WO2016017422A1 - Crosslinking nucleoside and nucleotide - Google Patents
Crosslinking nucleoside and nucleotide Download PDFInfo
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- WO2016017422A1 WO2016017422A1 PCT/JP2015/070201 JP2015070201W WO2016017422A1 WO 2016017422 A1 WO2016017422 A1 WO 2016017422A1 JP 2015070201 W JP2015070201 W JP 2015070201W WO 2016017422 A1 WO2016017422 A1 WO 2016017422A1
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- 0 CC(C(C1O)[C@@](N(C=C(C)C(N2)=O)C2=O)O[C@@]1(C*)[O-])=O Chemical compound CC(C(C1O)[C@@](N(C=C(C)C(N2)=O)C2=O)O[C@@]1(C*)[O-])=O 0.000 description 7
- AOUSVALLAUSDCJ-UHFFFAOYSA-N CC(C(N)=N1)=CN(C)C1=O Chemical compound CC(C(N)=N1)=CN(C)C1=O AOUSVALLAUSDCJ-UHFFFAOYSA-N 0.000 description 1
- GKMIDMKPBOUSBQ-UHFFFAOYSA-N CC(C(N1)=O)=CN(C)C1=O Chemical compound CC(C(N1)=O)=CN(C)C1=O GKMIDMKPBOUSBQ-UHFFFAOYSA-N 0.000 description 1
- PDWOASJWYRCPRX-VWPJIOGPSA-N CC(C(N1)=O)=CN([C@@H](C2OCC3)O[C@]3(CO)C2O)C1=O Chemical compound CC(C(N1)=O)=CN([C@@H](C2OCC3)O[C@]3(CO)C2O)C1=O PDWOASJWYRCPRX-VWPJIOGPSA-N 0.000 description 1
- ZWWQFRCBEVPEJK-VWPJIOGPSA-N CC(C(N1)=O)=CN([C@@H]2O[C@@]3(CO)OCCOC2C3O)C1=O Chemical compound CC(C(N1)=O)=CN([C@@H]2O[C@@]3(CO)OCCOC2C3O)C1=O ZWWQFRCBEVPEJK-VWPJIOGPSA-N 0.000 description 1
- HWPZZUQOWRWFDB-UHFFFAOYSA-N CN(C=CC(N)=N1)C1=O Chemical compound CN(C=CC(N)=N1)C1=O HWPZZUQOWRWFDB-UHFFFAOYSA-N 0.000 description 1
- XBCXJKGHPABGSD-UHFFFAOYSA-N CN(C=CC(N1)=O)C1=O Chemical compound CN(C=CC(N1)=O)C1=O XBCXJKGHPABGSD-UHFFFAOYSA-N 0.000 description 1
- UUWJNBOCAPUTBK-UHFFFAOYSA-N C[n]1c(N=C(N)NC2=O)c2nc1 Chemical compound C[n]1c(N=C(N)NC2=O)c2nc1 UUWJNBOCAPUTBK-UHFFFAOYSA-N 0.000 description 1
- WRXCXOUDSPTXNX-UHFFFAOYSA-N C[n]1c(ncnc2N)c2nc1 Chemical compound C[n]1c(ncnc2N)c2nc1 WRXCXOUDSPTXNX-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
Definitions
- the present invention relates to a bridged nucleoside and a nucleotide. More particularly, it relates to bridged nucleosides and nucleotides having high binding affinity for single-stranded RNA and double-stranded DNA, and high resistance to nucleases.
- anti-sense method anti-gene method, aptamer, siRNA and the like as treatment methods for diseases by nucleic acid medicine.
- the antisense method introduces an oligonucleotide (antisense strand) complementary to the mRNA involved in the disease from the outside to form a double strand, thereby inhibiting the translation process of the pathogenic RNA and treating the disease.
- siRNA is similar to this, and translation from mRNA to protein is inhibited by double-stranded RNA administered to a living body.
- the antigene method suppresses transcription from DNA to RNA by introducing from the outside a triplex-forming oligonucleotide corresponding to a DNA site that transcribes pathogenic RNA.
- aptamers are short nucleic acid molecules (oligonucleotides), they function by binding to biological components such as proteins that cause disease.
- S-oligo phosphorothioate
- 2 ′, 4′-BNA bridged nucleic acid
- LNA locked nucleic acid
- S-oligo is already marketed in the United States as an antisense drug against cytomegalovirus. Although this has high nuclease resistance, it has a drawback that its binding affinity to the target nucleic acid chain is low, and needs to be improved.
- 2 ′, 4′-BNA / LNA developed so far has the highest binding affinity to the target nucleic acid chain as a 2 ′, 4′-bridged artificial nucleoside, and is the most useful material for nucleic acid medicine in the future. It is an expected molecule. However, resistance to nucleases is not sufficient, and there is room for improvement in terms of stability in vivo.
- nucleoside based on the structure as shown in the above formula (a) is considered to have, for example, a very complicated and multi-step process for its synthesis, and there is room for improvement in resistance to nuclease. However, it was still not satisfactory. Further, it has been pointed out that the nucleoside serving as the base of the structure as shown in the formula (b) has a large seven-membered ring cross-linked structure, and thus it is considered that the binding affinity with the target nucleic acid chain is not satisfactory. It was. For this reason, it is desired to develop an oligonucleotide having performance equivalent to or higher than that of such an oligonucleotide and further excellent in industrial production efficiency.
- the present invention solves the above-mentioned problems, and the object of the present invention is to prevent degradation by nucleases in vivo, have high binding affinity and specificity for target mRNA, and express a specific gene. It is an object of the present invention to provide a novel molecule for antisense methods and nucleic acid pharmaceuticals, which can efficiently control the above-described molecules, and has excellent productivity.
- the present invention relates to a compound represented by the following formula (I) or (I ′) or a salt thereof:
- Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the ⁇ group, where The ⁇ group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis.
- R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring
- Chain or branch represents a chain alkylamino group];
- R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring.
- X is an oxygen atom or a sulfur atom).
- X is an oxygen atom.
- the Base is a 6-aminopurin-9-yl group, a 2,6-diaminopurin-9-yl group, a 2-amino-6- Chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-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-dimethoxypurine-9 -Yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group, 4-amino- 2-oxo-5-fluoro-1,
- the Base is a 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group.
- the present invention also provides an oligonucleotide containing at least one nucleoside structure represented by the following formula (II) or (II ′) or a pharmacologically acceptable salt thereof:
- Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the ⁇ group, where The ⁇ group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis.
- R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring;
- a group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ⁇ C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid
- Chain or branch represents a chain alkylamino group];
- R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring.
- X is an oxygen atom or a sulfur atom).
- X is an oxygen atom.
- novel 2 ', 4'-bridged 6-membered ring nucleosides and nucleotides having a hetero atom at the 6'-position are provided.
- This oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide has a binding affinity for single-stranded RNA and single-stranded DNA comparable to conventional oligonucleotides containing 2 ′, 4′-bridged artificial nucleotide.
- the oligonucleotide of the present invention is expected to be applied to, for example, nucleic acid medicine.
- the 2 ′, 4′-bridged nucleosides and nucleotides of the present invention can also introduce a heteroatom at the 6 ′ position in a 6-membered ring bridge structure, and through a simpler reaction process compared to the conventional one. Can be manufactured. For this reason, it is also possible to further increase industrial production efficiency.
- linear alkyl group having 1 to 6 carbon atoms refers to any linear alkyl group having 1 to 6 carbon atoms, specifically a methyl group, an ethyl group, an n-propyl group, An n-butyl group, an n-pentyl group, or an n-hexyl group.
- linear alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methyloxy group, an ethyloxy group, and an n-propyloxy group.
- a linear or branched alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having an arbitrary linear or branched alkyl group having 1 to 6 carbon atoms.
- Examples thereof include a methyloxy group, an ethyloxy group, an n-propyloxy group, an isopropyloxy group, an n-butyloxy group, an isobutyloxy group, a tert-butyloxy group, an n-pentyloxy group, and an isopentyloxy group.
- linear alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methylthio group, an ethylthio group, and an n-propylthio group.
- a linear or branched alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having an arbitrary linear or branched alkyl group having 1 to 6 carbon atoms.
- Examples include a methylthio group, an ethylthio group, an n-propylthio group, an isopropylthio group, an n-butylthio group, an isobutylthio group, a tert-butylthio group, an n-pentylthio group, and an isopentylthio group.
- C1-C6 cyanoalkoxy group refers to a group in which at least one hydrogen atom constituting the straight-chain alkoxy group having 1 to 6 carbon atoms is substituted with a cyano group.
- linear alkylamino group having 1 to 6 carbon atoms means a group in which one or two hydrogen atoms constituting the amino group are substituted with a linear alkyl group having 1 to 6 carbon atoms. Is included. Examples thereof include a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, and a diethylamino group.
- a linear or branched alkylamino group having 1 to 6 carbon atoms means any linear or branched group in which one or two hydrogen atoms constituting the amino group are 1 to 6 carbon atoms. Includes groups substituted with chain alkyl groups.
- Examples include methylamino group, dimethylamino group, ethylamino group, methylethylamino group, diethylamino group, n-propylamino group, di-n-propylamino group, isopropylamino group, diisopropylamino group and the like.
- an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring means any linear alkyl group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. Includes a chain alkyl group and any cyclic alkyl group having 3 to 7 carbon atoms. It may be simply referred to as “lower alkyl group”.
- arbitrary linear alkyl groups having 1 to 7 carbon atoms include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, and n-heptyl group.
- Examples of the branched alkyl group having 3 to 7 carbon atoms include isopropyl group, isobutyl group, tert-butyl group, isopentyl group and the like, and optional cyclic alkyl group having 3 to 7 carbon atoms include A cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. are mentioned.
- an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring means any linear alkenyl group having 2 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. Chain alkenyl groups, and any cyclic alkenyl group having 3 to 7 carbon atoms are included. It may be simply referred to as “lower alkenyl group”.
- Examples of the branched alkenyl group having 3 to 7 carbon atoms include isopropenyl group, 1-methyl-1-propenyl group, 1-methyl -2-propenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-methyl-2-butenyl group, etc., and any cyclic alkenyl group having 3 to 7 carbon atoms includes a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and the like.
- an alkoxy group having 1 to 7 carbon atoms which may form a branch or a ring means any linear alkoxy group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. It includes a chain alkoxy group and any cyclic alkoxy group having 3 to 7 carbon atoms. It may be simply referred to as “lower alkoxy group”.
- any linear alkoxy group having 1 to 7 carbon atoms includes a methoxy group, an ethoxy group, an n-propoxy group, an n-butoxy, an n-pentyloxy group, an n-hexyloxy group, and an n-heptyloxy group.
- Examples of the branched alkoxy group having 3 to 7 carbon atoms include isopropoxy group, isobutoxy group, tert-butoxy group, isopentyloxy group, etc., and any cyclic group having 3 to 7 carbon atoms
- Examples of the alkoxy group include a cyclobutoxy group, a cyclopentyloxy group, and a cyclohexyloxy group.
- an aryl group having 3 to 12 carbon atoms that may contain a heteroatom refers to any aryl group having 6 to 12 carbon atoms, which is composed of only hydrocarbons, and the aryl group. Including any heteroaryl group having 3 to 12 carbon atoms in which at least one carbon atom constituting the ring structure is substituted with a heteroatom (eg, a nitrogen atom, an oxygen atom, and a sulfur atom, and combinations thereof) To do.
- a heteroatom eg, a nitrogen atom, an oxygen atom, and a sulfur atom, and combinations thereof
- Examples of the aryl group having 6 to 12 carbon atoms include a phenyl group, a naphthyl group, an indenyl group, and an azulenyl group, and examples of the heteroaryl group having 3 to 12 carbon atoms include a pyridyl group, a pyrrolyl group, A quinolyl group, an indolyl group, an imidazolyl group, a furyl group, a thienyl group, and the like can be given.
- aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom examples include a benzyl group, a phenethyl group, a naphthylmethyl group, a 3-phenylpropyl group, -Phenylpropyl, 4-phenylbutyl, 2-phenylbutyl, pyridylmethyl, indolylmethyl, furylmethyl, thienylmethyl, pyrrolylmethyl, 2-pyridylethyl, 1-pyridylethyl, 3 -Thienylpropyl group and the like.
- examples of the term “acyl group” include aliphatic acyl groups and aromatic acyl groups.
- examples of the aliphatic acyl group include formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group, isovaleryl group, octanoyl group, nonanoyl group, decanoyl group, 3-methylnonanoyl group, 8-methylnonanoyl group, 3-ethyloctanoyl group, 3,7-dimethyloctanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, 1-methylpentadecanoyl group, 14-methylpentadecanoyl group, 13,13-
- aromatic acyl group examples include arylcarbonyl groups such as benzoyl group, ⁇ -naphthoyl group and ⁇ -naphthoyl group; halogenoarylcarbonyl groups such as 2-bromobenzoyl group and 4-chlorobenzoyl group; 2 , 4,6-trimethylbenzoyl group, lower alkylated arylcarbonyl group such as 4-toluoyl group; lower alkoxylated arylcarbonyl group such as 4-anisoyl group; 2-carboxybenzoyl group, 3-carboxybenzoyl group, 4 A carboxylated arylcarbonyl group such as a carboxybenzoyl group; a nitrated arylcarbonyl group such as a 4-nitrobenzoyl group or a 2-nitrobenzoyl group; a lower alkoxycarbonylated arylcarbonyl such as a 2- (methoxycarbonyl) benzoyl
- sil group examples include trimethylsilyl group, triethylsilyl group, isopropyldimethylsilyl group, t-butyldimethylsilyl group, methyldiisopropylsilyl group, methyldi-t-butylsilyl group, and triisopropylsilyl group.
- a tri-lower alkylsilyl group such as diphenylmethylsilyl group, butyldiphenylbutylsilyl group, diphenylisopropylsilyl group, tri-lower alkylsilyl group substituted with 1 to 2 aryl groups such as phenyldiisopropylsilyl group, etc.
- a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, and a t-butyldiphenylsilyl group are preferable, and a trimethylsilyl group is more preferable.
- halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferable is a fluorine atom or a chlorine atom.
- protecting group for amino group for nucleic acid synthesis is capable of stably protecting an amino group, a hydroxyl group, a phosphate group or a mercapto group during nucleic acid synthesis. If it is, it will not be restrict
- protecting group that is stable under acidic or neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis.
- protecting groups include lower alkyl groups, lower alkenyl groups, acyl groups, tetrahydropyranyl or tetrahydrothiopyranyl groups, tetrahydrofuranyl or tetrahydrothiofuranyl groups, silyl groups, lower alkoxymethyl groups, lower alkoxy groups.
- examples of the tetrahydropyranyl group or tetrahydrothiopyranyl group include a tetrahydropyran-2-yl group, a 3-bromotetrahydropyran-2-yl group, a 4-methoxytetrahydropyran-4-yl group, a tetrahydro Examples include a thiopyran-4-yl group and a 4-methoxytetrahydrothiopyran-4-yl group.
- examples of the tetrahydrofuranyl group or the tetrahydrothiofuranyl group include a tetrahydrofuran-2-yl group and a tetrahydrothiofuran-2-yl group.
- Examples of the lower alkoxymethyl group include a methoxymethyl group, a 1,1-dimethyl-1-methoxymethyl group, an ethoxymethyl group, a propoxymethyl group, an isopropoxymethyl group, a butoxymethyl group, and a t-butoxymethyl group.
- Examples of the lower alkoxylated lower alkoxymethyl group include 2-methoxyethoxymethyl group.
- Examples of the halogeno lower alkoxymethyl group include 2,2,2-trichloroethoxymethyl group and bis (2-chloroethoxy) methyl group.
- Examples of the lower alkoxylated ethyl group include 1-ethoxyethyl group and 1- (isopropoxy) ethyl group.
- Examples of the halogenated ethyl group include 2,2,2-trichloroethyl group.
- Examples of the methyl group substituted with 1 to 3 aryl groups include benzyl group, ⁇ -naphthylmethyl group, ⁇ -naphthylmethyl group, diphenylmethyl group, triphenylmethyl group, ⁇ -naphthyldiphenylmethyl group, 9-anne.
- Examples include a thrylmethyl group.
- Examples of the “methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkyl group, lower alkoxy group, halogen atom or cyano group” include 4-methylbenzyl group, 2,4,6- Trimethylbenzyl group, 3,4,5-trimethylbenzyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, 4,4'-dimethoxytriphenylmethyl group, 2-nitrobenzyl group, 4-nitrobenzyl group 4-chlorobenzyl group, 4-bromobenzyl group, 4-cyanobenzyl group and the like.
- Examples of the lower alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl group, and an isobutoxycarbonyl group.
- Examples of the “aryl group substituted with a halogen atom, lower alkoxy group or nitro group” include 4-chlorophenyl group, 2-fluorophenyl group, 4-methoxyphenyl group, 4-nitrophenyl group, 2,4-dinitrophenyl group Etc.
- Examples of the “lower alkoxycarbonyl group substituted with a halogen atom or tri-lower alkylsilyl group” include 2,2,2-trichloroethoxycarbonyl group, 2-trimethylsilylethoxycarbonyl group and the like.
- Examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group and an aryloxycarbonyl group.
- Examples of the “aralkyloxycarbonyl group whose aryl ring may be substituted with a lower alkoxy or nitro group” include benzyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 3,4-dimethoxybenzyloxycarbonyl group, 2-nitro Examples include benzyloxycarbonyl group, 4-nitrobenzyloxycarbonyl group and the like.
- the “hydroxyl-protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, “lower alkyl, lower alkoxy, halogen, cyano” A methyl group substituted with 1 to 3 aryl groups substituted with an aryl ring by a group ”, or a silyl group, and more preferably an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzoyl group, a dimethoxy group A trityl group, a monomethoxytrityl group or a tert-butyldiphenylsilyl group;
- Preferred examples of the protecting group for the “hydroxyl group protected with a protecting group for nucleic acid synthesis” include aliphatic acyl groups, aromatic acyl groups, “methyl groups substituted with 1 to 3 aryl groups
- the “protecting group for the amino group for nucleic acid synthesis” is preferably an acyl group, more preferably a benzoyl group.
- the “protecting group” of the “phosphate group protected with a protecting group for nucleic acid synthesis” is preferably a lower alkyl group, a lower alkyl group substituted with a cyano group, an aralkyl group, a “nitro group or a halogen atom”.
- the protecting group constituting the “phosphate group protected with a protecting group for nucleic acid synthesis” may be one or more.
- the “protecting group” of the “mercapto group protected with a protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group or an aromatic acyl group, and more preferably a benzoyl group.
- —P (R 4 ) R 5 wherein R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a protecting group for nucleic acid synthesis.
- the groups represented by the formula (1) represents a linear or branched alkylamino group of 1 to 6, the group that R 4 can represent as OR 4a and R 5 as NR 5a can be referred to as a “phosphoramidite group”. .
- the phosphoramidite group is preferably a group represented by the formula —P (OC 2 H 4 CN) (N (iPr) 2 ) or a formula —P (OCH 3 ) (N (iPr) 2 ). And the group represented.
- iPr represents an isopropyl group.
- nucleoside and nucleoside analog mean an unnatural type of “nucleoside” in which a purine or pyrimidine base and a sugar are bonded, and an aromatic heterocycle other than purine and pyrimidine. And an aromatic hydrocarbon ring capable of substituting with a purine or pyrimidine base and having a sugar bound thereto.
- the terms “artificial oligonucleotide” and “oligonucleotide analog” refer to an “oligonucleotide” in which 2 to 50 identical or different “nucleosides” or “nucleoside analogs” are linked by phosphodiester bonds.
- Such analogs preferably include a sugar derivative having a modified sugar moiety; a thioate derivative in which the phosphodiester moiety is thioated; an ester form in which the terminal phosphate moiety is esterified; Examples include amides in which the amino group is amidated, and more preferable examples include sugar derivatives in which the sugar moiety is modified.
- a salt thereof refers to a salt of a compound represented by the formula (I) or (I ′) of the present invention.
- examples of such salts include 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, 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 salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phene
- the term “pharmacologically acceptable salt thereof” refers to a salt of an oligonucleotide analog containing at least one nucleoside structure represented by the formula (II) or (II ′) of the present invention.
- salts include 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, 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 salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (Hydroxy Amine salts such as organic salts such as
- the compound of the present invention or a salt thereof is 2 ', 4'-bridged nucleoside and nucleotide or a salt thereof.
- the compound of the present invention or a salt thereof has the following formula (I) or (I ′):
- Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group optionally having one or more arbitrary substituents selected from ⁇ group
- the ⁇ group is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis.
- R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring
- Chain or branch represents a chain alkylamino group];
- R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring.
- X is an oxygen atom or a sulfur atom).
- Base is a purine base (ie, purin-9-yl group) or a pyrimidine base (ie, 2-oxo-1,2-dihydropyrimidin-1-yl group) ).
- bases are a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and 1 to carbon atoms. It may have one or more arbitrary substituents selected from the ⁇ group consisting of 6 linear alkylamino groups and halogen atoms.
- Base examples include 6-aminopurin-9-yl group (adenylyl group), 2,6-diaminopurine-9-yl group, 2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group (guaninyl group), 6-amino- 2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (cytosynyl group), 4-amino- 2-oxo-5-
- Base has the following structural formula from the viewpoint of introduction into nucleic acid medicine:
- 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group), 2-oxo-4-amino-1,2-dihydropyrimidine-1 -Yl group (cytosynyl group), 6-aminopurin-9-yl group (adenylyl group), 2-amino-6-hydroxypurin-9-yl group (guaninyl group), 4-amino-5-methyl-2-
- the oxo-1,2-dihydropyrimidin-1-yl group and the 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group are preferred, and in particular, the 2-oxo-4 A -hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group) is preferred.
- the hydroxyl group and amino group are protected by a protecting group.
- the 2 ′, 4′-bridged nucleoside of the present invention has a hetero atom such as an oxygen atom or a sulfur atom introduced into the 6 ′ position of the conventional 2 ′, 4′-bridge structure, which will be described later. Enzyme resistance performance of the oligonucleotide to be improved. In addition, such a heteroatom in the 2 ', 4'-crosslinked structure directly affects the conformation of the sugar moiety. For this reason, the 2 ', 4'-bridged nucleoside of the present invention can further improve the binding affinity of the resulting oligonucleotide to single-stranded RNA (ssRNA) due to the influence.
- ssRNA single-stranded RNA
- the 2 ′, 4′-bridged nucleoside of the present invention can be synthesized in a single step by introducing a heteroatom at the 6 ′ position by using a 4′-exoolefin compound.
- a radical cyclization reaction By utilizing a radical cyclization reaction, a crosslinked structure having a hetero atom at the 6′-position can be produced through very few steps. Therefore, the 2 ′, 4′-bridged nucleoside of the present invention can be synthesized more efficiently through fewer steps than conventional ENA or EoNA in which a hetero atom is introduced into the bridge structure. .
- Isomers (i) and R-form (ii) and S-form (iii) with controlled stereochemistry can be synthesized separately.
- oligonucleotides (2 ', 4'-bridged artificial nucleotides) can be prepared using such 2', 4'-bridged nucleosides.
- triphosphorylation can be easily performed according to the method described in Non-Patent Document 5.
- the oligonucleotide of the present invention or a pharmacologically acceptable salt thereof includes at least one nucleoside structure represented by the following formula (II) or (II ′):
- the oligonucleotide of the present invention has at least one nucleoside structure at any position.
- the position and number are not particularly limited, and can be appropriately designed according to the purpose.
- Oligonucleotide analogs containing such a nucleoside structure have a dramatic improvement in enzyme resistance compared to, for example, conventional EoNA. Moreover, it has a single-stranded RNA (ssRNA) binding affinity that exceeds the EoNA.
- ssRNA single-stranded RNA
- oligonucleotide analogues synthesized using the 2 ′, 4′-bridged nucleosides of the present invention inhibit the action of specific genes including antitumor agents and antiviral agents.
- both binding affinity to complementary sense strand RNA and resistance to in vivo DNA-degrading enzyme are required.
- the structure of the sugar part is constantly fluctuating between a shape close to a DNA duplex and a shape close to a DNA-RNA duplex or an RNA duplex. It has been known. When a single-stranded nucleic acid forms a double strand with a complementary RNA strand, its sugar structure is fixed.
- the sugar portion is fixed in the state in which a double strand is formed in advance, so that it is easy to form a double strand with a target RNA strand and is stable. Can be present. It is also known that nucleic acid duplexes are stabilized by hydrated water connected like a chain of water molecules.
- the oligonucleotide analog of the present invention contains, for example, adjuvants commonly used in the pharmaceutical formulation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and flavoring agents.
- adjuvants commonly used in the pharmaceutical formulation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and flavoring agents.
- it can be a parenteral preparation or a liposome preparation.
- a topical preparation such as a solution, cream, ointment, etc. can be prepared by blending a pharmaceutical carrier usually used in the art.
- anhydrous dimethylformamide solution 100 mL of compound 1 (9.30 g, 38.7 mmol) synthesized from 5-methyluridine by the method described in Non-Patent Document 8 was added at 0 ° C. to 1,4 Add diazabicyclo [2.2.2] octane (21.7 g, 194 mmol), tert-butyldimethylchlorosilane (7.00 g, 46.5 mmol), silver nitrate (7.90 g, 46.5 mmol) and add 12 at room temperature. Stir for hours. After completion of the reaction, the reaction solution was filtered through celite. Water was added to the filtrate and extracted with diethyl ether.
- Table 5 shows the physical property data of the obtained compound 5.
- Table 6 shows the physical property data of the obtained compound 6.
- Table 7 shows the physical property data of the obtained compound 7.
- N, N-diisopropylethylamine (0.21 mL, 1.18 mmol) was added to an anhydrous dichloromethane solution (3.0 mL) of the compound 7 (230 mg, 0.393 mmol) obtained above, and the mixture was brought to 0 ° C.
- 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.11 mL, 0.472 mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium hydrogen carbonate at 0 ° C., and extracted with ethyl acetate.
- Table 10 shows the physical property data of the obtained compound 10.
- N, N-diisopropylethylamine (0.21 mL, 1.15 mmol) was added to an anhydrous dichloromethane solution (3.0 mL) of the compound 10 obtained above (230 mg, 0.383 mmol), and 0 ° C. was added dropwise 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.10 mL, 0.460 mmol) and stirred at room temperature for 6 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
- Table 11 shows the physical property data of the obtained compound 11.
- Table 13 shows the physical property data of the obtained compound 13.
- Table 14 shows the physical property data of the obtained compound 14.
- Table 15 shows the physical property data of the obtained compound 15.
- N, N-diisopropylethylamine (0.55 mL, 3.10 mmol) was added to an anhydrous dichloromethane solution (5.0 mL) of the compound 15 obtained above (372 mg, 0.619 mmol), and 0 ° C.
- 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.21 mL, 0.929 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
- Table 16 shows the physical property data of the obtained compound 16.
- Example 4 Synthesis and purification of oligonucleotide (1) Compound 1.7 (Exomethylene), Compound 11 ((R) -methyl), Compound 16 ((S) obtained in Example 1.7, Example 2.3 and Example 3.5, and Comparative Example 1 ) - methyl body) and EoNA, used respectively as amidites blocks, these and d m C (Ac) and T phosphoramidites (both sigma - prepared Aldrich) and anhydrous acetonitrile 0.1M of Each of the oligonucleotides shown in Table 17 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where X is Corresponds to the structure derived from compound 8 (exomethylene) obtained in Example 1.7.
- nS-8 Oligonucleotides Synthesizer an oligonucle
- Y corresponds to the structure derived from the compound 11 ((R) -methyl form) obtained in Example 2.3
- Z represents the compound 16 ((S) -methyl form obtained in Example 3.5.
- W corresponds to the structure derived from EoNA obtained in Comparative Example 1).
- the synthesis scale in the synthesis was 0.2 ⁇ mol, and was performed under trityl-on conditions.
- As the activator 5- [3,5-bis (trifluoromethyl) phenyl] -1H-tetrazole (0.25 M anhydrous acetonitrile solution, Activator 42, Proligo (registered trademark)) was used.
- the condensation time is 10 minutes for the amidite blocks X, Y and Z obtained in Example 1.7, Example 2.3 and Example 3.5 and Comparative Example 1, and EoNA, and the natural amidite The block was 32 seconds.
- the mixture was treated with 28% aqueous ammonia at room temperature for 1.5 hours to cut out from the column carrier and to deprotect the base part and the phosphoric diester part. Subsequently, purification was performed by a simple gel filtration column (Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters), and further purified by reverse phase HPLC.
- a simple gel filtration column Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters
- Example 5 Measurement of melting temperature ( Tm ) (1) Sample solution containing 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 ⁇ M oligonucleotide obtained in Example 4 and 4 ⁇ M single-stranded RNA or single-stranded DNA shown in Table 17 (130 ⁇ L) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
- Table 17 shows the results when single-stranded RNA was used as the target strand and the results when single-stranded DNA was used as the target strand.
- Example 6 Measurement of melting temperature (T m ) (2) (Evaluation of triple chain forming ability)
- the final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 ⁇ M of the oligonucleotide obtained in Example 4, and 1.5 ⁇ M of the double-stranded DNA shown in Table 18.
- the containing sample solution (130 ⁇ L) was bathed in boiling water and slowly cooled to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C.
