WO2019009298A1 - α-SYNUCLEIN EXPRESSION INHIBITOR - Google Patents

Abstract

According to the present invention, a nucleic acid medicine, which has a higher effect of inhibiting α-synuclein expression and sustains this effect for a longer period of time, can be provided. The oligonucleotide according to the present invention or a pharmacologically acceptable salt thereof is characterized by: containing at least one nucleoside structure represented by formula (I) [wherein each of "Base" and "A" represents a definite substituent or structure]; being capable of binding to an α-synuclein gene; having an activity of inhibiting the expression of the α-synuclein gene; being complementary to the α-synuclein gene; having a specific sequence; being complementary to at least a part of α-synuclein represented by SEQ ID NO: 1; and having an oligonucleotide length of 12-20 bases.

Description

α-synuclein expression inhibitor

The present invention relates to a novel oligonucleotide exhibiting an α-synuclein expression inhibitory action, an α-synuclein expression inhibitor comprising the novel oligonucleotide, and more specifically, an α-synuclein expression inhibitor using a novel artificial nucleic acid.

Parkinson's disease (PD) can be classified as sporadic Parkinson's disease and hereditary Parkinson's disease.

Sporadic Parkinson's disease is a progressive neurodegenerative disease, with a prevalence of 1 in 1000, and progressive dementia and dementia. Such dementia is Lewy body dementia and the treatment is only symptomatic treatment. This is caused by aggregation and accumulation of α-synuclein in the brain.

Hereditary Parkinson's disease is 5 to 10% of Parkinson's disease, and involvement of the PARK4 gene is considered among the etiologic genes PARK1 to PARK20. Hereditary Parkinson's disease in which the PARK4 gene is involved exhibits autosomal dominant inheritance, and there are several dozen such patients in Japan. In hereditary Parkinson's disease in which the PARK4 gene is involved, the normal α-synuclein gene is in excess, resulting in the combination of Parkinson's disease and dementia.

Α-synuclein is a protein consisting of 140 amino acid residues and is an amyloid protein having no unique native structure. Alpha-synuclein is associated with synaptic vesicle accumulation and release. The α-synuclein knockout (KO) mouse is pathologically normal and shows no neuroprotection against neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) obtain.

Α-synuclein is a main component of Lewy bodies in diseases such as Parkinson's disease and Lewy body dementia (DLB). PD autopsy brain analysis For stage classification of Braak, α-synuclein staining of autopsy brain was performed and disease progress was compared with α-synuclein lesion, and α-synuclein aggregation in nerve cells is the main cause of lesion Is known. Also, when α-synuclein transgenic (Tg) mice were administered α-synuclein fibrils, the lesions extended to the nucleus and abnormal α-synuclein was also found outside the cells (prion-like cells) Outward propagation).

The pathological conditions of Parkinson's disease are as follows. Aggregation of aberrant α-synuclein in neurons denatures the midbrain nigral neurons, reduces the production of dopamine, and causes motor dysfunction or cognitive dysfunction. In conventional symptomatic treatment, neurodegeneration has gradually progressed, and administration of L-dopa preparation for dopamine replacement or administration of a dopamine agonist for promotion of dopamine secretion has been performed for dopamine replacement.

Attempts have been made to use nucleic acid drugs for α-synuclein knockdown, targeting the aggregation of aberrant α-synuclein in neuronal cells.

Use of Adeno-Associated Virus (AAV) Ribozymes in Rats for Nucleic Acid Drugs to Suppress Excess α-Synuclein (Non-patent Document 1), Use of Lentivirus-shRNA in Rats (Non-patent Document 2), AAV- Use of shRNA in rats (Non-patent documents 3 and 4), Use of naked siRNA in mice (Non-patent document 5), Use of exosomal siRNA in mice (Non-patent document 6), siRNA (2-O-Me And its use in monkeys (Non-patent Document 7) has been reported. However, non-patent documents 1 to 4 use viruses, the effects of siRNAs of non-patent documents 5 and 6 disappear early, and the effects of siRNA of non-patent document 7 are insufficient.

In addition, the use of artificial nucleic acids has been reported for suppressing the expression of α-synuclein gene (Patent Document 1). In Patent Document 1, a 2'-O-methoxyethyl (MOE) modified nucleoside is used. Also, in Patent Document 1, the oligonucleotide is injected and administered via intrastriatal bolus injection.

JP-A-2014-501507

Kinoh et al. BBRC, 2006, vol. 341, pp. 1088-95 Sapru et al. Exp Neurol, 2006, vol. 198, pp. 382-90 Gorbatyuk et al. MoI Ther, 2010, vol. 18, pp. 1450-7 Khodr et al. Brain Res, 2011, vol. 1395, pp. 94-107 Lewis et al. MoI Neurodegener, 2008, vol. 3, pp. 19 Cooper et al. Mov Disord, 2014, vol. 29, pp. 1476-85 McCormack et al. PLoS One, 2010, vol. 5, pp. E12122

The present invention solves the above-mentioned problems, and an object of the present invention is to provide a nucleic acid drug having higher efficacy and longer duration for suppression of α-synuclein expression.

The present invention provides a nucleoside structure represented by the following formula (I):

An oligonucleotide comprising at least one or a pharmacologically acceptable salt thereof,
here,
Base may be a purin-9-yl group which may have one or more optional substituents selected from α group, or may have one or more optional substituents selected from α group 2 -Oxo-1,2-dihydropyrimidin-1-yl group, wherein the α group is a hydroxyl group, a hydroxyl group protected by a protective group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, carbon A linear alkoxy group having a number of 1 to 6, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms , An amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom,
A is the following:

And a divalent group represented by
R ¹ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring, selected from the α group The aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a hetero atom, and has one or more optional substituents selected from the α group An aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may be and contain a hetero atom, or a protecting group of an amino group for nucleic acid synthesis;
R ² and R ³ are each independently a hydrogen atom; may be substituted with a C 3-12 aryl group which may contain a hetero atom, and may form a branch or a ring An alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom; or R ² and R ³ are taken together to form — (CH 2 ₂ ) _q- [wherein, q is an integer of 2 to 5];
R ⁴ and R ⁵ are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or, R ⁴ and R ⁵ together are CC (R ¹¹ ) R ¹² [wherein, R ¹¹ and R ¹² each independently represent a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a nucleic acid synthesis protecting group, a mercapto group, a mercapto group protected with a nucleic acid synthesis protecting group An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms Chain or branched alkyl amino Represents a group];
R ⁶ and R ⁷ each independently represent a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, or 1 to 7 carbons which may form a branch or a ring An alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms;
R ⁸ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, or 1 Represents 6 linear or branched alkylthio groups;
R ⁹ represents a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, an amino group, Or an amino group protected by a protecting group for nucleic acid synthesis;
R ¹⁰ is a hydrogen atom or a guanidino group;
R ¹³ and R ¹⁴ each independently represent a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis;
m is an integer of 0 to 2;
n is an integer of 0 to 1;
When R ¹⁰ is a hydrogen atom, p is 1; when R ¹⁰ is a guanidino group, p is 0;
X is an oxygen atom, a sulfur atom, or an amino group; and Y is an oxygen atom or a sulfur atom,
Here, the oligonucleotide is capable of binding to the α-synuclein gene, having an activity of suppressing the expression of the α-synuclein gene, and being complementary to the α-synuclein gene, and , 42 to 44, 72, 74 to 78, 82, 91, 97, 210, 215, 216, 227, 229, 232 to 234, 254 to 256, 262, 263, 266 to 269, 272 to 275, 277 A nucleotide complementary to any one nucleotide selected from the group consisting of positions 279, 281 to 289, 321, 367 to 369, and 412 to 415 is a 5 'end, and is complementary to at least a part of SEQ ID NO: 1 Or the pharmacologically acceptable salt thereof, wherein the length of the oligonucleotide is 12 to 20 bases To provide.

In one embodiment, the oligonucleotide is a gapmer consisting of a gap region of 6 to 10 bases, a 5 'wing of 3 to 5 bases and a 3' wing of 3 to 5 bases,
The gap region is located between the 5 'wing and the 3' wing, and the 5 'wing and the 3' wing comprise at least one nucleoside structure represented by the formula (I).

In a further embodiment, the gap region consists of 7-9 bases or 8-10 bases, the 5 'wing and the 3' wing each consist of 3 bases, and the 5 'wing and the 3' wing Each contains at least two nucleoside structures represented by the above formula (I).
The 5 'wing and the 3' wing may include a 2'-O-methyl modified nucleoside or a 2'-O-methoxyethyl (MOE) modified nucleoside.

In one embodiment, the nucleoside structure represented by formula (I) above is

［式中、Ｂａｓｅ、Ｒ¹、Ｘ、ｍおよびｎは、上述したとおりである。］

[Wherein, Base, R ¹ , X, m and n are as described above. ]

Is a structure represented by

In one embodiment, the nucleoside structure represented by the formula (I) is a structure represented by the formula (I ′), and in the formula (I ′), the m is 0, and R ¹ is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group or a benzyl group.

In one embodiment, the above oligonucleotide or a pharmacologically acceptable salt thereof is selected from 40, 42 to 44, 72, 74 to 78, 82, 91, 97, 210, 215, 216, 227 of SEQ ID NO: 1 Any one selected from the group consisting of positions 229, 232 to 234, 254 to 256, 262, 263, 266 to 269, 272 to 275, 277 to 279, 281 to 289, 321, 367 to 369, and 412 to 415; A nucleotide complementary to one nucleotide is at the 5 'end, is complementary to at least a part of SEQ ID NO: 1, and has a length of 13 to 15 bases.

In one embodiment, the above-mentioned oligonucleotide or a pharmacologically acceptable salt thereof comprises 42-44, 72, 74-78, 82, 91, 97, 210, 216, 229, 232, 233 of SEQ ID NO: 1 Complementary to any one nucleotide selected from the group consisting of positions 254 to 256, 262, 263, 266 to 269, 272 to 275, 278, 279, 281 to 289, 321, 367 to 369, and 413 to 415 And a complementary nucleotide at the 5 'end, which is complementary to at least a part of SEQ ID NO: 1 and has a length of 13 to 15 bases.

In one embodiment, the above-mentioned oligonucleotide or a pharmacologically acceptable salt thereof comprises 43, 44, 72, 74-78, 82, 91, 97, 210, 216, 229, 232, 233 of SEQ ID NO: 1 Any one selected from the group consisting of positions 254, 256, 262, 263, 266-269, 272-275, 278, 279, 281-284, 286-289, 321, 367-369, and 413-415; The nucleotide complementary to the nucleotide is at the 5 'end, is complementary to at least a part of SEQ ID NO: 1, and is 15 bases in length.

The present invention also provides an α-synuclein expression inhibitor comprising the above-mentioned oligonucleotide or a pharmacologically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides a pharmaceutical composition comprising the above oligonucleotide or a pharmacologically acceptable salt thereof as an active ingredient.

In one embodiment, the pharmaceutical composition is used for the treatment or prevention of alpha synuclein excess.

In one embodiment, the pharmaceutical composition is used for the treatment or prevention of Parkinson's disease or dementia with Lewy bodies.

Furthermore, the present invention provides a method for suppressing α-synuclein expression, comprising the step of administering the above-mentioned oligonucleotide or a pharmacologically acceptable salt thereof to an individual.

Furthermore, the present invention provides a method for treating or preventing alpha-synuclein excess, comprising administering the above-mentioned oligonucleotide or a pharmacologically acceptable salt thereof to an individual.

