JP2002255990A - 2',4'-bna oligonucleotide containing n3'-p5' bond - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new bicyclo nucleotide analog(BNA) having anti-AIDS activity and an intermediate for producing an oligonucleotide analog having excellent antisense or anti-gene activity and stability in an organism. SOLUTION: This compound is represented by structural formula (1) (R<1> is the same or different hydrogen atom or hydroxy group-protecting group; R<2> is an azide group, an amino group which may be protected or the like; B is purin-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl group which may contain a substituent group selected from the group α consisting of a halogen atom, a 1-6C alkyl group, a hydroxy group, a mercapto group, an amino group or the like) or its physiologically acceptable salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel bicyclonucleoside analog for producing a non-natural oligonucleotide analog having excellent antisense or antigene activity and being stable in vivo. .

[0002] The present invention also relates to novel oligonucleotide analogs containing one or more of the bicyclonucleoside structures.

[0003] The present invention further relates to novel modified bicyclonucleoside analogs having anti-AIDS activity.

[0004]

2. Description of the Related Art Oligonucleotides having excellent antisense or antigene activity and being stable in vivo are expected as useful drugs.

[0005] However, it is known that natural oligonucleotides are rapidly degraded by various nucleases present in blood and cells.

In order to overcome these drawbacks, various non-natural oligonucleotide analogs have been produced, and attempts have been made to develop them as pharmaceuticals. For example, those in which the oxygen atom bonded to the phosphorus atom in the phosphodiester bond of the oligonucleotide is substituted with a sulfur atom, those in which the oxygen atom is substituted with a methyl group, those in which the oxygen atom is substituted with a boron atom, those of the oligonucleotide Those obtained by chemically modifying a sugar moiety or a base moiety are known.

More specifically, ISIS has developed ISIS2922, a thioate-type oligonucleotide, as a therapeutic agent for human cytomegalovirus retinitis.
Sold in the United States as VitraveneTM.

[0008] However, the strength of antisense or antigene activity of the above-mentioned non-natural type oligonucleotide analogs, that is, the ability to form a stable complementary strand with m-RNA or DNA, the stability to various nucleases, Considering the occurrence of side effects due to non-specific binding to various proteins in the living body, etc. Oligonucleotide analogs of the type are desired.

The chemical structure related to the compound of the present invention, that is, a compound having the following dioxabicyclo [2,2,1] heptane structure is described in WO 98/39352. The substituent at the 'position differs from the compound of the present application. Further, it is not known that the compound has an anti-AIDS activity.

[0010]

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In the above formula, B ⁰ is a pyrimidine or purine nucleobase or an analog thereof, and X and Y are the same or different and are hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, It is an aryl group, an acyl group or a silyl group.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel bicyclo for producing a non-naturally occurring oligonucleotide analogue which has excellent antisense or antigene activity and is stable in vivo. An object of the present invention is to provide a nucleoside analog.

Another object of the present invention is to provide a novel oligonucleotide analog containing one or more of the bicyclonucleoside structures.

Another object of the present invention is to provide a novel bicyclonucleoside analog having an anti-AIDS activity.

The inventor of the present invention has been made to solve the above problem.
As a result of intensive studies, a novel bicyclonucleoside analog having 2'-O-4'-C-methylene has excellent antisense or antigene activity and is a non-natural type which is stable in vivo. Are found to be important intermediates for the production of oligonucleotide analogs, and the novel oligonucleotide analogs containing one or more bicyclonucleoside structures have excellent antisense or antigene activity. In addition, they have found that the bicyclonucleoside analog has excellent anti-AIDS activity, and have completed the present invention.

[0016]

The novel bicyclonucleoside analogs of the present invention include: 1) a compound represented by the following general formula (1):

[0017]

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[Wherein R ¹ is the same or different and is a hydrogen atom, a hydroxyl group-protecting group for nucleic acid synthesis, a phosphate group, a phosphate group protected with a protecting group for nucleic acid synthesis, or a compound of the formula -P (R ^4a ) R ^4b
(Wherein, R ^4a and R ^4b are the same or different and each is a hydroxyl group,
Hydroxyl group, mercapto group protected with a protecting group for nucleic acid synthesis,
Mercapto group and amino group protected with a protecting group for nucleic acid synthesis, amino group protected with a protecting group for nucleic acid synthesis, carbon number 1
Or an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 7 carbon atoms, or an amino group substituted with an alkyl group having 1 to 6 carbon atoms. R ² represents an azido group, an amino group or a group represented by the formula NH—R ³ (R ³ is the same or different and is protected by an amino acid-protecting group for nucleic acid synthesis, a phosphate group, or a protecting group for nucleic acid synthesis; Or a phosphate group of the formula -P ( ^R4a ) ^R4b , wherein ^R4a
And R ⁴ b are the same or different, a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group of the nucleic acid synthesis, an amino group, is protected with a protecting group of nucleic acid synthesis Amino group, C1 to C6 alkoxy group, C1 to C6 alkylthio group, C1 to C7 cyanoalkoxy group or C1 to C6
Represents an amino group substituted with an alkyl group), represents a group represented by), and B has at least one optional substituent selected from the following α group. Represents a purine-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl group. Or a pharmacologically acceptable salt thereof. (Α group) a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 6 carbon atoms, a mercapto group,
Mercapto group protected by a protecting group for nucleic acid synthesis, 1 carbon atom
To 6 alkylthio groups, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and Halogen atom.

Among the compounds of the present invention, preferred are: 2) a methyl group in which R ¹ is substituted by a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, a silyl group, or one to three aryl groups; Or 1 to 3 in which the aryl ring is substituted by lower alkyl, lower alkoxy, halogen or cyano group
3) a compound wherein R ¹ is a hydrogen atom, a silyl group, a methyl group substituted with 1 to 3 aryl groups, or lower alkyl, lower alkoxy, halogen Or a compound which is a methyl group substituted with one to three aryl groups in which an aryl ring is substituted with a cyano group. 4) R ¹ is a hydrogen atom, a trimethylsilyl group, a t-butyldimethylsilyl group, a t-butyl Diphenylsilyl group,
Benzyl group, triphenylmethyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group,
4,4′-dimethoxytriphenylmethyl group or 4,
A compound wherein 4 ′, 4 ″ -trimethoxytriphenylmethyl group; 5) R ¹ is a compound of the formula —P (OCH ₃ ) (N (CH (CH ₃ ) ₂ )
_2), or the formula _{-P (OCH 2 CH 2 CN)} (N (CH
(CH ₃ ) ₂ ) ₂ ) ₂ ) 6) R ² is an azide group, an amino group, or a formula NH—R ³ group, wherein R ³ is an aliphatic acyl group or an aromatic acyl group , 1
Methyl group, silyl group, phosphoramidite substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a methyl group, lower alkyl, lower alkoxy, halogen or cyano group substituted with 1 to 3 aryl groups A) a group, a phosphonyl group, a phosphate group or a phosphate group protected by a protecting group for nucleic acid synthesis) 7) R ² is an azide group, an amino group, a group of the formula NH—R ³ , R ³ is an acetyl group, a trifluoroacetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group,
rt-butyldiphenylsilyl group, formula -P (OC ₂ H ₄ C
N) a group represented by (N (CH (CH ₃ ) ₂ ) ₂ );
A group represented by (OCH ₃ ) (N (CH (CH ₃ ) ₂ ) ₂ ),
A phosphonyl group, or 2-chlorophenyl or 4-
A compound wherein R ² is an azido group or an amino group; 9) a compound wherein B is 6-aminopurin-9-yl (ie, adenyl) and the amino group is 6-aminopurin-9-yl and 2,6-diaminopurin-9-yl protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurin-9-yl where the amino group is protected with a protecting group for nucleic acid synthesis Il, 2-amino-6
Chloropurin-9-yl, 2-amino-6-chloropurin-9-yl and 2-amino-6-fluoropurin-9-yl in which the amino group is protected by a protecting group for nucleic acid synthesis, and amino group in the nucleic acid synthesis 2-amino-6 protected with a protecting group of
Fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6 in which the amino group is protected by a protecting group for nucleic acid synthesis -Hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl in which the amino group is protected by a protecting group for nucleic acid synthesis. 6-amino-2-methoxypurine-9-yl, 6-amino-2-chloropurine-9-, wherein the amino group is protected with a protecting group for nucleic acid synthesis.
6-amino-2-chloropurin-9-yl, 6-amino-2-yl having an yl or amino group protected with a protecting group for nucleic acid synthesis.
Fluoropurin-9-yl, 6-amino-2-fluoropurine-9- in which the amino group is protected with a protecting group for nucleic acid synthesis
Yl, 2,6-dimethoxypurin-9-yl, 2,6-
Dichloropurin-9-yl, 6-mercaptopurine-9
-Yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl) in which the mercapto group is protected by a protecting group for nucleic acid synthesis, amino group Is protected by a protecting group for nucleic acid synthesis, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-fluoro-1,2-dihydropyrimidin-1- 4-amino-2-oxo-5-fluoro-1,2-dihydropyrimidine-1 in which the yl or amino group is protected by a protecting group for nucleic acid synthesis
-Yl, 4-amino-2-oxo-5-chloro-1,2
-Dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy in which the amino group is protected with a protecting group for nucleic acid synthesis -1,2-dihydropyrimidine-1
-Yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2 in which the mercapto group is protected by a protecting group for nucleic acid synthesis.
-Dihydropyrimidin-1-yl, 2,4-dihydroxypyrimidin-1-yl (ie uracilyl),
2,4-dihydroxy-5-methylpyrimidin-1-yl (ie, thyminyl), 4-amino-5-methyl-
2-oxo-1,2-dihydropyrimidin-1-yl or a 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis 10) B is 6-benzoylaminopurin-9-yl,
Adenyl, 2-benzoylamino-6-hydroxypurin-9-yl, guaninyl, 2-oxo-4-benzoylamino-1,2-dihydropyrimidin-1-yl,
A compound which is a cytosinyl, uracilyl or thyminyl group.

R ¹ is selected from the above 2) to 5), R ² is selected from the above 6) to 8), and B is 9) to 10).
Any combination obtained by selecting from is also suitable,
Particularly preferred are combinations of 2), 6) and 9), 3),
A combination of 7) and 9), a combination of 5), 6) and 10) and a combination of 5), 7) and 10).

The novel oligonucleotide analogs of the present invention include: 1) the general formula (1a)

[0022]

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Wherein B represents a purine-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have a substituent selected from the following α group: . And pharmacologically acceptable salts thereof. However, when two or more of the above structures are contained, B is the same or different between the structures. (Α group) a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 6 carbon atoms, a mercapto group,
Mercapto group protected by a protecting group for nucleic acid synthesis, 1 carbon atom
To 6 alkylthio groups, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and Halogen atom.

Here, the term "oligonucleotide analog" refers to a non-natural type in which a nucleoside unit in a natural type oligonucleotide is substituted by one or more of the above-mentioned structures (1a), and such an analog is referred to. In, for example, a sugar derivative in which the sugar moiety is modified, a thioate derivative in which the phosphodiester binding moiety is thioated, an ester in which the terminal phosphate moiety is esterified, and an amino group on the purine base is amidated The amide form can also be contained as another nucleoside or nucleotide unit.

Among the novel oligonucleotide analogs of the present invention, preferred are: 2) B is a 6-aminopurin-9-yl group (ie,
Adenylyl group), 6-aminopurin-9-yl group, 2,6-diaminopurin-9-yl group, 2-amino-6-chloropurine- in which the amino group is protected by a protecting group for nucleic acid synthesis.
2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group in which 9-yl group and amino group are protected by a protecting group for nucleic acid synthesis, and amino group are nucleic acid synthesis. 2-amino-6-fluoropurin-9-yl group protected by a protecting group of 2-amino-6-bromopurine-9
-Yl and amino groups protected with a protecting group for nucleic acid synthesis;
-Amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group (that is, guaninyl group), 2-amino-6 in which the amino group is protected with a protecting group for nucleic acid synthesis. -Hydroxypurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group, 6-amino-2-methoxypurin-9-yl group in which an amino group and a hydroxyl group are protected with a protecting group for nucleic acid synthesis , 6-amino-2-
Chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurine-9-
Yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (that is, cytosinyl group) A 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis, 2-oxo-4-amino-5-
A fluoro-1,2-dihydropyrimidin-1-yl group,
2-oxo- wherein the amino group is protected by a protecting group for nucleic acid synthesis
4-amino-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-dihydropyrimidine-1-
An yl group, a 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl group, a 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group (that is, a uracinyl group), -Oxo-4-hydroxy-5
-Methyl-1,2-dihydropyrimidin-1-yl group (ie, thyminyl group), 4-amino-5-methyl-
4-amino-5-methyl-2-oxo-1, in which a 2-oxo-1,2-dihydropyrimidin-1-yl group (ie, a 5-methylcytosinyl group) or an amino group is protected with a protecting group for nucleic acid synthesis. Oligonucleotide analogs and pharmacologically acceptable salts thereof, which are 2-dihydropyrimidin-1-yl groups, 3) B is a 6-benzoylaminopurin-9-yl group, an adenylyl group, 2-isobutyryl An amino-6-hydroxypurin-9-yl group, a guaninyl group,
2-oxo-4-benzoylamino-1,2-dihydropyrimidin-1-yl group, cytosinyl group, 2-oxo-
Oligonucleotide analogs which are a 5-methyl-4-benzoylamino-1,2-dihydropyrimidin-1-yl group, a 5-methylcytosinyl group, a uracinyl group or a thyminyl group, and pharmacologically acceptable salts thereof.

The "protecting group for hydroxyl group synthesis of nucleic acid" in the definition of R ¹ is not particularly limited as long as it can stably protect the hydroxyl group during nucleic acid synthesis. Examples thereof include formyl, acetyl and propionyl. , Butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, decanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, tridecanoyl, hexadecanoyl, 14-methylpentadecanoyl, 13,13-
Alkylcarbonyl groups such as dimethyltetradecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl and henicosanoyl, carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl, adipoyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl A lower alkoxycarbonyl group such as a halogeno lower alkylcarbonyl group such as methoxyacetyl;
“Aliphatic acyl group” such as unsaturated alkylcarbonyl group such as -2-methyl-2-butenoyl; benzoyl, α
Arylcarbonyl groups such as -naphthoyl and β-naphthoyl, halogenoarylcarbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl, 2,4,6-
Lower alkylated arylcarbonyl groups such as trimethylbenzoyl and 4-toluoyl; lower alkoxylated arylcarbonyl groups such as 4-anisoyl; carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl and 4-carboxybenzoyl. Groups, nitrated arylcarbonyl groups such as 4-nitrobenzoyl and 2-nitrobenzoyl; lower alkoxycarbonylated arylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl, and arylated arylcarbonyl groups such as 4-phenylbenzoyl "Aromatic acyl group" such as tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-
2-yl, 4-methoxytetrahydrothiopyran-4-
“Tetrahydropyranyl or tetrahydrothiopyranyl group” such as yl; “tetrahydrofuranyl or tetrahydrothiofuranyl group” such as tetrahydrofuran-2-yl or tetrahydrothiofuran-2-yl; trimethylsilyl, triethylsilyl, isopropyldimethyl Tri-lower alkylsilyl groups such as silyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, diphenylmethylsilyl, t-butyldiphenylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl A "silyl group" such as a tri-lower alkylsilyl group substituted with one or two aryl groups; methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxy Shimechiru, isopropoxymethyl,
A "lower alkoxymethyl group" such as butoxymethyl and t-butoxymethyl; a "lower alkoxylated lower alkoxymethyl group" such as 2-methoxyethoxymethyl;
"Halogeno lower alkoxymethyl" such as 2,2,2-trichloroethoxymethyl and bis (2-chloroethoxy) methyl; "lower alkoxylated ethyl group" such as 1-ethoxyethyl and 1- (isopropoxy) ethyl ;
"Halogenated ethyl group" such as 2,2,2-trichloroethyl; benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α
“Methyl group substituted with 1 to 3 aryl groups” such as naphthyldiphenylmethyl, 9-anthrylmethyl; 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethyl Benzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl,
4,4'-dimethoxytriphenylmethyl, 4,4 ',
4 "-trimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl,
-Bromobenzyl, 4-cyanobenzyl, "a lower alkyl, a lower alkoxy, a halogen or a methyl group substituted with one to three aryl groups in which an aryl ring is substituted with a cyano group"; methoxycarbonyl, ethoxycarbonyl, "lower alkoxycarbonyl groups" such as t-butoxycarbonyl and isobutoxycarbonyl;
"Lower alkoxycarbonyl group substituted by halogen or tri-lower alkylsilyl group" such as 2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl; "alkenyloxycarbonyl group" such as vinyloxycarbonyl and allyloxycarbonyl; benzyl "One or two lower alkoxy or nitro groups such as oxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl Aralkyloxycarbonyl group which may be substituted '', preferably, an aliphatic acyl group, an aromatic acyl group,
A methyl group substituted with one to three aryl groups substituted with one to three aryl groups, a methyl group substituted with one to three aryl groups, a lower alkyl group, a lower alkoxy group, a halogen or cyano group, or a silyl group. And more preferably acetyl, benzoyl, benzyl, p-methoxybenzyl, dimethoxytrityl, monomethoxytrityl or tert-butyldiphenylsilyl.

