WO2024257861A1 - Lipid-binding oligonucleotide or complex thereof - Google Patents

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 oligonucleotide or complex thereof

The present invention relates to a lipid-bound oligonucleotide or a complex thereof.

Nucleic acid medicines are medicines consisting of nucleic acids (oligonucleotides) that form complementary base pairs with target DNA or RNA, and are expected to be new medicines. Representative nucleic acid medicines include antisense nucleic acids (ASOs) and siRNAs, but the development of a means to efficiently deliver these to target organs, especially to organs other than the liver, has long been recognized as a challenge.

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

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).

In this way, it has been shown that conjugating lipids to oligonucleotides can have an effect on organs other than the liver, but the effect is often limited, and there is still a need to develop lipids as a more effective means of delivery.

International Publication No. 2017/053999

Nucleic Acids Research, 2019, 47, 12, pp 6045-6058

The objective of the present invention is to provide a lipid-bound oligonucleotide or a complex thereof that improves the pharmacological effect on the liver and organs other than the liver.

The inventors conducted extensive research to solve the above problems and discovered that a novel oligonucleotide bound to a dialkyl lipid or a complex thereof improves the pharmacological effect on the liver and organs other than the liver. Based on these findings, the inventors have completed the present invention. In other words, the present invention encompasses the following aspects:

（式中、
Ｗは、オリゴヌクレオチド化合物又はオリゴヌクレオチド複合体に由来する基であり、
Ｌは、置換された若しくは置換されていないＣ_１－１０アルキレン基又は置換された若しくは置換されていないポリＣ_１－１０アルキレングリコールに由来する２価の基であり、
Ｒ^１及びＲ^２は、それぞれ独立に、置換された若しくは置換されていないＣ_５－３２アルキル基、又は置換された若しくは置換されていないＣ_５－３２アルケニル基であるか、あるいはＲ^１及びＲ^２は、互いに結合して環を形成していてもよい。）
［２］
　前記Ｒ^１及びＲ^２が、それぞれ独立に、置換されていないＣ_５－３２アルキル基、又は置換されていないＣ_５－３２アルケニル基であるか、あるいはＲ^１及びＲ^２は、互いに結合して環を形成していてもよい、［１］に記載の脂質結合オリゴヌクレオチド又はその複合体。
［３］
　前記Ｒ^１及びＲ^２が、それぞれ独立に、置換されていないＣ_５－３２アルキル基、又は置換されていないＣ_５－３２アルケニル基である、［１］又は［２］に記載の脂質結合オリゴヌクレオチド又はその複合体。
［４］
　前記Ｒ^１及びＲ^２が、それぞれ独立に、置換されていないＣ_{１０－２０}アルキル基である、［１］～［３］のいずれかに記載の脂質結合オリゴヌクレオチド又はその複合体。
［５］
　前記Ｒ^１及びＲ^２が、置換されていないＣ_１４アルキル基である、［１］～［４］のいずれかに記載の脂質結合オリゴヌクレオチド又はその複合体。 [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 divalent group derived from a substituted or unsubstituted C _1-10 alkylene group or a substituted or unsubstituted poly C _1-10 alkylene glycol,
R ¹ and R ² are each independently a substituted or unsubstituted C _5-32 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 lipid-bound oligonucleotide or its complex according to [1], wherein R ¹ and R ² are each independently an unsubstituted C _5-32 alkyl group or an unsubstituted C _5-32 alkenyl group, or R ¹ and R ² may be bonded to each other to form a ring.
[3]
The lipid-bound oligonucleotide or its complex according to [1] or [2], wherein R ¹ and R ² are each independently an unsubstituted C _5-32 alkyl group or an unsubstituted C _5-32 alkenyl group.
[4]
The lipid-bound oligonucleotide or its complex according to any one of [1] to [3], wherein R ¹ and R ² are each independently an unsubstituted C _10-20 alkyl group.
[5]
The lipid-linked oligonucleotide or its complex according to any one of [1] to [4], wherein R ¹ and R ² are unsubstituted C ₁₄ alkyl groups.

[6]
The lipid-linked oligonucleotide or a complex thereof according to any one of [1] to [5], wherein L is an unsubstituted C _1-10 alkylene group.
[7]
The lipid-linked oligonucleotide or its complex according to any one of [1] to [6], wherein L is an unsubstituted _C6 alkylene group.
[8]
The lipid-linked oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide compound is a gapmer-type antisense oligonucleotide.
[9]
The lipid-linked oligonucleotide according to [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 according to [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 [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 [8] to [11], wherein L is linked to the 5' end of the gapmer-type antisense oligonucleotide.
[13]
The lipid-linked oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide compound is a mixmer-type antisense oligonucleotide.
[14]
The lipid-linked oligonucleotide according to [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 [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 [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 [13] to [16], wherein L is bound to the 5' end of the mixmer-type antisense oligonucleotide.
[18]
the oligonucleotide complex being a double-stranded oligonucleotide complex comprising a first oligonucleotide and a second oligonucleotide,
the first oligonucleotide is a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides;
the second oligonucleotide comprises a sequence capable of hybridizing to at least a portion of the first oligonucleotide, and is composed 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;
The complex according to any one of [1] to [7], wherein the first oligonucleotide and the second oligonucleotide hybridize.
[19]
The conjugate according to claim 18, wherein the second oligonucleotide comprises at least four consecutive ribonucleosides.
[20]
The conjugate according to [18] or [19], wherein L is attached to the 5' end of the second oligonucleotide.

（式中、
Ｌは、置換された若しくは置換されていないＣ_１－１０アルキレン基又は置換された若しくは置換されていないポリＣ_１－１０アルキレングリコールに由来する２価の基であり、
Ｒ^１及びＲ^２は、それぞれ独立に、置換された若しくは置換されていないＣ_５－３２アルキル基、又は置換された若しくは置換されていないＣ_５－３２アルケニル基であるか、あるいはＲ^１及びＲ^２は、互いに結合して環を形成していてもよく、
Ｒ^３は、それぞれ独立に、Ｃ_１－１０アルキル基であるか、あるいはＲ^３の２つのアルキル基は、互いに結合して環を形成していてもよく、
Ｒ^４は、２－シアノエチル基である。）。 [21]
the oligonucleotide compound comprises a first oligonucleotide and a second oligonucleotide;
the first oligonucleotide is a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides;
the second oligonucleotide comprises a sequence capable of hybridizing to at least a portion of the first oligonucleotide, and is composed 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;
a first oligonucleotide and a second oligonucleotide are linked together;
The lipid-linked oligonucleotide according to any one of [1] to [7], wherein 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, aptamers and ribozymes.
[25]
The lipid-linked oligonucleotide according to any one of [1] to [7] and [24], wherein the oligonucleotide compound or oligonucleotide complex is an siRNA.
[26]
A pharmaceutical composition comprising the lipid-linked oligonucleotide or a complex thereof according to any one of [1] to [25] and a pharmacologically acceptable carrier.
[27]
A method for controlling a function of a target RNA, comprising a step of contacting a cell with the lipid-linked oligonucleotide or a complex thereof according to any one of [1] to [25].
[28]
A method for regulating a function of a target RNA in a mammal, comprising a step of administering to the mammal the pharmaceutical composition according to [26].
[29]
A method for controlling expression of a target gene, comprising a step of contacting a cell with the lipid-linked oligonucleotide or a 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 divalent group derived from a substituted or unsubstituted C _1-10 alkylene group or a substituted or unsubstituted poly C _1-10 alkylene glycol;
R ¹ and R ² are each independently a substituted or unsubstituted C _5-32 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;
Each R ³ is independently a C _1-10 alkyl group, or the two alkyl groups of R ³ may be bonded to each other to form a ring;
^R4 is a 2-cyanoethyl group.

The present invention provides lipid-bound oligonucleotides or complexes thereof that have enhanced pharmacological effects on the liver and organs other than the liver.

13 is a graph showing the results of the expression level of Malat1 10 days after administration of antisense oligonucleotides in antisense suppression of Malat1 in mice in Evaluation Example 3. 13 is a graph showing the results of the expression level of Malat1 5 days after administration of antisense oligonucleotides in antisense suppression of Malat1 in mice in Evaluation Example 3. 13 is a graph showing the results of Hprt1 expression levels in the liver of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in mice in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in the liver 10 days after administration of antisense oligonucleotides in antisense suppression of Hprt1 in mice in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in the hearts of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in mice in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in the heart 10 days after administration of antisense oligonucleotides in antisense suppression of Hprt1 in mice in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in the lungs of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in muscle of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in muscle 10 days after administration of antisense oligonucleotides in antisense suppression of Hprt1 in mice in Evaluation Example 4. 13 is a graph showing the results of Hprt1 expression levels in the hearts of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in mice in Evaluation Example 6. 13 is a graph showing the results of Hprt1 expression levels in the liver of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in mice in Evaluation Example 6. 13 is a graph showing the results of Hprt1 expression levels in muscle of mice administered 2.9 μmol/kg of antisense oligonucleotide in antisense suppression of Hprt1 in Evaluation Example 6. 13 is a graph showing the results of Sod1 expression levels in the cerebrum after administration of siRNA in RNA interference of Sod1 in mice in Evaluation Example 8. 13 is a graph showing the results of Sod1 expression levels in the hippocampus after administration of siRNA in RNA interference of Sod1 in mice in Evaluation Example 8.

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

"Nucleoside" is a term well known to those skilled in the art, and is generally understood to be 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 are described below. The nucleic acid base may be modified.

"Deoxyribonucleoside" means a molecule having a nucleobase at the carbon atom at position 1 of 2-deoxyribose. The deoxyribonucleoside in the present invention may be a naturally occurring deoxyribonucleoside or a deoxyribonucleoside in which the nucleobase portion 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 International Publication No. WO 2018/155450.