- Example 1.7 the compounds 8 (exomethylene) and 16 ((S) -methyl) obtained in Example 1.7, Example 2.3 and Example 3.5 were amidite.
- the oligonucleotide synthesized using the block showed a higher Tm value for double-stranded DNA and higher binding affinity than the oligonucleotide synthesized using EoNA as the amidite block. Recognize.
- Example 7 Oligonucleotide Synthesis and Purification (2) Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl), thymidine (naturally occurring), LNA and ENA were used as amidite blocks, respectively, in the same manner as in Example 4 according to the phosphoramidite method known in the art. Nucleotide synthesis was performed.
- Example 8 Enzyme resistance experiment (1) The final concentrations were respectively Tris-HCl buffer (pH 8.0) 50 mM, magnesium chloride 10 mM, each oligonucleotide 7.5 ⁇ M obtained in Example 7, and 3′-exonuclease (Crotalus Admanteus Venom Phosphoesterase: CAVP, Pharmacia Biotech) The sample solution (100 ⁇ L) adjusted to 1.5 ⁇ g / mL was kept at 37 ° C. for reaction. A part of the reaction solution (20 ⁇ L) was collected over time, heated at 90 ° C. for 2 minutes to inactivate the enzyme, and the remaining amount of oligonucleotide was determined by HPLC (LC-20AT, SPD-20A, CTO manufactured by SHIMADZU). -20A, CBM-20A).
- compound 8 (exomethylene) obtained in Example 1.7, compound 11 ((R) -methyl) obtained in Example 2.3, or Example 3.5
- the oligonucleotide synthesized using the compound 16 ((S) -methyl derivative) obtained in (1) as an amidite block has a residual amount of unreacted oligonucleotide as compared with those using natural thymidine or LNA. It can be seen that the result was extremely high. Further, even when compared with the oligonucleotides using ENA, the oligonucleotides using the exomethylene, (R) -methyl, or (S) -methyl are not left unreacted with time. It can be seen that the amount was not reduced, and had better enzyme resistance.
- Table 19 shows the physical property data of the obtained compound 17.
- 1,8-diazabicycloundecene (62 ⁇ L, 0.416 mmol) was added dropwise to an anhydrous tetrahydrofuran solution (2.0 mL) of the obtained crude product under a nitrogen stream, and iodine (43 0.7 mg, 0.166 mmol) was added and stirred at room temperature for 2 hours.
- the reaction solution was quenched by adding a saturated aqueous sodium thiosulfate solution at 0 ° C., and extracted with ethyl acetate. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (70.0 mg).
- N, N-diisopropylethylamine (0.23 mL, 1.28 mmol) was added to an anhydrous dichloromethane solution (5.0 mL) of the compound 19 (251 mg, 0.428 mmol) obtained above, 2-Cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.11 mL, 0.513 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
- the organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (3.49 g).
- Table 27 shows the physical property data of the obtained compound 25.
- N, N-diisopropylethylamine (0.61 mL, 3.42 mmol) was added to a solution of compound 25 (700 mg, 1.14 mmol) obtained in Example 10.5 in anhydrous dichloromethane (10 mL).
- 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.31 mL, 1.37 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
- Table 33 shows the physical property data of the obtained compound 31.
- N, N-diisopropylethylamine (0.35 mL, 1.96 mmol) was added to a solution of compound 31 (400 mg, 0.653 mmol) obtained in Example 11.5 in anhydrous dichloromethane (10 mL).
- 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.17 mL, 0.783 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
- Example 12 Synthesis and purification of oligonucleotide (3) Compound 9.4 (unsubstituted product), Compound 26 ((R) -methyl-exomethylene compound), and Compound 32 ((S)) obtained in Example 9.4, Example 10.6 and Example 11.6 -Methyl-exomethylene) were used as amidite blocks, respectively, to prepare 0.1M anhydrous acetonitrile solution of these with d m C (Ac) and T phosphoramidites (both from Sigma-Aldrich).
- Each of the oligonucleotides shown in Table 35 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where Q is This corresponds to the structure derived from compound 20 (unsubstituted product) obtained in Example 9.4, and R Corresponds to the structure derived from compound 26 ((R) -methyl-exomethylene) obtained in Example 10.6. S represents compound 32 ((S) -methyl obtained in Example 11.6. -Corresponds to a structure derived from an exomethylene).
- the synthesis scale in the synthesis was 0.2 ⁇ mol, and was performed under trityl-on conditions.
- As the activator 5- [3,5-bis (trifluoromethyl) phenyl] -1H-tetrazole (0.25 M anhydrous acetonitrile solution, Activator 42, Proligo (registered trademark)) was used.
- the condensation time was 10 minutes for the amidite blocks Q, R, and S obtained in Example 9.4, Example 10.6, and Example 11.6, and 32 seconds for the natural amidite block.
- the mixture was treated with 28% aqueous ammonia at room temperature for 1.5 hours to cut out from the column carrier and to deprotect the base part and the phosphoric diester part. Subsequently, purification was performed by a simple gel filtration column (Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters), and further purified by reverse phase HPLC.
- Tm melting temperature
- the final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 ⁇ M of the oligonucleotide obtained in Example 12, and a sample solution containing 4 ⁇ M of single-stranded RNA or single-stranded DNA shown in Table 35. (130 ⁇ L) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
- Table 35 shows the results when single-stranded RNA was used as the target strand and the results when single-stranded DNA was used as the target strand.
- Example 14 Measurement of melting temperature ( Tm ) (4) (Evaluation of triple chain forming ability) The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 ⁇ M oligonucleotide obtained in Example 12, and 1.5 ⁇ M hairpin double-stranded DNA shown in Table 36.
- the sample solution containing (130 ⁇ L) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C.
- Table 36 shows the results when double-stranded DNA was used as the target strand.
- Example 15 Oligonucleotide Synthesis and Purification (4)
- Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl form), compound 20 obtained in Example 9.4 (unsubstituted product), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene form), Example 11
- compound 32 ((S) -methyl-exomethylene), thymidine (natural product), and ENA obtained in .6 were used as amidite blocks, respectively.
- the following oligonucleotides were synthesized according to a known phosphoramidite method.
- Example 16 Enzyme Resistance Experiment (2) The final concentrations were Tris-HCl buffer (pH 8.0) 50 mM, magnesium chloride 10 mM, each oligonucleotide 7.5 ⁇ M obtained in Example 15, 3′-exonuclease (Crotalus Admanteus Venophophophosterase: CAVP, Pharmacia Biotech) (Product) 2.5 ⁇ g / mL sample solution (100 ⁇ L) was kept at 37 ° C. for reaction. A part of the reaction solution (20 ⁇ L) was collected over time, heated at 90 ° C. for 2 minutes to inactivate the enzyme, and the remaining amount of oligonucleotide was determined by HPLC (LC-20AT, SPD-20A, CTO manufactured by SHIMADZU). -20A, CBM-20A).
- Example 2 As shown in FIG. 2, in a system in which the concentration of 3′-exonuclease was higher than that in the experimental system described in Example 8, compound 8 (exomethylene) obtained in Example 1.7, Example 2 was obtained. Compound 11 ((R) -methyl compound) obtained in .3, Compound 16 ((S) -methyl compound) obtained in Example 3.5, and Compound 20 obtained in Example 9.4 (none) Substituent), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene), or compound 32 obtained in Example 11.6 ((S) -methyl-exomethylene) It can be seen that the oligonucleotide synthesized using amidite block showed a significantly higher residual amount of unreacted oligonucleotide compared to natural thymidine and ENA whose residual amount decreased over time.
- the remaining amount of the unreacted oligonucleotide of the oligonucleotide using (R) -methyl or (R) -methyl-exomethylene is less decreased with time. It can be seen that the enzyme had better enzyme resistance.
- Example 17 Oligonucleotide Synthesis and Purification (5)
- one of the following artificial nucleic acids was incorporated into an oligonucleotide composed of four types of bases (A, T, G and C).
- Compound 8 Exomethylene
- Compound 11 ((R) -methyl) obtained in Example 2.3
- Compound 16 obtained in Example 3.5
- compound 20 obtained in Example 9.4 (unsubstituted product)
- compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene form)
- Example 11 6 except that Compound 32 ((S) -methyl-exomethylene), thymidine (natural product), LNA and ENA obtained in .6 were used as amidite blocks, respectively.
- oligonucleotides were synthesized according to the known phosphoramidite method.
- sequence of the oligonucleotide is 5′-d (TTCAGCATTGGTATTC) -3 ′ (SEQ ID NO: 5).
- Example 18 Measurement of melting temperature (Tm) (4)
- each oligonucleotide obtained in Example 17 was double-stranded with a single-stranded oligo RNA of 5′-r (GAAUACCAAUGCUGAA) -3 ′ (SEQ ID NO: 6) as a target strand, and the RNA The melting temperature was measured.
- the final concentration is 10 mM sodium phosphate buffer (pH 7.2), 100 mM sodium chloride, 4 ⁇ M of the oligonucleotide obtained in Example 17, and the sample solution (130 ⁇ L) containing the single-stranded oligo RNA 4 ⁇ M is bathed in boiling water. Then, after slowly cooling to room temperature, each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C.
- any artificial oligonucleic acid can have sufficient binding ability to the target single-stranded RNA. Indicated.
- Example 19 Antisense evaluation in cell culture system Huh-7 (human hepatoma cell line: PS20) prepared to 4.51 ⁇ 10 5 cells / mL was seeded in a 96-well flat bottom microplate (IWAKI) and incubated at 37 ° C. For 24 hours under 5% CO 2 .
- Lipofectamine 3000 manufactured by Life Technologies
- Opti-MEM manufactured by Life Technologies were further added and mixed so that each antisense nucleic acid molecule had a final concentration of 100 nM or 400 nM and allowed to stand at room temperature for 15 minutes. Later, it was added to each well. Each oligonucleotide obtained in Example 17 was added, and cells were collected 24 hours later.
- CDNA was synthesized from the collected total RNA using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.). After the obtained cDNA was appropriately diluted, real-time PCR was performed to quantify the amount of ApoB mRNA. In real-time PCR, the amount of GAPDH mRNA of the housekeeping gene was also quantified. Thus, the amount of ApoB mRNA relative to the amount of GAPDH mRNA was quantitatively evaluated. For real-time PCR, Fast SYBR (registered trademark) Green Master Mix (Applied Biosystems) was used.
- Hs_ApoB_Fw GGCTCACCCTGAGAGAAGTG (SEQ ID NO: 8)
- Hs_ApoB_Rv GCTGCTTTCTGGGAACCTCAC (SEQ ID NO: 8)
- Hs_GAPDH_Fw GGCCTCCCAAGGAGTAAGACC (SEQ ID NO: 9)
- Hs_GAPDH_Rv AGGGTCTCATAGGGCACTG (SEQ ID NO: 10)
- EoDNA structure is an oligonucleotide comprising a structure derived from compound 20 (unsubstituted product) obtained in Example 9.4 among the oligonucleotides synthesized in Example 17;
- MeEoDNA structure is an oligonucleotide comprising a structure derived from compound 11 ((R) -methyl) obtained in Example 2.3 among the oligonucleotides synthesized in Example 17;
- S-MeEoDNA structure Is an oligonucleotide containing a structure derived from the compound 32 ((S) -methyl-exomethylene form) obtained in Example 11.6 among the oligonucleotides synthesized in Example 17;
- Methylene EoDNA structure "Is derived from compound 8 (exomethylene) obtained in Example 1.7 among the oligonucleotides synthesized in Example 17.
- R-Me-methylene structure is the same as the compound 26 ((R) -methyl-exomethylene form) obtained in Example 10.6 among the oligonucleotides synthesized in Example 17.
- novel 2 ', 4'-bridged 6-membered ring nucleosides and nucleotides having a hetero atom at the 6'-position are provided.
- This oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide has a binding affinity for single-stranded RNA and double-stranded DNA comparable to conventional oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide.
- the oligonucleotide of the present invention is useful as a material for nucleic acid medicine, for example.
Abstract
Disclosed are a crosslinking nucleoside and nucleotide. This nucleoside has a 2',4'-crosslinking structure, and is represented by formula (I) or (I'). An oligonucleotide containing this 2',4'-crosslinking artificial nucleotide exhibits a binding affinity to single-strand RNA comparable to that of oligonucleotides containing conventional 2',4'-crosslinking artificial nucleotides. An oligonucleotide according to the present invention is useful, for example, as a nucleic acid drug material.
Description
本発明は、架橋型ヌクレオシドおよびヌクレオチドに関する。より詳細には、一本鎖ＲＮＡおよび二本鎖ＤＮＡに対する高い結合親和性、ならびにヌクレアーゼに対する高い耐性を有する架橋型ヌクレオシドおよびヌクレオチドに関する。
The present invention relates to a bridged nucleoside and a nucleotide. More particularly, it relates to bridged nucleosides and nucleotides having high binding affinity for single-stranded RNA and double-stranded DNA, and high resistance to nucleases.
核酸医薬による疾病の治療法として、アンチセンス法、アンチジーン法、アプタマー、ｓｉＲＮＡなどがある。このうち、アンチセンス法は、疾病に関わるｍＲＮＡと相補的なオリゴヌクレオチド（アンチセンス鎖）を外部から導入し、二重鎖を形成させることにより、病原ＲＮＡの翻訳過程を阻害し、疾病の治療や予防を行う手法である。ｓｉＲＮＡもこれに類似しており、生体に投与した二重鎖ＲＮＡによりｍＲＮＡからタンパク質への翻訳を阻害する。一方、アンチジーン法は、病原ＲＮＡを転写するＤＮＡ部位に対応する三重鎖形成オリゴヌクレオチドを外部から導入することによりＤＮＡからＲＮＡへの転写を抑制する。また、アプタマーは、短い核酸分子（オリゴヌクレオチド）であるため、疾病の原因となるタンパク質などの生体成分と結合することにより機能を発揮する。
There are anti-sense method, anti-gene method, aptamer, siRNA and the like as treatment methods for diseases by nucleic acid medicine. Among these, the antisense method introduces an oligonucleotide (antisense strand) complementary to the mRNA involved in the disease from the outside to form a double strand, thereby inhibiting the translation process of the pathogenic RNA and treating the disease. And prevention. siRNA is similar to this, and translation from mRNA to protein is inhibited by double-stranded RNA administered to a living body. On the other hand, the antigene method suppresses transcription from DNA to RNA by introducing from the outside a triplex-forming oligonucleotide corresponding to a DNA site that transcribes pathogenic RNA. In addition, since aptamers are short nucleic acid molecules (oligonucleotides), they function by binding to biological components such as proteins that cause disease.
こうした核酸医薬の素材として、種々の人工核酸が開発されているが、未だ切札となるべき分子が存在しない。例えば、これまでに開発されてきた核酸医薬の素材として、Ｓ－オリゴ（ホスホロチオエート）、２’，４’－ＢＮＡ（bridged nucleic acid）／ＬＮＡ（locked nucleic acid）（特許文献１～３および非特許文献１～６参照）などがある。Ｓ－オリゴは、サイトメガロウイルスに対するアンチセンス医薬品として、既に米国で上市されている。これは、高いヌクレアーゼ耐性を有するものの、標的核酸鎖への結合親和性が低いという難点を有しており、改善が必要である。これまでに開発されている２’，４’－ＢＮＡ／ＬＮＡはいずれも、２’，４’－架橋型人工ヌクレオシドとして標的核酸鎖への結合親和性が高く、これからの核酸医薬の素材として最も期待される分子である。しかし、ヌクレアーゼへの耐性が十分ではなく、生体内での安定性という点で改良の余地を残している。
Various artificial nucleic acids have been developed as materials for such nucleic acid drugs, but there are still no molecules that should become trump cards. For example, S-oligo (phosphorothioate), 2 ′, 4′-BNA (bridged nucleic acid) / LNA (locked nucleic acid) (patent documents 1 to 3 and non-patent documents) References 1 to 6). S-oligo is already marketed in the United States as an antisense drug against cytomegalovirus. Although this has high nuclease resistance, it has a drawback that its binding affinity to the target nucleic acid chain is low, and needs to be improved. 2 ′, 4′-BNA / LNA developed so far has the highest binding affinity to the target nucleic acid chain as a 2 ′, 4′-bridged artificial nucleoside, and is the most useful material for nucleic acid medicine in the future. It is an expected molecule. However, resistance to nucleases is not sufficient, and there is room for improvement in terms of stability in vivo.
さらに、近年では、上記人工ヌクレオシドのうち、２’，４’－架橋構造内にヘテロ原子を導入して、例えば、標的核酸鎖への結合親和性をさらに向上させる開発が行われている。具体的には、以下の式（ａ）および式（ｂ）に示すようなヌクレオシド構造を有するオリゴヌクレオチド：
Furthermore, in recent years, development has been carried out to further improve the binding affinity to, for example, a target nucleic acid chain by introducing a heteroatom into the 2 ', 4'-bridged structure of the artificial nucleoside. Specifically, an oligonucleotide having a nucleoside structure as shown in the following formulas (a) and (b):
が提案されている（非特許文献７および８）。
Has been proposed (Non-Patent Documents 7 and 8).
しかし、上記式（ａ）に示すような構造をベースとするヌクレオシドは、例えば、その合成にあたり、非常に煩雑で多段階の工程を要し、ヌクレアーゼに対する耐性能に改善の余地があると考えられている点で未だ満足し得るものとは言えないものであった。また、式（ｂ）に示すような構造のベースとなるヌクレオシドは、大きな７員環架橋構造を有することにより標的核酸鎖との結合親和性が満足し得るものでないと考えられる点が指摘されていた。このため、このようなオリゴヌクレオチドと同等またはそれ以上の性能を有し、かつ工業的な生産効率が一層優れたオリゴヌクレオチドの開発が所望されている。
However, a nucleoside based on the structure as shown in the above formula (a) is considered to have, for example, a very complicated and multi-step process for its synthesis, and there is room for improvement in resistance to nuclease. However, it was still not satisfactory. Further, it has been pointed out that the nucleoside serving as the base of the structure as shown in the formula (b) has a large seven-membered ring cross-linked structure, and thus it is considered that the binding affinity with the target nucleic acid chain is not satisfactory. It was. For this reason, it is desired to develop an oligonucleotide having performance equivalent to or higher than that of such an oligonucleotide and further excellent in industrial production efficiency.
本発明は、上記課題を解決するものであり、その目的とするところは、生体内でヌクレアーゼによる分解を受けにくく、標的のｍＲＮＡに対する高い結合親和性および特異性を有し、特定の遺伝子の発現を効率よく制御することのできるアンチセンス法や核酸医薬用の新規な分子であって、生産性に優れた当該分子を提供することにある。
The present invention solves the above-mentioned problems, and the object of the present invention is to prevent degradation by nucleases in vivo, have high binding affinity and specificity for target mRNA, and express a specific gene. It is an object of the present invention to provide a novel molecule for antisense methods and nucleic acid pharmaceuticals, which can efficiently control the above-described molecules, and has excellent productivity.
本発明は、以下の式（Ｉ）または（Ｉ’）で表される化合物またはその塩：
The present invention relates to a compound represented by the following formula (I) or (I ′) or a salt thereof:
（式中、
Ｂａｓｅは、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ２およびＲ３は、それぞれ独立して、水素原子、核酸合成の水酸基の保護基、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数２から７のアルケニル基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール部分を有するアラルキル基、該α群から選択される任意の置換基を１以上有していてもよいアシル基、該α群から選択される任意の置換基を１以上有していてもよいシリル基、該α群から選択される任意の置換基を１以上有していてもよいリン酸基、核酸合成の保護基で保護されたリン酸基、－Ｐ（Ｒ４）Ｒ５［式中、Ｒ４およびＲ５は、それぞれ独立して、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）である。 (Where
Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the α group, where The α group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A functionalized mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protective group for nucleic acid synthesis, and a halogen atom;
R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring An alkenyl group having 2 to 7 carbon atoms, an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and may contain a hetero atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms, which may have one or more arbitrary substituents selected from the α group and may contain a hetero atom, any arbitrary selected from the α group An acyl group optionally having one or more substituents, a silyl group optionally having one or more arbitrary substituents selected from the α group, and an arbitrary substituent selected from the α group are 1 Protection of phosphate groups and nucleic acid synthesis that may have In protected phosphate group, -P (R 4) R 5 [ wherein, R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, nucleic acid synthesis A mercapto group protected with a protecting group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms Or a linear or branched alkylamino group having 1 to 6 carbon atoms]
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom).
Ｂａｓｅは、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ２およびＲ３は、それぞれ独立して、水素原子、核酸合成の水酸基の保護基、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数２から７のアルケニル基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール部分を有するアラルキル基、該α群から選択される任意の置換基を１以上有していてもよいアシル基、該α群から選択される任意の置換基を１以上有していてもよいシリル基、該α群から選択される任意の置換基を１以上有していてもよいリン酸基、核酸合成の保護基で保護されたリン酸基、－Ｐ（Ｒ４）Ｒ５［式中、Ｒ４およびＲ５は、それぞれ独立して、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）である。 (Where
Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the α group, where The α group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A functionalized mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protective group for nucleic acid synthesis, and a halogen atom;
R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring An alkenyl group having 2 to 7 carbon atoms, an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and may contain a hetero atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms, which may have one or more arbitrary substituents selected from the α group and may contain a hetero atom, any arbitrary selected from the α group An acyl group optionally having one or more substituents, a silyl group optionally having one or more arbitrary substituents selected from the α group, and an arbitrary substituent selected from the α group are 1 Protection of phosphate groups and nucleic acid synthesis that may have In protected phosphate group, -P (R 4) R 5 [ wherein, R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, nucleic acid synthesis A mercapto group protected with a protecting group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms Or a linear or branched alkylamino group having 1 to 6 carbon atoms]
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom).
１つの実施形態では、上記式（Ｉ）または（Ｉ’）において、Ｘは酸素原子である。
In one embodiment, in the above formula (I) or (I ′), X is an oxygen atom.
１つの実施形態では、上記式（Ｉ）または（Ｉ’）において、上記Ｂａｓｅは、６－アミノプリン－９－イル基、２，６－ジアミノプリン－９－イル基、２－アミノ－６－クロロプリン－９－イル基、２－アミノ－６－フルオロプリン－９－イル基、２－アミノ－６－ブロモプリン－９－イル基、２－アミノ－６－ヒドロキシプリン－９－イル基、６－アミノ－２－メトキシプリン－９－イル基、６－アミノ－２－クロロプリン－９－イル基、６－アミノ－２－フルオロプリン－９－イル基、２，６－ジメトキシプリン－９－イル基、２，６－ジクロロプリン－９－イル基、６－メルカプトプリン－９－イル基、２－オキソ－４－アミノ－１，２－ジヒドロピリミジン－１－イル基、４－アミノ－２－オキソ－５－フルオロ－１，２－ジヒドロピリミジン－１－イル基、４－アミノ－２－オキソ－５－クロロ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－メトキシ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－メルカプト－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－ヒドロキシ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基、または４－アミノ－５－メチル－２－オキソ－１，２－ジヒドロピリミジン－１－イル基である。
In one embodiment, in the above formula (I) or (I ′), the Base is a 6-aminopurin-9-yl group, a 2,6-diaminopurin-9-yl group, a 2-amino-6- Chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-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-dimethoxypurine-9 -Yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group, 4-amino- 2-oxo-5-fluoro-1,2-dihi Ropyrimidin-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, 2-oxo-4-hydroxy-5 -Methyl-1,2-dihydropyrimidin-1-yl group or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group.
さらなる実施形態では、上記式（Ｉ）または（Ｉ’）において、上記Ｂａｓｅは、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基である。
In a further embodiment, in the above formula (I) or (I ′), the Base is a 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group.
本発明はまた、以下の式（ＩＩ）または（ＩＩ’）で表されるヌクレオシド構造を少なくとも１つ含有するオリゴヌクレオチドまたはその薬理学上許容される塩：
The present invention also provides an oligonucleotide containing at least one nucleoside structure represented by the following formula (II) or (II ′) or a pharmacologically acceptable salt thereof:
（式中、
Ｂａｓｅは、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）である。 (Where
Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the α group, where The α group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A functionalized mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protective group for nucleic acid synthesis, and a halogen atom;
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom).
Ｂａｓｅは、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）である。 (Where
Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the α group, where The α group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A functionalized mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protective group for nucleic acid synthesis, and a halogen atom;
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom).
１つの実施形態では、上記式（ＩＩ）または（ＩＩ’）において、Ｘは酸素原子である。
In one embodiment, in the above formula (II) or (II ′), X is an oxygen atom.
本発明によれば、６’位にヘテロ原子を有する新規な２’，４’－架橋型６員環ヌクレオシドおよびヌクレオチドが提供される。この２’，４’－架橋型人工ヌクレオチドを含むオリゴヌクレオチドは、従来の２’，４’－架橋型人工ヌクレオチドを含むオリゴヌクレオチドを匹敵する一本鎖ＲＮＡおよび一本鎖ＤＮＡに対する結合親和性を有する。本発明のオリゴヌクレオチドは、例えば、核酸医薬への応用が期待される。本発明の２’，４’－架橋型ヌクレオシドおよびヌクレオチドはまた、６員環架橋構造内の６’位にヘテロ原子を導入することができ、かつ従来と比較して、より簡便な反応プロセスを通じて製造することができる。このため、工業的な生産効率を一層高めることも可能である。
According to the present invention, novel 2 ', 4'-bridged 6-membered ring nucleosides and nucleotides having a hetero atom at the 6'-position are provided. This oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide has a binding affinity for single-stranded RNA and single-stranded DNA comparable to conventional oligonucleotides containing 2 ′, 4′-bridged artificial nucleotide. Have. The oligonucleotide of the present invention is expected to be applied to, for example, nucleic acid medicine. The 2 ′, 4′-bridged nucleosides and nucleotides of the present invention can also introduce a heteroatom at the 6 ′ position in a 6-membered ring bridge structure, and through a simpler reaction process compared to the conventional one. Can be manufactured. For this reason, it is also possible to further increase industrial production efficiency.
まず、本明細書中で用いられる用語を定義する。
First, terms used in this specification are defined.
本明細書において、用語「炭素数１から６の直鎖アルキル基」は、炭素数１から６の任意の直鎖アルキル基をいい、具体的にはメチル基、エチル基、ｎ－プロピル基、ｎ－ブチル基、ｎ－ペンチル基、またはｎ－ヘキシル基をいう。
In the present specification, the term “linear alkyl group having 1 to 6 carbon atoms” refers to any linear alkyl group having 1 to 6 carbon atoms, specifically a methyl group, an ethyl group, an n-propyl group, An n-butyl group, an n-pentyl group, or an n-hexyl group.
本明細書において、用語「炭素数１から６の直鎖アルコキシ基」は、炭素数１から６の任意の直鎖アルキル基を有するアルコキシ基を包含する。例えば、メチルオキシ基、エチルオキシ基、ｎ－プロピルオキシ基などが挙げられる。本明細書において、用語「炭素数１から６の直鎖または分岐鎖アルコキシ基」は、炭素数１から６の任意の直鎖または分岐鎖アルキル基を有するアルコキシ基を包含する。例えば、メチルオキシ基、エチルオキシ基、ｎ－プロピルオキシ基、イソプロピルオキシ基、ｎ－ブチルオキシ基、イソブチルオキシ基、ｔｅｒｔ－ブチルオキシ基、ｎ－ペンチルオキシ基、イソペンチルオキシ基などが挙げられる。
In the present specification, the term “linear alkoxy group having 1 to 6 carbon atoms” includes an alkoxy group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methyloxy group, an ethyloxy group, and an n-propyloxy group. In this specification, the term “a linear or branched alkoxy group having 1 to 6 carbon atoms” includes an alkoxy group having an arbitrary linear or branched alkyl group having 1 to 6 carbon atoms. Examples thereof include a methyloxy group, an ethyloxy group, an n-propyloxy group, an isopropyloxy group, an n-butyloxy group, an isobutyloxy group, a tert-butyloxy group, an n-pentyloxy group, and an isopentyloxy group.
本明細書において、用語「炭素数１から６の直鎖アルキルチオ基」は、炭素数１から６の任意の直鎖アルキル基を有するアルキルチオ基を包含する。例えば、メチルチオ基、エチルチオ基、ｎ－プロピルチオ基などが挙げられる。本明細書において、用語「炭素数１から６の直鎖または分岐鎖アルキルチオ基」は、炭素数１から６の任意の直鎖または分岐鎖アルキル基を有するアルキルチオ基を包含する。例えば、メチルチオ基、エチルチオ基、ｎ－プロピルチオ基、イソプロピルチオ基、ｎ－ブチルチオ基、イソブチルチオ基、ｔｅｒｔ－ブチルチオ基、ｎ－ペンチルチオ基、イソペンチルチオ基などが挙げられる。
In the present specification, the term “linear alkylthio group having 1 to 6 carbon atoms” includes an alkylthio group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methylthio group, an ethylthio group, and an n-propylthio group. In the present specification, the term “a linear or branched alkylthio group having 1 to 6 carbon atoms” includes an alkylthio group having an arbitrary linear or branched alkyl group having 1 to 6 carbon atoms. Examples include a methylthio group, an ethylthio group, an n-propylthio group, an isopropylthio group, an n-butylthio group, an isobutylthio group, a tert-butylthio group, an n-pentylthio group, and an isopentylthio group.