In the definition of R ¹ and R ³ , the term “protecting group for nucleic acid synthesis” of “phosphate group protected with a protecting group for nucleic acid synthesis” refers to a group capable of stably protecting a phosphate group during nucleic acid synthesis. If so, there is no particular limitation, for example, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl Methylpentyl, 2-methylpentyl, 1
-Methylpentyl, 3,3-dimethylbutyl, 2,2-
"Lower alkyl groups" such as dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl; 2-cyanoethyl, 2-cyano "Cyanated lower alkyl group" such as -1,1-dimethylethyl; "silyl-substituted ethyl group" such as 2-methyldiphenylsilylethyl, 2-trimethylsilylethyl and 2-triphenylsilylethyl; 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloro-1,1-
A "halogenated lower alkyl group" such as dimethylethyl; ethenyl, 1-propenyl, 2-propenyl, 1
-Methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl -2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl, 1-
Ethyl-2-butenyl, 3-butenyl, 1-methyl-3
-Butenyl, 2-methyl-3-butenyl, 1-ethyl-
3-butenyl, 1-pentenyl, 2-pentenyl, 1-
Methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2
-Methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1
-Hexenyl, 2-hexenyl, 3-hexenyl, 4-
"Lower alkenyl groups" such as hexenyl, 5-hexenyl; cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl,
"Cycloalkyl group" such as adamantyl; "cyanoated lower alkenyl group" such as 2-cyanobutenyl; benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl , Diphenylmethyl, triphenylmethyl,
1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-
Phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-
Phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4-
Naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2
-Naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6
"Aralkyl groups" such as -phenylhexyl, 1-naphthylhexyl, 2-naphthylhexyl, 3-naphthylhexyl, 4-naphthylhexyl, 5-naphthylhexyl, 6-naphthylhexyl; 4-chlorobenzyl, 2-chlorobenzyl,
(4-nitrophenyl) ethyl, o-nitrobenzyl,
4-nitrobenzyl, 2,4-dinitrobenzyl, 4-
"Nitro group, aralkyl group having an aryl ring substituted with a halogen atom" such as chloro-2-nitrobenzyl; "aryl group" such as phenyl, indenyl, naphthyl, phenanthrenyl and anthracenyl; 2-methylphenyl, 2, 6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl,
2,5-dichlorophenyl, 2-bromophenyl, 4-
Examples include "lower alkyl group, halogen atom, aryl group substituted with nitro group" such as nitrophenyl and 4-chloro-2-nitrophenyl, and preferably,
"Lower alkyl group", "lower alkyl group substituted by cyano group", "aralkyl group", "nitro group, aralkyl group having aryl ring substituted by halogen atom" or "lower alkyl group, halogen atom, nitro group" And more preferably a 2-cyanoethyl group, a 2,2,2-trichloroethyl group, a benzyl group,
A chlorophenyl group or a 4-chlorophenyl group.

In the definition of the above α group, “C 1 to C 6
Examples of the `` alkyl group '' include, for example, methyl, ethyl,
Examples thereof include linear or branched alkyl groups having 1 to 6 carbon atoms such as n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, pentyl, and hexyl. , An alkyl group having 1 to 4 carbon atoms, more preferably an alkyl group having 1 or 2 carbon atoms, and most preferably a methyl group.

The term "protecting group for amino acid nucleic acid synthesis" in the above definition of R ² is not particularly limited as long as it can stably protect the amino group during nucleic acid synthesis.
Formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, decanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, tridecanoyl, hexadecanoyl, 14- Methyl pentadecanoyl, 13, 13
Alkylcarbonyl groups such as dimethyltetradecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl and henicosanoyl, succinoyl,
Glutaroyl, carboxylated alkylcarbonyl groups such as adipoyl, chloroacetyl, dichloroacetyl,
Trichloroacetyl, a halogeno lower alkylcarbonyl group such as trifluoroacetyl, a lower alkoxy lower alkylcarbonyl group such as methoxyacetyl,
(E) an “aliphatic acyl group” such as an unsaturated alkylcarbonyl group such as 2-methyl-2-butenoyl; an arylcarbonyl group such as benzoyl, α-naphthoyl and β-naphthoyl; 2-bromobenzoyl; A halogenoarylcarbonyl group such as chlorobenzoyl, 2,
Lower-alkylated arylcarbonyl groups such as 4,6-trimethylbenzoyl and 4-toluoyl; lower-alkoxylated arylcarbonyl groups such as 4-anisoyl;
-Carboxybenzoyl, 3-carboxybenzoyl,
Carboxylated arylcarbonyl groups such as 4-carboxybenzoyl, nitrated arylcarbonyl groups such as 4-nitrobenzoyl and 2-nitrobenzoyl;
“Aromatic acyl groups” such as lower alkoxycarbonylated arylcarbonyl groups such as (methoxycarbonyl) benzoyl and arylated arylcarbonyl groups such as 4-phenylbenzoyl; methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxy A "lower alkoxycarbonyl group" such as carbonyl;
"Lower alkoxycarbonyl groups substituted by halogen or tri-lower alkylsilyl groups" such as 2,2,2-trichloroethoxycarbonyl and 2-trimethylsilylethoxycarbonyl; "alkenyloxycarbonyl" such as vinyloxycarbonyl and allyloxycarbonyl Group "; 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl,
4,4'-dimethoxytriphenylmethyl, 4,4 ',
4 "-trimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl,
-Bromobenzyl, 4-cyanobenzyl, "a lower alkyl, a lower alkoxy, a methyl group substituted with 1 to 3 aryl groups in which an aryl ring is substituted with a halogen or a cyano group"; benzyloxycarbonyl, 4-cyanobenzyl Even when the aryl ring is substituted with one or two lower alkoxy or nitro groups such as methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl A good aralkyloxycarbonyl group '', preferably a lower alkyl,
"A methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkoxy, halogen or cyano group", "aliphatic acyl group", "aromatic acyl group", or "1 to 2 lower acyl groups" An aralkyloxycarbonyl group in which the aryl ring may be substituted with an alkoxy or nitro group ", and more preferably 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkyl, lower alkoxy, halogen or cyano group. Methyl group substituted with a group ",
An "aliphatic acyl group" or an "aralkyloxycarbonyl group in which the aryl ring may be substituted with one or two lower alkoxy or nitro groups", and particularly preferably
It is a 4,4'-dimethoxytrityl group, a 4-monomethoxytrityl group, a trifluoroacetyl group or a benzyloxycarbonyl group.

The above "phosphoramidite group" is a group represented by the formula -P (OR ^3a ) (NR ^3b ), wherein R ^3a is an alkyl group having 1 to 6 carbon atoms or a group having 1 carbon atom. And 7 to 7 cyanoalkyl groups, and R ^3b represents an alkyl group having 1 to 6 carbon atoms), and is preferably of the formula -P (OC ₂ H ₄
CN) a group represented by (N (CH (CH ₃ ) ₂ ) ₂ ) or a group represented by the formula —P (OCH ₃ ) (N (CH (CH ₃ ) ₂ ) ₂ ).

"Halogen atom" in the definition of the above α group
Examples thereof include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, and a fluorine atom or a chlorine atom is preferred.

The “alkyl group having 1 to 6 carbon atoms” in the definition of R ^4a and R ^4b and the α group includes, for example, methyl, ethyl, n-propyl, isopropyl, n
-Butyl, isobutyl, s-butyl, tert-butyl, pentyl, hexyl and the like, and a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms, preferably a methyl group or an ethyl group. is there.

As the protecting group for nucleic acid synthesis of the above-mentioned R ^4a and R ^4b and the “hydroxyl group protected by a protecting group for nucleic acid synthesis” in the definition of α group, “protecting group for the synthesis of hydroxyl group for nucleic acid in the definition of R ¹ ” The preferred groups are the same as the groups exemplified in "), preferably" aliphatic acyl group "or" aromatic acyl group ", more preferably benzoyl group.

Examples of the protecting group for the synthesis of nucleic acid such as “mercapto group protected by a protecting group for nucleic acid synthesis” in the definition of R ^4a and R ^4b and the α group include “a nucleic acid synthesis for hydroxyl group” in the definition of R ^1. Other than the above-mentioned `` protecting group, '' and `` a group forming a disulfide '' such as an alkylthio group such as methylthio, ethylthio, and tert-butylthio, and an arylthio group such as benzylthio. An aromatic acyl group "or an" aromatic acyl group ", and more preferably a benzoyl group.

As the protecting group for nucleic acid synthesis of the above-mentioned R ^4a and R ^4b and the “amino group protected by a protecting group for nucleic acid synthesis” in the definition of α group, “the amino group for amino acid synthesis in the definition of R ² ” The same groups as those exemplified for the "protecting group" can be mentioned, preferably an "aliphatic acyl group" or an "aromatic acyl group", and more preferably a benzoyl group.

Examples of the "alkoxy group having 1 to 6 carbon atoms" in the definition of R ^4a and R ^4b and the α group include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s- Butoxy, te
A straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms such as rt-butoxy, pentyloxy and hexyloxy can be mentioned, and a methoxy or ethoxy group is preferable.

Examples of the “alkylthio group having 1 to 6 carbon atoms” in the definition of R ^4a and R ^4b and the α group include, for example, methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, tert-butylthio, -Butylthio, pentylthio and hexylthio groups, and preferably a methylthio or ethylthio group.

The “alkylamino group substituted with an alkyl group having 1 to 6 carbon atoms” in the definition of R ^4a and R ^4b and the α group includes, for example, methylamino, ethylamino, propylamino, isopropylamino, Butylamino, isobutylamino, s-butylamino, tert-
Butylamino, pentylamino, hexylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di (s-butyl) amino, di (tert-butyl) amino, dipentylamino, dihexylamino And a methylamino, ethylamino, dimethylamino or diethylamino group.

As the “cyanoalkoxy group having 1 to 7 carbon atoms” in the definition of R ^4a and R ^4b , for example,
Cyanomethoxy, cyanoethoxy, cyanopropyloxy, cyanobutyloxy, cyanopentyloxy, cyanohexyloxy group and the like, preferably,
2-cyanoethoxy group.

The “pharmacologically acceptable salt thereof” refers to the nucleoside analog (1) of the present invention and the above-mentioned structure (1).
The oligonucleotide analog containing a) can be converted into a salt, and refers to a salt thereof. Such a salt is preferably an alkali metal salt such as a sodium salt, a potassium salt, or a lithium salt; Metal salts such as alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts; Dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, 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 (hydroxymethyl) Amine salts such as organic salts such as aminomethane salts; hydrofluorides, hydrochlorides, hydrobromides, hydrohalides such as hydroiodates, nitrates, perchlorates,
Inorganic salts such as sulfates and phosphates; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate, aryls such as benzenesulfonate and p-toluenesulfonate Organic acid salts such as sulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and glycine, lysine, arginine, and ornithine Salts, amino acids such as glutamate and aspartate can be mentioned. In the case of oligonucleotide analogs containing a nucleoside structure (1a), sodium salts, potassium salts and triethylamine salts are preferable. In the case of the analog (1), a free body is preferable.

The nucleoside analog (1) of the present invention
In addition, the oligonucleotide analog containing the above-mentioned structure (1a) absorbs water when left in the air to form adsorbed water or may become a hydrate. Included in the present invention.

Furthermore, the nucleoside analog (1) of the present invention and the oligonucleotide analog containing the above-mentioned structure (1a) may absorb some other solvent and form a solvate. Such salts are also included in the present invention.

Specific examples of the compound (1) of the present invention include:
For example, compounds as shown in Tables 1 and 2 below can be mentioned.

In Tables 1 and 2, "Bn" represents a benzyl group, "Bz" represents a benzoyl group, "Me" represents a methyl group, "PMBn" represents a p-methoxybenzyl group,
“MMTr” represents a 4-methoxytriphenylmethyl group, “DMTr” represents a 4,4′-dimethoxytriphenylmethyl group, and “TMTr” represents a 4,4 ′, 4 ″ -trimethoxytriphenylmethyl group. "TMS" is a trimethylsilyl group, "TBDMS" is a tert-butyldimethylsilyl group, "TBDPS" is a tert-butyldiphenylsilyl group, "Tfa" is a trifluoroacetyl group, and "Cbz "Is a benzyloxycarbonyl group,
Shown respectively.

[0045]

[Table 1]

[0046]

Embedded image

Example Compound No. R ¹ R ² Ra Rb −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− _{----------------------------- 1-1 H NH 2 HH 1-2 H} NH 2 H OH 1-3 H NH 2 H SH 1-4 H NH ₂ H NH ₂ 1-5 H NH ₂ H OMe 1-6 H NH ₂ FH 1-7 H NH ₂ F NH ₂ 1-8 H NH ₂ Cl H 1-9 H NH ₂ Cl NH ₂ 1-10 H NH ₂ Cl Cl 1-11 H NH ₂ Br H 1-12 H NH ₂ Br NH ₂ 1-13 H NH ₂ OH H 1-14 H NH ₂ OH OH 1-15 H NH ₂ OH NH ₂ 1-16 H NH ₂ OMe OMe 1-17 H NH ₂ OMe NH ₂ 1-18 H NH ₂ NH ₂ H 1-19 H NH ₂ NH ₂ F 1-20 H NH ₂ NH ₂ Cl 1-21 H NH ₂ NH ₂ Br 1-22 H NH ₂ NH ₂ OH 1-23 H NH ₂ NH ₂ NH ₂ 1-24 H NH ₂ NH ₂ OMe 1-25 HN ₃ HH 1-26 HN ₃ H OH 1-27 HN ₃ H SH 1-28 HN ₃ H NH ₂ 1-29 HN ₃ H OMe 1-30 HN ₃ FH 1-31 HN ₃ F NH ₂ 1-32 HN ₃ Cl H 1-33 HN ₃ Cl NH ₂ 1-34 HN ₃ Cl Cl 1-35 HN ₃ Br H 1-36 HN ₃ Br NH ₂ 1-37 HN ₃ OH H 1-38 HN ₃ OH OH 1-39 HN ₃ OH NH ₂ 1-40 HN ₃ OMe OMe 1- 41 HN ₃ OMe NH ₂ 1-42 HN ₃ NH ₂ H 1-43 HN ₃ NH ₂ F 1-44 HN ₃ NH ₂ Cl 1-45 HN ₃ NH ₂ Br 1-46 HN ₃ NH ₂ OH 1-47 HN ₃ NH ₂ NH ₂ 1-48 HN ₃ NH ₂ OMe 1-49 HN ₃ H NHBz 1-50 H NH ₂ H NHBz 1-51 HN ₃ Cl NHBz 1-52 HN ₃ OH NHBz 1-53 HN ₃ OMe NHBz 1-54 HN ₃ NHBz H 1-55 HN ₃ NHBz Cl 1-56 HN ₃ NHBz OH 1-57 H NH ₂ NHBz OH 1-58 HN ₃ NHBz NHBz 1-59 HN ₃ NHBz OMe 1-60 Bn N ₃ H NHBz 1-61 Bn N ₃ NHCOCH (CH ₃ ) ₂ OH 1-62 PMBn N ₃ H NHBz 1-63 PMBn N ₃ NHCOCH (CH ₃ ) ₂ OH 1-64 MMTr N ₃ H NHBz 1-65 MMTr N ₃ NHCOCH (CH ₃ ) ₂ OH 1-66 DMTr N ₃ H NHBz 1-67 DMTr N ₃ NHCOCH (CH ₃ ) ₂ OH 1-68 TMTr N ₃ H NHBz 1-69 TMTr N ₃ NHCOCH (CH ₃ ) ₂ OH 1-70 TMS N ₃ H NHBz 1-71 TMS N ₃ NHCOCH (CH ₃ ) ₂ OH 1- 72 TBDMS N ₃ H NHBz 1-73 TBDMS N ₃ NHCOCH (CH ₃ ) ₂ OH 1-74 TBDPS N ₃ H NHBz 1-75 TBDPS N ₃ NHBz OH 1-76 Bn NH ₂ H NHBz 1-77 Bn NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-78 PMBn NH ₂ H NHBz 1-79 PMBn NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-80 MMTr NH ₂ H NHBz 1-81 MMTr NH ₂ NHCOCH (CH ₃ ) ₂ OH 1- 82 DMTr NH ₂ H NHBz 1-83 DMTr NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-84 TMTr NH ₂ H NHBz 1-85 TMTr NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-86 TMS NH ₂ H NHBz 1-87 TMS NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-88 TBDMS NH ₂ H NHBz 1-89 TBDMS NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-90 TBDPS NH ₂ H NHBz 1-91 TBDPS NH ₂ NHCOCH (CH ₃ ) ₂ OH 1-92 TBDPS (MMTr) NH H NHBz 1-93 TBDPS (MMTr) NH NHCOCH (CH ₃ ) ₂ OH 1-94 H (MMTr) NH H NHBz 1-95 H ( MMTr) NH NHCOCH (CH ₃ ) ₂ OH 1-96 P (OCH ₂ CH ₂ CN)-(MMTr) NH H NHBz (N (iPr) ₂ ) 1-97 P (OCH ₂ CH ₂ CN)-(MMTr) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-98 P (OCH ₃ )-(MMTr) NH H NHBz (N (iPr) ₂ ) 1-99 P (OCH ₃ )-(MMTr) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-100 TBDPS (DMTr) NH H NHBz 1-101 TBDPS (DMTr) NH NHCOCH (CH ₃ ) ₂ OH 1-102 H (DMTr) NH H NHBz 1 -103 H (DMTr) NH NHCOCH (CH ₃ ) ₂ OH 1-104 P (OCH ₂ CH ₂ CN)-(DMTr) NH H NHBz (N (iPr) ₂ ) 1-105 P (OCH ₂ CH ₂ CN) -(DMTr) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-106 P (OCH ₃ )-(DMTr) NH H NHBz (N (iPr) ₂ ) 1-107 P (OCH ₃ )- (DMTr) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-108 TBDPS (Tfa) NH H NHBz 1-109 TBDPS (Tfa) NH NHCOCH (CH ₃ ) ₂ OH 1-110 H (Tfa) NH H NHBz 1-111 H (Tfa) NH NHCOCH (CH ₃ ) ₂ OH 1-112 P (OCH ₂ CH ₂ CN)-(T fa) NH H NHBz (N (iPr) ₂ ) 1-113 P (OCH ₂ CH ₂ CN)-(Tfa) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-114 P (OCH ₃ ) -(Tfa) NH H NHBz (N (iPr) ₂ ) 1-115 P (OCH ₃ )-(Tfa) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-116 TBDPS (Cbz) NH H NHBz 1-117 TBDPS (Cbz) NH NHCOCH (CH ₃ ) ₂ OH 1-118 H (Cbz) NH H NHBz 1-119 H (Cbz) NH NHCOCH (CH ₃ ) ₂ OH 1-120 P (OCH ₂ CH ₂ (CN)-(Cbz) NH H NHBz (N (iPr) ₂ ) 1-121 P (OCH ₂ CH ₂ CN)-(Cbz) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-122 P (OCH ₃ )-(Cbz) NH H NHBz (N (iPr) ₂ ) 1-123 P (OCH ₃ )-(Cbz) NH NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-124 DMTr NHP (CH ₂ CH ₂ CN) -H NHBz (N (iPr) ₂ ) 1-125 DMTr NHP (OCH ₃ )-H NHBz (N (iPr) ₂ ) 1-126 DMTr NHP (CH ₂ CH ₂ CN)-NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-127 DMTr NHP (OCH ₃ )-NHCOCH (CH ₃ ) ₂ OH (N (iPr) ₂ ) 1-128 MMTr NHP (CH ₂ CH ₂ CN)- H NHBz (N (iPr) ₂ ) 1-129 MMTr NHP (OCH ₃ )-H NHBz (N (iPr) ₂ ) 1-130 MMTr NHP (CH ₂ CH ₂ CN)-NHCOCH (CH ₃ ) ₂ OH (N _{(iPr) 2) 1-131 MMTr NHP} (OCH 3) - NHCOCH (CH 3) 2 OH (N (iPr) 2) ------------------- -------------