"Ribonucleoside" means a molecule having a nucleobase at the first carbon atom of ribose. The ribonucleoside in the present invention may be a naturally occurring ribonucleoside or a ribonucleoside in which the nucleobase portion of a naturally occurring ribonucleoside has been modified. A naturally occurring ribonucleoside is a ribonucleoside having a naturally occurring nucleobase. A combination of multiple types of modifications may be applied to one ribonucleoside. 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 International Publication No. WO 2018/155450.

"Modified sugar" refers to
(Z1) A molecule in which ribose or 2-deoxyribose is partially replaced by one or more substituents;
(Z2) Molecules in which ribose or 2-deoxyribose is replaced by a pentose or hexose sugar 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 with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.), or a partial structure capable of forming a 5- to 7-membered ring by hydrogen bonding (e.g., a peptide structure),
Or (Z4) means a molecule in which ribose or 2-deoxyribose is replaced with C2-6 alkylene glycol (eg, ethylene glycol, propylene glycol, etc.).
Modified sugars include "2-modified sugars" and "2-4 linked sugars," as defined below.
Examples of modified sugars and sugar-modified nucleosides described below include sugars and sugar-modified nucleosides disclosed as being suitable for use in the antisense method in JP-A-10-304889, WO 2005/021570, JP-A-10-195098, JP-T-2002-521310, WO 2007/143315, WO 2008/043753, WO 2008/029619, WO 2008/049085, and WO 2017/142054 (hereinafter, these documents are referred to as "documents related to the antisense method") and the like. 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, WO 2018/155450, and the like.

Examples of modified sugars that are partially substituted with a single substituent include a ribose or 2-deoxyribose substituted at any position of the sugar moiety with the following (i) or (ii):
(i) a C _1-6 alkyl group, where, when substitution with two or more C _1-6 alkyl groups is contained, the two or more C _1-6 alkyl groups may be joined together to form a 3- to 6-membered ring.
(ii) a C _{1-6 alkyl group substituted with at least one selected from the group consisting of a halogen atom, a C 1-6} alkoxy group, a halo C _1-6 alkoxy group, a mono- or di-C _1-6 alkylamino group, a 5- to 10-membered _heterocyclic group, a carboxy group, a carbamoyl group, and an N-substituted carbamoyl group.
Here, examples of the N-substituted carbamoyl group include an N-methyl-carbamoyl group and an N-ethyl-carbamoyl group, in which the methyl group and the ethyl group of the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group may be substituted with a 5- to 10-membered heterocyclic group or a mono- or di-C _1-6 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, an N-((benzimidazol-1-yl)ethyl)carbamoyl group, and the like.

"Sugar-modified nucleoside" refers to a molecule having the above-mentioned "modified sugar" in place of the sugar portion of a deoxyribonucleoside or ribonucleoside. For example, it includes the "2'-modified nucleoside" and "2'-4'-bridged nucleoside" described below. When the modified sugar is (Z3) as defined above, the sugar-modified nucleoside also includes a molecule in which the modified sugar and the nucleic acid base are linked via a methylene chain or the like.

"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 dimodified sugar, and examples include "2'-O-Me nucleoside,""2'-O-MOEnucleoside,""2'-O-MCEnucleoside,""2'-O-NMAnucleoside,""2'-O-APnucleoside,""2'-Fnucleoside,""2'-DMAECEnucleoside,""2'-MorECEnucleoside,""2'-PyECEnucleoside," and "2'-BimECE nucleoside."

"2-O-Me" (also called 2-O-methyl) means a sugar in which the hydroxy group at position 2 of ribose is replaced with a methoxy group.
A "2'-O-Me nucleoside" (also referred to as a 2'-O-methyl nucleoside) means a molecule having a nucleobase at the 1-position of "2-O-Me."

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

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

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

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

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

"2-DMAECE", "2-MorECE", "2-PyECE", and "2-BimECE" are modified sugars in which the hydroxyl 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 hydroxyl group at position 2 of ribose is bonded.

"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.

The term "2-4 bridged sugar" refers to a sugar in which a bridge unit is formed by substitution at two positions, the 2- and 4-positions of ribose. Examples of the bridge unit include a C _2-6 alkylene group (the alkylene group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are unsubstituted or independently substituted with a group selected from the group consisting of -O-, -NR ¹⁰ - (R ¹⁰ is a hydrogen atom, a C _1-6 alkyl group, or a haloC _1-6 alkyl group), and -S-).

"2',4'-bridged nucleoside"(2',4'-BNA) means a molecule having a nucleobase at the 1-position of the 2-4 bridged sugar. For example, β-D-methyleneoxy (4'-CH ₂ -O-2') BNA or α-L-methyleneoxy (4'-CH ₂ -O-2') BNA, also referred to as LNA (Locked Nucleic Acid (registered trademark)) described below, ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') BNA, also referred to as ENA, 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 ), oxyamino (4'-CH ₂ -N(R ¹² )-O-2') BNA (R ¹² is H or CH ₃ ), also referred to as 2',4'-BNA ^NC , 2',4'-BNA ^COC , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH ₃ )-O-2') BNA also referred to as cEt, (4'-CH(CH ₂ OCH ₃ )-O-2') BNA also referred to as cMOE-BNA, amide type BNA (4'-C(═O)-N(R ¹³ )-2') BNA also referred to as AmNA (R ¹³ is H or CH ₃ ), (4'-C(spiro-cyclopropyl)-O-2') BNA also referred to as scpBNA, (4'-CH ₂ -N(R ¹⁴ )-2') BNA also referred to as GuNA (R ¹⁴ is C(═NH ₂ ⁺ )NHR ¹⁵ , R ¹⁵ is H or CH ₃ ), and other BNAs known to those skilled in the art.

"5-modified sugar" refers to a non-bridged sugar in which the oxygen or carbon atom at position 5 of the ribose backbone is modified, and includes "5-CP,""5-methyl," and "5-aminopropyl." Positions 2 and 4 of the "5-modified sugar" are preferably not modified.
"5'-modified nucleoside" means a molecule having a nucleobase at the carbon atom at position 1 (position 1 of 2-deoxyribose before modification) of the "5-modified sugar" and includes, for example, "5'-CP nucleoside,""5'-methylnucleoside," and "5'-aminopropyl nucleoside." The 2' position of a 5'-modified nucleoside may or may not be modified. The 2' and 4' positions of a 5'-modified nucleoside may or may not be bridged, but are preferably not bridged.

　「５－メチル」は、リボース骨格の５位がメチル基で置換された糖である。
　「５－アミノプロピル」は、リボース骨格の５位が、３－アミノプロピル基で置換された糖である。
　「５’－メチルヌクレオシド」及び「５’－アミノプロピルヌクレオシド」は、それぞれ「５－メチル」及び「５－アミノプロピル」の１位（基となる２－デオキシリボースの１位）の炭素原子に核酸塩基を有する分子を意味する。 "5-CP" is a sugar in which the 5-position of the ribose backbone is substituted with two methyl groups, which together form a cyclopropane.
A "5'-CP nucleoside" is a molecule in which the sugar is the above-mentioned "5-CP" and which has a nucleic acid base at the carbon atom at position 1 (position 1 of the base 2-deoxyribose), and can be represented by the following structural formula. In the formula, Base is a nucleic acid base. The wavy line is understood to represent a bond to an adjacent nucleoside, a linker, etc., or to represent 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.
The terms "5'-methylnucleoside" and "5'-aminopropylnucleoside" refer to molecules having a nucleobase at the 1-position (the 1-position of the parent 2-deoxyribose) carbon atom of "5-methyl" and "5-aminopropyl," respectively.

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

Vinylphosphonate (VP) nucleosides and cyclopropanephosphonate (CPP) nucleosides, described below, are also included in the 5'-modified nucleosides.

In the above-mentioned "deoxyribonucleosides", "ribonucleosides", "2'-modified nucleosides", "2'-4'-bridged nucleosides" and "5'-modified nucleosides", the bond between the carbon atom at the 1' position 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.

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

"Halogen atom" or "halo" means a fluorine atom, chlorine atom, bromine atom, or iodine atom.

The term " _C1-6 alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and examples 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).

The term "C _1-10 alkyl group" means a straight-chain or branched alkyl group having 1 to 10 carbon atoms, and examples thereof include, in addition to the examples of the "C _1-6 alkyl group" mentioned above, a heptyl group, an octyl group, a nonyl group, a decyl group (including various isomers), etc.

The term " _C5-32 alkyl group" means a straight-chain 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 (including various isomers) groups.

The term " _C8-28 alkyl group" means a straight-chain or branched alkyl group having 8 to 28 carbon atoms, and examples thereof include octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, docosyl, tetracosyl, hexacosyl, and octacosyl (including various isomers).

The term " _C10-20 alkyl group" means a straight-chain or branched alkyl group having 10 to 20 carbon atoms, and examples thereof include 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, and an icosyl group (including various isomers).
The term "C _12-14 alkyl group" refers to a straight or branched alkyl group having 12 to 14 carbon atoms, and examples thereof include dodecyl, tridecyl, and tetradecyl groups (including various isomers).
The term " _C12 alkyl group" means a straight or branched alkyl group having 12 carbon atoms, and examples include 1-dodecyl, 6-dodecyl, 1-methylundecyl, 6-methylundecyl, and the like.
The term " _C14 alkyl group" means a straight or branched alkyl group having 14 carbon atoms, and examples include 1-tetradecyl group, 7-tetradecyl group, 1-methyltridecyl group, 12-methyltridecyl group, and the like.

The term "C _5-32 alkenyl group" means a straight-chain or branched unsaturated hydrocarbon group containing one or more carbon-carbon double bonds and having 5 to 32 carbon atoms, and examples thereof include a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, 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, an icosenyl group, a docosenyl group, a tetracosenyl group, a hexacosenyl group, an octacosenyl group, a triacontenyl group, a hentriacontenyl group, and a dotriacontenyl group (including various isomers).
The term " _C8-28 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 (including various isomers).
The term " _C10-20 alkenyl group" means a straight-chain or branched unsaturated hydrocarbon group containing one or more carbon-carbon double bonds and having 10 to 20 carbon atoms, 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 icosenyl group (including various isomers).
The term "C ₁₄ 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.

