a linking group containing a non-nucleotide structure, or single-stranded oligonucleotide compounds directly linked together (single-stranded heteroduplex nucleic acid).

[0117] W in the general formula (I) of the present invention is preferably a group derived from a gapmer antisense oligonucleotide. A group derived from a gapmer antisense oligonucleotide refers to a partial structure formed by removing a hydrogen atom, hydroxyl group, or the like from the 3'- or 5'-end of a gapmer, and is, for example, composed of 7 to 100 nucleosides, preferably 10 to 40 nucleosides, and more preferably 13 to 25 nucleosides, covalently bonded to L at the 3'- or 5'-end and covalently bonded to L at the 5'-end. Furthermore, gapmer antisense oligonucleotides preferably contain at least four consecutive deoxyribonucleosides. Furthermore, gapmer antisense oligonucleotides preferably contain at least one selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

[0118] W in the general formula (I) of the present invention is preferably a group derived from a mixmer-type antisense oligonucleotide. The group derived from a mixmer-type antisense oligonucleotide refers to a partial structure formed by removing a hydrogen atom, a hydroxyl group, or the like from the 3'- or 5'-end of a mixmer, and is, for example, composed of 7 to 100 nucleosides, preferably 10 to 40 nucleosides, and more preferably 13 to 25 nucleosides, and is preferably covalently bonded to L at its 3'- or 5'-end and L at its 5'-end. Furthermore, the mixmer-type antisense oligonucleotide preferably does not contain at least four consecutive deoxyribonucleosides. Furthermore, the mixmer-type antisense oligonucleotide preferably contains at least one selected from the group consisting of a 2'-modified nucleoside and a 2'-4'-bridged nucleoside.

[0119] Each internucleoside bond in gapmer-type and mixmer-type antisense oligonucleotides may be a phosphodiester bond or a modified internucleoside bond. Each internucleoside bond is, for example, independently and preferably a phosphodiester bond or a phosphorothioate bond. More preferably, the 5'-most internucleoside bond is a phosphorothioate bond, and the 3'-most internucleoside bond is a phosphorothioate bond. Preferably, 50 to 100% (more preferably, 70 to 100%, 80 to 100%, 90 to 100%, or 95 to 100%) of the internucleoside bonds in the antisense oligonucleotide are phosphorothioate bonds. The remaining internucleoside bonds are phosphodiester bonds.

[0120] Alternatively, W in general formula (I) of the present invention is preferably an oligonucleotide conjugate derived from a double-stranded nucleic acid. Double-stranded nucleic acids are described as heteroduplexes (HDOs), for example, in International Publication No. WO 2013 / 089283, the entire contents of which are incorporated herein by reference. Preferably, the oligonucleotide conjugate is a double-stranded oligonucleotide conjugate comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is a gapmer antisense oligonucleotide or a mixmer antisense oligonucleotide consisting of 7 to 100 nucleosides, and the second oligonucleotide comprises a sequence capable of hybridizing with at least a portion of the first oligonucleotide and consists of 4 to 100 nucleosides independently selected from deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, and the first oligonucleotide and the second oligonucleotide hybridize to each other. Preferably, the oligonucleotide conjugate is covalently linked to L via the 3' or 5' end of the first or second oligonucleotide, more preferably via the 3' or 5' end of the second oligonucleotide, and especially preferably via the 5' end of the second oligonucleotide.

[0121] Alternatively, W in the general formula (I) of the present invention is preferably a group derived from a single-stranded oligonucleotide compound in which the first and second oligonucleotides of the oligonucleotide conjugate are linked by a nucleic acid linker. As the nucleic acid linker, a group derived from an oligonucleotide consisting of 2 to 10 nucleosides is preferred, a group derived from an oligonucleotide consisting of 3 to 7 nucleosides is more preferred, a group derived from an oligonucleotide consisting of 4 or 5 nucleosides is even more preferred, and a group derived from an oligonucleotide consisting of 4 nucleosides is particularly preferred. The internucleoside bonds linking the nucleosides constituting the linker may independently be phosphodiester bonds or modified internucleoside bonds, but are preferably independently phosphodiester bonds or phosphorothioate bonds. More preferably, the linker contains one or two phosphorothioate bonds, with the other internucleoside bond being a phosphodiester bond. Particularly preferably, the internucleoside bonds linking the nucleosides constituting the linker are phosphodiester bonds. The nucleosides constituting the linker are preferably independently adenosine, uridine, cytidine, guanosine, 2'-deoxyadenosine, thymidine, 2'-deoxycytidine, or 2'-deoxyguanosine, with adenosine being particularly preferred. Alternatively, W in the general formula (I) of the present invention is preferably a group derived from a single-stranded oligonucleotide compound in which the first and second oligonucleotides of the oligonucleotide conjugate are linked via a linking group containing a non-nucleotide structure. Examples of linking groups containing a non-nucleotide structure include alkylene groups having 2 to 50 carbon atoms and groups derived from polyalkylene glycol. Alternatively, W in the general formula (I) of the present invention is preferably a group derived from a single-stranded oligonucleotide compound in which the first and second oligonucleotides of the oligonucleotide conjugate are directly linked.The first oligonucleotide of the oligonucleotide conjugate is preferably a gapmer antisense oligonucleotide, and the above-described embodiments of the gapmer antisense oligonucleotide apply to the first oligonucleotide of the oligonucleotide conjugate. When the first oligonucleotide is a gapmer, the number of nucleosides constituting the second oligonucleotide is preferably 7 to 50, more preferably 7 to 27, even more preferably 14 to 22, and particularly preferably 16, as in the case of a gapmer. When the first oligonucleotide is a mixmer, the number of nucleosides constituting the second oligonucleotide is preferably 10 to 40, more preferably 13 to 25, as in the case of a mixmer. The second oligonucleotide preferably contains at least one ribonucleoside, more preferably at least four consecutive ribonucleosides, and even more preferably 1 to 6 (even more preferably 2 to 4, even more preferably 2 to 3) sugar-modified nucleosides (even more preferably 2'-modified nucleosides), and the other nucleosides contained in the second oligonucleotide are ribonucleosides. Even more preferably, both or one of the 3'- and 5'-ends of the second oligonucleotide is a sugar-modified nucleoside. Particularly preferably, all nucleosides constituting the second oligonucleotide are ribonucleosides. The internucleoside linkages connecting the nucleosides constituting the second oligonucleotide may independently be phosphodiester linkages or modified internucleoside linkages, but are preferably independently phosphodiester linkages or phosphorothioate linkages, more preferably contain 1 to 6 (even more preferably 2 to 4, 2 to 3) phosphorothioate linkages, and the remaining internucleoside linkages are phosphodiester linkages. Particularly preferably, the internucleoside linkages connecting the nucleosides constituting the second oligonucleotide are phosphodiester linkages. The above-mentioned embodiments are described, for example, in WO 2017 / 131124, WO 2018 / 143475, and WO 2019 / 022196, the entire contents of which are incorporated herein by reference.