[0048]

[Table 2]

[0049]

Embedded image

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Exemplified Compound No. R ¹ R ² R ⁵ R ⁶ −− −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 2-1 H NH ₂ HH 2-2 H NH ₂ Cl H 2-3 H NH ₂ OH H 2-4 H NH ₂ OH Me 2-5 H NH ₂ SH H 2-6 H NH ₂ NH ₂ H 2-7 H NH ₂ NH ₂ F 2-8 H NH ₂ NH ₂ Cl 2-9 H NH ₂ NH ₂ Me 2-10 H NH ₂ OMe H 2-11 HN ₃ HH 2-12 HN ₃ Cl H 2-13 HN ₃ OH H 2-14 HN ₃ OH Me 2-15 HN ₃ SH H 2- 16 HN ₃ NH ₂ H 2-17 HN ₃ NH ₂ F 2-18 HN ₃ NH ₂ Cl 2-19 HN ₃ NH ₂ Me 2-20 HN ₃ OMe H 2-21 HN ₃ NHBz H 2-22 H NH ₂ NHBz H 2-23 HN ₃ NHBz F 2-24 HN ₃ NHBz Cl 2-25 HN ₃ NHBz Me 2-26 Bn N ₃ OH H 2-27 Bn N ₃ OH Me 2-28 Bn N ₃ NHBz H 2-29 PMBn N ₃ OH H 2-30 PMBn N ₃ OH Me 2-31 PMBn N ₃ NHBz H 2-32 MMTr N ₃ OH H 2-33 MMTr N ₃ OH Me 2-34 MMTr N ₃ NHBz H 2-35 DMTr N ₃ OH H 2-36 DMTr N ₃ OH Me 2-37 DMTr N ₃ NHBz H 2-38 TMTr N ₃ OH H 2-39 TMTr N ₃ OH Me 2-40 TMTr N ₃ NHBz H 2-41 TMS N ₃ OH H 2-42 TMS N ₃ OH Me 2-43 TMS N ₃ NHBz H 2-44 TBDMS N ₃ OH H 2-45 TBDMS N ₃ OH Me 2-46 TBDMS N ₃ NHBz H 2-47 TBDPS N ₃ OH H 2-48 TBDPS N ₃ OH Me 2-49 TBDPS N ₃ NHBz H 2-50 Bn NH ₂ OH H 2-51 Bn NH ₂ OH Me 2-52 Bn NH ₂ NHBz H 2-53 PMBn NH ₂ OH H 2-54 PMBn NH ₂ OH Me 2-55 PMBn NH ₂ NHBz H 2-56 MMTr NH ₂ OH H 2-57 MMTr NH ₂ OH Me 2-58 MMTr NH ₂ NHBz H 2-59 DMTr NH ₂ OH H 2-60 DMTr NH ₂ OH Me 2- 61 DMTr NH ₂ NHBz H 2-62 TMTr NH ₂ OH H 2-63 TMTr NH ₂ OH Me 2-64 TMTr NH ₂ NHBz H 2-65 TMS NH ₂ OH H 2-66 TMS NH ₂ OH Me 2-67 TMS NH ₂ NHBz H 2-68 TBDMS NH ₂ OH H 2-69 TBDMS NH ₂ OH Me 2-70 TBDMS NH ₂ NHBz H 2-71 TBDPS NH ₂ OH H 2-72 TBDPS NH ₂ OH Me 2-73 TBDPS NH ₂ NHBz H 2-74 TBDPS (MMTr) NH OH H 2-75 TBDPS (MMTr) NH OH Me 2-76 TBDPS (MMTr) NH NHBz H 2-77 TBDPS (MMTr) NH NHBz Me 2-78 H (MMTr) NH OH H 2-79 H (MMTr) NH OH Me 2-80 H (MMTr) NH NHBz H 2-81 H (MMTr) NH NHBz Me 2-82 P (OCH ₂ CH ₂ CN)-(MMTr) NH OH H (N (iPr) ₂ ) 2-83 P (OCH ₂ CH ₂ CN)-(MMTr) NH OH Me (N (iPr) ₂ ) 2-84 P (OCH ₂ CH ₂ CN)-(MMTr) NH NHBz H (N (iPr) ₂ ) 2-85 P (OCH ₂ CH ₂ CN)-(MMTr) NH NHBz Me (N (iPr) ₂ ) 2-86 P (OCH ₃ )-(MMTr) NH OH H (N (iPr) ₂ ) 2- 87 P (OCH ₃ )-(MMTr) NH OH Me (N (iPr) ₂ ) 2-88 P (OCH ₃ )-(MMTr) NH NHBz H (N (iPr) ₂ ) 2-89 P (OCH ₃ ) -(MMTr) NH NHBz Me (N (iPr) ₂ ) 2-90 TBDPS (DMTr) NH OH H 2-91 TBDPS (DMTr) NH OH Me 2-92 TBDPS (DMTr) NH NHBz H 2-93 TBDPS (DMTr ) NH NHBz Me 2-94 H (DMTr) NH OH H 2-95 H (DMTr) NH OH Me 2-96 H (DMTr) NH NHBz H 2-97 H (DMTr) NH NHBz Me 2-98 P (OCH ₂ CH ₂ CN)-(DMTr) NH OH H (N (iPr) ₂ ) 2-99 P (OCH ₂ CH ₂ CN)-(DMTr) NH OH Me (N (iPr) ₂ ) 2-100 P (OCH ₂ CH ₂ CN)-(DMTr) NH NHBz H (N (iPr) ₂ ) 2-101 P (OCH ₂ CH ₂ CN)-(DMTr) NH NHBz Me (N (iPr) ₂ ) 2-102 P (OCH ₃ )-(DMTr) NH OH H (N (iPr) ₂ ) 2-103 P (OCH ₃ )-(DMTr) NH OH Me (N (iPr) ₂ ) 2-104 P (OCH ₃ )-(DMTr) NH NHBz H (N (iPr) ₂ ) 2-105 P (OCH ₃ )-(DMTr) NH NHBz Me (N (iPr) ₂ ) 2-106 TBDPS (Tfa) NH OH H 2-107 TBDPS (Tfa) NH OH Me 2-108 TBDPS (Tfa) NH NHBz H 2-109 TBDPS (Tfa) NH NHBz Me 2-110 H (Tfa) NH OH H 2-111 H (Tfa) NH OH Me 2-112 H (Tfa) NH NHBz H 2-113 H (Tfa) NH NHBz Me 2-114 P (OCH ₂ CH ₂ CN)-(Tfa) NH OH H (N (iPr) ₂ ) 2-115 P (OCH ₂ CH ₂ CN)-(Tfa) NH OH Me (N (iPr) ₂ ) 2-116 P (OCH ₂ CH ₂ CN)-(Tfa) NH NHBz H (N (iPr) ₂ ) 2-117 P (OCH ₂ CH ₂ CN)-(Tfa) NH NHBz Me (N (iPr) ₂ ) 2-118 P (OCH ₃ )-(Tfa) NH OH H (N (iPr) ₂ ) 2-119 P (OCH ₃ )-(Tfa) NH OH Me (N (iPr) ₂ ) 2-120 P ( OCH ₃ )-(Tfa) NH NHBz H (N (iPr) ₂ ) 2-121 P (OCH ₃ )-(Tfa) NH NHBz Me (N (iPr) ₂ ) 2-122 TBDPS (Cbz) NH OH H 2 -123 TBDPS (Cbz) NH OH Me 2-124 TBDPS (Cbz) NH NHBz H 2-125 TBDPS (Cbz) NH NHBz Me 2-126 H (Cbz) NH OH H 2-127 H (Cbz) NH OH Me 2 -128 H (Cbz) NH NHBz H 2-129 H (Cbz) NH NHBz Me 2-130 P (OCH ₂ CH ₂ CN)-(Cbz) NH OH H (N (iPr) ₂ ) 2-131 P (OCH ₂ CH ₂ CN)-(Cbz) NH OH Me (N (iPr) ₂ ) 2-132 P (OCH ₂ CH ₂ CN)-(Cbz) NH NHBz H (N (iPr) ₂ ) 2-133 P (OCH ₂ CH ₂ CN)-(Cbz) NH NHBz Me (N (iPr) ₂ ) 2-134 P (OCH ₃ )-(Cbz) NH OH H (N (iPr) ₂ ) 2-135 P (OCH ₃ )- (Cbz) NH OH Me (N (iPr) ₂ ) 2-136 P (OCH ₃ )-(Cbz) NH NHBz H (N (iPr) ₂ ) 2-137 P (OCH ₃ )-(Cbz) NH NHBz Me (N (iPr) ₂ ) 2-138 DMTr NHP (OCH ₂ CH ₂ CN)-OH H (N (iPr) ₂ ) 2 -139 DMTr NHP (OCH ₂ CH ₂ CN)-OH Me (N (iPr) ₂ ) 2-140 DMTr NHP (OCH ₂ CH ₂ CN)-NHBz H (N (iPr) ₂ ) 2-141 DMTr NHP (OCH ₂ CH ₂ CN)-NHBz Me (N (iPr) ₂ ) 2-142 DMTr NHP (OCH ₃ )-OH H (N (iPr) ₂ ) 2-143 DMTr NHP (OCH ₃ )-OH Me (N (iPr ) ₂ ) 2-144 DMTr NHP (OCH ₃ )-NHBz H (N (iPr) ₂ ) 2-145 DMTr NHP (OCH ₃ )-NHBz Me (N (iPr) ₂ ) 2-146 MMTr NHP (OCH ₂ CH ₂ CN)-OH H (N (iPr) ₂ ) 2-147 MMTr NHP (OCH ₂ CH ₂ CN)-OH Me (N (iPr) ₂ ) 2-148 MMTr NHP (OCH ₂ CH ₂ CN)-NHBz H (N (iPr) ₂ ) 2-149 MMTr NHP (OCH ₂ CH ₂ CN)-NHBz Me (N (iPr) ₂ ) 2-150 MMTr NHP (OCH ₃ )-OH H (N (iPr) ₂ ) 2- 151 MMTr NHP (OCH ₃ ) -OH Me (N (iPr) ₂ ) 2-152 MMTr NHP (OCH ₃ )-NHBz H (N (iPr) ₂ ) 2-153 MMTr NHP (OCH ₃ )-NHBz Me (N (iPr) ₂ ) ------------------------ In the above table, the preferred compound is Compound No. 1 −
3, 1-4, 1-7, 1-9, 1-10, 1-16, 1
-17, 1-19, 1-20, 1-21, 1-22, 1
-23, 1-27, 1-28, 1-31, 1-33, 1
-34, 1-40, 1-41, 1-43, 1-44, 1
−45, 1-46, 1-47, 1-49, 1-50, 1
-56, 1-57, 1-82, 1-83, 1-92, 1
-93, 1-94, 1-95, 1-96, 1-97, 1
-98, 1-99, 2-3, 2-4, 2-5, 2-6,
2-7, 2-8, 2-9, 2-10, 2-13, 2-1
4, 2-15, 2-16, 2-17, 2-18, 2-1
9, 2-20, 2-21, 2-22, 2-48, 2-5
9, 2-60, 2-61, 2-74, 2-75, 2-7
6, 2-77, 2-78, 2-79, 2-80, 2-8
1, 2-82, 2-83, 2-84, 2-85, 2-8
6, 2-87, 2-88, and 2-89, and more preferable compounds are Compound No. 1-
4, 1-22, 1-28, 1-46, 1-49, 1-5
0, 1-56, 1-57, 1-82, 1-83, 1-9
6, 1-97, 1-98, 1-99, 2-3, 2-4,
2-6, 2-13, 2-14, 2-16, 2-21, 2
-22, 2-48, 2-59, 2-60, 2-61, 2
-82, 2-83, 2-84, 2-85, 2-86, 2
-87, 2-88, and 2-89, and the most preferred compound is Compound No. 2-4: 3'-amino-3'-deoxy-
2'-O, 4'-C-methylene-5-methyluridine, Compound No. 2-14: 3'-azido-3'-deoxy-
2'-O, 4'-C-methylene-5-methyluridine, Compound No. 2-36: 3'-azido-3'-deoxy-
5'-O- (4,4'-dimethoxytrityl) -2'-
O, 4'-C-methylene-5-methyluridine, compound No. 2-48: 3'-azido-5'-O-tert-butyldiphenylsilyl-3'-deoxy-2'-O,
4'-C-methylene-5-methyluridine, and Compound No. 2-60: 3'-amino-3'-deoxy-
5'-O- (4,4'-dimethoxytrityl) -2'-
O, 4'-C-methylene-5-methyluridine Compound No. 2-83: 3'-amino-3'-deoxy-3'-N
-(4-monomethoxytrityl) -2'-O, 4'-C
-Methylene-5-methyluridine-5'-O- (2-cyanoethyl N, N-diisopropyl) phosphoramidite.

[0051]

BEST MODE FOR CARRYING OUT THE INVENTION The compound (1) of the present invention can be produced by the following method A.

[0052]

Embedded image

In the above process chart, R ¹ , R ² and B have the same meanings as described above.

R ⁷ represents a hydroxyl-protecting group, preferably an arylcarbonyl group such as benzoyl, α-naphthoyl or β-naphthoyl, or a lower group such as 2,4,6-trimethylbenzoyl or 4-toluoyl. “Aromatic acyl group” such as an alkylated arylcarbonyl group and an arylated arylcarbonyl group such as 4-phenylbenzoyl
And more preferably a benzoyl group.

R ⁸ represents a protecting group for a hydroxyl group, preferably, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl,
Tri-lower alkylsilyl groups such as triisopropylsilyl, tri-lower alkylsilyl groups substituted with one or two aryl groups such as diphenylmethylsilyl, t-butyldiphenylsilyl, diphenylisopropylsilyl, and phenyldiisopropylsilyl; "Silyl group";
Benzyl, α-naphthylmethyl, β-naphthylmethyl,
“1” such as diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl
A methyl group substituted with 3 to 3 aryl groups "; 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,
4,5-trimethylbenzyl, 4-methoxybenzyl,
4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4 ', 4 "-trimethoxytriphenylmethyl, 2-nitrobenzyl,
"Lower alkyl, lower alkoxy, halogen, methyl substituted with one to three aryl groups in which the aryl ring is substituted with a cyano group such as nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, and 4-cyanobenzyl. Group "
More preferably, a trimethylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group,
Benzyl group, triphenylmethyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group,
4,4′-dimethoxytriphenylmethyl group or 4,
4 ', 4 "-trimethoxytriphenylmethyl group.

R ⁹ represents a leaving group, preferably a lower alkylsulfonyl group such as methanesulfonyl and ethanesulfonyl, and a trifluoromethanesulfonyl group such as trifluoromethanesulfonyl.
It is an arylsulfonyl group such as a halogen-substituted lower alkylsulfonyl group or p-toluenesulfonyl, and more preferably a methanesulfonyl or p-toluenesulfonyl group.

R ¹⁰ represents a protecting group for a hydroxyl group, and is preferably formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, decanoyl, 1-methylheptadecanoyl, nonadecanoyl, or eicosanoyl. And a carboxylated alkylcarbonyl group such as succinoyl, glutaroyl, and adipoyl; a halogeno lower alkylcarbonyl group such as chloroacetyl, dichloroacetyl, trichloroacetyl, and trifluoroacetyl; a lower group such as methoxyacetyl. “Aliphatic acyl group” such as an alkoxy lower alkylcarbonyl group, an unsaturated alkylcarbonyl group such as (E) -2-methyl-2-butenoyl; benzoyl, α-naph Yl, arylcarbonyl groups such as β- naphthoyl, 2-bromobenzoyl, 4-chlorobenzoyl halogenoaryl carbonyl groups such as yl,
Lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl and 4-toluoyl, lower alkoxylated arylcarbonyl groups such as 4-anisoyl, 2-carboxybenzoyl, 3-carboxybenzoyl and 4-carboxybenzoyl A carboxylated arylcarbonyl group, a nitrated arylcarbonyl group such as 4-nitrobenzoyl or 2-nitrobenzoyl; a lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl,
An `` aromatic acyl group '' such as an arylated arylcarbonyl group such as phenylbenzoyl, and more preferably,
It is an "aliphatic acyl group", particularly preferably an acetyl group.

