JP2004307464A - New artificial nucleic acid with saccharide s-type conformation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an intermediate for preparing an oligonucleotide that has excellent antisense or antigene activity or decoy nucleic acid, DNA enzyme and RNA interference function and is stable in vivo. <P>SOLUTION: This compound is represented by general formula (I) (wherein R<SP>1</SP>is H, a hydroxy-protecting group in the nucleic acid synthesis, R<SP>2</SP>is H or a 1-6C alkyl or amino group-protecting group in the nucleic acid synthesis, B is purin-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl group which may be substituted). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention has an excellent antisense or antigene activity, or a decoy nucleic acid, a DNA enzyme and an RNA interference function, and is an intermediate for producing a non-natural type oligonucleotide analogue that is stable in vivo. And a novel crosslinked nucleic acid derivative.

  Oligonucleotides which have excellent antisense or antigene activity or decoy nucleic acid, DNA enzyme and RNA interference functions and are stable in vivo are expected as useful medicines.

  Since natural oligonucleotides are known to be rapidly degraded by various nucleases present in blood and cells, in order to overcome this drawback, various non-natural oligonucleotide analogs are used. Attempts have been made to produce them and develop them as medicaments.

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 a thioate-type oligonucleotide, ISIS2922, as a therapeutic agent for human cytomegalovirus retinitis and sells it in the United States as Vitravene ^™ .

  However, the strength of antisense or antigene activity in the above-mentioned non-natural type oligonucleotide analogs, that is, the ability to form a stable complementary strand with mRNA or DNA, the stability to various nucleases, and the various proteins in vivo Considering the occurrence of side effects caused by non-specific binding to the non-natural oligonucleotide analogs, which have even better antisense or antigene activity, are stable in vivo, and have few side effects. The body is desired.

  It is known that the conformation of the sugar moiety of a nucleic acid has an S-type and an N-type, and it is known that the former conforms to DNA and the latter conforms to RNA (J Am. Chem. Soc., 94, 8205 (1992) and J. Am. Chem. Soc., 95, 2333 (1993)). Therefore, a nucleic acid having S-type selectively binds to DNA, and has an advantage of DNA selectivity as compared with a normal nucleic acid when used for the above-mentioned antisense application or the like. Furthermore, nucleic acid derivatives that adopt the S-type conformation and mimic DNA may be particularly useful as decoy nucleic acids (Gene Thrapy, 8, 1635 (2001)) and DNA enzymes (Nature, 375, 611 (1995)). Be expected. Further, a nucleic acid derivative having a high binding property to DNA is useful as a detection agent, a diagnostic agent or a primer for starting amplification of a specific gene such as a DNA chip.

  A compound which is an intermediate for producing a non-natural type oligonucleotide analog and is related to the intermediate of the present invention is described in JP-A-2001-64297. That is, the following general formula

(In the above formula, R ¹⁰¹ and R ¹⁰³ are the same or different and each represent a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, etc., and R ¹⁰² represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or an amino group. A protecting group for nucleic acid synthesis, wherein B ¹⁰⁰ is a purin-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl group which may have a substituent selected from the following α ¹⁰⁰ group: Is shown.).
(Α ¹⁰⁰ group) halogen atom, alkyl group having 1 to 6 carbon atoms, hydroxyl group, mercapto group, amino group and the like.

However, among the above compounds, a hydroxyl group or a derivative thereof is essential at the 2'-position of the nucleic acid, and a compound substituted with a hydrogen atom has not been known.
JP-A-2001-64297

  An object of the present invention is to provide a novel intermediate having excellent antisense or antigene activity, or a decoy nucleic acid, a DNA enzyme and an RNA interference function, and serving as an intermediate for producing an in vivo stable oligonucleotide. Another object of the present invention is to provide a bicyclonucleoside derivative.

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

  The inventor of the present invention has conducted intensive studies to solve the problem, and as a result, has 3′-amino-3′-N, 4′-C-methylene, and the 2′-position is substituted with a hydrogen atom. The novel bicyclonucleoside derivative is useful as an intermediate for producing an oligonucleotide analog having the property of forming a double-stranded or triple-stranded nucleic acid with respect to DNA. That the novel oligonucleotide analogue containing one or two or more has excellent nuclease resistance and can be an excellent antisense drug, antigene drug, decoy nucleic acid, DNA enzyme or RNA interference nucleic acid; The present inventors have found that a nucleic acid derivative having a high molecular weight is useful as a detection agent, a diagnostic agent, or a primer for initiating amplification of a specific gene such as a DNA chip, and completed the present invention.

The crosslinked nucleic acid derivative of the present invention,
1) General formula (1)

[Wherein, R ¹ is a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or a group represented by the formula —P (R ^3a ) R ^3b. (Wherein R ^3a and R ^3b are the same or different and each is a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, An amino group protected by a protecting 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. Represents a substituted amino group).
R ² represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a protecting group for nucleic acid synthesis of an amino group,
B represents a purine-9-yl or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have a substituent selected from the following α group. ]
And a pharmacologically acceptable salt thereof.
(Α group)
Hydroxyl group,
A hydroxyl group protected with a protecting group for nucleic acid synthesis,
An alkoxy group having 1 to 6 carbon atoms,
Mercapto group,
A mercapto group protected with a protecting group for nucleic acid synthesis,
An alkylthio group having 1 to 6 carbon atoms,
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 or pharmacologically acceptable salts thereof, preferred are:
2) When R ¹ is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, a lower alkyl, a lower alkoxy, a halogen or a cyano group, the aryl ring is substituted. A compound or a pharmacologically acceptable salt thereof, which is a methyl group or a silyl group substituted with 1 to 3 aryl groups,
3) R ¹ is a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a dimethoxytrityl group, a monomethoxytrityl group, a tert-butyldiphenylsilyl group, a -P (OC ₂ H ₄ CN) ( NCH (CH ₃ ) ₂ ), -P (OCH ₃ ) (NCH (CH ₃ ) ₂ ), a phosphonyl group, or a 2-chlorophenyl or 4-chlorophenyl phosphate group, or a pharmaceutically acceptable salt thereof ,
4) R ² is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aliphatic acyl group, an aromatic acyl group, a monomethoxytrityl group, an aryloxycarbonyl group, or 1 to 2 lower alkoxy or nitro An aralkyloxycarbonyl group in which the aryl ring may be substituted with a group, a compound or a pharmaceutically acceptable salt thereof,
5) R ² is a hydrogen atom, an alkyl group having 1 or 2 carbon atoms, an aliphatic acyl group, a monomethoxytrityl group, an aryloxycarbonyl group, or 1 to 2 lower alkoxy or nitro groups and an aryl ring An aralkyloxycarbonyl group which may be substituted, a compound or a pharmaceutically acceptable salt thereof,
6) a compound or a pharmaceutically acceptable salt thereof, wherein R ² is a hydrogen atom, a methyl group, a trifluoroacetyl group, a phenoxyacetyl group, a monomethoxytrityl group, or a benzyloxycarbonyl group;
7) B is a 6-aminopurin-9-yl group (that is, an adenyl group), a 6-aminopurin-9-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurine- 2,6-diaminopurin-9-yl group, 2-amino-6-chloropurin-9-yl group in which 9-yl group and amino group are protected with a protecting group for nucleic acid synthesis, and amino group are protection for nucleic acid synthesis. 2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group and 2-amino-6 in which the amino group is protected with a protecting group for nucleic acid synthesis. -Fluoropurin-9-yl group, 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-hydroxypurin-9-yl group (i.e. , 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, and 2-amino-6 in which an amino group and a hydroxyl group are protected with a protecting group for nucleic acid synthesis. -Hydroxypurin-9-yl group, 6-amino-2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidine- 1-yl group (that is, cytosinyl group), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group in which amino group is protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino -5-fluoro- , 2-Dihydropyrimidin-1-yl group, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis, 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), 2-oxo-4-hydroxy-5-methyl-1 , 2-dihydropyrimidin-1-yl group (ie, thyminyl group), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group (ie, 5-methylcytosini) Or a pharmacologically acceptable compound wherein the amino group is a 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group protected with a protecting group for nucleic acid synthesis. salt,
8) B is a 6-benzoylaminopurin-9-yl group, an adenyl group, a 2-isobutyrylamino-6-hydroxypurin-9-yl group, a guaninyl group, 2-oxo-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, uracinyl group or thyminyl group Or a pharmacologically acceptable salt thereof.

In the above, any combination obtained by selecting R ¹ from the above 2) or 3), selecting R ² from the above 4) to 6), and selecting B from the above 7) to 8) is also suitable. And particularly preferably a combination of 2), 4) and 7) and a combination of 3), 6) and 8).

Nucleic acid reagents and medicaments containing the novel oligonucleotide analogues of the present invention
1) General formula (Ia)

[Where,
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)
Hydroxyl group,
A hydroxyl group protected with a protecting group for nucleic acid synthesis,
An alkoxy group having 1 to 6 carbon atoms,
Mercapto group,
A mercapto group protected with a protecting group for nucleic acid synthesis,
An alkylthio group having 1 to 6 carbon atoms,
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 “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 structure (Ia). In such an analog, 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 amide in which the amino group on the purine base is amidated As other nucleoside or nucleotide units.

Among the nucleic acid reagents and medicaments containing the novel oligonucleotide analogs of the present invention, preferred are:
2) B is a 6-aminopurin-9-yl group (that is, an adenyl group), a 6-aminopurin-9-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurine- 9-yl group, 2-amino-6-chloropurin-9-yl group, 2-amino-6-chloropurin-9-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 2-amino-6 -Fluoropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, amino group protected with a protecting group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl group, amino A 2-amino-6-bromopurin-9-yl group, a 2-amino-6-hydroxypurin-9-yl group (that is, a guaninyl group) in which the group is protected by a protecting group for nucleic acid synthesis, and an amino group wherein 2-amino-6 protected with a protecting group of A 2-amino-6-hydroxypurin-9-yl group, a 6-amino-2-methoxypurin-9-yl group in which a droxypurin-9-yl group, an amino group and a hydroxyl group are protected by a protecting group for nucleic acid synthesis, 6 -Amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group , A 6-mercaptopurin-9-yl group, a 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (ie, a cytosinyl group), and an amino group protected by a protecting group for nucleic acid synthesis. -Oxo-4-amino-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group and amino group protect nucleic acid synthesis Protected by 2-oxo-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-dihydropyrimidin-1-yl group, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-hydroxy-1,2 -Dihydropyrimidin-1-yl group (ie, uracinyl group), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (ie, thyminyl group), 4-amino-5 -Methyl-2-oxo-1,2-dihydropyrimidin-1-yl group (that is, a 5-methylcytosinyl group) or 4-amino-5-methyl-2 in which an amino group is protected with a protecting group for nucleic acid synthesis. Oxo-1,2-dihydro-pyrimidin-1-yl group, oligonucleotide analogues and their pharmaceutically acceptable salts,
3) B is a 6-benzoylaminopurin-9-yl group, an adenyl group, a 2-isobutyrylamino-6-hydroxypurin-9-yl group, a guaninyl group, 2-oxo-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, uracinyl group or thyminyl group , Oligonucleotide analogs and pharmacologically acceptable salts thereof.