本明細書において、用語「炭素数１から６のシアノアルコキシ基」は、上記炭素数１から６の直鎖アルコキシ基を構成する少なくとも１つの水素原子がシアノ基で置換された基をいう。
In this specification, the term “C1-C6 cyanoalkoxy group” refers to a group in which at least one hydrogen atom constituting the straight-chain alkoxy group having 1 to 6 carbon atoms is substituted with a cyano group.
本明細書において、用語「炭素数１から６の直鎖アルキルアミノ基」は、アミノ基を構成する水素原子の１つまたは２つが、炭素数１から６の直鎖アルキル基で置換された基を包含する。例えば、メチルアミノ基、ジメチルアミノ基、エチルアミノ基、メチルエチルアミノ基、ジエチルアミノ基などが挙げられる。本明細書において、用語「炭素数１から６の直鎖または分岐鎖アルキルアミノ基」は、アミノ基を構成する水素原子の１つまたは２つが、炭素数１から６の任意の直鎖または分岐鎖アルキル基で置換された基を包含する。例えば、メチルアミノ基、ジメチルアミノ基、エチルアミノ基、メチルエチルアミノ基、ジエチルアミノ基、ｎ－プロピルアミノ基、ジｎ－プロピルアミノ基、イソプロピルアミノ基、ジイソプロピルアミノ基などが挙げられる。
In the present specification, the term “linear alkylamino group having 1 to 6 carbon atoms” means a group in which one or two hydrogen atoms constituting the amino group are substituted with a linear alkyl group having 1 to 6 carbon atoms. Is included. Examples thereof include a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, and a diethylamino group. In this specification, the term “a linear or branched alkylamino group having 1 to 6 carbon atoms” means any linear or branched group in which one or two hydrogen atoms constituting the amino group are 1 to 6 carbon atoms. Includes groups substituted with chain alkyl groups. Examples include methylamino group, dimethylamino group, ethylamino group, methylethylamino group, diethylamino group, n-propylamino group, di-n-propylamino group, isopropylamino group, diisopropylamino group and the like.
本明細書において、用語「分岐または環を形成していてもよい炭素数１から７のアルキル基」は、炭素数１から７の任意の直鎖アルキル基、炭素数３から７の任意の分岐鎖アルキル基、および炭素数３から７の任意の環状アルキル基を包含する。単に、「低級アルキル基」という場合もある。例えば、炭素数１から７の任意の直鎖アルキル基としては、メチル基、エチル基、ｎ－プロピル基、ｎ－ブチル基、ｎ－ペンチル基、ｎ－ヘキシル基、およびｎ－ヘプチル基が挙げられ、炭素数３から７の任意の分岐鎖アルキル基としては、イソプロピル基、イソブチル基、ｔｅｒｔ－ブチル基、イソペンチル基などが挙げられ、そして炭素数３から７の任意の環状アルキル基としては、シクロブチル基、シクロペンチル基、シクロヘキシル基などが挙げられる。
In the present specification, the term “an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring” means any linear alkyl group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. Includes a chain alkyl group and any cyclic alkyl group having 3 to 7 carbon atoms. It may be simply referred to as “lower alkyl group”. For example, arbitrary linear alkyl groups having 1 to 7 carbon atoms include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, and n-heptyl group. Examples of the branched alkyl group having 3 to 7 carbon atoms include isopropyl group, isobutyl group, tert-butyl group, isopentyl group and the like, and optional cyclic alkyl group having 3 to 7 carbon atoms include A cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. are mentioned.
本明細書において、用語「分岐または環を形成していてもよい炭素数２から７のアルケニル基」は、炭素数２から７の任意の直鎖アルケニル基、炭素数３から７の任意の分岐鎖アルケニル基、および炭素数３から７の任意の環状アルケニル基を包含する。単に、「低級アルケニル基」という場合もある。例えば、炭素数２から７の任意の直鎖アルケニル基としては、エテニル基、１－プロペニル基、２－プロペニル基、１－ブテニル基、２－ブテニル基、１－ペンテニル基、２－ペンテニル基、３－ペンテニル基、４－ペンテニル基、１－ヘキセニル基などが挙げられ、炭素数３から７の任意の分岐鎖アルケニル基としては、イソプロペニル基、１－メチル－１－プロペニル基、１－メチル－２－プロペニル基、２－メチル－１－プロペニル基、２－メチル－２－プロペニル基、１－メチル－２－ブテニル基などが挙げられ、そして炭素数３から７の任意の環状アルケニル基としては、シクロブテニル基、シクロペンテニル基、シクロヘキセニル基などが挙げられる。
In this specification, the term “an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring” means any linear alkenyl group having 2 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. Chain alkenyl groups, and any cyclic alkenyl group having 3 to 7 carbon atoms are included. It may be simply referred to as “lower alkenyl group”. For example, as an arbitrary straight chain alkenyl group having 2 to 7 carbon atoms, ethenyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1-hexenyl group and the like. Examples of the branched alkenyl group having 3 to 7 carbon atoms include isopropenyl group, 1-methyl-1-propenyl group, 1-methyl -2-propenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-methyl-2-butenyl group, etc., and any cyclic alkenyl group having 3 to 7 carbon atoms Includes a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and the like.
本明細書において、用語「分岐または環を形成していてもよい炭素数１から７のアルコキシ基」は、炭素数１から７の任意の直鎖アルコキシ基、炭素数３から７の任意の分岐鎖アルコキシ基、および炭素数３から７の任意の環状アルコキシ基を包含する。単に、「低級アルコキシ基」という場合もある。例えば、炭素数１から７の任意の直鎖アルコキシ基としては、メトキシ基、エトキシ基、ｎ－プロポキシ基、ｎ－ブチロキシ、ｎ－ペンチルオキシ基、ｎ－ヘキシルオキシ基、およびｎ－ヘプチルオキシ基が挙げられ、炭素数３から７の任意の分岐鎖アルコキシ基としては、イソプロポキシ基、イソブチロキシ基、ｔｅｒｔ－ブチロキシ基、イソペンチルオキシ基などが挙げられ、そして炭素数３から７の任意の環状アルコキシ基としては、シクロブチロキシ基、シクロペンチルオキシ基、シクロヘキシルオキシ基などが挙げられる。
In this specification, the term “an alkoxy group having 1 to 7 carbon atoms which may form a branch or a ring” means any linear alkoxy group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. It includes a chain alkoxy group and any cyclic alkoxy group having 3 to 7 carbon atoms. It may be simply referred to as “lower alkoxy group”. For example, any linear alkoxy group having 1 to 7 carbon atoms includes a methoxy group, an ethoxy group, an n-propoxy group, an n-butoxy, an n-pentyloxy group, an n-hexyloxy group, and an n-heptyloxy group. Examples of the branched alkoxy group having 3 to 7 carbon atoms include isopropoxy group, isobutoxy group, tert-butoxy group, isopentyloxy group, etc., and any cyclic group having 3 to 7 carbon atoms Examples of the alkoxy group include a cyclobutoxy group, a cyclopentyloxy group, and a cyclohexyloxy group.
本明細書において、用語「ヘテロ原子を含んでいてもよい炭素数３から１２のアリール基」は、炭化水素のみで構成された、炭素数６から１２の任意のアリール基と、当アリール基の環構造を構成する少なくとも１つの炭素原子がヘテロ原子（例えば、窒素原子、酸素原子、および硫黄原子、ならびにこれらの組合せ）で置換された、炭素数３から１２の任意のヘテロアリール基とを包含する。当該炭素数６から１２のアリール基としては、フェニル基、ナフチル基、インデニル基、アズレニル基などが挙げられ、そして当該炭素数３から１２の任意のヘテロアリール基としては、ピリジル基、ピロリル基、キノリル基、インドリル基、イミダゾリル基、フリル基、チエニル基などが挙げられる。
In this specification, the term “an aryl group having 3 to 12 carbon atoms that may contain a heteroatom” refers to any aryl group having 6 to 12 carbon atoms, which is composed of only hydrocarbons, and the aryl group. Including any heteroaryl group having 3 to 12 carbon atoms in which at least one carbon atom constituting the ring structure is substituted with a heteroatom (eg, a nitrogen atom, an oxygen atom, and a sulfur atom, and combinations thereof) To do. Examples of the aryl group having 6 to 12 carbon atoms include a phenyl group, a naphthyl group, an indenyl group, and an azulenyl group, and examples of the heteroaryl group having 3 to 12 carbon atoms include a pyridyl group, a pyrrolyl group, A quinolyl group, an indolyl group, an imidazolyl group, a furyl group, a thienyl group, and the like can be given.
本明細書において、用語「ヘテロ原子を含んでいてもよい炭素数３から１２のアリール部分を有するアラルキル基」の例としては、ベンジル基、フェネチル基、ナフチルメチル基、３－フェニルプロピル基、２－フェニルプロピル基、４－フェニルブチル基、２－フェニルブチル基、ピリジルメチル基、インドリルメチル基、フリルメチル基、チエニルメチル基、ピロリルメチル基、２－ピリジルエチル基、１－ピリジルエチル基、３－チエニルプロピル基などが挙げられる。
In this specification, examples of the term “aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom” include a benzyl group, a phenethyl group, a naphthylmethyl group, a 3-phenylpropyl group, -Phenylpropyl, 4-phenylbutyl, 2-phenylbutyl, pyridylmethyl, indolylmethyl, furylmethyl, thienylmethyl, pyrrolylmethyl, 2-pyridylethyl, 1-pyridylethyl, 3 -Thienylpropyl group and the like.
本明細書において、用語「アシル基」の例としては、脂肪族アシル基および芳香族アシル基が挙げられる。具体的には、脂肪族アシル基の例としては、ホルミル基、アセチル基、プロピオニル基、ブチリル基、イソブチリル基、ペンタノイル基、ピバロイル基、バレリル基、イソバレリル基、オクタノイル基、ノナノイル基、デカノイル基、３－メチルノナノイル基、８－メチルノナノイル基、３－エチルオクタノイル基、３，７－ジメチルオクタノイル基、ウンデカノイル基、ドデカノイル基、トリデカノイル基、テトラデカノイル基、ペンタデカノイル基、ヘキサデカノイル基、１－メチルペンタデカノイル基、１４－メチルペンタデカノイル基、１３，１３－ジメチルテトラデカノイル基、ヘプタデカノイル基、１５－メチルヘキサデカノイル基、オクタデカノイル基、１－メチルヘプタデカノイル基、ノナデカノイル基、アイコサノイル基およびヘナイコサノイル基のようなアルキルカルボニル基；スクシノイル基、グルタロイル基、アジポイル基のようなカルボキシ化アルキルカルボニル基；クロロアセチル基、ジクロロアセチル基、トリクロロアセチル基、トリフルオロアセチル基のようなハロゲノ低級アルキルカルボニル基；メトキシアセチル基のような低級アルコキシ低級アルキルカルボニル基；（Ｅ）－２－メチル－２－ブテノイル基のような不飽和アルキルカルボニル基が挙げられる。また、芳香族アシル基の例としては、ベンゾイル基、α－ナフトイル基、β－ナフトイル基のようなアリールカルボニル基；２－ブロモベンゾイル基、４－クロロベンゾイル基のようなハロゲノアリールカルボニル基；２，４，６－トリメチルベンゾイル基、４－トルオイル基のような低級アルキル化アリールカルボニル基；４－アニソイル基のような低級アルコキシ化アリールカルボニル基；２－カルボキシベンゾイル基、３－カルボキシベンゾイル基、４－カルボキシベンゾイル基のようなカルボキシ化アリールカルボニル基；４－ニトロベンゾイル基、２－ニトロベンゾイル基のようなニトロ化アリールカルボニル基；２－（メトキシカルボニル）ベンゾイル基のような低級アルコキシカルボニル化アリールカルボニル基；４－フェニルベンゾイル基のようなアリール化アリールカルボニル基などが挙げられる。好適には、ホルミル基、アセチル基、プロピオニル基、ブチリル基、イソブチリル基、ペンタノイル基、ピバロイル基、ベンゾイル基である。
In the present specification, examples of the term “acyl group” include aliphatic acyl groups and aromatic acyl groups. Specifically, examples of the aliphatic acyl group include formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group, isovaleryl group, octanoyl group, nonanoyl group, decanoyl group, 3-methylnonanoyl group, 8-methylnonanoyl group, 3-ethyloctanoyl group, 3,7-dimethyloctanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, 1-methylpentadecanoyl group, 14-methylpentadecanoyl group, 13,13-dimethyltetradecanoyl group, heptadecanoyl group, 15-methylhexadecanoyl group, octadecanoyl group, 1-methylheptadecanoyl group, Nonadecanoyl group, icosanoyl group And alkylcarbonyl groups such as heniacosanoyl group; carboxylated alkylcarbonyl groups such as succinoyl group, glutaroyl group and adipoyl group; halogeno lower alkylcarbonyl groups such as chloroacetyl group, dichloroacetyl group, trichloroacetyl group and trifluoroacetyl group Groups; lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl group; and unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl group. Examples of the aromatic acyl group include arylcarbonyl groups such as benzoyl group, α-naphthoyl group and β-naphthoyl group; halogenoarylcarbonyl groups such as 2-bromobenzoyl group and 4-chlorobenzoyl group; 2 , 4,6-trimethylbenzoyl group, lower alkylated arylcarbonyl group such as 4-toluoyl group; lower alkoxylated arylcarbonyl group such as 4-anisoyl group; 2-carboxybenzoyl group, 3-carboxybenzoyl group, 4 A carboxylated arylcarbonyl group such as a carboxybenzoyl group; a nitrated arylcarbonyl group such as a 4-nitrobenzoyl group or a 2-nitrobenzoyl group; a lower alkoxycarbonylated arylcarbonyl such as a 2- (methoxycarbonyl) benzoyl group Group; 4-phenyl Examples include arylated arylcarbonyl groups such as benzoyl groups. Preferred are formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group and benzoyl group.
本明細書において、用語「シリル基」の例としては、トリメチルシリル基、トリエチルシリル基、イソプロピルジメチルシリル基、ｔ－ブチルジメチルシリル基、メチルジイソプロピルシリル基、メチルジ－ｔ－ブチルシリル基、トリイソプロピルシリル基のようなトリ低級アルキルシリル基；ジフェニルメチルシリル基、ブチルジフェニルブチルシリル基、ジフェニルイソプロピルシリル基、フェニルジイソプロピルシリル基のような１～２個のアリール基で置換されたトリ低級アルキルシリル基などが挙げられる。好適には、トリメチルシリル基、トリエチルシリル基、トリイソプロピルシリル基、ｔ－ブチルジメチルシリル基、ｔ－ブチルジフェニルシリル基であり、さらに好適にはトリメチルシリル基である。
In this specification, examples of the term “silyl group” include trimethylsilyl group, triethylsilyl group, isopropyldimethylsilyl group, t-butyldimethylsilyl group, methyldiisopropylsilyl group, methyldi-t-butylsilyl group, and triisopropylsilyl group. A tri-lower alkylsilyl group such as diphenylmethylsilyl group, butyldiphenylbutylsilyl group, diphenylisopropylsilyl group, tri-lower alkylsilyl group substituted with 1 to 2 aryl groups such as phenyldiisopropylsilyl group, etc. Can be mentioned. A trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, and a t-butyldiphenylsilyl group are preferable, and a trimethylsilyl group is more preferable.
本明細書において、用語「ハロゲン原子」としては、例えば、フッ素原子、塩素原子、臭素原子、またはヨウ素原子が挙げられる。好適には、フッ素原子または塩素原子である。
In the present specification, examples of the term “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferable is a fluorine atom or a chlorine atom.
本明細書において、用語「核酸合成のアミノ基の保護基」、「核酸合成の水酸基の保護基」、「核酸合成の保護基で保護された水酸基」、「核酸合成の保護基で保護されたリン酸基」、「核酸合成の保護基で保護されたメルカプト基」の「保護基」とは、核酸合成の際に安定してアミノ基、水酸基、リン酸基またはメルカプト基を保護し得るものであれば、特に制限されない。具体的には、酸性または中性条件で安定であり、加水素分解、加水分解、電気分解、および光分解のような化学的方法により開裂し得る保護基のことをいう。このような保護基としては、例えば、低級アルキル基、低級アルケニル基、アシル基、テトラヒドロピラニルまたはテトラヒドロチオピラニル基、テトラヒドロフラニルまたはテトラヒドロチオフラニル基、シリル基、低級アルコキシメチル基、低級アルコキシ化低級アルコキシメチル基、ハロゲノ低級アルコキシメチル基、低級アルコキシ化エチル基、ハロゲン化エチル基、１～３個のアリール基で置換されたメチル基、「低級アルキル基、低級アルコキシ基、ハロゲン原子またはシアノ基でアリール環が置換された１～３個のアリール基で置換されたメチル基」、低級アルコキシカルボニル基、「ハロゲン原子、低級アルコキシ基またはニトロ基で置換されたアリール基」、「ハロゲン原子またはトリ低級アルキルシリル基で置換された低級アルコキシカルボニル基」、アルケニルオキシカルボニル基、「低級アルコキシまたはニトロ基でアリール環が置換されていてもよいアラルキルオキシカルボニル基」などが挙げられる。
In the present specification, the terms “protecting group for amino group for nucleic acid synthesis”, “protecting group for hydroxyl group for nucleic acid synthesis”, “hydroxyl group protected with protecting group for nucleic acid synthesis”, “protected group for protecting nucleic acid synthesis” “Protecting group” of “phosphate group” or “mercapto group protected with a protecting group for nucleic acid synthesis” is capable of stably protecting an amino group, a hydroxyl group, a phosphate group or a mercapto group during nucleic acid synthesis. If it is, it will not be restrict | limited in particular. Specifically, it refers to a protecting group that is stable under acidic or neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis. Examples of such protecting groups include lower alkyl groups, lower alkenyl groups, acyl groups, tetrahydropyranyl or tetrahydrothiopyranyl groups, tetrahydrofuranyl or tetrahydrothiofuranyl groups, silyl groups, lower alkoxymethyl groups, lower alkoxy groups. Lower alkoxymethyl group, halogeno lower alkoxymethyl group, lower alkoxylated ethyl group, ethyl halide group, methyl group substituted with 1 to 3 aryl groups, “lower alkyl group, lower alkoxy group, halogen atom or cyano” A methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a group ”, a lower alkoxycarbonyl group, an“ aryl group substituted with a halogen atom, a lower alkoxy group or a nitro group ”, a“ halogen atom or Substituted with a tri-lower alkylsilyl group Grade alkoxycarbonyl group ", alkenyloxycarbonyl groups, such as" lower alkoxy or aralkyloxy carbonyl group optionally aryl ring is substituted by nitro group "can be mentioned.
より具体的には、テトラヒドロピラニル基またはテトラヒドロチオピラニル基としては、テトラヒドロピラン－２－イル基、３－ブロモテトラヒドロピラン－２－イル基、４－メトキシテトラヒドロピラン－４－イル基、テトラヒドロチオピラン－４－イル基、４－メトキシテトラヒドロチオピラン－４－イル基などが挙げられる。テトラヒドロフラニル基またはテトラヒドロチオフラニル基としては、テトラヒドロフラン－２－イル基、テトラヒドロチオフラン－２－イル基が挙げられる。低級アルコキシメチル基としては、メトキシメチル基、１，１－ジメチル－１－メトキシメチル基、エトキシメチル基、プロポキシメチル基、イソプロポキシメチル基、ブトキシメチル基、ｔ－ブトキシメチル基などが挙げられる。低級アルコキシ化低級アルコキシメチル基としては、２－メトキシエトキシメチル基などが挙げられる。ハロゲノ低級アルコキシメチル基としては、２，２，２－トリクロロエトキシメチル基、ビス（２－クロロエトキシ）メチル基などが挙げられる。低級アルコキシ化エチル基としては、１－エトキシエチル基、１－（イソプロポキシ）エチル基などが挙げられる。ハロゲン化エチル基としては、２，２，２－トリクロロエチル基などが挙げられる。１～３個のアリール基で置換されたメチル基としては、ベンジル基、α－ナフチルメチル基、β－ナフチルメチル基、ジフェニルメチル基、トリフェニルメチル基、α－ナフチルジフェニルメチル基、９－アンスリルメチル基などが挙げられる。「低級アルキル基、低級アルコキシ基、ハロゲン原子またはシアノ基でアリール環が置換された１～３個のアリール基で置換されたメチル基」としては、４－メチルベンジル基、２，４，６－トリメチルベンジル基、３，４，５－トリメチルベンジル基、４－メトキシベンジル基、４－メトキシフェニルジフェニルメチル基、４，４’－ジメトキシトリフェニルメチル基、２－ニトロベンジル基、４－ニトロベンジル基、４－クロロベンジル基、４－ブロモベンジル基、４－シアノベンジル基などが挙げられる。低級アルコキシカルボニル基としては、メトキシカルボニル基、エトキシカルボニル基、ｔ－ブトキシカルボニル基、イソブトキシカルボニル基などが挙げられる。「ハロゲン原子、低級アルコキシ基またはニトロ基で置換されたアリール基」としては、４－クロロフェニル基、２－フロロフェニル基、４－メトキシフェニル基、４－ニトロフェニル基、２，４－ジニトロフェニル基などが挙げられる。「ハロゲン原子またはトリ低級アルキルシリル基で置換された低級アルコキシカルボニル基」としては、２，２，２－トリクロロエトキシカルボニル基、２－トリメチルシリルエトキシカルボニル基などが挙げられる。アルケニルオキシカルボニル基としては、ビニルオキシカルボニル基、アリールオキシカルボニル基などが挙げられる。「低級アルコキシまたはニトロ基でアリール環が置換されていてもよいアラルキルオキシカルボニル基」としては、ベンジルオキシカルボニル基、４－メトキシベンジルオキシカルボニル基、３，４－ジメトキシベンジルオキシカルボニル基、２－ニトロベンジルオキシカルボニル基、４－ニトロベンジルオキシカルボニル基などが挙げられる。
More specifically, examples of the tetrahydropyranyl group or tetrahydrothiopyranyl group include a tetrahydropyran-2-yl group, a 3-bromotetrahydropyran-2-yl group, a 4-methoxytetrahydropyran-4-yl group, a tetrahydro Examples include a thiopyran-4-yl group and a 4-methoxytetrahydrothiopyran-4-yl group. Examples of the tetrahydrofuranyl group or the tetrahydrothiofuranyl group include a tetrahydrofuran-2-yl group and a tetrahydrothiofuran-2-yl group. Examples of the lower alkoxymethyl group include a methoxymethyl group, a 1,1-dimethyl-1-methoxymethyl group, an ethoxymethyl group, a propoxymethyl group, an isopropoxymethyl group, a butoxymethyl group, and a t-butoxymethyl group. Examples of the lower alkoxylated lower alkoxymethyl group include 2-methoxyethoxymethyl group. Examples of the halogeno lower alkoxymethyl group include 2,2,2-trichloroethoxymethyl group and bis (2-chloroethoxy) methyl group. Examples of the lower alkoxylated ethyl group include 1-ethoxyethyl group and 1- (isopropoxy) ethyl group. Examples of the halogenated ethyl group include 2,2,2-trichloroethyl group. Examples of the methyl group substituted with 1 to 3 aryl groups include benzyl group, α-naphthylmethyl group, β-naphthylmethyl group, diphenylmethyl group, triphenylmethyl group, α-naphthyldiphenylmethyl group, 9-anne. Examples include a thrylmethyl group. Examples of the “methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkyl group, lower alkoxy group, halogen atom or cyano group” include 4-methylbenzyl group, 2,4,6- Trimethylbenzyl group, 3,4,5-trimethylbenzyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, 4,4'-dimethoxytriphenylmethyl group, 2-nitrobenzyl group, 4-nitrobenzyl group 4-chlorobenzyl group, 4-bromobenzyl group, 4-cyanobenzyl group and the like. Examples of the lower alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl group, and an isobutoxycarbonyl group. Examples of the “aryl group substituted with a halogen atom, lower alkoxy group or nitro group” include 4-chlorophenyl group, 2-fluorophenyl group, 4-methoxyphenyl group, 4-nitrophenyl group, 2,4-dinitrophenyl group Etc. Examples of the “lower alkoxycarbonyl group substituted with a halogen atom or tri-lower alkylsilyl group” include 2,2,2-trichloroethoxycarbonyl group, 2-trimethylsilylethoxycarbonyl group and the like. Examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group and an aryloxycarbonyl group. Examples of the “aralkyloxycarbonyl group whose aryl ring may be substituted with a lower alkoxy or nitro group” include benzyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 3,4-dimethoxybenzyloxycarbonyl group, 2-nitro Examples include benzyloxycarbonyl group, 4-nitrobenzyloxycarbonyl group and the like.
「核酸合成の水酸基の保護基」としては、好適には、脂肪族アシル基、芳香族アシル基、１～３個のアリール基で置換されたメチル基、「低級アルキル、低級アルコキシ、ハロゲン、シアノ基でアリール環が置換された１～３個のアリール基で置換されたメチル基」、またはシリル基であり、さらに好適には、アセチル基、ベンゾイル基、ベンジル基、ｐ－メトキシベンゾイル基、ジメトキシトリチル基、モノメトキシトリチル基またはｔｅｒｔ－ブチルジフェニルシリル基である。「核酸合成の保護基で保護された水酸基」の保護基としては、好適には、脂肪族アシル基、芳香族アシル基、「１～３個のアリール基で置換されたメチル基」、「ハロゲン原子、低級アルコキシ基またはニトロ基で置換されたアリール基」、低級アルキル基、または低級アルケニル基であり、さらに好適には、ベンゾイル基、ベンジル基、２－クロロフェニル基、４－クロロフェニル基または２－プロペニル基である。「核酸合成のアミノ基の保護基」としては、好適には、アシル基であり、さらに好適には、ベンゾイル基である。「核酸合成の保護基で保護されたリン酸基」の「保護基」としては、好適には、低級アルキル基、シアノ基で置換された低級アルキル基、アラルキル基、「ニトロ基またはハロゲン原子でアリール環が置換されたアラルキル基」または「低級アルキル基、ハロゲン原子、またはニトロ基で置換されたアリール基」であり、さらに好適には、２－シアノエチル基、２，２，２－トリクロロエチル基、ベンジル基、２－クロロフェニル基または４－クロロフェニル基である。「核酸合成の保護基で保護されたリン酸基」を構成する保護基は１つまたはそれ以上であり得る。「核酸合成の保護基で保護されたメルカプト基」の「保護基」としては、好適には、脂肪族アシル基または芳香族アシル基であり、さらに好適には、ベンゾイル基である。
The “hydroxyl-protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, “lower alkyl, lower alkoxy, halogen, cyano” A methyl group substituted with 1 to 3 aryl groups substituted with an aryl ring by a group ”, or a silyl group, and more preferably an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzoyl group, a dimethoxy group A trityl group, a monomethoxytrityl group or a tert-butyldiphenylsilyl group; Preferred examples of the protecting group for the “hydroxyl group protected with a protecting group for nucleic acid synthesis” include aliphatic acyl groups, aromatic acyl groups, “methyl groups substituted with 1 to 3 aryl groups”, “halogen” An aryl group substituted with an atom, a lower alkoxy group or a nitro group ”, a lower alkyl group, or a lower alkenyl group, and more preferably a benzoyl group, a benzyl group, a 2-chlorophenyl group, a 4-chlorophenyl group, or 2- Propenyl group. The “protecting group for the amino group for nucleic acid synthesis” is preferably an acyl group, more preferably a benzoyl group. The “protecting group” of the “phosphate group protected with a protecting group for nucleic acid synthesis” is preferably a lower alkyl group, a lower alkyl group substituted with a cyano group, an aralkyl group, a “nitro group or a halogen atom”. An aralkyl group substituted with an aryl ring "or an" aryl group substituted with a lower alkyl group, a halogen atom or a nitro group ", more preferably a 2-cyanoethyl group, a 2,2,2-trichloroethyl group Benzyl group, 2-chlorophenyl group or 4-chlorophenyl group. The protecting group constituting the “phosphate group protected with a protecting group for nucleic acid synthesis” may be one or more. The “protecting group” of the “mercapto group protected with a protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group or an aromatic acyl group, and more preferably a benzoyl group.
本明細書において、－Ｐ（Ｒ４）Ｒ５［式中、Ｒ４およびＲ５は、それぞれ独立して、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］で表される基のうち、Ｒ４がＯＲ４ａそしてＲ５がＮＲ５ａとして表すことができる基は、「ホスホロアミダイト基」という。ホスホロアミダイト基としては、好適には、式－Ｐ（ＯＣ２Ｈ４ＣＮ）（Ｎ（ｉＰｒ）２）で表される基、または式－Ｐ（ＯＣＨ３）（Ｎ（ｉＰｒ）２）で表される基が挙げられる。ここで、ｉＰｒはイソプロピル基を表す。
In the present specification, —P (R 4 ) R 5 wherein R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a protecting group for nucleic acid synthesis. Protected with a mercapto group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or carbon Among the groups represented by the formula (1) represents a linear or branched alkylamino group of 1 to 6, the group that R 4 can represent as OR 4a and R 5 as NR 5a can be referred to as a “phosphoramidite group”. . The phosphoramidite group is preferably a group represented by the formula —P (OC 2 H 4 CN) (N (iPr) 2 ) or a formula —P (OCH 3 ) (N (iPr) 2 ). And the group represented. Here, iPr represents an isopropyl group.
