WO2015125845A1 - Phosphate site modification of nucleic acid containing nitrogen-containing non-aromatic heterocycle - Google Patents

Phosphate site modification of nucleic acid containing nitrogen-containing non-aromatic heterocycle Download PDF

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WO2015125845A1
WO2015125845A1 PCT/JP2015/054539 JP2015054539W WO2015125845A1 WO 2015125845 A1 WO2015125845 A1 WO 2015125845A1 JP 2015054539 W JP2015054539 W JP 2015054539W WO 2015125845 A1 WO2015125845 A1 WO 2015125845A1
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substituted
unsubstituted
oligonucleotide
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salt
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釘宮　啓
典一 黒田
中村　淳
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塩野義製薬株式会社
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    • 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
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • 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
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone

Definitions

  • the present invention relates to a nucleic acid having a novel phosphate site modification. More specifically, the present invention relates to an oligonucleotide having a phosphate site modification containing a monocyclic or bicyclic nitrogen-containing non-aromatic heterocycle. Further, the present invention relates to an amidite used for preparing the oligonucleotide.
  • the antisense oligonucleotide is an oligonucleotide complementary to ncRNA (non-coding RNA) such as mRNA, mRNA precursor or ribosomal RNA, transfer RNA, miRNA and the like of a target gene, and consists of about 8 to 30 bases. Strand DNA, RNA and / or structural analogs thereof. The function of mRNA, mRNA precursor or ncRNA is suppressed by forming a double strand with the mRNA, mRNA precursor or ncRNA targeted by the antisense oligonucleotide.
  • ncRNA non-coding RNA
  • SiRNA is a small double-stranded RNA consisting of about 19 to 25 base pairs homologous to the target gene. It is involved in a phenomenon called RNA interference and suppresses gene expression by degrading mRNA in a base sequence-specific manner.
  • Ribozymes are RNAs that have enzymatic activity to cleave nucleic acids. It forms a double strand with the mRNA of the target gene and specifically cleaves the mRNA.
  • Antigenes are oligonucleotides that correspond to the double stranded DNA site of the target gene. Transcription from DNA to mRNA is suppressed by forming a triplex with the DNA site and oligonucleotide.
  • Aptamers are DNA, RNA and / or structural analogs thereof that specifically bind to a specific molecule. By binding to the target protein, the function of the protein is inhibited.
  • Decoy nucleic acids are short DNAs that contain the same sequence as the binding site for a particular transcriptional regulator. The binding between the transcriptional regulatory factor and the gene is inhibited, and the expression of a target gene activated by the transcriptional regulatory factor is suppressed.
  • nucleic acid drugs are difficult to put to practical use because they are easily degraded by nucleases in living bodies and the efficiency of incorporation into target cells is low.
  • chemical modification of the base part, sugar part and phosphate part of nucleic acid, which is an active ingredient has been studied for many years. By performing the modification, the target gene expression suppression activity increases, that is, there is a modification that can be used as an active ingredient of a nucleic acid pharmaceutical.
  • Patent Document 3 describes an oligonucleotide having a phosphate site modification of a nucleic acid containing a nitrogen-containing chain. However, the oligonucleotide is used as a prodrug of a nucleic acid pharmaceutical, and the modification is eliminated when metabolized in the body.
  • Non-Patent Document 1 describes a nucleic acid in which a terminal phosphate site is substituted with a phosphate protecting group having phenothiazine. The protecting group is used as a label.
  • amidites used for preparing nucleic acids amidites having a protecting group containing pyrrolidine at the phosphate site (Patent Document 4) and amidites having a protecting group containing pyridine (Non-Patent Document 2) are known. Yes.
  • the protecting group is a modification for improving stability during synthesis of the oligonucleotide, and the protecting group is removed when the oligonucleotide is separated from the carrier. Therefore, these phosphate site modifications do not have a function as an active ingredient of a nucleic acid pharmaceutical, that is, a function of increasing the target gene expression suppressing activity or the target protein function inhibiting activity.
  • An object of the present invention is to synthesize an oligonucleotide having a novel phosphate site modification that can be used as a nucleic acid drug including an antisense oligonucleotide, siRNA, ribozyme, antigene, aptamer, decoy nucleic acid, and the like, and the oligonucleotide. It is to provide a novel amidite that can be used as a material.
  • oligonucleotide of the present invention has been found to have improved target gene expression-suppressing activity as compared to an oligonucleotide in which all phosphate sites are phosphorothioates.