The “protecting group for the synthesis of a nucleic acid of a hydroxyl group” in the definition of R ¹ is not particularly limited as long as it can stably protect a hydroxyl group during the synthesis of a nucleic acid, and examples thereof include formyl, acetyl, 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, Halogeno lower alkylcarbonyl groups such as loroacetyl, trichloroacetyl and trifluoroacetyl; lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl; and unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl. "Aliphatic acyl group";
Arylcarbonyl groups such as benzoyl, α-naphthoyl and β-naphthoyl; halogenoarylcarbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl; lower alkyl groups such as 2,4,6-trimethylbenzoyl and 4-toluoyl Arylcarbonyl group, lower alkoxylated arylcarbonyl group such as 4-anisoyl, carboxylated arylcarbonyl group such as 2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl, 4-nitrobenzoyl, 2-nitrobenzoyl A lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl; an arylated arylcarbonyl group such as 4-phenylbenzoyl; A sil group ";
"Tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl, 4-methoxytetrahydrothiopyran-4-yl, such as" tetrahydropyran-2-yl A "pyranyl or tetrahydrothiopyranyl group"; a "tetrahydrofuranyl or tetrahydrothiofuranyl group" such as tetrahydrofuran-2-yl, tetrahydrothiofuran-2-yl;
Tri-lower alkylsilyl groups such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, diphenylmethylsilyl, t-butyldiphenylsilyl, diphenylisopropylsilyl A "silyl group" such as a tri-lower alkylsilyl group substituted with one or two aryl groups such as phenyldiisopropylsilyl;
"Lower alkoxymethyl groups" such as methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl;
A "lower alkoxylated lower alkoxymethyl group" such as 2-methoxyethoxymethyl;
"Halogeno lower alkoxymethyl" such as 2,2,2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl;
A "lower alkoxylated ethyl group" such as 1-ethoxyethyl, 1- (isopropoxy) ethyl;
"Halogenated ethyl groups" such as 2,2,2-trichloroethyl;
"A methyl group substituted with one to three aryl groups" such as benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl;
4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl, 4,4 ′, Aryl rings with lower alkyl, lower alkoxy, halogen or cyano groups such as 4 "-trimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl A methyl group substituted with one to three aryl groups substituted with
A "lower alkoxycarbonyl group" such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl;
A "lower alkoxycarbonyl group substituted with a halogen or a tri-lower alkylsilyl group" such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl;
"Alkenyloxycarbonyl groups" such as vinyloxycarbonyl and allyloxycarbonyl;
"One or two lower alkoxy or nitro groups such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl May be substituted aralkyloxycarbonyl group ''.
In R ¹ , an aryl ring is preferably substituted with an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, a lower alkyl, a lower alkoxy, a halogen or a cyano group. A methyl group or a silyl group substituted with 1 to 3 aryl groups, more preferably an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a dimethoxytrityl group, a monomethoxytrityl group, or tert-butyldiphenylsilyl group.

The protecting group for nucleic acid synthesis of the above-mentioned "phosphate group protected with a protecting group for nucleic acid synthesis" is not particularly limited as long as it can stably protect a phosphate group during nucleic acid synthesis. 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, -Methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3 "Lower alkyl" such as dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl;
"Cyanated lower alkyl groups" such as 2-cyanoethyl, 2-cyano-1,1-dimethylethyl;
"Ethyl group substituted by silyl group" such as 2-methyldiphenylsilylethyl, 2-trimethylsilylethyl, 2-triphenylsilylethyl;
"Halogenated lower" such as 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloro-1,1-dimethylethyl Alkyl group ";
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-hexenyl, 5-A "lower alkenyl group" such as hexenyl;
"Cycloalkyl groups" such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl;
"Cyanated lower alkenyl groups", such as 2-cyanobutenyl;
Benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthyl Ethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenyl Butyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthyl Pentyl, 2-naphthyl pentyl , 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylhexyl "Aralkyl groups" such as, 2-naphthylhexyl, 3-naphthylhexyl, 4-naphthylhexyl, 5-naphthylhexyl, 6-naphthylhexyl;
“Nitro group, halogen atom such as 4-chlorobenzyl, 2- (4-nitrophenyl) ethyl, o-nitrobenzyl, 4-nitrobenzyl, 2,4-dinitrobenzyl, 4-chloro-2-nitrobenzyl An aralkyl group substituted with an aryl ring ";
"Aryl groups" such as phenyl, indenyl, naphthyl, phenanthrenyl, anthracenyl;
2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2-bromophenyl, 4-nitrophenyl, 4-chloro-2-nitrophenyl Such as a "lower alkyl group, a halogen atom, an aryl group substituted with a nitro group",
Preferably, a "lower alkyl group", a "lower alkyl group substituted with a cyano group", an "aralkyl group", a "nitro group, an aralkyl group having an aryl ring substituted with a halogen atom" or a "lower alkyl group, halogen" An aryl group substituted with an atom or a nitro group ", and more preferably a 2-cyanoethyl group, a 2,2,2-trichloroethyl group, a benzyl group, a 2-chlorophenyl group or a 4-chlorophenyl group.

  Examples of the “alkyl group having 1 to 6 carbon atoms” in the above include, for example, a carbon number such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, pentyl, and hexyl. Examples thereof include a linear or branched alkyl group having 1 to 6 carbon atoms, preferably an alkyl group having 1 to 4 carbon atoms, and 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. For example, formyl, acetyl, propionyl , Butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, decanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, tridecanoyl, hexadecanoyl, 14-methylpentadecanoyl, Alkylcarbonyl groups such as 13,13-dimethyltetradecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl and henicosanoyl, carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl, adipoyl, chloroacetyl Halogeno lower alkylcarbonyl groups such as dichloroacetyl, trichloroacetyl, and trifluoroacetyl; lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl; and unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl. "Aliphatic acyl group";
Arylcarbonyl groups such as benzoyl, α-naphthoyl and β-naphthoyl; halogenoarylcarbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl; lower alkyl groups such as 2,4,6-trimethylbenzoyl and 4-toluoyl Arylcarbonyl group, lower alkoxylated arylcarbonyl group such as 4-anisoyl, carboxylated arylcarbonyl group such as 2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl, 4-nitrobenzoyl, 2-nitrobenzoyl An aromatic group such as a lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl and an arylated arylcarbonyl group such as 4-phenylbenzoyl; Sill group ";
A "lower alkoxycarbonyl group" such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl;
A "lower alkoxycarbonyl group substituted with a halogen or a tri-lower alkylsilyl group" such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl;
"Alkenyloxycarbonyl groups" such as vinyloxycarbonyl and allyloxycarbonyl;
"One or two lower alkoxy or nitro groups such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl Is optionally substituted aralkyloxycarbonyl group ";
4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl, 4,4 ′, Aryl rings with lower alkyl, lower alkoxy, halogen or cyano groups such as 4 "-trimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl A "methyl group substituted with 1 to 3 aryl groups" and "aryloxycarbonyl group",
Preferably, an "aliphatic acyl group", an "aromatic acyl group", an "aralkyloxycarbonyl group in which the aryl ring may be substituted with one or two lower alkoxy or nitro groups" or a "1-2 aralkyloxy group" An aralkyloxycarbonyl group which may be substituted on the aryl ring with a lower alkoxy or nitro group of
More preferably, "an aliphatic acyl group", "an aralkyloxycarbonyl group in which the aryl ring may be substituted with one or two lower alkoxy or nitro groups" or "one or two lower alkoxy or nitro groups" An aralkyloxycarbonyl group which may be substituted with an aryl ring;
Particularly preferred is a trifluoroacetyl group, a monomethoxytrityl group, or a benzyloxycarbonyl group.

  The “halogen atom” in the definition of the above α group includes a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and is preferably a fluorine atom or a chlorine atom.

As the “alkyl group having 1 to 6 carbon atoms” in the definition of R ^3a and R ^3b and the α group, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert- Examples thereof include a linear or branched alkyl group having 1 to 6 carbon atoms such as butyl, pentyl and hexyl, preferably a methyl group or an ethyl group.

  Examples of the protecting group for nucleic acid synthesis of the above-mentioned "hydroxyl group protected by a protecting group for nucleic acid synthesis" include the same groups as those exemplified in the above-mentioned "protecting group for nucleic acid synthesis of a hydroxyl group". , An "aliphatic acyl group" or an "aromatic acyl group", and more preferably a benzoyl group.

  Examples of the protecting group for nucleic acid synthesis of the “mercapto group protected with a protecting group for nucleic acid synthesis” in the above include, for example, those described above as the “protecting group for hydroxyl group nucleic acid synthesis”, methylthio, ethylthio, and tert-butylthio. Such an alkylthio group, an arylthio group such as benzylthio, and the like, a `` group forming a disulfide '' can be mentioned, preferably an `` aliphatic acyl group '' or an `` aromatic acyl group '', and more preferably , A benzoyl group.

Examples of the protecting group for nucleic acid synthesis of the “amino group protected by a protecting group for nucleic acid synthesis” in the definition of R ^3a and R ^3b and the α group include the groups exemplified in the above “protecting group for nucleic acid synthesis for amino group”. The same groups can be mentioned, preferably an "aliphatic acyl group" or an "aromatic acyl group", and more preferably a benzoyl group.

  As the “alkoxy group having 1 to 6 carbon atoms” in the above, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like And a straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms, preferably a methoxy or ethoxy group.

  Examples of the “alkyl having 1 to 6 carbon atoms” include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, tert-butylthio, pentylthio, and hexylthio groups. Is a methylthio or ethylthio group.