[0122] As W in the general formula (I) of the present invention, a group derived from siRNA is also preferred. Either the antisense strand or the sense strand may be bound to L, but it is preferred that the sense strand be bound to L. The 5'-end or 3'-end of the antisense strand is bound to L, and more preferably the 3'-end is bound to L. The 5'-end or 3'-end of the sense strand is bound to L, and more preferably the 5'-end is bound to L.

[0123] The group derived from siRNA may, for example, be linked to L via an internucleoside linkage, preferably via a phosphodiester or phosphorothioate linkage, more preferably via a phosphorothioate linkage.

[0124] The lipid-linked oligonucleotides and the like according to this embodiment can be produced by the method shown below. However, the production method below shows an example of a general production method and does not limit the method for producing the oligonucleotides and the like according to this embodiment.

[0125]

[0126] The symbols in the formula are the same as defined above, and step means a process.

[0127] Compound A, which is the starting material in Step I, can be synthesized, for example, according to the method described in JP-A-2018-532990 or Japanese Patent No. 6356171. １ , R ２ Compound A having the formula: can be synthesized from Compound A-1 described below by combining oxidation reactions known to those skilled in the art (for oxidation reactions, see, for example, Comprehensive Organic Transformations, Second Edition, by R. C. Larock, Wiley-VCH (1999)).

[0128]

[0129] In the formula, R １ and R ２is the same as defined above. For example, R １ and R ２ In order to synthesize Compound A-1 in which R is an alkyl group or an alkenyl group, the primary hydroxy group of Compound A-2 is converted to R １ The desired R1 can be introduced by alkylation or alkenylation using an alkyl halide reagent corresponding to the formula: ２ By alkylating or alkenylating the compound with an alkyl halide reagent corresponding to the compound, the desired compound R ２ The hydroxy group protected by P1 in the resulting compound is deprotected to introduce R １ and R ２ is an alkyl group, an alkenyl group, or the like.

[0130] R １ and R ２ Compound A-1 is a compound in which R １ and R ２ The desired R １ and R ２ After introducing the above, the saturated ring compound can be obtained by an olefin metathesis reaction. Specific examples of the olefin metathesis reaction include a method in which a second-generation Grubbs catalyst, such as dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine)ruthenium(II), is reacted in a solvent. Furthermore, for example, the saturated ring compound can be obtained by hydrogenating the olefin ring compound obtained by the olefin metathesis reaction with hydrogen and palladium carbon in a solvent.

[0131] In the formula, P １ and represent a hydroxy-protecting group, and the other symbols are as defined above.

[0132] (Step I) Amidation Reaction with a Primary Amine A primary alkylamine bound to L can be used to carry out a condensation exchange reaction with a carboxylic acid (compound A) to obtain compound B. For example, compound A can be reacted with 1 to 10 equivalents of the primary alkylamine in a solvent in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate.

[0133] (Step II) Phosphitylation Reaction The hydroxy group of Compound B can be phosphitylated by a reaction known to those skilled in the art (for example, a reaction using a disubstituted alkoxyphosphine) to obtain Compound C. A specific example of the phosphitylation reaction includes a method of reacting 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite in a solvent in the presence of 4,5-dicyanoimidazole.

[0134] (Step III) Conjugation reaction between oligonucleotide and lipid The lipid-conjugated oligonucleotide can be synthesized using Compound C and commercially available nucleotides, phosphoramidite reagents, etc., necessary for producing an oligonucleotide compound of a desired nucleotide sequence, using an automatic nucleic acid synthesizer (e.g., M-8-SE (manufactured by Nippon Techno Co., Ltd.), nS-8II (manufactured by Gene Design Co., Ltd.), etc.).

[0135] The desired oligonucleotide compound can be produced using commercially available phosphoramidite reagents corresponding to the nucleotide sequence, by solid-phase synthesis using an automatic nucleic acid synthesizer, or by reactions using enzymes (polymerase, ligase, restriction enzyme, etc.). When the oligonucleotide compound or its complex contains a double-stranded structure that hybridizes with each other, the method may include mixing the oligonucleotide compounds in an appropriate buffer, denaturing them at 90 to 98°C for several minutes (e.g., 5 minutes), and then hybridizing them at 30 to 70°C for 1 to 8 hours.

[0136] One embodiment of the present invention is a compound of the formula (II): wherein L is a substituted or unsubstituted C １－１０is an alkylene group, R １ and R ２ are each independently a substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ may be bonded to each other to form a ring, R ３ are each independently C １－１０ alkyl group or R ３ The two alkyl groups may be bonded to each other to form a ring, ４ is a 2-cyanoethyl group. The compound of formula (II) is useful for producing the lipid-linked oligonucleotide of the present invention.

[0137] One embodiment of the present invention is a pharmaceutical composition comprising the lipid-linked oligonucleotide compound of the general formula (I) or a complex thereof and a pharmacologically acceptable carrier.

[0138] The pharmaceutical composition of the present invention containing the lipid-linked oligonucleotide of general formula (I) or a complex thereof can be formulated by known pharmaceutical methods, for example, into 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., and can be used enterally (orally, etc.) or parenterally.

[0139] 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.

[0140] The administration route of the pharmaceutical composition containing the lipid-linked oligonucleotide of the general formula (I) of the present invention or a complex thereof is not particularly limited, and includes enteral (oral, etc.) or parenteral administration. More preferred administration routes 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.

[0141] Diseases that can be treated, prevented, or ameliorated by a nucleic acid drug using a pharmaceutical composition containing the lipid-linked oligonucleotide of the general formula (I) of the present invention or a complex thereof are not particularly limited, and examples thereof 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.

[0142] In addition to primates such as humans, various mammalian diseases can be treated, prevented, or ameliorated by pharmaceutical compositions containing the lipid-linked oligonucleotide of the general formula (I) of the present invention or its complex. For example, but not limited to, diseases of mammalian species, including 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).

[0143] When a pharmaceutical composition containing the lipid-linked oligonucleotide of the general formula (I) of the present invention or a complex thereof is administered to or ingested by an animal including a human, the dosage or intake amount is appropriately selected depending on the age, body weight, symptoms, health condition, type of composition (drug, food or drink, etc.) of the subject, etc., and the dosage or intake amount is preferably 0.0001 mg / kg / day to 100 mg / kg / day in terms of lipid-linked oligonucleotide.

[0144] The lipid-linked oligonucleotide of the present invention represented by the general formula (I) or a complex thereof is delivered to a target organ more efficiently than conventional oligonucleotides, and therefore, an enhanced pharmacological effect can be expected. Therefore, a method for safely regulating target gene expression can be provided by administering the lipid-linked oligonucleotide of the present invention represented by the general formula (I) or a complex thereof to an animal, including a human. Furthermore, a method for treating, preventing, or ameliorating various diseases involving target gene regulation can be provided, which comprises administering a composition containing the lipid-linked oligonucleotide of the present invention represented by the general formula (I) or a complex thereof to a mammal, including a human.