本明細書において、用語「ヌクレオシド」および「ヌクレオシド類縁体」とは、プリンまたはピリミジン塩基と糖とが結合した「ヌクレオシド」のうち非天然型のもの、ならびに、プリンおよびピリミジン以外の芳香族複素環および芳香族炭化水素環でプリンまたはピリミジン塩基との代用が可能なものと糖が結合したものをいう。
In the present specification, the terms “nucleoside” and “nucleoside analog” mean an unnatural type of “nucleoside” in which a purine or pyrimidine base and a sugar are bonded, and an aromatic heterocycle other than purine and pyrimidine. And an aromatic hydrocarbon ring capable of substituting with a purine or pyrimidine base and having a sugar bound thereto.
本明細書において、用語「人工オリゴヌクレオチド」および「オリゴヌクレオチド類縁体」とは、同一または異なる「ヌクレオシド」または「ヌクレオシド類縁体」がリン酸ジエステル結合で２～５０個結合した「オリゴヌクレオチド」の非天然型誘導体をいう。そのような類縁体としては、好適には、糖部分が修飾された糖誘導体；リン酸ジエステル部分がチオエート化されたチオエート誘導体；末端のリン酸部分がエステル化されたエステル体；プリン塩基上のアミノ基がアミド化されたアミド体が挙げられ、さらに好適には、糖部分が修飾された糖誘導体が挙げられる。
As used herein, the terms “artificial oligonucleotide” and “oligonucleotide analog” refer to an “oligonucleotide” in which 2 to 50 identical or different “nucleosides” or “nucleoside analogs” are linked by phosphodiester bonds. A non-natural derivative. Such analogs preferably include a sugar derivative having a modified sugar moiety; a thioate derivative in which the phosphodiester moiety is thioated; an ester form in which the terminal phosphate moiety is esterified; Examples include amides in which the amino group is amidated, and more preferable examples include sugar derivatives in which the sugar moiety is modified.
本明細書において、用語「その塩」とは、本発明の式（Ｉ）または（Ｉ’）で表される化合物の塩をいう。そのような塩としては、例えば、ナトリウム塩、カリウム塩、リチウム塩のようなアルカリ金属塩、カルシウム塩、マグネシウム塩のようなアルカリ土類金属塩、アルミニウム塩、鉄塩、亜鉛塩、銅塩、ニッケル塩、コバルト塩などの金属塩；アンモニウム塩のような無機塩、ｔ－オクチルアミン塩、ジベンジルアミン塩、モルホリン塩、グルコサミン塩、フェニルグリシンアルキルエステル塩、エチレンジアミン塩、Ｎ－メチルグルカミン塩、グアニジン塩、ジエチルアミン塩、トリエチルアミン塩、ジシクロヘキシルアミン塩、Ｎ，Ｎ’－ジベンジルエチレンジアミン塩、クロロプロカイン塩、プロカイン塩、ジエタノールアミン塩、Ｎ－ベンジル－フェネチルアミン塩、ピペラジン塩、テトラメチルアンモニウム塩、トリス（ヒドロキシメチル）アミノメタン塩のような有機塩等のアミン塩；フッ化水素酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩のようなハロゲン原子化水素酸塩、硝酸塩、過塩素酸塩、硫酸塩、リン酸塩等の無機酸塩；メタンスルホン酸塩、トリフルオロメタンスルホン酸塩、エタンスルホン酸塩のような低級アルカンスルホン酸塩、ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩のようなアリールスルホン酸塩、酢酸塩、リンゴ酸塩、フマル酸塩、コハク酸塩、クエン酸塩、酒石酸塩、シュウ酸塩、マレイン酸塩等の有機酸塩；および、グリシン塩、リジン塩、アルギニン塩、オルニチン塩、グルタミン酸塩、アスパラギン酸塩のようなアミノ酸塩が挙げられる。
In the present specification, the term “a salt thereof” refers to a salt of a compound represented by the formula (I) or (I ′) of the present invention. Examples of such salts include 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, 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 salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (Hydroxy Amine salts such as organic salts such as methyl) aminomethane; hydrohalogenated hydrohalates such as hydrofluorates, hydrochlorides, hydrobromides, hydroiodates, nitrates, perchloric acid Inorganic salts such as salts, sulfates and phosphates; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate Such as aryl sulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc .; and glycine salt, lysine salt, Examples thereof include amino acid salts such as arginine salt, ornithine salt, glutamate salt, and aspartate salt.
本明細書において、用語「その薬理学上許容される塩」としては、本発明の式（ＩＩ）または（ＩＩ’）で表されるヌクレオシド構造を少なくとも１つ含有するオリゴヌクレオチド類縁体の塩をいう。そのような塩としては、例えば、ナトリウム塩、カリウム塩、リチウム塩のようなアルカリ金属塩、カルシウム塩、マグネシウム塩のようなアルカリ土類金属塩、アルミニウム塩、鉄塩、亜鉛塩、銅塩、ニッケル塩、コバルト塩などの金属塩；アンモニウム塩のような無機塩、ｔ－オクチルアミン塩、ジベンジルアミン塩、モルホリン塩、グルコサミン塩、フェニルグリシンアルキルエステル塩、エチレンジアミン塩、Ｎ－メチルグルカミン塩、グアニジン塩、ジエチルアミン塩、トリエチルアミン塩、ジシクロヘキシルアミン塩、Ｎ，Ｎ’－ジベンジルエチレンジアミン塩、クロロプロカイン塩、プロカイン塩、ジエタノールアミン塩、Ｎ－ベンジル－フェネチルアミン塩、ピペラジン塩、テトラメチルアンモニウム塩、トリス（ヒドロキシメチル）アミノメタン塩のような有機塩等のアミン塩；フッ化水素酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩のようなハロゲン原子化水素酸塩、硝酸塩、過塩素酸塩、硫酸塩、リン酸塩等の無機酸塩；メタンスルホン酸塩、トリフルオロメタンスルホン酸塩、エタンスルホン酸塩のような低級アルカンスルホン酸塩、ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩のようなアリールスルホン酸塩、酢酸塩、リンゴ酸塩、フマル酸塩、コハク酸塩、クエン酸塩、酒石酸塩、シュウ酸塩、マレイン酸塩等の有機酸塩；および、グリシン塩、リジン塩、アルギニン塩、オルニチン塩、グルタミン酸塩、アスパラギン酸塩のようなアミノ酸塩が挙げられる。
In this specification, the term “pharmacologically acceptable salt thereof” refers to a salt of an oligonucleotide analog containing at least one nucleoside structure represented by the formula (II) or (II ′) of the present invention. Say. Examples of such salts include 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, 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 salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (Hydroxy Amine salts such as organic salts such as methyl) aminomethane; hydrohalogenated hydrohalates such as hydrofluorates, hydrochlorides, hydrobromides, hydroiodates, nitrates, perchloric acid Inorganic salts such as salts, sulfates and phosphates; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate Such as aryl sulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc .; and glycine salt, lysine salt, Examples thereof include amino acid salts such as arginine salt, ornithine salt, glutamate salt, and aspartate salt.
以下、本発明について詳述する。
Hereinafter, the present invention will be described in detail.
本発明の化合物はまたはその塩は、２’，４’－架橋型ヌクレオシドおよびヌクレオチドまたはその塩である。本発明の化合物またはその塩は、以下の式（Ｉ）または（Ｉ’）：
The compound of the present invention or a salt thereof is 2 ', 4'-bridged nucleoside and nucleotide or a salt thereof. The compound of the present invention or a salt thereof has the following formula (I) or (I ′):
（式中、
「Ｂａｓｅ」は、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ２およびＲ３は、それぞれ独立して、水素原子、核酸合成の水酸基の保護基、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数２から７のアルケニル基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール部分を有するアラルキル基、該α群から選択される任意の置換基を１以上有していてもよいアシル基、該α群から選択される任意の置換基を１以上有していてもよいシリル基、該α群から選択される任意の置換基を１以上有していてもよいリン酸基、核酸合成の保護基で保護されたリン酸基、－Ｐ（Ｒ４）Ｒ５［式中、Ｒ４およびＲ５は、それぞれ独立して、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）で表される構造を有する。 (Where
“Base” represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group optionally having one or more arbitrary substituents selected from α group, The α group is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A mercapto group protected with 1, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom ;
R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring An alkenyl group having 2 to 7 carbon atoms, an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and may contain a hetero atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms, which may have one or more arbitrary substituents selected from the α group and may contain a hetero atom, any arbitrary selected from the α group An acyl group optionally having one or more substituents, a silyl group optionally having one or more arbitrary substituents selected from the α group, and an arbitrary substituent selected from the α group are 1 Protection of phosphate groups and nucleic acid synthesis that may have In protected phosphate group, -P (R 4) R 5 [ wherein, R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, nucleic acid synthesis A mercapto group protected with a protecting group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms Or a linear or branched alkylamino group having 1 to 6 carbon atoms]
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom).
「Ｂａｓｅ」は、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ２およびＲ３は、それぞれ独立して、水素原子、核酸合成の水酸基の保護基、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数２から７のアルケニル基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール部分を有するアラルキル基、該α群から選択される任意の置換基を１以上有していてもよいアシル基、該α群から選択される任意の置換基を１以上有していてもよいシリル基、該α群から選択される任意の置換基を１以上有していてもよいリン酸基、核酸合成の保護基で保護されたリン酸基、－Ｐ（Ｒ４）Ｒ５［式中、Ｒ４およびＲ５は、それぞれ独立して、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）で表される構造を有する。 (Where
“Base” represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group optionally having one or more arbitrary substituents selected from α group, The α group is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A mercapto group protected with 1, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protecting group for nucleic acid synthesis, and a halogen atom ;
R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring An alkenyl group having 2 to 7 carbon atoms, an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and may contain a hetero atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms, which may have one or more arbitrary substituents selected from the α group and may contain a hetero atom, any arbitrary selected from the α group An acyl group optionally having one or more substituents, a silyl group optionally having one or more arbitrary substituents selected from the α group, and an arbitrary substituent selected from the α group are 1 Protection of phosphate groups and nucleic acid synthesis that may have In protected phosphate group, -P (R 4) R 5 [ wherein, R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, nucleic acid synthesis A mercapto group protected with a protecting group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms Or a linear or branched alkylamino group having 1 to 6 carbon atoms]
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group, or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom).
上記式（Ｉ）または（Ｉ’）において、「Ｂａｓｅ」は、プリン塩基（すなわち、プリン－９－イル基）またはピリミジン塩基（すなわち、２－オキソ－１，２－ジヒドロピリミジン－１－イル基）である。これらの塩基は、水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、およびハロゲン原子からなるα群より選択される任意の置換基を１以上有していてもよい。
In the above formula (I) or (I ′), “Base” is a purine base (ie, purin-9-yl group) or a pyrimidine base (ie, 2-oxo-1,2-dihydropyrimidin-1-yl group) ). These bases are a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and 1 to carbon atoms. It may have one or more arbitrary substituents selected from the α group consisting of 6 linear alkylamino groups and halogen atoms.
上記「Ｂａｓｅ」の具体例としては、６－アミノプリン－９－イル基（アデニニル基）、２，６－ジアミノプリン－９－イル基、２－アミノ－６－クロロプリン－９－イル基、２－アミノ－６－フルオロプリン－９－イル基、２－アミノ－６－ブロモプリン－９－イル基、２－アミノ－６－ヒドロキシプリン－９－イル基（グアニニル基）、６－アミノ－２－メトキシプリン－９－イル基、６－アミノ－２－クロロプリン－９－イル基、６－アミノ－２－フルオロプリン－９－イル基、２，６－ジメトキシプリン－９－イル基、２，６－ジクロロプリン－９－イル基、６－メルカプトプリン－９－イル基、２－オキソ－４－アミノ－１，２－ジヒドロピリミジン－１－イル基（シトシニル基）、４－アミノ－２－オキソ－５－フルオロ－１，２－ジヒドロピリミジン－１－イル基、４－アミノ－２－オキソ－５－クロロ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－メトキシ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－メルカプト－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－ヒドロキシ－１，２－ジヒドロピリミジン－１－イル基（ウラシリル基）、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基（チミニル基）、および４－アミノ－５－メチル－２－オキソ－１，２－ジヒドロピリミジン－１－イル基が挙げられる。
Specific examples of the above “Base” include 6-aminopurin-9-yl group (adenylyl group), 2,6-diaminopurine-9-yl group, 2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group (guaninyl group), 6-amino- 2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (cytosynyl group), 4-amino- 2-oxo-5-fluoro-1 2-dihydropyrimidin-1-yl group, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-methoxy-1,2-dihydropyrimidine-1 -Yl group, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl group, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group (urasilyl group), 2- Oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group) and 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group Is mentioned.
中でも、「Ｂａｓｅ」は、核酸医薬への導入という観点から、以下の構造式：
Among them, “Base” has the following structural formula from the viewpoint of introduction into nucleic acid medicine:
でそれぞれ表される、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基（チミニル基）、２－オキソ－４－アミノ－１，２－ジヒドロピリミジン－１－イル基（シトシニル基）、６－アミノプリン－９－イル基（アデニニル基）、２－アミノ－６－ヒドロキシプリン－９－イル基（グアニニル基）、４－アミノ－５－メチル－２－オキソ－１，２－ジヒドロピリミジン－１－イル基、および２－オキソ－４－ヒドロキシ－１，２－ジヒドロピリミジン－１－イル基（ウラシリル基）が好適であり、特に、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基（チミニル基）が好適である。また、オリゴヌクレオチドの合成の際には、水酸基およびアミノ基が保護基により保護されていることが好ましい。
2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group), 2-oxo-4-amino-1,2-dihydropyrimidine-1 -Yl group (cytosynyl group), 6-aminopurin-9-yl group (adenylyl group), 2-amino-6-hydroxypurin-9-yl group (guaninyl group), 4-amino-5-methyl-2- The oxo-1,2-dihydropyrimidin-1-yl group and the 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group (urasilyl group) are preferred, and in particular, the 2-oxo-4 A -hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group) is preferred. Further, in the synthesis of the oligonucleotide, it is preferable that the hydroxyl group and amino group are protected by a protecting group.
本発明の２’，４’－架橋型ヌクレオシドは、従来の２’，４’－の架橋構造の６’位に、酸素原子または硫黄原子のようなヘテロ原子が導入されていることにより、後述するオリゴヌクレオチドにおける酵素耐性能が向上する。また、このような２’，４’－の架橋構造内のヘテロ原子は、糖部のコンホメーションに直接的に影響を及ぼすものである。このため、本発明の２’，４’－架橋型ヌクレオシドは、得られたオリゴヌクレオチドに対し、当該影響による一本鎖ＲＮＡ（ｓｓＲＮＡ）との結合親和性をも一層向上させ得る。
The 2 ′, 4′-bridged nucleoside of the present invention has a hetero atom such as an oxygen atom or a sulfur atom introduced into the 6 ′ position of the conventional 2 ′, 4′-bridge structure, which will be described later. Enzyme resistance performance of the oligonucleotide to be improved. In addition, such a heteroatom in the 2 ', 4'-crosslinked structure directly affects the conformation of the sugar moiety. For this reason, the 2 ', 4'-bridged nucleoside of the present invention can further improve the binding affinity of the resulting oligonucleotide to single-stranded RNA (ssRNA) due to the influence.
さらに、本発明の２’，４’－架橋型ヌクレオシドは、その合成について、６’位へのヘテロ原子の導入を４’－エキソオレフィン体を利用することにより単工程で実現可能であり、さらに、ラジカル環化反応を利用することによって、６’位にヘテロ原子を有する架橋構造を極めて少ない工程を経て製造することができる。このため、本発明の２’，４’－架橋型ヌクレオシドは、従来より当該架橋構造内にヘテロ原子を導入したＥＮＡやＥｏＮＡと比較して、より少ない工程を通じて一層効率的に合成することができる。
Furthermore, the 2 ′, 4′-bridged nucleoside of the present invention can be synthesized in a single step by introducing a heteroatom at the 6 ′ position by using a 4′-exoolefin compound. By utilizing a radical cyclization reaction, a crosslinked structure having a hetero atom at the 6′-position can be produced through very few steps. Therefore, the 2 ′, 4′-bridged nucleoside of the present invention can be synthesized more efficiently through fewer steps than conventional ENA or EoNA in which a hetero atom is introduced into the bridge structure. .
これにより、本発明の２’，４’－架橋型ヌクレオシドとして、例えば、以下の（ｉ）～（ｉｉｉ）：
Thereby, as the 2 ', 4'-bridged nucleoside of the present invention, for example, the following (i) to (iii):
のようなエキソオレフィン体（ｉ）、ならびに立体化学が制御されたＲ体（ｉｉ）およびＳ体（ｉｉｉ）をそれぞれ別々に合成することができる
Isomers (i) and R-form (ii) and S-form (iii) with controlled stereochemistry can be synthesized separately.
本発明において、オリゴヌクレオチド（２’，４’－架橋型人工ヌクレオチド）は、このような２’，４’－架橋型ヌクレオシドを用いて調製することができる。例えば、三リン酸化は、非特許文献５に記載の方法に従って容易に行われ得る。
In the present invention, oligonucleotides (2 ', 4'-bridged artificial nucleotides) can be prepared using such 2', 4'-bridged nucleosides. For example, triphosphorylation can be easily performed according to the method described in Non-Patent Document 5.
本発明のオリゴヌクレオチドまたはその薬理学上許容される塩は、以下の式（ＩＩ）または（ＩＩ’）で表されるヌクレオシド構造を少なくとも１つ含む：
The oligonucleotide of the present invention or a pharmacologically acceptable salt thereof includes at least one nucleoside structure represented by the following formula (II) or (II ′):
（式中、「Ｂａｓｅ」、Ｒ６、Ｒ７、Ｒ８およびＲ９は、上記式（Ｉ）または（Ｉ’）で定義されるものと同様である）。
(In the formula, “Base”, R 6 , R 7 , R 8 and R 9 are the same as defined in the above formula (I) or (I ′)).
本発明のオリゴヌクレオチドは、上記ヌクレオシド構造を、任意の位置に少なくとも１つ有する。その位置および数は、特に限定されず、目的に応じて適宜設計され得る。
The oligonucleotide of the present invention has at least one nucleoside structure at any position. The position and number are not particularly limited, and can be appropriately designed according to the purpose.
このようなヌクレオシド構造を含むオリゴヌクレオチド類縁体（アンチセンス分子）は、例えば、従来のＥｏＮＡを用いる場合と比較して、酵素耐性能が飛躍的に向上する。また、当該ＥｏＮＡを上回る一本鎖ＲＮＡ（ｓｓＲＮＡ）結合親和性を有する。
Oligonucleotide analogs (antisense molecules) containing such a nucleoside structure have a dramatic improvement in enzyme resistance compared to, for example, conventional EoNA. Moreover, it has a single-stranded RNA (ssRNA) binding affinity that exceeds the EoNA.
これらのことから、本発明の２’，４’－架橋型ヌクレオシドを用いて合成されたオリゴヌクレオチド類縁体は、抗腫瘍剤、抗ウイルス剤をはじめとした、特定の遺伝子の働きを阻害して疾病を治療する医薬品（アンチセンス分子）としての有用性が期待される。
From these facts, oligonucleotide analogues synthesized using the 2 ′, 4′-bridged nucleosides of the present invention inhibit the action of specific genes including antitumor agents and antiviral agents. Expected to be useful as a drug (antisense molecule) for treating diseases.
特に、アンチセンス法では、相補センス鎖ＲＮＡに対する結合親和性および生体内ＤＮＡ分解酵素への耐性の両方が必要とされる。一般的に、核酸は、一本鎖状態では、糖部の構造が絶えずＤＮＡ二重鎖に近い形と、ＤＮＡ－ＲＮＡ二重鎖やＲＮＡ二重鎖に近い形との間で揺らいでいることが知られている。一本鎖核酸が相補的なＲＮＡ鎖と二重鎖を形成する場合、その糖部構造は固定される。そこで、本発明の２’，４’－架橋型ヌクレオシドでは、糖部を予め二重鎖を形成する場合の状態に固定されているため、目的のＲＮＡ鎖と二重鎖を形成しやすく、安定に存在させることができる。また、核酸二重鎖は、水分子のネットワークと呼ばれる鎖のようにつながった水和水により安定化されていることも知られている。
In particular, in the antisense method, both binding affinity to complementary sense strand RNA and resistance to in vivo DNA-degrading enzyme are required. In general, in single-stranded nucleic acids, the structure of the sugar part is constantly fluctuating between a shape close to a DNA duplex and a shape close to a DNA-RNA duplex or an RNA duplex. It has been known. When a single-stranded nucleic acid forms a double strand with a complementary RNA strand, its sugar structure is fixed. Therefore, in the 2 ′, 4′-bridged nucleoside of the present invention, the sugar portion is fixed in the state in which a double strand is formed in advance, so that it is easy to form a double strand with a target RNA strand and is stable. Can be present. It is also known that nucleic acid duplexes are stabilized by hydrated water connected like a chain of water molecules.
本発明のオリゴヌクレオチド類縁体は、例えば、賦形剤、結合剤、防腐剤、酸化安定剤、崩壊剤、滑沢剤、矯味剤などの医薬の製剤技術分野において通常用いられる補助剤を配合して、非経口投与製剤またはリポソーム製剤とすることができる。また、例えば、当該技術分野で通常用いられる医薬用担体を配合して、液剤、クリーム剤、軟膏剤などの局所用の製剤を調製することができる。
The oligonucleotide analog of the present invention contains, for example, adjuvants commonly used in the pharmaceutical formulation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and flavoring agents. Thus, it can be a parenteral preparation or a liposome preparation. Also, for example, a topical preparation such as a solution, cream, ointment, etc. can be prepared by blending a pharmaceutical carrier usually used in the art.
以下、実施例により本発明をより具体的に説明するが、本発明はこれらの実施例により限定されるものではない。
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
（実施例１）２’，４’－架橋型ヌクレオシド（エキソメチレン体）の合成
Example 1 Synthesis of 2 ', 4'-Bridged Nucleoside (Exomethylene Form)
（実施例１．１）２’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－４’，５’－デヒドロ－５’－デオキシ－５－メチルウリジン（２ａ）および３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－４’，５’－デヒドロ－５’－デオキシ－５－メチルウリジン（２ｂ）の合成
Example 1.1 2′-O-tert-butyldimethylsilyl-4 ′, 5′-dehydro-5′-deoxy-5-methyluridine (2a) and 3′-O-tert-butyldimethylsilyl- Synthesis of 4 ′, 5′-dehydro-5′-deoxy-5-methyluridine (2b)
窒素気流下にて、非特許文献８に記載の方法により５－メチルウリジンから合成した化合物１（９．３０ｇ，３８．７ｍｍｏｌ）の無水ジメチルホルムアミド溶液（１００ｍＬ）に、０℃にて１，４－ジアザビシクロ［２．２．２］オクタン （２１．７ｇ，１９４ｍｍｏｌ）、ｔｅｒｔ－ブチルジメチルクロロシラン（７．００ｇ，４６．５ｍｍｏｌ）、硝酸銀（７．９０ｇ，４６．５ｍｍｏｌ）を添加し、室温で１２時間撹拌した。反応終了後、反応液をセライトで濾過した。ろ液に水を添加し、ジエチルエーテルで抽出した。有機層を飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（１４．８ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：１から３：２）で精製し、標題の化合物２ａ（５．９０ｇ，４３％）および化合物２ｂ（５．３５ｇ，３９％）をそれぞれ白色泡状固体として得た。
Under a nitrogen stream, an anhydrous dimethylformamide solution (100 mL) of compound 1 (9.30 g, 38.7 mmol) synthesized from 5-methyluridine by the method described in Non-Patent Document 8 was added at 0 ° C. to 1,4 Add diazabicyclo [2.2.2] octane (21.7 g, 194 mmol), tert-butyldimethylchlorosilane (7.00 g, 46.5 mmol), silver nitrate (7.90 g, 46.5 mmol) and add 12 at room temperature. Stir for hours. After completion of the reaction, the reaction solution was filtered through celite. Water was added to the filtrate and extracted with diethyl ether. The organic layer was washed once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (14.8 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 1 to 3: 2) to give the title compound 2a (5.90 g, 43%) and compound 2b (5.35 g, 39). %) As white foamy solids.
次いで、得られた化合物２ａ（５．９０ｇ，１６．６ｍｍｏｌ）の無水テトラヒドロフラン溶液（１００ｍＬ）に、窒素気流下にて、０℃で水素化ナトリウム（７９９ｍｇ，２０．０ｍｍｏｌ）を添加し、室温で１時間撹拌した。反応終了後、０℃にて反応液に塩化アンモニウム水溶液を添加し、酢酸エチルで抽出した。有機層を水で１回、かつ飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（５．６６ｇ）を得た。粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：１から３：２）で精製し、標題の化合物２ｂ（３．１９ｇ，５４％）を白色泡状固体として得た。
Next, sodium hydride (799 mg, 20.0 mmol) was added to an anhydrous tetrahydrofuran solution (100 mL) of the obtained compound 2a (5.90 g, 16.6 mmol) at 0 ° C. in a nitrogen stream at room temperature. Stir for 1 hour. After completion of the reaction, an aqueous ammonium chloride solution was added to the reaction solution at 0 ° C., and the mixture was extracted with ethyl acetate. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (5.66 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 1 to 3: 2) to give the title compound 2b (3.19 g, 54%) as a white foamy solid.
得られた化合物２ａおよび２ｂの物性データをそれぞれ表１および表２に示す。
The physical property data of the obtained compounds 2a and 2b are shown in Table 1 and Table 2, respectively.
（実施例１．２）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－４’－（プロプ－２－イン－１－イルオキシ）－５－メチルウリジン（３）の合成
Example 1.2 Synthesis of 3'-O-tert-butyldimethylsilyl-4 '-(prop-2-yn-1-yloxy) -5-methyluridine (3)
窒素気流下、上記で得られた化合物２ｂ（２．０８ｇ，５．８６ｍｍｏｌ）の２－プロピン－１－オール溶液（５０ｍＬ）に、０℃で硫酸マグネシウム処理したｍ－クロロ過安息香酸（７５ｗ／ｗ％，１．２２ｇ，７．０３ｍｍｏｌ）を添加し、室温で３時間撹拌した。反応終了後、反応液を飽和重曹水で中和し、水を添加して酢酸エチルで抽出した。有機層を飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（３．０９ｇ）を得た。粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン/酢酸エチル＝２：１から２：３）で精製し、標題の化合物３（１．００ｇ，４０％）を白色泡状固体として得た。
Under a nitrogen stream, m-chloroperbenzoic acid (75 w / min) treated with magnesium sulfate at 0 ° C. was added to a 2-propyn-1-ol solution (50 mL) of the compound 2b (2.08 g, 5.86 mmol) obtained above. w%, 1.22 g, 7.03 mmol) was added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction mixture was neutralized with saturated aqueous sodium hydrogen carbonate, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (3.09 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 2: 3) to give the title compound 3 (1.00 g, 40%) as a white foamy solid.
得られた化合物３の物性データを表３に示す。
The physical property data of the obtained compound 3 are shown in Table 3.
（実施例１．３）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－４’－（プロプ－２－イン－１－イルオキシ）－５－メチルウリジン（４）の合成
Example 1.3 3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -4 ′-(prop-2-yn-1-yloxy) -5 Synthesis of methyluridine (4)
窒素気流下、上記で得られた化合物３（１．００ｇ，２．３４ｍｍｏｌ）の無水ジクロロメタン溶液（１７．５ｍＬ）に、２，６－ルチジン（０．５４ｍＬ，４．６８ｍｍｏｌ）を添加した後、０℃で４，４’－ジメトキシトリチルトリフルオロメタンスルホニルの無水ジクロロメタン溶液（１Ｍ，２．６ｍＬ，２．６ｍｍｏｌ）を滴下し、室温で１時間撹拌した。反応終了後、０℃にてメタノールを添加してクエンチし、ジクロロメタンで希釈した。有機層を水で１回、かつ飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（１．７７ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から１：１）で精製し、標題の化合物４（１．３８ｇ，８１％）を白色泡状固体として得た。
Under a nitrogen stream, 2,6-lutidine (0.54 mL, 4.68 mmol) was added to an anhydrous dichloromethane solution (17.5 mL) of the compound 3 obtained above (1.00 g, 2.34 mmol), An anhydrous dichloromethane solution (1M, 2.6 mL, 2.6 mmol) of 4,4′-dimethoxytrityltrifluoromethanesulfonyl was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, it was quenched by adding methanol at 0 ° C. and diluted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (1.77 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 1: 1) to give the title compound 4 (1.38 g, 81%) as a white foamy solid.
得られた化合物４の物性データを表４に示す。
The physical property data of the obtained compound 4 are shown in Table 4.