Still further, the present invention provides a method for treating or preventing Parkinson's disease or dementia with Lewy bodies, comprising the step of administering the above oligonucleotide or a pharmacologically acceptable salt thereof to an individual.

According to the present invention, there is provided an oligonucleotide having a sustained expression inhibitory effect on α-synuclein. According to the present invention, the α-synuclein expression inhibitory effect of the oligonucleotide can be exhibited also in intrathecal administration which is a administration route usually used at the time of clinical application.

FIG. 16 is a graph showing the amount of α-synuclein mRNA after transfection of antisense oligonucleotide (ASO) to HEK293T cells in Example 3. FIG. FIG. 16 is a graph showing the amount of α-synuclein mRNA after transfection of antisense oligonucleotide (ASO) to HEK293T cells in Example 4. FIG. FIG. 16 is a graph showing the amount of α-synuclein mRNA after transfection of antisense oligonucleotide (ASO) to HEK293T cells in Example 5. FIG. FIG. 16 is a graph showing the amount of α-synuclein mRNA in the substantia nigra (a) and striatum (b) after antisense oligonucleotide (ASO) administration to SNCA Tg mice in Example 6. FIG. 16 is a graph showing the amount of α-synuclein mRNA after transfection of antisense oligonucleotide (ASO) to HEK293A cells in Test Example 1. FIG. FIG. 16 is a graph showing the amount of α-synuclein mRNA after transfection of antisense oligonucleotide (ASO) to HEK293A cells in Test Example 2. FIG. FIG. 16 is a graph showing the amount of α-synuclein mRNA after transfection of antisense oligonucleotide (ASO) to HEK293A cells in Test Example 3. FIG.

First, the terms used in the present specification will be defined.

In the present specification, the term "C1-C6 linear alkyl group" refers to any C1-C6 linear alkyl group, and more specifically methyl, ethyl, n-propyl, An n-butyl group, an n-pentyl group or an n-hexyl group.

As used herein, the term "C1-C6 linear alkoxy group" includes alkoxy groups having any C1-C6 linear alkyl group. For example, methyloxy group, ethyloxy group, n-propyloxy group and the like can be mentioned. As used herein, the term "C1-C6 linear or branched alkoxy group" includes alkoxy groups having any C1-C6 linear or branched alkyl group. For example, methyloxy, ethyloxy, n-propyloxy, isopropyloxy, n-butyloxy, isobutyloxy, tert-butyloxy, n-pentyloxy, isopentyloxy and the like can be mentioned.

As used herein, the term "C1-C6 linear alkylthio group" includes alkylthio groups having any C1-C6 linear alkyl group. For example, a methylthio group, an ethylthio group, an n-propylthio group and the like can be mentioned. As used herein, the term "C1-C6 linear or branched alkylthio group" includes alkylthio groups having any C1-C6 linear or branched alkyl group. For example, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio, isopentylthio and the like can be mentioned.

In the present specification, the term "C1-C6 cyanoalkoxy group" refers to a group in which at least one hydrogen atom constituting the C1-C6 linear alkoxy group is substituted with a cyano group.

In the present specification, the term "C1-C6 linear alkylamino group" is a group in which one or two hydrogen atoms constituting an amino group are substituted with a C1-C6 linear alkyl group. Includes For example, methylamino group, dimethylamino group, ethylamino group, methylethylamino group, diethylamino group and the like can be mentioned. In the present specification, the term "C1-C6 linear or branched alkylamino group" is any linear or branched C1-C6 linear or branched hydrogen atom constituting an amino group. It includes a group substituted with a chain alkyl group. For example, methylamino group, dimethylamino group, ethylamino group, methylethylamino group, diethylamino group, n-propylamino group, di (n-propyl) amino group, isopropylamino group, diisopropylamino group and the like can be mentioned.

In the present specification, the term "C1-C7 alkyl group which may form a branch or a ring" is any C1-C7 linear alkyl group and any C3-C7 branched chain. It includes a chain alkyl group and any cyclic alkyl group having 3 to 7 carbon atoms. It may be simply referred to as "lower alkyl group". For example, as the arbitrary linear alkyl group having 1 to 7 carbon atoms, methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, and n-heptyl group can be mentioned. And the optionally branched alkyl group having 3 to 7 carbon atoms includes isopropyl, isobutyl, tert-butyl, isopentyl and the like, and the optional cyclic alkyl group having 3 to 7 carbon atoms includes A cyclobutyl group, a cyclopentyl group, a cyclohexyl group etc. are mentioned.

In the present specification, the term "alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring" is any linear alkenyl group having 2 to 7 carbon atoms and any branch having 3 to 7 carbon atoms. It includes a chain alkenyl group and any cyclic alkenyl group having 3 to 7 carbon atoms. It may be simply referred to as "lower alkenyl group". For example, arbitrary linear alkenyl group having 2 to 7 carbon atoms includes ethenyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 1-pentenyl group, 2-pentenyl group, Examples thereof include 3-pentenyl group, 4-pentenyl group, 1-hexenyl group and the like, and as the arbitrary branched alkenyl group having 3 to 7 carbon atoms, isopropenyl group, 1-methyl-1-propenyl group, 1-methyl -2-propenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-methyl-2-butenyl group and the like, and as an optional cyclic alkenyl group having 3 to 7 carbon atoms And cyclobutenyl group, cyclopentenyl group, cyclohexenyl group and the like.

In the present specification, the term “C 1 -C 7 alkoxy group which may form a branch or a ring” is any C 1 -C 7 linear alkoxy group, and any C 3 -C 7 branched group It includes a chain alkoxy group, and any cyclic alkoxy group having 3 to 7 carbon atoms. It may be simply referred to as "lower alkoxy group". For example, as the arbitrary linear alkoxy group having 1 to 7 carbon atoms, a methoxy group, an ethoxy group, an n-propoxy group, an n-butyloxy group, an n-pentyloxy group, an n-hexyloxy group, and an n-heptyloxy group Groups, and examples of the optionally branched alkoxy group having 3 to 7 carbon atoms include isopropoxy group, isobutyroxy group, tert-butyloxy group, isopentyloxy group and the like, and any of 3 to 7 carbon atoms Examples of the cyclic alkoxy group include cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group and the like.

In the present specification, the term "aryl group having 3 to 12 carbon atoms which may contain a hetero atom" refers to any aryl group having 6 to 12 carbon atoms, which is composed only of hydrocarbon, Including any heteroaryl group having 3 to 12 carbon atoms in which at least one carbon atom constituting the ring structure is substituted with a heteroatom (for example, a nitrogen atom, an oxygen atom, and a sulfur atom, and a combination thereof) Do. Examples of the aryl group having 6 to 12 carbon atoms include a phenyl group, a naphthyl group, an indenyl group, and an azulenyl group, and an arbitrary heteroaryl group having 3 to 12 carbon atoms includes a pyridyl group, a pyrrolyl group, A quinolyl group, an indolyl group, an imidazolyl group, a furyl group, a thienyl group etc. are mentioned.

In the present specification, examples of the term “aralkyl group having a C 3-12 aryl moiety which may contain a hetero atom” include benzyl group, phenethyl group, naphthylmethyl group, 3-phenylpropyl group, 2 -Phenylpropyl, 4-phenylbutyl, 2-phenylbutyl, pyridylmethyl, indolylmethyl, furylmethyl, thienylmethyl, pyrrolylmethyl, 2-pyridylethyl, 1-pyridylethyl, 3 -A thienylpropyl group etc. are mentioned.

In the present specification, examples of the term "acyl group" include aliphatic acyl groups and aromatic acyl groups. Specifically, examples of aliphatic acyl groups include formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group, isovaleryl group, octanoyl group, nonanoyl group, decanoyl group, 3-methylnonanoyl group, 8-methylnonanoyl group, 3-ethyloctanoyl group, 3,7-dimethyloctanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, 1-methylpentadecanoyl group, 14-methylpentadecanoyl group, 13,13-dimethyltetradecanoyl group, heptadecanoyl group, 15-methylhexadecanoyl group, octadecanoyl group, 1-methylheptadecanoyl group, Nonadecanoyl group, Eikosanoyl group And alkylcarbonyl groups such as henicosanoyl group; carboxylated alkylcarbonyl groups such as succinoyl group, glutaroyl group and adipoyl group; halogeno lower alkyl carbonyl groups such as chloroacetyl group, dichloroacetyl group, trichloroacetyl group and trifluoroacetyl group Lower alkoxy lower alkylcarbonyl group such as methoxyacetyl group; unsaturated alkylcarbonyl group such as (E) -2-methyl-2-butenoyl group. Also, examples of the aromatic acyl group include arylcarbonyl groups such as benzoyl group, α-naphthoyl group and β-naphthoyl group; halogenoarylcarbonyl groups such as 2-bromobenzoyl group and 4-chlorobenzoyl group; Lower alkylated arylcarbonyl group such as 4,6-trimethylbenzoyl group, 4-toluoyl group; lower alkoxylated arylcarbonyl group such as 4-anisoyl group; 2-carboxybenzoyl group, 3-carboxybenzoyl group, 4 -Carboxylated arylcarbonyl group such as -carboxybenzoyl group; 4-nitrobenzoyl group, nitrated arylcarbonyl group such as 2-nitrobenzoyl group; lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl group Group; 4-phenyl And arylated arylcarbonyl groups such as benzoyl group. Preferably, it is formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group or benzoyl group.

In the present specification, examples of the term "silyl group" include trimethylsilyl group, triethylsilyl group, isopropyldimethylsilyl group, t-butyldimethylsilyl group, methyldiisopropylsilyl group, methyldi-t-butylsilyl group, triisopropylsilyl group Tri lower alkylsilyl groups such as: diphenylmethyl silyl group, butyl diphenyl butyl silyl group, diphenyl isopropyl silyl group, tri lower alkyl silyl group substituted by 1 to 2 aryl groups such as phenyl diisopropyl silyl group, etc. It can be mentioned. Preferably, it is a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, and more preferably a trimethylsilyl group.

In the present specification, the term "halogen atom" includes, for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. Preferably, it is a fluorine atom or a chlorine atom.

As used herein, the terms "amino group protecting group for nucleic acid synthesis", "hydroxy acid protecting group for nucleic acid synthesis", "hydroxy group protected with nucleic acid synthesis protecting group", "nucleic acid synthesis protecting group protected" The “protecting group” of the “phosphate group” and the “mercapto group protected by a protecting group for nucleic acid synthesis” respectively stably protects an amino group, a hydroxyl group, a phosphate group or a mercapto group during nucleic acid synthesis. There is no particular limitation as long as it can be obtained. Specifically, it refers to a protecting group that is stable under acidic or neutral conditions and is cleavable by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis. As such a protective group, for example, lower alkyl group, lower alkenyl group, acyl group, tetrahydropyranyl group or tetrahydrothiopyranyl group, tetrahydrofuranyl group or tetrahydrothiofuranyl group, silyl group, lower alkoxymethyl group, Lower alkoxyated lower alkoxy methyl group, halogeno lower alkoxy methyl group, lower alkoxyated ethyl group, halogenated ethyl group, methyl group substituted with 1 to 3 aryl groups, “lower alkyl group, lower alkoxy group, halogen atom Or a methyl group substituted by 1 to 3 aryl groups wherein the aryl ring is substituted by a cyano group, a lower alkoxycarbonyl group, an "aryl group substituted by a halogen atom, a lower alkoxy group or a nitro group", a halogen Substituted with an atom or tri-lower alkylsilyl group Lower alkoxycarbonyl group, alkenyloxycarbonyl group, “aralkyloxycarbonyl group wherein the aryl ring may be substituted with lower alkoxy or nitro group”, “lower alkoxycarbonyl group substituted with cyano group”, “1 to And benzenesulfonyl group substituted by 4 nitro groups.