[0145] Preferred methods for using the lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex thereof include the following: - A method for regulating the function of a target RNA, comprising the step of contacting a cell with the lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex 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 lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex thereof. - Use of the lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex thereof for regulating the function of a target RNA in a mammal. - Use of the lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex thereof for producing a drug 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 lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex 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 lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex thereof. - Use of the lipid-linked oligonucleotide of the present invention represented by general formula (I) or a complex thereof for regulating the expression of a target gene in a mammal. Use of the lipid-linked oligonucleotide of the present invention of said general formula (I) or a complex thereof for the manufacture of a drug for controlling the expression of a target gene in a mammal.

[0146] In the present invention, control of the function of a target RNA means, for example, regulation or conversion of a splicing function such as translation inhibition or exon skipping, which occurs when an antisense sequence portion coats a part of the target RNA through hybridization, or suppression of the function of the target RNA through degradation of the target RNA, which may occur when a part where the antisense sequence portion hybridizes with a part of the target RNA is recognized.

[0147] The mammal is preferably a human. The route of administration is preferably enteral. In another embodiment, the route of administration is parenteral.

[0148] The present invention will be described in more detail below with reference to examples, which are not intended to limit the scope of the present invention. In the examples, "NMR" means nuclear magnetic resonance, and "(v / v)" means (volume / volume). １ When H NMR data is listed, it was measured at 300 MHz (JNM-ECX300; JEOL Ltd., or JNM-ECP400; JEOL Ltd.), and the chemical shift δ (unit: ppm) (splitting pattern, integrated value) of the signal was measured using tetramethylsilane as an internal standard. "d" means doublet, "t" means triplet, "dd" means doublet of doublets, "m" means multiplet, "J" means coupling constant, and "CDCl" means deuterated chloroform. Purification by silica gel column chromatography was performed using Purif-Pack (registered trademark)-EX (SI-50 μm) manufactured by SHOKO SCIENCE.

[0149] [Production Example 1]

[0150] Under an argon atmosphere, sodium hydride (55% by weight liquid paraffin, 8.38 g, 192 mmol) and 1-bromotetradecane (67.1 mL, 247 mmol) were added to a solution of compound 1 (10.0 g, 54.9 mmol) in dimethylformamide (100 mL), and the mixture was heated and stirred at 80°C overnight (approximately 16 to 20 hours). After cooling to room temperature, water was added to the reaction mixture, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate and washed with dilute hydrochloric acid, water, and brine, successively. After concentrating the organic layer, the resulting crude compound 2 was dissolved in ethyl acetate (100 mL) and methanol (100 mL), and 10% palladium-carbon (5.0 g) was added. The mixture was stirred overnight (approximately 16 to 20 hours) under a hydrogen atmosphere. The reaction solution was filtered through Celite, and the filter cake was washed with a mixed solution of ethyl acetate, ethanol, and water. The filtrate and the washings were then concentrated to obtain a crude product, which was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 3 (17.8 g, yield 67%) as a white solid. 1H NMR (CDCl3, 300 MHz) δ 0.88 (3H, t, 6.6 Hz), 1.19-1.38 (44H, m), 1.50-1.62 (4H, m), 2.16 (1H, t, 6.3Hz), 3.40-3.66 (8H, m), 3.68-3.77 (1H, m).

[0151] To a mixed solution of compound 3 (17.4 g, 35.9 mmol) in methylene chloride (120 mL) and water (60 mL), 2-azaadamantane-N-hydroxyl (AZADOL®) (457 mg, 1.08 mmol) and diacetoxyiodobenzene (34.7 g, 108 mmol) were added, and the mixture was stirred overnight (approximately 16 to 20 hours) at room temperature. Subsequently, an aqueous sodium thiosulfate solution was added to the reaction solution, followed by extraction with methylene chloride. The organic layer was washed with water and dried with sodium sulfate. After concentration, methanol was added to the resulting crude product, which was then vigorously stirred and filtered. This procedure was repeated three times to obtain compound 4 (16.3 g, yield 91%) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (3H, t, 7.2 Hz), 1.20-1.37 (44H, m), 1.53-1.68 (4H, m), 3.43-3.53 (2H, m), 3.61-3.67 (2H, m), 3.71 (1H, dd, 10.6Hz, 5.0Hz), 3.80 (1H, dd, 10.6Hz, 3.2Hz), 4.04 (1H, dd, 5.0Hz, 3.2Hz).

[0152] Under an argon atmosphere, triethylamine (4.59 mL, 66.4 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (10.1 g, 26.6 mmol), and 6-amino-1-hexanol (3.11 g, 26.6 mmol) were added to a solution of compound 4 (11.0 g, 22.1 mmol) in methylene chloride (100 mL), and the mixture was stirred overnight (approximately 16 to 20 hours) at room temperature. Subsequently, water was added to the reaction mixture, which was then extracted with methylene chloride. The organic layer was washed with dilute hydrochloric acid and brine. After concentration, ethyl acetate was added to the resulting crude product, which was vigorously stirred and then filtered. This procedure was repeated three times to obtain the desired compound 5 (11.8 g, 87% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (3H, t, 7.2Hz), 1.21-1.44 (48H, m), 1.49-1.64 (9H, m), 3.28 (1H, dd, 13.6Hz, 6.8Hz), 3.38-3.53 (3H, m), 3.38-3.53 (3H, m), 3.59-3.66 (4H, m), 3.77 (1H, dd, 10.6Hz, 2.8Hz), 3.87 (1H, dd, 5.4Hz, 2.4Hz), 6.70 (1H, t, 6.0Hz).

[0153] Toluene was added to compound 5 (4.58 g, 7.66 mmol), and the mixture was concentrated. Under an argon atmosphere, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (3.40 mL, 10.7 mmol) was added to a methylene chloride (40 mL) solution of the concentrated compound 5, and the mixture was stirred at room temperature. Subsequently, an acetonitrile solution of 4,5-dicyanoimidazole (628 mg, 5.36 mmol) was added, and the mixture was stirred for 2 hours. After concentration, methylene chloride was added, and the mixture was filtered to remove the white precipitate. The filtrate was concentrated and then purified by silica gel column chromatography (hexane / ethyl acetate / triethylamine), yielding the desired compound 6 (3.22 g, 53%) as a white solid. 1H NMR (CDCl3, 300 MHz) δ 0.88 (3H, t, 6.6Hz), 1.17 (3H, d, 3.9Hz), 1.19 (3H, d, 3.6Hz), 1.23-1.38 (48H, m), 1.47-1.66 (8H, m), 2.64 (1H, t, 6.9Hz), 3.26 (1H, dd, 13.7Hz, 5.7Hz), 3.36-3.68 (9H,m), 3.74-3.89 (4H, m), 6.68 (1H, t, 6.3Hz).