  • the present inventors have devised the modification position and the number of modifications of the oligonucleotide of the present invention, so that compared to oligonucleotides in which all phosphate sites are phosphorothioates, in the biological experiment using mice, It was found that the retention was improved and the organ (liver and lung) distribution changed greatly, that is, the pharmacokinetics changed significantly.
  • oligonucleotides having the same sequence but containing a bond represented by the following formula (I) are: As the amount of the bond represented by the following formula (I) increases, accumulation in mouse organs (liver and lung) increases.
  • the bond represented by the following formula (I) By utilizing the oligonucleotide of the present invention, that is, the bond represented by the following formula (I), a result different from the organ accumulation property of the original sequence can be obtained, so that it can be efficiently delivered to the target organ. It was suggested that the expression of the target gene or target protein can be effectively suppressed.
  • the oligonucleotide of this invention is very useful as a nucleic acid pharmaceutical containing an antisense oligonucleotide etc.
  • the oligonucleotide of the present invention is stable without any by-product by a general synthesis method using a compound containing a group represented by the following formula (II) and a nucleoside structure (hereinafter, “amidite of the present invention”). And can be synthesized.
  • the amidite of the present invention is very useful as a material for synthesizing nucleic acid pharmaceuticals containing antisense oligonucleotides and the like.
  • X 2 is substituted or unsubstituted aziridinyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted piperidyl, substituted or unsubstituted piperidino, substituted or unsubstituted piperazinyl, substituted Or the substituted piperazino, substituted or unsubstituted morpholinyl, substituted or unsubstituted morpholino, substituted or unsubstituted azepanyl, or substituted or unsubstituted quinuclidinyl, or the pharmaceutically acceptable oligonucleotide thereof (1) salt.
  • oligonucleotide or pharmaceutically acceptable salt thereof according to (1) or (2), wherein X 1 is absent or unsubstituted alkylene.
  • (4) The oligonucleotide according to any one of (1) to (3) or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide has a length of 8 to 25 bases.
  • (5) containing one or more sugar-modified nucleosides having a substituent at the 2′-position of the sugar or a sugar-modified nucleoside having a cross-linked structure between the 4′-position and the 2′-position of the sugar; (1) to the oligonucleotide according to any one of (4) or a pharmaceutically acceptable salt thereof.
  • the crosslinked structure is 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′—C ( ⁇ O) —NR 1 -2 ′ (R 1 is The oligonucleotide or pharmaceutically acceptable salt thereof according to (5), which is a hydrogen atom or alkyl).
  • the nucleoside structure has the formula (III): (Where: Bx is a hydrogen or nucleobase moiety; Z is a hydrogen atom or a hydroxyl protecting group, R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, R 5 is hydrogen; R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy, Here, R 5 and R 6 may be combined to form a crosslinked structure, R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl)
  • R 6 is F, O
  • the crosslinked structure is 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′—C ( ⁇ O) —NR 1 -2 ′ (R 1 is The compound according to (10), which is a hydrogen atom or alkyl, or a salt thereof.
  • the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl.
  • 2,4-dinitrophenyl benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, levulinoyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl , Triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl, 9-fluorenylmethyloxycarbonyl, methanesulfonyl, p-toluenesulfur Nyl, trifluoromethanesulfonyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phen
  • the oligonucleotide of the present invention comprises Exhibit superior expression suppression activity against the target gene, and
  • the oligonucleotide of the present invention improves plasma retention by devising the modification position and the number of modifications, for example, can increase accumulation in organs such as the liver or lung, and efficiently target the target organ Since it is delivered and it is possible to effectively suppress the expression of the target gene or target protein, application to nucleic acid pharmaceuticals is expected.
  • Nucleic acid is a high-molecular substance composed of a nucleotide, which is a compound in which a phosphate moiety is bonded to a sugar molecule of a nucleoside, which is a compound in which a nucleobase moiety and a sugar moiety are covalently bonded.
  • Nucleobase moiety means a substituent comprising a nucleobase or an analog thereof. Natural nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) or uracil (U). The nucleobases of the present invention are not limited to these, but also include other artificial or natural nucleobases. For example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone Etc.
  • the “nucleobase moiety” in the present invention is a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted carbocyclic group that constitutes the base part of a nucleic acid (DNA, RNA).
  • the heterocyclic ring includes a single ring or a condensed ring having one or more of the same or different heteroatoms arbitrarily selected from O, S and N in the ring.
  • the carbocycle includes a monocyclic or condensed hydrocarbon ring. Examples thereof include benzene, naphthalene, anthracene, phenanthrene, indane, indene, tetrahydronaphthylene, biphenylene and the like. Preferred is benzene or naphthalene.