The term "halo C _1-6 alkyl group" means a group in which a hydrogen atom at any position of the above "C _1-6 alkyl group" is substituted with one or more of the above "halogen atoms".

The term "C _2-6 alkylene group" means a divalent group (alkanediyl group) obtained by removing two hydrogen atoms at any position from a straight-chain or branched saturated hydrocarbon group having 2 to 6 carbon atoms, and examples 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.

The term "C _1-10 alkylene group" means a straight-chain or branched alkylene group having 1 to 10 carbon atoms, and examples thereof include, in addition to the examples of the "C _2-6 alkylene group" above, a methylene group, 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, an octamethylene group, and a decamethylene group.

The term "C _2-8 alkylene group" means a straight-chain or branched alkylene group having 2 to 8 carbon atoms, and examples thereof include, in addition to the examples of the "C _2-6 alkylene group" above, a methylene group, 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, and an octamethylene group.

The term "C _3-6 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.

The term " _C6 alkylene group" means a straight or branched alkylene group having 6 carbon atoms, and examples include hexamethylene, 1-methyl-pentamethylene, 2-methyl-pentamethylene, 1,1-dimethyl-tetramethylene, and the like.

The term "C _1-6 alkoxy group" means a group in which the above "C _1-6 alkyl group" is bonded to an oxy group.

The term "halo C _1-6 alkoxy group" means a group in which a hydrogen atom at any position of the above "C _1-6 alkoxy group" is substituted with one or more of the above "halogen atoms".

The "mono- or di-C _1-6 alkylamino group" means a group in which one hydrogen atom of an amino group is replaced by one "C _1-6 alkyl group", or a group in which two hydrogen atoms of an amino group are replaced by the same or different two "C _1-6 alkyl groups", 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.

"3- to 6-membered ring" means a monocyclic saturated or unsaturated hydrocarbon ring having 3 to 6 carbon atoms, such as cyclopropane, cyclobutane, and cyclohexane.

The term "5- to 10-membered heterocyclic group" means a 5- to 10-membered monocyclic or condensed 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.

The term "oxo group" refers to a group in which an oxygen atom is substituted via a double bond (=O). When an oxo group is substituted for a carbon atom, it forms a carbonyl group together with the carbon atom.
The term "thioxo group" refers to a group in which a sulfur atom is substituted via a double bond (=S). When a thioxo group is substituted for a carbon atom, it forms a thiocarbonyl group together with the carbon atom.

"Nucleic acid base" refers to 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 "nucleic acid base" includes naturally occurring nucleobases and the "modified nucleobases" described below.

Examples of modifications of nucleic acid bases in "modified nucleic acid bases" include halogenation, methylation, ethylation, n-propylation, isopropylation, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, cyclobutylation, hydroxylation, amination, thiolation, demethylation, etc. More specifically, 5-methylation, 5-fluoroation, 5-bromination, 5-iodination, and N4-methylation of cytosine; 2-thiolation, 5-demethylation, 5-fluoroation, 5-bromination, and 5-iodination of thymine; 2-thiolation, 5-fluoroation, 5-bromination, and 5-iodination of uracil; N6-methylation and 8-bromination of adenine; N2-methylation and 8-bromination of guanine, etc. Furthermore, examples of modifications of the nucleic acid base moiety in nucleosides 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, etc.

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.

"5-methylcytosine" means cytosine with a methyl group at the 5th position.

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

"Consecutive nucleobases" refers to a sequence from the 5' to the 3' end of a contiguous portion of nucleobases in the "nucleobase sequence."

"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" described below.

"Modified internucleoside bond" means a modified phosphodiester bond, such as a phosphorothioate bond, a methylphosphonate bond (including a chiral-methylphosphonate bond), a methylthiophosphonate bond, a phosphorodithioate bond, a phosphoramidate bond, a phosphorodiamidate bond, a phosphoroamidothioate 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.

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

"Oligonucleotide" refers to a molecule having a structure in which two or more "nucleosides" that are the same or different are linked together by "internucleoside bonds" (e.g., phosphodiester bonds or modified phosphodiester bonds) that are independently selected from each other.
The term "oligodeoxyribonucleotide" refers to a polynucleotide or oligonucleotide in which two or more of the same or different "deoxyribonucleosides" are linked by the "internucleoside bonds" selected independently from each other.
The term "oligoribonucleotide" refers to a polynucleotide or oligonucleotide in which two or more of the same or different "ribonucleosides" are linked by the above-mentioned "internucleoside linkages" which are 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 through 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 through an internucleoside bond.

"DNA" refers to a polynucleotide or oligonucleotide in which two or more naturally occurring "deoxyribonucleosides" as defined above are linked by phosphodiester bonds. The naturally occurring deoxyribonucleosides that make up DNA may be the same or different.
"RNA" refers to a polynucleotide or oligonucleotide in which two or more naturally occurring "ribonucleosides" are linked by phosphodiester bonds. The naturally occurring ribonucleosides that make up the RNA may be the same or different.

"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.

"Target RNA" means mRNA, mRNA precursor, or ncRNA, and includes mRNA transcribed from genomic DNA encoding the target gene, mRNA with no base modifications, mRNA precursor and ncRNA that have not been spliced, etc. There are no particular limitations on the "target RNA" whose function is controlled by the antisense effect, 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.

The "antisense effect" means that the function of a target RNA is controlled by hybridization of 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 an mRNA, this means inhibition of translation of the target RNA by hybridization, a splicing function conversion effect such as exon skipping, or degradation of the target RNA by recognition of the hybridized portion.

An "antisense oligonucleotide" (ASO) is an oligonucleotide that produces the antisense effect. Examples include, but are not limited to, DNA, oligodeoxyribonucleotides, gapmer-type antisense oligonucleotides (or simply "gapmers"), and mixmer-type antisense oligonucleotides (or simply "mixmers"). The ASO may also be RNA, oligoribonucleotides, or oligonucleotides designed to normally produce an antisense effect.

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

"Complementary" means that two nucleobases can form Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds. Two oligonucleotides or portions thereof can "hybridize" if their sequences are complementary. Although two oligonucleotides or portions thereof do not need to be completely complementary to hybridize, the complementarity for two oligonucleotides or portions thereof to hybridize is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more). Sequence complementarity is determined by using a computer program that automatically identifies partial sequences of oligonucleotides. For example, OligoAnalyzer is one such software and is provided by Integrated DNA Technologies. This program is also available on the website. When two oligonucleotides have the above-described preferred complementarity, a person skilled in the art can determine that the two oligonucleotides hybridize.

"Gapmer" refers to an oligonucleotide that includes a "gap segment," a "5' wing segment," and a "3' wing segment," as described below.

The term "gap segment" refers to a region that includes "at least four consecutive nucleosides that are recognized by RNase H" and is not particularly limited as long as it includes 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.

The "5' wing segment" is a region that is linked to the 5' side of the gap segment and contains "at least one nucleoside" without containing the "at least four consecutive nucleosides recognized by RNase H", and 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 difference in sugar moiety identifies the boundary between the 5' wing segment and the gap segment. In one 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 here, as defined above, a "2-modified sugar" is a non-bridged sugar in which the oxygen 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 replaced by substitutions at two positions, the 2-position and the 4-position of ribose, as defined above, and therefore the 2-position is modified in both cases. (For example, the nucleoside at the 5' end of the gap segment is a deoxyribonucleoside and the nucleoside at the 3' end 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 nucleoside at the 5' end of the gap segment is a 5'-modified nucleoside and the nucleoside at the 3' end of the 5' wing segment is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside.) The nucleoside at the 3' end of the 5' wing segment is generally a sugar-modified nucleoside, 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 deoxyribonucleosides and sugar-modified nucleosides, and includes at least one sugar-modified nucleoside. The 5' wing segment is preferably independently selected from sugar-modified nucleosides, and more preferably independently selected from 2'-4'-bridged nucleosides and 2'-modified nucleosides.

The "3' wing segment" is a region that is linked to the 3' side of the gap segment and contains "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 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 nucleoside at the 5' end of the 3' wing segment is generally a sugar-modified nucleoside, 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 deoxyribonucleosides and sugar-modified nucleosides, and includes at least one sugar-modified nucleoside. The 3' wing segment is preferably independently selected from sugar-modified nucleosides, and more preferably independently selected from 2'-4'-bridged nucleosides and 2'-modified nucleosides.

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 nucleoside at the 3' end of the 5' wing segment is linked to another nucleoside (deoxyribonucleoside, ribonucleoside, etc.) other than 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'-end nucleoside of the 3' wing segment is linked to another nucleoside (deoxyribonucleoside, ribonucleoside, etc.) other than the 2'-modified nucleoside or 2'-4'-bridged nucleoside is the boundary between the 3' wing segment and the gap segment.

"RNase H" is generally known as a ribonuclease that recognizes a double strand in which DNA and RNA are hybridized in a living body, cleaves the RNA, and produces single-stranded DNA. RNase H can recognize not only a double strand in which DNA and RNA are hybridized, but also a double strand in which at least one of the nucleic acid base portion, the phosphodiester bond portion, and the sugar portion of at least one of DNA and RNA is modified. For example, it can recognize a double strand in which DNA modified with phosphorothioate bonds is hybridized with RNA. It can also recognize a double strand in which an oligodeoxyribonucleotide is hybridized with an oligoribonucleotide.
Therefore, when DNA hybridizes with RNA, it can be recognized by RNase H. Also, when RNA hybridizes with DNA, it can be cleaved by RNase H. The same applies when at least one of the nucleic acid base portion, the phosphodiester bond portion, and the sugar portion is modified in at least one of DNA and RNA. For example, a representative example is an oligonucleotide in which the phosphodiester bond portion of DNA is modified to phosphorothioate.
Examples of modifications of DNA and/or RNA that can be recognized by RNase H are described, for example, in 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, and the like.
In the present invention, RNase H is preferably mammalian RNase H, more preferably human RNase H, and particularly preferably human RNase H1.