B ¹ represents a purin-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl group optionally having one or more optional substituents selected from the following α1 group: . (Α1 group) a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 6 carbon atoms, a mercapto group,
Mercapto group protected by a protecting group for nucleic acid synthesis, 1 carbon atom
To 6 alkylthio groups, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and
Halogen atom.

In method A, starting from compound (2),
In this method, cyclization is performed after the introduction of the substituent B to obtain the target compounds (1a), (1b) and (1c).

Here, the starting compound (2) was prepared by using a commercially available diacetone-D-glucose as a starting material in the literature (O.
T. Schmidt, Methods in Carbohydr. Chem., 4, 318 (1
964); JS Brimacombe and OA Ching, Carbhyd. R
es., 8, 82 (1968); TF Tam and B. Fraser-Reid, C
an. J. Chem., 57, 2818 (1979); SA Suzhkov, Nucl.
eosides & Nucleotides, 13, 2283 (1994)).

Hereinafter, each step of the method A will be described in detail. <Method A> (Step A-1) This step is a step of producing a compound (3) by deprotecting the protecting group of the primary hydroxyl group of the starting compound (2) in an inert solvent in the presence of a base. is there.

The solvent to be used is not particularly limited as long as it is used in a usual hydrolysis reaction, and examples thereof include water; alcohols such as methanol, ethanol and n-propanol; tetrahydrofuran and dioxane. Organic solvents such as ethers or a mixed solvent of water and the above organic solvent are used, preferably alcohols.

The base used is not particularly limited as long as it does not affect the other parts of the compound, but is preferably a metal alkoxide such as sodium methoxide; sodium carbonate, potassium carbonate, or the like. Alkali metal carbonates such as lithium carbonate; alkali metal hydroxides or aqueous ammonia such as sodium hydroxide, potassium hydroxide, lithium hydroxide and barium hydroxide, concentrated ammonia
Ammonia such as methanol is used, preferably an alkali metal carbonate.

The reaction temperature and the reaction time vary depending on the starting material, the solvent and the base used, and are not particularly limited.
To suppress side reactions, usually at 0 ° C to 150 ° C,
It is performed for 1 to 10 hours.

After completion of the reaction, the target compound (3) of this reaction is collected from the reaction mixture according to a conventional method. For example, neutralize the reaction solution, concentrate the reaction mixture, add an immiscible organic solvent such as water and ethyl acetate, wash with water, separate the organic layer containing the target compound, and dry over anhydrous sodium sulfate and the like. It is obtained by distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

(Step A-2) This step is carried out in an inert solvent
In this step, compound (3) obtained in step A-1 is reacted with a protecting agent for a hydroxyl group in the presence of a base to produce compound (4).

The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent. Examples thereof include aliphatic hydrocarbons such as hexane and heptane; benzene, toluene, Aromatic hydrocarbons such as xylene; methylene chloride, chloroform, carbon tetrachloride, halogenated hydrocarbons such as dichloroethane, chlorobenzene, dichlorobenzene; ethyl formate;
Esters such as ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; Formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-
Amides such as methyl-2-pyrrolidone, N-methylpyrrolidinone, and hexamethylphosphorotriamide can be mentioned, and methylene chloride is preferred.

The base to be used is not particularly limited as long as it is used as a base in a usual reaction. For example, N-methylmorpholine, triethylamine, tributylamine, diisopropylethylamine,
Dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4- (N,
Organic bases such as N-dimethylamino) pyridine, 2,6-di (tert-butyl) -4-methylpyridine, quinoline, N, N-dimethylaniline and N, N-diethylaniline can be mentioned. And preferably triethylamine.

The protecting agent used includes, for example, t
-Butyldimethylsilyl chloride, trimethylsilyl chloride, triethylsilyl chloride, triethylsilyl bromide, triisopropylsilyl chloride, dimethylisopropylsilyl chloride, diethylisopropylsilyl chloride, t-butyldiphenylsilyl chloride,
Silyl halides such as diphenylmethylsilyl chloride and triphenylsilyl chloride, 4-methoxytriphenylmethyl chloride, 4,4'-dimethoxytriphenylmethyl chloride, 4,4 ', 4''-trimethoxytriphenylmethyl chloride And aralkyl halides such as benzyl chloride, benzyl bromide and p-methoxybenzyl bromide, and t-butyldiphenylsilyl chloride is preferred.

The reaction temperature is usually from -20 ° C to the reflux temperature of the solvent used, preferably from 0 ° C to the reflux temperature of the solvent used.

The reaction time mainly depends on the reaction temperature, starting compound,
Although it depends on the type of the base or the solvent used, it is usually from 10 minutes to 3 days, preferably from 1 to 3 days.
Hours to 24 hours.

After completion of the reaction, the target compound (4) of this reaction is collected from the reaction mixture according to a conventional method. For example, neutralizing the reaction solution, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous sodium sulfate, etc., and distilling off the solvent. Is obtained.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography or the like, particularly when a compound having R8 introduced into an undesired hydroxyl group is formed.

(Step A-3) This step is carried out in an inert solvent
Compound (4) obtained in step A-2 in the presence of a base catalyst
And reacting the compound with a leaving group-introducing reagent to produce a compound (5).

Examples of the solvent to be used include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxy Ethers such as ethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; formamide , N, N
Amides such as -dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, hexamethylphosphorotriamide; sulfoxides such as sulfolane; and pyridines. And preferably methylene chloride.

The base catalyst used is preferably
Bases such as triethylamine, pyridine, dimethylaminopyridine.

Examples of the leaving group-introducing reagent to be used include alkylsulfonyl halides such as methanesulfonyl chloride and ethanesulfonyl bromide; and arylsulfonyl halides such as p-toluenesulfonyl chloride. Are methanesulfonyl chloride and p-toluenesulfonyl chloride.

The reaction temperature depends on the starting compound used, the solvent, the leaving group-introducing reagent, and the base catalyst.
The temperature is 0 ° C to 50 ° C, preferably 10 ° C to 40 ° C.

The reaction time varies depending on the starting compound used, the solvent, the leaving group-introducing reagent, the base catalyst and the reaction temperature, but is usually from 10 minutes to 24 hours, preferably from 1 to 15 hours. .

After completion of the reaction, the desired compound (5)
Is collected from the reaction mixture according to a conventional method. For example, neutralize the reaction solution, concentrate the reaction mixture, add an immiscible organic solvent such as water and ethyl acetate, wash with water, separate the organic layer containing the target compound, and dry over anhydrous sodium sulfate and the like. It is obtained by distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

(Step A-4) This step is a step of producing a compound (6) by reacting the compound (5) obtained in the step A-3 with an acid anhydride in a solvent in the presence of an acid catalyst. It is.

Examples of the solvent used include ethers such as diethyl ether, dioxane and tetrahydrofuran; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethyl Amides such as acetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, and hexamethyl phosphorotriamide; organic acids such as acetic acid; and the like, preferably acetic acid.

The acid catalyst to be used includes, for example, inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and preferred is sulfuric acid (particularly concentrated sulfuric acid).

Examples of the acid anhydride to be used include anhydrides of lower aliphatic carboxylic acids such as acetic anhydride and propionic anhydride, and acetic anhydride is preferred.

The reaction temperature varies depending on the starting compound, solvent, acid catalyst and acid anhydride used, but is usually 0 ° C. to 50 ° C., preferably 10 ° C. to 40 ° C.

The reaction time varies depending on the used starting compound, solvent, acid catalyst, acid anhydride and reaction temperature, but is usually from 10 minutes to 12 hours, preferably from 30 minutes to 6 hours.

After completion of the reaction, the target compound (6) of this reaction is collected from the reaction mixture according to a conventional method. For example, it is obtained by adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating an organic layer containing the target compound, drying over anhydrous sodium sulfate or the like, and distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

(Step A-5) This step is carried out in an inert solvent
Compound (6) obtained in step A-4 in the presence of an acid catalyst
References (H. Vorbrueggen, K. Krolikiewicz and B, B
ennua, Chem. Ber., 114, 1234-1255 (1981)), and reacting a trimethylsilylated compound corresponding to a purine or pyrimidine which may have a desired substituent to give compound (7). This is the manufacturing process.

Solvents used include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, chlorobenzene and dichlorobenzene. Nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N
-Methyl-2-pyrrolidone, N-methylpyrrolidinone,
Amides such as hexamethyl phosphorotriamide; carbon sulfide and the like can be mentioned, and preferred is 1,2-dichloroethane.

Examples of the acid catalyst used include, for example, AlCl
₃ , Lewis acid catalysts such as SnCl ₄ , TiCl ₄ , ZnCl ₂ , BF ₃ , and trimethylsilyl trifluoromethanesulfonate, and the like, preferably tin tetrachloride (SnCl ₄ ).

The reaction temperature depends on the starting compound, solvent and acid catalyst used, but is usually 0 to 100 ° C., preferably 30 to 80 ° C.

The reaction time varies depending on the used starting compound, solvent, acid catalyst and reaction temperature, but is usually 1 hour to 3 days, preferably 1 hour to 2 days.

After completion of the reaction, the target compound (7) of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction solution is neutralized, water and an immiscible organic solvent such as ethyl acetate or methylene chloride are added, and after washing with water, the organic layer containing the target compound is separated, dried over anhydrous sodium sulfate or the like, and then the solvent is distilled off. Obtained by leaving.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

(Step A-6) This step is carried out in an inert solvent
Compound (7) obtained in step A-5 in the presence of a base catalyst
In which cyclization is carried out to produce compound (1a).

The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent. Isobutanol, t-butanol, isoamyl alcohol,
Alcohols such as diethylene glycol, glycerin, octanol, cyclohexanol, and methyl cellosolve can be mentioned, and methanol is more preferable.

Examples of the base catalyst used include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; sodium methoxide and sodium ethoxide. Such alkali metal alkoxides; ammonia water and the like can be mentioned, preferably an alkali metal carbonate, and more preferably potassium carbonate.

The reaction temperature varies depending on the starting compound, solvent and base catalyst used, but is usually 0 ° C to 50 ° C, preferably 10 ° C to 30 ° C.

The reaction time varies depending on the used starting compound, solvent, base catalyst and reaction temperature, but is usually 1 hour to 3 days, preferably 3 hours to 2 days.

After completion of the reaction, the desired compound (1a)
Is collected from the reaction mixture according to a conventional method. For example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous sodium sulfate or the like, and distilling off the solvent. can get.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

(Step A-7) This step is carried out in an inert solvent
In this step, compound (1b) is produced by reacting compound (1a) obtained in step A-6 with a deprotection reagent. However, if deprotection is not required, the process can proceed to the next step without performing this step.

The method of deprotection varies depending on the type of the protecting group, but is not particularly limited as long as it does not cause other side reactions.
thesis ”(Theodora W. Greene, 1981, A Wiley-
Interscience Publication)).

When a plurality of different types of protecting groups are present, deprotection can be performed sequentially by appropriately combining these methods.

In particular, when the protecting group is (1) an “aliphatic acyl group or an aromatic acyl group”, (2) a “methyl group substituted with 1 to 3 aryl groups” or a “lower alkyl, lower alkoxy, A methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a halogen or cyano group ",
(3) In the case of a "silyl group", it can be carried out by the following method. (1) In the case of an aliphatic acyl group or an aromatic acyl group, the reaction is usually carried out by reacting a base in an inert solvent.

The solvent to be used is not particularly limited as long as it is one used in a usual hydrolysis reaction. For example, water; alcohols such as methanol, ethanol and n-propanol; tetrahydrofuran and dioxane. Organic solvents such as ethers or a mixed solvent of water and the above-mentioned organic solvents are used, preferably alcohols.

The base used is not particularly limited as long as it does not affect the other parts of the compound, but is preferably a metal alkoxide such as sodium methoxide; sodium carbonate, potassium carbonate, or the like. Alkali metal carbonates such as lithium carbonate; alkali metal hydroxides or aqueous ammonia such as sodium hydroxide, potassium hydroxide, lithium hydroxide and barium hydroxide, concentrated ammonia
Ammonia such as methanol is used, preferably an alkali metal carbonate.

The reaction temperature and the reaction time vary depending on the starting material, the solvent and the base used, and are not particularly limited.
To suppress side reactions, usually at 0 ° C to 150 ° C,
It is performed for 1 to 10 hours.

After the completion of the reaction, the target compound (1b) of this reaction
Is collected from the reaction mixture according to a conventional method. For example,
It is obtained by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying over anhydrous sodium sulfate, and evaporating the solvent. .

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization or silica gel column chromatography. (2) the protecting group is substituted with “1 to 3 aryl groups substituted with 1 to 3 aryl groups” or “1 to 3 aryl groups in which the aryl ring is substituted with lower alkyl, lower alkoxy, halogen, and cyano groups; In the case of "a methyl group", the reaction is performed using a reducing agent in an inert solvent.

The solvent used is preferably an alcohol such as methanol, ethanol or isopropanol; an ether such as diethyl ether, tetrahydrofuran or dioxane; or an aromatic hydrocarbon such as toluene, benzene or xylene. Aliphatic hydrocarbons such as hexane and cyclohexane; esters such as ethyl acetate and propyl acetate; organic acids such as acetic acid; and mixed solvents of these organic solvents and water.

The reducing agent to be used is not particularly limited as long as it is usually used in a catalytic reduction reaction, but is preferably palladium carbon, Raney nickel, platinum oxide,
Examples include platinum black, rhodium-aluminum oxide, triphenylphosphine-rhodium chloride, and palladium-barium sulfate.

Although the pressure is not particularly limited, it is usually 1 to 1
Performed at 0 atm.

The reaction temperature is from 0 ° C. to 60 ° C., preferably from 20 ° C. to 40 ° C.

The reaction time is from 10 minutes to 24 hours,
Preferably, it is 1 to 3 hours.

After completion of the reaction, the desired compound (1b)
Is collected from the reaction mixture according to a conventional method. For example,
From the reaction mixture, remove the reducing agent, add an immiscible organic solvent such as water and ethyl acetate, wash with water, separate the organic layer containing the target compound, and dry over anhydrous sodium sulfate and the like.
It is obtained by distilling off the solvent.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

In the case of "a methyl group substituted with three aryl groups", that is, a trityl group, the reaction can be carried out using an acid.

In this case, examples of the solvent to be used include aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, chlorobenzene and dichlorobenzene. Halogenated hydrocarbons; alcohols such as methanol, ethanol, isopropanol and tert-butanol; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethyl Acetamide,
Amides such as N-methyl-2-pyrrolidone, N-methylpyrrolidinone, hexamethyl phosphorotriamide; organic acids such as acetic acid; and organic acids (particularly acetic acid) or alcohols are preferred. (Especially te
rt-butanol).

The acid used is preferably acetic acid or trifluoroacetic acid.

The reaction temperature is from 0 ° C. to 60 ° C., preferably from 20 ° C. to 40 ° C.

The reaction time is from 10 minutes to 24 hours,
Preferably, it is 1 to 3 hours.

After completion of the reaction, the desired compound (1b)
Is collected from the reaction mixture according to a conventional method. For example,
Neutralize the reaction mixture, add an immiscible organic solvent such as water and ethyl acetate, wash with water, separate the organic layer containing the target compound, dry over anhydrous sodium sulfate, etc., and evaporate the solvent. It is.

The obtained compound can be further purified, if necessary, by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (3) When the protecting group is a “silyl group”, it is usually treated with a compound that generates a fluorine anion such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, and potassium fluoride. Or acetic acid, methanesulfonic acid,
It can be removed by treatment with an organic acid such as paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid or an inorganic acid such as hydrochloric acid.

When removing with a fluorine anion,
The reaction may be accelerated by adding organic acids such as formic acid, acetic acid, propionic acid.

The solvent used is not particularly limited as long as it does not hinder the reaction and dissolves the starting materials to some extent, but is preferably diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol Ethers such as dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; water; organic acids such as acetic acid; and mixed solvents thereof.

The reaction temperature is from 0 ° C. to 100 ° C., preferably from 20 ° C. to 70 ° C.

The reaction time is 5 minutes to 48 hours, preferably 1 to 24 hours.

After completion of the reaction, the desired compound (1b) of this reaction
Is collected from the reaction mixture according to a conventional method. For example,
It is obtained by distilling off the solvent and purifying by silica gel column chromatography.

(Step A-8) This step is carried out in an inert solvent
A step of reducing the azide group of the compound (1b) obtained in step A-7 to an amino group in the presence of hydrogen and a catalyst, and optionally protecting the amino group to produce a compound (1c) It is.

The solvent used is not particularly limited as long as it does not participate in the reaction, but is preferably an alcohol such as methanol, ethanol or isopropanol, or an ether such as diethyl ether, tetrahydrofuran or dioxane. , Aromatic hydrocarbons such as toluene, benzene, xylene, aliphatic hydrocarbons such as hexane and cyclohexane, esters such as ethyl acetate and propyl acetate, formamide, dimethylformamide, dimethylacetamide, N-methyl Amides such as -2-pyrrolidone and hexamethyl phosphorotriamide, fatty acids such as formic acid and acetic acid, water, or a mixed solvent thereof is used.

The catalyst to be used is not particularly limited as long as it is usually used in a catalytic reduction reaction, but is preferably palladium carbon, palladium black, Raney nickel, platinum oxide, platinum black, rhodium- Aluminum oxide,
Triphenylphosphine-rhodium chloride, palladium-
Barium sulfate is used.

Although the pressure is not particularly limited, it is usually 1 to 1
Performed at 0 atm.

The reaction temperature and the reaction time vary depending on the starting material, the solvent, the type of the catalyst and the like.
0 ° C. (preferably 20 ° C. to 40 ° C.), 5 minutes to 48 hours (preferably 30 minutes to 10 hours).

After completion of the reaction, the desired compound (1c) of this reaction
Is collected from the reaction mixture according to a conventional method. For example, it is obtained by removing the catalyst by filtration and distilling off the solvent.