  As the “alkylamino group substituted with an alkyl group having 1 to 6 carbon atoms” in the above, 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 preferably a methylamino, ethylamino, dimethylamino or diethylamino group.

  Examples of the cyanoalkoxy group having 1 to 7 carbon atoms include, for example, cyanomethoxy, cyanoethoxy, cyanopropyloxy, cyanobutyloxy, cyanopentyloxy, cyanohexyloxy, and the like. 2-cyanoethoxy group.

  The "pharmacologically acceptable salt thereof" refers to a salt of the compound (I) of the present invention and the oligonucleotide analog containing the structure (Ia), which can be converted into a salt. Preferred salts are alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, and the like. Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts , Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine Amine salts such as organic salts such as salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylammonium salts, tris (hydroxymethyl) aminomethane salts; hydrofluorides , Hydrochlorides, hydrobromides, inorganic salts such as hydrohalides such as hydroiodide, nitrates, perchlorates, sulfates, phosphates; methanesulfonate, trifluoromethanesulfone Acid salts, lower alkane sulfonates such as ethane sulfonate, benzene sulfonate, aryl sulfonates such as p-toluene sulfonate, acetate, malate, fumarate, succinate, Organic acid salts such as citrate, tartrate, oxalate and maleate; and glycine, lysine, arginine, ornithine, Amino acid salts such as tamates and aspartates can be mentioned. In the case of oligonucleotide analogs having a bicyclonucleoside structure (Ia), sodium salts, potassium salts and triethylamine salts are preferred. In the case of (I), a free body is preferable.

  In addition, the oligonucleotide (A) containing the compound (I) of the present invention and the above-mentioned structure (Ia) absorbs water when left in the air to form adsorbed water or forms a hydrate. In some cases, such salts are also included in the present invention.

  Further, the oligonucleotide analogue containing the compound (I) of the present invention and the above-mentioned structure (Ia) may absorb some other solvent to form a solvate. Included in the invention.

  Specific examples of the bicyclonucleoside derivative (I) of the present invention include, for example, compounds shown in Tables 1 and 2 below.

In Tables 1 and 2,
“Bn” is a benzyl group, “Bz” is a benzoyl group, “PhOAc” is a phenoxyacetyl group, “Me” is a methyl group, “PMBn” is a p-methoxybenzyl group, “iPr” is isopropyl A group, "MMTr" is a 4-methoxytriphenylmethyl group, "DMTr" is a 4,4'-dimethoxytriphenylmethyl group, "TES" is a triethylsilyl group, "Tfa" is a trifluoroacetyl group, “TMTr” is a 4,4 ′, 4 ″ -trimethoxytriphenylmethyl group, “TMS” is a trimethylsilyl group, “TBDMS” is a tert-butyldimethylsilyl group, “TBDPS” is tert-butyldiphenylsilyl Group, “PCN” is cyanoethoxy (diisopropylamino) phosphino group, “PMN” is methoxy (diisopropylamino) phosphino group Shows the amino group.
(Table 1)

--------------------------------------------------------------------------
Exemplary compound No. R ¹ R ² R ⁴ R ⁵
--------------------------------------------------------------------------
1-1 HHHH
1-2 HHH OH
1-3 HHH SH
1-4 HHH NH ₂
1-5 H Me H NH ₂
1-6 HHH OMe
1-7 HHFH
1-8 HHF NH ₂
1-9 HH Cl H
1-10 HH Cl NH ₂
1-11 HH Cl Cl
1-12 HH Br H
1-13 HH Br NH ₂
1-14 HH OH H
1-15 HH OH OH
1-16 HH OH NH ₂
1-17 HH OMe OMe
1-18 HH OMe NH ₂
1-19 HH NH ₂ H
1-20 HH NH ₂ F
1-21 HH NH ₂ Cl
1-22 HH NH ₂ Br
1-23 HH NH ₂ OH
1-24 H Me NH ₂ OH
1-25 HH NH ₂ NH ₂
1-26 HH NH ₂ OMe
1-27 H PhOAc H NHBz
1-28 H Tfa H NHBz
1-29 H PhOAc NHBz OH
1-30 H Tfa NHBz OH
1-31 Bn PhOAc H NHBz
1-32 PMBn PhOAc H NHBz
1-33 Bn Tfa H NHBz
1-34 PMBn Tfa H NHBz
1-35 Bn PhOAc NHBz OH
1-36 PMBn PhOAc NHBz OH
1-37 Bn Tfa NHBz OH
1-38 PMBn Tfa NHBz OH
1-39 MMTr PhOAc H NHBz
1-40 DMTr PhOAc H NHBz
1-41 TMTr PhOAc H NHBz
1-42 MMTr Tfa H NHBz
1-43 DMTr Tfa H NHBz
1-44 TMTr Tfa H NHBz
1-45 MMTr PhOAc NHBz OH
1-46 DMTr PhOAc NHBz OH
1-47 TMTr PhOAc NHBz OH
1-48 MMTr Tfa NHBz OH
1-49 DMTr Tfa NHBz OH
1-50 TMTr Tfa NHBz OH
1-51 TBDPS PhOAc H NHBz
1-52 TBDMS PhOAc H NHBz
1-53 TMS PhOAc H NHBz
1-54 TBDPS Tfa H NHBz
1-55 TBDMS Tfa H NHBz
1-56 TMS Tfa H NHBz
1-57 TBDPS PhOAc NHBz OH
1-58 TBDMS PhOAc NHBz OH
1-59 TMS PhOAc NHBz OH
1-60 TBDPS Tfa NHBz OH
1-61 TBDMS Tfa NHBz OH
1-62 TMS Tfa NHBz OH
1-63 DMTr Tfa NHCOCH (CH ₃ ) ₂ OH
1-64 DMTr PhOAc NHCOCH (CH ₃ ) ₂ OH
1-65 DMTr Me NHCOCH (CH ₃ ) ₂ OH
1-66 DMTr Me H NHBz
1-67 TBDMS Tfa NHCOCH (CH ₃ ) ₂ OH
1-68 H Me NHCOCH (CH ₃ ) ₂ OH
1-69 H Me H NHBz
1-70 TBDPS MMTr H NHBz
1-71 TBDPS MMTr NHCOCH (CH ₃ ) ₂ OH
1-72 TBDMS MMTr H NHBz
1-73 TBDMS MMTr NHCOCH (CH ₃ ) ₂ OH
1-74 H MMTr H NHBz
1-75 H MMTr NHCOCH (CH ₃ ) ₂ OH
1-76 PCN MMTr H NHBz
1-77 PCN MMTr NHCOCH (CH ₃ ) ₂ OH
1-78 PMN MMTr H NHBz
1-79 PMN MMTr NHCOCH (CH ₃ ) ₂ OH
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
(Table 2)

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Exemplary compound No. R ¹ R ² R ⁶ R ⁷
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
2-1 HHHH
2-2 HH Cl H
2-3 HH OH H
2-4 H Me OH H
2-5 HH OH Me
2-6 H Me OH Me
2-7 HH SH H
2-8 HH NH ₂ H
2-9 H Me NH ₂ H
2-10 HH NH ₂ F
2-11 HH NH ₂ Cl
2-12 HH NH ₂ Me
2-13 HH OMe H
2-14 H PhOAc OH H
2-15 H Tfa OH H
2-16 H PhOAc OH Me
2-17 H Tfa OH Me
2-18 H PhOAc NHBz H
2-19 H Tfa NHBz H
2-20 Bn PhOAc OH H
2-21 PMBn PhOAc OH H
2-22 Bn Tfa OH H
2-23 PMBn Tfa OH H
2-24 Bn PhOAc OH Me
2-25 PMBn PhOAc OH Me
2-26 Bn Tfa OH Me
2-27 PMBn Tfa OH Me
2-28 Bn PhOAc NHBz H
2-29 PMBn PhOAc NHBz H
2-30 Bn Tfa NHBz H
2-31 PMBn Tfa NHBz H
2-32 MMTr PhOAc OH H
2-33 DMTr PhOAc OH H
2-34 TMTr PhOAc OH H
2-35 MMTr Tfa OH H
2-36 DMTr Tfa OH H
2-37 TMTr Tfa OH H
2-38 MMTr PhOAc OH Me
2-39 DMTr PhOAc OH Me
2-40 TMTr PhOAc OH Me
2-41 MMTr Tfa OH Me
2-42 DMTr Tfa OH Me
2-43 TMTr Tfa OH Me
2-44 MMTr PhOAc NHBz H
2-45 DMTr PhOAc NHBz H
2-46 TMTr PhOAc NHBz H
2-47 MMTr Tfa NHBz H
2-48 DMTr Tfa NHBz H
2-49 TMTr Tfa NHBz H
2-50 TBDPS PhOAc OH H
2-51 TBDMS PhOAc OH H
2-52 TMS PhOAc OH H
2-53 TBDPS Tfa OH H
2-54 TBDMS Tfa OH H
2-55 TMS Tfa OH H
2-56 TBDPS PhOAc OH Me
2-57 TBDMS PhOAc OH Me
2-58 TMS PhOAc OH Me
2-59 TBDPS Tfa OH Me
2-60 TBDMS Tfa OH Me
2-61 TMS Tfa OH Me
2-62 TBDPS PhOAc NHBz H
2-63 TBDMS PhOAc NHBz H
2-64 TMS PhOAc NHBz H
2-65 TBDPS Tfa NHBz H
2-65 TBDMS Tfa NHBz H
2-67 TMS Tfa NHBz H
2-68 DMTr Tfa NHBz Me
2-69 H Me NHBz Me
2-70 TBDPS (MMTr) NH OH H
2-71 TBDPS (MMTr) NH OH Me
2-72 TBDPS (MMTr) NH NHBz H
2-73 TBDPS (MMTr) NH NHBz Me
2-74 TBDMS (MMTr) NH OH H
2-75 TBDMS (MMTr) NH OH Me
2-76 TBDMS (MMTr) NH NHBz H
2-77 TBDMS (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 PCN (MMTr) NH OH H
2-83 PCN (MMTr) NH OH Me
2-84 PCN (MMTr) NH NHBz H
2-85 PCN (MMTr) NH NHBz Me
2-86 PMN (MMTr) NH OH H
2-87 PMN (MMTr) NH OH Me
2-88 PMN (MMTr) NH NHBz H
2-89 PMN (MMTr) NH NHBz Me
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
In the above table, preferred compounds include compound numbers 1-3, 1-4, 1-5, 1-8, 1-10, 1-11, 1-17, 1-18, 1-20, 1- 21, 1-22, 1-23, 1-24, 1-25, 1-28, 1-30, 1-54, 1-60, 1-70 to 1-79, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-16, 2-17, 2-19, 2-42, 2-56, 2- 59, 2-65, 2-70 to 2-90,
Further preferred compounds include Compound Nos. 1-4, 1-23, 1-76 to 1-79, 2-5, 2-6, 2-8, 2-16, 2-17, 2-42, 2 -56, 2-59, 2-83 to 2-90.