"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.
Whether or not a certain sequence of at least four consecutive nucleosides is "at least four consecutive nucleosides recognized by RNase H" can be determined by a person skilled in the art based on the structure of the sugar moieties of the consecutive nucleosides.

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.

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.

A mixmer is an oligonucleotide that contains multiple sugar-modified nucleosides and produces an antisense effect without having the gap segment, and examples of such oligonucleotides 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, and an oligonucleotide consisting only of sugar-modified nucleosides as nucleosides. In one embodiment, the mixmer is 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.

"siRNA" (small interfering RNA) is a small double-stranded oligoribonucleotide. siRNA is involved in a phenomenon called RNA interference (RNAi) and suppresses gene expression by destroying the target mRNA. siRNA includes an antisense strand and a sense strand.
In one embodiment, the antisense strand and the sense strand of the siRNA comprise RNA. The siRNA typically consists of 15 to 30 base pairs, and preferably 21 to 23 base pairs. The 3' ends of the two nucleic acid strands often have 1 to 5 (preferably 2) overhanging deoxythymidines (generally written as tt, dTdT, etc.) (generally called an overhang). Only the 3' end of the sense strand may have two overhanging deoxythymidines, or only the 3' end of the antisense strand may have two overhanging deoxythymidines.

In some embodiments, neither the antisense 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 ratio is, 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.

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 antisense strand hybridizing portion. The other nucleosides are 2'-O-Me nucleosides.

Preferably, the sense strand contains a 2'-fluoro nucleoside at at least one position selected from positions 7, 9, 10, and 11, counting from the 5' end. Particularly preferably, the sense strand contains 2'-fluoro nucleosides at positions 7, 9, 10, and 11, counting from the 5' end. The other nucleosides are 2'-O-Me nucleosides.

The internucleoside bonds of the siRNA may be phosphodiester bonds or modified internucleoside bonds, but each internucleoside bond is preferably independently a phosphodiester bond or a phosphorothioate bond. More preferably, 1 to 5 internucleoside bonds from the 5' and 3' ends of each of the antisense and sense strands are phosphorothioate bonds, and the other internucleoside bonds are phosphodiester bonds. Even more preferably, 1 to 3 internucleoside bonds from the 5' and 3' ends of each of the antisense and sense strands are phosphorothioate bonds, and the other internucleoside bonds are phosphodiester bonds. Even more preferably, 2 to 3 internucleoside bonds from the 5' and 3' ends of each of the antisense and sense strands are phosphorothioate bonds, and the other internucleoside bonds are phosphodiester bonds. Particularly preferably, two internucleoside bonds at the 5' and 3' ends of each of the antisense and sense strands are phosphorothioate bonds, and the other internucleoside bonds are phosphodiester bonds.

In some embodiments, the sense strand or/and the antisense strand comprising 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 (S)P-O-5'), 5'-monodithiophosphate (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'-alkyl phosphonates (R'P(OH)(O)-O-5'- (R'=alkyl, e.g. methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkenyl phosphonates (alkenyl is e.g. vinyl ((OH) ₂ (O)P-5'-CH=CH-), substituted vinyl), 5'-alkoxyalkyl phosphonates (R"P(OH)(O)-O-5'- (R" = alkoxyalkyl, e.g., methoxymethyl, ethoxymethyl, etc.), 5'-cycloalkylphosphonate ((OH) ₂ (O)P-5'-(cycloalkane-diyl)-, e.g., (OH) ₂ (O)P-5'-(cyclopropane-1,2-diyl)-). Modifications may be located within the antisense strand of the siRNA. For example, the antisense strand may include a 5'-vinyl phosphonate nucleoside at the 5' terminus, preferably a 5'-E-vinyl phosphonate nucleoside. Also, for example, the antisense strand may include a 5'-cyclopropane phosphonate nucleoside at the 5' terminus, preferably a 5'-(trans)-cyclopropane phosphonate nucleoside.

（式（X）におけるＢａｓｅは、式（Ｉ）におけるＸ及びＢａｓｅと同義である）で示される構造（５’－ビニルホスホネート（ＶＰ）化２’－Ｏ－Ｍｅヌクレオシド）が５’－Ｅ－ビニルホスホネートとして好ましい。 In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises a 5'-E-vinyl phosphonate or a 5' cyclopropane phosphonate, for example, the following formula (X):

A structure represented by the following formula (5'-vinylphosphonate (VP)-modified 2'-O-Me nucleoside) (Base in formula (X) has the same meaning as X and Base in formula (I)) is preferred as the 5'-E-vinylphosphonate.

（式（XI）におけるＢａｓｅは、式（Ｉ）におけるX及びＢａｓｅと同義である。シクロプロパンへの置換基はｔｒａｎｓ体である）で示される構造（５’－シクロプロパンホスホネート（ＣＰＰ）化２’－Ｏ－Ｍｅヌクレオシド）が好ましい。 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.

Chemical modifications in siRNA are well known in the field, and reference can be made, for example, to 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.

An "aptamer" is an oligonucleotide that binds to specific extracellular proteins and inhibits their function.

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

"miRNA" (micro-RNA) is RNA that is coded in the genome but is not translated into protein (non-coding RNA), and has the effect of suppressing gene expression. It is typically RNA of 21 to 25 bases.

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

"Decoy nucleic acid" is a double-stranded oligodeoxyribonucleotide that binds to a transcription factor, among other proteins, and inhibits the transcription stage. Decoy nucleic acid can inhibit gene expression of the transcription factor. Decoy nucleic acid typically consists of 15 to 30 base pairs.

（式中、
Ｗは、オリゴヌクレオチド化合物又はオリゴヌクレオチド複合体に由来する基であり、
Ｌは、置換された若しくは置換されていないＣ_１－１０アルキレン基又は置換された若しくは置換されていないポリＣ_１－１０アルキレングリコールに由来する２価の基であり、
Ｒ^１及びＲ^２は、それぞれ独立に、置換された若しくは置換されていないＣ_５－３２アルキル基、又は置換された若しくは置換されていないＣ_５－３２アルケニル基であるか、あるいはＲ^１及びＲ^２は、互いに結合して環を形成していてもよい）
で表される。 <Lipid-bound oligonucleotide or complex thereof>
The present invention provides a lipid-linked oligonucleotide or a complex thereof. The lipid-linked oligonucleotide or a complex thereof of the present invention has the following general formula (I):

(Wherein,
W is a group derived from an oligonucleotide compound or an oligonucleotide conjugate;
L is a divalent group derived from a substituted or unsubstituted C _1-10 alkylene group or a substituted or unsubstituted poly C _1-10 alkylene glycol;
R ¹ and R ² are each independently a substituted or unsubstituted C _5-32 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.
It is expressed as:

In the present invention, a "substituted or unsubstituted C _1-10 alkylene group" means an (unsubstituted) C _1-10 alkylene group or a C _1-10 alkylene group substituted with one or more arbitrary substituents.

In the present invention, the term "substituted or unsubstituted" means that the group is unsubstituted or substituted with at least one substituent selected from a given group of substituents, for example, a halogen atom, a hydroxy group, a carboxy group, a C _1-6 alkoxy group, and an aryl group.

The term "substituted C _1-10 alkylene group" means that any one or more hydrogen atoms of the alkylene group are substituted with a substituent selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, a C _1-6 alkoxy group, and an aryl group.

Examples of divalent groups derived from poly C _1-10 alkylene glycol include groups 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 a C _1-10 alkylene group which is optionally substituted with a group selected from the group consisting of a hydroxy group, a protected hydroxy group, a halogen atom, a hydroxy group, a carboxy group, a C _1-6 alkoxy group, and an aryl group). The protected hydroxy group is not particularly limited as long as it is stable when bound to an oligonucleotide, and examples of the protected hydroxy group 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, etc.; 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 (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), 9-(p-methoxyphenyl)xanthen-9-yl (MOX group), etc. The protecting group for a "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.

Among these, as L in general formula (I), an unsubstituted C _1-10 alkylene group is preferable, a C _2-8 alkylene group is more preferable, a C _3-6 alkylene group is even more preferable, and a C ₆ alkylene group is particularly preferable. 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 preferable, a trimethylene group, a tetramethylene group, or a hexamethylene group (including various isomers) is even more preferable, and a hexamethylene group (including various isomers) is particularly preferable. Furthermore, a 1,6-hexamethylene group (1,6-hexanediyl group) is particularly preferable.

In the present invention, "substituted or unsubstituted C _5-32 alkyl group" means an (unsubstituted) C _5-32 alkyl group or a C _5-32 alkyl group substituted with one or more optional substituents.

The term "substituted C _5-32 alkyl group" means that any one or more hydrogen atoms of the alkyl group are substituted with a substituent selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, a C _1-6 alkoxy group, and an aryl group.

Among these, R ¹ and R ² in the general formula (I) are preferably unsubstituted C _5-32 alkyl groups, more preferably C _8-28 alkyl groups, even more preferably C _10-20 alkyl groups, even more preferably C _12-14 alkyl groups, and particularly preferably C ₁₄ alkyl groups. Also, C ₁₂ alkyl groups are particularly preferred. For example, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, and icosyl group (including various isomers) are more preferred, and tetradecyl group (including various isomers) is particularly preferred. As the alkyl groups of R ¹ and R ² in the general formula (I), linear alkyl groups are preferred.

In the present invention, "substituted or unsubstituted C _5-32 alkenyl group" means an (unsubstituted) C _5-32 alkenyl group or a C _5-32 alkenyl group substituted by one or more optional substituents.

The term "substituted _C5-32 alkenyl group" means that any one or more hydrogen atoms of the alkenyl group are substituted with a substituent selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, a _C1-6 alkoxy group, and an aryl group.

Among these, R ¹ and R ² in general formula (I) are preferably unsubstituted C _5-32 alkenyl groups, more preferably C _8-28 alkenyl groups, even more preferably C _10-20 alkenyl groups, and particularly preferably C ₁₄ alkenyl groups. For example, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, and icosenyl groups (including various isomers) are more preferred, and tetradecenyl groups (including various isomers) are particularly preferred.