（実施例１．４）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－Ｏ－（１－イミダゾリルチオカルボニル）－４’－（プロプ－２－イン－１－イルオキシ）－５－メチルウリジン（５）の合成
Example 1.4 3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -2′-O- (1-imidazolylthiocarbonyl) -4 ′-( Synthesis of prop-2-yn-1-yloxy) -5-methyluridine (5)
窒素気流下、上記で得られた化合物４（２．１２ｇ，２．９１ｍｍｏｌ）の無水テトラヒドロフラン溶液（１５ｍＬ）に、１，１’－チオカルボニルジイミダゾール（１．０４ｇ，５．８２ｍｍｏｌ）を添加し、反応液を３時間加熱還流した。反応終了後、溶媒を減圧留去して粗成績体（３．０９ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：２）で精製し、標題の化合物５（２．２０ｇ，９０％）を白色泡状固体として得た。
Under a nitrogen stream, 1,1′-thiocarbonyldiimidazole (1.04 g, 5.82 mmol) was added to an anhydrous tetrahydrofuran solution (15 mL) of the compound 4 (2.12 g, 2.91 mmol) obtained above. The reaction solution was heated to reflux for 3 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (3.09 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 2) to give the title compound 5 (2.20 g, 90%) as a white foamy solid.
得られた化合物５の物性データを表５に示す。
Table 5 shows the physical property data of the obtained compound 5.
（実施例１．５）１－［（１Ｒ，５Ｒ，６Ｒ，８Ｓ）－８－ｔｅｒｔ－ブチルジメチルシリルオキシ－１－（４，４’－ジメトキシトリチルオキシメチル）－４－メチレン－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（６）の合成
Example 1.5 1-[(1R, 5R, 6R, 8S) -8-tert-butyldimethylsilyloxy-1- (4,4′-dimethoxytrityloxymethyl) -4-methylene-2,7 -Synthesis of dioxabicyclo [3.2.1] octane-6-yl] -thymine (6)
窒素気流下、上記で得られた化合物５（２．２０ｇ，２．６２ｍｍｏｌ）の無水トルエン溶液（５０ｍＬ）に、９０℃にてトリストリメチルシリルシラン（２．４ｍＬ，７．８６ｍｍｏｌ）とアゾビスイソブチロニトリル（８６．１ｍｇ，０．５２４ｍｍｏｌ）とを添加し、同温度にて１時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（２．４１ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：１から３：２）で精製し、標題の化合物６（１．０７ｇ，５７％）を白色泡状固体として得た。
Under a nitrogen stream, tristrimethylsilylsilane (2.4 mL, 7.86 mmol) and azobisisobutyrate were added to an anhydrous toluene solution (50 mL) of the compound 5 (2.20 g, 2.62 mmol) obtained above at 90 ° C. Ronitrile (86.1 mg, 0.524 mmol) was added and stirred at the same temperature for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (2.41 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 1 to 3: 2) to give the title compound 6 (1.07 g, 57%) as a white foamy solid.
得られた化合物６の物性データを表６に示す。
Table 6 shows the physical property data of the obtained compound 6.
（実施例１．６）１－［（１Ｒ，５Ｒ，６Ｒ，８Ｓ）－１－（４，４’－ジメトキシトリチルオキシメチル）－８－ヒドロキシ－４－メチレン－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（７）の合成
Example 1.6 1-[(1R, 5R, 6R, 8S) -1- (4,4′-dimethoxytrityloxymethyl) -8-hydroxy-4-methylene-2,7-dioxabicyclo [ 3.2.1] Synthesis of Octane-6-yl] -thymine (7)
上記で得られた化合物６（３２１ｍｇ，０．４５０ｍｍｏｌ）のテトラヒドロフラン溶液（５．０ｍＬ）に室温でテトラブチルアンモニウムフルオリド（１Ｍテトラヒドロフラン溶液，０．５０ｍＬ，０．５０ｍｍｏｌ）を添加し、１５時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（３８８ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：２）で精製し、標題の化合物７（２３４ｍｇ，８７％）を白色泡状固体として得た。
Tetrabutylammonium fluoride (1M tetrahydrofuran solution, 0.50 mL, 0.50 mmol) was added to a tetrahydrofuran solution (5.0 mL) of the compound 6 (321 mg, 0.450 mmol) obtained above at room temperature and stirred for 15 hours. did. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (388 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 2) to give the title compound 7 (234 mg, 87%) as a white foamy solid.
得られた化合物７の物性データを表７に示す。
Table 7 shows the physical property data of the obtained compound 7.
（実施例１．７）１－［（１Ｒ，５Ｒ，６Ｒ，８Ｓ）－８－［２－シアノエトキシ（ジイソプロピルアミノ）ホスフィノキシ］－１－（４，４’－ジメトキシトリチルオキシメチル）－４－メチレン－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（８）（エキソメチレン体）の合成
Example 1.7 1-[(1R, 5R, 6R, 8S) -8- [2-Cyanoethoxy (diisopropylamino) phosphinoxy] -1- (4,4′-dimethoxytrityloxymethyl) -4- Synthesis of methylene-2,7-dioxabicyclo [3.2.1] octane-6-yl] -thymine (8) (exomethylene)
窒素気流下、上記で得られた化合物７（２３０ｍｇ，０．３９３ｍｍｏｌ）の無水ジクロロメタン溶液（３．０ｍＬ）にＮ，Ｎ－ジイソプロピルエチルアミン（０．２１ｍＬ，１．１８ｍｍｏｌ）を添加し、０℃にて２－シアノエチル－Ｎ，Ｎ－ジイソプロピルクロロホスホロアミジド（０．１１ｍＬ，０．４７２ｍｍｏｌ）を滴下し、室温で２時間撹拌した。反応終了後、０℃で反応液に飽和重曹水を添加してクエンチし、酢酸エチルで抽出した。有機層を水で１回、かつ飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（３１２ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：２）で精製し、標題の化合物８（２５６ｍｇ，７８％；エキソメチレン体）を白色泡状固体として得た。
Under a nitrogen stream, N, N-diisopropylethylamine (0.21 mL, 1.18 mmol) was added to an anhydrous dichloromethane solution (3.0 mL) of the compound 7 (230 mg, 0.393 mmol) obtained above, and the mixture was brought to 0 ° C. 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.11 mL, 0.472 mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium hydrogen carbonate at 0 ° C., and extracted with ethyl acetate. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (312 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 2) to give the title compound 8 (256 mg, 78%; exomethylene) as a white foamy solid. It was.
得られた化合物８の物性データを表８に示す。
The physical property data of the obtained compound 8 are shown in Table 8.
（実施例２）２’，４’－架橋型ヌクレオシド（（Ｒ）－メチル体）の合成
Example 2 Synthesis of 2 ', 4'-Bridged Nucleoside ((R) -Methyl Form)
（実施例２．１）１－［（１Ｒ，４Ｒ，５Ｒ，６Ｒ，８Ｓ）－８－ｔｅｒｔ－ブチルジメチルシリルオキシ－１－（４，４’－ジメトキシトリチルオキシメチル）－４－メチル－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（９）の合成
Example 2.1 1-[(1R, 4R, 5R, 6R, 8S) -8-tert-butyldimethylsilyloxy-1- (4,4′-dimethoxytrityloxymethyl) -4-methyl-2 , 7-dioxabicyclo [3.2.1] octane-6-yl] -thymine (9)
酸化白金（４１４ｍｇ，１．８２ｍｍｏｌ）のテトラヒドロフラン懸濁液（５．０ｍＬ）に、実施例１．５で得られた化合物６（１．３０ｇ，１．８２ｍｍｏｌ）のテトラヒドロフラン溶液（１０ｍＬ）を添加し、水素気流下で、室温にて２４時間撹拌した。反応終了後、酸化白金をセライトろ過によって取り除き、溶媒を減圧留去して粗成績体（１．３８ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から３：２）で精製し、標題の化合物９（１．１３ｇ，８７％）を白色泡状固体として得た。
To a tetrahydrofuran suspension (5.0 mL) of platinum oxide (414 mg, 1.82 mmol) was added a tetrahydrofuran solution (10 mL) of the compound 6 (1.30 g, 1.82 mmol) obtained in Example 1.5. The mixture was stirred at room temperature for 24 hours under a hydrogen stream. After completion of the reaction, platinum oxide was removed by celite filtration, and the solvent was distilled off under reduced pressure to obtain a crude product (1.38 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 3: 2) to give the title compound 9 (1.13 g, 87%) as a white foamy solid.
得られた化合物９の物性データを表９に示す。
The physical property data of the obtained compound 9 are shown in Table 9.
（実施例２．２）１－［（１Ｒ，４Ｒ，５Ｒ，６Ｒ，８Ｓ）－１－（４，４’－ジメトキシトリチルオキシメチル）－８－ヒドロキシ－４－メチル－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（１０）の合成
Example 2.2 1-[(1R, 4R, 5R, 6R, 8S) -1- (4,4′-dimethoxytrityloxymethyl) -8-hydroxy-4-methyl-2,7-dioxa Synthesis of bicyclo [3.2.1] octane-6-yl] -thymine (10)
上記で得られた化合物９（１．１３ｇ，１．５９ｍｍｏｌ）のテトラヒドロフラン溶液（１０ｍＬ）に、室温でテトラブチルアンモニウムフルオリド（１Ｍテトラヒドロフラン溶液，１．８ｍＬ，１．８０ｍｍｏｌ）を添加し、９時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（１．１８ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から２：３）で精製し、標題の化合物１０（９０７ｍｇ，９５％）を白色泡状固体として得た。
Tetrabutylammonium fluoride (1M tetrahydrofuran solution, 1.8 mL, 1.80 mmol) was added to a tetrahydrofuran solution (10 mL) of the compound 9 (1.13 g, 1.59 mmol) obtained above at room temperature for 9 hours. Stir. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (1.18 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 2: 3) to give the title compound 10 (907 mg, 95%) as a white foamy solid.
得られた化合物１０の物性データを表１０に示す。
Table 10 shows the physical property data of the obtained compound 10.
（実施例２．３）１－［（１Ｒ，４Ｒ，５Ｒ，６Ｒ，８Ｓ）－８－［２－シアノエトキシ（ジイソプロピルアミノ）ホスフィノキシ］－１－（４，４’－ジメトキシトリチルオキシメチル）－４－メチル－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（１１）（（Ｒ）－メチル体）の合成
Example 2.3 1-[(1R, 4R, 5R, 6R, 8S) -8- [2-Cyanoethoxy (diisopropylamino) phosphinoxy] -1- (4,4′-dimethoxytrityloxymethyl)- Synthesis of 4-methyl-2,7-dioxabicyclo [3.2.1] octane-6-yl] -thymine (11) ((R) -methyl)
窒素気流下、上記で得られた化合物１０（２３０ｍｇ，０．３８３ｍｍｏｌ）の無水ジクロロメタン溶液（３．０ｍＬ）に、Ｎ，Ｎ－ジイソプロピルエチルアミン（０．２１ｍＬ，１．１５ｍｍｏｌ）を添加し、０℃で２－シアノエチル－Ｎ，Ｎ－ジイソプロピルクロロホスホロアミジド（０．１０ｍＬ，０．４６０ｍｍｏｌ）を滴下し、室温で６時間撹拌した。反応終了後、０℃で反応液に飽和重曹水を添加してクエンチし、ジクロロメタンで抽出した。有機層を水で１回、かつ飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（３５８ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から２：３）で精製し、標題の化合物１１（１６６ｍｇ，５４％；（Ｒ）－メチル体）を白色泡状固体として得た。
Under a nitrogen stream, N, N-diisopropylethylamine (0.21 mL, 1.15 mmol) was added to an anhydrous dichloromethane solution (3.0 mL) of the compound 10 obtained above (230 mg, 0.383 mmol), and 0 ° C. Was added dropwise 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.10 mL, 0.460 mmol) and stirred at room temperature for 6 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (358 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 2: 3) to give the title compound 11 (166 mg, 54%; (R) -methyl compound) as a white foam Obtained as a solid.
得られた化合物１１の物性データを表１１に示す。
Table 11 shows the physical property data of the obtained compound 11.
（実施例３）２’，４’－架橋型ヌクレオシド（（Ｓ）－メチル体）の合成
Example 3 Synthesis of 2 ', 4'-Bridged Nucleoside ((S) -Methyl Form)
（実施例３．１）５’－Ｏ－（４，４’－ジメトキシトリチル）－４’－（プロプ－２－エン－１－イルオキシ）－３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５－メチルウリジン（１２）の合成
Example 3.1 5′-O- (4,4′-Dimethoxytrityl) -4 ′-(prop-2-en-1-yloxy) -3′-O-tert-butyldimethylsilyl-5 Synthesis of methyluridine (12)
リンドラー触媒（５５５ｍｇ）の酢酸エチル懸濁液（３０ｍＬ）に、上記実施例１．３で得られた化合物４（９．５０ｇ，１３．０ｍｍｏｌ）の酢酸エチル溶液（１２０ｍＬ）を添加し、室温で３時間撹拌した。反応終了後、セライトろ過によってパラジウム触媒を除き、溶媒を減圧留去して粗成績体（９．４０ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：２）で精製し、標題の化合物１２（８．５７ｇ，９０％）を白色泡状固体として得た。
To a suspension of Lindlar catalyst (555 mg) in ethyl acetate (30 mL) was added an ethyl acetate solution (120 mL) of compound 4 (9.50 g, 13.0 mmol) obtained in Example 1.3 above, and at room temperature. Stir for 3 hours. After completion of the reaction, the palladium catalyst was removed by Celite filtration, and the solvent was distilled off under reduced pressure to obtain a crude product (9.40 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 2) to give the title compound 12 (8.57 g, 90%) as a white foamy solid.
得られた化合物１２の物性データを表１２に示す。
The physical property data of the obtained compound 12 are shown in Table 12.
（実施例３．２）５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－Ｏ－（１－イミダゾリルチオカルボニル）－４’－（プロプ－２－エン－１－イルオキシ）－３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５－メチルウリジン（１３）の合成
Example 3.2 5′-O- (4,4′-dimethoxytrityl) -2′-O- (1-imidazolylthiocarbonyl) -4 ′-(prop-2-en-1-yloxy)- Synthesis of 3′-O-tert-butyldimethylsilyl-5-methyluridine (13)
窒素気流下、上記で得られた化合物１２（２．００ｇ，２．７４ｍｍｏｌ）の無水テトラヒドロフラン溶液（２０ｍＬ）に、１，１’－チオカルボニルジイミダゾール（９２５ｍｇ，５．４７ｍｍｏｌ）を添加し、反応液を３時間加熱還流した。反応終了後、溶媒を減圧留去して粗成績体（２．９０ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：２）で精製し、標題の化合物１３（１．９２ｇ，８３％）を白色泡状固体として得た。
Under a nitrogen stream, 1,1′-thiocarbonyldiimidazole (925 mg, 5.47 mmol) was added to an anhydrous tetrahydrofuran solution (20 mL) of the compound 12 obtained above (2.00 g, 2.74 mmol) and reacted. The solution was heated to reflux for 3 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (2.90 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 2) to give the title compound 13 (1.92 g, 83%) as a white foamy solid.
得られた化合物１３の物性データを表１３に示す。
Table 13 shows the physical property data of the obtained compound 13.
（実施例３．３）１－［（１Ｒ，４Ｓ，５Ｒ，６Ｒ，８Ｓ）－８－ｔｅｒｔ－ブチルジメチルシリルオキシ－１－（４，４’－ジメトキシトリチルオキシメチル）－４－メチル－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（１４）の合成
Example 3.3 1-[(1R, 4S, 5R, 6R, 8S) -8-tert-butyldimethylsilyloxy-1- (4,4′-dimethoxytrityloxymethyl) -4-methyl-2 , 7-dioxabicyclo [3.2.1] octane-6-yl] -thymine (14)
窒素気流下、上記で得られた化合物１３（１．９２ｇ，２．２８ｍｍｏｌ）の無水トルエン溶液（２０ｍＬ）に、９０℃にてトリストリメチルシリルシラン（１．１ｍＬ，３．４２ｍｍｏｌ）とアゾビスイソブチロニトリル（７５．０ｍｇ，０．４５７ｍｍｏｌ）とを添加し、同温度で１．５時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（１．９９ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：１から３：２）で精製し、標題の化合物１４（１．０８ｇ，６６％）を白色泡状固体として得た。
Under a nitrogen stream, tristrimethylsilylsilane (1.1 mL, 3.42 mmol) and azobisisobutyrate were added to an anhydrous toluene solution (20 mL) of the compound 13 (1.92 g, 2.28 mmol) obtained above at 90 ° C. Ronitrile (75.0 mg, 0.457 mmol) was added and stirred at the same temperature for 1.5 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (1.99 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 1 to 3: 2) to give the title compound 14 (1.08 g, 66%) as a white foamy solid.
得られた化合物１４の物性データを表１４に示す。
Table 14 shows the physical property data of the obtained compound 14.
（実施例３．４）１－［（１Ｒ，４Ｓ，５Ｒ，６Ｒ，８Ｓ）－１－（４，４’－ジメトキシトリチルオキシメチル）－８－ヒドロキシ－４－メチル－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（１５）の合成
Example 3.4 1-[(1R, 4S, 5R, 6R, 8S) -1- (4,4′-dimethoxytrityloxymethyl) -8-hydroxy-4-methyl-2,7-dioxa Synthesis of bicyclo [3.2.1] octane-6-yl] -thymine (15)
上記で得られた化合物１４（９００ｍｇ，１．２６ｍｍｏｌ）のテトラヒドロフラン溶液（１０ｍＬ）に室温でテトラブチルアンモニウムフルオリド（１Ｍテトラヒドロフラン溶液，１．４ｍＬ，１．４０ｍｍｏｌ）を添加し、９時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（１．０３ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：２）で精製し、標題の化合物１５（６１２ｍｇ，８１％）を白色泡状固体として得た。
Tetrabutylammonium fluoride (1M tetrahydrofuran solution, 1.4 mL, 1.40 mmol) was added to a tetrahydrofuran solution (10 mL) of the compound 14 (900 mg, 1.26 mmol) obtained above at room temperature and stirred for 9 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (1.03 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 2) to give the title compound 15 (612 mg, 81%) as a white foamy solid.
得られた化合物１５の物性データを表１５に示す。
Table 15 shows the physical property data of the obtained compound 15.
（実施例３．５）１－［（１Ｒ，４Ｓ，５Ｒ，６Ｒ，８Ｓ）－８－［２－シアノエトキシ（ジイソプロピルアミノ）ホスフィノキシ］－１－（４，４’－ジメトキシトリチルオキシメチル）－４－メチル－２，７－ジオキサビシクロ［３．２．１］オクタン－６－イル］－チミン（１６）（（Ｓ）－メチル体）の合成
Example 3.5 1-[(1R, 4S, 5R, 6R, 8S) -8- [2-Cyanoethoxy (diisopropylamino) phosphinoxy] -1- (4,4′-dimethoxytrityloxymethyl)- Synthesis of 4-methyl-2,7-dioxabicyclo [3.2.1] octane-6-yl] -thymine (16) ((S) -methyl)
窒素気流下、上記で得られた化合物１５（３７２ｍｇ，０．６１９ｍｍｏｌ）の無水ジクロロメタン溶液（５．０ｍＬ）に、Ｎ，Ｎ－ジイソプロピルエチルアミン（０．５５ｍＬ，３．１０ｍｍｏｌ）を添加し、０℃で２－シアノエチル－Ｎ，Ｎ－ジイソプロピルクロロホスホロアミジド（０．２１ｍＬ，０．９２９ｍｍｏｌ）を滴下し、室温で４時間撹拌した。反応終了後、０℃で反応液に飽和重曹水を添加してクエンチし、ジクロロメタンで抽出した。有機層を水で１回、かつ飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（５６０ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から２：３）で精製し、標題の化合物１６（２８７ｍｇ，５８％；（Ｓ）－メチル体）を白色泡状固体として得た。
Under a nitrogen stream, N, N-diisopropylethylamine (0.55 mL, 3.10 mmol) was added to an anhydrous dichloromethane solution (5.0 mL) of the compound 15 obtained above (372 mg, 0.619 mmol), and 0 ° C. Then, 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.21 mL, 0.929 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (560 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 2: 3) to give the title compound 16 (287 mg, 58%; (S) -methyl compound) as a white foam Obtained as a solid.
得られた化合物１６の物性データを表１６に示す。
Table 16 shows the physical property data of the obtained compound 16.
（比較例１）２’－Ｏ，４’－Ｃ－エチレンオキシ架橋した５－メチルウリジン（ＥｏＮＡ）の合成
(Comparative Example 1) Synthesis of 2'-O, 4'-C-ethyleneoxy-crosslinked 5-methyluridine (EoNA)
非特許文献８に記載の方法にしたがって、標題の化合物（ＥｏＮＡ）を合成した。
According to the method described in Non-Patent Document 8, the title compound (EoNA) was synthesized.
（実施例４）オリゴヌクレオチドの合成および精製（１）
実施例１．７、実施例２．３および実施例３．５、ならびに比較例１で得られた化合物８（エキソメチレン体）、化合物１１（（Ｒ）－メチル体）、化合物１６（（Ｓ）－メチル体）ならびにＥｏＮＡを、それぞれアミダイトブロックとして用い、これらとｄｍＣ（Ａｃ）およびＴのホスホロアミダイト（いずれもシグマ－アルドリッチ社製）との０．１Ｍの無水アセトニトリル溶液を調製し、ｎＳ－８ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ Ｓｙｎｔｈｅｓｉｚｅｒ（株式会社ジーンデザイン製オリゴヌクレオチド合成装置）を用いて、当該分野において公知のホスホロアミダイト法に従って、表１７に示す各オリゴヌクレオチドの合成を行った（ここで、Ｘは実施例１．７で得られた化合物８（エキソメチレン体）に由来する構造に相当し、Ｙは実施例２．３で得られた化合物１１（（Ｒ）－メチル体）に由来する構造に相当し、Ｚは実施例３．５で得られた化合物１６（（Ｓ）－メチル体）に由来する構造に相当し、Ｗは比較例１で得られたＥｏＮＡに由来する構造に相当する）。 (Example 4) Synthesis and purification of oligonucleotide (1)
Compound 1.7 (Exomethylene), Compound 11 ((R) -methyl), Compound 16 ((S) obtained in Example 1.7, Example 2.3 and Example 3.5, and Comparative Example 1 ) - methyl body) and EoNA, used respectively as amidites blocks, these and d m C (Ac) and T phosphoramidites (both sigma - prepared Aldrich) and anhydrous acetonitrile 0.1M of Each of the oligonucleotides shown in Table 17 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where X is Corresponds to the structure derived from compound 8 (exomethylene) obtained in Example 1.7. , Y corresponds to the structure derived from the compound 11 ((R) -methyl form) obtained in Example 2.3, and Z represents the compound 16 ((S) -methyl form obtained in Example 3.5. ), And W corresponds to the structure derived from EoNA obtained in Comparative Example 1).
実施例１．７、実施例２．３および実施例３．５、ならびに比較例１で得られた化合物８（エキソメチレン体）、化合物１１（（Ｒ）－メチル体）、化合物１６（（Ｓ）－メチル体）ならびにＥｏＮＡを、それぞれアミダイトブロックとして用い、これらとｄｍＣ（Ａｃ）およびＴのホスホロアミダイト（いずれもシグマ－アルドリッチ社製）との０．１Ｍの無水アセトニトリル溶液を調製し、ｎＳ－８ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ Ｓｙｎｔｈｅｓｉｚｅｒ（株式会社ジーンデザイン製オリゴヌクレオチド合成装置）を用いて、当該分野において公知のホスホロアミダイト法に従って、表１７に示す各オリゴヌクレオチドの合成を行った（ここで、Ｘは実施例１．７で得られた化合物８（エキソメチレン体）に由来する構造に相当し、Ｙは実施例２．３で得られた化合物１１（（Ｒ）－メチル体）に由来する構造に相当し、Ｚは実施例３．５で得られた化合物１６（（Ｓ）－メチル体）に由来する構造に相当し、Ｗは比較例１で得られたＥｏＮＡに由来する構造に相当する）。 (Example 4) Synthesis and purification of oligonucleotide (1)
Compound 1.7 (Exomethylene), Compound 11 ((R) -methyl), Compound 16 ((S) obtained in Example 1.7, Example 2.3 and Example 3.5, and Comparative Example 1 ) - methyl body) and EoNA, used respectively as amidites blocks, these and d m C (Ac) and T phosphoramidites (both sigma - prepared Aldrich) and anhydrous acetonitrile 0.1M of Each of the oligonucleotides shown in Table 17 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where X is Corresponds to the structure derived from compound 8 (exomethylene) obtained in Example 1.7. , Y corresponds to the structure derived from the compound 11 ((R) -methyl form) obtained in Example 2.3, and Z represents the compound 16 ((S) -methyl form obtained in Example 3.5. ), And W corresponds to the structure derived from EoNA obtained in Comparative Example 1).
当該合成における合成スケールは０．２μｍｏｌであり、トリチルオン条件で行った。活性化剤には５－［３，５－ビス（トリフルオロメチル）フェニル］－１Ｈ－テトラゾール（０．２５Ｍ無水アセトニトリル溶液，Ａｃｔｉｖａｔｏｒ ４２，Ｐｒｏｌｉｇｏ（登録商標））を使用した。縮合時間は、実施例１．７、実施例２．３および実施例３．５ならびに比較例１で得られたアミダイトブロックＸ、ＹおよびＺ、ならびにＥｏＮＡについては１０分間であり、天然体のアミダイトブロックでは３２秒間とした。合成完了後、２８％アンモニア水で室温下にて１．５時間処理して、カラム担体からの切り出しおよび塩基部、リン酸ジエステル部の脱保護を行った。次いで、簡易ゲルろ過カラム（Ｗａｔｅｒｓ社製Ｓｅｐ－Ｐａｋ（登録商標） Ｐｌｕｓ Ｃ１８ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｃａｒｔｒｉｄｇｅｓ）により精製し、さらに逆相ＨＰＬＣにて精製を行った。
The synthesis scale in the synthesis was 0.2 μmol, and was performed under trityl-on conditions. As the activator, 5- [3,5-bis (trifluoromethyl) phenyl] -1H-tetrazole (0.25 M anhydrous acetonitrile solution, Activator 42, Proligo (registered trademark)) was used. The condensation time is 10 minutes for the amidite blocks X, Y and Z obtained in Example 1.7, Example 2.3 and Example 3.5 and Comparative Example 1, and EoNA, and the natural amidite The block was 32 seconds. After completion of the synthesis, the mixture was treated with 28% aqueous ammonia at room temperature for 1.5 hours to cut out from the column carrier and to deprotect the base part and the phosphoric diester part. Subsequently, purification was performed by a simple gel filtration column (Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters), and further purified by reverse phase HPLC.
なお、このＨＰＬＣの条件は以下の通りであった。
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １６～３２％（３０分間）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（１０×５０ｍｍ）(精製)
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（４．６×５０ｍｍ）(純度確認)
流速
・３．０ｍＬ／分（精製）
・１．０ｍＬ／分（純度確認）
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 16-32% (30 minutes)
Column-Waters X Bridge ™ OST C 18 2.5 μm (10 × 50 mm) (purification)
-Waters XBridge TM OST C 18 2.5 μm (4.6 × 50 mm) (purity confirmation)
Flow rate ・ 3.0mL / min (purification)
・ 1.0 mL / min (purity confirmation)
Column temperature ・ 50 ℃
Detection ・ UV (260nm)
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １６～３２％（３０分間）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（１０×５０ｍｍ）(精製)
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（４．６×５０ｍｍ）(純度確認)
流速
・３．０ｍＬ／分（精製）
・１．０ｍＬ／分（純度確認）
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 16-32% (30 minutes)
Column-Waters X Bridge ™ OST C 18 2.5 μm (10 × 50 mm) (purification)
-Waters XBridge TM OST C 18 2.5 μm (4.6 × 50 mm) (purity confirmation)
Flow rate ・ 3.0mL / min (purification)
・ 1.0 mL / min (purity confirmation)
Column temperature ・ 50 ℃
Detection ・ UV (260nm)
（実施例５）融解温度（Ｔｍ）の測定（１）
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、実施例４で得られたオリゴヌクレオチド４μＭ、および表１７に示される一本鎖ＲＮＡまたは一本鎖ＤＮＡ４μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９５℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 5) Measurement of melting temperature ( Tm ) (1)
Sample solution containing 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 μM oligonucleotide obtained in Example 4 and 4 μM single-stranded RNA or single-stranded DNA shown in Table 17 (130 μL) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、実施例４で得られたオリゴヌクレオチド４μＭ、および表１７に示される一本鎖ＲＮＡまたは一本鎖ＤＮＡ４μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９５℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 5) Measurement of melting temperature ( Tm ) (1)
Sample solution containing 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 μM oligonucleotide obtained in Example 4 and 4 μM single-stranded RNA or single-stranded DNA shown in Table 17 (130 μL) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
一本鎖ＲＮＡを標的鎖とした場合の結果と、一本鎖ＤＮＡを標的鎖とした場合の結果をそれぞれ表１７に示す。
Table 17 shows the results when single-stranded RNA was used as the target strand and the results when single-stranded DNA was used as the target strand.