  The bicyclonucleoside derivative (I) of the present invention can be produced by Method A described below.

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

R ⁸ represents a protecting group for nucleic acid synthesis of a hydroxyl group, and is preferably t-butyldimethylsilyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, t-butyldiphenylsilyl, diphenylmethylsilyl. A silyl group such as triphenylsilyl, a trityl group such as 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4 ', 4 "-trimethoxytriphenylmethyl, benzyl, p- An aralkyl group such as methoxybenzyl, and more preferably a t-butyldiphenylsilyl group.

R ⁹ represents a protecting group for nucleic acid synthesis of an amino group, preferably an “aliphatic acyl group”, an “aromatic acyl group”, or an aryl ring substituted with one or two lower alkoxy or nitro groups. An aralkyloxycarbonyl group which may be substituted or an aralkyloxycarbonyl group which may be substituted on the aryl ring with one or two lower alkoxy or nitro groups, and more preferably an "aliphatic acyl group" , "An aralkyloxycarbonyl group in which an aryl ring may be substituted with one or two lower alkoxy or nitro groups" or "an aralkyl in which an aryl ring may be substituted with one or two lower alkoxy or nitro groups" Oxycarbonyl group '', particularly preferably a trifluoroacetyl group, a monomethoxytrityl group, or a benzyloxycarbonyl group.

B ¹ represents a purin-9-yl or 2-oxo-1,2-dihydropyrimidin-1-yl group which may have a substituent selected from the following α1 group.
(Group α1)
Halogen atom,
An alkyl group having 1 to 6 carbon atoms,
Hydroxyl group,
A hydroxyl group protected with a protecting group for nucleic acid synthesis,
Mercapto group,
A mercapto group protected with a protecting group for nucleic acid synthesis,
Amino group,
An amino group protected with a protecting group for nucleic acid synthesis,
An alkoxy group having 1 to 6 carbon atoms,
An alkylthio group having 1 to 6 carbon atoms, and
A mono- or dialkylamino group substituted with an alkyl group having 1 to 6 carbon atoms;

  Method A is a method for obtaining the target compound (I) using the compound (III) as a starting material.

  Hereinafter, each step of the method A will be described in detail.

<Method A>
(Step A-1)
This step is a step of converting a hydroxyl group of compound (II), which can be produced by a known method, into a hydrogen atom in an inert solvent in the presence of hydrogen and a catalyst to produce compound (III).

  Compound (II) is the same as compound (1) in JP-A-2001-64297, and can be obtained according to the method described in JP-A-2001-64297.

This step is performed by performing an addition reaction (A-1a) between a hydroxyl group and a thiocarbonyl group, and then performing a reduction reaction (A-1b).
(A-1a) Addition reaction of hydroxyl group and thiocarbonyl group The solvent to be used is not particularly limited as long as it does not participate in this reaction, but ethers such as diethyl ether, tetrahydrofuran and dioxane are preferred. And amides such as formamide, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone and hexamethylphosphorotriamide, and nitriles such as acetonitrile and isobutyronitrile, and preferably nitriles. .

  The reagent used is not particularly limited as long as it is generally used for a thiocarbonyl group addition reaction, but is preferably a halogenophenoxythiocarbonyl, and more preferably phenoxythiocarbonyl chloride. Used.

  The reaction temperature and the reaction time vary depending on the type of the starting material, the solvent and the catalyst, but are usually 0 ° C to 100 ° C (preferably 20 ° C to 40 ° C), and 5 minutes to 48 hours (preferably 1 Hours to 10 hours).

  After completion of the reaction, the target compound of the present reaction is, for example, 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, anhydrous magnesium sulfate, etc. And dried to remove the solvent.

If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, silica gel column chromatography or the like. Further, it can be used for the next reaction without purification.
(A-1b) Reduction reaction Examples of the solvent used include aromatic hydrocarbons such as benzene, toluene, and xylene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether. Examples of such ethers include aromatic hydrocarbons, and toluene is more preferable.

  As the reducing agent used, for example, azobisisobutyronitrile, using a radical initiator such as triphenylboron as a catalyst, tributyltin hydride, triphenyltin hydride, radical reducing agent such as dibutyltin hydride And preferably a combination of azobisisobutyronitrile and tributyltin hydride.

  The reaction temperature varies depending on the starting compound and the reaction reagent, but it is carried out at 20 ° C to 200 ° C, preferably 50 ° C to 120 ° C.

  The reaction time varies depending on the reaction temperature, the starting compound, the reaction reagent or the type of the solvent used, but is usually 1 hour to 3 days, preferably 3 hours to 24 hours.

  After completion of the reaction, the target compound of the present reaction is, for example, 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, anhydrous magnesium sulfate, etc. And dried to remove the solvent.

  If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, silica gel column chromatography or the like.

(Step A-2)
In this step, a compound (II) produced in step A-1 is reacted with a deprotecting reagent in an inert solvent, and if necessary, alkylation is carried out to produce compound (I). It is.

  The method of deprotection varies depending on the type of protecting group, but is not particularly limited as long as it does not cause other side reactions. For example, "Protective Groups in Organic Synthesis" (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 “lower alkyl, lower alkoxy, halogen, cyano, In the case of "a methyl group substituted with one to three aryl groups having an aryl ring substituted with a group" and (3) a "silyl group", the reaction can be performed by the following method.
(1) In the case of an aliphatic acyl group and 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. Examples thereof include water; alcohols such as methanol, ethanol, and n-propanol; and ethers such as tetrahydrofuran and dioxane. And the like, or a mixed solvent of water and the above-mentioned organic solvent, and 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, and lithium carbonate. Alkali metal carbonates; alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, and barium hydroxide or ammonia water, and ammonia such as concentrated ammonia-methanol. , An alkali metal carbonate.

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

  After completion of the reaction, the target compound (I) of this reaction 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.

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

  As the solvent to be used, preferably, alcohols such as methanol, ethanol and isopropanol; ethers such as diethyl ether, tetrahydrofuran and dioxane; aromatic hydrocarbons such as toluene, benzene and xylene; hexane; Examples thereof include aliphatic hydrocarbons such as 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 generally used for a catalytic reduction reaction, but is preferably palladium carbon, Raney nickel, platinum oxide, platinum black, rhodium-aluminum oxide, triphenylphosphine. -Rhodium chloride and palladium-barium sulfate.

  The pressure is not particularly limited, but it is usually 1 to 10 atm.

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

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

  After completion of the reaction, the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method. For example, the reducing agent is removed from the reaction mixture, 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 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.

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

  In this case, examples of the solvent used include aromatic hydrocarbons such as benzene, toluene, and xylene; and halogenated solvents such as methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, chlorobenzene, and dichlorobenzene. Hydrocarbons; alcohols such as methanol, ethanol, isopropanol and tert-butanol; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N Amides such as -methyl-2-pyrrolidone, N-methylpyrrolidinone, and hexamethylphosphorotriamide; and organic acids such as acetic acid. Preferably, organic acids (particularly acetic acid) or alcohols ( Particularly, tert-butanol).

  The acid used is preferably acetic acid or trifluoroacetic acid.

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

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

  After completion of the reaction, the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method. For example, neutralizing 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, 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 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. Alternatively, it can be removed by treatment with an organic acid such as acetic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, or an inorganic acid such as hydrochloric acid.

  In addition, when removing with a fluorine anion, the reaction may be accelerated by adding an organic acid such as formic acid, acetic acid, or propionic acid.

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

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

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

  After completion of the reaction, the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method. For example, it can be obtained by removing the solvent and purifying by silica gel column chromatography.

The compound in which R ² is an alkyl group having 1 to 6 carbon atoms can be produced by deprotecting R ^{10 by the} above-mentioned method and then using an alkylating agent and a base.

  The solvent to be used is not particularly limited as long as it does not hinder the reaction and dissolves the starting material to some extent, but preferably, ethers such as diethyl ether, dioxane and tetrahydrofuran, methylene chloride, chloroform and tetrahydrofuran are used. Chlorinated hydrocarbons such as carbon chloride, dichloroethane, chlorobenzene and dichlorobenzene, and mixed solvents thereof.

  As the alkylating agent used, alkyl iodide is preferred.

  As a base to be used, an organic base such as triethylamine, pyridine, dimethylaminopyridine, 1,8-diazabicyclo [5,4,0] undec-7-ene is preferable.

  The reaction temperature is from -30C to 40C, preferably from -10C to 20C.

  The reaction time is 1 to 100 hours, preferably 12 to 48 hours.

  After completion of the reaction, the target compound (I) of this reaction is collected from the reaction mixture according to a conventional method. For example, it can be obtained by removing the solvent and purifying by silica gel column chromatography.

When R ¹ is a phosphate group or a protected phosphate group, the following method is additionally performed.

That is, a desired phosphorylating reagent (for example, 2-chlorophenylphosphorobistriazolide) is reacted with the compound (I) in which R ¹ is hydrogen in an inert solvent.

  The solvent to be used is not particularly limited as long as it does not inhibit the reaction, but usually, an aromatic amine such as pyridine is used.

  The reaction temperature is not particularly limited from −20 to 100 ° C., but the reaction is usually performed at room temperature. The reaction time varies depending on the solvent and the reaction temperature, but is 1 hour when pyridine is used as the reaction solvent and the reaction is performed at room temperature.

  After completion of the reaction, the target 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, and washing with water. The organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off.

  If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.

  The protecting group on the phosphate group can be removed by a method usually used in nucleic acid synthesis.

When R ¹ is a group represented by the above formula —P (R ^3a ) R ^3b , the following method is additionally performed.