[0154] [Production Example 2]

[0155] Under an argon atmosphere, sodium hydride (55 wt % liquid paraffin, 2.51 g, 57.6 mmol) and 1-bromohexadecane (22.6 mL, 74.1 mmol) were added to a solution of compound 1 (3.00 g, 16.5 mmol) in dimethylformamide (30 mL), and the mixture was heated and stirred at 80°C overnight (approximately 16 to 20 hours). After cooling to room temperature, water was added to the reaction mixture, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate and washed successively with dilute hydrochloric acid, water, and brine. After concentrating the organic layer, the resulting crude compound 7 was dissolved in ethyl acetate (30 mL) and methanol (30 mL), and 10% palladium-carbon (1.5 g) was added. The mixture was stirred overnight (approximately 16 to 20 hours) under a hydrogen atmosphere. The reaction mixture was filtered through Celite, and the filter cake was washed with a mixed solution of ethyl acetate, ethanol, and water. The filtrate and washings were concentrated to obtain a crude product, which was then added with ethyl acetate and ethanol, stirred vigorously, and filtered. This procedure was repeated three times to obtain the desired compound 8 (7.79 g, yield 87%) as a white solid. 1 H NMR (CDCl3, 300 MHz) δ 0.88 (6H, t, 6.5 Hz), 1.20-1.36 (52H, m), 1.50-1.62 (4H, m), 2.16 (1H, dd, 6.8Hz, 5.8Hz), 3.39-3.66 (8H, m), 3.68-3.77 (1H, m).

[0156] Compound 9 (2.68 g, yield 68%) was obtained as a white solid using compound 8 (3.87 g, 7.15 mmol) as a starting material in the same manner as in the synthesis of compound 4. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.2 Hz), 1.19-1.38 (52H, m), 1.52-1.68 (4H, m), 3.42-3.54 (2H, m), 3.59-3.73 (3H, m), 3.80 (1H, dd, 10.6Hz, 3.3Hz), 4.04 (1H, dd, 5.1Hz, 3.2Hz).

[0157] Compound 10 (1.61 g, 85% yield) was obtained as a white solid using compound 9 (1.60 g, 2.88 mmol) as a starting material in the same manner as in the synthesis of compound 5. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 7.0Hz), 1.19-1.44 (56H, m), 1.48-1.66 (9H, m), 3.28 (2H, dd, 13.9Hz, 7.0Hz), 3.38-3.54 (3H, m), 3.59-3.66 (4H, m), 3.77 (1H, dd, 10.6Hz, 2.8Hz), 3.87 (1H, dd, 5.1Hz, 2.2Hz), 6.70 (1H, t, 6.2Hz).

[0158] Compound 11 (1.93 g, 92% yield) was obtained as a white solid using compound 10 (1.60 g, 2.45 mmol) as a starting material in the same manner as in the synthesis of compound 6. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.6Hz), 1.11-1.42 (68H, m), 1.47-1.66 (8H, m), 2.64 (2H, t, 6.6Hz), 3.18-3.32 (2H, m), 3.36-3.70 (9H, m), 3.71-3.90 (4H, m), 6.78 (1H, t, 5.9Hz).

[0159] [Production Example 3]

[0160] Under an argon atmosphere, sodium hydride (55 wt % liquid paraffin, 2.51 g, 57.6 mmol) and 1-bromododecane (17.8 mL, 74.1 mmol) were added to a solution of compound 1 (3.00 g, 16.5 mmol) in dimethylformamide (30 mL), and the mixture was heated and stirred at 80°C overnight (approximately 16 to 20 hours). After cooling to room temperature, water was added to the reaction mixture, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate and washed with dilute hydrochloric acid, water, and brine, successively. After concentrating the organic layer, the resulting crude compound 12 was dissolved in ethyl acetate (30 mL) and methanol (30 mL), and 10% palladium-carbon (1.5 g) was added. The mixture was stirred overnight (approximately 16 to 20 hours) under a hydrogen atmosphere. The reaction solution was filtered through Celite, and the filter cake was washed with a mixed solution of ethyl acetate, ethanol, and water. The filtrate and the washings were then concentrated to obtain a crude product, which was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 13 (4.62 g, yield 65%) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.6 Hz), 1.20-1.37 (36H, m), 1.52-1.61 (4H, m), 2.19 (1H, t, 5.5Hz), 3.41-3.65 (8H, m), 3.69-3.76 (1H, m).

[0161] Starting from compound 13 (4.00 g, 9.33 mmol), crude compound 14 was obtained in the same manner as in the synthesis of compound 4. Methanol was added to the crude compound obtained, and the mixture was stirred vigorously and then filtered to obtain compound 14 (2.00 g, yield 48%) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.6 Hz), 1.20-1.38 (36H, m), 1.51-1.68 (4H, m), 3.42-3.54 (2H, m), 3.64 (2H, t, 6.6Hz), 3.71 (1H, dd, 10.6Hz, 5.1Hz), 3.80 (1H, dd, 10.6Hz, 3.3Hz), 4.05 (1H, dd, 5.1Hz, 3.3Hz).

[0162] Starting from compound 14 (2.00 g, 4.51 mmol), crude compound 15 was obtained in the same manner as in the synthesis of compound 5. Ethyl acetate was added to the crude compound obtained, and the mixture was stirred vigorously and then filtered to obtain compound 15 (1.23 g, yield 50%) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.6Hz), 1.21-1.44 (40H, m), 1.48-1.65 (9H, m), 3.28 (2H, dd, 12.8Hz, 6.6Hz), 3.38-3.54 (3H, m), 3.59-3.66 (4H, m), 3.77 (1H, dd, 10.6Hz, 2.6Hz), 3.87 (1H, dd, 5.1Hz, 2.6Hz), 6.70 (1H, t, 6.2Hz).

[0163] Compound 16 (1.21 g, yield 74%) was obtained as a white solid using compound 15 (1.20 g, 2.21 mmol) as a starting material in the same manner as in the synthesis of compound 6. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.6Hz), 1.17 (3H, d, 5.5Hz), 1.19 (3H, d, 5.1Hz), 1.22-1.43 (40H, m), 1.48-1.65 (8H, m), 2.64 (2H, t, 7.0Hz), 3.22-3.30 (2H, m), 3.38-3.68 (9H,m), 3.74-3.89 (4H, m), 6.69 (1H, t, 5.9Hz).

[0164] [Production Example 4]

[0165] Under an argon atmosphere, sodium hydride (55 wt % liquid paraffin, 2.51 g, 57.6 mmol) and 1-bromodecane (15.9 mL, 76.8 mmol) were added to a solution of compound 1 (3.50 g, 19.2 mmol) in dimethylformamide (30 mL), and the mixture was heated and stirred at 80°C overnight (approximately 16 to 20 hours). After cooling to room temperature, water was added to the reaction mixture, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate and washed successively with dilute hydrochloric acid, water, and brine. After concentrating the organic layer, the resulting crude compound 17 was dissolved in ethyl acetate (30 mL) and methanol (30 mL), and 10% palladium-carbon (1.5 g) was added. The mixture was stirred overnight (approximately 16 to 20 hours) under a hydrogen atmosphere. The reaction solution was filtered through Celite, and the filter cake was washed with a mixed solution of ethyl acetate and methanol. The filtrate and washings were then concentrated to obtain a crude product, which was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 18 (6.08 g, yield 85%) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.88 (6H, t, 6.6 Hz), 1.22-1.36 (28H, m), 1.52-1.62 (4H, m), 2.19 (1H, t, 6.6Hz), 3.41-3.66 (8H, m), 3.70-3.76 (1H, m).