表１７から明らかなように、実施例１．７、実施例２．３および実施例３．５で得られた化合物８（エキソメチレン体）、化合物１１（（Ｒ）－メチル体）および化合物１６（（Ｓ）－メチル体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、コントロールのオリゴヌクレオチドと比較して、一本鎖オリゴＲＮＡに対してＴｍ値が高く、より高い結合親和性を示していたことがわかる。また、これらの実施例１．７、実施例２．３および実施例３．５で得られた化合物をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、ＥｏＮＡをアミダイトブロックとして用いて合成したオリゴヌクレオチドを比較しても、一本鎖オリゴＲＮＡに対してＴｍ値が高く、より高い結合親和性を示していたことがわかる。
As is clear from Table 17, Compound 8 (exomethylene), Compound 11 ((R) -methyl) and Compound 16 obtained in Example 1.7, Example 2.3 and Example 3.5 Oligonucleotides synthesized using ((S) -methyl) as amidite blocks have a higher Tm value and higher binding affinity for single-stranded oligo RNA compared to control oligonucleotides. I understand that it was. In addition, the oligonucleotides synthesized using the compounds obtained in Example 1.7, Example 2.3 and Example 3.5 as amidite blocks are the same as those synthesized using EoNA as the amidite block. Even if it compares, it turns out that Tm value was high with respect to single stranded oligo RNA, and showed higher binding affinity.
（実施例６）融解温度（Ｔｍ）の測定（２）（三重鎖形成能評価）
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、塩化マグネシウム５ｍＭ、実施例４で得られたオリゴヌクレオチド１．５μＭ、および表１８に示される二本鎖ＤＮＡ１．５μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９０℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 6) Measurement of melting temperature (T m ) (2) (Evaluation of triple chain forming ability)
The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 μM of the oligonucleotide obtained in Example 4, and 1.5 μM of the double-stranded DNA shown in Table 18. The containing sample solution (130 μL) was bathed in boiling water and slowly cooled to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、塩化マグネシウム５ｍＭ、実施例４で得られたオリゴヌクレオチド１．５μＭ、および表１８に示される二本鎖ＤＮＡ１．５μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９０℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 6) Measurement of melting temperature (T m ) (2) (Evaluation of triple chain forming ability)
The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 μM of the oligonucleotide obtained in Example 4, and 1.5 μM of the double-stranded DNA shown in Table 18. The containing sample solution (130 μL) was bathed in boiling water and slowly cooled to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
二本鎖ＤＮＡを標的鎖とした場合の結果を表１８に示す。
The results when double-stranded DNA is used as the target strand are shown in Table 18.
表１８から明らかなように、実施例１．７、実施例２．３および実施例３．５で得られた化合物８（エキソメチレン体）、および化合物１６（（Ｓ）－メチル体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、ＥｏＮＡをアミダイトブロックとして用いて合成したオリゴヌクレオチドと比較しても、二本鎖ＤＮＡに対するＴｍ値が遜色なく、より高い結合親和性を示していたことがわかる。
As is apparent from Table 18, the compounds 8 (exomethylene) and 16 ((S) -methyl) obtained in Example 1.7, Example 2.3 and Example 3.5 were amidite. The oligonucleotide synthesized using the block showed a higher Tm value for double-stranded DNA and higher binding affinity than the oligonucleotide synthesized using EoNA as the amidite block. Recognize.
（実施例７）オリゴヌクレオチドの合成および精製（２）
実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、チミジン（天然体）、ＬＮＡおよびＥＮＡを、それぞれアミダイトブロックとして用いたこと以外は、実施例４と同様にして当該分野において公知のホスホロアミダイト法に従って、以下に示すオリゴヌクレオチドの合成を行った。 Example 7 Oligonucleotide Synthesis and Purification (2)
Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl), thymidine (naturally occurring), LNA and ENA were used as amidite blocks, respectively, in the same manner as in Example 4 according to the phosphoramidite method known in the art. Nucleotide synthesis was performed.
実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、チミジン（天然体）、ＬＮＡおよびＥＮＡを、それぞれアミダイトブロックとして用いたこと以外は、実施例４と同様にして当該分野において公知のホスホロアミダイト法に従って、以下に示すオリゴヌクレオチドの合成を行った。 Example 7 Oligonucleotide Synthesis and Purification (2)
Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl), thymidine (naturally occurring), LNA and ENA were used as amidite blocks, respectively, in the same manner as in Example 4 according to the phosphoramidite method known in the art. Nucleotide synthesis was performed.
（実施例８）酵素耐性実験（１）
終濃度をそれぞれＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）５０ｍＭ、塩化マグネシウム１０ｍＭ、実施例７で得られた各オリゴヌクレオチド７．５μＭ、３’－エキソヌクレアーゼ（Ｃｒｏｔａｌｕｓ Ａｄｍａｎｔｅｕｓ Ｖｅｎｏｍ Ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ：ＣＡＶＰ，Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）１．５μｇ／ｍＬとしたサンプル溶液（１００μＬ）を、３７℃に保ち反応を行った。経時的に反応液の一部（２０μＬ）を採取し、９０℃で２分間加熱して酵素を失活させ、オリゴヌクレオチドの残量を、ＨＰＬＣ（ＳＨＩＭＡＤＺＵ社製ＬＣ－２０ＡＴ、ＳＰＤ－２０Ａ、ＣＴＯ－２０Ａ、ＣＢＭ－２０Ａ）により定量した。 (Example 8) Enzyme resistance experiment (1)
The final concentrations were respectively Tris-HCl buffer (pH 8.0) 50 mM,magnesium chloride 10 mM, each oligonucleotide 7.5 μM obtained in Example 7, and 3′-exonuclease (Crotalus Admanteus Venom Phosphoesterase: CAVP, Pharmacia Biotech) The sample solution (100 μL) adjusted to 1.5 μg / mL was kept at 37 ° C. for reaction. A part of the reaction solution (20 μL) was collected over time, heated at 90 ° C. for 2 minutes to inactivate the enzyme, and the remaining amount of oligonucleotide was determined by HPLC (LC-20AT, SPD-20A, CTO manufactured by SHIMADZU). -20A, CBM-20A).
終濃度をそれぞれＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）５０ｍＭ、塩化マグネシウム１０ｍＭ、実施例７で得られた各オリゴヌクレオチド７．５μＭ、３’－エキソヌクレアーゼ（Ｃｒｏｔａｌｕｓ Ａｄｍａｎｔｅｕｓ Ｖｅｎｏｍ Ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ：ＣＡＶＰ，Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）１．５μｇ／ｍＬとしたサンプル溶液（１００μＬ）を、３７℃に保ち反応を行った。経時的に反応液の一部（２０μＬ）を採取し、９０℃で２分間加熱して酵素を失活させ、オリゴヌクレオチドの残量を、ＨＰＬＣ（ＳＨＩＭＡＤＺＵ社製ＬＣ－２０ＡＴ、ＳＰＤ－２０Ａ、ＣＴＯ－２０Ａ、ＣＢＭ－２０Ａ）により定量した。 (Example 8) Enzyme resistance experiment (1)
The final concentrations were respectively Tris-HCl buffer (pH 8.0) 50 mM,
なお、このＨＰＬＣの条件は以下の通りであった。
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １２～２４％（１５分間） （天然体、ＬＮＡ）
１２～２４％（３０分間） （ＥＮＡ）
１４～２６％（１５分間） （エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）（天然体、ＬＮＡ、エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体）
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）を２本連結（ＥＮＡのみ）
流速
・１．０ｍＬ／分
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 12-24% (15 minutes) (natural body, LNA)
12-24% (30 minutes) (ENA)
14-26% (15 minutes) (Exomethylene, (R) -methyl, (S) -methyl)
Column XBridge ™ Shield RP18 (4.6 × 50 mm) manufactured by Waters (Natural, LNA, Exomethylene, (R) -Methyl, (S) -Methyl)
-Two XBridge ™ Shield RP18 (4.6 x 50 mm) manufactured by Waters are connected (ENA only)
Flow rate ・ 1.0mL / min Column temperature ・ 50 ℃
Detection ・ UV (260nm)
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １２～２４％（１５分間） （天然体、ＬＮＡ）
１２～２４％（３０分間） （ＥＮＡ）
１４～２６％（１５分間） （エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）（天然体、ＬＮＡ、エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体）
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）を２本連結（ＥＮＡのみ）
流速
・１．０ｍＬ／分
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 12-24% (15 minutes) (natural body, LNA)
12-24% (30 minutes) (ENA)
14-26% (15 minutes) (Exomethylene, (R) -methyl, (S) -methyl)
Column XBridge ™ Shield RP18 (4.6 × 50 mm) manufactured by Waters (Natural, LNA, Exomethylene, (R) -Methyl, (S) -Methyl)
-Two XBridge ™ Shield RP18 (4.6 x 50 mm) manufactured by Waters are connected (ENA only)
Flow rate ・ 1.0mL / min Column temperature ・ 50 ℃
Detection ・ UV (260nm)
得られた結果を図１に示す。
The results obtained are shown in FIG.
図１に示すように、実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、または実施例３．５で得られた化合物１６（（Ｓ）－メチル体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、天然体のチミジンまたはＬＮＡを用いたものと比較して、未反応のオリゴヌクレオチドの残存量が著しく高い結果を示していたことがわかる。さらに、ＥＮＡを用いたオリゴヌクレオチドと比較しても、当該エキソメチレン体、（Ｒ）－メチル体、または（Ｓ）－メチル体を用いたオリゴヌクレオチドは、経時的に未反応のオリゴヌクレオチドの残存量が低下するものでもなく、より優れた酵素耐性を有していたことがわかる。
As shown in FIG. 1, compound 8 (exomethylene) obtained in Example 1.7, compound 11 ((R) -methyl) obtained in Example 2.3, or Example 3.5 The oligonucleotide synthesized using the compound 16 ((S) -methyl derivative) obtained in (1) as an amidite block has a residual amount of unreacted oligonucleotide as compared with those using natural thymidine or LNA. It can be seen that the result was extremely high. Further, even when compared with the oligonucleotides using ENA, the oligonucleotides using the exomethylene, (R) -methyl, or (S) -methyl are not left unreacted with time. It can be seen that the amount was not reduced, and had better enzyme resistance.
（実施例９）ホスホロアミダイト化された２’，４’－架橋型ヌクレオシド（無置換体）の合成
Example 9 Synthesis of Phosphoramiditeized 2 ', 4'-Bridged Nucleoside (Unsubstituted Product)
（実施例９．１）（２’Ｒ）－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－（１－オキソ－エチレンオキシ）チミジン（１７）の合成
Example 9.1 Synthesis of (2'R) -5'-O- (4,4'-dimethoxytrityl) -2 ', 4'-(1-oxo-ethyleneoxy) thymidine (17)
実施例１．６で得られた化合物７（１．１７ｇ，１．９５ｍｍｏｌ）のジクロロメタン溶液（２０ｍＬ）に、アセトン（１０ｍＬ）および飽和重層水（４００ｍＬ）を添加し、０℃でオキソン（登録商標）（１８．０ｇ，２９．３ｍｍｏｌ）の水溶液（１００ｍＬ）を滴下して、室温で２４時間撹拌した。反応終了後、ジクロロメタンで抽出し、有機層を飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（１．１０ｇ）を得た。
Acetone (10 mL) and saturated multistory water (400 mL) are added to a dichloromethane solution (20 mL) of compound 7 (1.17 g, 1.95 mmol) obtained in Example 1.6, and Oxone (registered trademark) is added at 0 ° C. ) (18.0 g, 29.3 mmol) in water (100 mL) was added dropwise and stirred at room temperature for 24 hours. After completion of the reaction, the mixture was extracted with dichloromethane, and the organic layer was washed once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (1.10 g).
次いで、この粗成績体のジオキサン溶液（２０ｍＬ）に１Ｍ水酸化カリウム水溶液（２０ｍＬ，２０ｍｍｏｌ）を滴下し、室温で１２時間撹拌した。反応終了後、０℃で反応液に飽和塩化アンモニウム水溶液を添加することによりクエンチし、ジクロロメタンで抽出した。有機層を水で１回、飽和食塩水で１回それぞれ洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（１．４８ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー (クロロホルム/メタノール＝２５：１から７．１)で精製し、化合物７（５８０ｍｇ，５０％）と、トリオール（４２２ｍｇ）とをそれぞれ白色泡状固体として得た。
Next, a 1M aqueous potassium hydroxide solution (20 mL, 20 mmol) was added dropwise to the dioxane solution (20 mL) of the crude product, and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, the reaction solution was quenched by adding a saturated aqueous ammonium chloride solution at 0 ° C., and extracted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (1.48 g). The crude product was purified by silica gel column chromatography (chloroform / methanol = 25: 1 to 7.1) to obtain compound 7 (580 mg, 50%) and triol (422 mg) as white foam solids, respectively. .
次いで、得られたトリオールのテトラヒドロフラン／水溶液（３：１，１０ｍＬ）に０℃にて過ヨウ素酸ナトリウム（２８５ｍｇ，１．３３ｍｍｏｌ）を添加し、室温で２時間撹拌した。反応終了後、０℃で反応液に飽和チオ硫酸ナトリウム水溶液を添加してクエンチし、酢酸エチルで抽出した。有機層を水で１回、飽和食塩水で１回それぞれ洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（５６０ｍｇ）を得た。粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：２）で精製し、標題の化合物１７（３４６ｍｇ，３０％，３工程）を白色泡状固体として得た。
Next, sodium periodate (285 mg, 1.33 mmol) was added to the resulting tetrahydrofuran / water solution of triol (3: 1, 10 mL) at 0 ° C., and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was quenched by adding a saturated aqueous sodium thiosulfate solution at 0 ° C., and extracted with ethyl acetate. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (560 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 2) to give the title compound 17 (346 mg, 30%, 3 steps) as a white foamy solid.
得られた化合物１７の物性データを表１９に示す。
Table 19 shows the physical property data of the obtained compound 17.
（実施例９．２）（２’Ｒ）－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－（１－ヨード－ビニレンオキシ）チミジン（１８）の合成
Example 9.2 Synthesis of (2'R) -5'-O- (4,4'-dimethoxytrityl) -2 ', 4'-(1-iodo-vinyleneoxy) thymidine (18)
上記で得られた化合物１７（５０．０ｍｇ，０．０８３２ｍｍｏｌ）のエタノール溶液（２．０ｍＬ）に、トリエチルアミン（５８μＬ，０．４１６ｍｍｏｌ）およびヒドラジン一水和物（８．０μＬ，０．１６６ｍｍｏｌ）を滴下し、室温で４時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（５３．２ｍｇ）を得た。
To an ethanol solution (2.0 mL) of the compound 17 (50.0 mg, 0.0832 mmol) obtained above, triethylamine (58 μL, 0.416 mmol) and hydrazine monohydrate (8.0 μL, 0.166 mmol) were added. The solution was added dropwise and stirred at room temperature for 4 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (53.2 mg).
次いで、窒素気流下にて、得られた粗成績体の無水テトラヒドロフラン溶液（２．０ｍＬ）に１，８－ジアザビシクロウンデセン（６２μＬ，０．４１６ｍｍｏｌ）を滴下し、０℃でヨウ素（４３．７ｍｇ，０．１６６ｍｍｏｌ）を添加し、室温で２時間撹拌した。反応終了後、０℃で反応液に飽和チオ硫酸ナトリウム水溶液を添加してクエンチし、酢酸エチルで抽出した。有機層を水で１回、飽和食塩水で１回それぞれ洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（７０．０ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から２：３）で精製し、標題の化合物１８（３７．８ｍｇ，６４％，２工程）を白色泡状固体として得た。
Next, 1,8-diazabicycloundecene (62 μL, 0.416 mmol) was added dropwise to an anhydrous tetrahydrofuran solution (2.0 mL) of the obtained crude product under a nitrogen stream, and iodine (43 0.7 mg, 0.166 mmol) was added and stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was quenched by adding a saturated aqueous sodium thiosulfate solution at 0 ° C., and extracted with ethyl acetate. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (70.0 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 2: 3) to give the title compound 18 (37.8 mg, 64%, 2 steps) as a white foamy solid. Obtained.
得られた化合物１８の物性データを表２０に示す。
The physical property data of the obtained compound 18 are shown in Table 20.
（実施例９．３）（２’Ｒ）－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－（エチレンオキシ）チミジン（１９）の合成
Example 9.3 Synthesis of (2'R) -5'-O- (4,4'-dimethoxytrityl) -2 ', 4'-(ethyleneoxy) thymidine (19)
酸化白金（５１．６ｍｇ，０．２２８ｍｍｏｌ）のテトラヒドロフラン懸濁液（１．０ｍＬ）に、上記で得られた化合物１８（３２３ｍｇ，０．４５５ｍｍｏｌ）のテトラヒドロフラン溶液（４．０ｍＬ）および１，８－ジアザビシクロウンデセン（０．２０ｍＬ，１．３６ｍｍｏｌ）を添加し、水素気流下にて、室温で１２時間撹拌した。反応終了後、酸化白金をセライトろ過によって取り除き、水を添加し、酢酸エチルで抽出した。有機層を飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（２６８ｍｇ）を得た。得られた粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：４）で精製し、標題の化合物１９（２５１ｍｇ，９４％）を白色泡状固体として得た。
To a tetrahydrofuran suspension (1.0 mL) of platinum oxide (51.6 mg, 0.228 mmol), a tetrahydrofuran solution (4.0 mL) of the compound 18 (323 mg, 0.455 mmol) obtained above and 1,8- Diazabicycloundecene (0.20 mL, 1.36 mmol) was added, and the mixture was stirred at room temperature for 12 hours under a hydrogen stream. After completion of the reaction, platinum oxide was removed by celite filtration, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (268 mg). The obtained crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 4) to give the title compound 19 (251 mg, 94%) as a white foamy solid.
得られた化合物１９の物性データを表２１に示す。
The physical property data of the obtained compound 19 are shown in Table 21.
（実施例９．４）（２’Ｒ）－３’－Ｏ－［２－シアノエトキシ（ジイソプロピルアミノ）ホスフィノ］－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－（エチレンオキシ）チミジン（２０）（無置換体）（ホスホロアミダイト化された２’，４’－架橋型ヌクレオシド）の合成
Example 9.4 (2′R) -3′-O- [2-cyanoethoxy (diisopropylamino) phosphino] -5′-O- (4,4′-dimethoxytrityl) -2 ′, 4 ′ Synthesis of — (ethyleneoxy) thymidine (20) (unsubstituted product) (phosphoramiditeized 2 ′, 4′-bridged nucleoside)
窒素気流下にて、上記で得られた化合物１９（２５１ｍｇ，０．４２８ｍｍｏｌ）の無水ジクロロメタン溶液（５．０ｍＬ）に、Ｎ，Ｎ－ジイソプロピルエチルアミン（０．２３ｍＬ，１．２８ｍｍｏｌ）を添加し、０℃で２－シアノエチル－Ｎ，Ｎ－ジイソプロピルクロロホスホロアミジド（０．１１ｍＬ，０．５１３ｍｍｏｌ）を滴下し、室温で３時間撹拌した。反応終了後、０℃で反応液に飽和重曹水を添加してクエンチし、ジクロロメタンで抽出した。有機層を水で１回、飽和食塩水で１回それぞれ洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（３５０ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から１：２）で精製し、標題の化合物２０（無置換体）（２８３ｍｇ，８４％）を白色泡状固体として得た。
Under a nitrogen stream, N, N-diisopropylethylamine (0.23 mL, 1.28 mmol) was added to an anhydrous dichloromethane solution (5.0 mL) of the compound 19 (251 mg, 0.428 mmol) obtained above, 2-Cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.11 mL, 0.513 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (350 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 1: 2) to give the title compound 20 (unsubstituted product) (283 mg, 84%) as a white foamy solid. Obtained.
得られた化合物２０の物性データを表２２に示す。
The physical property data of the obtained compound 20 are shown in Table 22.
（実施例１０）２’，４’－架橋型ヌクレオシド（（Ｒ）－メチル－エキソメチレン体）の合成
Example 10 Synthesis of 2 ', 4'-Bridged Nucleoside ((R) -Methyl-Exomethylene)
（実施例１０．１）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－４’－Ｃ－［（１Ｒ）－メチル－２－プロピン－１－イルオキシ］－５－メチルウリジン（２１）の合成
Example 10.1 Synthesis of 3'-O-tert-butyldimethylsilyl-4'-C-[(1R) -methyl-2-propyn-1-yloxy] -5-methyluridine (21)
実施例１．１で得られた化合物２ｂ（２．５３ｇ，７．１３ｍｍｏｌ）のジクロロメタン溶液（５０ｍＬ）にアセトン（５０ｍＬ）と飽和重層水（５０ｍＬ）とを添加し、０℃でオキソン（登録商標）（１３．１ｇ，２１．４ｍｍｏｌ）の水溶液（５０ｍＬ）を滴下し、室温で３時間撹拌した。反応終了後、ジクロロメタンで抽出し、有機層を飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（２．６６ｇ）を得た。
Acetone (50 mL) and saturated multistory water (50 mL) were added to a dichloromethane solution (50 mL) of compound 2b (2.53 g, 7.13 mmol) obtained in Example 1.1, and Oxone (registered trademark) was added at 0 ° C. ) (13.1 g, 21.4 mmol) in water (50 mL) was added dropwise and stirred at room temperature for 3 hours. After completion of the reaction, the mixture was extracted with dichloromethane, and the organic layer was washed once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (2.66 g).
次いで、アルゴン気流下にて、この粗成績体の無水ジエチルエーテル溶液（１０ｍＬ）に、－４０℃で（Ｒ）－３－ブチン－２－オール（４．５ｇ，６４．２ｍｍｏｌ）、塩化亜鉛（１Ｍジエチルエーテル溶液の７．１ｍＬ，７．１ｍｍｏｌ）を滴下し、室温で1時間撹拌した。反応終了後、反応液を飽和重曹水でクエンチし、セライトでろ過した。ろ液から酢酸エチルで抽出し、有機層を水で１回、および飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（２．８８ｇ）を得た。その後、この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から１：２）で精製し、標題の化合物２１（１．８６ｇ，５９％，２工程）を白色泡状固体として得た。
Then, under an argon stream, the crude product was added to an anhydrous diethyl ether solution (10 mL) at −40 ° C. at (R) -3-butyn-2-ol (4.5 g, 64.2 mmol), zinc chloride ( 1 mL of diethyl ether solution (7.1 mL, 7.1 mmol) was added dropwise, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was quenched with saturated aqueous sodium hydrogen carbonate and filtered through celite. The filtrate was extracted with ethyl acetate, the organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (2.88 g). It was. The crude product was then purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 1: 2) to give the title compound 21 (1.86 g, 59%, 2 steps) as a white foam Obtained as a solid.
得られた化合物２１の物性データを表２３に示す。
The physical property data of the obtained compound 21 are shown in Table 23.
（実施例１０．２）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－４’－Ｃ－［（１Ｒ）－メチル－２－プロピン－１－イルオキシ］－５－メチルウリジン（２２）の合成
Example 10.2 3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -4′-C-[(1R) -methyl-2-propyne-1 -Iloxy] -5-methyluridine (22) synthesis
アルゴン気流下にて、実施例１０．１で得られた化合物２１（１．８２ｇ，４．１３ｍｍｏｌ）の無水ジクロロメタン溶液（２５ｍＬ）に、２，６－ルチジン（０．９６ｍＬ，８．２６ｍｍｏｌ）を添加した後、０℃にて４，４’－ジメトキシトリチルトリフルオロメタンスルホニルの無水ジクロロメタン溶液（１Ｍ，４．５ｍＬ，４．５ｍｍｏｌ）を滴下し、室温で１時間撹拌した。反応終了後、０℃でメタノールを添加してクエンチし、ジクロロメタンで希釈した。有機層を水で１回、および飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（３．４９ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：１から１：１）で精製し、標題の化合物２２（２．９６ｇ，９６％）を淡黄色泡状固体として得た。
Under an argon stream, 2,6-lutidine (0.96 mL, 8.26 mmol) was added to an anhydrous dichloromethane solution (25 mL) of the compound 21 (1.82 g, 4.13 mmol) obtained in Example 10.1. After the addition, 4,4′-dimethoxytrityltrifluoromethanesulfonyl in anhydrous dichloromethane (1M, 4.5 mL, 4.5 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, it was quenched by adding methanol at 0 ° C. and diluted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (3.49 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 1 to 1: 1) to give the title compound 22 (2.96 g, 96%) as a pale yellow foamy solid. .
得られた化合物２２の物性データを表２４に示す。
The physical property data of the obtained compound 22 are shown in Table 24.
（実施例１０．３）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－Ｏ－（１－イミダゾリルチオカルボニル）－４’－Ｃ－［（１Ｒ）－メチル－２－プロピン－１－イルオキシ］－５－メチルウリジン（２３）の合成
Example 10.3 3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -2′-O- (1-imidazolylthiocarbonyl) -4′-C Synthesis of [(1R) -methyl-2-propyn-1-yloxy] -5-methyluridine (23)
アルゴン気流下にて、実施例１０．２で得られた化合物２２（２．９２ｇ，３．９３ｍｍｏｌ）の無水テトラヒドロフラン溶液（３０ｍＬ）に、１，１’－チオカルボニルジイミダゾール（１．４０ｇ，７．８６ｍｍｏｌ）を添加し、得られた反応液を３時間加熱下にて還流した。反応終了後、溶媒を減圧留去して粗成績体（４．４１ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：２から２：３）で精製し、標題の化合物２３（２．６８ｇ，８０％）を白色泡状固体として得た。
Under an argon gas stream, 1,1′-thiocarbonyldiimidazole (1.40 g, 7) was added to an anhydrous tetrahydrofuran solution (30 mL) of the compound 22 (2.92 g, 3.93 mmol) obtained in Example 10.2. .86 mmol) was added and the resulting reaction solution was refluxed with heating for 3 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (4.41 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 2 to 2: 3) to give the title compound 23 (2.68 g, 80%) as a white foamy solid.
得られた化合物２３の物性データを表２５に示す。
The physical property data of the obtained compound 23 are shown in Table 25.
（実施例１０．４）（２’Ｒ）－３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－［（２Ｒ）－メチル－１－メチレン－エチレンオキシ］チミジン（２４）の合成
Example 10.4 (2′R) -3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -2 ′, 4 ′-[(2R) — Synthesis of methyl-1-methylene-ethyleneoxy] thymidine (24)
アルゴン気流下にて、実施例１０．３で得られた化合物２３（２．６４ｇ，３．０９ｍｍｏｌ）の無水トルエン溶液（３０ｍＬ）に、９０℃にてトリストリメチルシリルシラン（１．９ｍＬ，６．１８ｍｍｏｌ）とアゾビスイソブチロニトリル（１０２ｍｇ，０．６１９ｍｍｏｌ）とを添加し、同温度で１時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（４．０１ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝５：１から２：１）で精製し、標題の化合物２４（１．２３ｇ，５５％）を白色泡状固体として得た。
Under an argon stream, tristrimethylsilylsilane (1.9 mL, 6.18 mmol) was added to an anhydrous toluene solution (30 mL) of compound 23 (2.64 g, 3.09 mmol) obtained in Example 10.3 at 90 ° C. ) And azobisisobutyronitrile (102 mg, 0.619 mmol) were added and stirred at the same temperature for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (4.01 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 5: 1 to 2: 1) to give the title compound 24 (1.23 g, 55%) as a white foamy solid.
得られた化合物２４の物性データを表２６に示す。
The physical property data of the obtained compound 24 are shown in Table 26.
（実施例１０．５）（２’Ｒ）－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－［（２Ｒ）－メチル－１－メチレン－エチレンオキシ］チミジン（２５）の合成
Example 10.5 (2′R) -5′-O- (4,4′-dimethoxytrityl) -2 ′, 4 ′-[(2R) -methyl-1-methylene-ethyleneoxy] thymidine ( 25) Synthesis
実施例１０．４で得られた化合物２４（１．１８ｇ，１．６２ｍｍｏｌ）のテトラヒドロフラン溶液（１０ｍＬ）に、室温でテトラブチルアンモニウムフルオリド（１Ｍテトラヒドロフラン溶液，１．８ｍＬ，１．８ｍｍｏｌ）を添加し、１２時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（１．９５ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：２）で精製し、標題の化合物２５（８８９ｍｇ，８９％）を白色泡状固体として得た。
To a tetrahydrofuran solution (10 mL) of compound 24 (1.18 g, 1.62 mmol) obtained in Example 10.4, tetrabutylammonium fluoride (1M tetrahydrofuran solution, 1.8 mL, 1.8 mmol) was added at room temperature. And stirred for 12 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (1.95 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 2) to give the title compound 25 (889 mg, 89%) as a white foamy solid.
得られた化合物２５の物性データを表２７に示す。
Table 27 shows the physical property data of the obtained compound 25.
（実施例１０．６）（２’Ｒ）－３’－Ｏ－[２－シアノエトキシ（ジイソプロピルアミノ）ホスフィノ]－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－［（２Ｒ）－メチル－１－メチレン－エチレンオキシ］チミジン（２６）（（Ｒ）－メチル－エキソメチレン体）の合成
Example 10.6 (2′R) -3′-O- [2-cyanoethoxy (diisopropylamino) phosphino] -5′-O- (4,4′-dimethoxytrityl) -2 ′, 4 ′ Synthesis of — [(2R) -methyl-1-methylene-ethyleneoxy] thymidine (26) ((R) -methyl-exomethylene)
アルゴン気流下にて、実施例１０．５で得られた化合物２５（７００ｍｇ，１．１４ｍｍｏｌ）の無水ジクロロメタン溶液（１０ｍＬ）に、Ｎ，Ｎ－ジイソプロピルエチルアミン（０．６１ｍＬ，３．４２ｍｍｏｌ）を添加し、０℃にて２－シアノエチル－Ｎ，Ｎ－ジイソプロピルクロロホスホロアミジド（０．３１ｍＬ，１．３７ｍｍｏｌ）を滴下し、室温で３時間撹拌した。反応終了後、０℃で反応液に飽和重曹水を添加してクエンチし、ジクロロメタンで抽出した。有機層を水で１回、および飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（８１５ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から２：３）で精製し、標題の化合物２６（６５６ｍｇ，７１％）を白色泡状固体として得た。
Under an argon stream, N, N-diisopropylethylamine (0.61 mL, 3.42 mmol) was added to a solution of compound 25 (700 mg, 1.14 mmol) obtained in Example 10.5 in anhydrous dichloromethane (10 mL). Then, 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.31 mL, 1.37 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (815 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 2: 3) to give the title compound 26 (656 mg, 71%) as a white foamy solid.