In this step, a compound in which R ¹ is hydrogen in an inert solvent in the presence of a deoxidizing agent is added to a compound of the formula ^XP (R ^3a2 ) R ^3b2 (where R ^3a2 and R ^3b2 are the same or different. A hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group protected with a protecting group for nucleic acid synthesis, an alkoxy group having 1 to 6 carbon atoms, A phosphorylating agent represented by 6 alkylthio groups, 1-7 carbon atoms of a cyanoalkoxy group or an amino group substituted by 1-6 carbon atoms, and X represents a halogen atom) Is reacted.

  Preferred phosphating agents used are chloromorpholinomethoxyphosphine, chloromorpholinocyanoethoxyphosphine, chlorodimethylaminomethoxyphosphine, chlorodimethylaminocyanoethoxyphosphine, chlorodiisopropylaminomethoxyphosphine, chlorodiisopropylaminocyanoethoxyphosphine. And more preferably, chloromorpholinomethoxyphosphine, chloromorpholino cyanoethoxyphosphine, chlorodiisopropylaminomethoxyphosphine, and chlorodiisopropylaminocyanoethoxyphosphine.

  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.

  Examples of the deoxidizing agent used include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine and diisopropylethylamine. Preferably, aliphatic amines (particularly, Diisopropylethylamine).

  The reaction temperature is not particularly limited, but is usually -50 to 50 ° C, and preferably room temperature. 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, and preferably 30 minutes when reacted at room temperature.

  After completion of the reaction, the target 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, and washing with water. Thereafter, 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.

  If desired, protecting groups on each functional group can be removed by a commonly used method during nucleic acid synthesis.

  Using the compound (I) of the present invention, an oligonucleotide analog having the above structure (Ia) can be produced by the method B described below.

In the above process chart, R ¹⁰ represents a protecting group for nucleic acid synthesis of an amino group (particularly, a trityl group which may be substituted with a methoxy group), R ¹¹ represents a phosphonyl group, a monosubstituted chloro (alkoxy) ) Represents a group formed by reacting phosphines or disubstituted-alkoxyphosphines.

Hereinafter, each step of the method B will be described in detail.
<Method B>
(Step B-1)
In this step, amiditization is carried out on a compound (Ib) produced by the method A in an inert solvent (the compound corresponds to the compound (I) obtained by deprotecting R ¹ in the step A-2). This is a step of producing a compound (IV) by reacting a mono-substituted-chloro (alkoxy) phosphine or a di-substituted-alkoxyphosphine which is usually used for the above.

  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.

  Examples of the monosubstituted-chloro (alkoxy) phosphines used include, for example, chloro (morpholino) methoxyphosphine, chloro (morpholino) cyanoethoxyphosphine, chloro (dimethylamino) methoxyphosphine, chloro (dimethylamino) cyanoethoxyphosphine, and chloro. Examples thereof include phosphines such as (diisopropylamino) methoxyphosphine and chloro (diisopropylamino) cyanoethoxyphosphine, and preferred are chloro (morpholino) methoxyphosphine, chloro (morpholino) cyanoethoxyphosphine, and chloro (diisopropylamino) methoxyphosphine. , Chloro (diisopropylamino) cyanoethoxyphosphine.

  When mono-substituted-chloro (alkoxy) phosphines are used, a deoxidizing agent is used. In this case, the deoxidizing agent used is a heterocyclic amine such as pyridine, dimethylaminopyridine, trimethylamine, Aliphatic amines such as triethylamine and diisopropylethylamine are exemplified, and aliphatic amines (particularly diisopropylethylamine) are preferred.

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

  When disubstituted-alkoxyphosphines are used, an acid is used, in which case the acid used is preferably tetrazole, acetic acid or p-toluenesulfonic acid.

  The reaction temperature is not particularly limited, but is usually -50 to 50 ° C, and preferably room temperature.

  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, and preferably 30 minutes when reacted at room temperature.

  After completion of the reaction, the target compound (13) of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, 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 added. And dried over anhydrous sodium sulfate or the like, and then the solvent is distilled off.

  If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or silica gel column chromatography.

  Alternatively, in this step, the compound (Ib) is reacted with tris- (1,2,4-triazolyl) phosphite in an inert solvent (preferably, a halogenated hydrocarbon such as methylene chloride). , Water and H-phosphonation to produce compound (IV).

  The reaction temperature is not particularly limited, but is usually -20 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, and preferably 30 minutes when reacted at room temperature.

  After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, 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 added. And dried over anhydrous sodium sulfate or the like, and then the solvent is distilled off.

If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
(Step B-2)
In this step, at least one compound (IV) produced with B-1 and a commercially available phosphoramidite reagent or the like necessary for producing an oligonucleotide having a desired nucleotide sequence, This is a step of producing an oligonucleotide analog containing the target structure (Ia) on an automatic DNA synthesizer by an ordinary method.

Oligonucleotide analogs having a desired nucleotide sequence can be obtained from a literature (Nucleic Acid) using a DNA synthesizer, for example, Model 392 by the phosphoramidite method of PerkinElmer.
Acids Research, 12, 4539 (1984)).

  If desired, when thioate is used, a reagent that reacts with trivalent phosphoric acid such as tetraethylthiuram disulfide (TETD, Applied Biosystems) or Beaucage reagent (Millipore) in addition to sulfur to use thioate is used. The thioate derivative can be produced according to the method described in the literature (Tetarhedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990)).

  The resulting crude oligonucleotide can be purified using an Oligopack (reverse phase chromatography column), and the purity of the purified product can be confirmed by HPLC analysis.

  The chain length of the obtained oligonucleotide analog is usually 2 to 50, preferably 10 to 30, as nucleoside units.

  The obtained oligonucleotide analog is hardly degraded 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 a triplex is formed between the double-stranded DNA in the genome and transcription to mRNA. Or inhibit the growth of viruses that infect cells. In addition, the obtained oligonucleotide analog takes an S-type conformation, selectively binds to DNA in a situation where DNA and RNA are mixed, and is useful as, for example, a DNA enzyme.

  The oligonucleotide analogue of the present invention is expected as a drug for inhibiting diseases of specific genes, including antitumor agents and antiviral agents, and for treating diseases.

  The oligonucleotide analogue of the present invention can be formulated into a parenteral administration preparation or a liposome preparation by blending a conventional auxiliary agent 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 amount used depends on the symptoms, age, administration method, etc., but for example, the lower limit is 0.001 mg / kg body weight (preferably 0.01 mg / kg
It is desirable to use 100 mg / kg body weight (preferably 10 mg / kg body weight) once or several times a day depending on the symptom.

  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.

(Example 1) 3 '-(4-methoxytrityl) amino-3'-deoxy-3'-N, 4'-C-methylenethymidine
(1a) 5'-O-tert-butyldiphenylsilyl-3'-phenoxyacetylamino-2'-O-phenoxythiocarbonyl-3'-deoxy-3'-N, 4'-C-methylene-5-methyl Uridine 5'-O-tert-butyldiphenylsilyl-3'-phenoxyacetylamino-3'-deoxy-3'-N, 4'-C-methylene-5-methyleneuridine under nitrogen stream (obtained in Reference Example 4) To a solution of the obtained compound (69 mg, 0.108 mmol) in anhydrous acetonitrile (10 ml) were added 4- (dimethylamino) pyridine (32 mg, 0.259 mmol) and phenoxythiocarbonyl chloride (18 ml, 0.130 mmol). For 3 hours. After adding water, the solvent was distilled off, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogen carbonate, water, and saturated saline. After the organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off to obtain the desired compound (104 mg). The compound was used for the next reaction without purification.

(1b) 5'-O-tert-butyldiphenylsilyl-3'-phenoxyacetylamino-3'-deoxy-3'-N, 4'-C-methylenethymidine 1a obtained in the previous reaction under a stream of nitrogen (104 mg) in anhydrous toluene solution (10 ml) were added with tributyltin hydride (69 ml, 0.258 mmol) and 2,2'-azobisisobutyronitrile (11 mg, 0.065 mmol) and refluxed for 13 hours. . Further, tributyltin hydride (69 ml, 0.258 mmol) and 2,2′-azobisisobutyronitrile (11 mg, 0.065 mmol) were added, and the mixture was stirred for 4 hours. The solvent was distilled off, and the residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 1: 1) to obtain the desired compound (37 mg, 0.059 mmol, 55% from 5 in 2 steps) as a colorless oily substance.
¹ H-NMR (DMSO-d ₆ , 150 ° C): δ 0.81 (s, 9H), 1.38 (s, 3H), 1.85 (s, 1H), 2.36 (dd, 1H, J = 6, 14 Hz), 3.71, 4.01 (AB, 2H, J = 11 Hz), 3.74, 3.80 (AB, 2H, J = 11 Hz), 4.33 (s, 2H), 4.70 (d, 1H, J = 5 Hz), 6.18 (t , 1H, J = 8 Hz), 6.67-7.39 (m, 16H).

(1c) 5'-O-tert-butyldiphenylsilyl-3 '-(4-methoxytrityl) amino-3'-deoxy-3'-N, 4'-C-methylenethymidine
To a 1,4-dioxane solution (5 ml) of 1b (136 mg, 0.22 mmol) was added a 28% aqueous ammonia solution (1 ml), and the mixture was stirred at 55 ° C. Further, a 28% aqueous ammonia solution was added 0.8 ml after 3 hours, 1 ml after 16 hours, 1 ml after 20 hours, and 2 ml after 26 hours, followed by stirring for a total of 40 hours. The solvent was distilled off to obtain 11 crude products (200 mg). Subsequently, under a nitrogen stream, 4-methoxytrityl chloride (51 mg, 0.17 mmol) was added to an anhydrous pyridine solution (3 ml) of the obtained 11 crude product (100 mg of 200 mg was used) at room temperature. Was added and stirred at room temperature for 14 hours. Saturated aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and 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 (n-hexane: ethyl acetate = 3: 1 → 1: 1) to give the desired compound (12 mg, 0.016 mmol). Obtained as a colorless oil.
¹ H-NMR (CDCl ₃ ): δ 0.95 (s, 9H), 1.24 (d, 3H, J = 1 Hz), 1.67 (m, 1H), 2.61, 3.50 (AB, 2H, J = 10 Hz), 2.90, 3.58 (AB, 2H, J = 10 Hz), 2.80 (dd, 1H, J = 5, 13 Hz), 3.79 (s, 3H), 4.15 (d, 1H, J = 5 Hz), 6.84 (d , 2H, J = 9 Hz), 7.11 (dd, 1H, J = 5, 10 Hz), 7.17-7.56 (m, 23H), 8.27 (brs, 1H).