More specifically, as a tetrahydropyranyl group or a tetrahydrothiopyranyl group, tetrahydropyran-2-yl group, 3-bromotetrahydropyran-2-yl group, 4-methoxytetrahydropyran-4-yl group, tetrahydro Thiopyran-4-yl group, 4-methoxytetrahydrothiopyran-4-yl group and the like can be mentioned. Examples of the tetrahydrofuranyl group or the tetrahydrothiofuranyl group include tetrahydrofuran-2-yl group and tetrahydrothiofuran-2-yl group. Examples of lower alkoxymethyl groups include methoxymethyl group, 1,1-dimethyl-1-methoxymethyl group, ethoxymethyl group, propoxymethyl group, isopropoxymethyl group, butoxymethyl group, t-butoxymethyl group and the like. The lower alkoxylated lower alkoxymethyl group includes a 2-methoxyethoxymethyl group and the like. As the halogeno lower alkoxymethyl group, a 2,2,2-trichloroethoxymethyl group, a bis (2-chloroethoxy) methyl group and the like can be mentioned. As the lower alkoxylated ethyl group, 1-ethoxyethyl group, 1- (isopropoxy) ethyl group and the like can be mentioned. Examples of the halogenated ethyl group include 2,2,2-trichloroethyl group and the like. Examples of the methyl group substituted by 1 to 3 aryl groups include benzyl group, α-naphthylmethyl group, β-naphthylmethyl group, diphenylmethyl group, triphenylmethyl group, α-naphthyldiphenylmethyl group, 9-an Thryl methyl group etc. are mentioned. Examples of “a lower alkyl group, a lower alkoxy group, a methyl group substituted by one to three aryl groups in which an aryl ring is substituted by a halogen atom or a cyano group” include a 4-methylbenzyl group, 2,4,6- Trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl And 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl and the like. The lower alkoxycarbonyl group includes a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl group, an isobutoxycarbonyl group and the like. As the "aryl group substituted with a halogen atom, lower alkoxy group or nitro group", 4-chlorophenyl group, 2-fluorophenyl group, 4-methoxyphenyl group, 4-nitrophenyl group, 2,4-dinitrophenyl group Etc. Examples of the "lower alkoxycarbonyl group substituted with a halogen atom or a trilower alkylsilyl group" include a 2,2,2-trichloroethoxycarbonyl group, a 2-trimethylsilylethoxycarbonyl group and the like. Examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group and an aryloxycarbonyl group. Examples of “aralkyloxycarbonyl group in which the aryl ring may be substituted with lower alkoxy or nitro group” include benzyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 3,4-dimethoxybenzyloxycarbonyl group, 2-nitro Examples thereof include benzyloxycarbonyl group and 4-nitrobenzyloxycarbonyl group. Examples of the "lower alkoxycarbonyl group substituted with cyano group" include cyanoethoxycarbonyl group and the like. Examples of the “benzenesulfonyl group substituted with 1 to 4 nitro groups” include 2-nitrobenzenesulfonyl group, 2,4-dinitrobenzenesulfonyl group and the like.

As the “hydroxy group-protecting group for nucleic acid synthesis”, an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, “lower alkyl, lower alkoxy, halogen, cyano,” are preferably used. Group is a methyl group substituted with one to three aryl groups, in which the aryl ring is substituted, or a silyl group, and more preferably an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzoyl group, a dimethoxy group It is a trityl group, a monomethoxytrityl group or a tert-butyldiphenylsilyl group. As the protective group for the “hydroxyl group protected with a protective group for nucleic acid synthesis”, an aliphatic acyl group, an aromatic acyl group, “a methyl group substituted with 1 to 3 aryl groups”, and “halogen” are preferably used. An aryl group substituted with an atom, a lower alkoxy group or a nitro group, a lower alkyl group or a lower alkenyl group, and more preferably a benzoyl group, a benzyl group, a 2-chlorophenyl group, a 4-chlorophenyl group or 2- It is a propenyl group. The “amino group-protecting group for nucleic acid synthesis” is preferably an acyl group, more preferably a benzoyl group. The "protecting group" of the "phosphate group protected by a protecting group for nucleic acid synthesis" is preferably a lower alkyl group, a lower alkyl group substituted with a cyano group, an aralkyl group, a "nitro group or a halogen atom An aralkyl group substituted with an aryl ring or an aryl group substituted with a lower alkyl group, a halogen atom, or a nitro group, more preferably a 2-cyanoethyl group or a 2,2,2-trichloroethyl group , Benzyl group, 2-chlorophenyl group or 4-chlorophenyl group. There may be one or more protecting groups that constitute "a phosphate group protected with a nucleic acid synthesis protecting group". The "protecting group" of the "mercapto group protected by a protecting group for nucleic acid synthesis" is preferably an aliphatic acyl group or an aromatic acyl group, and more preferably a benzoyl group.

As used herein, the term "nucleoside" refers to a "nucleoside" in which a purine or pyrimidine base is linked to a sugar, and an aromatic heterocyclic ring and aromatic hydrocarbon ring other than purine and pyrimidine substitute for purine or pyrimidine base And “sugar” linked “nucleoside”. Natural type nucleosides are also referred to as "natural nucleosides". The modified non-naturally occurring nucleoside is also referred to as "modified nucleoside", and in particular, the sugar moiety-modified nucleotide is referred to as "sugar modified nucleoside". "Nucleotide" means a compound in which a phosphate group is linked to a sugar of a nucleoside.

As used herein, the term "oligonucleotide" is a polymer of "nucleotides" in which the same or different "nucleosides" are linked by 2 to 50 phosphodiester bonds or other linkages, both naturally occurring and non-naturally occurring. Including the type thing. As the non-naturally occurring "oligonucleotide", preferably, a sugar derivative having a sugar moiety modified; a thioate derivative having a thiophosphate-like thioether moiety; an ester having an esterified phosphate moiety at its terminal; The amide form which the amino group on the base was amidated is mentioned, The sugar derivative in which the sugar part was modified is mentioned more suitably.

As used herein, the term "antisense oligonucleotide" (AON) refers to an oligonucleotide complementary to a target gene mRNA, pre-mRNA or ncRNA (non-coding RNA), single-stranded DNA, Composed of RNA and / or their analogues. The action of mRNA, pre-mRNA or ncRNA is suppressed by forming a double strand with the mRNA, pre-mRNA or ncRNA targeted by the antisense oligonucleotide. "Antisense oligonucleotides" include those which are perfectly complementary to the target mRNA, pre-mRNA or ncRNA. In addition, as long as they can bind to mRNA, pre-mRNA or ncRNA and suppress their function, those containing one or several mismatches and those containing a base forming a wobble base pair are also included. By DNA or RNA analogue is meant a molecule having a structure similar to DNA or RNA. For example, peptide nucleic acid (PNA) etc. are mentioned. ncRNA (non-coding RNA) is a generic term for RNA that functions without being translated into protein. For example, ribosomal RNA, transfer RNA, miRNA and the like can be mentioned.

As used herein, the term "salt thereof" refers to a salt of a compound represented by Formula (II) described below. Such salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, iron salts, zinc salts, copper salts, Nickel salts, metal salts such as cobalt salts; inorganic salts such as ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts , Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (Hydroki Amine salts such as methyl) aminomethane salt; Hydrohalic acid salts such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide; Nitrate, perchlorate, sulfate, phosphorus Inorganic acid salts such as acid salts; lower alkanesulphonates such as methanesulphonate, trifluoromethanesulphonate and ethanesulphonate; arylsulphonates such as benzenesulphonate and p-toluenesulphonate Organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates and maleates; and glycine salts, lysine salts, arginine salts, ornithine salts, Amino acid salts such as glutamate and aspartate are included.

As used herein, the term "a pharmacologically acceptable salt thereof" refers to a salt of an oligonucleotide containing at least one nucleoside structure represented by formula (I) of the present invention Physiologically and pharmaceutically acceptable salts of nucleotides, that is, salts that retain the desired biological activity of the oligonucleotide and do not produce undesired toxicological effects there. Such salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, iron salts, zinc salts, copper salts, Nickel salts, metal salts such as cobalt salts; inorganic salts such as ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts , Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (Hydroki Amine salts such as methyl) aminomethane salt; Hydrohalic acid salts such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide; Nitrate, perchlorate, sulfate, phosphorus Inorganic acid salts such as acid salts; lower alkanesulphonates such as methanesulphonate, trifluoromethanesulphonate and ethanesulphonate; arylsulphonates such as benzenesulphonate and p-toluenesulphonate Organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates and maleates; and glycine salts, lysine salts, arginine salts, ornithine salts, Amino acid salts such as glutamate and aspartate are included.

Hereinafter, the present invention will be described in detail.

The oligonucleotides of the present invention include oligonucleotides in which naturally occurring DNA or RNA is chemically modified. Such modifications alter the activity of the oligonucleotide. For example, it increases the affinity for the target nucleic acid, increases the resistance to nucleolytic enzymes (nucleases), and alters the pharmacokinetics or tissue distribution of the oligonucleotide. Increasing the affinity of the oligonucleotide for its target may allow the use of shorter oligonucleotides.

The present invention includes oligonucleotides as described below and their pharmacologically acceptable salts.

The oligonucleotide of the present invention contains at least one sugar-modified nucleoside at any position. The sugar modified nucleoside has a predetermined bridge between the 2 and 4 positions of the sugar ring. The sugar modified nucleoside in the present invention is described below.

The sugar-modified nucleoside in the present invention is a nucleoside structure represented by the following formula (I):

here,
Base may be a purin-9-yl group which may have one or more optional substituents selected from α group, or may have one or more optional substituents selected from α group 2 -Oxo-1,2-dihydropyrimidin-1-yl group, wherein the α group is a hydroxyl group, a hydroxyl group protected by a protective group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, carbon A linear alkoxy group having a number of 1 to 6, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms , An amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom,
A is the following:

And a divalent group represented by
R ¹ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring, selected from the α group The aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a hetero atom, and has one or more optional substituents selected from the α group An aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may be and contain a hetero atom, or a protecting group of an amino group for nucleic acid synthesis;
R ² and R ³ are each independently a hydrogen atom; may be substituted with a C 3-12 aryl group which may contain a hetero atom, and may form a branch or a ring An alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom; or R ² and R ³ are taken together to form — (CH 2 ₂ ) _q- [wherein, q is an integer of 2 to 5];
R ⁴ and R ⁵ are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or, R ⁴ and R ⁵ together are CC (R ¹¹ ) R ¹² [wherein, R ¹¹ and R ¹² each independently represent a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a nucleic acid synthesis protecting group, a mercapto group, a mercapto group protected with a nucleic acid synthesis protecting group An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms Chain or branched alkyl amino Represents a group];
R ⁶ and R ⁷ each independently represent a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, or 1 to 7 carbons which may form a branch or a ring An alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms;
R ⁸ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, or 1 Represents 6 linear or branched alkylthio groups;
R ⁹ represents a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, an amino group, Or an amino group protected by a protecting group for nucleic acid synthesis;
R ¹⁰ is a hydrogen atom or a guanidino group;
R ¹³ and R ¹⁴ each independently represent a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis;
m is an integer of 0 to 2;
n is an integer of 0 to 1;
When R ¹⁰ is a hydrogen atom, p is 1; when R ¹⁰ is a guanidino group, p is 0;
X is an oxygen atom, a sulfur atom, or an amino group; and Y is an oxygen atom or a sulfur atom.