In the present invention, "may be bonded to each other to form a ring" means that the substituted or unsubstituted C _5-32 alkyl group or substituted or unsubstituted C _5-32 alkenyl group of R ¹ and R ² may be bonded to each other at a part thereof to form a 10-64 membered ring including the "-O-CH ₂ -CH-O-" portion in general formula (I) to which they are bonded. The 10-64 membered ring is preferably a 10-40 membered ring, more preferably a 12-30 membered ring, even more preferably a 20-30 membered ring, and particularly preferably a 22 membered ring. The ring is even more preferably unsubstituted.

The "group derived from an oligonucleotide compound or oligonucleotide complex" of the present invention means a partial structure of an oligonucleotide compound formed by removing a hydrogen atom, a hydroxyl group, etc. from the 3'-end or 5'-end of at least one oligonucleotide compound constituting the oligonucleotide compound or oligonucleotide complex. Thus, one of the 3'-end or 5'-end of the oligonucleotide compound is covalently bonded 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.

The oligonucleotide compound or oligonucleotide complex 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 the 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).

The oligonucleotide compound of the present invention is preferably an antisense oligonucleotide, and examples of the antisense oligonucleotide include, but are not limited to, DNA, oligodeoxyribonucleotide, gapmer-type antisense oligonucleotide (or simply referred to as "gapmer"), and mixmer-type antisense oligonucleotide (or simply referred to as "mixmer"), and may be RNA, oligoribonucleotide, or an oligonucleotide designed to normally produce an antisense effect.
Alternatively, the "oligonucleotide complex" of the present invention may be a heteroduplex (HDO) comprising an antisense oligonucleotide (first oligonucleotide) which is a single-stranded DNA and a complementary RNA oligonucleotide (second oligonucleotide).
Alternatively, the first and second oligonucleotides may be linked via a nucleic acid linker, via a linking group containing a non-nucleotide structure, or directly (single-stranded heteroduplex nucleic acid).

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

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 means a partial structure formed by removing a hydrogen atom, a hydroxyl group, or the like from the 3'-end or 5'-end of a mixmer, and is, for example, composed of 7 to 100 nucleosides, preferably 10 to 40 nucleosides, more preferably 13 to 25 nucleosides, and is preferably covalently bonded to L at its 3'-end or 5'-end and covalently bonded to L at its 5'-end.
Also, the mixmer-type antisense oligonucleotide preferably does not contain at least four consecutive deoxyribonucleosides.
Moreover, 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.

Each internucleoside bond in the gapmer and mixmer antisense oligonucleotides may be a phosphodiester bond or a modified internucleoside bond. Each internucleoside bond is, for example, preferably independently 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. Also, preferably, 50-100% (more preferably 70-100%, 80-100%, 90-100%, 95-100%) of the internucleoside bonds in the antisense oligonucleotide are phosphorothioate bonds. The remaining internucleoside bonds are phosphodiester bonds.

Alternatively, W in the general formula (I) of the present invention is preferably a group derived from a double-stranded nucleic acid in an oligonucleotide complex. The double-stranded nucleic acid is, for example, described as HDO (heteroduplex nucleic acid) in WO 2013/089283, the entire contents of which are incorporated herein by reference.
Preferably, 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, and the second oligonucleotide comprises a sequence that allows hybridization 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 with 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.

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 complex 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 preferable, a group derived from an oligonucleotide consisting of 3 to 7 nucleosides is more preferable, a group derived from an oligonucleotide consisting of 4 or 5 nucleosides is even more preferable, and a group derived from an oligonucleotide consisting of 4 nucleosides is particularly preferable.
The internucleoside bonds linking the nucleosides constituting the linker may be independently phosphodiester bonds or modified internucleoside bonds, but are preferably independently phosphodiester bonds or phosphorothioate bonds. The linker further preferably contains one or two phosphorothioate bonds, and the other internucleoside bonds are phosphodiester bonds, and particularly preferably the internucleoside bonds linking the nucleosides constituting the linker are phosphodiester bonds. The nucleosides constituting the linker are independently preferably adenosine, uridine, cytidine, guanosine, 2'-deoxyadenosine, thymidine, 2'-deoxycytidine, or 2'-deoxyguanosine, and particularly preferably adenosine.
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 complex are linked by a linking group containing a non-nucleotide structure. Examples of the linking group containing a non-nucleotide structure include an alkylene group having 2 to 50 carbon atoms and a group derived from a 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 complex are directly linked together.
The first oligonucleotide of said oligonucleotide complex is preferably a gapmer-type antisense oligonucleotide, and the above-mentioned embodiments of gapmer-type antisense oligonucleotides are applied to the first oligonucleotide of the oligonucleotide complex.
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, similar to the 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, similar to the mixmer.
The second oligonucleotide preferably contains at least one ribonucleoside, more preferably contains at least 4 consecutive ribonucleosides, and further preferably contains 1 to 6 (even more preferably 2 to 4, 2 to 3) sugar-modified nucleosides (even more preferably 2'-modified nucleosides), and 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 are sugar-modified nucleosides. Particularly preferably, all nucleosides constituting the second oligonucleotide are ribonucleosides. The internucleoside bonds linking the nucleosides constituting the second oligonucleotide may be independently phosphodiester bonds or modified internucleoside bonds, but are preferably independently phosphodiester bonds or phosphorothioate bonds, more preferably contains 1 to 6 (even more preferably 2 to 4, 2 to 3) phosphorothioate bonds, and other internucleoside bonds are phosphodiester bonds. Particularly preferably, the internucleoside bond linking the nucleosides constituting the second oligonucleotide is a phosphodiester bond.
The above aspects are described, for example, in WO 2017/131124, WO 2018/143475, and WO 2019/022196, the entireties of which are incorporated herein by reference.

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' or 3' end of the antisense strand is bound to L, particularly preferably the 3' end. The 5' or 3' end of the sense strand is bound to L, particularly preferably the 5' end.

The group derived from the siRNA may be bound to L, for example, via an internucleoside bond, preferably via a phosphodiester bond or a phosphorothioate bond, more preferably via a phosphorothioate bond.

The lipid-bound oligonucleotides and the like according to this embodiment can be produced by the method shown below, but the production method below is an example of a general production method and does not limit the method for producing the oligonucleotides and the like according to this embodiment.

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

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. Specifically, compound A having various R ¹ and R ² can be synthesized from compound A-1 described below by combining oxidation reactions known to those skilled in the art (for the oxidation reaction, see, for example, Comprehensive Organic Transformations, Second Edition, by R.C. Larock, Wiley-VCH (1999)).

In the formula, R ¹ and R ² are as defined above.
For example, to synthesize compound A-1 in which R ¹ and R ² are alkyl or alkenyl groups, the desired R 1 can be introduced by alkylating or alkenylating the primary hydroxy group of compound A-2 using an alkyl halide reagent or the like corresponding to R ^1. Also, the desired R ² can be introduced by alkylating or alkenylating the secondary hydroxy group using an alkyl halide reagent or the like corresponding to R ^2. The hydroxy group protected by P1 of the obtained compound is deprotected to synthesize compound A-1 in which R ¹ and R ² are alkyl or alkenyl groups, or the like.

Compound A-1, in which R ¹ and R ² are bonded to each other to form a ring, can be obtained by introducing desired R ¹ and R ² to the terminals of R ¹ and R ² using an alkyl halide reagent having a double bond, and then subjecting the compound to an olefin metathesis reaction. A specific example of the olefin metathesis reaction is a method in which a second generation Grubbs catalyst, for example, dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine)ruthenium(II) is reacted in a solvent. In addition, for example, an olefin ring compound obtained by the olefin metathesis reaction can be hydrogenated in a solvent with hydrogen and palladium on carbon to obtain a saturated ring compound.

In the formula, P1 and ^P2 each represent a hydroxy-protecting group, and the other symbols are as defined above.

(Step I) Amidation Reaction with 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, a method can be exemplified in which compound A is 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.

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

(Step III) Conjugation reaction between oligonucleotide and lipid The lipid-conjugated oligonucleotide can be synthesized using compound C, commercially available nucleotides necessary for producing an oligonucleotide compound of a desired nucleotide sequence, phosphoramidite reagents, etc., with 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.).

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

（式中、
Ｌは、置換された若しくは置換されていないＣ_１－１０アルキレン基であり、
Ｒ^１及びＲ^２は、それぞれ独立に、置換された若しくは置換されていないＣ_５－３２アルキル基、又は置換された若しくは置換されていないＣ_５－３２アルケニル基であるか、あるいはＲ^１及びＲ^２は、互いに結合して環を形成していてもよく、
Ｒ^３は、それぞれ独立に、Ｃ_１－１０アルキル基であるか、あるいはＲ^３の２つのアルキル基は、互いに結合して環を形成していてもよく、
Ｒ^４は、２－シアノエチル基である）
で表される化合物である。
　前記式（II）の化合物は、本発明の脂質結合オリゴヌクレオチドの製造に有用である。 One embodiment of the present invention is a compound represented by the following formula (II):

(Wherein,
L is a substituted or unsubstituted C _1-10 alkylene group;
R ¹ and R ² are each independently a substituted or unsubstituted C _5-32 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;
Each R ³ is independently a C _1-10 alkyl group, or the two alkyl groups of R ³ may be bonded to each other to form a ring;
^R4 is a 2-cyanoethyl group.
It is a compound represented by the formula:
The compound of formula (II) is useful for producing the lipid-linked oligonucleotide of the present invention.

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

The pharmaceutical composition of the present invention containing the lipid-linked oligonucleotide of the general formula (I) or a complex thereof can be formulated by known pharmaceutical methods. For example, it can be used enterally (orally, etc.) or parenterally as a capsule, tablet, pill, liquid, powder, granule, fine granule, film coating agent, pellet, troche, sublingual, chewable agent, buccal agent, paste, syrup, suspension, elixir, emulsion, liniment, ointment, plaster, cataplasm, transdermal preparation, lotion, inhalant, aerosol, injection, suppository, etc.