(1d) 3c (10 mg, 0.013 mmol), a solution of 3 '-(4-methoxytrityl) amino-3'-deoxy-3'-N, 4'-C-methylenethymidine in a stream of nitrogen in anhydrous tetrahydrofuran (1 ml), tetrabutylammonium fluoride (1.0 M in tetrahydrofuran, 14 μl, 0.014 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Further, tetrabutylammonium fluoride (1.0 M in tetrahydrofuran, 14 μl, 0.014 mmol) was added, and the mixture was stirred for 20 hours. After evaporating the solvent, the residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 1: 1) to obtain the desired compound (5 mg, 0.010 mmol, 77%) as a colorless oil.
¹ H-NMR (CDCl ₃ ): δ 1.80 (m, 1H), 1.90 (d, 3H, J = 1 Hz), 2.52, 3.26 (AB, 2H, J = 12 Hz), 2.72 (dd, 1H, J = 5, 14 Hz), 2.97, 3.60 (AB, 2H, J = 10 Hz), 3.79 (s, 3H), 3.98 (d, 1H, J = 5 Hz), 6.84 (d, 2H, J = 9 Hz) ), 6.89 (dd, 1H, J = 5, 9 Hz), 7.18-7.53 (m, 13H).

(1e) 5′-O- (N, N-diisopropylamino-β-cyanoethoxyphosphino) -3 ′-(4-methoxytrityl) amino-3′-deoxy-3′-N, 4′-C- Methylene thymidine
A mixture of 1d (5 mg, 0.010 mmol) and diisopropylammonium tetrazolide (2 mg, 0.012 mmol) was azeotroped three times with anhydrous acetonitrile, and then anhydrous acetonitrile-anhydrous tetrahydrofuran solution (3: 1, v / v, 2 ml). Under a nitrogen stream, 2-cyanoethyl N, N, N ', N'-tetraisopropylphosphoramidite (5 µl, 0.017 mmol) was added dropwise at room temperature, and the mixture was stirred at room temperature for 11 hours. After evaporating the solvent, the residue was purified by silica gel column chromatography (n-hexane: ethyl acetate = 2: 1) to obtain the desired compound (7 mg, 100%) as a colorless oil.
³¹ P-NMR (acetone-d ₆ ): δ 149.7, 149.4.

Reference Example 1 5-O-tert-butyldiphenylsilyl-3-deoxy-1,2-O-isopropylidene-3-phenoxyacetylamino-3-N, 4-C-methylene-α-D-ribofuranose Under a nitrogen stream, 3-amino-5-O-tert-butyldiphenylsilyl-3-deoxy-1,2-O-isopropylidene-3-N, 4-C-methylene-α-D-ribofuranose ( A compound described as Reference Example 6 in 2001-64297: 1.70 g, 3.87 mmol) in anhydrous methylene chloride solution (70 ml) at 0 ° C., phenoxyacetyl chloride (0.59 ml, 4.26 mmol), triethylamine ( 0.81 ml, 5.81 mmol) and stirred at room temperature for 30 minutes. Saturated aqueous sodium hydrogen carbonate was added to the reaction solution, extracted with methylene chloride, and washed with water and 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 (n-hexane: ethyl acetate = 4: 1) to give the title compound (2.02 g, yield 91%) as a colorless oil. Obtained as material.

[α] _D ²⁵ +75.2 (c 1.05, CHCl ₃ ).
IRνmax (KBr) cm ^-1 : 2936, 2856, 1654, 1595, 1432, 1380, 1221, 1108.
¹ H-NMR (CDCl ₃ ) δppm: 1.01 (27 / 4H, s), 1.03 (9 / 4H, s), 1.44 (3 / 4H, s), 1.56 (9 / 4H, s), 3.64, 3.79 ( 6 / 4H, AB, J = 11Hz), 3.66, 3.77 (2 / 4H, AB, J = 11Hz), 3.88, 4.05 (6 / 4H, AB, J = 11Hz), 4.18, 4.28 (2 / 4H, AB , J = 10Hz), 4.53-4.85 (4H, m), 6.04 (1 / 4H, d, J = 5Hz), 6.08 (3 / 4H, d, J = 5Hz), 6.85-7.04 (3H, m), 7.24-7.45 (8H, m), 7.54-7.61 (4H, m).
¹ H-NMR (DMSO-d ₆ , 150 ° C) δppm: 1.06 (9H, s), 1.35 (3H, s), 1.48 (3H, s), 3.81-4.01 (4H, m), 4.53, 4.61 (2H , AB, J = 15Hz), 4.75-4.78 (2H, m), 6.01 (1H, d, J = 3Hz), 6.93-6.98 (3H, m), 7.24-7.30 (2H, m), 7.38-7.47 ( 6H, m), 7.62-7.64 (4H, m).
¹³ C-NMR (DMSO-d ₆ , 150 ° C) δppm: 18.1, 18.5, 25.9, 26.0, 64.4, 66.1, 78.9, 85.1, 109.0, 112.3, 114.0, 120.3, 126.8, 128.4, 128.8, 132.3, 134.1, 157.4 .
Mass (FAB) m / z 574 (MH ⁺ ).
Anal.calced for C ₃₃ H ₃₉ NO ₆ Si ・ H ₂ O: C, 69.08; H, 6.85; N, 2.44. Found: C, 68.87; H, 6.86; N, 2.43.
Reference Example 2 5-O-tert-butyldiphenylsilyl-3-deoxy-1,2-di-O-acetyl-3-phenoxyacetylamino-3-N, 4-C-methylene-α and β-D -Ribofuranose Under a nitrogen stream, a solution of the compound obtained in Reference Example 1 (2.00 g, 3.49 mmol) in acetic acid (15 ml) was added at room temperature to acetic anhydride (3.63 ml, 38.4 mmol) and concentrated sulfuric acid (0%). (0.032 ml, 0.63 mmol) and stirred for 1 hour. The reaction solution was neutralized by dropping the reaction solution at 0 ° C. in a saturated aqueous solution of sodium bicarbonate, and the solution was filtered. The mother liquor was extracted with ethyl acetate, and washed with saturated aqueous sodium hydrogen carbonate and 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 (n-hexane: ethyl acetate = 2: 1) to give the title compound (1.60 g, yield 74%) as a colorless oil. Obtained as material.

alpha isomer
[α] _D ²⁴ +60.7 (c 1.03, CHCl ₃ ).
IRνmax (KBr) cm ^-1 : 3059, 2936, 2859, 1755, 1659, 1595, 1432, 1368, 1220, 1109, 1011.
¹ H-NMR (CDCl ₃ ) δppm: 1.02 (9 / 2H, s), 1.05 (9 / 2H, s), 1.76 (3 / 2H, s), 1.95 (3 / 2H, s), 2.07 (3 / 2H, s), 2.10 (3 / 2H, s), 3.71, 3.77 (2 / 2H, AB, J = 12Hz), 3.76 (2 / 2H, s), 4.08 (2 / 2H, s), 4.29, 4.35 (2 / 2H, AB, J = 10Hz), 4.55, 4.57 (2 / 2H, AB, J = 15Hz), 4.61, 4.74 (2 / 2H, AB, J = 15Hz), 5.04 (1 / 2H, d, J = 6Hz), 5.10 (1 / 2H, d, J = 6Hz), 5.17 (1 / 2H, dd, J = 5, 6Hz), 5.30 (1 / 2H, dd, J = 5, 6Hz), 6.57 ( 1 / 2H, d, J = 5Hz), 6.59 (1 / 2H, d, J = 5Hz), 6.85-7.02 (3H, m), 7.24-7.45 (8H, m), 7.54-7.64 (4H, m) .
¹ H-NMR (DMSO-d ₆ , 150 ° C) δppm: 1.07 (9H, s), 1.95 (3H, s), 2.00 (3H, s), 3.94 (2H, s), 4.08, 4.25 (2H, AB , J = 9Hz), 4.56, 4.64 (2H, AB, J = 14Hz), 5.01 (1H, brs), 5.22 (1H, brs), 6.93-6.96 (3H, m), 7.25-7.31 (2H, m) , 7.43 (6H, brs), 7.63 (4H, brs).
¹³ C-NMR (DMSO-d ₆ , 150 ° C) δppm: 18.2, 19.0, 19.6, 26.0, 63.3, 65.9, 71.3, 83.4, 95.0, 114.0, 120.4, 126.8, 126.8, 128.4, 128.8, 132.2, 132.3, 134.1 , 134.2, 157.3, 166.9, 167.9, 168.0.
Mass (FAB) m / z 618 (MH ⁺ ).
Anal.calced for C ₃₃ H ₃₉ NO ₆ Si: C, 66.10; H, 6.36; N, 2.27. Found: C, 66.19; H, 6.42; N, 2.24.
β isomer
[α] _D ²⁴ +17.0 (c 1.02, CHCl ₃ ).
IRνmax (KBr) cm ^-1 : 3048, 2936, 2859, 1753, 1660, 1595, 1431, 1367, 1220, 1109.
¹ H-NMR (CDCl ₃ ) δppm: 1.04 (27 / 8H, s), 1.06 (45 / 8H, s), 1.86 (9 / 8H, s), 1.93 (15 / 8H, s), 2.02 (3H, s), 3.68, 3.76 (6 / 8H, AB, J = 11Hz), 3.72, 3.73 (10 / 8H, AB, J = 11Hz), 3.96, 4.30 (6 / 8H, AB, J = 11Hz), 4.18, 4.44 (10 / 8H, AB, J = 10Hz), 4.58 (2H, s), 5.05 (3 / 8H, d, J = 5Hz), 5.08 (5 / 8H, d, J = 6Hz), 5.27 (5 / 8H, dd, J = 3, 6Hz), 5.32 (3 / 8H, dd, J = 1, 6Hz), 6.22 (5 / 8H, d, J = 3Hz), 6.23 (3 / 8H, d, J = 1Hz) ), 6.84-7.02 (3H, m), 7.24-7.45 (8H, m), 7.61-7.63 (4H, m).
¹ H-NMR (DMSO-d ₆ , 150 ° C) δppm: 1.07 (9H, s), 1.92 (3H, s), 1.97 (3H, s), 3.91 (2H, s), 3.99, 4.31 (2H, AB , J = 10Hz), 4.55, 4.59 (2H, AB, J = 15Hz), 5.00 (1H, d, J = 5Hz), 5.26 (1H, brs), 6.23 (1H, brs), 6.92-7.00 (3H, m), 7.25-7.31 (2H, m), 7.40-7.43 (6H, m), 7.63-7.65 (4H, m).
¹³ C-NMR (DMSO-d ₆ , 150 ° C) δppm: 18.2, 19.2, 19.5, 26.0, 64.0, 65.9, 74.5, 83.9, 100.0, 114.0, 120.5, 126.8, 128.4, 128.8, 132.2, 134.2, 157.2, 167.2 , 167.3, 167.8.
Mass (FAB) m / z 618 (MH ⁺ ).
Anal.calced for C ₃₃ H ₃₉ NO ₆ Si ・ 1 / 10H ₂ O: C, 66.10; H, 6.36; N, 2.27. Found: C, 65.91; H, 6.38; N, 2.26.