  • the substituent of the heterocyclic group or carbocyclic group include substituents included in the substituent group ⁇ . The carbon atom at any position may be bonded to one or more substituents selected from the substituent group ⁇ .
  • Substituent group ⁇ halogen, hydroxy, hydroxyl group protected with a protecting group used for nucleic acid synthesis, alkyl, alkyloxy, alkylthio, alkylamino, alkenyl, alkynyl, mercapto, mercapto protected with a protecting group used for nucleic acid synthesis , Amino, amino protected with a protecting group used in nucleic acid synthesis.
  • the protecting group of “hydroxyl group protected by a protecting group used for nucleic acid synthesis” is not particularly limited as long as it can stably protect a hydroxyl group during nucleic acid synthesis. Specifically, it is 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 include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, formyl, and the following protecting groups.
  • Aliphatic acyl alkylcarbonyl (eg, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyl Octanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexayl Decanoyl, octadecanoyl, 1-methylheptadecanoyl, non
  • Aromatic acyl Aromatic carbocyclic group carbonyl (eg, benzoyl, ⁇ -naphthoyl, ⁇ -naphthoyl, etc.), aromatic carbocyclic group carbonyl substituted with halogen (eg, 2-bromobenzoyl, 4-chlorobenzoyl) Etc.), an aromatic carbocyclic group carbonyl substituted with alkyl (eg 2,4,6-trimethylbenzoyl, 4-toluoyl etc.), an aromatic carbocyclic group carbonyl substituted with alkyloxy (eg 4 -Anisoyl etc.), carboxy substituted aromatic carbocyclic groups carbonyl (2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl etc.), nitro substituted aromatic carbocyclic groups carbonyl (4- Nitrobenzoyl, 2-nitrobenzoyl, etc.) Aromatic carbocyclic substituted with alkyloxycarbonyl
  • Tetrahydropyranyl tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl and the like.
  • Tetrahydrothiopyranyl tetrahydrothiopyran-2-yl, 4-methoxytetrahydrothiopyran-4-yl and the like.
  • Tetrahydrofuranyl tetrahydrofuran-2-yl and the like.
  • Tetrahydrothiofuranyl tetrahydrothiofuran-2-yl and the like.
  • Trialkylsilyl (trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, etc.) substituted with 1 to 2 aromatic carbocyclic groups
  • Trialkylsilyl (diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl, etc.) and the like.
  • Alkyloxymethyl methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl and the like.
  • Alkyloxylated alkyloxymethyl 2-methoxyethoxymethyl and the like.
  • Halogenoalkyloxymethyl 2,2,2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl and the like.
  • Alkyloxylated ethyl 1-ethoxyethyl, 1- (isopropoxy) ethyl and the like.
  • Ethyl halide 2,2,2-trichloroethyl and the like.
  • Methyl substituted with 1 to 3 aromatic carbocyclic groups benzyl, ⁇ -naphthylmethyl, ⁇ -naphthylmethyl, diphenylmethyl, triphenylmethyl, ⁇ -naphthyldiphenylmethyl, 9-anthrylmethyl and the like.
  • Methyl substituted with 1 to 3 aromatic carbocyclic groups in which the aromatic carbocycle is substituted with alkyl, alkyloxy, halogen or cyano 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3, 4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4 -Cyanobenzyl and the like.
  • Alkyloxycarbonyl methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl and the like.
  • Aromatic carbocyclic groups substituted with halogen, alkyloxy or nitro 4-chlorophenyl, 2-fluorophenyl, 4-methoxyphenyl, 4-nitrophenyl, 2,4-dinitrophenyl and the like.
  • Alkyloxycarbonyl substituted with a halogen or trialkylsilyl group 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl and the like.
  • Alkenyloxycarbonyl vinyloxycarbonyl, aromatic carbocyclic group oxycarbonyl and the like.
  • Aralkyloxycarbonyl optionally substituted with 1 to 2 alkyloxy or nitro aromatic carbocycles: benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxy Carbonyl, 4-nitrobenzyloxycarbonyl and the like.
  • Preferred protecting groups include alkyl, alkenyl, “aliphatic acyl”, “aromatic acyl”, “methyl substituted with 1 to 3 aromatic carbocyclic groups”, “substituted with halogen, alkyloxy or nitro” Aromatic carbocyclic group "and the like. More preferred are benzoyl, benzyl, 2-chlorophenyl, 4-chlorophenyl, 2-propenyl and the like.