Here, if desired, the aforementioned literature (Protec
The amino group can also be protected according to the method described in “Tive Groups in Organic Synthesis”.

Further, if desired, monosubstituted-chloro (alkoxy) which is usually used for amidite formation in an inert solvent.
By reacting phosphines or disubstituted-alkoxyphosphines, the 5′-hydroxyl group is H-phosphonated, or
It can be phosphoramidite.

The solvent used is not particularly limited as long as it does not affect the reaction, but is preferably an ether such as tetrahydrofuran, diethyl ether or dioxane; methylene chloride, chloroform or tetrachloride. Examples include halogenated hydrocarbons such as carbon, dichloroethane, chlorobenzene, and dichlorobenzene.

The mono-substituted chloro (alkoxy) phosphines used include, for example, chloro (morpholino) methoxyphosphine, chloro (morpholino) cyanoethoxyphosphine, chloro (dimethylamino) methoxyphosphine, chloro (dimethylamino) cyanoethoxy. Phosphines such as phosphine, chloro (diisopropylamino) methoxyphosphine, chloro (diisopropylamino) cyanoethoxyphosphine;
Preferably, chloro (morpholino) methoxyphosphine,
Chloro (morpholino) cyanoethoxyphosphine, chloro (diisopropylamino) methoxyphosphine, and chloro (diisopropylamino) cyanoethoxyphosphine.

When a monosubstituted chloro (alkoxy) phosphine is used, a deoxidizing agent is used. In this case, the deoxidizing agent used is a heterocyclic amine such as pyridine or dimethylaminopyridine. And aliphatic amines such as trimethylamine, triethylamine and diisopropylamine, preferably aliphatic amines (particularly diisopropylamine).

Examples of the di-substituted alkoxyphosphines used include, for example, bis (diisopropylamino) cyanoethoxyphosphine, bis (diethylamino) methanesulfonylethoxyphosphine, bis (diisopropylamino) (2,2,2-trichloroethoxy) Examples thereof include phosphines such as phosphine and bis (diisopropylamino) (4-chlorophenylmethoxy) phosphine, and preferred is bis (diisopropylamino) cyanoethoxyphosphine.

When di-substituted alkoxyphosphines are used, an acid is used. In this case, the acid used is preferably tetrazole, acetic acid or p-toluenesulfonic acid.

The reaction temperature is not particularly limited, but is usually 0 to 80 ° C., preferably room temperature.

The reaction time varies depending on the starting materials, reagents, temperature and the like used, but is usually 5 minutes to 30 hours, preferably 30 minutes to 10 hours when the reaction is carried out at room temperature.

After completion of the reaction, the desired compound is neutralized, for example, by appropriately neutralizing the reaction mixture.
After removal by filtration, an immiscible organic solvent such as water and ethyl acetate is added, and after washing with water, the organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then obtained by distilling off the solvent. . If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.

Further, if desired, 5'-
After the hydroxyl group is reacted with tris- (1,2,4-triazolyl) phosphite, water can be added to form an H-phosphonate.

The reaction temperature is not particularly limited, but is usually -2.
It is 0 to 100 ° C, preferably 10 to 40 ° C.

The reaction time varies depending on the starting materials, reagents, temperature and the like to be used, but is usually 5 minutes to 30 hours, preferably 30 minutes when the reaction is carried out at room temperature. The compound is, for example, appropriately neutralizing the reaction mixture, and, if insolubles are present, removing by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, and washing the target compound. It is obtained by separating an organic layer containing the organic layer, drying the organic layer with anhydrous magnesium sulfate or the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.

Using the compound (1d) of the present invention, an N3′-P5 ′ type compound of the present invention in which a nitrogen atom at the 3′-position and an oxygen atom at the 5′-position are bonded with phosphoric acid by the method B described below. Oligonucleotide analogs can be synthesized.

[0153]

Embedded image

[0154] In the above process chart, B ¹ and R ⁸ is the same as defined above, and the B ¹ of formula (1d) B ¹ and equation (8) may be the same or different.

R ¹¹ is succinic acid CPG (succinyl Contr.
Shows resins commonly used for oligonucleotide synthesis, such as olled Pore Glass or Tentagel.

CEO represents a 2-cyanoethoxy group.

Hereinafter, each step of the method B will be described in detail. (Step B-1) In this step, oxidative phosphorylation coupling is carried out by reacting compound (1d) with compound (8) to produce compound (9).
Reference (1) (Nucleic Acids Research, Vol. 2
3, No. 14, pp. 2661-2668, 1995).

Compound (1d) is the same as compound (1c) obtained in step A-8, except that the 5′-hydroxyl group is protected and that base B has an amino group. Is a protected compound.

Compound (8) is a compound that can be synthesized from compound (1c) obtained in step A-8 in the same manner as in Reference (1). (Step B-2) This step is a step of synthesizing an oligonucleotide from the compound (9) obtained in Step B-1.

First, the protecting group R ⁸ for the hydroxyl group of the compound (9) is deprotected by using the method of the above-mentioned step A-7.
Phosphorylation is carried out in the same manner as in the document (1), and the compound (1d) is reacted in the same manner as in the above step B-1.
By repeating this cycle, an oligonucleotide can be synthesized.

The length of the resulting oligonucleotide analog is usually 2 to 50, preferably 5 to 30, as nucleoside units.

Using the compound (1e) of the present invention, an N3′-P5 ′ type compound of the present invention in which a 3 ′ nitrogen atom and a 5 ′ oxygen atom are bonded with phosphoric acid also by the method C described below. Can be synthesized.

[0163]

Embedded image

In the above process chart, B¹, R^ThreeAnd R¹¹Is
Is the same as that of the formula (1e), ¹And B in equation (10)¹
And may be the same or different.

Hereinafter, each step of the method C will be described in detail. (Step C-1) In this step, the protecting group R ³ of the 3′-amino group of the compound (10) is deprotected by using the method of the above-mentioned step A-8, and the compound obtained is obtained by adding the compound (1e) To produce a compound (11) by reacting
(Nucleic Acids Research, Vol. 26, No. 18, pp. 4160
-4167, 1998).

The compound (1e) is the same as the compound (1c) obtained in the step A-8, except that the compound represented by the formula
It is a compound having P (R ^4a ) R ^4b (R ^4a and R ^4b have the same meanings as described above), and when the amino group is present in the base B, the amino group is protected.

Compound (10) was prepared from compound (1c) obtained in step A-8 by reference (1) or reference (2) (Oligonulcotide synthesis. A practical appr.
oach, edited by MJGait, (1984) IRL Press Limited
pp35-81). (Step C-2) This step is a step of synthesizing an oligonucleotide from the compound (11) obtained in Step C-1.

Compound (1) was prepared in the same manner as in the above-mentioned Step C-1.
The compound (1e) is reacted with 1) obtained by deprotecting the protecting group R ³ of the 3′-amino group in 1). Oligonucleotides can be synthesized by repeating this cycle, and high-purity oligonucleotides can be obtained by deprotection and purification in the same manner as in Reference (1) or Reference (2). In place of compound (11) used in Steps C-1 and C-2, Universal Q Supp
A desired oligonucleotide can also be synthesized using ort or Universal Support (manufactured by Glen Research).

The chain length of the obtained oligonucleotide analog is usually 2 to 50, preferably 5 to 30, as nucleoside units.

The obtained oligonucleotide analog is hardly decomposed by various nucleases, and can exist in the living body for a long time after administration to the living body. Further, for example, a stable double strand is formed with mRNA to inhibit the biosynthesis of a pathogenic protein, or the double-stranded DN
A can form a triplex with A to inhibit transcription into mRNA or suppress the growth of virus that infects cells.

Therefore, the oligonucleotide analogues of the present invention are expected to be used as anti-tumor agents, anti-viral agents and other pharmaceuticals that inhibit the action of specific genes and treat diseases.

The oligonucleotide analog of the present invention can be formulated into a parenteral administration preparation or a liposome preparation by blending conventional auxiliaries such as a buffer and / or a stabilizer. In addition, as a topical preparation, a common pharmaceutical carrier can be compounded to prepare an ointment, cream, liquid, salve or the like.

The dosage varies depending on the condition, age, administration method and the like.
It is desirable to use 1 mg / kg body weight (preferably 0.01 mg / kg body weight) and, as an upper limit, 100 mg / kg body weight (preferably 10 mg / kg body weight) once or several times a day depending on the symptoms.

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the scope of the present invention is not limited thereto.

[0175]

EXAMPLES (Example 1) 3'-azido-5'-O-tert-butyldiphenylsilyl-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine (exemplary compound number 2-48) To a methanol solution (7 ml) of the compound of Reference Example 5 (200 mg, 0.27 mmol) was added potassium carbonate (41 mg, 0.29 mmo) at 0 ° C.
l) was added, and the mixture was stirred at room temperature for 4.5 hours. Then, potassium carbonate (34 mg, 0.25 mmol) was further added, and the mixture was stirred for 23 hours. After methanol was distilled off, water was added to the residue, extracted with ethyl acetate, and washed with saturated saline. The organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off, and the residue was purified by silica gel column chromatography (ethyl acetate: n-hexane = 2: 1) to give the target compound as colorless crystals (142 mg, 0.27 mmol,
100%). mp 93-95 ° C. IR νmax (KBr): 3169, 3047, 2956, 2888, 2859, 211
. 7, 1696, 1275, 1109 cm - 1 1 H-NMR (CDCl 3) δ: 1.12 (9H, s), 1.65 (3H, s), 3.7
8, 3.84 (2H, AB, J = 8Hz), 3.90, 4.08 (2H, AB, J =
12.5 Hz), 4.02 (1H, s), 4.67 (1H, s), 5.67 (1H, s)
s), 7.54 (1H, s), 7.39-7.48 (6H, m), 7.67-7.71 (4
H, m), 8.46 (1H, br s). ¹³ C-NMR (CDCl ₃ ) δ: 12.3, 19.5, 27.0, 58.7, 60.3,
71.4, 77.2, 78.6, 87.2, 90.1, 110.8, 128.0, 130.1,
130.2, 131.7, 132.3, 133.7, 135.1, 135.4, 149.6,
163.6. (Example 2) 3'-Azido-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine (Exemplary Compound No. 2-14) The compound of Example 1 under a nitrogen stream. (140 mg, 0.26 mmol)
In anhydrous tetrahydrofuran (5 ml) was added tetrabutylammonium fluoride (1.0 M in THF, 290 μl,
0.29 mmol) and stirred at room temperature for 1 hour. After evaporating the solvent, the residue was purified by silica gel column chromatography (ethyl acetate: n-hexane = 25: 1) to obtain the desired product as a white powder (65.7 mg, 0.22 mmol, 85%). mp 94-96 ° C. IR νmax (KBr): 3163, 3046, 2118, 1692, 1468, 127
^{. 3, 1062 cm -1 1 H} -NMR (CD 3 OD) δ: 1.89 (3H, s), 3.76, 3.86 (2H, A
(B, J = 8 Hz), 3.85, 3.95 (2H, AB, J = 13 Hz), 4.03
(1H, s), 4.58 (1H, s), 5.58 (1H, s), 7.70 (1H, s)
s). ¹³ C-NMR (CD ₃ OD) δ: 12.8, 57.3, 61.2, 72.4, 79.8,
88.3, 91.0, 110.8, 136.3, 151.5, 166.1. (Example 3) 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine (Exemplary Compound No. 2-4) The compound of Example 2 (64 ml) was added to a solution of 10% palladium carbon (28 mg) in anhydrous tetrahydrofuran (5 ml) under a stream of hydrogen.
mg, 0.22 mmol) in ethanol (3 ml) and stirred at room temperature for 0.5 hour. After filtration of the reaction solution, the solvent was distilled off, and the target product was white powder (59 mg, 0.22 mmol, 100%).
As obtained. mp 243-246 ° C. IR νmax (KBr): 3459, 3365, 1699, 1447, 1273, 1054
^{. cm -1 1 H-NMR (} C 5 D 5 N) δ: 1.83 (3H, s), 3.62 (1H, s), 3.9
2, 4.14 (2H, AB, J = 8Hz), 4.24 (2H, s), 4.54 (1H,
s), 5.97 (1H, s), 7.90 (1H, s). ¹³ C-NMR (C ₅ D ₅ N) δ: 12.8, 54.2, 57.2, 71.6, 81.4,
91.1, 109.5, 150.8, 164.3. (Example 4) 3'-azido-3'-deoxy-5'-O- (4,4'-dimethoxytrityl) -2'-O, 4'-C-methylene- 5-Methyluridine (Exemplified Compound No. 2-36) Dimethoxytrityl chloride (4 ml) was added to a pyridine solution (6 ml) of the compound of Example 2 (300 mg, 1.02 mmol) under a nitrogen stream.
15 mg, 1.22 mmol) and dimethylaminopyridine (12.5
mg, 0.10 mmol) and stirred at room temperature for 20.5 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (n-hexane: ethyl acetate = 2: 1 → 1: 1) to give the title compound as a pale yellow foam (462 mg, (0.78 mmol, 76%). mp 125-128 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.66 (3H, s), 3.32, 3.65 (2H, AB
q, J = 11 Hz), 3.78 (2H, s), 3.80 (6H, s), 4.13 (1H,
s), 4.63 (1H, s), 5.67 (1H, s), 6.86 (4H, dd, J = 2
Hz, 9 Hz), 7.23-7.45 (9H, m), 7.73 (1H, s), 8.04
(1H, brs). (Example 5) 3'-amino-3'-deoxy-5'-O- (4,4'-dimethoxytrityl) -2'-O, 4'-C-methylene-5- Methyluridine (Exemplary Compound No. 2-60) The compound of Example 4 (110 mg, 0.18 mmol) under a nitrogen stream
Triphenylphosphine in pyridine solution (2.5 ml)
(94.0 ml, 0.36 mmol) was added and stirred at room temperature for 3.5 hours. Subsequently, a 28% aqueous ammonia solution (5.5 ml) was added, and the mixture was stirred at room temperature for 24 hours. The solvent was evaporated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (chloroform: ethanol = 20: 1) to give the title compound (103 mg, 0.18 mmol, 97) as a pale yellow foam.
%). mp 131-134 ° C. ¹ H-NMR (Pyridine-d ₅ ) δ: 1.89 (3H, s), 3.71 (6H,
s), 3.77 (1H, s), 3.84 (2H, s), 3.99, 4.10 (2H, A
Bq, J = 8 Hz), 4.69 (1H, s), 6.04 (1H, s), 7.03-7.
87 (13H, m), 8.58 (1H, s). (Example 6) 3'-amino-3'-deoxy-5'-O- (4,4'-dimethoxytrityl) -2'-O, 4 '-C-methylene-5-methyluridinyl- (3' → 5 ')
-3'-O- (tert-butyldimethylsilyl) thymidine 2-cyanoethyl ester The compound of Reference Example 6 (14.5 mg, 0.28 μmo
l) Acetonitrile solution (0.3 ml) was added with the compound of Example 5 (10.0 mg, 18 μmol) in carbon tetrachloride (0.3 ml), triethylamine (0.05 ml, 0.36 mmol), and acetonitrile solution (0.2 ml). Stirred for 14.5 hours. The solvent was distilled off under reduced pressure, and the obtained crude product was subjected to silica gel column chromatography (n-hexane: ethyl acetate = 1: 1 → 0: 1)
The title compound (13.0 mg, 1
2.5 μmol, 71%). ^{. mp 101-105 ℃ 31 P-NMR} (CDCl 3) δ:. 7.68, 8.24 Mass (FAB):. m / z 1043 (M + + H) ( Example 7) 3'-amino-3'-deoxy -5'-O- (4,4'-dimethoxytrityl) -2'-O, 4'-C-methylene-5-methyluridinyl- (3 '→ 5')
-3'-O- (tert-butyldimethylsilyl) thymidine methyl ester Compound of Reference Example 7 (22.1 mg, 51 μmol) under nitrogen stream
To acetonitrile solution (0.3 ml) of Example 5 was added a solution of the compound of Example 5 (10.0 mg, 18 μmol) in carbon tetrachloride (0.3 ml), triethylamine (0.05 ml, 0.36 mmol) and acetonitrile solution (0.2 ml), and the mixture was stirred at room temperature for 18 hours. Stirred. After water was added to the reaction solution, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was subjected to silica gel column chromatography (dichloromethane: methanol = 20:
1,30: 1) to give the title compound as a white powder.
(6.9 mg, 6.87 μmol, 39%). mp 118-122 ° C. ³¹ P-NMR (CDCl ₃ ) δ: 11.20, 11.30. Mass (FAB): m / z 10
Example 8 3'-Amino-3'-deoxy-5'-O- (4,4'-dimethoxytrityl) -2'-O, 4'-C-methylene- 5-methyluridinyl- (3 '→ 5')
-Thymidine methyl ester The compound of Example 7 (13.9 mg, 14 μmol) under nitrogen stream
In tetrahydrofuran solution (1 ml) in tetrabutylammonium fluoride (1.0 M,
15 μl, 15 μmol) and stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (ethyl acetate: ethanol = 5: 1) to give the title compound (9.7 mg, 10.9 mg) as colorless crystals.
μmol, 78%). ^{. mp 157-160 ℃ 31 P-NMR} (CD 3 OD) δ:. 11.15, 11.23 Mass (FAB):. m / z 912 (M + + Na) ( Example 9) 3'-amino-3'- Deoxy-5'-O- (4,4'-dimethoxytrityl) -2'-O, 4'-C-methylene-5-methyluridinyl- (3 '→ 5')
-2 '-[Cyanoethoxy (diisopropylamino) phosphino] thymidine methyl ester The compound of Example 8 (10.0 mg, 11 μm
l), and diisopropylammonium tetrazolide (15.5
mg, 77 μmol) in acetonitrile solution (0.6 ml), tetrahydrofuran (0.2 ml) was added, and 2-cyanoethyldiisopropylchlorophosphoramidite (39.8 mg, 132 μm) was added.
mol) was added and the mixture was stirred at room temperature for 25 hours. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (ethyl acetate: triethylamine = 99: 1 → ethyl acetate: ethanol: triethylamine = 100: 10: 1), and then dichloromethane, n- Reprecipitate with hexane to give the title compound (3.8 mg, 3.5 μmol,
31%). mp 113-116 ° C. ³¹ P-NMR (CD ₃ OD) δ: 8.67, 8.77, 9.07, 9.28, 148.53,
148.93, 148.99, 149.03. (Example 10) Synthesis of oligonucleotide analog Nucleic acid synthesizer (Pharmacia Gene Assembler Plus)
Was performed on a 0.2 μmol scale. The concentration of solvents, reagents and phosphoramidites in each synthesis cycle is the same as in the case of natural oligonucleotide synthesis, and all solvents, reagents and phosphoramidites of natural nucleosides are Ph
The one manufactured by armacia was used. UniversalQ CPG (0.2μmo
l, manufactured by Glen Research), the DMTr group was deprotected with trichloroacetic acid, and the resulting hydroxyl group was repeatedly subjected to a condensation reaction using the compound of Example 9 and an amidite for the synthesis of natural nucleotides. Analogs were synthesized. The synthesis cycle is as follows.