(Reference Example 3) 2'-O-acetyl-5'-O-tert-butyldiphenylsilyl-3'-deoxy-3'-phenoxyacetylamino-3'-N, 4'-C-methylene-5-methyl Uridine Under a nitrogen stream, thymine (59 mg, 0.47 mmol) and N, O-bis (trimethylsilyl) acetamide (0.31 ml) were added to a solution of the compound obtained in Reference Example 2 (189 mg, 0.31 mmol) in anhydrous dichloroethane (3 ml). , 1.24 mmol) and heated to reflux for 2 hours. After cooling to 0 ° C., trimethylsilyltrifluoromethanesulfonic acid (0.06 ml, 0.34 mmol) was added, and the mixture was refluxed for 7 hours. Saturated aqueous sodium hydrogen carbonate was added to the reaction solution, the solvent was distilled off, and the mixture was extracted with ethyl acetate, and washed with water and 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 (n-hexane: ethyl acetate = 1: 1) to obtain the title compound (215 mg, yield 100%) as a white powder. Was.

mp 99-101 ° C.
[α] _D ²⁴ +35.6 (c 0.80, CHCl ₃ ).
IRνmax (KBr) cm ^-1 : 3199, 3068, 2954, 2859, 1691, 1595, 1463, 1374, 1225, 1110.
¹ H-NMR (CDCl ₃ ) δppm: 1.06 (9 / 3H, s), 1.10 (18 / 3H, s), 1.61 (6 / 3H, s), 1.65 (3 / 3H, s), 1.98 (3 / 3H, s), 2.08 (6/3, s), 3.71, 3.93 (2 / 3H, AB, J = 12Hz), 3.78, 3.92 (4 / 3H, AB, J = 11Hz), 4.04, 4.11 (2 / 3H, AB, J = 11Hz), 4.29, 4.33 (4 / 3H, AB, J = 11Hz), 4.54-4.76 (2H, m), 5.08 (1 / 3H, d, J = 6Hz), 5.09 (2 / 3H, dd, J = 6, 8Hz), 5.21 (2 / 3H, d, J = 6Hz), 5.37 (1 / 3H, dd, J = 6, 8Hz), 6.48 (1H, d, J = 8Hz), 6.84-7.17 (4H, m), 7.29-7.47 (7H, m), 7.54-7.65 (4H, m), 9.19 (1 / 3H, s), 9.23 (2 / 3H, s).
¹ H-NMR (DMSO-d ₆ , 150 ° C) δppm: 1.08 (9H, s), 1.66 (3H, s), 1.93 (3H, s), 4.03, 4.07 (2H, AB, J = 11Hz), 4.10 , 4.40 (2H, AB, J = 11Hz), 4.58, 4.65 (2H, AB, J = 15Hz), 5.07 (1H, d, J = 6Hz), 5.41 (1H, dd, J = 6, 7Hz), 6.28 (1H, J = 7Hz), 6.93-7.00 (3H, m), 7.25-7.42 (8H, m), 7.63-7.65 (4H, m), 10.73 (1H, s).
¹³ C-NMR (DMSO-d ₆ , 150 ° C) δppm: 11.5, 19.1, 20.1, 27.0, 64.5, 66.8, 73.2, 82.4, 87.0, 110.6, 115.0, 121.4, 127.7, 129.3, 129.8, 133.0, 133.3, 135.0 , 135.1, 135.7, 150.4, 158.1, 163.0, 168.2, 169.1.
Mass (FAB) m / z 684 (MH ⁺ ).
Anal.calced for C ₃₇ H ₄₁ N ₃ O ₈ Si ・ 1 / 7H ₂ O: C, 64.99; H, 6.04; N, 6.14. Found: C, 64.74; H, 6.06; N, 6.12.

(Reference Example 4) 5'-O-tert-butyldiphenylsilyl-3'-phenoxyacetylamino-3'-deoxy-3'-N, 4'-C-methylene-5-methyluridine Reference example under a nitrogen stream. Potassium carbonate (6 mg, 0.043 mmol) was added at 0 ° C. to an anhydrous methanol solution (10 ml) of the compound obtained in 3 (100 mg, 0.15 mmol), and the mixture was stirred at room temperature for 3 hours. After neutralization with acetic acid, the solvent was distilled off, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and 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 (n-hexane: ethyl acetate = 1: 2) to obtain the title compound (92 mg, yield 98%) as colorless crystals. Was.

mp 89-92 ° C.
[α] _D ²⁴ +64.5 (c 0.97, CHCl ₃ ).
IRνmax (KBr) cm ^-1 : 3207, 3068, 2933, 2859, 1692, 1467, 1240, 1109.
¹ H-NMR (CDCl ₃ ) δppm: 1.08 (9H, s), 1.59 (9 / 5H, s), 1.62 (6 / 5H, s), 1.94 (3 / 5H, s), 2.35 (2 / 5H, s), 3.72, 4.01 (2H, m), 4.18-4.37 (2H, m), 4.59-4.82 (2H, m), 4.88-4.95 (1H, m), 5.10 (3 / 5H, d, J = 5Hz ), 5.26 (2 / 5H, d, J = 8Hz), 6.38 (3 / 5H, d, J = 8Hz), 6.44 (2 / 5H, d, J = 7Hz), 6.85-7.03 (3H, m), 7.11-7.47 (8H, m), 7.60-7.62 (4H, m), 9.22 (3 / 5H, s), 9.71 (2 / 5H, s).
Mass (FAB) m / z 642 (MH ⁺ ).

(Example 2) (Synthesis of oligonucleotide analog)
Using a nucleic acid synthesizer (Expedite (TM) 8909, manufactured by Applied Biosystems), the reaction was performed on a 2.0 μmol scale. The concentration of solvents, reagents and phosphoramidite in each synthesis cycle is the same as in the case of natural oligonucleotide synthesis, and all solvents, reagents and 5'-phosphoramidites of natural nucleosides are from GLEN RESEARCH Was. GLEN RESEARCH Universal Support (0.2 μmol) was used as the solid phase. The 3'-O-DMTr-thymidine in which the 5'-hydroxyl group is bonded to the solid phase is deprotected with trichloroacetic acid, and the 3'-hydroxyl group is a phosphoramidite for natural nucleotide synthesis and the compound of Example 1e. Was used to repeatedly perform a condensation reaction to synthesize a modified oligonucleotide analog having each sequence. The synthesis cycle is as follows.
Synthesis cycle (when using natural 5'-phosphoramidite)
1) detritylation trichloroacetic acid / dichloromethane; 70sec
2) coupling phosphoramidite (25eq), tetrazole / acetonitrile; 300sec
3) capping 1-methylimidazole / tetrahydrofuran, acetic anhydride / pyridine / tetrahydrofuran; 30sec
4) oxidation iodine / water / pyridine / tetrahydrofuran; 5sec

Synthesis cycle (when using the compound of Example 1e)
1) detritylation trichloroacetic acid / dichloromethane; 120sec
2) coupling phosphoramidite (25eq), tetrazole / acetonitrile; 1200sec
3) capping 1-methylimidazole / tetrahydrofuran, acetic anhydride / pyridine / tetrahydrofuran; 30sec
4) oxidation iodine / water / pyridine / tetrahydrofuran; 5sec

After synthesizing the oligonucleotide having the target sequence, detritylation was performed with trichloroacetic acid / dichloromethane to deprotect the 3′-DMTr group. Then, according to a conventional method, the oligomer was cut out from the support by treatment with concentrated aqueous ammonia, the cyanoethyl group on the phosphorus atom was removed, and the protecting group on the nucleobase was removed.

  The obtained natural oligonucleotides and modified oligonucleotide analogs were purified by reverse phase HPLC to obtain target oligonucleotides.

According to this synthesis method, the following sequence:
5'-gcgttntttgct-3 '(SEQ ID NO: 1 in Sequence Listing): oligonucleotide 1
5'-gcgttntntgct-3 '(SEQ ID NO: 2 in Sequence Listing): oligonucleotide 2
5'-gcgntntntgct-3 '(SEQ ID NO: 3 in Sequence Listing): oligonucleotide 3
5'-gcgttnnntgct-3 '(SEQ ID NO: 4 in Sequence Listing): oligonucleotide 4
5'-ttttttttnt-3 '(SEQ ID NO: 5 in Sequence Listing): oligonucleotide 5

In the above sequence, n is the following using the phosphoramidite form of Example 1e

Represents a nucleoside unit having the structure:
Purification of the resulting modified oligonucleotide analog was performed by reversed-phase HPLC (HPLC: LC-VP manufactured by Shimadzu Corporation; column: WAKO WAKOPAK WS-DNA (10 × 250 mm); 0.1 M aqueous triethylamine acetate (TEAA), pH 7; 8) → 16% CH ₃ CN / 30 min, linear gradient; 50 ° C .; 3.0 ml / min; 254 nm).

The structure of the obtained oligonucleotide was confirmed by MALDI-TOF-MS. Table 3 shows the results.
(Table 3)

MH
Test compound Cald. Found

Oligonucleotide 1 3643.45 3643.76
Oligonucleotide 2 3654.47 3654.41
Oligonucleotide 3 3665.50 3665.30
Oligonucleotide 4 3665.50 3665.44
Oligonucleotide 5 2990.08 2990.14


(Test Example 1) Measurement of nuclease enzyme resistance 3′-exonuclease (phosphodiesterase from Crotalus durissus (Boehringer Mannheim)) 0.24 in 400 μl of a buffer solution containing oligonucleotide 5 (10 μg) (50 mM Tris (pH 8.0) and 10 mM MgCl 2) μg was added, and the mixture was kept at 37 ° C. to perform a reaction. After a certain time, a part of the mixture is taken and heated at 90 ° C. for 2 minutes to inactivate the enzyme and stop the reaction. 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. As a control, a natural DNA having the same sequence as oligonucleotide 5 was synthesized and compared. Table 4 shows the results.
(Table 4)
Oligonucleotide residual rate (%)

Start of test compound (0 min) 20 min 90 min

Oligonucleotide 100 90 83
5

Oligonucleotide 100 Not detected Not detected
(Natural type)

As is evident from the above, the compounds of the present invention exhibited significant nuclease resistance.