In one embodiment, the nucleoside structure represented by formula (I) above is

Is a structure represented by Base, R ¹ , X, m and n in the formulas (I ′) and (I ′ ′) are as described above.

In the formulas (I ′) and (I ′ ′), R ¹ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, or a carbon number which may form a branch or a ring 2 to 7 alkenyl group, aryl group having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and may contain a hetero atom, or the α group It is an aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may have one or more optional substituents selected and may contain a hetero atom, more preferably R ¹ is hydrogen It is an atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group or a benzyl group, and more preferably, R ¹ is a hydrogen atom or a methyl group.

In formula (I ′), m is an integer of 0 to 2; and in formula (I ′ ′), n is an integer of 0 to 1. That is, 2 ′ position, 3 ′ position, 4 ′ position And the ring containing the bridge portion is a 5- to 7-membered ring.

In the formula (I ′ ′), X is an oxygen atom, a sulfur atom, an amino group or a methylene group. Preferably, X is an oxygen atom or an amino group. In some cases, it may be substituted by a lower alkyl group.

In one embodiment, the nucleoside structure represented by Formula (I) above is a structure represented by Formula (I ') above, and in this Formula (I'), m is 0, and R is ¹ is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group or a benzyl group. Such a nucleoside structure is also referred to as an amide bridged nucleic acid, an amide BNA (Bridged Nucleic Acid), or AmNA.

In the compounds represented by the formulas (I ′) and (I ′ ′), an amide bond is formed between the amino group at the 2 ′ position of the sugar moiety and the carbonyl group extended from the 4 ′ position. Thus, the structure of the sugar moiety of the nucleoside is immobilized by crosslinking because it has an amide bond with less structural fluctuation and excellent hydrophilicity.

As the nucleoside structure represented by the above formula (I), in addition to the above formulas (I ′) and (I ′ ′), for example, the following may be mentioned:

In each of the above formulas, Base, R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹³ , R ¹⁴ , p and X are as described above. Here, when R ¹³ and R ¹⁴ are both hydrogen atoms, they correspond to a nucleoside structure called 2 ′, 4′-BNA or LNA (Locked Nucleic Acid).

The above "Base" is a purine base (ie, a purine-9-yl group) or a pyrimidine base (ie, a 2-oxo-1,2-dihydropyrimidin-1-yl group). These bases include a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group and 1 to 6 carbon atoms. It may have one or more optional substituents selected from the linear alkylamino group of 6 and the α group consisting of halogen atoms.

Specific examples of the above-mentioned "Base" include adenynyl group, guaninyl group, sitocynyl group, urasilyl group, and thyminyl group, and 6-aminopurin-9-yl group, 2,6-diaminopurin-9-yl group, 2 -Amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurine- 9-yl group, 6-amino-2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2, 6 -Dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group , 4-a No-2-oxo-5-fluoro-1,2-dihydropyrimidin-1-yl group, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl group, 2-oxo- 4-methoxy-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-hydroxy-1,2-dihydropyrimidine -1-yl group, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group, and 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidine- 1-yl group is mentioned.

Among them, “Base” has the following structural formula from the viewpoint of introduction to nucleic acid medicine:

And 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidine, each of which is represented by the following groups (ie, thyminyl group, sitocynyl group, adenynyl group, guaninyl group, 5-methylsitocinyl group and urasilyl group); 1-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group, 6-aminopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group, 4 -Amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl and 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl are preferred, in particular The 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group and the thyminyl group are preferred. Further, at the time of synthesis of the oligonucleotide, it is preferable that the hydroxyl group and the amino group be protected by a protecting group.

The oligonucleotide containing at least one sugar-modified nucleoside structure as described above can be obtained, for example, by using a sugar-modified nucleoside compound, for example, WO 2011/052436, JP 2014-043462 and WO 2014 /. It can be synthesized using the method as described in US Pat.

As an example of the sugar modified nucleoside compound, a compound represented by the following formula (II) or a salt thereof:

(here,
Base may be a purin-9-yl group which may have one or more optional substituents selected from α group, or may have one or more optional substituents selected from α group 2 -Oxo-1,2-dihydropyrimidin-1-yl group, wherein the α group is a hydroxyl group, a hydroxyl group protected by a protective group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, carbon A linear alkoxy group having a number of 1 to 6, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms , An amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom,
As A, the following:

And a divalent group represented by
R ¹ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring, selected from the α group The aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a hetero atom, and has one or more optional substituents selected from the α group An aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may be and contain a hetero atom, or a protecting group of an amino group for nucleic acid synthesis;
R ² and R ³ are each independently a hydrogen atom; may be substituted with a C 3-12 aryl group which may contain a hetero atom, and may form a branch or a ring An alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom; or R ² and R ³ are taken together to form — (CH 2 ₂ ) _q- [wherein, q is an integer of 2 to 5];
R ⁴ and R ⁵ are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or, R ⁴ and R ⁵ together are CC (R ¹¹ ) R ¹² [wherein, R ¹¹ and R ¹² each independently represent a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a nucleic acid synthesis protecting group, a mercapto group, a mercapto group protected with a nucleic acid synthesis protecting group An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms Chain or branched alkyl amino Represents a group];
R ⁶ and R ⁷ each independently represent a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, or 1 to 7 carbons which may form a branch or a ring An alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms;
R ⁸ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, or 1 Represents 6 linear or branched alkylthio groups;
R ⁹ represents a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, an amino group, Or an amino group protected by a protecting group for nucleic acid synthesis;
R ¹⁰ is a hydrogen atom or a guanidino group;
R ¹³ and R ¹⁴ each independently represent a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis;
m is an integer of 0 to 2;
n is an integer of 0 to 1;
When R ¹⁰ is a hydrogen atom, p is 1; when R ¹⁰ is a guanidino group, p is 0;
X is an oxygen atom, a sulfur atom, or an amino group; and Y is an oxygen atom or a sulfur atom,
Can be mentioned.

Sugar modified nucleotides can be readily prepared from sugar modified nucleosides as described above. For example, triphosphorylation can be easily carried out according to the method described in M. Kuwahara et al., Nucleic Acids Res., 2008, vol. 36, No. 13, pp. 4257-65.

The oligonucleotides of the invention can be linked to the α-synuclein gene. In the present specification, “binding of the oligonucleotide of the present invention to the α-synuclein gene” refers to direct binding of the oligonucleotide of the present invention to the α-synuclein gene, and to the mRNA of the α-synuclein gene of the oligonucleotide of the present invention. And binding of the oligonucleotide of the present invention to the pre-mRNA of the α-synuclein gene.

Here, "capable of binding" means that different single-stranded oligonucleotides or nucleic acids can form a double-stranded or more-stranded nucleic acid by the complementarity of nucleobases. Preferably, it means that double-stranded nucleic acid can be formed. The melting temperature (T _m ) of the nucleic acid of two or more strands, which is an indicator of the thermal stability of binding, is not particularly limited. Double-stranded nucleic acid melting temperature (T _m) may be determined, for example, as follows: buffer _{(8.1mM Na 2 HPO 4, 2.68mM} KCl, 1.47mM KH 2 PO 4, pH7.2 The oligonucleotide and the target RNA are mixed equimolarly, heated at 95 ° C. for 5 minutes, and gradually cooled to room temperature for annealing to form a double stranded nucleic acid. The temperature of double-stranded nucleic acid is heated from 20 ° C. to 95 ° C. at a rate of 0.5 ° C./min, and the change of absorbance (A) at 260 nm with temperature (T) is measured. / dT draw a graph of vs T, the value of dA / dT is the largest becomes the temperature, i.e. the temperature change by T of a is maximized, and the T _m of a double-stranded nucleic acid. The melting temperature (T _m ) is, for example, 40 ° C. or more, preferably 50 ° C. or more.

The oligonucleotide of the present invention is complementary to the α-synuclein gene, but does not need to have perfect complementarity, and may have a mismatch or a wobble base pair. For example, the oligonucleotides of the present invention and the α-synuclein gene do not need to have complete complementarity in the base sequence of the region forming the double strand, and can form a double-stranded nucleic acid, and the expression suppression effect As long as it has, it may have one or several mismatches. One or several mismatches may depend on the length of the oligonucleotide, but mean 1 to 4, preferably 1 to 3, and more preferably 1 or 2 mismatches. The oligonucleotide of the present invention is preferably one having complete (100%) complementarity to the base sequence of the region forming a double strand.

Examples of the α-synuclein (SNCA) gene which is a target gene of the oligonucleotide of the present invention include, but are not limited to, human SNCA (“hSNCA”) gene, mouse SNCA (“mSNCA”) gene and the like.

Α-synuclein ("SNCA") is a protein consisting of 140 amino acid residues and is an amyloid protein having no unique native structure. Alpha-synuclein is associated with synaptic vesicle accumulation and release. The DNA sequence (base sequence) of the coding region (GenBank accession number: NM — 000345) of human SNCA (hSNCA) is set forth in SEQ ID NO: 1 and the amino acid sequence is set forth in SEQ ID NO: 2 in the sequence listing. The "SNCA" in the present invention is not limited to the sequence of SEQ ID NO: 2, and as long as the function of the protein of SEQ ID NO: 2 is maintained, the number of mutations of amino acids or DNA and the mutation site are not limited. .

The oligonucleotide of the present invention has an activity of suppressing the expression of the α-synuclein gene. The SNCA expression suppression activity (knockdown activity) can be measured by a known method. For example, it is determined by the method of transfection of antisense oligonucleotide (ASO) to HEK293T cells described below (Examples 3 to 5) or intracerebroventricular administration of α-synuclein transgenic mouse (SNCA Tg mouse) (Example 6) be able to.

The oligonucleotide of the present invention is, for example, 12 to 20 bases in length, preferably 13 to 20 bases in length, more preferably 14 to 20 bases in length, still more preferably 15 to 19 bases in length, particularly preferably 15 to 18 bases in length. is there. More specifically, oligonucleotides such as 15 bases, 16 bases, 17 bases and the like can be mentioned. By the oligonucleotide being as described above, binding to the SNCA target gene, binding of the SNCA target gene to mRNA or mRNA precursor, and SNCA expression suppression (knockdown) can be performed more effectively.

In one embodiment, the oligonucleotide of the present invention can bind to a target region consisting of the nucleotide sequence of positions 99 to 123 of SEQ ID NO: 1. The target region is a region associated with human SNCA gene, in particular, an activity to suppress the expression of α-synuclein gene or knockdown activity.

In the present invention, the “target region” is a region on the SNCA gene to be targeted (eg, a target region consisting of the indicated nucleotide sequence (eg, the nucleotide sequence of positions 99 to 123 of SEQ ID NO: 1)) It includes the region on the mRNA or pre-mRNA of the SNCA gene corresponding to the region on the gene. In addition, “binding to a target region” does not necessarily form two or more strands (preferably a double strand) with the entire target region, but a region that is a part of the region and two or more strands ( Preferably, it may form a double strand). The oligonucleotide of the present invention is, for example, complementary to at least a part of the target region, and preferably has complete complementarity. Here, “a part” is a region of 12 to 20 bases in length in the target region. Preferably, a "partial" region such that the 3 'end of this target region is at position 118, 121 or 123 of the nucleotide sequence of SEQ ID NO: 1 can be selected as the target region. “Complementary to at least a portion of the target region” means complementary to the base of at least a portion of the target region on the SNCA gene (eg, consisting of the nucleotide sequence of positions 99 to 123 of SEQ ID NO: 1) And complementary to the base of the region on the mRNA or pre-mRNA corresponding to the at least one region.