Synthesis cycle 1) detritylation trichloroacetic acid / dichloromethane; 60
sec 2) coupling phosphoramidite (25eq), tetrazole
3) capping 1-methylimidazole / acetonitrile, acetic anhydride / 2,4,6-collidine / acetonitrile; 36sec 4) oxidation iodine / water / pyridine / acetonitrile; 6s
ec In the above, when performing the reaction using the compound of Example 10 in cycle 2), the reaction was performed for 30 minutes, and when using another phosphoramidite, the reaction was performed for 2 minutes.

After synthesizing the oligonucleotide having the target sequence and performing the synthesis cycle up to 1) to deprotect the 5′-dimethoxytrityl group, the oligomer is cut out from the support by treatment with concentrated aqueous ammonia according to a conventional method. The protecting group on the phosphorus atom, the cyanoethyl group, was removed, and the protecting group on the nucleobase was removed.

Purification was performed by reverse phase HPLC to obtain the target oligonucleotide.

According to this synthesis method, it has a sequence represented by the following sequence: 5′-ttttttttttnt-3 ′ (SEQ ID NO: 1 in the sequence listing), and n of base number 11 is 3′-amino-
An oligonucleotide analog that was 3′-deoxy-2′-O, 4′-C-methylene-5-methyluridine (hereinafter referred to as “oligonucleotide (1)”) was obtained. (Yield 8.5 nmol (4.3% yield)) Purification of the resulting modified oligonucleotide analog was performed by reversed-phase HPLC (HPLC: GILSON
Company; Model 302, Column: CHEMCO CHEMCOBOND 5-ODS-H
(7.8 x 300 mm); 0.1 M triethylamine acetate aqueous solution (TEA
A), pH7; 10 → 12.5% CH ₃ CN / 40min, linear gradient;
(50 ° C; 2.5 ml / min; 254 nm), and the fraction eluted at 25.4 minutes was collected. Example 11 Synthesis of Oligonucleotide Analog 5'-O-dimethoxytrityl-N-4-benzoyl-5-methyl-
2'-deoxycytidine-3'-O- (2-cyanoethyl) N,
Using N-diisopropyl phosphoramidite (manufactured by Pharmacia), in the same manner as in Example 11, the following sequence: 5′-tttttmtntmtmtmt-3 ′ (SEQ ID NO: 2 in the sequence listing)
Wherein m is 5-methyl 2'-deoxycytidine and n is 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine An oligonucleotide analog (hereinafter, referred to as “oligonucleotide (2)”) was obtained. (Yield 7.1 nmol (3.5% yield)) Purification of the resulting modified oligonucleotide analog was performed by reversed-phase HPLC (HPLC: GILSON
Company; Model 302, Column: CHEMCO CHEMCOBOND 5-ODS-H
(7.8 x 300 mm); 0.1 M triethylamine acetate aqueous solution (TEA
A), pH7; 10 → 12% CH ₃ CN / 40min, linear gradient; 50
C .; 2.5 ml / min; 254 nm) and fractions eluted at 22.5 minutes were collected. (Example 12) 3'-amino-3'-deoxy-5'-O-tert-butyldiphenylsilyl-2'-O, 4'-C-methylene-5-methyluridine (Exemplary Compound No. 2-72) Under a nitrogen stream, triphenylphosphine (4 ml) was added to a pyridine solution (2 ml) of the compound of Example 1 (50 mg, 0.09 mmol).
9.2 mg, 0.19 mmol) and stirred at room temperature for 100 minutes. 28%
Aqueous ammonia (5 ml) was added, and the mixture was stirred at room temperature for 20 hours. After distilling off the solvent, the residue was subjected to column chromatography.
(Chloroform: methanol = 30: 1) to give the target compound (49 mg, 100%) as a colorless powder. mp 89-92 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.12 (9H, s), 1.70 (3H, s), 3.3
3 (1H, s), 3.75, 3.80 (2H, ABq, J = 8 Hz), 3.95, 4.
07 (2H, ABq, J = 8Hz), 7.26-7.73 (10H, m), 8.08 (1H,
s). (Example 13) 3'-amino-3'-deoxy-5'-O-tert-butyldiphenylsilyl-3'-N- (4-monomethoxytrityl) -2'-O, 4'- C-methylene-5-methyluridine (Exemplary Compound No. 2-75) The compound of Example 12 (102 mg, 0.20 mmol) under a nitrogen stream
4-methoxytrityl chloride (93.4 mg, 0.30 mmol) was added to an anhydrous pyridine solution (3 ml), and the mixture was stirred at room temperature for 10 hours. After adding saturated aqueous sodium hydrogen carbonate, the organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. After the solvent was distilled off, the residue was subjected to column chromatography (ethyl acetate:
Purification with hexane = 1: 3) gave the desired compound (154 mg, 9
8%) as a colorless powder. mp 102-105 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.13 (9H, s), 1.62 (3H, s), 1.9
4 (1H, d, J = 10 Hz), 2.48 (1H, s), 2.74 (1H, d, J = 10 Hz
Hz), 3.73 (3H, s), 3.83, 3.91 (2H, ABq, J = 8 Hz),
4.25, 4.35 (2H, ABq, J = 12 Hz), 5.36 (1H, s), 6.70
(2H, d, J = 9 Hz), 7.02-7.75 (22H, m), 8.05 (1H,
s). (Example 14) 3'-amino-3'-deoxy-3'-N- (4-monomethoxytrityl) -2'-O, 4'-C-methylene-5-methyluridine (exemplary compound No. 2-79) Compound of Example 13 (147 mg, 0.19 mmol) under a nitrogen stream
To a THF solution (4 ml) of was added tetrabutylammonium fluoride (1 M in THF, 0.21 ml, 0.21 mmol), and the mixture was stirred at room temperature for 4 hours. After the solvent was distilled off, the residue was subjected to column chromatography (ethyl acetate: hexane = 1: 1 → 2:
1 → 1: 0) to give the desired compound (97 mg, 96%)
Was obtained as a colorless powder. mp 136-141 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.77 (3H, s), 1.98 (1H, d, J = 11
Hz), 2.36 (1H, s), 2.92 (1H, d, J = 10 Hz), 3.77, 3.
91 (2H, ABq, J = 7 Hz), 3.78 (1H, s), 4.19,4.33 (2
H, ABq, J = 14 Hz), 5.37 (1H, s), 6.78 (2H, d, J = 9 H
z), 7.20-7.45 (12H, m), 7.94 (1H, s). (Example 15) 3'-amino-3'-deoxy-3'-N- (4-monomethoxytrityl) -2'- O, 4'-C-methylene-5-methyluridine 5'-O- (2-
Cyanoethyl N, N-diisopropyl) phosphoramidite (Exemplified Compound No. 2-83) The compound of Example 14 (95 mg, 0.18 mmol) under a nitrogen stream
THF (1 ml) was added to a solution of tetrazole N, N-diisopropylamine salt (42.5 mg, 0.25 mmol) in acetonitrile (3 ml) to a THF solution (4 ml) of 2-cyanoethyl N, N,
N ', N'-tetraisopropyl phosphoramidite (126.
9 mg, 0.43 mmol) and stirred at room temperature for 3 hours. After evaporating the solvent, the residue was purified by column chromatography (ethyl acetate: hexane = 1: 1) to obtain the desired compound (140 m
g, quant) as a colorless oil. ³¹ P-NMR (CDCl ₃ , standard substance H ₃ PO ₄ )) δ: 148.61, 148.
76. (Example 16) Synthesis of Oligonucleotide Analogue Nucleic Acid Synthesizer (Applied Biosystems Expedi)
te 8909) on a 0.2 μmol scale. The concentration of the solvent, the reagent and the phosphoramidite in each synthesis cycle were the same as in the case of the synthesis of the natural oligonucleotide, and the solvent and the reagent were all manufactured by Applied Biosystems. Oligonucleotide synthesis is usually 3 '
The procedure was performed in the direction from the end to the 5 'end, but this time, the procedure was performed from the 5' end to the 3 'end. 5'-O-amidite form of natural thymidine (dT-5'-CE-phosphoramidite, catalog No. 10-
[0101-05] A product manufactured by Glen Research was used. Universa
l DMTr of QSupport (0.2μmol, manufactured by Glen Research)
The group was deprotected with trichloroacetic acid, and the resulting hydroxyl group was added to the compound of Example 15 and the 5′-O- of the natural thymidine.
The condensation reaction was repeated using the amidite to synthesize a modified oligonucleotide analog having each sequence.
The synthesis cycle is as follows.

Synthesis cycle 1) detritylation trichloroacetic acid / dichloromethane; 49
sec 2) coupling phosphoramidite (ca. 35 eq), tetrazole / acetonitrile; 1.5 min or 10.5 min 3) capping 1-methylimidazole / tetrahydrofuran
/ Pyridine, acetic anhydride / tetrahydrofuran; 15 sec 4) oxidation iodine / water / pyridine / tetrahydrofuran; 6 sec 5) capping 1-methylimidazole / tetrahydrofuran
/ Pyridine / acetic anhydride / tetrahydrofuran; 2.5 sec In the above, when the reaction is performed using the compound of Example 15 in cycle 2), the reaction is performed for 10.5 minutes, and when the other phosphoramidite is used, the reaction is performed for 1.5 minutes. went.

After synthesizing an oligonucleotide having the target sequence and performing the synthesis cycle 1) to deprotect the 3′-position monomethoxytrityl group, the oligomer is cut out from the support by a concentrated ammonia water treatment according to a conventional method. At the same time, the cyanoethyl group as the protecting group on the phosphorus atom was removed, and the protecting group on the nucleobase was further removed.

Purification was performed by reverse phase HPLC to obtain the target oligonucleotide.