得られた化合物２６の物性データを表２８に示す。
The physical property data of the obtained compound 26 are shown in Table 28.
（実施例１１）２’，４’－架橋型ヌクレオシド（（Ｓ）－メチル－エキソメチレン体）の合成
Example 11 Synthesis of 2 ', 4'-Bridged Nucleoside ((S) -Methyl-Exomethylene)
（実施例１１．１）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－４’－Ｃ－[（１Ｓ）－メチル－２－プロピン－１－イルオキシ]－５－メチルウリジン（２７）の合成
Example 11.1 Synthesis of 3'-O-tert-butyldimethylsilyl-4'-C-[(1S) -methyl-2-propyn-1-yloxy] -5-methyluridine (27)
実施例１．１で得られた化合物２ｂ（２．５３ｇ，７１３ｍｍｏｌ）のジクロロメタン溶液（５０ｍＬ）に、アセトン（５０ｍＬ）と飽和重層水（５００ｍＬ）とを添加し、０℃にてオキソン（登録商標）（１３．１ｇ，２１．４ｍｍｏｌ）の水溶液（５０ｍＬ）を滴下し、室温で３時間撹拌した。反応終了後、ジクロロメタンで抽出し、有機層を飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（２．６６ｇ）を得た。
Acetone (50 mL) and saturated multistory water (500 mL) are added to a dichloromethane solution (50 mL) of compound 2b (2.53 g, 713 mmol) obtained in Example 1.1, and Oxone (registered trademark) is added at 0 ° C. ) (13.1 g, 21.4 mmol) in water (50 mL) was added dropwise and stirred at room temperature for 3 hours. After completion of the reaction, the mixture was extracted with dichloromethane, and the organic layer was washed once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (2.66 g).
次いで、アルゴン気流下にて、この粗成績体の無水ジエチルエーテル溶液（１０ｍＬ）に、－４０℃にて（Ｓ）－３－ブチン－２－オール（４．５ｇ，６４．２ｍｍｏｌ）、塩化亜鉛（１Ｍジエチルエーテル溶液、７．１ｍＬ，７．１ｍｍｏｌ）を滴下し、室温で１時間撹拌した。反応終了後、反応液を飽和重曹水でクエンチし、セライトでろ過した。ろ液から酢酸エチルで抽出し、有機層を水で１回、および飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（３．２５ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から２：３）で精製し、標題の化合物２７（２．４２ｇ，７７％，２工程）を白色泡状固体として得た。
Next, under an argon stream, this crude product was added to an anhydrous diethyl ether solution (10 mL) at −40 ° C. at (S) -3-butyn-2-ol (4.5 g, 64.2 mmol), zinc chloride. (1M diethyl ether solution, 7.1 mL, 7.1 mmol) was added dropwise and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was quenched with saturated aqueous sodium hydrogen carbonate and filtered through celite. The filtrate was extracted with ethyl acetate, the organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (3.25 g). It was. The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 2: 3) to give the title compound 27 (2.42 g, 77%, 2 steps) as a white foamy solid. Obtained.
得られた化合物２７の物性データを表２９に示す。
The physical property data of the obtained compound 27 are shown in Table 29.
（実施例１１．２）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－４’－Ｃ－[（１Ｓ）－メチル－２－プロピン－１－イルオキシ]－５－メチルウリジン（２８）の合成
Example 11.2 3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -4′-C-[(1S) -methyl-2-propyne-1 -Iloxy] -5-methyluridine (28) synthesis
アルゴン気流下にて、実施例１１．１で得られた化合物２７（２．４０ｇ，５．４５ｍｍｏｌ）の無水ジクロロメタン溶液（２５ｍＬ）に、２，６－ルチジン（１．３ｍＬ，１０．９ｍｍｏｌ）を添加した後、０℃で４，４’－ジメトキシトリチルトリフルオロメタンスルホニルの無水ジクロロメタン溶液（１Ｍ，６．０ｍＬ，６．０ｍｍｏｌ）を滴下し、室温で１時間撹拌した。反応終了後、０℃にてメタノールを添加してクエンチし、ジクロロメタンで希釈した。有機層を水で１回、および飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（４．０８ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：１から３：２）で精製し、標題の化合物２８（３．４５ｇ，８５％）を淡黄色泡状固体として得た。
Under an argon stream, 2,6-lutidine (1.3 mL, 10.9 mmol) was added to an anhydrous dichloromethane solution (25 mL) of the compound 27 (2.40 g, 5.45 mmol) obtained in Example 11.1. After the addition, a solution of 4,4′-dimethoxytrityltrifluoromethanesulfonyl in anhydrous dichloromethane (1M, 6.0 mL, 6.0 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, it was quenched by adding methanol at 0 ° C. and diluted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (4.08 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 1 to 3: 2) to give the title compound 28 (3.45 g, 85%) as a pale yellow foamy solid. .
得られた化合物２８の物性データを表３０に示す。
The physical property data of the obtained compound 28 are shown in Table 30.
（実施例１１．３）３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－Ｏ－（１－イミダゾリルチオカルボニル）－４’－Ｃ－[（１Ｓ）－メチル－２－プロピン－１－イルオキシ]－５－メチルウリジン（２９）の合成
Example 11.3 3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -2′-O- (1-imidazolylthiocarbonyl) -4′-C Synthesis of — [(1S) -methyl-2-propyn-1-yloxy] -5-methyluridine (29)
アルゴン気流下にて、実施例１１．２で得られた化合物２８（３．４０ｇ，４．５８ｍｍｏｌ）の無水テトラヒドロフラン溶液（３０ｍＬ）に、１，１’－チオカルボニルジイミダゾール（１．６３ｇ，９．１５ｍｍｏｌ）を添加し、反応液を３時間加熱還流した。反応終了後、溶媒を減圧留去して粗成績体（５．０１ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝３：２から２：３）で精製し、標題の化合物２９（３．０６ｇ，７８％）を白色泡状固体として得た。
Under a stream of argon, 1,2′-thiocarbonyldiimidazole (1.63 g, 9) was added to an anhydrous tetrahydrofuran solution (30 mL) of compound 28 (3.40 g, 4.58 mmol) obtained in Example 11.2. .15 mmol) was added and the reaction was heated to reflux for 3 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (5.01 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3: 2 to 2: 3) to give the title compound 29 (3.06 g, 78%) as a white foamy solid.
得られた化合物２９の物性データを表３１に示す。
The physical property data of the obtained compound 29 are shown in Table 31.
（実施例１１．４）（２’Ｒ）－３’－Ｏ－ｔｅｒｔ－ブチルジメチルシリル－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－[（２Ｓ）－メチル－１－メチレン－エチレンオキシ]チミジン（３０）の合成
Example 11.4 (2′R) -3′-O-tert-butyldimethylsilyl-5′-O- (4,4′-dimethoxytrityl) -2 ′, 4 ′-[(2S) — Synthesis of methyl-1-methylene-ethyleneoxy] thymidine (30)
アルゴン気流下にて、実施例１１．３で得られた化合物２９（３．００ｇ，３．５２ｍｍｏｌ）の無水トルエン溶液（３０ｍＬ）に、９０℃でトリストリメチルシリルシラン（２．２ｍＬ，７．０４ｍｍｏｌ）とアゾビスイソブチロニトリル（１１５ｍｇ，０．７０３ｍｍｏｌ）とを添加し、同温度で２時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（４．４８ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー （ｎ－ヘキサン／酢酸エチル＝５：１から２：１）で精製し、標題の化合物３０（１．０５ｇ，４１％）を白色泡状固体として得た。
Under an argon stream, tristrimethylsilylsilane (2.2 mL, 7.04 mmol) was added to an anhydrous toluene solution (30 mL) of the compound 29 (3.00 g, 3.52 mmol) obtained in Example 11.3 at 90 ° C. And azobisisobutyronitrile (115 mg, 0.703 mmol) were added and stirred at the same temperature for 2 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (4.48 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 5: 1 to 2: 1) to give the title compound 30 (1.05 g, 41%) as a white foamy solid.
得られた化合物３０の物性データを表３２に示す。
The physical property data of the obtained compound 30 are shown in Table 32.
（実施例１１．５）（２’Ｒ）－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－[（２Ｓ）－メチル－１－メチレン－エチレンオキシ]チミジン（３１）の合成
Example 11.5 (2′R) -5′-O- (4,4′-dimethoxytrityl) -2 ′, 4 ′-[(2S) -methyl-1-methylene-ethyleneoxy] thymidine ( 31) Synthesis
実施例１１．４で得られた化合物３０（１．００ｇ，１．３８ｍｍｏｌ）のテトラヒドロフラン溶液（１０ｍＬ）に、室温でテトラブチルアンモニウムフルオリド（１Ｍテトラヒドロフラン溶液，１．５ｍＬ，１．５ｍｍｏｌ）を添加し、１２時間撹拌した。反応終了後、溶媒を減圧留去して粗成績体（１．８０ｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝１：１から１：２）で精製し、標題の化合物３１（４８５ｍｇ，５８％）を白色泡状固体として得た。
Tetrabutylammonium fluoride (1M tetrahydrofuran solution, 1.5 mL, 1.5 mmol) was added to a tetrahydrofuran solution (10 mL) of compound 30 (1.00 g, 1.38 mmol) obtained in Example 11.4 at room temperature. And stirred for 12 hours. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain a crude product (1.80 g). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1: 1 to 1: 2) to give the title compound 31 (485 mg, 58%) as a white foamy solid.
得られた化合物３１の物性データを表３３に示す。
Table 33 shows the physical property data of the obtained compound 31.
（実施例１１．６）（２’Ｒ）－３’－Ｏ－［２－シアノエトキシ（ジイソプロピルアミノ）ホスフィノ］－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’，４’－[（２Ｓ）－メチル－１－メチレン－エチレンオキシ]チミジン（３２）（（Ｓ）－メチル－エキソメチレン体）の合成
Example 11.6 (2'R) -3'-O- [2-Cyanoethoxy (diisopropylamino) phosphino] -5'-O- (4,4'-dimethoxytrityl) -2 ', 4' Synthesis of — [(2S) -methyl-1-methylene-ethyleneoxy] thymidine (32) ((S) -methyl-exomethylene)
アルゴン気流下にて、実施例１１．５で得られた化合物３１（４００ｍｇ，０．６５３ｍｍｏｌ）の無水ジクロロメタン溶液（１０ｍＬ）に、Ｎ，Ｎ－ジイソプロピルエチルアミン（０．３５ｍＬ，１．９６ｍｍｏｌ）を添加し、０℃で２－シアノエチル－Ｎ，Ｎ－ジイソプロピルクロロホスホロアミジド（０．１７ｍＬ，０．７８３ｍｍｏｌ）を滴下し、室温で３時間撹拌した。反応終了後、０℃で反応液に飽和重曹水を添加してクエンチし、ジクロロメタンで抽出した。有機層を水で１回、および飽和食塩水で１回洗浄し、硫酸ナトリウムで乾燥し、溶媒を減圧留去して粗成績体（５２８ｍｇ）を得た。この粗成績体をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン／酢酸エチル＝２：１から２：３）で精製し、標題の化合物３２（３３２ｍｇ，６３％）を白色泡状固体として得た。
Under an argon stream, N, N-diisopropylethylamine (0.35 mL, 1.96 mmol) was added to a solution of compound 31 (400 mg, 0.653 mmol) obtained in Example 11.5 in anhydrous dichloromethane (10 mL). Then, 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.17 mL, 0.783 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (528 mg). The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2: 1 to 2: 3) to give the title compound 32 (332 mg, 63%) as a white foamy solid.
得られた化合物３２の物性データを表３４に示す。
The physical property data of the obtained compound 32 are shown in Table 34.
（実施例１２）オリゴヌクレオチドの合成および精製（３）
実施例９．４、実施例１０．６および実施例１１．６で得られた化合物２０（無置換体）、化合物２６（（Ｒ）－メチル－エキソメチレン体）、および化合物３２（（Ｓ）－メチル－エキソメチレン体）を、それぞれアミダイトブロックとして用い、これらとｄｍＣ（Ａｃ）およびＴのホスホロアミダイト（いずれもシグマ－アルドリッチ社製）との０．１Ｍの無水アセトニトリル溶液を調製し、ｎＳ－８ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ Ｓｙｎｔｈｅｓｉｚｅｒ（株式会社ジーンデザイン製オリゴヌクレオチド合成装置）を用いて、当該分野において公知のホスホロアミダイト法に従って、表３５に示す各オリゴヌクレオチドの合成を行った（ここで、Ｑは実施例９．４で得られた化合物２０（無置換体）に由来する構造に相当し、Ｒは実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）に由来する構造に相当し、Ｓは実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）に由来する構造に相当する）。 (Example 12) Synthesis and purification of oligonucleotide (3)
Compound 9.4 (unsubstituted product), Compound 26 ((R) -methyl-exomethylene compound), and Compound 32 ((S)) obtained in Example 9.4, Example 10.6 and Example 11.6 -Methyl-exomethylene) were used as amidite blocks, respectively, to prepare 0.1M anhydrous acetonitrile solution of these with d m C (Ac) and T phosphoramidites (both from Sigma-Aldrich). Each of the oligonucleotides shown in Table 35 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where Q is This corresponds to the structure derived from compound 20 (unsubstituted product) obtained in Example 9.4, and R Corresponds to the structure derived from compound 26 ((R) -methyl-exomethylene) obtained in Example 10.6. S represents compound 32 ((S) -methyl obtained in Example 11.6. -Corresponds to a structure derived from an exomethylene).
実施例９．４、実施例１０．６および実施例１１．６で得られた化合物２０（無置換体）、化合物２６（（Ｒ）－メチル－エキソメチレン体）、および化合物３２（（Ｓ）－メチル－エキソメチレン体）を、それぞれアミダイトブロックとして用い、これらとｄｍＣ（Ａｃ）およびＴのホスホロアミダイト（いずれもシグマ－アルドリッチ社製）との０．１Ｍの無水アセトニトリル溶液を調製し、ｎＳ－８ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ Ｓｙｎｔｈｅｓｉｚｅｒ（株式会社ジーンデザイン製オリゴヌクレオチド合成装置）を用いて、当該分野において公知のホスホロアミダイト法に従って、表３５に示す各オリゴヌクレオチドの合成を行った（ここで、Ｑは実施例９．４で得られた化合物２０（無置換体）に由来する構造に相当し、Ｒは実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）に由来する構造に相当し、Ｓは実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）に由来する構造に相当する）。 (Example 12) Synthesis and purification of oligonucleotide (3)
Compound 9.4 (unsubstituted product), Compound 26 ((R) -methyl-exomethylene compound), and Compound 32 ((S)) obtained in Example 9.4, Example 10.6 and Example 11.6 -Methyl-exomethylene) were used as amidite blocks, respectively, to prepare 0.1M anhydrous acetonitrile solution of these with d m C (Ac) and T phosphoramidites (both from Sigma-Aldrich). Each of the oligonucleotides shown in Table 35 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where Q is This corresponds to the structure derived from compound 20 (unsubstituted product) obtained in Example 9.4, and R Corresponds to the structure derived from compound 26 ((R) -methyl-exomethylene) obtained in Example 10.6. S represents compound 32 ((S) -methyl obtained in Example 11.6. -Corresponds to a structure derived from an exomethylene).
当該合成における合成スケールは０．２μｍｏｌであり、トリチルオン条件で行った。活性化剤には５－［３，５－ビス（トリフルオロメチル）フェニル］－１Ｈ－テトラゾール（０．２５Ｍ無水アセトニトリル溶液，Ａｃｔｉｖａｔｏｒ ４２，Ｐｒｏｌｉｇｏ（登録商標））を使用した。縮合時間は、実施例９．４、実施例１０．６および実施例１１．６で得られたアミダイトブロックＱ、ＲおよびＳについて１０分間であり、天然体のアミダイトブロックでは３２秒間とした。合成完了後、２８％アンモニア水で室温下にて１．５時間処理して、カラム担体からの切り出しおよび塩基部、リン酸ジエステル部の脱保護を行った。次いで、簡易ゲルろ過カラム（Ｗａｔｅｒｓ社製Ｓｅｐ－Ｐａｋ（登録商標） Ｐｌｕｓ Ｃ１８ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｃａｒｔｒｉｄｇｅｓ）により精製し、さらに逆相ＨＰＬＣにて精製を行った。
The synthesis scale in the synthesis was 0.2 μmol, and was performed under trityl-on conditions. As the activator, 5- [3,5-bis (trifluoromethyl) phenyl] -1H-tetrazole (0.25 M anhydrous acetonitrile solution, Activator 42, Proligo (registered trademark)) was used. The condensation time was 10 minutes for the amidite blocks Q, R, and S obtained in Example 9.4, Example 10.6, and Example 11.6, and 32 seconds for the natural amidite block. After completion of the synthesis, the mixture was treated with 28% aqueous ammonia at room temperature for 1.5 hours to cut out from the column carrier and to deprotect the base part and the phosphoric diester part. Subsequently, purification was performed by a simple gel filtration column (Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters), and further purified by reverse phase HPLC.
なお、このＨＰＬＣの条件は以下の通りであった。
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １６～３２％（３０分間）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（１０×５０ｍｍ）(精製)
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（４．６×５０ｍｍ）(純度確認)
流速
・３．０ｍＬ／分（精製）
・１．０ｍＬ／分（純度確認）
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 16-32% (30 minutes)
Column-Waters X Bridge ™ OST C 18 2.5 μm (10 × 50 mm) (purification)
-Waters XBridge TM OST C 18 2.5 μm (4.6 × 50 mm) (purity confirmation)
Flow rate ・ 3.0mL / min (purification)
・ 1.0 mL / min (purity confirmation)
Column temperature ・ 50 ℃
Detection ・ UV (260nm)
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １６～３２％（３０分間）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（１０×５０ｍｍ）(精製)
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ ＯＳＴ Ｃ１８ ２．５μｍ（４．６×５０ｍｍ）(純度確認)
流速
・３．０ｍＬ／分（精製）
・１．０ｍＬ／分（純度確認）
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 16-32% (30 minutes)
Column-Waters X Bridge ™ OST C 18 2.5 μm (10 × 50 mm) (purification)
-Waters XBridge TM OST C 18 2.5 μm (4.6 × 50 mm) (purity confirmation)
Flow rate ・ 3.0mL / min (purification)
・ 1.0 mL / min (purity confirmation)
Column temperature ・ 50 ℃
Detection ・ UV (260nm)
（実施例１３）融解温度（Ｔｍ）の測定（３）
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、実施例１２で得られたオリゴヌクレオチド４μＭ、および表３５に示される一本鎖ＲＮＡまたは一本鎖ＤＮＡ４μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９５℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 13) Measurement of melting temperature ( Tm ) (3)
The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 μM of the oligonucleotide obtained in Example 12, and a sample solution containing 4 μM of single-stranded RNA or single-stranded DNA shown in Table 35. (130 μL) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、実施例１２で得られたオリゴヌクレオチド４μＭ、および表３５に示される一本鎖ＲＮＡまたは一本鎖ＤＮＡ４μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９５℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 13) Measurement of melting temperature ( Tm ) (3)
The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 μM of the oligonucleotide obtained in Example 12, and a sample solution containing 4 μM of single-stranded RNA or single-stranded DNA shown in Table 35. (130 μL) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
一本鎖ＲＮＡを標的鎖とした場合の結果と、一本鎖ＤＮＡを標的鎖とした場合の結果をそれぞれ表３５に示す。
Table 35 shows the results when single-stranded RNA was used as the target strand and the results when single-stranded DNA was used as the target strand.
表３５から明らかなように、実施例９．４、実施例１０．６および実施例１１．６で得られた化合物２０（無置換体）、化合物２６（（Ｒ）－メチル－エキソメチレン体）および化合物３２（（Ｓ）－メチル－エキソメチレン体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、コントロールのオリゴヌクレオチドと比較して、いずれも一本鎖オリゴＲＮＡに対してＴｍ値が高く、より高い結合親和性を示していたことがわかる。
As is apparent from Table 35, Compound 20 (unsubstituted product) and Compound 26 ((R) -methyl-exomethylene compound) obtained in Example 9.4, Example 10.6 and Example 11.6 And compound 32 ((S) -methyl-exomethylene) as an amidite block, both Tm values were higher than single-stranded oligo RNA compared to control oligonucleotides. It can be seen that it showed higher binding affinity.
（実施例１４）融解温度（Ｔｍ）の測定（４）（三重鎖形成能評価）
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、塩化マグネシウム５ｍＭ、実施例１２で得られたオリゴヌクレオチド１．５μＭ、および表３６に示されるヘアピン二本鎖ＤＮＡ１．５μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９０℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 14) Measurement of melting temperature ( Tm ) (4) (Evaluation of triple chain forming ability)
The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 μM oligonucleotide obtained in Example 12, and 1.5 μM hairpin double-stranded DNA shown in Table 36. The sample solution containing (130 μL) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
終濃度をカコジル酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化カリウム１４０ｍＭ、塩化マグネシウム５ｍＭ、実施例１２で得られたオリゴヌクレオチド１．５μＭ、および表３６に示されるヘアピン二本鎖ＤＮＡ１．５μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９０℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。 (Example 14) Measurement of melting temperature ( Tm ) (4) (Evaluation of triple chain forming ability)
The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 μM oligonucleotide obtained in Example 12, and 1.5 μM hairpin double-stranded DNA shown in Table 36. The sample solution containing (130 μL) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
二本鎖ＤＮＡを標的鎖とした場合の結果を表３６に示す。
Table 36 shows the results when double-stranded DNA was used as the target strand.
表３６から明らかなように、実施例９．４、実施例１０．６および実施例１１．６で得られた化合物２０（無置換体）、化合物２６（（Ｒ）－メチル－エキソメチレン体）および化合物３２（（Ｓ）－メチル－エキソメチレン体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、コントロールのオリゴヌクレオチドと比較して、その多くが二本鎖ＤＮＡに対してＴｍ値が高く、より高い結合親和性を示していたことがわかる。
As is apparent from Table 36, Compound 20 (unsubstituted product) and Compound 26 ((R) -methyl-exomethylene compound) obtained in Example 9.4, Example 10.6 and Example 11.6 And most of the oligonucleotides synthesized using compound 32 ((S) -methyl-exomethylene) as an amidite block had a higher Tm value than the double-stranded DNA compared to the control oligonucleotide. It can be seen that it showed higher binding affinity.
（実施例１５）オリゴヌクレオチドの合成および精製（４）
実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、実施例９．４で得られた化合物２０（無置換体）、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）、実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）、チミジン（天然体）、およびＥＮＡを、それぞれアミダイトブロックとして用いたこと以外は、実施例４と同様にして当該分野において公知のホスホロアミダイト法に従って、以下に示すオリゴヌクレオチドの合成を行った。 Example 15 Oligonucleotide Synthesis and Purification (4)
Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl form),compound 20 obtained in Example 9.4 (unsubstituted product), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene form), Example 11 In the art in the same manner as in Example 4 except that compound 32 ((S) -methyl-exomethylene), thymidine (natural product), and ENA obtained in .6 were used as amidite blocks, respectively. The following oligonucleotides were synthesized according to a known phosphoramidite method.
実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、実施例９．４で得られた化合物２０（無置換体）、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）、実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）、チミジン（天然体）、およびＥＮＡを、それぞれアミダイトブロックとして用いたこと以外は、実施例４と同様にして当該分野において公知のホスホロアミダイト法に従って、以下に示すオリゴヌクレオチドの合成を行った。 Example 15 Oligonucleotide Synthesis and Purification (4)
Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl form),
（実施例１６）酵素耐性実験（２）
終濃度をそれぞれＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）５０ｍＭ、塩化マグネシウム１０ｍＭ、実施例１５で得られた各オリゴヌクレオチド７．５μＭ、３’－エキソヌクレアーゼ（Ｃｒｏｔａｌｕｓ Ａｄｍａｎｔｅｕｓ Ｖｅｎｏｍ Ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ：ＣＡＶＰ，Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）２．５μｇ／ｍＬとしたサンプル溶液（１００μＬ）を、３７℃に保ち反応を行った。経時的に反応液の一部（２０μＬ）を採取し、９０℃で２分間加熱して酵素を失活させ、オリゴヌクレオチドの残量を、ＨＰＬＣ（ＳＨＩＭＡＤＺＵ社製ＬＣ－２０ＡＴ、ＳＰＤ－２０Ａ、ＣＴＯ－２０Ａ、ＣＢＭ－２０Ａ）により定量した。 Example 16 Enzyme Resistance Experiment (2)
The final concentrations were Tris-HCl buffer (pH 8.0) 50 mM,magnesium chloride 10 mM, each oligonucleotide 7.5 μM obtained in Example 15, 3′-exonuclease (Crotalus Admanteus Venophophophosterase: CAVP, Pharmacia Biotech) (Product) 2.5 μg / mL sample solution (100 μL) was kept at 37 ° C. for reaction. A part of the reaction solution (20 μL) was collected over time, heated at 90 ° C. for 2 minutes to inactivate the enzyme, and the remaining amount of oligonucleotide was determined by HPLC (LC-20AT, SPD-20A, CTO manufactured by SHIMADZU). -20A, CBM-20A).
終濃度をそれぞれＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ８．０）５０ｍＭ、塩化マグネシウム１０ｍＭ、実施例１５で得られた各オリゴヌクレオチド７．５μＭ、３’－エキソヌクレアーゼ（Ｃｒｏｔａｌｕｓ Ａｄｍａｎｔｅｕｓ Ｖｅｎｏｍ Ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ：ＣＡＶＰ，Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）２．５μｇ／ｍＬとしたサンプル溶液（１００μＬ）を、３７℃に保ち反応を行った。経時的に反応液の一部（２０μＬ）を採取し、９０℃で２分間加熱して酵素を失活させ、オリゴヌクレオチドの残量を、ＨＰＬＣ（ＳＨＩＭＡＤＺＵ社製ＬＣ－２０ＡＴ、ＳＰＤ－２０Ａ、ＣＴＯ－２０Ａ、ＣＢＭ－２０Ａ）により定量した。 Example 16 Enzyme Resistance Experiment (2)
The final concentrations were Tris-HCl buffer (pH 8.0) 50 mM,
なお、このＨＰＬＣの条件は以下の通りであった。
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １２～２４％（１５分間） （天然体、無置換体）
１２～２４％（３０分間） （ＥＮＡ）
１４～２６％（１５分間） （エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体、（Ｒ）－メチル－エキソメチレン体、（Ｓ）－メチル－エキソメチレン体）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）（天然体、エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体、（Ｒ）－メチル－エキソメチレン体、（Ｓ）－メチル－エキソメチレン体）
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）を２本連結（ＥＮＡのみ）
流速
・１．０ｍＬ／分
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 12-24% (15 minutes) (natural, non-substituted)
12-24% (30 minutes) (ENA)
14 to 26% (15 minutes) (Exomethylene, (R) -methyl, (S) -methyl, (R) -methyl-exomethylene, (S) -methyl-exomethylene)
Column: XBridge ™ Shield RP18 (4.6 × 50 mm) manufactured by Waters (natural, exomethylene, (R) -methyl, (S) -methyl, (R) -methyl-exomethylene, (S ) -Methyl-Exomethylene)
-Two XBridge ™ Shield RP18 (4.6 x 50 mm) manufactured by Waters are connected (ENA only)
Flow rate ・ 1.0mL / min Column temperature ・ 50 ℃
Detection ・ UV (260nm)
溶離液
・Ａ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液（ｐＨ７．０）
・Ｂ液：０．１Ｍ酢酸トリエチルアンモニウム緩衝液／アセトニトリル＝１：１
グラジエント
・Ｂ液濃度： １２～２４％（１５分間） （天然体、無置換体）
１２～２４％（３０分間） （ＥＮＡ）
１４～２６％（１５分間） （エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体、（Ｒ）－メチル－エキソメチレン体、（Ｓ）－メチル－エキソメチレン体）
カラム
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）（天然体、エキソメチレン体、（Ｒ）－メチル体、（Ｓ）－メチル体、（Ｒ）－メチル－エキソメチレン体、（Ｓ）－メチル－エキソメチレン体）
・Ｗａｔｅｒｓ社製ＸＢｒｉｄｇｅＴＭ Ｓｈｉｅｌｄ ＲＰ１８（４．６×５０ｍｍ）を２本連結（ＥＮＡのみ）
流速
・１．０ｍＬ／分
カラム温度
・５０℃
検出
・ＵＶ（２６０ｎｍ） The HPLC conditions were as follows.