In these formulations, they can be appropriately combined with carriers that are pharmacologically or food and beverage acceptable, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, dissolution aid, or other additives.

The administration form of the pharmaceutical composition containing the lipid-linked oligonucleotide of the general formula (I) or a complex thereof of the present invention is not particularly limited, and examples thereof include enteral (oral, etc.) and parenteral administration. More preferred examples include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, intranasal administration, and intravitreal administration, as well as administration by infusion.

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 metabolic diseases, cardiovascular diseases, tumors, infectious diseases, eye diseases, inflammatory diseases, autoimmune diseases, rare genetic diseases, and other diseases caused by gene expression. More specifically, hypercholesterolemia, hypertriglyceridemia, spinal muscular atrophy, muscular dystrophy (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, Alzheimer's disease, and other conditions. These include rheumatoid arthritis, 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 causing the disease is set as the target gene, and the expression control sequence (e.g., antisense sequence) can be appropriately set according to the sequence of the target gene.

In addition to primates such as humans, various other mammalian diseases can be treated, prevented, or ameliorated by pharmaceutical compositions containing the lipid-linked oligonucleotides of the general formula (I) of the present invention or complexes thereof. For example, diseases of mammalian species including, but not limited to, cow, sheep, goat, horse, dog, cat, guinea pig, or other rodent species such as bovine, ovine, equine, canine, feline, and mouse can be treated. Compositions containing antisense oligonucleotides can also be applied in other species such as birds (e.g., chickens).

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

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 is therefore expected to have enhanced pharmacological effects. Therefore, a method for controlling the expression of a target gene more safely 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. In addition, a method for treating, preventing, and ameliorating various diseases involving the control of a target gene can also be provided, which includes 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.

Preferred methods for using the lipid-linked oligonucleotide of the present invention represented by the above general formula (I) or a complex thereof are as follows.
- 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 of 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 said mammal a pharmaceutical composition comprising the lipid-linked oligonucleotide of the present invention of general formula (I) or a complex thereof.
The use of the lipid-linked oligonucleotide of the present invention of said general formula (I) or a complex thereof for controlling the function of a target RNA in a mammal.
The use of the lipid-linked oligonucleotide of said general formula (I) of the invention or a complex thereof for the manufacture of a drug for controlling the function of a target RNA in a mammal.
- a method for controlling the expression of a target gene, comprising the step of contacting a cell with the lipid-linked oligonucleotide of the present invention of general formula (I) or a complex thereof.
A method for controlling the expression of a target gene in a mammal, comprising the step of administering to said mammal a pharmaceutical composition comprising the lipid-linked oligonucleotide of the present invention of general formula (I) or a complex thereof.
The use of the lipid-linked oligonucleotide of said general formula (I) of the present invention or a complex thereof for controlling the expression of a target gene in a mammal.
The use of the lipid-linked oligonucleotide of said general formula (I) of the invention or a complex thereof for the manufacture of a drug for controlling the expression of a target gene in a mammal.

In the present invention, control of the function of the target RNA means, for example, the regulation or conversion of the splicing function, such as translation inhibition or exon skipping, which occurs when the antisense sequence portion covers a part of the target RNA through hybridization, or the suppression of the function of the target RNA through degradation of the target RNA, which may occur when the hybridized part of the antisense sequence portion and a part of the target RNA is recognized.

The mammal is preferably a human.
The route of administration is preferably enteral. In other embodiments, the route of administration is parenteral.

The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the scope of the present invention in any way.
In the examples, "NMR" means nuclear magnetic resonance, and "(v/v)" means (volume/volume).
When ¹ H NMR data is given, it is measured at 300 MHz (JNM-ECX300; manufactured by JEOL Ltd., or JNM-ECP400; manufactured by JEOL Ltd.), and the chemical shift δ (unit: ppm) (splitting pattern, integral value) of the signal is shown 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 " _CDCl3 " means deuterated chloroform.
Purification by silica gel column chromatography was performed using Purif-Pack (registered trademark)-EX (SI-50 μm) manufactured by SHOKO SCIENCE.

[Production Example 1]

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 (about 16 to 20 hours). After cooling to room temperature, water was added to the reaction solution, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate, and then washed with dilute hydrochloric acid, water, and brine in that order. After concentrating the organic layer, the obtained crude compound 2 was dissolved in ethyl acetate (100 mL) and methanol (100 mL), 10% palladium-carbon (5.0 g) was added, and the mixture was stirred overnight (about 16 to 20 hours) under a hydrogen atmosphere. The reaction liquid 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.
¹ H NMR (CDCl ₃ , 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).

2-Azaadamantane-N-hydroxyl (AZADOL®) (457 mg, 1.08 mmol) and diacetoxyiodobenzene (34.7 g, 108 mmol) were added to a mixed solution of compound 3 (17.4 g, 35.9 mmol) in methylene chloride (120 mL) and water (60 mL), and the mixture was stirred overnight (about 16 to 20 hours) at room temperature. Successively, 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 with sodium sulfate. After concentration, methanol was added to the obtained crude product, which was vigorously stirred and then filtered. This operation was repeated three times to obtain compound 4 (16.3 g, yield 91%) as a white solid.
¹ H NMR (CDCl ₃ , 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).

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 (about 16 to 20 hours) at room temperature. Subsequently, water was added to the reaction solution, which was then extracted with methylene chloride, and the organic layer was washed with dilute hydrochloric acid and saline. After concentration, ethyl acetate was added to the obtained crude product, which was vigorously stirred and then filtered. This operation was repeated three times to obtain the desired compound 5 (11.8 g, 87% yield) as a white solid.
¹ H NMR (CDCl ₃ , 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).

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. Then, 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) to obtain the target compound 6 (3.22 g, 53%) as a white solid.
¹ H NMR (CDCl ₃ , 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).

[Production Example 2]

Under an argon atmosphere, sodium hydride (55% by weight 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 (about 16 to 20 hours). After cooling to room temperature, water was added to the reaction solution, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate, and then washed with dilute hydrochloric acid, water, and saline in that order. After concentrating the organic layer, the obtained crude compound 7 was dissolved in ethyl acetate (30 mL) and methanol (30 mL), and 10% palladium-carbon (1.5 g) was added and the mixture was stirred overnight (about 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 washings were concentrated to obtain a crude product, which was then added with ethyl acetate and ethanol, stirred vigorously, and filtered. This operation was repeated three times to obtain the desired compound 8 (7.79 g, yield 87%) as a white solid.
¹ H NMR (CDCl ₃ , 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).

Compound 9 (2.68 g, 68% yield) was obtained as a white solid using compound 8 (3.87 g, 7.15 mmol) as a starting material and in a similar manner to the synthesis of compound 4.
¹ H NMR (CDCl ₃ , 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).

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 and in a similar manner to the synthesis of compound 5.
¹ H NMR (CDCl ₃ , 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).

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 and a method similar to that for the synthesis of compound 6.
¹ H NMR (CDCl ₃ , 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).

[Production Example 3]

Under an argon atmosphere, sodium hydride (55% by weight 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 (about 16 to 20 hours). After cooling to room temperature, water was added to the reaction solution, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate, and then washed with dilute hydrochloric acid, water, and brine in that order. After concentrating the organic layer, the obtained crude compound 12 was dissolved in ethyl acetate (30 mL) and methanol (30 mL), 10% palladium-carbon (1.5 g) was added, and the mixture was stirred overnight (about 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 (CDCl ₃ , 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).

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 (CDCl ₃ , 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).

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 obtained crude compound, 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 (CDCl ₃ , 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).

Compound 16 (1.21 g, 74% yield) was obtained as a white solid using compound 15 (1.20 g, 2.21 mmol) as a starting material and a method similar to that for the synthesis of compound 6.
¹ H NMR (CDCl ₃ , 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).

[Production Example 4]

Under an argon atmosphere, sodium hydride (55% by weight 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 (about 16 to 20 hours). After cooling to room temperature, water was added to the reaction solution, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate, and then washed with dilute hydrochloric acid, water, and brine in that order. After concentrating the organic layer, the obtained crude compound 17 was dissolved in ethyl acetate (30 mL) and methanol (30 mL), 10% palladium-carbon (1.5 g) was added, and the mixture was stirred overnight (about 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 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 18 (6.08 g, yield 85%) as a white solid.
¹ H NMR (CDCl ₃ , 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).

2-Azaadamantane-N-hydroxyl (AZADOL (registered trademark)) (956 mg, 2.25 mmol) and diacetoxyiodobenzene (7.26 g, 22.5 mmol) were added to a mixed solution of compound 18 (4.00 g, 10.7 mmol) in methylene chloride (40 mL) and water (20 mL), and the mixture was stirred at room temperature overnight (about 16 to 20 hours). 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 with 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 obtained compound 19, and the mixture was stirred overnight (about 16 to 20 hours) at room temperature. Subsequently, water was added to the reaction solution, followed by extraction with methylene chloride, and the organic layer was washed with dilute hydrochloric acid and saline. After concentration, the obtained crude product was purified by silica gel column chromatography (hexane/ethyl acetate) to obtain the target compound 20 (4.64 g, 89% yield) as a white solid.
¹ H NMR (CDCl ₃ , 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).

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 and a method similar to that for the synthesis of compound 6.
¹ H NMR (CDCl ₃ , 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).

[Production Example 5]

Under an argon atmosphere, sodium hydride (55% by weight 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 (about 16 to 20 hours). After cooling to room temperature, water was added to the reaction solution, which was then concentrated. The residue was extracted with a mixed solvent of hexane and ethyl acetate, and then washed with water and brine in that order. After concentrating the organic layer, the obtained crude product was purified by silica gel column chromatography (hexane/ethyl acetate) to obtain the target compound 22 (6.16 g, yield 61%) as a colorless oil.
¹ H NMR (CDCl ₃ , 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).