According to this synthesis method, it has a sequence represented by the following sequence: 5′-nnnnnnnnnn-3 ′ (SEQ ID NO: 7 in the sequence listing), and n is 3′-amino-3′-deoxy-2 ′.
Oligonucleic acid which is -O, 4'-C-methylene-5-methyluridine
Reotide analogs (hereinafter “oligonucleotide (6)”
I do. ) Got. (Yield: 9.4 nmol (4.6% yield)) Purification of the resulting modified oligonucleotide analog was performed in reverse phase.
HPLC (HPLC: GILSON; Model 302, column: CHEMCOSO
RB 300-5C18 (7.5 × 250mm); 0.1M triethylamine acetate
Aqueous solution (TEAA), pH7; 8 → 10% CH_ThreeCN / 30min, linear g
radient; 50 ° C; 2.5 ml / min; 254 nm) for 9.7 minutes
Fractions eluting with were collected. Example 17 Synthesis of Oligonucleotide Analogs As in Example 16, the following sequence: 5′-ntntntntnt-
3 ′ (SEQ ID NO: 8 in the sequence listing)
Gonucleotide analogs (hereinafter “oligonucleotides”
(7) ". ) Got. (Yield 20 nmol (10% yield)) Purification of the resulting modified oligonucleotide analog
HPLC (HPLC: GILSON; Model 302, column: CHEMCOSO
RB 300-5C18 (7.5 × 250mm); 0.1M triethylamine acetate
Aqueous solution (TEAA), pH7; 8 → 11% CH_ThreeCN / 45 min, linear
gradient; 50 ° C; 2.5 ml / min; 254 nm), 19.6
Fractions eluting in minutes were collected. (Example 18) Synthesis of oligonucleotide analog As in Example 16, the following sequence: 5'-tntntntntn-
3 ′ (SEQ ID NO: 9 in the sequence listing).
Gonucleotide analogs (hereinafter “oligonucleotides”
(8) ". ) Got. (Yield 30 nmol (15% yield))
Purification of nucleotide analogs is performed by reversed-phase HPLC (HPLC: GILSON
Company; Model 302, Column: CHEMCOSORB 300-5C18 (7.5 ×
250mm); 0.1M triethylamine acetate aqueous solution (TEAA), pH
7; 8 → 11% CH_ThreeCN / 45 min, linear gradient; 50 ℃;
 (2.5 ml / min; 254 nm), fraction eluted at 22.2 minutes
Collected. (Reference Example 1) 3-azido-3-deoxy-4-hydroxymethyl-1,2-O-iso
Propylidene-α-D-ribofuranose Article (Surzhykov S.A., Krayevsky A.A, Nucleosides N
ucleotides, 13, 2283-2305 (1994))
Azido-4-benzoyloxymethyl-5-O-benzoyl-3-
Deoxy-1,2-O-isopropylidene-α-D-ribofurano
Solution (4.13 g, 9.15 mmol) in methanol (85 ml)
Potassium carbonate (380 mg, 2.75 mmol) and water (15
 ml) and stirred at the same temperature for 4.5 hours. 10% at 0 ° C
Neutralized with hydrochloric acid, methanol was distilled off. Add water to the residue
Then, the mixture was extracted with ethyl acetate and washed with saturated saline. Organic
After drying the layer over anhydrous sodium sulfate, the solvent was distilled off.
Wash the washed white solid with cold n-hexane to
Obtained as a powder (1.93 g, 7.87 mmol, 86%). mp 113-115 ° C (toluene). IR νmax (KBr): 3460, 3417, 2989, 2951, 2907, 2111
 cm^-1.¹ H-NMR (CDCl_Three) Δ: 1.62 (3H, s), 1.35 (3H, s) 2.65
 (2H, br s), 3.81, 3.65 (2H, AB, J = 12 Hz), 3.59,
 4.00 (2H, AB, J = 12.5 Hz), 4.28 (1H, d, J = 5.5 H
z), 4.82 (1H, dd, J = 4 Hz, 5.5 Hz), 5.85 (1H, d,
J = 4 Hz).¹³ C-NMR (CDCl_Three) Δ: 25.7, 26.2, 61.9, 62.1, 63.2,
79.9, 87.3, 104.4, 113.6. (Reference Example 2) 3-azido-5-O-tert-butyldiphenylsilyl-3-deoxy
C-4-hydroxymethyl-1,2-O-isopropylidene-α-D-
Ribofuranose Compound of Reference Example 1 (2.56 g, 10.5 mmol) under nitrogen stream
In anhydrous methylene chloride solution (73 ml) at 0 ° C
Tylamine (3.5g, 4.82 ml, 34.6 mmol), t-butyl chloride
Rudiphenylsilane (9.75 g, 9.22 ml, 35.46 mmol)
The mixture was stirred at room temperature for 24 hours. Saturated aqueous sodium bicarbonate solution
Was added, extracted with ethyl acetate, and washed with saturated saline.
The organic layer was dried over anhydrous sodium sulfate.
Ricagel column chromatography (ethyl acetate: n-f
The product was purified by using xanthane (1: 6) and the target product was white powder (3.
13 g, 6.47 mmol, 62%). mp 99.5-100.5 ° C (n-hexane). IR νmax (KBr): 3504, 2936, 2852, 2111 cm^-1.¹ H-NMR (CDCl_Three) Δ: 1.07 (9H, s), 1.36 (3H, s), 1.6
2 (3H, s), 3.62, 3.92 (2H, AB, J = 12 Hz), 4.38 (1
H, d, J = 6 Hz), 4.84 (1H, dd, J = 4 Hz, 5.5 Hz),
3.82, 3.70 (2H, AB, J = 11 Hz), 4.84 (1H, dd, J =
4 Hz, 5.5 Hz), 5.86 (1H, d, J = 4 Hz), 7.36-7.44
(6H, m), 7.64-7.67 (4H, m).¹³ C-NMR (CDCl_Three) Δ: 19.2, 26.1, 26.3, 26.8, 62.2,
62.3, 65.2, 80.4, 88.0, 104.5, 113.7, 127.7, 127.
8, 129.8, 129.9, 132.7, 132.8, 135.5. (Reference Example 3) 3-Azido-5-O-tert-butyldiphenylsilyl-3-deoxy
C-4- (p-toluenesulfonyloxymethyl) -1,2-O-iso
Propylidene-α-D-ribofuranose The compound of Reference Example 2 (100 mg, 0.2
1 mmol) in anhydrous methylene chloride solution (2 ml).
Amine (137 mg, 180 μl, 1.29 mmol), p-toluene chloride
Ensulfonyl (63.3 mg, 0.33 mmol), 4-dimethyl
Add aminopyridine (4 mg, 0.03 mmol) and add 14
Stirred for hours. Saturated aqueous sodium bicarbonate was added to the reaction solution, and ethyl acetate was added.
And washed with saturated saline. Organic layer is sulfuric anhydride
After drying with sodium and evaporating the solvent,
Chromatography (ethyl acetate: n-hexane = 1: 6)
And purified with white powder (130 mg, 0.20 mmo
l, 98%). mp 122-124 ° C (ethyl acetate-n-hexane). IR νmax (KBr): 3069, 2935, 2114, 1366, 1183, 1109
 cm^-1.¹ H-NMR (CDCl_Three) Δ: 1.03 (9H, s), 1.27 (3H, s), 1.3
1 (3H, s), 2.41 (3H, s), 3.60, 3.72 (2H, AB, J = 1
0.5 Hz), 4.33, 4.40 (2H, AB, J = 10 Hz), 4.55 (1H,
 d, J = 5.5 Hz), 5.00 (1H, dd, J = 3.7 Hz, 5.5 H
z), 5.82 (1H, d, J = 3.7 Hz), 7.23 (2H, d, J = 8.5
Hz), 7.36-7.45 (6H, m), 7.61-7.63 (4H, m), 7.72 (2
(H, d, J = 8.5 Hz).¹³ C-NMR (CDCl_Three) Δ: 19.1, 21.5, 25.9, 26.0, 26.7,
63.1, 64.7, 68.9, 80.1, 85.6, 104.4, 113.8, 127.8,
 128.0, 129.6, 129.9, 132.4, 132.5, 135.4, 144.6. (Reference Example 4) 3-Azido-5-O-tert-butyldiphenylsilyl-3-deoxy
C-4- (p-toluenesulfonyloxymethyl) -1,2-di-O-
Acetyl-D-ribofuranose Compound of Reference Example 3 (230 mg, 0.36 mmol) under nitrogen stream
Acetic anhydride (406 mg, 375 μl,
 3.98 mmol), concentrated sulfuric acid (6.5 mg, 3.5 μl, 0.066 mmo
l) was added and the mixture was stirred at room temperature for 5 hours. Reaction solution in ice water
Stir for 30 minutes, add saturated saline, add ethyl acetate
Extracted. The organic layer was dried over anhydrous sodium sulfate and dissolved.
After distilling off the solvent, silica gel column chromatography (acetic acid)
Purified by ethyl: n-hexane = 4: 1), α: β =
The target substance, which is a mixture of about 3: 7, was converted to a colorless oily substance (230 m
g, 0.34 mmol, 94%). IR νmax (KBr): 3048, 2935, 2864, 2117, 1756.cm^-1.¹ H-NMR (CDCl_Three) [Β-form] δ: 1.06 (9H, s), 1.83 (3H,
 s), 2.08 (3H, s), 2.40 (3H, s), 3.54, 3.80 (2H, A
B, J = 11 Hz), 4.12, 4.26 (2H, AB, J = 10 Hz), 4.3
7 (1H, d, J = 5.5 Hz), 5.32 (1H, d, J = 5.5 Hz),
5.98 (1H, s), 7.29 (2H, d, J = 8 Hz), 7.37-7.46 (6
H, m), 7.59-7.65 (4H, m), 7.76 (2H, d, J = 8 Hz). [Α-form] δ: 1.05 (9H, s), 2.02 (3H, s), 2.13 (3H,
s), 2.39 (3H, s), 3.51,3.68 (2H, AB, J = 11 Hz),
4.12, 4.21 (2H, AB, J = 10.5 Hz), 4.40 (1H, d, J =
 7 Hz), 5.32 (1H, m), 6.31 (1H, d, J = 4.5 Hz), 7.
25 (2H, d, J = 8.5 Hz), 7.37-7.46 (6H, m), 7.59-7.
65 (4H, m), 7.70 (2H, d, J = 8.5 Hz).¹³ C-NMR (CDCl_Three) Δ: 19.0, 19.1, 20.0, 20.6, 20.9,
21.1, 21.5, 26.6, 61.0, 63.2, 65.1, 68.4, 68.8, 7
2.2, 75.5, 85.4, 86.5, 93.6, 96.0, 97.3, 127.8, 12
7.9, 128.0, 129.6, 129.9, 130.0, 132.0, 132.3, 13
2.4, 135.4, 144.7, 168.5, 169.2, 169.3, 169.4. (Reference Example 5) 2'-O-acetyl-3'-azido-5'-O-tert-butyldiphenyl
Silyl-3'-deoxy-4 '-(p-toluenesulfonyloxy
Methyl) -5-methyluridine The compound of Reference Example 4 (300 mg, 0.44
mmol) in anhydrous 1,2-dichloroethane (6 ml).
O'-bis (trimethylsilyl) thymine (240 mg, 0.93 mmo
l), tin tetrachloride (253 mg, 114 μl, 0.97 mmol)
And stirred at room temperature for 43 hours. Dichloromethane under ice-cooling
After dilution with water, add saturated aqueous sodium bicarbonate
Extracted. After washing with saturated saline, the organic layer was dried over sulfuric anhydride.
After drying with sodium and evaporating the solvent,
Chromatography (ethyl acetate: n-hexane = 1: 2-
1: 0) to give the desired product as a white powder (300 mg, 0.4
 mmol, 91%). mp 158.5-159.5 ℃ (ethyl acetate-n-hexane). IR νmax (KBr): 3185, 3067, 2956, 2116, 1752, 169
5, 1369, 1100 cm^-1.¹ H-NMR (CDCl_Three) Δ: 1.11 (9H, s), 1.59 (3H, s), 2.1
5 (3H, s), 2.41 (3H, s), 3.80, 3.84 (2H, AB, J = 1
1.5 Hz), 4.04, 4.10 (2H, AB, J = 11 Hz), 4.47 (1H,
 d, J = 6 Hz), 5.53 (1H, t, J = 6.5 Hz), 5.94 (1H,
 d, J = 7 Hz), 7.18 (1H, s), 7.28 (2H, d, J = 7.5
Hz), 7.37-7.47 (6H, m), 7.61-7.65 (4H, m), 7.71 (2
H, d, J = 7.5 Hz), 9.68 (1H, br s).¹³ C-NMR (CDCl_Three) Δ: 11.8, 19.2, 20.9, 21.5, 26.9,
62.3, 65.9, 68.3, 74.2, 84.8, 86.1, 118.9, 127.9,
128.0, 129.7, 130.1, 131.5, 132.2, 135.2, 135.3, 1
35.5, 145.0, 150.4, 163.6, 169.9. (Reference Example 6) 3'-O- (tert-butyldimethylsilyl) thymidine 5 '-(2-
Cyanoethyl) phosphonate 3'-O- (tert-butyldimethylsilyl) thymine under nitrogen stream
Gin (K. M. Fries, C. Joswing and R. F. Borch, J. M.
ed. Chem., 38, 2672 (1995)) (100 m
g, 0.34 mmol) in acetonitrile (4 ml).
Noethyltetraisopropyl phosphorodiamidite (13
(2 mg, 0.44 mmol) over 5 minutes and stirred at room temperature for 2.2 hours.
Stirred. Subsequently, tetrazole (30.8 mg, 0.44 mmol)
Acetonitrile solution (0.88 ml) was added and the mixture was allowed to stand at room temperature for 1 hour.
Stirred. After adding water to the reaction solution, dichloroethane
Extracted. After washing the organic layer with saturated saline, anhydrous sodium sulfate
It was dried with lium. The solvent was distilled off under reduced pressure to obtain the crude product.
Silica gel column chromatography (chloropho
Lum: methanol = 30: 1, n-hexane: ethyl acetate = 1: 5
→ 0: 1) to give the title compound as a colorless oil.
(98.4 mg, 0.21 mmol, 70%).¹ H-NMR (CDCl_Three) δ: 0.10 (6H, s), 0.90 (9H, s), 1.96
(3H, s), 2.16-2.28 (2H, m), 2.77-2.82 (2H, m),
4.09-4.41 (6H, m), 6.28 (1H, dd, J = 7 Hz, 11 Hz),
 6.98 (1H, d, J = 720 Hz), 7.36 (1H, d, J = 8 Hz),
 8.20 (1H, brs). ³¹ P-NMR (CDCl_Three) δ: 7.70, 8.94. (Reference Example 7) 3'-O- (tert-butyldimethylsilyl) thymidine 5'-methyl
3'-O- (tert-butyldimethylsilyl) thymine
Gin (100 mg, 0.28 mmol) in dichloromethane (2 m
l) chlorodiisopropylaminomethoxyphosphine
(69.2 mg, 0.35 mmol) over 5 minutes and at room temperature for 1 hour
Stirred. Subsequently, tetrazole (56.0 mg, 0.80 mmo
l) in acetonitrile (2.0 ml) and stirred at room temperature for 40 minutes.
Stirred. After adding water to the reaction solution, extract with dichloroethane.
Issued. After washing the organic layer with saturated saline, anhydrous sodium sulfate was added.
And dried with um. The solvent was distilled off under reduced pressure and the obtained crude results
The silica gel column chromatography (n-hexa
(1: 1 → 0: 1, n-hexane: ethyl acetate = 1: 4)
To give the title compound as a colorless oil (109 m
g, 0.25 mmol, 91%).³¹ P-NMR (CDCl_Three) δ: 9.13, 10.07. (Test Example 1) (Tm measurement for measuring triple-strand forming ability) 140 mM KCl and 10
 mM MgCl_Two7 mM sodium phosphate buffer (pH 7.
0) (or its MgCl_TwoSolution which is not added)
Oligonucleotide (2) that forms triple strand and two natural types
Equimolar amount of each strand DNA oligonucleotide (each oligonucleotide
Final solution of otide is 1.5 microM)
In boiling water and slowly warm to room temperature over 12 hours
It was cooled and left at 4 ° C. for 1 hour. Spectrophotometer (B
In the cell room of Duc 650 manufactured by eckman, sample solution was
Temperature is gradually increased from 0.5 to 85 ° C (0.5 ° C / min), 2
Ultraviolet absorption at 60 nm was measured. Natural double-stranded D
The NA oligonucleotide has the sequence: 5′-gctaaaaagaaagagagatcg-3 ′ (SEQ ID NO:
3) and its complementary strand. Sequence: 5'-cgatctctctttctttttagc-3 '(SEQ ID NO:
4) The following was used. In addition, a natural type that forms a triple strand
The oligonucleotide has the following sequence: 5'-tttttmtttmtmtmt-3 '(SEQ ID NO: 5 in the sequence listing), wherein m is 5-methyl-2'-deoxycytidine.
(Hereinafter referred to as “oligonucleotide (3)”)
Was.

Table 3 shows the Tm measurement results of the oligonucleotides (2) and (3) and the double-stranded DNA.

[0185]

[Table 3] ――――――――――――――――――――――――――――― Tm (℃) Oligonucleotide (2) Oligonucleotide (3) Implementation Example 11 ――――――――――――――――――――――――――――― MgCl ₂ added 55 44 MgCl ₂ not added 44 32 ―――――― As is clear from the above, the oligonucleotide analogue of the present invention has 3 The Tm value of the main chain was high, indicating an extremely good triple-strand forming ability.

An example in which a triple strand dissolved in a 10 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl and 10 mM MgCl ₂ (or a solution to which MgCl _{2 is} not added) is formed. The oligonucleotide (6) according to 16,
A sample solution containing the same molar amount of DNA oligonucleotides that form a hairpin type (final concentration of each oligonucleotide is 2 μM) is bathed in boiling water, slowly cooled to room temperature over 12 hours, and further cooled to 4 ° C. For 1 hour. The temperature of the sample solution was gradually increased from 5 ° C. to 80 ° C. (0.5 ° C./min) in a cell chamber of a spectrophotometer (Du 650 manufactured by Beckman), and ultraviolet absorption at 260 nm and 284 nm was measured.

As the DNA oligonucleotide forming the hairpin type, the sequence: 5'-aaaaaaaaaacccctttttttttt-3 '(SEQ ID NO: 10 in the sequence listing) (hereinafter referred to as "oligonucleotide (8)") was used.

Table 4 shows the Tm measurement results of the oligonucleotide (6) and the oligonucleotide (8).

[0189]

[Table 4] ---------------------------------------------- -------------------- Tm (℃) MgCl ₂ not added MgCl ₂ added ------------------- ----------------------------------------------- Oligonucleotide ( 8) only (55.3) (59.8) Oligonucleotide (6) and (55.5), 63.4 (60.3), 73.6 Oligonucleotide (8) 10-mer oligothymidylic acid having N3'-P5 'bond and (55.7) 32.0 (61.5), 47.2 Oligonucleotide (8) ^* -------------------------------------- ---------------------------- The values in parentheses indicate the T of the double-stranded portion of oligonucleotide (8).
Represents the m value. As is clear from the above, it was found that the oligonucleotide analog of the present invention binds to the double-stranded portion of the hairpin DNA oligonucleotide as a triple-stranded nucleic acid. This value is higher than the Tm value of a 10-mer oligothymidylic acid having a N3'-P5 'bond and a hairpin-type DNA oligonucleotide (8) known in the literature ( ^* indicates that of the literature SMGryaznov and H. Winter). Nucleic Acid
s Res. (1998) 26, 4160-4167), showing that the oligonucleotide analogues of the present invention have very good triple-strand forming ability. (Test Example 2) (Measurement of nuclease enzyme resistance) 320 μl of a buffer solution containing various oligonucleotides (10 μg) (50 mM Tris (pH
8.0) and 10mM MgCl ₂ ) with 3'-exonuclease (pho
sphodiesterase from Crotalus durissus (Boehringer
Mannheim)) was added, and the mixture was kept at 37 ° C to carry out a reaction. After a certain period of time, remove a part of the mixed solution, and
By heating for minutes, the enzyme was inactivated and the reaction was stopped. The remaining amount of the oligonucleotide in the mixture at each time was quantified by reversed-phase high performance liquid column chromatography, and the time-dependent change in the amount of the oligonucleotide in the presence of nuclease was measured. The results are shown in FIG. Oligonucleotides used for the test 1. Oligonucleotide (1) obtained in Example 10 2. Sequence: 5′-ttttttttttnt-3 ′ (SEQ ID NO: 1 in Sequence Listing), where n is 2 Oligonucleotide that is' -O, 4'-C-methylene-5-methyluridine (hereinafter referred to as “oligonucleotide (4)”) 3. Sequence: 5′-tttttttttttttt-3 ′ (SEQ ID NO: in Sequence Listing) 6) A natural oligonucleotide having the sequence represented by (hereinafter, referred to as “oligonucleotide (5)”) The oligonucleotide analogue of the present invention showed remarkable nuclease resistance as compared with the natural oligonucleotide. Furthermore, it showed remarkable nuclease resistance even when compared with known non-natural oligonucleotide analogs.

The hybridization ability and anti-HIV activity of the oligonucleotide analogue of the present invention can be measured according to the following methods.

(Test Method 1) The melting temperature (Tm value) of the various oligonucleotide analogs obtained as an antisense strand, which was annealed with a natural DNA or RNA sense strand, was measured. Complementary DNA and R for oligonucleotide analogues of the invention
The ability to hybridize to NA is examined.

Each final concentration was 100 ml of NaCl.
M, sodium phosphate buffer (pH 7.2) 10 mM,
A sample solution (500 μL) containing 4 μM of the antisense strand and 4 μM of the sense strand is bathed in boiling water, and slowly cooled to room temperature over 10 hours. A nitrogen stream is passed through the cell chamber of the spectrophotometer (eg, Shimadzu UV-2100PC) to prevent dew condensation, the sample solution is gradually cooled to 5 ° C, and the measurement is started after keeping it at 5 ° C for 20 minutes. I do. The sample temperature was increased by 0.2 ° C per minute to 90 ° C,
The UV absorption at 260 nm is measured at intervals.

In order to prevent the sample concentration from changing with an increase in temperature, a cell with a lid is used, and measurement is performed by adding one drop of mineral oil to the surface of the sample solution.

(Test Method 2) Measurement of Anti-HIV Activity The anti-HIV activity of the compound of the present invention was measured by the method of R. Pauwel et al. (J. Virological Method 20, p. 309-321 (1988)).
Perform according to. That is, the cell pellet obtained by centrifuging (1000 × g, 5 minutes) the MT-4 cells was transferred to a cell suspension suspended in a serum-free RPMI-1640 medium.
After inoculating HIV and culturing at 37 ° C. for 1 hour, 10%
Washing and centrifugation (1000 × g, 5 minutes) in addition to RPMI-1640 medium supplemented with fetal calf serum (hereinafter referred to as serum medium)
I do. HIV-infected cells and HI thus obtained
V non-infected cells are suspended in a serum medium at 4 × 105 / ml, and 100 μl is dispensed into each well of a 96-well multiwell for tissue culture. After dispensing 100 μl into each of these wells, the cells are cultured at 37 ° C. for 5 days in the presence of 5% carbon dioxide. Similarly, HIV-infected cells and non-HIV-infected cells without compound are cultured. After completion of the culture, the number of living cells was measured using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide), and the cytotoxicity-suppressing activity (anti-HIV activity) upon compound addition was measured. Ask for. In addition, confirm that mycoplasma is not contained in the cell solution and the inoculated virus solution.

Assuming that the cytotoxicity-suppressing activity of the HIV-infected cells without addition of the compound is 100% and the cytotoxicity-suppressing activity of the HIV-infected cells without the compound is 0%, the cytotoxicity of the HIV-infected cells is 50%. The concentration of the compound showing the activity (EC50) is determined.

[0196]

Industrial Applicability The novel bicyclonucleoside analogs of the present invention have excellent antisense or antigene activity and are useful as intermediates for producing oligonucleotide analogs that are stable in vivo. .

The novel oligonucleotide analogs of the present invention are stable in vivo and are useful as antisense or antigene drugs.

Further, the novel bicyclonucleoside analog of the present invention has an anti-AIDS activity and is useful as an agent for preventing or treating AIDS.

[Brief description of the drawings]

FIG. 1 shows the time course of the amount of oligonucleotide in the presence of nuclease. The vertical axis indicates the residual ratio (%) of each oligonucleotide relative to the amount of each oligonucleotide at 0 (minute). The horizontal axis shows the elapsed time (minutes) from the start of the reaction.