[0166] To a mixed solution of compound 18 (4.00 g, 10.7 mmol) in methylene chloride (40 mL) and water (20 mL), 2-azaadamantane-N-hydroxyl (AZADOL (registered trademark)) (956 mg, 2.25 mmol) and diacetoxyiodobenzene (7.26 g, 22.5 mmol) were added, and the mixture was stirred overnight (approximately 16 to 20 hours) at room temperature. Subsequently, an aqueous sodium thiosulfate solution was added to the reaction solution, followed by extraction with methylene chloride, and the organic layer was washed with water and dried over sodium sulfate. After concentration, methylene chloride (40 mL), triethylamine (1.66 mL, 12.0 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (4.90 g, 12.9 mmol), and 6-amino-1-hexanol (1.51 g, 12.9 mmol) were added to the resulting compound 19, and the mixture was stirred overnight (approximately 16 to 20 hours) at room temperature. Subsequently, water was added to the reaction mixture, followed by extraction with methylene chloride. The organic layer was washed with dilute hydrochloric acid and brine. After concentration, the resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 20 (4.64 g, 89% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 0.86-0.91 (6H, m), 1.20-1.45 (32H, m), 1.48-1.65 (9H, m), 3.28 (2H, dd, 13.6Hz, 6.2Hz), 3.38-3.54 (3H, m), 3.59-3.66 (4H, m), 3.77 (1H, dd, 10.6Hz, 2.6Hz), 3.87 (1H, dd, 5.1Hz, 2.6Hz), 6.71 (1H, t, 5.9Hz).

[0167] Compound 21 (1.11 g, 52% yield) was obtained as a white solid using compound 20 (1.50 g, 2.29 mmol) as a starting material in the same manner as in the synthesis of compound 6. 1H NMR (CDCl3, 400 MHz) δ 0.85-0.91 (6H, m), 1.17 (3H, d, 5.1Hz), 1.19 (3H, d, 5.5Hz), 1.21-1.41 (32H, m), 1.48-1.66 (8H, m), 2.65 (2H, t, 6.6Hz), 3.23-3.31 (2H, m), 3.38-3.69 (9H, m), 3.74-3.90 (4H, m), 6.69 (1H, t, 5.9Hz).

[0168] [Production Example 5]

[0169] Under an argon atmosphere, sodium hydride (55 wt % liquid paraffin, 2.87 g, 65.9 mmol) and 10-bromo-1-decene (17.7 mL, 87.8 mmol) were added to a solution of compound 1 (4.00 g, 22.0 mmol) in dimethylformamide (40 mL), and the mixture was heated and stirred at 80°C overnight (approximately 16 to 20 hours). After cooling to room temperature, water was added to the reaction mixture, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate and washed with water and then brine. The organic layer was concentrated, and the resulting crude product was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 22 (6.16 g, yield 61%) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ 1.23-1.41 (20H, m), 1.50-1.61 (4H, m), 2.00-2.08 (4H, m), 3.43 (2H, t, 6.6Hz), 3.46-3.63 (7H, m), 4.55 (2H, s), 4.90-4.96 (2H, m), 4.95-5.03 (2H, m), 5.75-5.87 (2H, m), 7.27-7.36 (5H, m).

[0170] Under an argon atmosphere, (1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(ortho-isopropoxyphenylmethylene)ruthenium (Hoveyda-Grubbs catalyst (registered trademark)) (444 mg, 0.523 mmol) was added to a solution of compound 22 (4.80 g, 10.5 mmol) in 1,2-dichloromethane (1.05 L), and the mixture was stirred at 80°C overnight (approximately 16 to 20 hours). After completion of the reaction, the reaction solution was allowed to cool to room temperature and concentrated. The resulting residue was filtered through silica gel and washed with a mixed solution of ethyl acetate and hexane. Crude compound 23 obtained after concentration was dissolved in ethyl acetate (43 mL) and methanol (43 mL), and 10% palladium-carbon (2.4 g) was added, followed by stirring overnight (approximately 16 to 20 hours) under a hydrogen atmosphere. The reaction solution was filtered through Celite, and the filter cake was washed with a mixed solution of ethyl acetate and methanol. The filtrate and washings were then concentrated to obtain a crude product, which was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 24 (2.32 g, yield 65%) as a colorless oil. 1 H NMR (CDCl3, 300 MHz) δ 1.23-1.44 (28H, m), 1.48-1.70 (4H, m), 2.14 (1H, s), 3.38-3.73 (9H, m).

[0171] Compound 26 (975 mg, yield 49%) was obtained as a colorless oil using compound 24 (1.50 g, 4.38 mmol) as a starting material in the same manner as in the synthesis of compound 20. 1 H NMR (CDCl3, 400 MHz) δ 1.29-1.50 (32H, m), 1.53-1.71 (8H, m), 3.32 (1H, dd, 13.9Hz, 7.0Hz), 3.47-3.55 (9H, m), 3.59-3.66 (9H, m), 3.68 (1H, t, 6.6Hz), 3.80-3.87 (1H, m), 3.95 (1H, dd, 6.2Hz, 2.2Hz), 6.71-6.81 (1H, m).

[0172] Compound 27 (980 mg, yield 70%) was obtained as a colorless oil using Compound 26 (970 mg, 2.13 mmol) as a starting material and in the same manner as in the synthesis of Compound 6. 1 H NMR (CDCl3, 400 MHz) δ 1.17 (3H, d, 4.8Hz), 1.19 (3H, d, 5.1Hz), 1.27-1.43 (32H, m), 1.48-1.66 (8H, m), 2.65 (2H, t, 6.6Hz), 3.19-3.32 (2H, m), 3.41-3.51 (2H, m), 3.52-3.70 (7H, m), 3.75-3.93 (4H, m), 6.66-6.75 (1H, m).