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 (about 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. The crude compound 23 obtained by concentration was dissolved in ethyl acetate (43 mL) and methanol (43 mL), 10% palladium-carbon (2.4 g) was added, and the mixture was stirred overnight (about 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 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 24 (2.32 g, yield 65%) as a colorless oil.
¹ H NMR (CDCl ₃ , 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).

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 and in a similar manner to the synthesis of compound 20.
¹ H NMR (CDCl ₃ , 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).

Compound 27 (980 mg, 70% yield) was obtained as a colorless oil using compound 26 (970 mg, 2.13 mmol) as a starting material and in a similar manner to the synthesis of compound 6.
¹ H NMR (CDCl ₃ , 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).

（式中、＊は、オリゴヌクレオチドＷとの結合位置を表す。（＊がホスホジエステル結合の酸素原子である））で表される基が結合していることを意味する。「J2-」は、５’末端のヒドロキシ基の酸素原子に、ホスホジエステル結合を介して下記式（IＶ）

（式中、＊は、オリゴヌクレオチドＷとの結合位置を表す。（＊がホスホジエステル結合の酸素原子である））で表される基が結合していることを意味する。
　なお、実施例中の化学構造の表記（表１、４及び６）において、隣り合う２つのヌクレオシドの間に、「＾」が記載されていないとき、その２つの核酸塩基の間のヌクレオシド間結合は、ホスホジエステル結合である。例えば、「Ａ（Ｌ）Ｔ（Ｌ）」との表記では、ＡとＴとの間のヌクレオシド間結合は、ホスホジエステル結合であり、「ＡＧ（Ｌ）」との表記では、ＡとＧの間のヌクレオシド間結合は、ホスホジエステル結合である。 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)" means LNA (β-D-methyleneoxy BNA), lowercase alphabets mean deoxyribonucleosides, uppercase alphabets (excluding the alphabets with (L) above and (I) below) mean ribonucleosides, "^" means phosphorothioate bond, "5(x)" means that the nucleobase of the deoxyribonucleoside is 5-methylcytosine, and "5" in "5(L)" means that the nucleobase of the nucleoside is 5-methylcytosine. "J1-" means that the oxygen atom of the hydroxyl group at the 5'-terminus is linked to the nucleotide of the following formula (III) via a phosphodiester bond.

(wherein * represents the bonding position with oligonucleotide W. (* is the oxygen atom of the phosphodiester bond)) is bonded to the oxygen atom of the hydroxyl group at the 5' end. "J2-" means that a group represented by the following formula (IV):

(wherein * represents the bonding position with oligonucleotide W. (* is the oxygen atom of a phosphodiester bond)) is bonded.
In addition, in the notation of the chemical structure 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.

[Production Example 6]
The antisense oligonucleotides (compounds represented by chemical structures corresponding to the compound numbers) listed in Table 1 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.). 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 Co., Ltd.) containing single-stranded DNA with 15, 20, 30, 40, 50, 60, and 80 nucleotides and double-stranded RNA size markers (manufactured by Gene Design Co., Ltd.) with 17, 21, 25, and 29 base pairs were used as markers, and a single band was confirmed for each single-stranded oligonucleotide.

[Evaluation Example 1] Antisense inhibition of mouse Malat1 in 3T3-L1 cells 3T3-L1 cells were seeded in a 96-well plate at a density of 20,000 cells/well. After about 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 amount of the housekeeping gene Gapdh [glyceraldehyde-3-phosphate dehydrogenase] was also quantified at the same time, 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 Table 2 as the percentage expression of Malat1 relative to untreated control cells.

[Evaluation Example 2] Antisense inhibition of mouse Malat1 in N1E-115 cells N1E-115 cells were seeded in a 96-well plate at a density of 20,000 cells/well. After about 24 hours, the culture supernatant of the N1E-115 cells was replaced with D-MEM containing no FBS, and P21790027 and P22790171 were added to a final concentration of 1000 nM (Free-Uptake). After 24 hours, a cell lysate containing RNA was prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio), and then 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 amount of mRNA of the housekeeping gene Gapdh was also quantified at the same time, and the amount of mRNA of Malat1 relative to the amount of mRNA of Gapdh was evaluated as the expression level of Malat1. The results are presented in Table 3 as the percentage expression of Malat1 relative to untreated control cells.

[Evaluation Example 3] Antisense inhibition of Malat1 in mice C57BL/6J mice (male, 6 weeks old, Jackson Laboratory Japan Co., Ltd.) were intravenously administered with P21790027, P22790171, and P22790172 dissolved in saline (Otsuka saline injection, Otsuka Pharmaceutical Factory) so that the dose per mouse was 0.95 μmol/kg in terms of the amount of antisense oligonucleotide. As a control, saline alone (Otsuka saline injection, Otsuka Pharmaceutical Factory) was administered. Heart, muscle, lung, liver, and kidney tissues were collected under isoflurane anesthesia 5 and 10 days after administration (P22790172 was collected only 5 days after administration). After isolating RNA from each organ using MagMAX (registered trademark) mirVana (registered trademark) Total RNA Isolation Kit (Thermo Fisher Scientific), the expression level of mouse Malat1 gene was measured by quantitative real-time PCR using One Step PrimeScript (registered trademark) III RT-qPCR Mix (Takara Bio) and TaqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of mRNA of the housekeeping gene Gapdh was also quantified at the same time, and the amount of mRNA of Malat1 relative to the amount of mRNA of Gapdh was evaluated as the expression level of Malat1. The results are shown in FIG. 1 (Day 10) and FIG. 2 (Day 5) as the percentage expression of Malat1 relative to the untreated control group (control).

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.

[Evaluation Example 4] Antisense inhibition of Hprt1 in mice C57BL/6J mice (male, 6 weeks old, Jackson Laboratory Japan, Inc.) were administered with P22790163, P23790243, P23790244, or P23790246 dissolved in physiological saline (Otsuka saline injection, Otsuka Pharmaceutical Factory, Ltd.) at a dose of 0.95 μmol/kg (P2379024 The mice were intravenously administered with 1.2 μmol/kg of saline (Otsuka saline injection, Otsuka Pharmaceutical Factory) as a control. Five, ten and twenty days after administration, cardiac (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 (P23790246 was collected only 10 days after administration). After isolating RNA from each organ using MagMAX (registered trademark) mirVana (registered trademark) 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 (registered trademark) III RT-qPCR Mix (Takara Bio) and TaqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of mRNA of the housekeeping gene Gapdh was also quantified at the same time, 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).

As is clear from Figures 3 to 9, P23790244 and P23790246 showed a higher inhibitory effect on Hprt1 expression in the liver, heart, lungs, and muscles than P22790163 and P23790243.

（式中、＊は、オリゴヌクレオチドＷとの結合位置を表す。（＊がホスホジエステル結合の酸素原子である））で表される基が結合していることを意味し、「J4-」は、５’末端のヒドロキシ基の酸素原子に、ホスホジエステル結合を介して下記式（VI）

（式中、＊は、オリゴヌクレオチドＷとの結合位置を表す。（＊がホスホジエステル結合の酸素原子である））で表される基が結合していることを意味し、「J5-」は、５’末端のヒドロキシ基の酸素原子に、ホスホジエステル結合を介して下記式（VII）

（式中、＊は、オリゴヌクレオチドＷとの結合位置を表す。（＊がホスホジエステル結合の酸素原子である））で表される基が結合していることを意味し、「J6-」は、５’末端のヒドロキシ基の酸素原子に、ホスホジエステル結合を介して下記式（VIII）

（式中、＊は、オリゴヌクレオチドＷとの結合位置を表す。（＊がホスホジエステル結合の酸素原子である））で表される基が結合していることを意味する。 [Production Example 7]
The antisense oligonucleotides (compounds represented by chemical structures corresponding to compound numbers) shown in Table 4 were prepared using automated nucleic acid synthesizers nS-8II (Gene Design) and M-8-SE (Nihon Techno Service). The target gene is mouse hypoxanthine phosphoribosyltransferase 1 (Hprt1). In Table 4, "Cmpd No", "Chemical Structure" and sequence notation are the same as in Table 1. In the sequence notation in Table 4, "J3-" indicates that the following formula (V) is linked to the oxygen atom of the hydroxyl group at the 5' end via a phosphodiester bond.

(wherein * represents the bonding position with oligonucleotide W. (* is the oxygen atom of the phosphodiester bond)) is bonded to the oxygen atom of the hydroxyl group at the 5' end, and "J4-" means that a group represented by the following formula (VI) is bonded to the oxygen atom of the hydroxyl group at the 5' end via a phosphodiester bond.

(wherein * represents the bonding position with oligonucleotide W. (* is the oxygen atom of the phosphodiester bond)) is bonded to the oxygen atom of the hydroxyl group at the 5' end, and "J5-" means that a group represented by the following formula (VII) is bonded to the oxygen atom of the hydroxyl group at the 5' end via a phosphodiester bond.

(wherein * represents the bonding position with oligonucleotide W. (* is the oxygen atom of the phosphodiester bond)) is bonded to the oxygen atom of the hydroxyl group at the 5' end, and "J6-" means that a group represented by the following formula (VIII) is bonded to the oxygen atom of the hydroxyl group at the 5' end via a phosphodiester bond.

(wherein * represents the bonding position with oligonucleotide W. (* is the oxygen atom of a phosphodiester bond)) is bonded.

[Evaluation Example 5] Antisense inhibition of mouse Hprt1 in 3T3-L1 cells 3T3-L1 cells were seeded in a 96-well plate at a density of 15,000 cells/well. After about 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, a cell lysate containing RNA was prepared using CellAmp® Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio), and then 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 amount of mRNA of the housekeeping gene Gapdh was also quantified at the same time, and the amount of Hprt1 mRNA relative to the amount of Gapdh mRNA was evaluated as the expression level of Hprt1. The results are presented in Table 5 as percent expression of Hprt1 relative to untreated control cells.