Eluent-Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0)
-Solution B: 0.1 M triethylammonium acetate buffer / acetonitrile = 1: 1
Gradient-B solution concentration: 12-24% (15 minutes) (natural, non-substituted)
12-24% (30 minutes) (ENA)
14 to 26% (15 minutes) (Exomethylene, (R) -methyl, (S) -methyl, (R) -methyl-exomethylene, (S) -methyl-exomethylene)
Column: XBridge ™ Shield RP18 (4.6 × 50 mm) manufactured by Waters (natural, exomethylene, (R) -methyl, (S) -methyl, (R) -methyl-exomethylene, (S ) -Methyl-Exomethylene)
-Two XBridge ™ Shield RP18 (4.6 x 50 mm) manufactured by Waters are connected (ENA only)
Flow rate ・ 1.0mL / min Column temperature ・ 50 ℃
Detection ・ UV (260nm)
得られた結果を図２に示す。
The obtained results are shown in FIG.
図２に示すように、実施例８に記載の実験系よりも３’－エキソヌクレアーゼの濃度を高めた系において、実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、実施例９．４で得られた化合物２０（無置換体）、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）、または実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、天然体のチミジンおよび残存量が経時的に低下したＥＮＡと比較して、未反応のオリゴヌクレオチドの残存量が著しく高い結果を示していたことがわかる。特に当該酵素および／または濃度においては、（Ｒ）－メチル体または（Ｒ）－メチル－エキソメチレン体を用いたオリゴヌクレオチドの未反応のオリゴヌクレオチドの残存量の経時的な低下が少ないものであり、より優れた酵素耐性を有していたことがわかる。
As shown in FIG. 2, in a system in which the concentration of 3′-exonuclease was higher than that in the experimental system described in Example 8, compound 8 (exomethylene) obtained in Example 1.7, Example 2 was obtained. Compound 11 ((R) -methyl compound) obtained in .3, Compound 16 ((S) -methyl compound) obtained in Example 3.5, and Compound 20 obtained in Example 9.4 (none) Substituent), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene), or compound 32 obtained in Example 11.6 ((S) -methyl-exomethylene) It can be seen that the oligonucleotide synthesized using amidite block showed a significantly higher residual amount of unreacted oligonucleotide compared to natural thymidine and ENA whose residual amount decreased over time. . In particular, in the enzyme and / or concentration, the remaining amount of the unreacted oligonucleotide of the oligonucleotide using (R) -methyl or (R) -methyl-exomethylene is less decreased with time. It can be seen that the enzyme had better enzyme resistance.
（実施例１７）オリゴヌクレオチドの合成および精製（５）
本実施例では、４種の塩基（Ａ、Ｔ、ＧおよびＣ）で構成されるオリゴヌクレオチドにおいて、以下の人工核酸のいずれかを取り込ませた。実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、実施例９．４で得られた化合物２０（無置換体）、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）、実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）、チミジン（天然体）、ＬＮＡおよびＥＮＡを、それぞれアミダイトブロックとして用いたこと以外は、実施例４と同様にして当該分野において公知のホスホロアミダイト法に従って、以下に示すオリゴヌクレオチドの合成を行った。天然体の場合、オリゴヌクレオチドの配列は、５’－d(TTCAGCATTGGTATTC)－３’（配列番号５）である。 Example 17 Oligonucleotide Synthesis and Purification (5)
In this example, one of the following artificial nucleic acids was incorporated into an oligonucleotide composed of four types of bases (A, T, G and C). Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl form),compound 20 obtained in Example 9.4 (unsubstituted product), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene form), Example 11 6 except that Compound 32 ((S) -methyl-exomethylene), thymidine (natural product), LNA and ENA obtained in .6 were used as amidite blocks, respectively. The following oligonucleotides were synthesized according to the known phosphoramidite method. In the case of a natural product, the sequence of the oligonucleotide is 5′-d (TTCAGCATTGGTATTC) -3 ′ (SEQ ID NO: 5).
本実施例では、４種の塩基（Ａ、Ｔ、ＧおよびＣ）で構成されるオリゴヌクレオチドにおいて、以下の人工核酸のいずれかを取り込ませた。実施例１．７で得られた化合物８（エキソメチレン体）、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）、実施例３．５で得られた化合物１６（（Ｓ）－メチル体）、実施例９．４で得られた化合物２０（無置換体）、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）、実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）、チミジン（天然体）、ＬＮＡおよびＥＮＡを、それぞれアミダイトブロックとして用いたこと以外は、実施例４と同様にして当該分野において公知のホスホロアミダイト法に従って、以下に示すオリゴヌクレオチドの合成を行った。天然体の場合、オリゴヌクレオチドの配列は、５’－d(TTCAGCATTGGTATTC)－３’（配列番号５）である。 Example 17 Oligonucleotide Synthesis and Purification (5)
In this example, one of the following artificial nucleic acids was incorporated into an oligonucleotide composed of four types of bases (A, T, G and C). Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl form),
（実施例１８）融解温度（Ｔｍ）の測定（４）
本実施例では、実施例１７で得られた各オリゴヌクレオチドについて、標的鎖として５’－ｒ（ＧＡＡＵＡＣＣＡＡＵＧＣＵＧＡＡ）－３’（配列番号６）の一本鎖オリゴＲＮＡと二重鎖形成させ、当該ＲＮＡとの融解温度を測定した。 (Example 18) Measurement of melting temperature (Tm) (4)
In this example, each oligonucleotide obtained in Example 17 was double-stranded with a single-stranded oligo RNA of 5′-r (GAAUACCAAUGCUGAA) -3 ′ (SEQ ID NO: 6) as a target strand, and the RNA The melting temperature was measured.
本実施例では、実施例１７で得られた各オリゴヌクレオチドについて、標的鎖として５’－ｒ（ＧＡＡＵＡＣＣＡＡＵＧＣＵＧＡＡ）－３’（配列番号６）の一本鎖オリゴＲＮＡと二重鎖形成させ、当該ＲＮＡとの融解温度を測定した。 (Example 18) Measurement of melting temperature (Tm) (4)
In this example, each oligonucleotide obtained in Example 17 was double-stranded with a single-stranded oligo RNA of 5′-r (GAAUACCAAUGCUGAA) -3 ′ (SEQ ID NO: 6) as a target strand, and the RNA The melting temperature was measured.
終濃度をリン酸ナトリウム緩衝液（ｐＨ ７．２）１０ｍＭ、塩化ナトリウム１００ｍＭ、実施例１７で得られたオリゴヌクレオチド４μＭ、および上記一本鎖オリゴＲＮＡ４μＭを含むサンプル溶液（１３０μＬ）を沸騰水中に浴し、室温までゆっくりと冷ました後、各サンプル溶液を１５℃まで冷却して融解温度（Ｔｍ）の測定を開始した。毎分０．５℃の割合で９５℃まで昇温し、０．５℃間隔で２６０ｎｍにおける吸光度をＳＨＩＭＡＤＺＵ ＵＶ－１６５０ＰＣ、ＳＨＩＭＡＤＺＵ ＵＶ－１８００ ｓｐｅｃｔｒｏｍｅｔｅｒｓ（株式会社島津製作所製）を用いて測定し、プロットした。Ｔｍ値は全て中線法で算出し、３回の独立した測定結果の平均値とした。結果を表３７に示す。
The final concentration is 10 mM sodium phosphate buffer (pH 7.2), 100 mM sodium chloride, 4 μM of the oligonucleotide obtained in Example 17, and the sample solution (130 μL) containing the single-stranded oligo RNA 4 μM is bathed in boiling water. Then, after slowly cooling to room temperature, each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results. The results are shown in Table 37.
表３７から明らかなように、化合物８（エキソメチレン体）、化合物１１（（Ｒ）－メチル体）、化合物１６（（Ｓ）－メチル体）、化合物２０（無置換体）、化合物２６（（Ｒ）－メチル－エキソメチレン体）、および化合物３２（（Ｓ）－メチル－エキソメチレン体）のいずれをアミダイトブロックとして用いて合成したオリゴヌクレオチドは、コントロールの天然体を用いたオリゴヌクレオチドと比較して、標的鎖の一本鎖オリゴＲＮＡに対してＴｍ値が高く、より高い結合親和性を示していたことがわかる。化合物８（エキソメチレン体）、化合物１６（（Ｓ）－メチル体）、化合物２０（無置換体）、化合物２６（（Ｒ）－メチル－エキソメチレン体）、および化合物３２（（Ｓ）－メチル－エキソメチレン体）については、ＬＮＡおよびＥＮＡと同程度のＴｍ値を示し、よって、これらに匹敵する高い結合親和性を示していたことがわかる。
As is apparent from Table 37, compound 8 (exomethylene), compound 11 ((R) -methyl), compound 16 ((S) -methyl), compound 20 (unsubstituted), compound 26 (( R) -methyl-exomethylene) and compound 32 ((S) -methyl-exomethylene) were used as amidite blocks, compared with oligonucleotides using control natural products. Thus, it can be seen that the Tm value was high with respect to the single-stranded oligo RNA of the target strand, indicating a higher binding affinity. Compound 8 (Exomethylene), Compound 16 ((S) -Methyl), Compound 20 (Unsubstituted), Compound 26 ((R) -Methyl-Exomethylene), and Compound 32 ((S) -Methyl) It can be seen that -exomethylene form) showed a T m value comparable to that of LNA and ENA, and thus showed a high binding affinity comparable to these.
よって、４種の塩基（Ａ、Ｔ、ＧおよびＣ）で構成されるオリゴヌクレオチドの場合であっても、いずれの人工オリゴ核酸も標的一本鎖ＲＮＡに対して十分な結合能を持つことが示された。
Therefore, even in the case of an oligonucleotide composed of four types of bases (A, T, G and C), any artificial oligonucleic acid can have sufficient binding ability to the target single-stranded RNA. Indicated.
（実施例１９）細胞培養系におけるアンチセンス評価
４．５１×１０５細胞／ｍＬに調製したＨｕｈ－７（ヒト肝癌細胞株：ＰＳ２０）を９６ウェル平底マイクロプレート（ＩＷＡＫＩ）に播種し、３７℃にて５％ＣＯ２下で２４時間培養した。各アンチセンス核酸分子が最終濃度１００ｎＭもしくは４００ｎＭとなるように、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ３０００（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）およびＯｐｔｉ－ＭＥＭ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）をさらに添加して混合し、室温にて１５分間静置した後、各ウェルに添加した。実施例１７で得られた各オリゴヌクレオチドを添加し、２４時間後に細胞を回収した。なお、コントロールとして、オリゴヌクレオチド無添加で同じ処理を行った。細胞の回収は、ＳｕｐｅｒＰｒｅｐ（登録商標） Ｃｅｌｌ Ｌｙｓｉｓ ＆ ＲＴ Ｋｉｔ ｆｏｒ ｑＰＣＲ（東洋紡績株式会社製）を用いて行った。また、このキットを用いてトータルＲＮＡの回収を行った。 (Example 19) Antisense evaluation in cell culture system Huh-7 (human hepatoma cell line: PS20) prepared to 4.51 × 10 5 cells / mL was seeded in a 96-well flat bottom microplate (IWAKI) and incubated at 37 ° C. For 24 hours under 5% CO 2 . Lipofectamine 3000 (manufactured by Life Technologies) and Opti-MEM (manufactured by Life Technologies) were further added and mixed so that each antisense nucleic acid molecule had a final concentration of 100 nM or 400 nM and allowed to stand at room temperature for 15 minutes. Later, it was added to each well. Each oligonucleotide obtained in Example 17 was added, and cells were collected 24 hours later. As a control, the same treatment was performed without addition of oligonucleotide. The cells were collected using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.). In addition, total RNA was collected using this kit.
４．５１×１０５細胞／ｍＬに調製したＨｕｈ－７（ヒト肝癌細胞株：ＰＳ２０）を９６ウェル平底マイクロプレート（ＩＷＡＫＩ）に播種し、３７℃にて５％ＣＯ２下で２４時間培養した。各アンチセンス核酸分子が最終濃度１００ｎＭもしくは４００ｎＭとなるように、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ３０００（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）およびＯｐｔｉ－ＭＥＭ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）をさらに添加して混合し、室温にて１５分間静置した後、各ウェルに添加した。実施例１７で得られた各オリゴヌクレオチドを添加し、２４時間後に細胞を回収した。なお、コントロールとして、オリゴヌクレオチド無添加で同じ処理を行った。細胞の回収は、ＳｕｐｅｒＰｒｅｐ（登録商標） Ｃｅｌｌ Ｌｙｓｉｓ ＆ ＲＴ Ｋｉｔ ｆｏｒ ｑＰＣＲ（東洋紡績株式会社製）を用いて行った。また、このキットを用いてトータルＲＮＡの回収を行った。 (Example 19) Antisense evaluation in cell culture system Huh-7 (human hepatoma cell line: PS20) prepared to 4.51 × 10 5 cells / mL was seeded in a 96-well flat bottom microplate (IWAKI) and incubated at 37 ° C. For 24 hours under 5% CO 2 . Lipofectamine 3000 (manufactured by Life Technologies) and Opti-MEM (manufactured by Life Technologies) were further added and mixed so that each antisense nucleic acid molecule had a final concentration of 100 nM or 400 nM and allowed to stand at room temperature for 15 minutes. Later, it was added to each well. Each oligonucleotide obtained in Example 17 was added, and cells were collected 24 hours later. As a control, the same treatment was performed without addition of oligonucleotide. The cells were collected using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.). In addition, total RNA was collected using this kit.
ＳｕｐｅｒＰｒｅｐ（登録商標） Ｃｅｌｌ Ｌｙｓｉｓ ＆ ＲＴ Ｋｉｔ ｆｏｒ ｑＰＣＲ（東洋紡績株式会社製）を用いて、回収したトータルＲＮＡよりｃＤＮＡを合成した。得られたｃＤＮＡを適宜希釈した後、リアルタイムＰＣＲを行い、ＡｐｏＢのｍＲＮＡ量を定量した。リアルタイムＰＣＲでは、ハウスキーピング遺伝子のＧＡＰＤＨのｍＲＮＡ量も同時に定量した。これにより、ＧＡＰＤＨのｍＲＮＡ量に対するＡｐｏＢのｍＲＮＡ量を定量的に評価した。リアルタイムＰＣＲにはＦａｓｔ ＳＹＢＲ（登録商標） Ｇｒｅｅｎ Ｍａｓｔｅｒ Ｍｉｘ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を用いた。
CDNA was synthesized from the collected total RNA using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.). After the obtained cDNA was appropriately diluted, real-time PCR was performed to quantify the amount of ApoB mRNA. In real-time PCR, the amount of GAPDH mRNA of the housekeeping gene was also quantified. Thus, the amount of ApoB mRNA relative to the amount of GAPDH mRNA was quantitatively evaluated. For real-time PCR, Fast SYBR (registered trademark) Green Master Mix (Applied Biosystems) was used.
以下に使用したプライマーのシーケンスを示す：
Ｈｓ＿ＡｐｏＢ＿Ｆｗ ：ＧＧＣＴＣＡＣＣＣＴＧＡＧＡＧＡＡＧＴＧ（配列番号８）
Ｈｓ＿ＡｐｏＢ＿Ｒｖ ：ＧＣＴＧＣＴＴＴＣＴＧＧＡＡＣＣＴＣＡＣ（配列番号８）
Ｈｓ＿ＧＡＰＤＨ＿Ｆｗ：ＧＧＣＣＴＣＣＡＡＧＧＡＧＴＡＡＧＡＣＣ（配列番号９）
Ｈｓ＿ＧＡＰＤＨ＿Ｒｖ：ＡＧＧＧＧＴＣＴＡＣＡＴＧＧＣＡＡＣＴＧ（配列番号１０） The primer sequences used are shown below:
Hs_ApoB_Fw: GGCTCACCCTGAGAGAAGTG (SEQ ID NO: 8)
Hs_ApoB_Rv: GCTGCTTTCTGGGAACCTCAC (SEQ ID NO: 8)
Hs_GAPDH_Fw: GGCCTCCCAAGGAGTAAGACC (SEQ ID NO: 9)
Hs_GAPDH_Rv: AGGGTCTCATAGGGCACTG (SEQ ID NO: 10)
Ｈｓ＿ＡｐｏＢ＿Ｆｗ ：ＧＧＣＴＣＡＣＣＣＴＧＡＧＡＧＡＡＧＴＧ（配列番号８）
Ｈｓ＿ＡｐｏＢ＿Ｒｖ ：ＧＣＴＧＣＴＴＴＣＴＧＧＡＡＣＣＴＣＡＣ（配列番号８）
Ｈｓ＿ＧＡＰＤＨ＿Ｆｗ：ＧＧＣＣＴＣＣＡＡＧＧＡＧＴＡＡＧＡＣＣ（配列番号９）
Ｈｓ＿ＧＡＰＤＨ＿Ｒｖ：ＡＧＧＧＧＴＣＴＡＣＡＴＧＧＣＡＡＣＴＧ（配列番号１０） The primer sequences used are shown below:
Hs_ApoB_Fw: GGCTCACCCTGAGAGAAGTG (SEQ ID NO: 8)
Hs_ApoB_Rv: GCTGCTTTCTGGGAACCTCAC (SEQ ID NO: 8)
Hs_GAPDH_Fw: GGCCTCCCAAGGAGTAAGACC (SEQ ID NO: 9)
Hs_GAPDH_Rv: AGGGTCTCATAGGGCACTG (SEQ ID NO: 10)
得られた結果を図３に示す。図３において、「ＥｏＤＮＡ構造」は、実施例１７で合成されたオリゴヌクレオチドのうち、実施例９．４で得られた化合物２０（無置換体）に由来する構造を含むオリゴヌクレオチド；「Ｒ－ＭｅＥｏＤＮＡ構造」は、実施例１７で合成されたオリゴヌクレオチドのうち、実施例２．３で得られた化合物１１（（Ｒ）－メチル体）に由来する構造を含むオリゴヌクレオチド；「Ｓ－ＭｅＥｏＤＮＡ構造」は、実施例１７で合成されたオリゴヌクレオチドのうち、実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）に由来する構造を含むオリゴヌクレオチド；「メチレンＥｏＤＮＡ構造」は、実施例１７で合成されたオリゴヌクレオチドのうち、実施例１．７で得られた化合物８（エキソメチレン体）に由来する構造を含むオリゴヌクレオチド；「Ｒ－Ｍｅ－メチレン構造」は、実施例１７で合成されたオリゴヌクレオチドのうち、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）に由来する構造を含むオリゴヌクレオチド；そして「Ｓ－Ｍｅ－メチレン構造」は、実施例１７で合成されたオリゴヌクレオチドのうち、実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）に由来する構造を含むオリゴヌクレオチドを表す。
The obtained results are shown in FIG. In FIG. 3, “EoDNA structure” is an oligonucleotide comprising a structure derived from compound 20 (unsubstituted product) obtained in Example 9.4 among the oligonucleotides synthesized in Example 17; “MeEoDNA structure” is an oligonucleotide comprising a structure derived from compound 11 ((R) -methyl) obtained in Example 2.3 among the oligonucleotides synthesized in Example 17; “S-MeEoDNA structure” Is an oligonucleotide containing a structure derived from the compound 32 ((S) -methyl-exomethylene form) obtained in Example 11.6 among the oligonucleotides synthesized in Example 17; “Methylene EoDNA structure” "Is derived from compound 8 (exomethylene) obtained in Example 1.7 among the oligonucleotides synthesized in Example 17. The “R-Me-methylene structure” is the same as the compound 26 ((R) -methyl-exomethylene form) obtained in Example 10.6 among the oligonucleotides synthesized in Example 17. An oligonucleotide containing the derived structure; and “S-Me-methylene structure” refers to the compound 32 ((S) -methyl-exo) obtained in Example 11.6 of the oligonucleotides synthesized in Example 17. It represents an oligonucleotide containing a structure derived from a methylene form.
図３に示すように、最終濃度が１００ｎＭの場合、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）をアミダイトブロックとして用いて合成したオリゴヌクレオチドは、ＬＮＡを用いたものよりも強いＡｐｏＢ遺伝子発現抑制効果（アンチセンス効果）を示し、その他の新規架橋型核酸においてもＥＮＡまたはＬＮＡを用いたものと同様のアンチセンス効果を示した。最終濃度が４００ｎＭの場合、実施例１０．６で得られた化合物２６（（Ｒ）－メチル－エキソメチレン体）および実施例１１．６で得られた化合物３２（（Ｓ）－メチル－エキソメチレン体）を用いた各オリゴヌクレオチドは、ＬＮＡを用いたものよりも強いアンチセンス効果を示し、その他の新規架橋型核酸においてもＥＮＡまたはＬＮＡを用いたものと同様のアンチセンス効果を示した。
As shown in FIG. 3, when the final concentration was 100 nM, an oligonucleotide synthesized using the compound 26 ((R) -methyl-exomethylene) obtained in Example 10.6 as an amidite block, The ApoB gene expression suppression effect (antisense effect) was stronger than that used, and the other novel cross-linked nucleic acids also showed the same antisense effect as that using ENA or LNA. When the final concentration was 400 nM, Compound 26 ((R) -methyl-exomethylene) obtained in Example 10.6 and Compound 32 ((S) -methyl-exomethylene obtained in Example 11.6) Each of the oligonucleotides using the body exhibited a stronger antisense effect than that using LNA, and other novel cross-linked nucleic acids also showed the same antisense effect as that using ENA or LNA.
本発明によれば、６’位にヘテロ原子を有する新規な２’，４’－架橋型６員環ヌクレオシドおよびヌクレオチドが提供される。この２’，４’－架橋型人工ヌクレオチドを含むオリゴヌクレオチドは、従来の２’，４’－架橋型人工ヌクレオチドを含むオリゴヌクレオチドを匹敵する一本鎖ＲＮＡおよび二本鎖ＤＮＡに対する結合親和性を有する。本発明のオリゴヌクレオチドは、例えば、核酸医薬への素材として有用である。
According to the present invention, novel 2 ', 4'-bridged 6-membered ring nucleosides and nucleotides having a hetero atom at the 6'-position are provided. This oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide has a binding affinity for single-stranded RNA and double-stranded DNA comparable to conventional oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide. Have. The oligonucleotide of the present invention is useful as a material for nucleic acid medicine, for example.
Claims (6)
- 以下の式（Ｉ）または（Ｉ’）で表される化合物またはその塩：
Ｂａｓｅは、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ２およびＲ３は、それぞれ独立して、水素原子、核酸合成の水酸基の保護基、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数２から７のアルケニル基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール基、該α群から選択される任意の置換基を１以上有していてもよくそしてヘテロ原子を含んでいてもよい炭素数３から１２のアリール部分を有するアラルキル基、該α群から選択される任意の置換基を１以上有していてもよいアシル基、該α群から選択される任意の置換基を１以上有していてもよいシリル基、該α群から選択される任意の置換基を１以上有していてもよいリン酸基、核酸合成の保護基で保護されたリン酸基、－Ｐ（Ｒ４）Ｒ５［式中、Ｒ４およびＲ５は、それぞれ独立して、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）。 A compound represented by the following formula (I) or (I ′) or a salt thereof:
Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the α group, where The α group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A functionalized mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protective group for nucleic acid synthesis, and a halogen atom;
R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring An alkenyl group having 2 to 7 carbon atoms, an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents selected from the α group and may contain a hetero atom, an aralkyl group having an aryl moiety having 3 to 12 carbon atoms, which may have one or more arbitrary substituents selected from the α group and may contain a hetero atom, any arbitrary selected from the α group An acyl group optionally having one or more substituents, a silyl group optionally having one or more arbitrary substituents selected from the α group, and an arbitrary substituent selected from the α group are 1 Protection of phosphate groups and nucleic acid synthesis that may have In protected phosphate group, -P (R 4) R 5 [ wherein, R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, nucleic acid synthesis A mercapto group protected with a protecting group, an amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms Or a linear or branched alkylamino group having 1 to 6 carbon atoms]
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom). - 前記式（Ｉ）または（Ｉ’）において、Ｘが酸素原子である、請求項１に記載の化合物またはその塩。 The compound or a salt thereof according to claim 1, wherein, in the formula (I) or (I '), X is an oxygen atom.
- 前記式（Ｉ）または（Ｉ’）において、前記Ｂａｓｅが、６－アミノプリン－９－イル基、２，６－ジアミノプリン－９－イル基、２－アミノ－６－クロロプリン－９－イル基、２－アミノ－６－フルオロプリン－９－イル基、２－アミノ－６－ブロモプリン－９－イル基、２－アミノ－６－ヒドロキシプリン－９－イル基、６－アミノ－２－メトキシプリン－９－イル基、６－アミノ－２－クロロプリン－９－イル基、６－アミノ－２－フルオロプリン－９－イル基、２，６－ジメトキシプリン－９－イル基、２，６－ジクロロプリン－９－イル基、６－メルカプトプリン－９－イル基、２－オキソ－４－アミノ－１，２－ジヒドロピリミジン－１－イル基、４－アミノ－２－オキソ－５－フルオロ－１，２－ジヒドロピリミジン－１－イル基、４－アミノ－２－オキソ－５－クロロ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－メトキシ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－メルカプト－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－ヒドロキシ－１，２－ジヒドロピリミジン－１－イル基、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基、または４－アミノ－５－メチル－２－オキソ－１，２－ジヒドロピリミジン－１－イル基である、請求項１または２に記載の化合物またはその塩。 In the formula (I) or (I ′), the Base is a 6-aminopurin-9-yl group, a 2,6-diaminopurin-9-yl group, or a 2-amino-6-chloropurin-9-yl group. Group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-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-dihydropyrimidin-1-yl group, 4-amino-2-oxo-5- Fluoro-1,2-dihydropyrimidine-1- 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, 2-oxo-4-hydroxy-5-methyl-1 The compound or a salt thereof according to claim 1 or 2, which is a 1,2-dihydropyrimidin-1-yl group or a 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group. .
- 前記式（Ｉ）または（Ｉ’）において、前記Ｂａｓｅが、２－オキソ－４－ヒドロキシ－５－メチル－１，２－ジヒドロピリミジン－１－イル基である、請求項３に記載の化合物またはその塩。 The compound according to claim 3, wherein in the formula (I) or (I '), the Base is a 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group. Its salt.
- 以下の式（ＩＩ）または（ＩＩ’）で表されるヌクレオシド構造を少なくとも１つ含有するオリゴヌクレオチドまたはその薬理学上許容される塩：
Ｂａｓｅは、α群から選択される任意の置換基を１以上有していてもよいプリン－９－イル基または２－オキソ－１，２－ジヒドロピリミジン－１－イル基を表し、ここで、該α群は、水酸基、核酸合成の保護基で保護された水酸基、炭素数１から６の直鎖アルキル基、炭素数１から６の直鎖アルコキシ基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、炭素数１から６の直鎖アルキルチオ基、アミノ基、炭素数１から６の直鎖アルキルアミノ基、核酸合成の保護基で保護されたアミノ基、およびハロゲン原子からなり；
Ｒ６およびＲ７は、それぞれ独立して、水素原子；水酸基；分岐または環を形成していてもよい炭素数１から７のアルキル基；分岐または環を形成していてもよい炭素数１から７のアルコキシ基；アミノ基；および核酸合成の保護基で保護されたアミノ基；からなる群から選択される基であるか、あるいは、Ｒ６およびＲ７は一緒になって、＝Ｃ（Ｒ１０）Ｒ１１［式中、Ｒ１０およびＲ１１は、それぞれ独立して、水素原子、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１から６の直鎖または分岐鎖アルコキシ基、炭素数１から６の直鎖または分岐鎖アルキルチオ基、炭素数１から６のシアノアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルアミノ基を表す］を表し；
Ｒ８およびＲ９は、それぞれ独立して、水素原子、分岐または環を形成していてもよい炭素数１から７のアルキル基、分岐または環を形成していてもよい炭素数１から７のアルコキシ基、または炭素数１から６の直鎖または分岐鎖アルキルチオ基を表し；そして
Ｘは酸素原子または硫黄原子である）。 Oligonucleotide or pharmacologically acceptable salt thereof containing at least one nucleoside structure represented by the following formula (II) or (II ′):
Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the α group, where The α group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis. A functionalized mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, a linear alkylamino group having 1 to 6 carbon atoms, an amino group protected with a protective group for nucleic acid synthesis, and a halogen atom;
R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring; A group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ═C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid synthesis. An amino group, a linear or branched alkoxy group having 1 to 6 carbon atoms, a linear or branched alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a straight chain having 1 to 6 carbon atoms. Chain or branch Represents a chain alkylamino group];
R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring. Represents an alkoxy group or a linear or branched alkylthio group having 1 to 6 carbon atoms; and X is an oxygen atom or a sulfur atom). - 前記式（ＩＩ）または（ＩＩ’）において、Ｘが酸素原子である、請求項５に記載のオリゴヌクレオチドまたはその薬理学上許容される塩。 The oligonucleotide or a pharmacologically acceptable salt thereof according to claim 5, wherein, in the formula (II) or (II '), X is an oxygen atom.
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| WO2018155450A1 (en) | 2017-02-21 | 2018-08-30 | 国立大学法人大阪大学 | Antisense oligonucleic acid |
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| WO2019009298A1 (en) * | 2017-07-05 | 2019-01-10 | 国立大学法人大阪大学 | α-SYNUCLEIN EXPRESSION INHIBITOR |
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