[0173] In Table (1) in the Examples, "Compd No." means the compound number, and "Chemical Structure" means the chemical structure of each compound. The target gene is mouse Metastasis Associated in Lung Adenocarcinoma Transcript-1 (Malat1) or mouse Hypoxanthine Phosphoribosyltransferase 1 (Hprt1). In the sequence notations in the Examples (Table 1), unless otherwise specified, "(L)" represents LNA (β-D-methyleneoxy BNA), lowercase letters represent deoxyribonucleosides, uppercase letters (excluding the above (L) and the letters marked with (I) below) represent ribonucleosides, "^" represents a phosphorothioate bond, "5(x)" represents that the nucleobase of the deoxyribonucleoside is 5-methylcytosine, and the "5" in "5(L)" represents that the nucleobase of the nucleoside is 5-methylcytosine. "J1-" represents that the nucleotide represented by the following formula (III) is linked to the oxygen atom of the hydroxy group at the 5'-end via a phosphodiester bond. (where * represents the bonding position with oligonucleotide W. (* represents the oxygen atom of the phosphodiester bond)) is bonded to the oxygen atom of the hydroxy group at the 5'-end. "J2-" means that a group represented by the following formula (IV): (where * represents the bonding position with the oligonucleotide W. (* is the oxygen atom of the phosphodiester bond)) means that a group represented by the following is bonded. In the notation of chemical structures in the examples (Tables 1, 4 and 6), when there is no "^" between two adjacent nucleosides, the internucleoside bond between the two nucleic acid bases is a phosphodiester bond. For example, in the notation "A(L)T(L)", the internucleoside bond between A and T is a phosphodiester bond, and in the notation "AG(L)", the internucleoside bond between A and G is a phosphodiester bond.

[0174] [Preparation Example 6] The antisense oligonucleotides (compounds represented by chemical structures corresponding to the compound numbers) listed in Table 1 were prepared using an automated nucleic acid synthesizer nS-8II (manufactured by Gene Design) and M-8-SE (manufactured by Nippon Techno Service Co., Ltd.). Furthermore, intramolecular hybridization of the single-stranded oligonucleotides P22790172 and P22790146 was confirmed by non-denaturing polyacrylamide gel electrophoresis. In non-denaturing polyacrylamide gel electrophoresis, single-stranded DNA size markers (manufactured by Gene Design) containing single-stranded DNA with 15, 20, 30, 40, 50, 60, and 80 nucleotides and double-stranded RNA size markers (manufactured by Gene Design) with 17, 21, 25, and 29 base pairs were used as markers, and a single band was confirmed for each single-stranded oligonucleotide.

[0175]

[0176] Evaluation Example 1 Antisense Inhibition of Mouse Malat1 in 3T3-L1 Cells 3T3-L1 cells were seeded onto a 96-well plate at a density of 20,000 cells / well. After approximately 24 hours, the culture supernatant of the cells was replaced with D-MEM (Dulbecco's Modified Eagle Medium) containing 2% FBS, and P21790027 and P22790171 were added to a final concentration of 1000 nM (Free-Uptake). After 72 hours, RNA-containing cell lysates were prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio), and the expression level of the mouse Malat1 gene was 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 (glyceraldehyde-3-phosphate dehydrogenase) was also quantified, and the Malat1 mRNA level relative to the Gapdh mRNA level was evaluated as the Malat1 expression level. The results are shown in Table 2 as the percentage expression of Malat1 relative to untreated control cells.

[0177]

[0178] Evaluation Example 2 Antisense Inhibition of Mouse Malat1 in N1E-115 Cells N1E-115 cells were seeded into a 96-well plate at a density of 20,000 cells / well. After approximately 24 hours, the culture supernatant of the cells was replaced with FBS-free D-MEM, and P21790027 and P22790171 were added to a final concentration of 1000 nM (Free-Uptake). After 24 hours, RNA-containing cell lysates were prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio). Then, mouse Malat1 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 Malat1 mRNA level relative to the Gapdh mRNA level was evaluated as the Malat1 expression level. The results are presented in Table 3 as percent expression of Malat1 relative to untreated control cells.

[0179]

[0180] Evaluation Example 3: Antisense inhibition of Malat1 in mice C57BL / 6J mice (male, 6 weeks old, Jackson Laboratory Japan, Inc.) were intravenously administered with P21790027, P22790171, and P22790172 dissolved in saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory, Inc.) at a dose of 0.95 μmol / kg of antisense oligonucleotide per mouse. As a control, saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory, Inc.) alone was administered. Five and ten days after administration, heart, muscle, lung, liver, and kidney tissues were collected under isoflurane anesthesia (P22790172 was collected only five days after administration). After isolating RNA from each organ using the MagMAX® mirVana® Total RNA Isolation Kit (Thermo Fisher Scientific), the expression level of the mouse Malat1 gene was 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 amount of the housekeeping gene Gapdh was also quantified simultaneously, and the amount of Malat1 mRNA relative to the amount of Gapdh mRNA was evaluated as the expression level of Malat1. The results are shown in Figure 1 (Day 10) and Figure 2 (Day 5) as the percentage expression of Malat1 relative to the untreated control group (control).

[0181] As is clear from Figures 1 and 2, P22790171 and P22790172 showed a higher inhibitory effect on Malat1 expression in the heart, muscle, lung, and liver than P21790027.

[0182] Evaluation Example 4 Antisense Suppression of Hprt1 in Mice C57BL / 6J mice (male, 6 weeks old, Jackson Laboratory Japan, Inc.) were administered P22790163, P23790243, P23790244, and P23790246 dissolved in physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory, Ltd.) at a dose of 0.95 μmol / kg (P23790244) in terms of the amount of antisense oligonucleotide per mouse. The concentrations were administered intravenously to the mice at 2.9 μmol / kg (P22790163, P23790243, and P23790244), 9.5 μmol / kg (P22790163, P23790243, and P23790244), and 19.5 μmol / kg (P22790163 only). As a control, only saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) was administered. Heart (P22790163 and P23790244), liver (P22790163, P23790243, P23790244, and P23790246), lung (P22790163, P23790243, and P23790244), and muscle (P22790163, P23790244, and P23790246) tissues were collected under isoflurane anesthesia 5, 10, and 20 days after administration (P23790246 was collected only 10 days after administration). After isolating RNA from each organ using the MagMAX® mirVana® Total RNA Isolation Kit (Thermo Fisher Scientific), the expression level of the mouse Hprt1 gene was 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 amount of the housekeeping gene Gapdh was also quantified simultaneously, and the amount of Hprt mRNA relative to the amount of Gapdh mRNA was evaluated as the expression level of Hprt1.The results are shown as the percentage expression of Hprt1 relative to the untreated control group (control) in Figure 3 (2.9 μmol / kg administration, liver), Figure 4 (Day 10, liver), Figure 5 (2.9 μmol / kg administration, heart), Figure 6 (Day 10, heart), Figure 7 (2.9 μmol / kg administration, lung), Figure 8 (2.9 μmol / kg administration, muscle), and Figure 9 (Day 10, muscle).

[0183] As is clear from FIGS. 3 to 9, P23790244 and P23790246 showed a higher inhibitory effect on Hprt1 expression in the liver, heart, lung, and muscle than P22790163 and P23790243.