A preferred base sequence of the oligonucleotide of the present invention is a base sequence shown in SEQ ID NO: 3 (antisense oligonucleotide for a region on mRNA corresponding to a target region consisting of the base sequence of positions 99 to 123 of SEQ ID NO: 1) The base sequence which consists of a part of is mentioned. The sequence of the antisense oligonucleotide corresponds to the number of bases constituting the antisense oligonucleotide (corresponding to the base length of the oligonucleotide), in the direction from 3 'to 5' (3 'to 5'), in SEQ ID NO: 1 It can be designed to align the bases complementary to the bases of the indicated target region. When the base sequence of the antisense oligonucleotide is represented in the 5 'to 3' direction (5 'to 3'), it can be reverse complement to the base sequence of the target region shown in SEQ ID NO: 1. The oligonucleotide of the present invention may have one or several bases deleted, substituted, added or inserted as long as it has SNCA gene expression suppression activity in these sequences. Preferably, 1 to 3, more preferably 1 to 2 and even more preferably 1 base may be deleted, substituted, added or inserted.

As an oligonucleotide according to the present invention or a pharmacologically acceptable salt thereof, for example,
40, 42 to 44, 72, 74 to 78, 82, 91, 97, 210, 215, 216, 227, 229, 232 to 234, 254 to 256, 262, 263, 266 to 269, 272 to SEQ ID NO: 1 A nucleotide complementary to any one nucleotide selected from the group consisting of positions 275, 277 to 279, 281 to 289, 321, 367 to 369, and 412 to 415 is a 5 'end, at least one of SEQ ID NO: 1 Complementary to the part and 13 to 15 bases long;
42 to 44, 72, 74 to 78, 82, 91, 97, 210, 216, 229, 232, 233, 254 to 256, 262, 263 to 266, 269 to 272, 275 to 278, 279, SEQ ID NO: 1 A nucleotide complementary to any one nucleotide selected from the group consisting of positions 281 to 289, 321, 367 to 369, and 413 to 415 is a 5 ′ end, which is complementary to at least a part of SEQ ID NO: 1, 13 to 15 bases long; and
43, 44, 72, 74 to 78, 82, 91, 97, 210, 216, 229, 232, 254, 256, 262, 263 to 266, 269 to 272, 275, 278, 279, SEQ ID NO: 1 A nucleotide complementary to any one nucleotide selected from the group consisting of 281 to 284, 286 to 289, 321, 367 to 369, and 413 to 415 is a 5 'end, and at least a part of SEQ ID NO: 1 Those which are complementary and 15 bases in length can be mentioned as preferred.
These oligonucleotides or their pharmacologically acceptable salts have particularly high affinity to the α-synuclein gene, and exhibit excellent α-synuclein gene expression inhibitory action.

As a preferred oligonucleotide of the present invention, the 5 'end thereof is the first (corresponding to position 123 of SEQ ID NO: 1), the third (corresponding to position 121 of SEQ ID NO: 1), or the sixth (SEQ ID NO: 1). And oligonucleotides which are bases corresponding to position 118 of SEQ ID NO: 1). For example, SEQ ID NO: 4 (15 bases long), SEQ ID NO: 5 (14 bases long), SEQ ID NO: 6 (16 bases long) and SEQ ID NO: 7 (20 bases long) (position 121); SEQ ID NO: 8 (15 bases long) And at least one sugar-modified nucleoside in the sequence of SEQ ID NO: 9 (length of 15 bases) (position 123) (the position number in parentheses indicates the target in the nucleotide sequence of SEQ ID NO: 1) This is the position of the 3 'terminal base of the region, and the base sequence is shown in the sequence listing without distinguishing between the natural nucleoside and the modified nucleoside. For example, the oligonucleotide of SEQ ID NO: 4 has a 5 'end of the third base of SEQ ID NO: 3 and a length of 15 bases. Moreover, the oligonucleotide of SEQ ID NO: 4 is 5'-GTGTTCTCTATGTAG-3 '(SEQ ID NO: 10) which is a region of 107-121, which is a region of 15 bases with a 3' end at position 121 of SEQ ID NO: 1 The target region is an antisense oligonucleotide which is complementary to and can bind to the corresponding region on mRNA. In these sequences, one or several bases may be deleted, substituted, added or inserted as long as it has SNCA expression suppression activity. Preferably, 1 to 3, more preferably 1 to 2 and even more preferably 1 base may be deleted, substituted, added or inserted.

Any nucleotide modification known in the art other than the above sugar modification can be used for the oligonucleotide of the present invention. As modification of nucleotides, phosphate modification and nucleobase modification are known. Such nucleic acid modifications can be performed based on methods known in the art.

Examples of phosphate modification include phosphodiester bond possessed by natural nucleic acid, S-oligo (phosphorothioate), D-oligo (phosphodiester), M-oligo (methyl phosphonate), boranophosphate and the like . S-oligo (phosphorothioate) has a PS skeleton in which the oxygen atom of the phosphate moiety of the phosphodiester bond between nucleosides is substituted with a sulfur atom. This modification is incorporated into the oligonucleotide according to known methods. This modification is referred to as S-oligo type (phosphorothioate type) antisense oligonucleotide having one or more in the oligonucleotide.

Examples of nucleobase modifications include 5-methylcytosine, 5-hydroxymethylcytosine, 5-propynylcytosine and the like.

In the oligonucleotide of the present invention, the position and the number of sugar-modified nucleosides are not particularly limited, and may be appropriately designed depending on the purpose. The two or more sugar modified nucleosides may be the same as or different from one another.

The oligonucleotide of the present invention is preferably a gapmer. A gapmer is an oligonucleotide comprising a "gap" as a central region and regions on both sides of the gap, two wings, ie, a "5 'wing" at the 5' side and a "3 'wing" at the 3' side. means.

The gap region of the gapmer in the present invention may be 6 to 10 bases long, preferably 7 to 10 bases long, more preferably 7 to 9 bases long, still more preferably 8 to 9 bases long, particularly preferably 9 bases long . The gap is composed of natural nucleosides.

The wing region of the gapmer in the present invention may be 3 to 5 bases in length, preferably 3 to 4 bases in length, more preferably 3 bases in length. The oligonucleotide of the present invention contains at least one sugar-modified nucleoside in the "5 'wing" and / or the "3' wing". Preferably, the "5 'wing" contains at least one sugar-modified nucleoside, preferably 1 to 5, more preferably 2 to 4, more preferably 2 to 3, particularly preferably 3. Preferably, the “3 ′ wing” contains at least one sugar-modified nucleoside, preferably 1 to 5, more preferably 2 to 4, more preferably 2 to 3, particularly preferably 2.

In one embodiment, the gap region is comprised between the 5 'wing and the 3' wing, consisting of a gap region of 6 to 10 bases, a 5 'wing of 3 to 5 bases and a 3' wing of 3 to 5 bases, The 5 'wing and the 3' wing may comprise at least one nucleoside structure represented by formula (I) above. Furthermore, phosphate modification, base modification and the like may be included. The type, number and location of modifications in one wing may be the same or different from the type, number and location of modifications in the other wing.

In one preferred embodiment, the gap region of 7 to 9 bases, the 5 'wing of 3 bases and the 3' wing of 3 bases, wherein the 5 'wing and the 3' wing each have at least two of the above formula (I) It may contain the depicted nucleoside structure.

In a more preferred embodiment, it comprises a gap region of 9 bases, a 5 'wing of 3 bases and a 3' wing of 3 bases, wherein 3 bases of the 5 'wing are nucleosides represented by the above formula (I), 3' Of the three bases of the wing, two bases may include a nucleoside structure represented by the above formula (I).

Examples of such gapmers include 3-9-2-1, 3-8-2-1, 3-10-2-1, 3-10-3, 5-10-5, and the like. Here, in the notation of "ABC" or "ABCD", "A" indicates the number of bases in the 5 'wing, "B" indicates the number of bases in the gap, "C" “Indicates the number of sugar-modified nucleosides among the bases forming the 3 ′ wing, and“ D ”indicates the number of natural nucleosides among the bases forming the 3 ′ wing. For example, in the case of 3-9-2-1, 9 bases of the gap are natural nucleosides (DNA), the 5 'wing (3 bases from the 5' end) is a sugar-modified nucleoside, and the 3 'wing ( Of the three bases from the 3 'end, two bases from the central side are sugar modified nucleosides, and the last one base (3' terminal base) is a natural nucleoside (DNA). Although depending on the sequence, 3-9-2-1 is preferred.

The oligonucleotides of the present invention include, for example, (position 121) SEQ ID NO: 11 (3-9-2-1), SEQ ID NO: 12 (3-8-2-1), SEQ ID NO: 13 (3-10-3, 3-10-2-1), and SEQ ID NO: 14 (5-10-5); (position 118) SEQ ID NO: 15 (3-9-2-1); and (position 123) SEQ ID NO: 16 (3-9) (The position number in parentheses is the position of the 3 'terminal base of the target region in the base sequence of SEQ ID NO: 1, and indicates the region of the capmer together). In these sequences, one or several bases may be deleted, substituted, added or inserted as long as it has SNCA expression suppression activity. Preferably, 1 to 3, more preferably 1 to 2 and even more preferably 1 base may be deleted, substituted, added or inserted.

The oligonucleotide of the present invention can be synthesized by a conventional method using a sugar-modified nucleoside as described above and a natural nucleoside, for example, a commercially available automatic nucleic acid synthesizer (eg, Applied Biosystems, Gene Design, Inc.) Can be easily synthesized. The synthesis methods include solid phase synthesis methods using phosphoroamidites, solid phase synthesis methods using hydrogen phosphonates, and the like. For example, it is disclosed in Tetrahedron Letters, 1981, vol. 22. pp. 1859-1862, WO 2011/052436 and the like.

The present invention also encompasses an α-synuclein expression inhibitor containing the oligonucleotide of the present invention. In the present disclosure, the “alpha-synuclein expression inhibitor” inhibits biosynthesis of alpha-synuclein by binding to the alpha-synuclein gene and suppressing the expression of the alpha-synuclein gene. The invention further encompasses pharmaceutical compositions containing the oligonucleotides of the invention. Any administration method and formulation known in the art can be used as the administration method and formulation of the α-synuclein expression inhibitor or pharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention can be administered by a variety of methods depending on the local or systemic treatment or the area to be treated. The method of administration may be, for example, topically (including eye drops, intravaginal, intrarectal, intranasal and transdermal), orally or parenterally. Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, intracerebroventricular administration and the like.

When the pharmaceutical composition of the present invention is topically administered, preparations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders and the like can be used.

Compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets and the like.

Compositions for parenteral, intrathecal or intracerebroventricular administration include sterile aqueous solutions and the like containing buffers, diluents and other suitable additives.

The pharmaceutical composition of the present invention comprises various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents and the like suitable for its dosage form in an effective amount of the oligonucleotide of the present invention. It can be obtained by mixing as needed. In the case of an injection, it may be sterilized with a suitable carrier to give a preparation.