[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 so that the dose per mouse was 2.9 μmol/kg in terms of the amount of antisense oligonucleotide. As a control, only physiological saline (Otsuka Saline Injection, Otsuka Pharmaceutical Factory) was administered. The amount of Hprt1 mRNA relative to the amount of Gapdh mRNA in the heart, liver, and muscle tissues 10 days after administration was evaluated as the expression level of Hprt1. The results are shown in Figure 10 (heart), Figure 11 (liver), and Figure 12 (muscle) as the percentage expression of Hprt1 relative to the untreated control group (control).

As is clear from Figures 10 to 12, P23790460 showed a higher inhibitory effect on Hprt1 expression in the heart, liver, and muscle than P22790163.

（式中、＊は、ホスホジエステル結合やホスホロチオエート結合などのヌクレオシド間結合の酸素原子である））で表されるビニルホスホネート化２’－ＯＭｅヌクレオシドを意味する。
　「J1-＾」、「J2-＾」、「J3-＾」、「J4-＾」及び「J6-＾」は、それぞれ前記「J1-」、「J2-」、「J3-」、「J4-」及び「J6-」と同じ基が結合するが、当該基は５’末端のヒドロキシ基の酸素原子にホスホロチオエート結合を介して結合し、対応する式（III）、式（IＶ）、式（V）、式（VI）及び式（VIII）における＊は、ホスホロチオエート結合の酸素原子である。 [Production Example 8]
The siRNAs (compounds represented by chemical structures corresponding to compound numbers) shown in Table 6 were prepared using an automatic nucleic acid synthesizer M-8-SE (manufactured by Nippon Techno Service Co., Ltd.). The target gene is mouse superoxide dismutase 1 (Sod1). In Table 6, "Compd No,""ChemicalStructure," 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'-fluoro nucleoside. "VPU(M)" at the 5' end is a compound represented by the following formula (IX):

(wherein * is an oxygen atom of an internucleoside bond such as a phosphodiester bond or a phosphorothioate bond)) means a vinylphosphonate-2'-OMe nucleoside represented by the formula:
"J1-^", "J2-^", "J3-^", "J4-^" and "J6-^" are bonded to the same group as "J1-", "J2-", "J3-", "J4-" and "J6-", respectively, but the group is bonded to the oxygen atom of the hydroxy group at the 5' end via a phosphorothioate bond, and * in the corresponding formulas (III), (IV), (V), (VI) and (VIII) is the oxygen atom of the phosphorothioate bond.

Hybridization of each sense strand (S) and antisense strand (AS) was performed 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.

[Evaluation Example 7] Sod1 knockdown effect of siRNA in mouse primary hepatocytes Mouse primary hepatocytes were seeded in a 96-well plate at a density of 20,000 cells/well. After about 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 MagMAX® mirVana® Total RNA Isolation Kit (Thermo Fisher Scientific), and then the expression level of 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 amount of mRNA of the housekeeping gene Ppia [peptidylprolyl isomerase A] was also quantified at the same time, and the amount of Sod1 mRNA relative to the amount of Ppia mRNA was evaluated as the expression level of Sod1. The results are shown in Table 7 as the percentage expression of Sod1 relative to the untreated control cells.

[Evaluation Example 8] Sod1 knockdown effect of siRNA in mice P24791052, P24791054, P24791055 and P24791056 (4.0 nmol each) were dissolved in 10 μl of physiological saline (Otsuka saline injection, Otsuka Pharmaceutical Factory) to prepare various nucleic acid agents. C57BL/6J mice (male, 6 weeks old, Jackson Laboratory Japan Co., Ltd.) were fixed in a brain stereotaxic apparatus under 2.5-4% isoflurane anesthesia. Then, the skin was incised 2-3 cm anterior-posterior between the ears, and a hole was made 1 mm to the right and 0.5 mm posterior to the bregma with a 1 mm diameter drill. Various nucleic acid agents or physiological saline as a negative control were filled in a Hamilton syringe. A needle was inserted about 2 mm into the perforated area, and 10 μl of the solution per mouse was administered into the right lateral ventricle at a rate of 10 μl/min, and the skin was sutured with nylon thread. Two weeks later, the mice were dissected to remove the cerebrum and hippocampus. RNA was isolated using 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 amount of mRNA of the housekeeping gene Tbp (TATA binding protein) was also quantified at the same time, and the amount of Sod1 mRNA relative to the amount of Tbp mRNA was evaluated as the expression level of Sod1. The results are shown in Figure 13 (cerebrum) and Figure 14 (hippocampus) as the percentage expression of Sod1 relative to the negative control (vehicle).

As is clear from Figures 13 and 14, P24791054, P24791055, and P24791056 showed a higher inhibitory effect on Sod1 expression in the cerebrum and hippocampus than P24791052.

P22790171, P23790244, P23790458, P23790459, P23790460, P24791053, P24791054, and P24791055 demonstrated improved knockdown activity of target RNA under Free-Uptake conditions, which are highly applicable to animal experiments (in vivo testing) (Evaluation Examples 1, 2, 5, and 7).

The lipid-bound oligonucleotide of the present invention has 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, and lungs. Therefore, the lipid portion of the present invention is expected to be a means of delivering various oligonucleotides.

Claims (31)

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 divalent group derived from a substituted or unsubstituted C _1-10 alkylene group or a substituted or unsubstituted poly C _1-10 alkylene glycol,
R ¹ and R ² are each independently a substituted or unsubstituted C _5-32 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. The lipid-bound oligonucleotide or its complex according to claim 1, wherein R ¹ and R ² are each independently an unsubstituted C _5-32 alkyl group or an unsubstituted C _5-32 alkenyl group, or R ¹ and R ² may be bonded to each other to form a ring. The lipid-bound oligonucleotide or its complex according to claim 1 or 2, wherein R ¹ and R ² are each independently an unsubstituted C _5-32 alkyl group or an unsubstituted C _5-32 alkenyl group. The lipid-linked oligonucleotide or its complex according to any one of claims 1 to 3, wherein R ¹ and R ² are each independently an unsubstituted C _10-20 alkyl group. The lipid-linked oligonucleotide or its complex according to any one of claims 1 to 4, wherein R ¹ and R ² are unsubstituted C ₁₄ alkyl groups. The lipid-linked oligonucleotide or a complex thereof according to any one of claims 1 to 5, wherein said L is an unsubstituted C _1-10 alkylene group. The lipid-linked oligonucleotide or its complex according to any one of claims 1 to 6, wherein L is an unsubstituted _C6 alkylene group. The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide compound is a gapmer-type antisense oligonucleotide. 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. The lipid-linked oligonucleotide of claim 8 or 9, wherein the gapmer-type antisense oligonucleotide comprises at least four consecutive deoxyribonucleosides. The lipid-linked oligonucleotide according to any one of claims 8 to 10, wherein the gapmer-type antisense oligonucleotide is composed of 13 to 25 nucleosides. The lipid-linked oligonucleotide according to any one of claims 8 to 11, wherein L is bound to the 5' end of the gapmer-type antisense oligonucleotide. The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide compound is a mixmer-type antisense oligonucleotide. The lipid-linked oligonucleotide according to 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. 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. The oligonucleotide compound according to any one of claims 13 to 15, wherein the mixmer-type antisense oligonucleotide consists of 13 to 25 nucleosides. The lipid-linked oligonucleotide compound according to any one of claims 13 to 16, wherein L is bound to the 5' end of the mixmer-type antisense oligonucleotide. the oligonucleotide complex being a double-stranded oligonucleotide complex comprising a first oligonucleotide and a second oligonucleotide,
the first oligonucleotide is a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides;
the second oligonucleotide comprises a sequence capable of hybridizing to at least a portion of the first oligonucleotide, and is composed 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;
The complex of any one of claims 1 to 7, wherein the first oligonucleotide and the second oligonucleotide hybridize. The complex of claim 18, wherein the second oligonucleotide comprises at least four consecutive ribonucleosides. 20. The complex of claim 18 or 19, wherein L is attached to the 5' end of the second oligonucleotide. the oligonucleotide compound comprises a first oligonucleotide and a second oligonucleotide;
the first oligonucleotide is a gapmer-type antisense oligonucleotide or a mixmer-type antisense oligonucleotide consisting of 7 to 100 nucleosides;
the second oligonucleotide comprises a sequence capable of hybridizing to at least a portion of the first oligonucleotide, and is composed 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;
a first oligonucleotide and a second oligonucleotide are linked together;
The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the first oligonucleotide and the second oligonucleotide hybridize. 22. The lipid-linked oligonucleotide of claim 21, wherein the second oligonucleotide comprises at least four consecutive ribonucleosides. The lipid-linked oligonucleotide of claim 21 or 22, wherein L is attached to the 5' end of the second oligonucleotide. The lipid-linked oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide compound or oligonucleotide complex is selected from siRNA, aptamers and ribozymes. The lipid-linked oligonucleotide according to any one of claims 1 to 7 and 24, wherein the oligonucleotide compound or oligonucleotide complex is an siRNA. A pharmaceutical composition comprising the lipid-linked oligonucleotide or complex thereof according to any one of claims 1 to 25 and a pharmacologically acceptable carrier. A method for controlling the function of a target RNA, comprising a step of contacting a cell with the lipid-linked oligonucleotide or a complex thereof according to any one of claims 1 to 25. A method for regulating the function of a target RNA in a mammal, comprising administering to the mammal the pharmaceutical composition according to claim 26. A method for controlling expression of a target gene, comprising a step of contacting a cell with the lipid-bound oligonucleotide or a complex thereof according to any one of claims 1 to 25. A method for controlling expression of a target gene in a mammal, comprising administering to the mammal the pharmaceutical composition according to claim 26. A compound represented by the following formula (II):

(Wherein,
L is a divalent group derived from a substituted or unsubstituted C _1-10 alkylene group or a substituted or unsubstituted poly C _1-10 alkylene glycol;
R ¹ and R ² are each independently a substituted or unsubstituted C _5-32 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;
Each R ³ is independently a C _1-10 alkyl group, or the two alkyl groups of R ³ may be bonded to each other to form a ring;
^R4 is a 2-cyanoethyl group.