[0184] [Preparation Example 7] The antisense oligonucleotides (compounds represented by chemical structures corresponding to the compound numbers) listed in Table 4 were prepared using automated nucleic acid synthesizers nS-8II (manufactured by Gene Design Co., Ltd.) and M-8-SE (manufactured by Nippon Techno Service Co., Ltd.). The target gene was mouse hypoxanthine phosphoribosyltransferase 1 (Hprt1). In Table 4, "Compd No.", "Chemical Structure", and sequence notation are the same as in Table 1. In the sequence notation in Table 4, "J3-" indicates that the 5'-terminal hydroxyl group is linked to the oxygen atom via a phosphodiester bond by a nucleotide of the following formula (V): (wherein * represents the bonding position with oligonucleotide W. (* represents the oxygen atom of the phosphodiester bond)) means that a group represented by the following formula (VI) is bonded to the oxygen atom of the hydroxy group at the 5' end via a phosphodiester bond. (where * represents the bonding position with oligonucleotide W. (* represents the oxygen atom of the phosphodiester bond)) means that a group represented by the following formula (VII) is bonded to the oxygen atom of the hydroxy group at the 5' end via a phosphodiester bond. (where * represents the bonding position with oligonucleotide W. (* represents the oxygen atom of the phosphodiester bond)) is bonded to the oxygen atom of the hydroxy group at the 5'-end, and "J6-" represents a bond to the oxygen atom of the hydroxy group at the 5'-end via a phosphodiester bond, and (wherein * represents the bonding position with oligonucleotide W. (* represents the oxygen atom of the phosphodiester bond)) is bonded.

[0185]

[0186] Evaluation Example 5 Antisense Suppression of Mouse Hprt1 in 3T3-L1 Cells 3T3-L1 cells were seeded onto a 96-well plate at a density of 15,000 cells / well. After approximately 24 hours, the culture supernatant of the cells was replaced with D-MEM containing 2% FBS, and P22790163, P23790243, P23790244, P23790458, P23790459, and P23790460 were added to a final concentration of 300 nM (Free-Uptake). After 72 hours, cell lysates containing RNA were prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio). Mouse Hprt1 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). The mRNA levels of the housekeeping gene Gapdh were also quantified in real-time PCR, and the relative amount of Hprt1 mRNA relative to Gapdh mRNA was used to assess Hprt1 expression levels. The results are presented in Table 5 as percent expression of Hprt1 relative to untreated control cells.

[0187]

[0188] Evaluation Example 6: Antisense Suppression of Hprt1 in Mice The same evaluation method as in Evaluation Example 4 was used. P22790163 and P23790460 were administered intravenously to mice at a dose of 2.9 μmol / kg in terms of antisense oligonucleotide amount per mouse. As a control, saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) alone was administered. Ten days after administration, the amount of Hprt1 mRNA relative to the amount of Gapdh mRNA in heart, liver, and muscle tissues was evaluated as the Hprt1 expression level. The results are shown in Figure 10 (heart), Figure 11 (liver), and Figure 12 (muscle) as the percent expression of Hprt1 relative to the untreated control group (control).

[0189] As is clear from FIGS. 10 to 12, P23790460 exhibited a higher inhibitory effect on Hprt1 expression in the heart, liver, and muscle than P22790163.

[0190] [Preparation Example 8] The siRNAs (compounds represented by chemical structures corresponding to the compound numbers) listed in Table 6 were prepared using an automated nucleic acid synthesizer M-8-SE (manufactured by Nippon Techno Service Co., Ltd.). The target gene was mouse superoxide dismutase 1 (Sod1). In Table 6, the "Compd No.", "Chemical Structure," and sequence notation are the same as in Tables 1 and 4. "Strand" indicates whether each strand is a sense strand (S) or an antisense strand (AS). "(M)" means 2'-OMe nucleoside, and "(F)" means 2'-fluoronucleoside. "VPU(M)" at the 5' end is a nucleotide represented by the following formula (IX): (wherein * represents an oxygen atom of an internucleoside bond such as a phosphodiester bond or a phosphorothioate bond)). "J1-^", "J2-^", "J3-^", "J4-^", and "J6-^" are each bonded to the same group as "J1-", "J2-", "J3-", "J4-", and "J6-", respectively, but the group is bonded to the oxygen atom of the 5'-terminal hydroxy group via a phosphorothioate bond, and * in the corresponding formulas (III), (IV), (V), (VI), and (VIII) represents an oxygen atom of the phosphorothioate bond.

[0191] Hybridization of each sense strand (S) and antisense strand (AS) was carried out by heating at 95°C for 5 minutes and then leaving at a constant temperature of 37°C for 1 hour. Hybridization was confirmed by non-denaturing polyacrylamide gel electrophoresis.

[0192]

[0193] Evaluation Example 7 Sod1 Knockdown Effect of siRNA in Mouse Primary Hepatocytes Mouse primary hepatocytes were seeded onto a 96-well plate at a density of 20,000 cells / well. After approximately 24 hours, the culture supernatant was replaced with FBS-free William's E Medium, and P24791052, P24791053, P24791054, and P24791055 were added to a final concentration of 1000 nM (Free-Uptake). After 24 hours, RNA was isolated using the MagMAX® mirVana® Total RNA Isolation Kit (Thermo Fisher Scientific), and then the expression level of the mouse Sod1 gene was 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 Ppia (peptidylprolyl isomerase A) was also quantified, and the expression level of Sod1 was evaluated as the ratio of the mRNA level of Sod1 to the mRNA level of Ppia. The results are shown in Table 7 as the percent expression of Sod1 relative to the untreated control cells.

[0194]

[0195] Evaluation Example 8: Sod1 Knockdown Effect of siRNA in Mice. Various nucleic acid agents were prepared by dissolving 4.0 nmol each of P24791052, P24791054, P24791055, and P24791056 in 10 μL of physiological saline (Otsuka Normal Saline Injection, Otsuka Pharmaceutical Factory). C57BL / 6J mice (male, 6 weeks old, Jackson Laboratory Japan, Inc.) were anesthetized with 2.5-4% isoflurane and fixed in a stereotaxic apparatus. Subsequently, a 2-3 cm incision was made in the skin between the ears, and a hole was drilled 1 mm to the right and 0.5 mm posterior to bregma with a 1 mm drill bit. Various nucleic acid agents or physiological saline as a negative control were filled into a Hamilton syringe. A needle was inserted approximately 2 mm into the hole, and 10 μL of the solution per mouse was administered into the right lateral ventricle at a rate of 10 μL / min. The skin was then sutured with nylon thread. Two weeks later, the mice were dissected, and the cerebrum and hippocampus were removed. RNA was isolated using the MagMAX® mirVana® Total RNA Isolation Kit (Thermo Fisher Scientific). Mouse Sod1 gene expression levels were then 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 Tbp (TATA binding protein) was also quantified, and the expression level of Sod1 was evaluated as the ratio of the mRNA level of Sod1 to the mRNA level of Tbp. The results are shown in Figure 13 (cerebrum) and Figure 14 (hippocampus) as the percent expression of Sod1 relative to the negative control (vehicle).

[0196] As is clear from FIGS. 13 and 14, P24791054, P24791055 and P24791056 showed a higher effect of suppressing the expression of Sod1 in the cerebrum and hippocampus than P24791052.