Excipients include lactose, sucrose, glucose, starch, calcium carbonate or crystalline cellulose and the like. The binder includes methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, polyvinyl pyrrolidone and the like. Disintegrants include carboxymethylcellulose, carboxymethylcellulose sodium, starch, sodium alginate, agar powder, sodium lauryl sulfate and the like. The lubricant may, for example, be talc, magnesium stearate or macrogol. Cocoa butter, macrogol or methylcellulose can be used as a base for suppositories. In addition, when prepared as a solution or emulsion, suspension injection, a solubilizing agent, a suspending agent, an emulsifying agent, a stabilizer, a preservative, an isotonic agent, etc. which are usually used are appropriately added. You may In the case of oral administration, a flavoring agent, a fragrance and the like may be added.

The pharmaceutical composition of the present invention can be used for the treatment or prevention of diseases associated with the α-synuclein (SNCA) gene. The pharmaceutical composition of the present invention can be used, for example, for treatment or prevention based on SNCA expression suppression activity (knockdown activity). Diseases in which the pharmaceutical composition of the present invention can be used include, for example, excess α-synuclein. According to the pharmaceutical composition of the present invention, prevention of progression of neurodegeneration and prevention of development of dementia (in particular, DLB and the like) can be expected by its SNCA expression suppression activity (knockdown activity). The pharmaceutical composition of the present invention can be used, for example, for the treatment or prevention of Parkinson's disease or dementia with Lewy bodies.

The present invention provides a method for suppressing α-synuclein expression. The invention further provides methods of treating or preventing alpha synuclein excess; and methods of treating or preventing Parkinson's disease or Lewy body dementia. These methods comprise the steps of administering to an oligonucleotide individual of the invention. The "individual" is preferably a mammal, more preferably a human, a monkey, a dog, a cat, a rat and a mouse, and further preferably a human. In these methods, any administration method and dosage form can be used as long as an effective amount of the oligonucleotide of the present invention is administered. The dose effective amount depends on the individual to be administered, but can be arbitrarily determined according to the sex, age, weight, symptoms etc of the individual, and the method, route, frequency etc of the administration. For example, a dose of 0.1 to 10 mg / kg can be mentioned. The administration method and the like are as described above.

The present application claims the benefit of priority based on Japanese Patent Application No. 2017-132288 filed on July 5, 2017. The entire content of the specification of Japanese Patent Application No. 2017-132288 filed on July 5, 2017 is incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described by way of examples, but the present invention is not limited by these examples.

Example 1: Oligonucleotide Synthesis Oligonucleotides related to the present invention were synthesized by the method described in Tetrahedron Letters 22, 1859-1862 (1981), WO 2011/052436, and the like.

Specifically, regarding the oligonucleotide containing LNA represented by the formula (a), the synthesis was commissioned to Gene Design Inc.

(In the formula, Base is 5-methylsitocinyl group, thyminyl group, adenynyl group or guaninyl group.)

With respect to the oligonucleotide containing amido BNA (AmNA) represented by formula (b), it was synthesized with reference to the method described in WO 2011/025436.

(In the formula, Base is 5-methylsitocinyl group, thyminyl group, adeninyl group or guaninyl group, and Me is methyl.)

A 14-mer to 20-mer oligonucleotide containing LNA represented by formula (a) or amide BNA (AmNA) represented by formula (b) was produced using a nucleic acid automatic synthesizer ("nS-8 type" manufactured by Gene Design) And synthesized on a 0.2 μmol scale. The chain length extension is carried out using a standard phosphoroamidite protocol (solid phase carrier: CPG resin, and sulfurization using DDT (3H-1, 2-benzodithiole-3-one, 1, 1-dioxide) etc.). Then, the hydroxyl group at the terminal 5 'position was protected by a DMTr (dimethoxytrityl) group, and the 3' position was supported on a solid phase to obtain an oligonucleotide. Subsequently, after the DMTr group was removed by acid treatment, the target product was cut out from the solid phase carrier by base treatment. After neutralization with dilute acid, the solvent was distilled off, and the obtained crude product was purified by gel filtration column chromatography and reverse phase HPLC to obtain the desired product.

The crosslinked structure of LNA or AmNA used in this example, and the purity and structure of each oligonucleotide obtained were confirmed by HPLC and MALDI-TOF-MS (manufactured by BRUKER DALTONICS).

Example 2: Human Antisense Oligonucleotide Sequences Antisense oligonucleotides (AON) were designed to target the human alpha synuclein (hSNCA) gene coding region (GenBank: NM-000345 (SEQ ID NO: 1)).

The reference number ("X" of hSNCA-X) of the antisense oligonucleotide corresponds to the number of the base position corresponding to the 3 'end of the target region in SEQ ID NO: 1. For example, in the case of hSNCA-121, position 121 of the nucleotide sequence of SEQ ID NO: 1 is the 3 'end of the target region.

The sequence of the antisense oligonucleotide determines the 3 'end position of the target region based on the base sequence of SEQ ID NO: 1, and the base complementary to the base in the target region is the length of the antisense oligonucleotide It was designed to align in the direction from 3 'to 5' (3 'to 5') by the number of bases. Therefore, when the sequence of the antisense oligonucleotide is represented in the 5 'to 3' direction (5 'to 3'), it becomes a reverse complement to the target sequence represented by the base sequence of SEQ ID NO: 1. For example, when the antisense oligonucleotide is 15 mer in hSNCA-121, it is extended from position 121 to 15 bases by 5 bases of the nucleotide sequence of SEQ ID NO: 1, ie, complementary to the bases from position 121 to position 107 The sequence was designed in such a manner that the basic bases were arranged in the order described in this description. In the case of hSNCA-121 (3-9-2-1), 5'-GTGTTCTCTATGTAG-3 'at position 107 to position 121, which is a region of 15 bases long with the position 121 of the base sequence of SEQ ID NO: 1 as the 3' end. It is an antisense oligonucleotide targeted to the sequence of (SEQ ID NO: 10), and the sequence of hSNCA-121 (3-9-2-1) is 5'-CTACATAGGAACAC-3 '(SEQ ID NO: 11).

The gapmer configuration of the antisense oligonucleotide was designed to place the sugar modified nucleoside and the natural nucleoside as shown in the oligonucleotide notation. For example, in the case of hSNCA-121 (3-9-2-1), 9 bases of the gap region are natural nucleosides (DNA), the 5 'wing (3 bases from 5' end) is a sugar-modified nucleoside, and Of the 3 'wing (3 bases from the 3' end), 2 bases from the central side are sugar modified nucleosides, and the last 1 base (3 'terminal base) is a natural nucleoside (DNA). In the case of hSNCA-121 (5-10-5), the 10 bases in the central region are naturally occurring nucleosides (DNA), the 5 'wing (5 bases from the 5' end) is a sugar-modified nucleoside, and 3 The 'Wing (5 bases from the 3' end) is a sugar modified nucleoside.

Furthermore, for comparison, oligonucleotides of oligo ID 387985, 387986, and 388038 of Patent Document 1 were prepared. All of these oligonucleotides were designed as 5-10-5 AmNA modified gapmers.

In each case, the internucleoside linkage was phosphorothioate (P = S) throughout the oligonucleotide.

Example 3 Primary Screening Based on Transfection of Antisense Oligonucleotide (ASO) to HEK 293 T Cells In this example, the numbers shown in the horizontal axis of FIG. 1 (corresponding to the numbers of the base positions in SEQ ID NO: 1) An antisense oligonucleotide at the 3 'end of was designed as described in Example 2 and prepared as described in Example 1. The antisense oligonucleotide contains LNA, and the gapmer configuration is 3-8-2-1.

Details of the prepared oligonucleotides are shown in Table 1 below. A, T, c, C and G in the base sequences in Table 1 represent the following bases (except for c and C, they are common in upper and lower case letters, and the “Base” in the formula (a) is enclosed in parentheses. Groups are shown): c = cytosine (cytidinyl group); C = 5-methylcytosine (5-methylcytidinyl group); T = thymine (thyminyl group); A = adenine (adeninyl group); and G = guanine (guaninyl group) .

HEK293T cells (obtained from "ATCC CRL-1573" ATCC in 1 mL of DMEM medium (manufactured by Thermo Fisher Scientific) added with 10% FBS (without antibiotics) in a 12-well plate (manufactured by Thermo Fisher Scientific) on the previous day ) I saw 2.5 x 10 ⁵ . Three wells + control were prepared per oligonucleotide.

Prepare tube 1: 75 μL Opti-MEM (manufactured by Thermo Fisher Scientific) + 5 μL Lipofectamine 2000 (manufactured by Invitrogen) per tube, and tube 2: 75 μL Opti-MEM + 1 μL 50 μM antisense oligonucleotide per tube. And left for 5 minutes at room temperature and then added to the cells. Media was changed after 4 hours and RNA extraction was performed after approximately 24 hours.

RNA extraction was performed using Rneasy Plus Mini Kit (manufactured by Thermo Fisher Scientific) as follows: 50 μL of buffer RLT was added to each well and mixed. The mixture was transferred to a gDNA eliminater tube and centrifuged at 10000 rpm for 30 seconds. The tube column was then removed, 350 μL of 70% ethanol was added, mixed and transferred to a spin column. The mixture was centrifuged at 10000 rpm for 15 seconds, and the filtrate was discarded. Then, 700 μL buffer RW1 was added, centrifuged at 10000 rpm for 15 seconds, 500 μL buffer RPE was added, centrifuged at 10000 rpm for 15 seconds, 500 μL buffer RPE was added, and centrifuged at 10000 rpm for 120 seconds. The column was placed in an eppen tube, 40 μl of RNase-free water was added, and the mixture was centrifuged at 10000 rpm for 60 seconds.

Reverse transcription was performed using Superscript III kit (manufactured by Thermo Fisher Scientific) as follows: 1 μg of RNA was added with redistilled water (ddW) to make a total of 8 μL, and 1 μL of random hexamer primer ( Thermo Fisher Scientific) and 1 μl of 10 mM dNTP were added to make a total of 10 μl.

In the case of the extracted RNA concentration of 400 ng / μL, 2.5 μL was taken per 1 μg of RNA, and 5.5 μL of ddW was added, incubated at 65 ° C. for 5 minutes, and allowed to stand on ice.

The following Master Mix 1 was prepared: 2 μL 10 × RT buffer; 4 μL 25 mM MgCl ₂ ; 2 μL 0.1 M DTT; 1 μL RNase OUT; and 1 μL Superscript III. 10 μL of this master mix was added to the sample and incubated at 25 ° C. for 10 minutes, 50 ° C. for 50 minutes, and 85 ° C. for 5 minutes. 1 μL RNase H was added and incubated at 37 ° C. for 20 minutes. Thus, cDNA was obtained.

Quantitative PCR was performed using TaqMan® Gene Expression Assays (Applied Biosystems) as follows: The resulting cDNA was diluted 10-fold with ddW (1 μL of cDNA and 9 μL of ddW). Master Mix 2 was prepared by mixing Taqman probe mix (manufactured by Applied Biosystems): ddw: SNCA primer (TaqMan (registered trademark) Gene Expression Assays, ID: Hs01103383_m1) = 10: 7: 1. In each cDNA, 3.3 μL of diluted DNA (3.3 μL ddW for NTC) was added to 29.7 μL of Master Mix 2. The same was performed for the 18s primer ("Eukaryotic 18S rRNA Endogenous Control", Applied Biosystems). 10 μL of each was dispensed to a PCR plate (384 wells) and centrifuged at 2000 rpm for 5 minutes. Real-time PCR was performed by ABI PRISM 7900HT real-time PCR analysis system (manufactured by Applied Biosystems) to quantify the mRNA amount of SNCA.

Cells not subjected to the transfection procedure were used as a control.