[Sequence List Free Text]

[SEQ ID NO: 1] <223> Description of artificial sequence: Synthetic oligonucleotide for testing nuclease resistance <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 11 <223> Feature description: Modified uridine

[SEQ ID NO: 2] <223> Description of artificial sequence: Synthetic oligonucleotide for testing triple-strand forming ability <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 6 <223> Feature description: 5-methyl 2'-deoxycytidine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 8 <223> Feature description: 3'-amino -3'-deoxy-2'-O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 10 <223> Characteristic description: 5-methyl 2'-deoxycytidine <220> sequence Characteristics of <221> Sequence type: Modified base <222> Characteristic position: 12 <223> Description of feature: 5-methyl 2'-deoxycytidine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 14 <223> Characteristic description: 5-methyl 2'-deoxycytidine

[SEQ ID NO: 3] <223> Description of artificial sequence: Synthetic oligonucleotide for testing triple-strand forming ability

[SEQ ID NO: 4] <223> Description of artificial sequence: Synthetic oligonucleotide for testing triple-strand forming ability

[SEQ ID NO: 5] <223> Description of artificial sequence: Synthetic oligonucleotide for testing triple strand-forming ability <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 6 <223> Feature description: 5-methyl 2'-deoxycytidine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 10 <223> Feature description: 5-methyl 2 '-Deoxycytidine <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 12 <223> Character description: 5-methyl 2'-deoxycytidine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 14 <223> Characteristic description: 5-methyl 2'-deoxycytidine

[SEQ ID NO: 6] <223> Description of artificial sequence: Synthetic oligonucleotide for testing nuclease resistance

[SEQ ID NO: 7] <223> Description of artificial sequence: Synthetic oligonucleotide for testing nuclease resistance <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 1 <223> Description of features: 3'-amino-3'-deoxy-2'-O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 2 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 3 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 4 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 5 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 6 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 7 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 8 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 9 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 10 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine

[SEQ ID NO: 8] <223> Description of artificial sequence: Synthetic oligonucleotide for testing triple-strand forming ability <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 1 <223> Description of features: 3'-amino-3'-deoxy-2'-O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 3 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 5 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 7 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 9 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine

[SEQ ID NO: 9] <223> Description of artificial sequence: Synthetic oligonucleotide for testing triple-strand forming ability <220> Sequence characteristics <221> Sequence type: modified base <222> Characteristic position: 2 <223> Description of features: 3'-amino-3'-deoxy-2'-O, 4'-C
-Methylene-5-methyluridine <220> Sequence features <221> Sequence type: Modified base <222> Feature position: 4 <223> Feature description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 6 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 8 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine <220> Sequence characteristics <221> Sequence type: Modified base <222> Characteristic position: 10 <223> Characteristic description: 3'-amino-3'-deoxy-2 ' -O, 4'-C
-Methylene-5-methyluridine

[SEQ ID NO: 10] <223> Description of artificial sequence: Synthetic oligonucleotide that forms hairpin form

[Sequence List] SEQUENCE LISTING <110> Sankyo Company, Limited <120> N3'-P5'binding 2 ', 4'-Bicyclo-Nucleoside Analogues <130> 2001130SL <140> <141> <150> JP 2000-354326 < 151> 2000-11-21 <160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the nuclease resistance <220> <221> modified_base <222> (11) <223> modified uridine <400> 1 ttttttttttnt 12 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand <220> <221> modified_base <222> (6) <223> 5-methyl 2'-deoxycytidine <220> <221> modified_base <222> ( 8) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (10) <223> 5-methyl 2 '-deoxycytidine <220> <221> modified_base <222> (12) <223> 5-methyl 2'-deoxycytidine <220> <221> modified_base <222> (14) <223> 5-methyl 2'-deoxycytidine <400> 2 tttttntntn tntnt 15 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand < 400> 3 gctaaaaaga aagagagatc g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand <400> 4 cgatctctct ttctttttag c 21 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand <220> <221> modified_base <222 > (6) <223> 5-methyl 2'-deoxycytidine <220> <221> modified_base <222> (10) <223> 5-methyl 2'-deoxycytidine <220> <221> modified_base <222> (12) <223> 5-methyl 2'-deoxycytidine <220> <221> modified_base <222> (14) <223> 5-methyl 2'-deoxycytidine <400> 5 tttttntttn tntnt 15 <210> 6 <211> 12 <212 > DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the nuclease resistance <400> 6 tttttttttt tt 12 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand <220> <221> modified_base <222> (1) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene- 5-methyluridine <220> <221> modified_base <222> (2) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (3) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (4) <223 > 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (5) <223> 3'-amino-3'- deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (6) <223> 3'-amino-3'-deoxy-2'-O, 4 ' -C-methylene-5-methyluridine <220> <221> modified_base <222> (7) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine < 220> <22 1> modified_base <222> (8) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (9) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (10) <223> 3'-amino-3 '-deoxy-2'-O, 4'-C-methylene-5-methyluridine <400> nnnnnnnnnn 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand <220> <221> modified_base <222> (1) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene- 5-methyluridine <220> <221> modified_base <222> (3) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (5) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (7) <223 > 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (9) <223> 3'-amino-3'- deoxy-2'-O, 4'-C-methylene-5-methyluridine <400> ntntntntnt 10 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized oligonucleotide for testing the formability of a triple strand <220> <221> modified_base <222> (2) <223> 3'-amino -3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (4) <223> 3'-amino-3'-deoxy-2'- O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (6) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene- 5-methyluridine <220> <221> modified_base <222> (8) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <220> <221> modified_base <222> (10) <223> 3'-amino-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine <400> tntntntntn 10 <210> 10 <211> 24 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized and hairpin-formed oligonucleotide <400> 24 aaaaaaaaaa cccctttttt tttt 24

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C07H 21/00 C07H 21/00 C12N 15/09 ZNA C12N 15/00 ZNAA

Claims (17)

[Claims] 1. A compound of the general formula (1) [Wherein, R ^{1 s} are the same or different and each is a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, a phosphate group, a phosphate group protected with a protecting group for nucleic acid synthesis, or a formula -P (R ^4a ) R ^4b. (Wherein R ^4a and R ^4b
Are the same or different, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, Which represents an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 7 carbon atoms or an amino group substituted with an alkyl group having 1 to 6 carbon atoms) R ² represents an azido group, an amino group or a formula NH—R ³ (R ³ is
The same or different, a protecting group for nucleic acid synthesis of amino group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or a formula-
P (R ^4a ) R ^4b (wherein R ^4a and R ^4b are the same or different and each is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis,
Mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, amino group, amino group protected with a protecting group for nucleic acid synthesis, alkoxy group having 1 to 6 carbon atoms, 1 to 6 carbon atoms
Represents an alkylthio group, a cyanoalkoxy group having 1 to 7 carbon atoms or an amino group substituted with an alkyl group having 1 to 6 carbon atoms)). B is purin-9-yl or 2-oxo-1, optionally having one or more optional substituents selected from the following α group.
2 represents a 2-dihydropyrimidin-1-yl group. Or a pharmacologically acceptable salt thereof. (Α group) hydroxyl group, hydroxyl group protected with a protecting group for nucleic acid synthesis, alkoxy group having 1 to 6 carbon atoms, mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, alkylthio group having 1 to 6 carbon atoms A group, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a halogen atom. 2. The method according to claim 1, wherein R ¹ is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, a silyl group, a methyl group substituted with 1 to 3 aryl groups, a lower alkyl group or a lower alkyl group. A compound which is a methyl group substituted with one to three aryl groups in which the aryl ring is substituted with an alkoxy, halogen or cyano group. 3. The method according to claim 1, wherein R ¹ is a hydrogen atom, a silyl group, a methyl group substituted with 1 to 3 aryl groups, or a lower alkyl, lower alkoxy, halogen or cyano group, and the aryl ring is A compound which is a methyl group substituted with one to three substituted aryl groups. 4. The method according to claim 1, wherein R ¹ is hydrogen, trimethylsilyl, t-butyldimethylsilyl, t-
Butyldiphenylsilyl, benzyl, triphenylmethyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl or 4,4 ', 4 "-trimethoxytriphenyl A compound which is a methyl group. 5. The method of claim 1, wherein R ² is an azido group, an amino group, or a group of the formula NH—R ³ , wherein R ³ is an aliphatic acyl group, an aromatic acyl group, A methyl group substituted with an aryl group, a lower alkyl, a lower alkoxy group, a methyl group substituted with 1 to 3 aryl groups each having an aryl ring substituted with a halogen or a cyano group, a silyl group,
A phosphoramidite group, a phosphonyl group, a phosphate group or a phosphate group protected with a protecting group for nucleic acid synthesis).
Compound. 6. The method according to claim 1, wherein R^TwoIs an azide group,
Mino group, formula NH-R^ThreeGroup (wherein R^ThreeIs an acetyl group,
Trifluoroacetyl group, benzoyl group, benzyl group, p
-Methoxybenzyl group, tert-butyldiphenylsilyl
Group, formula -P (OC_TwoH_FourCN) (N (CH (C
H_Three)_Two)_Two), A group represented by the formula -P (OCH_Three) (N (C
H (CH _Three)_Two)_Two), A phosphonyl group, or
Is 2-chlorophenyl or 4-chlorophenyl
Which is an acid group). 7. The compound according to claim 1, wherein R ² is an azide group or an amino group. 8. The method according to claim 1, wherein B is 6-aminopurin-9-yl (ie, adenyl), 6-aminopurin-9-yl wherein the amino group is protected by a protecting group for nucleic acid synthesis, 6-diaminopurin-9-yl, 2,6-diaminopurine-in which the amino group is protected with a protecting group for nucleic acid synthesis
9-yl, 2-amino-6-chloropurin-9-yl,
2-amino- wherein the amino group is protected by a protecting group for nucleic acid synthesis
6-chloropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl in which the amino group is protected by a protecting group for nucleic acid synthesis,
2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl in which the amino group is protected by a protecting group for nucleic acid synthesis (I.e., guaninyl), 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurine- in which the amino group is protected with a protecting group for nucleic acid synthesis.
9-yl, whose amino group is protected with a protecting group for nucleic acid synthesis 6
-Amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-chloropurine-9 in which the amino group is protected by a protecting group for nucleic acid synthesis
-Yl, 6-amino-2-fluoropurin-9-yl,
6-amino- wherein the amino group is protected with a protecting group for nucleic acid synthesis
2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl,
6-mercaptopurin-9-yl, 6-mercaptopurine-9- in which the mercapto group is protected with a protecting group for nucleic acid synthesis.
Yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (ie, cytosinyl), 2-oxo-4-amino-1,2- having an amino group protected with a protecting group for nucleic acid synthesis. Dihydropyrimidin-1-yl, 4-amino-2-oxo-5-fluoro-1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo- wherein the amino group is protected by a protecting group for nucleic acid synthesis 5-fluoro-1,2
-Dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-
4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2 in which the yl and amino groups are protected with a protecting group for nucleic acid synthesis.
-Dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2 in which the mercapto group is protected by a protecting group for nucleic acid synthesis. -Dihydropyrimidine-1-
Yl, 2,4-dihydroxypyrimidin-1-yl (ie, uracilyl), 2,4-dihydroxy-5-methylpyrimidin-1-yl (ie, thyminyl),
-Amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl or 4-amino-5-methyl-2-oxo- having an amino group protected with a protecting group for nucleic acid synthesis
A compound which is a 1,2-dihydropyrimidin-1-yl group. 9. The method according to claim 1, wherein B is 6-benzoylaminopurin-9-yl, adenyl, 2-benzoylamino-6-hydroxypurin-9-yl, guaninyl, 2-oxo-4-benzoylamino-. A compound which is a 1,2-dihydropyrimidin-1-yl, cytosinyl, uracilyl or thyminyl group. 10. A compound selected from the following compound group: 3'-amino-3'-deoxy-2'-O, 4'-C-
Methylene-5-methyluridine, 3'-azido-3'-deoxy-2'-O, 4'-C-
Methylene-5-methyluridine, 3'-azido-5'-O-tert-butyldiphenylsilyl-3'-deoxy-2'-O, 4'-C-methylene-5-methyluridine, 3'-azido- 3'-deoxy-5'-O- (4,4 '
-Dimethoxytrityl) -2'-O, 4'-C-methylene-5-methyluridine and 3'-amino-3'-deoxy-5'-O- (4,4 '
-Dimethoxytrityl) -2'-O, 4'-C-methylene-5-methyluridine. 11. A compound of the general formula (1a) [In the formula, B represents a purine-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have a substituent selected from the following α group. And pharmacologically acceptable salts thereof. However, the above structure is 2
In the case where B is contained, B is the same or different between the structures. (Α group) hydroxyl group, hydroxyl group protected with a protecting group for nucleic acid synthesis, alkoxy group having 1 to 6 carbon atoms, mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, alkylthio group having 1 to 6 carbon atoms A group, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, and a halogen atom. 12. The method according to claim 11, wherein B is a 6-aminopurin-9-yl group (ie, an adenyl group), and the amino group is a 6-aminopurin-9-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis. A 2,6-diaminopurin-9-yl group,
2-amino-6-chloropurin-9-yl group, 2-amino-6-chloropurin-9-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis, 2-amino-6-fluoropurine-9 A 2-amino-6-fluoropurin-9-yl group in which an yl group and an amino group are protected by a protecting group for nucleic acid synthesis, 2
-Amino-6-bromopurin-9-yl group, 2-amino-6-bromopurin-9-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis, 2-amino-6-hydroxypurine-9- An yl group (that is, a guaninyl group), a 2-amino-6-hydroxypurin-9-yl group in which an amino group is protected with a protecting group for nucleic acid synthesis, an amino group and a hydroxyl group protected with a protecting group for nucleic acid synthesis. -Amino-6-hydroxypurin-9-yl group, 6-amino-2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurine- A 9-yl group,
2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurine-9-
2-oxo-4 in which an yl group, a 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (that is, a cytosinyl group), and an amino group are protected with a protecting group for nucleic acid synthesis.
An amino-1,2-dihydropyrimidin-1-yl group,
2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-amino-5-fluoro-1, in which the amino group is protected with a protecting group for nucleic acid synthesis, 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-dihydropyrimidin-1-yl group (that is, uracinyl group),
-Oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (that is, a thyminyl group), 4-amino-5-methyl-2-oxo-1,2-
4-amino-5-methyl-2-oxo-1,2 in which a dihydropyrimidin-1-yl group (ie, a 5-methylcytosinyl group) or an amino group is protected with a protecting group for nucleic acid synthesis.
-An oligonucleotide analogue, which is a dihydropyrimidin-1-yl group, and a pharmaceutically acceptable salt thereof. 13. The method according to claim 11, wherein B is a 6-benzoylaminopurin-9-yl group, an adenyl group, a 2-isobutyrylamino-6-hydroxypurin-9-yl group, a guaninyl group, a 2-oxo group. -4-benzoylamino-1,2-dihydropyrimidin-1-yl group, cytosinyl group, 2-oxo-5-methyl-4-benzoylamino-1,2-dihydropyrimidin-1-yl group, 5-methylcytosinyl group Is a uracinyl group or a thyminyl group,
Oligonucleotide analogs and pharmacologically acceptable salts thereof. 14. The method according to claim 1, wherein R ¹ is a phosphate group protected with a protecting group for nucleic acid synthesis, or a compound of the formula —P (R ^4a ) R
^4b (wherein R ^4a and R ^4b are the same or different and each is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, An amino group, an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 7 carbon atoms or an alkyl group having 1 to 6 carbon atoms And R ² is a formula NH—R ³ (R ³ is a protecting group for nucleic acid synthesis of an amino group). 15. The method according to claim 1, wherein R ¹ is a group represented by the formula -P (R
^4a ) R ^{4b wherein} R ^4a and R ^4b are the same or different,
A hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 7 carbon atoms or an amino group substituted with an alkyl group having 1 to 6 carbon atoms);
² is a compound of the formula NH—R ³ (R ³ is a protecting group for nucleic acid synthesis of an amino group). 16. The method according to claim 1, wherein R ¹ is a group represented by the formula -P (R
^4a ) R ^{4b wherein} R ^4a and R ^4b are the same or different,
A hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 7 carbon atoms or an amino group substituted with an alkyl group having 1 to 6 carbon atoms);
² is a formula NH—R ³ (R ³ is a protecting group for nucleic acid synthesis of an amino group), and B is 6-benzoylaminopurine-9-
Yl group, adenyl group, 2-isobutyrylamino-6-
Hydroxypurin-9-yl group, guaninyl group, 2-oxo-4-benzoylamino-1,2-dihydropyrimidin-1-yl group, cytosinyl group, 2-oxo-5-methyl-4-benzoylamino-1, A compound which is a 2-dihydropyrimidin-1-yl group, a 5-methylcytosinyl group, a uracinyl group or a thyminyl group. 17. The method of claim 1, wherein R ¹ is of the formula -P (O
CH ₃ ) (N (CH (CH ₃ ) ₂ ) ₂ ) or the formula —P (O
CH ₂ CH ₂ CN) (N (CH (CH ₃ ) ₂ ) ₂ )
R ² is a formula NH—R ³ (R ³ is a trifluoroacetyl group, a benzyloxycarbonyl group, a 4,4′-dimethoxytrityl group or a 4-monomethoxytrityl group), and B is 6- Benzoylaminopurin-9-yl group, adenyl group, 2-isobutyrylamino-6-hydroxypurin-9-yl group, guaninyl group, 2-oxo-4-benzoylamino-1,2-dihydropyrimidine-1- A compound which is an yl group, a cytosinyl group, a 2-oxo-5-methyl-4-benzoylamino-1,2-dihydropyrimidin-1-yl group, a 5-methylcytosinyl group, a uracinyl group or a thyminyl group.