(Test Example 2) Measurement of Conformation As a means for confirming the conformation of a nucleic acid, the Altona equation using the coupling constant (J) of NMR is known (J. Am. Chem. Soc., 94, 8205 (1992) and J. Am. Chem. Soc., 95, 2333 (1993).

Calculating the compound of Example 1 according to the above formula _, the value of J _{1 ′, 2′β} is 10, and S (%) is 100 or more. Therefore, it was found that the compound of Example 1 had an S-type conformation and had DNA selectivity.

(Test Example 3) Measurement of DNA Selectivity The melting temperature (Tm) of DNA and RNA having a sequence complementary to oligonucleotides 1 to 4 was measured according to the following method. That is, a sample solution (100 mM NaCl, 10 mM Na ₂ HPO ₄ buffer (pH 7.2), 400 μL) containing 4 μM of oligonucleotide and DNA or RNA having a complementary sequence was bathed in boiling water, and it took about 8 hours. And slowly cooled to room temperature. The sample solution was heated and measured using a spectrophotometer (BECKMAN DU650). The temperature was increased from 5 ° C. to 90 ° C. at 0.5 ° C./min, and the ultraviolet absorption intensity at an absorption maximum wavelength near 260 nm was measured at 0.5 ° C. intervals. The temperature at which the amount of change per 1 ° C. became the maximum was defined as Tm (melting temperature), and the complementary chain form performance of the compound of the present invention was evaluated at this temperature. As a control, the melting temperature of native DNA was also measured. Table 5 shows the results.
(Table 5)
Oligonucleotide residual rate (%)

Tm (ΔTm) ℃
Test compound DNA RNA

Natural DNA 51.7 47.0
Oligonucleotide 1 51.9 (+0.2) 45.7 (-1.3)
Oligonucleotide 2 52.4 (+0.4) 46.0 (-0.5)
Oligonucleotide 3 52.9 (+0.4) 43.9 (-1.0)
Oligonucleotide 4 52.2 (+0.2) 45.2 (-0.6)

As is evident from the above, the compounds of the present invention showed significant DNA selectivity.

  The nucleic acid reagent containing the novel bicyclonucleoside derivative of the present invention has excellent antisense or antigene activity, or has decoy nucleic acid, DNA enzyme and RNA interference functions, and produces an in vivo stable oligonucleotide. It is useful as an intermediate for

The novel oligonucleotide analogs of the present invention are stable in vivo, and are useful as antisense drugs, antigene drugs, decoy nucleic acids, DNA enzymes, RNA interference nucleic acids, and for detecting specific genes (probes) And also useful as a primer (primer) for starting amplification of a specific gene.

SEQ ID NO 1: Synthetic oligonucleotide for testing DNA selectivity SEQ ID NO 2: Synthetic oligonucleotide for testing DNA selectivity SEQ ID NO 3: Synthetic oligonucleotide for testing DNA selectivity SEQ ID NO 4: DNA Synthetic oligonucleotides for testing selectivity SEQ ID NO: 5: synthetic oligonucleotides for testing nuclease resistance

Claims (11)

General formula (I)

[Where,
R ¹ represents a hydrogen atom, a protecting group for protecting a hydroxyl group for nucleic acid synthesis, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or a group represented by the formula —P (R ^3a ) R ^3b , wherein R ^3a and R ^3b are the same or different and are protected with 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, and a protecting group for nucleic acid synthesis. 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 amino substituted with an alkyl group having 1 to 6 carbon atoms Represents a group),
R ² represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a protecting group for nucleic acid synthesis of an amino group,
B represents a purine-9-yl or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have a substituent selected from the following α group. ]
Or a pharmacologically acceptable salt thereof.
(Α group)
Hydroxyl group,
A hydroxyl group protected with a protecting group for nucleic acid synthesis,
An alkoxy group having 1 to 6 carbon atoms,
Mercapto group,
A mercapto group protected with a protecting group for nucleic acid synthesis,
An alkylthio group having 1 to 6 carbon atoms,
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. In claim 1,
R ¹ is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with one to three aryl groups, a lower alkyl, a lower alkoxy, a halogen atom or a cyano group, wherein the aryl ring is substituted with 1 to A compound or a pharmacologically acceptable salt thereof, which is a methyl group or a silyl group substituted with three aryl groups. In claim 1,
R ¹ is a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a dimethoxytrityl group, a monomethoxytrityl group, a tert-butyldiphenylsilyl group, a -P (OC ₂ H ₄ CN) (NCH ( CH ₃ ) ₂ ), —P (OCH ₃ ) (NCH (CH ₃ ) ₂ ), a phosphonyl group, or a 2-chlorophenyl or 4-chlorophenylphosphate group. In any one of claims 1 to 3,
R ² is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aliphatic acyl group, an aromatic acyl group, a monomethoxytrityl group, an aryloxycarbonyl group, or 1 to 2 lower alkoxy or nitro groups; A compound or a pharmaceutically acceptable salt thereof, wherein the aryl ring is an optionally substituted aralkyloxycarbonyl group. In any one of claims 1 to 3,
R ² represents a hydrogen atom, an alkyl group having 1 or 2 carbon atoms, an aliphatic acyl group, a monomethoxytrityl group, an aryloxycarbonyl group, or an aryl ring substituted with 1 or 2 lower alkoxy or nitro groups; Or a pharmacologically acceptable salt thereof, which is an aralkyloxycarbonyl group which may be substituted. In any one of claims 1 to 3,
A compound or a pharmaceutically acceptable salt thereof, wherein R ² is a hydrogen atom, a methyl group, a trifluoroacetyl group, a phenoxyacetyl group, a monomethoxytrityl group or a benzyloxycarbonyl group. In any one of claims 1 to 6,
B is a 6-aminopurin-9-yl group (that is, an adenyl group), a 6-aminopurin-9-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis, and 2,6-diaminopurine-9- 2,6-diaminopurin-9-yl group, 2-amino-6-chloropurine-9-yl group in which an yl group and an amino group are protected with a protecting group for nucleic acid synthesis, and an amino group is a protecting group for nucleic acid synthesis. Protected 2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-fluoro wherein the amino group is protected by a protecting group for nucleic acid synthesis Purine-9-yl group, 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-hydroxypurin-9-yl group (ie, Anilinyl group), a 2-amino-6-hydroxypurin-9-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, and 2-amino-6 in which the amino group and the hydroxyl group are protected with a protecting group for nucleic acid synthesis. A hydroxypurin-9-yl group, a 6-amino-2-methoxypurin-9-yl group, a 6-amino-2-chloropurin-9-yl group, a 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidine-1 -Yl group (ie, cytosinyl group), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino- 5-fluoro-1, -Dihydropyrimidin-1-yl group, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 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), 2-oxo-4-hydroxy-5-methyl-1,2 -Dihydropyrimidin-1-yl group (ie, thyminyl group), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group (ie, 5-methylcytosinyl group) Or salt amino group is protected 4-amino-5-methyl-2-oxo-1,2-dihydro-pyrimidin-1-yl group with a protective group for nucleic acid synthesis, acceptable compound or a pharmacologically. In any one of claims 1 to 6,
B is a 6-benzoylaminopurin-9-yl group, an adenyl group, a 2-isobutyrylamino-6-hydroxypurin-9-yl group, a guaninyl group, 2-oxo-4-benzoylamino-1,2- A compound which is a dihydropyrimidin-1-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; Or a pharmacologically acceptable salt thereof. General formula (Ia)

[Where,
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. Or a pharmacologically acceptable salt thereof, comprising one or more of the structures represented by the formula:
(Α group)
Hydroxyl group,
A hydroxyl group protected with a protecting group for nucleic acid synthesis,
An alkoxy group having 1 to 6 carbon atoms,
Mercapto group,
A mercapto group protected with a protecting group for nucleic acid synthesis,
An alkylthio group having 1 to 6 carbon atoms,
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. In claim 9,
B is a 6-aminopurin-9-yl group (that is, an adenyl group), a 6-aminopurin-9-yl group in which the amino group is protected by a protecting group for nucleic acid synthesis, and 2,6-diaminopurine-9- 2,6-diaminopurin-9-yl group, 2-amino-6-chloropurine-9-yl group in which an yl group and an amino group are protected with a protecting group for nucleic acid synthesis, and an amino group is a protecting group for nucleic acid synthesis. Protected 2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-fluoro wherein the amino group is protected by a protecting group for nucleic acid synthesis Purine-9-yl group, 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-hydroxypurin-9-yl group (ie, Anilinyl group), a 2-amino-6-hydroxypurin-9-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, and 2-amino-6 in which the amino group and the hydroxyl group are protected with a protecting group for nucleic acid synthesis. A hydroxypurin-9-yl group, a 6-amino-2-methoxypurin-9-yl group, a 6-amino-2-chloropurin-9-yl group, a 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidine-1 -Yl group (ie, cytosinyl group), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 2-oxo-4-amino- 5-fluoro-1, -Dihydropyrimidin-1-yl group, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group in which the amino group is protected with a protecting group for nucleic acid synthesis, 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), 2-oxo-4-hydroxy-5-methyl-1,2 -Dihydropyrimidin-1-yl group (ie, thyminyl group), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group (ie, 5-methylcytosinyl group) Alternatively, an oligonucleotide analog or a pharmacologically acceptable amino acid in which the amino group is a 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group protected with a protecting group for nucleic acid synthesis. salt. In claim 9,
B is a 6-benzoylaminopurin-9-yl group, an adenyl group, a 2-isobutyrylamino-6-hydroxypurin-9-yl group, a guaninyl group, 2-oxo-4-benzoylamino-1,2- An oligo which is a dihydropyrimidin-1-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; A nucleotide analog or a pharmacologically acceptable salt thereof.