The results are shown in FIG. The vertical axis in FIG. 1 indicates the amount of mRNA. The amount of mRNA after transfection of the antisense oligonucleotide was relatively represented, assuming that the amount of mRNA in the control ("Cont" in Fig. 1) was 1.0. Based on this result, in the next example, an antisense oligonucleotide in which position 121 of the base sequence of SEQ ID NO: 1 was used as the 3 'end of the target region.

Example 4: Secondary screening based on transfection of antisense oligonucleotide (ASO) to HEK293T cells Transfection of antisense oligonucleotide (ASO) to HEK293T cells is performed in the same manner as in Example 3 except for the following. The amount of mRNA was measured.

In this example, position 121 of the base sequence of SEQ ID NO: 1 is the 3 'end of the target region and contains AmNA, and the gapmer configuration is 3-10-3, 3-10-2-1, 3-9, respectively. Antisense oligonucleotides that are -2-1 and 5-10-5 were designed as described in Example 2 and prepared as described in Example 1. For comparison, the AmNA modified gapmer 5-10-5 of oligo ID 387985, 387986, and 388038 described in Example 2 was also used (oligo ID 388038 is a 4-base fragment at position 122 to 125 of SEQ ID NO: 1) Is a mismatch).

Details of the prepared oligonucleotides are shown in Table 2 below. A, T, c, C and G of the base sequences in Table 2 represent the following bases (except for c and C, they are common to upper and lower case letters, and the “Base” in the formula (a) is enclosed in parentheses. Groups are shown): c = cytosine (cytidinyl group); C = 5-methylcytosine (5-methylcytidinyl group); T = thymine (thyminyl group); A = adenine (adeninyl group); and G = guanine (guaninyl group) .
Details of the prepared oligonucleotides are shown in Table 2 below. A, T, c, C and G of the base sequences in Table 2 represent the following bases (except for c and C, they are common to upper and lower case letters, and in parentheses, “Base” in the formula (b) Groups are shown): c = cytosine (cytidinyl group); C = 5-methylcytosine (5-methylcytidinyl group); T = thymine (thyminyl group); A = adenine (adeninyl group); and G = guanine (guaninyl group) .

The results are shown in FIG. The vertical axis in FIG. 2 indicates the amount of mRNA. Assuming that the amount of mRNA in the control ("Cont" in Fig. 2) is 1.0, the amount of mRNA after transfection of the antisense oligonucleotide is relatively represented. Among the antisense oligonucleotides examined, the 3-9-2-1 gapmer showed particularly excellent α-synuclein inhibitory effect. For gapmer 5-10-5, an antisense oligonucleotide having the position 121 of the base sequence of SEQ ID NO: 1 at the 3 'end of the target region is superior to any of ID 387985, 387986, and 380383 described in Patent Document 1 Showed an α-synuclein inhibitory effect.

Example 5: Tertiary Screening Based on Transfection of Antisense Oligonucleotide (ASO) to HEK293T Cells The transfection of antisense oligonucleotide (ASO) to HEK293T cells is carried out in the same manner as in Example 3 except for the following and mRNA The amount was measured.

In this example, the antisense oligonucleotide has the number shown on the horizontal axis of FIG. 3 (corresponding to the number of the base position in SEQ ID NO: 1) as the 3 'end of the target region, containing AmNA, and the gapmer configuration Designed as described in Example 2 and prepared as described in Example 1 as being 3-9-2-1.

Details of the prepared oligonucleotides are shown in Table 3 (Tables 3-1 to 3-2) below. The base sequences a, g, c, t, A, T, C and G in Tables 3-1 to 3-2 are the same as those in Table 2.

The results are shown in FIG. The vertical axis in FIG. 3 indicates the amount of mRNA. The amount of mRNA after transfection of the antisense oligonucleotide was relatively represented, assuming that the amount of mRNA in the control ("Cont" in Fig. 3) was 1.0. Among the antisense oligonucleotides examined, an antisense oligonucleotide having the position 118, 121 and 123 of the base sequence of SEQ ID NO: 1 at the 3 'end of the target region shows particularly excellent α-synuclein expression inhibitory effect The

Example 6: Intraventricular administration of α-synuclein transgenic mice (SNCA Tg mice) Of the oligonucleotides in Table 3, positions 118, 121, and 123 of the base sequence of SEQ ID NO: 1 are used as the 3 'end of the target region. , And AmNA, and an antisense oligonucleotide having a gapmer configuration of 3-9-2-1.

Anesthesia under SNCA Tg mice (obtained from Osaka University Immunology Frontier Center Rikisei Kashiyama, Neurobiology of Aging, 2008, vol. 29, pp. 574-585) under three types of mixed anesthesia, then from the anterior apex (Bregma) A needle was inserted at a depth of 3 mm at a site 0.2 mm on the dorsal side and 1 mm on the left. The needle was once removed and reinserted after confirming the cerebrospinal fluid leakage, and 10 μL of antisense oligonucleotide (about 1.3 mM) was injected over approximately 5 minutes, then allowed to stand for 2 minutes, and removed. Then, one week after suturing and confirming anesthesia awakening, mice were sacrificed, and left and right striatum, substantia nigra and cortex were taken out of the brain and rapidly frozen in liquid nitrogen.

RNA extraction was performed using ISOGEN (manufactured by Nippon Gene Co., Ltd.) as follows: Brain tissue was ground in liquid nitrogen, dissolved in 950 μL ISOGEN, pipeted several times with a 21 G needle, and 200 μL chloroform And vortexed and centrifuged at 12000 g for 15 minutes at 4 ° C. The supernatant (aqueous layer) was transferred to another tube, 500 μl of isopropanol was added, 3 μl ethacinmate (manufactured by Nippon Gene Co., Ltd.) and 10 μl of sodium acetate were added, and the mixture was centrifuged at 12000 g for 10 minutes at 4 ° C. The supernatant was discarded, and the precipitate was washed with 500 μL 75% ethanol and centrifuged at 7500 g for 5 minutes at 4 ° C. The supernatant was discarded and the precipitate was dissolved in 30 μL ddW. Reverse transcription and quantitative PCR were performed as in Example 3.

The results are shown in FIG. FIG. 4 shows mRNA levels in the substantia nigra (A) and striatum (B) after antisense oligonucleotide administration to SNCA Tg mice. The vertical axes in FIGS. 4A and 4B indicate the amount of mRNA. The case where the antisense oligonucleotide was not administered was used as a control, and the amount of mRNA after administration of the antisense oligonucleotide was represented relative to the amount of mRNA in this control ("Cont" in Fig. 4) as 1.0.

Even when any of the antisense oligonucleotides was administered, a decrease in the amount of substantia nigra and striatal mRNA was observed as compared to the control. In this example, intracerebroventricular administration was performed in consideration of clinical application, but a decrease in the amount of mRNA was observed in the striatum. Therefore, AmNA modified oligonucleotides show excellent tissue transferability. Thus, AmNA modified oligonucleotides are also expected to be applied via intrathecal administration for delivery into the brain.

Example 7: Synthesis of 5'-TCCctccttggcCTt-3 '(AmNA-hSNCA-42, SEQ ID NO: 85) The base sequence of this compound is coding for human α-synuclein (hSNCA) gene (GenBank: NM_000345 (SEQ ID NO: 1)) Complementary to region 28-42. In the sequences, upper case letters represent AmNA and lower case letters represent naturally occurring DNA nucleosides. The linkage between all the nucleosides is a phosphorothioate linkage. "C" represents 5-methylcytosine.
Using a nucleic acid automatic synthesizer ("ABI 394 or 392 DNA / RNA Synthesizer" manufactured by Applied Biosystems), synthesis was performed using a program of 1 μmol scale. However, for condensation, 5-benzylthio-1H-tetrazole (manufactured by Wako Pure Chemical Industries, Ltd.) is reacted for 30 minutes in the case of AmNA and for 1 minute in the case of DNA, and sulfidation is 0.2 M phenylacetyll disulfide / pyridine: acetolitrile (1: 1 v / v) The solution was reacted in 10 minutes for AmNA and 2.5 minutes for DNA. Chain length extension was performed using thymidine-bound CPG (Glen Research, 0.8 μmol) as a solid phase carrier. The terminal 5'-hydroxyl group was protected with a DMTr (dimethoxytrityl) group, and the 3'-position was supported on a solid phase to obtain an oligonucleotide. Subsequently, the desired product was cleaved from the solid phase support by base treatment, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed, and the resulting mixture was concentrated. Clarity QSP DNA Loading Buffer (manufactured by Phenomenex): 900 μL of water = 1: 1 (v / v) solution was added and mixed, and charged onto Clarity 30 μ QSP (manufactured by Phenomenex). Clarity QSP DNA Loading Buffer: 1 mL of water = 1: 1 (v / v) solution, 0.1 M aqueous solution of tetrabutylammonium bromide: 2 mL of a solution of acetonitrile = 8: 2 (v / v), 3 mL of 3% aqueous solution of dichloroacetic acid (DCA) After sequentially adding 4 mL of water and 2 mL of 20 mM Tris aqueous solution, the components to be extracted were collected with a 20 mM Tris aqueous solution: acetonitrile = 9: 1 (v / v) solution. After distilling off the solvent, the desired product was obtained. The compound is reverse phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100 A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM aqueous triethylamine solution, solution B: methanol, B%: It was eluted at 2.433 minutes when analyzed at 10% to 25% (4 min, linear gradient); 60 ° C .; 0.5 mL / min; 260 nm. The compound was identified by negative ion ESI mass spectrometry (calculated: 49845.51, found: 4984.50).

Examples 8 to 52
The compounds of Examples 8 to 52 were synthesized in the same manner as in Example 7. The sequences of the compounds are shown in Table 4. In the sequences, upper case letters represent AmNA and lower case letters represent naturally occurring DNA nucleosides. The linkage between all the nucleosides is a phosphorothioate linkage. In Table 4, a, g, c, t, A, T, C and G are the same as in Table 2.