[0197] P22790171, P23790244, P23790458, P23790459, P23790460, P24791053, P24791054, and P24791055 showed improved target RNA knockdown activity under free-uptake conditions, which are highly applicable to animal experiments (in vivo tests) (Evaluation Examples 1, 2, 5, and 7).

[0198] The lipid-conjugated oligonucleotides of the present invention have been confirmed to have enhanced pharmacological effects not only on the liver but also on organs other than the liver, such as the heart, muscles, lungs, etc. Therefore, the lipid moieties of the present invention are expected to be useful as a means of delivering various oligonucleotides.

Claims

1. A lipid-linked oligonucleotide or a complex thereof represented by the following general formula (I): wherein W is a group derived from an oligonucleotide compound or an oligonucleotide conjugate; L is a substituted or unsubstituted C １－１０ Alkylene group or substituted or unsubstituted polyC １－１０ is a divalent group derived from alkylene glycol, R １ and R ２ each independently represents a substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ may be bonded to each other to form a ring.) 2. The above R １ and R ２ each independently represents an unsubstituted C ５－３２ An alkyl group or an unsubstituted C ５－３２ alkenyl group, or R １ and R ２ The lipid-bound oligonucleotide or a complex thereof according to claim 1, wherein the lipid-bound oligonucleotide and the lipid-bound oligonucleotide may be linked to each other to form a ring.

3. The above R １ and R ２ each independently represents an unsubstituted C ５－３２ An alkyl group or an unsubstituted C ５－３２ The lipid-linked oligonucleotide or complex thereof according to claim 1 or 2, which is an alkenyl group.

4. The above R １ and R ２ each independently represents an unsubstituted C １０－２０ The lipid-linked oligonucleotide or complex thereof according to any one of claims 1 to 3, which is an alkyl group.

5. The above R １ and R ２ is not substituted C １４ The lipid-linked oligonucleotide or complex thereof according to any one of claims 1 to 4, which is an alkyl group.

6. The L is an unsubstituted C １－１０ The lipid-linked oligonucleotide or its complex according to any one of claims 1 to 5, which is an alkylene group.

7. The L is an unsubstituted C ６ The lipid-linked oligonucleotide or its complex according to any one of claims 1 to 6, which is an alkylene group.

8. The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide compound is a gapmer-type antisense oligonucleotide.

9. The lipid-linked oligonucleotide of claim 8, wherein the gapmer-type antisense oligonucleotide comprises at least one selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

10. The lipid-linked oligonucleotide of claim 8 or 9, wherein the gapmer-type antisense oligonucleotide comprises at least four consecutive deoxyribonucleosides.

11. The lipid-linked oligonucleotide according to any one of claims 8 to 10, wherein the gapmer-type antisense oligonucleotide consists of 13 to 25 nucleosides.

12. The lipid-linked oligonucleotide according to any one of claims 8 to 11, wherein L is attached to the 5' end of the gapmer-type antisense oligonucleotide.

13. The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide compound is a mixmer-type antisense oligonucleotide.

14. The lipid-linked oligonucleotide of claim 13, wherein the mixmer-type antisense oligonucleotide comprises at least one selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

15. The lipid-linked oligonucleotide according to claim 13 or 14, wherein the mixmer-type antisense oligonucleotide is an oligonucleotide in which nucleosides or oligonucleotides consisting of 1 to 20 sugar-modified nucleosides and nucleosides or oligonucleotides consisting of 1 to 3 deoxyribonucleosides are alternately linked, or an oligonucleotide consisting only of sugar-modified nucleosides as nucleosides.

16. The oligonucleotide compound according to any one of claims 13 to 15, wherein the mixmer-type antisense oligonucleotide consists of 13 to 25 nucleosides.

17. The lipid-linked oligonucleotide compound according to any one of claims 13 to 16, wherein L is attached to the 5' end of the mixmer-type antisense oligonucleotide.

18. The complex according to any one of claims 1 to 7, wherein the oligonucleotide complex is a double-stranded oligonucleotide complex comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides, wherein the second oligonucleotide comprises a sequence capable of hybridizing with at least a portion of the first oligonucleotide and consists of 4 to 100 nucleosides independently selected from deoxyribonucleosides, ribonucleosides and sugar-modified nucleosides, and wherein the first oligonucleotide or the second oligonucleotide is bound to L, and the first oligonucleotide and the second oligonucleotide hybridize to each other.

19. The conjugate of claim 18, wherein the second oligonucleotide comprises at least four consecutive ribonucleosides.

20. The conjugate of claim 18 or 19, wherein L is attached to the 5' end of the second oligonucleotide.

21. The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide compound comprises a first oligonucleotide and a second oligonucleotide, the first oligonucleotide being a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides, the second oligonucleotide comprising a sequence capable of hybridizing with at least a portion of the first oligonucleotide and consisting of 4 to 100 nucleosides independently selected from deoxyribonucleosides, ribonucleosides and sugar-modified nucleosides, and the first oligonucleotide or the second oligonucleotide is linked to L, and the first oligonucleotide and the second oligonucleotide hybridize to each other.

22. The lipid-linked oligonucleotide of claim 21, wherein the second oligonucleotide comprises at least four consecutive ribonucleosides.

23. The lipid-linked oligonucleotide of claim 21 or 22, wherein L is attached to the 5' end of the second oligonucleotide.

24. The lipid-linked oligonucleotide of any one of claims 1 to 7, wherein the oligonucleotide compound or oligonucleotide conjugate is selected from siRNA, aptamers and ribozymes.

25. The lipid-linked oligonucleotide of any one of claims 1 to 7 and 24, wherein the oligonucleotide compound or oligonucleotide conjugate is an siRNA.

26. A pharmaceutical composition comprising the lipid-linked oligonucleotide or a complex thereof according to any one of claims 1 to 25 and a pharmacologically acceptable carrier.

27. A method for regulating the function of a target RNA, comprising the step of contacting a cell with the lipid-linked oligonucleotide or a complex thereof according to any one of claims 1 to 25.

28. A method for regulating the function of a target RNA in a mammal, comprising the step of administering to said mammal the pharmaceutical composition according to claim 26.

29. A method for controlling expression of a target gene, comprising the step of contacting a cell with the lipid-linked oligonucleotide or a complex thereof according to any one of claims 1 to 25.

30. A method for regulating expression of a target gene in a mammal, comprising the step of administering to said mammal the pharmaceutical composition according to claim 26.

31. A compound represented by the following formula (II): (Wherein, L is a substituted or unsubstituted C １－１０ Alkylene group or substituted or unsubstituted polyC １－１０ is a divalent group derived from alkylene glycol, R １ and R ２ each independently represents a substituted or unsubstituted C ５－３２ an alkyl group or a substituted or unsubstituted C ５－３２ alkenyl group, or R １ and R ２ may be bonded to each other to form a ring, R ３ are each independently １－１０ is an alkyl group or R ３ The two alkyl groups may be bonded to each other to form a ring, ４ is a 2-cyanoethyl group.

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