Test Example 1: Additional screening based on transfection of ASO to HEK293A cells (1)
On the day before transfection, HEK293A cells (ATCC) in 0.5 mL of DMEM medium (manufactured by Thermo Fisher Scientific) added with 10% FBS (manufactured by HyClone) using a 24-well plate (manufactured by Thermo Fisher Scientific) 5 × 10 ⁴ pieces manufactured by Co., Ltd., model number R705-07). One well was prepared for each type of ASO, and two wells were prepared for distilled deionized water (ddW: manufactured by Nacalai Tesque) as a control containing no ASO.
Using a 96-well plate (Applied Biosystems), 2.5 μL ASO (concentration 10 μM) added to 35 μL Opti-MEM (manufactured by Thermo Fisher Scientific), 37.5 μL OptiMEM and 0.9 μL Lipofectamine One to which RNAiMAX (manufactured by Invitrogen) was added was prepared as one well, left at room temperature for about 20 minutes, and then added to cells. For comparison, ISIS 387985, which is an antisense oligonucleotide against α-synuclein described in WO 2012/068405, was used. Control wells without ASO were prepared as follows. After adding 2.5 μL ddW to 35 μL Opti-MEM and further adding 0.9 μL Lipofectamine RNAiMAX to 37.5 μL OptiMEM as one well, prepare 2 wells and allow to stand at room temperature for about 20 minutes , Added to the cells. After approximately 24 hours, each well was washed twice with PBS (manufactured by Thermo Fisher Scientific).
RNA extraction was performed according to the protocol of the kit using RNeasy Mini Kit (manufactured by Qiagen). Reverse transcription reaction was performed as follows using High capacity RNA to cDNA kit (manufactured by Invitrogen). ddW was added to 0.5 μg of total RNA to make a total of 9 μL, and 1 μL of 10 × RT Enzyme and 10 μL of 2 × RT buffer were added to make a total of 20 μL. This master mix was incubated at 37 ° C. for 60 minutes and at 95 ° C. for 5 minutes using a thermal cycler (manufactured by Applied Biosystems). Thus, cDNA was obtained.
Quantitative PCR was performed using TagMan Gene Expression Assay (manufactured by Applied Biosystems) as follows. To 20 μL of the obtained cDNA, 80 μL of ddW was added to dilute 5 times. 2 × Taqman probe master mix: ddW: 5 times diluted cDNA per well of 384 PCR plate (manufactured by Applied Biosystems): hSNCA primer (“Hs01103383” manufactured by Applied Biosystems): β-Actin primer (“Hs99999903”) A total of 10 μL was prepared by mixing at a ratio of 5: 2: 2: 0.5: 0.5 (μL) manufactured by Applied Biosystems, and 2 wells were prepared for each cDNA. Real-time PCR was performed by Viia 7 (manufactured by Applied Biosystems), and the amount of SNCA mRNA was quantified as an average value between 2 wells. An average of duplicate averages of cDNAs of 2 wells transfected with ddW containing no ASO was used as a control.
The above experiment was performed four times to calculate the mean value and standard deviation (SD) between experiments. The results are shown in FIG. The vertical axis in FIG. 5 indicates the amount of mRNA. Assuming that the amount of mRNA in the control (“No ASO” in FIG. 5) was 1.0, the amount of mRNA after transfection of ASO was relatively represented. As shown in FIG. 5, ASO having the positions 283 and 369 of the base sequence of SEQ ID NO: 1 at the 3 'end of the target region showed the effect of suppressing excellent α-synuclein mRNA. These sequences showed a significantly higher mRNA suppression effect on ISIS 387985.

Test Example 2: Additional screening based on transfection of ASO to HEK293A cells (2)
As in Test Example 1, HEK293A cells were transfected with ASO, and the amount of mRNA was quantified. However, the following method was performed by changing the method of Test Example 1.
HEK293A cells were seeded at 1 × 10 ^{4 in} 0.1 mL of the same medium as in Test Example 1 using a 96-well plate the day before transfection. One well was prepared for each type of ASO, and two wells were prepared for control without ASO.
What added 9.76 μL OptiMEM and 0.24 μL Lipofectamine RNAiMAX (manufactured by Invitrogen) to 7.5 μL Opti-MEM to which 2.5 μL ASO (concentration 2 μM) was added using a 96-well plate They were made and left at room temperature for about 20 minutes before being added to cells. For the wells of ddW not containing ASO, 2 wells were prepared using ddW instead of ASO, left for about 20 minutes at room temperature, and then added to the cells. After approximately 24 hours, each well was washed twice with PBS.
RNA extraction and reverse transcription were performed according to the protocol of the kit using SuperPrep Cell Lysis RT Kit for qPCR (manufactured by Takara Bio Inc.). Thus, the obtained cDNA was diluted 3-fold with ddW. Using the diluted cDNA, the amount of hSNCA mRNA was quantified by real-time PCR as in Example 7.
The results are shown in FIG. The vertical axis in FIG. 6 indicates the amount of mRNA. The amount of mRNA in the control (“No ASO” in FIG. 6) was relatively expressed. Among the examined ASOs, 42, 72, 74, 75, 76, 77, 78, 82, 91, 97, 210, 229, 233, 254, 263, 266, 269 of the nucleotide sequence of SEQ ID NO: 1 ASO with position 3 'of the target region at positions 272, 275, 278, 281, 282, 282, 284, 285, 286, 287, 288, 289, 321, 367, 368 and 415 suppresses superior α-synuclein mRNA It showed the effect.

Test Example 3: Additional screening based on transfection of ASO to HEK293A cells (3)
As in Test Example 3, HEK293A cells were transfected with ASO, and the amount of mRNA was quantified.
The results are shown in FIG. The vertical axis in FIG. 7 indicates the amount of mRNA. The amount of mRNA after transfection with ASO was relatively expressed, assuming that the mRNA in the control (“No ASO” in FIG. 7) was 1.0. Α-synuclein mRNA excellent in ASO having positions 43, 44, 232, 255, 256, 262, 267, 268, 273, 274, 279, 413 and 414 of the nucleotide sequence of SEQ ID NO: 1 as the 3 'end of the target region Showed the effect of suppressing

Test Example 4
The compounds listed in Table 5 can be synthesized in the same manner as Example 7. As a 2′-O-Me RNA amidite reagent, a phosphoroamidite of 2′-O-Me nucleoside (adenosine product product No. ANP-575, cytidine product product No. ANP-5752, guanosine product product No. ANP-5753 , Uridine product product No. ANP-5754) uses ChemGenes. In the sequences, upper case letters represent AmNA, lower case letters represent naturally occurring DNA nucleosides, and underlines represent 2'-O-methyl RNA. The linkage between all the nucleosides is a phosphorothioate linkage. In Table 5, a, g, c, t, A, T, C and G are the same as in Table 2.

According to the present invention, an oligonucleotide useful for suppressing the expression of α-synuclein is provided. The oligonucleotide of the present invention is expected to be useful as a nucleic acid medicine useful for, for example, treatment or prevention of α-synuclein excess and treatment or prevention of Parkinson's disease, dementia with Lewy bodies, and the like.

Claims (12)

Nucleoside structure represented by the following formula (I):

An oligonucleotide comprising at least one or a pharmacologically acceptable salt thereof,
here,
Base may be a purin-9-yl group which may have one or more optional substituents selected from α group, or may have one or more optional substituents selected from α group 2 -Oxo-1,2-dihydropyrimidin-1-yl group, wherein the α group is a hydroxyl group, a hydroxyl group protected by a protective group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, carbon A linear alkoxy group having a number of 1 to 6, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms , An amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom,
A is the following:

And a divalent group represented by
R ¹ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring, selected from the α group The aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a hetero atom, and has one or more optional substituents selected from the α group An aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may be and contain a hetero atom, or a protecting group of an amino group for nucleic acid synthesis;
R ² and R ³ are each independently a hydrogen atom; may be substituted with a C 3-12 aryl group which may contain a hetero atom, and may form a branch or a ring An alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom; or R ² and R ³ are taken together to form — (CH 2 ₂ ) _q- [wherein, q is an integer of 2 to 5];
R ⁴ and R ⁵ are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or, R ⁴ and R ⁵ together are CC (R ¹¹ ) R ¹² [wherein, R ¹¹ and R ¹² each independently represent a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a nucleic acid synthesis protecting group, a mercapto group, a mercapto group protected with a nucleic acid synthesis protecting group An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms Chain or branched alkyl amino Represents a group];
R ⁶ and R ⁷ each independently represent a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, or 1 to 7 carbons which may form a branch or a ring An alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms;
R ⁸ is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, or 1 Represents 6 linear or branched alkylthio groups;
R ⁹ represents a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkoxy group having 1 to 7 carbons which may form a branch or a ring, an amino group, Or an amino group protected by a protecting group for nucleic acid synthesis;
R ¹⁰ is a hydrogen atom or a guanidino group;
R ¹³ and R ¹⁴ each independently represent a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring; 1 carbon having an carbon number which may form a branch or a ring A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis;
m is an integer of 0 to 2;
n is an integer of 0 to 1;
When R ¹⁰ is a hydrogen atom, p is 1; when R ¹⁰ is a guanidino group, p is 0;
X is an oxygen atom, a sulfur atom, or an amino group; and Y is an oxygen atom or a sulfur atom,
Here, the oligonucleotide is capable of binding to the α-synuclein gene, having an activity of suppressing the expression of the α-synuclein gene, and being complementary to the α-synuclein gene, and , 42 to 44, 72, 74 to 78, 82, 91, 97, 210, 215, 216, 227, 229, 232 to 234, 254 to 256, 262, 263, 266 to 269, 272 to 275, 277 A nucleotide complementary to any one nucleotide selected from the group consisting of positions 279, 281 to 289, 321, 367 to 369, and 412 to 415 is a 5 'end, and is complementary to at least a part of SEQ ID NO: 1 Or the pharmacologically acceptable salt thereof, wherein the length of the oligonucleotide is 12 to 20 bases The oligonucleotide is a gapmer comprising a gap region of 6 to 10 bases, a 5 'wing of 3 to 5 bases and a 3' wing of 3 to 5 bases,
The gap region is located between the 5 'wing and the 3' wing, and the 5 'wing and the 3' wing comprise at least one nucleoside structure represented by the formula (I)
The oligonucleotide according to claim 1 or a pharmacologically acceptable salt thereof. The gap region consists of 7 to 9 bases, the 5 'wing and the 3' wing each consist of 3 bases, and the 5 'wing and the 3' wing each have at least two of the formula (I) The oligonucleotide according to claim 2, comprising the depicted nucleoside structure or a pharmacologically acceptable salt thereof. The nucleoside structure represented by the formula (I) is

[Wherein, Base, R ¹ , X, m and n are as described above. ]
The oligonucleotide according to any one of claims 1 to 3, or a pharmacologically acceptable salt thereof, which has a structure represented by The nucleoside structure represented by the formula (I) is a structure represented by the formula (I ′), and in the formula (I ′), the m is 0, and the R ¹ is hydrogen The oligonucleotide according to claim 4, which is an atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, or a pharmacologically acceptable salt thereof. 40, 42 to 44, 72, 74 to 78, 82, 91, 97, 210, 215, 216, 227, 229, 232 to 234, 254 to 256, 262, 263, 266 to 269, 272 to SEQ ID NO: 1 A nucleotide complementary to any one nucleotide selected from the group consisting of positions 275, 277 to 279, 281 to 289, 321, 367 to 369, and 412 to 415 is a 5 'end, at least one of SEQ ID NO: 1 The oligonucleotide according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof, which is complementary to a part and has a length of 13 to 15 bases. 42 to 44, 72, 74 to 78, 82, 91, 97, 210, 216, 229, 232, 233, 254 to 256, 262, 263 to 266, 269 to 272, 275, 278, 279, SEQ ID NO: 1 A nucleotide complementary to any one nucleotide selected from the group consisting of positions 281 to 289, 321, 367 to 369, and 413 to 415 is a 5 'end, which is complementary to at least a part of SEQ ID NO: 1, The oligonucleotide according to any one of claims 1 to 5 having a length of 13 to 15 bases, or a pharmacologically acceptable salt thereof. 43, 44, 72, 74 to 78, 82, 91, 97, 210, 216, 229, 232, 254, 256, 262, 263 to 266, 269 to 272, 275, 278, 279, SEQ ID NO: 1 A nucleotide complementary to any one nucleotide selected from the group consisting of 281 to 284, 286 to 289, 321, 367 to 369, and 413 to 415 is a 5 'end, and at least a part of SEQ ID NO: 1 The oligonucleotide according to any one of claims 1 to 5, which is complementary and 15 bases long, or a pharmacologically acceptable salt thereof. An α-synuclein expression inhibitor comprising the oligonucleotide according to any one of claims 1 to 8 or a pharmacologically acceptable salt thereof as an active ingredient. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1 to 8 or a pharmacologically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 10, which is used for the treatment or prevention of alpha-synuclein excess. The pharmaceutical composition according to claim 10, which is used for the treatment or prevention of Parkinson's disease or dementia with Lewy bodies.

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