  • the protecting group of “mercapto protected with a protecting group used for nucleic acid synthesis” is not particularly limited as long as it can stably protect mercapto during nucleic acid synthesis. Specifically, it is 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. For example, in addition to those mentioned as the protective group for the hydroxyl group, the following are also included. Groups that form disulfides: alkylthio (methylthio, ethylthio, tert-butylthio, etc.), aromatic carbocyclic groups thio (benzylthio, etc.), etc.
  • Preferable protecting groups include “aliphatic acyl”, “aromatic acyl” and the like. More preferably, benzoyl etc. are mentioned.
  • the protecting group of “amino protected with a protecting group used for nucleic acid synthesis” is not particularly limited as long as it can stably protect amino during nucleic acid synthesis. Specifically, it is 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.
  • aliphatic acyl formyl, “aliphatic acyl”, “aromatic acyl”, “alkyloxycarbonyl”, “alkyloxycarbonyl substituted with a halogen or trialkylsilyl group”, “alkenyloxy” mentioned as the protective group for the above hydroxyl group Carbonyl ",” aralkyloxycarbonyl optionally substituted with 1 to 2 alkyloxy or nitro aromatic carbocycle ".
  • Preferable protecting groups include “aliphatic acyl”, “aromatic acyl” and the like. More preferably, benzoyl etc. are mentioned.
  • nucleobase moiety is preferably substituted or unsubstituted purin-9-yl, substituted or unsubstituted 2-oxo-pyrimidin-1-yl, and the like.
  • ring substituent contained in the nucleobase moiety include those contained in the above substituent group ⁇ .
  • the carbon atom at any position may be bonded to one or more substituents selected from the substituent group ⁇ . More preferred is purin-9-yl or 2-oxo-pyrimidin-1-yl substituted with one or more substituents selected from the above substituent group ⁇ .
  • 6-aminopurin-9-yl ie, adeninyl
  • 6-aminopurin-9-yl 2,6-diaminopurin-9-yl
  • amino protected with a protecting group used in nucleic acid synthesis amino 2,6-diaminopurin-9-yl, 6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, amino protected with a protecting group used for nucleic acid synthesis
  • 2-Amino-6-chloropurin-9-yl, 6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl, amino protected with the protecting groups used are used for nucleic acid synthesis
  • R a is a hydrogen atom or alkyl
  • R b is a hydrogen atom or alkyl.
  • a group represented by R a is preferably a hydrogen atom or C1-C5 alkyl. More preferably, they are a hydrogen atom or methyl.
  • R b is preferably a hydrogen atom.
  • R c is a hydrogen atom, halogen or alkyl, R d is amino, mercapto, alkyloxy, NHCOR e , NHCOCH 2 OR e or N ⁇ NR f ; R e is a substituted or unsubstituted alkyl or a substituted or unsubstituted aromatic carbocyclic group; R f is a hydrogen atom or alkyl)
  • a group represented by R c is preferably a hydrogen atom or C1-C5 alkyl. More preferably, they are a hydrogen atom or methyl.
  • R d is preferably NHCOPh, NHCOCH 3 , NHCOCH 2 OPh, NHCOCH 2 O— (4-tBu) Ph.
  • R h is a hydrogen atom, halogen, amino or alkyloxy, R i is a substituted or unsubstituted alkyl or a substituted or unsubstituted aromatic carbocyclic group;
  • R j is a hydrogen atom or alkyl
  • a group represented by R g is preferably NHCOPh, NHCOCH 3 , NHCOCH 2 OPh, NHCOCH 2 O— (4-tBu) Ph.
  • R h is preferably a hydrogen atom.
  • R k is amino, NHCOR m , NHCOCH 2 OR m or N ⁇ NR n ;
  • R m is a substituted or unsubstituted alkyl or a substituted or unsubstituted aromatic carbocyclic group;
  • R n is a hydrogen atom or alkyl
  • a group represented by R k is preferably NHCOPh, NHCOCH 3 , NHCOCH (CH 3 ) 2 , NHCOCH 2 OPh, NHCOCH 2 O— (4-tBu) Ph.
  • R ′ is a hydrogen atom or an amino protecting group used for nucleic acid synthesis. Examples thereof include isobutyl, acetyl, benzoyl, phenoxyacetyl, etc.
  • “Sugar moiety” means a substituent containing a sugar or analog thereof contained in a natural nucleic acid. “Substituents of sugars contained in natural nucleic acids” include the following sugar sites of DNA And the following sugar sites of RNA: Is mentioned.
  • substituted containing a sugar analogue examples include a modified sugar containing a 5-membered ring, a modified sugar containing a 6-membered ring such as tetrahydropyran, and the like used in this field. Modified examples of sugars containing a ring or 3 rings (see, for example, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
  • modified sugar containing a 5-membered ring examples include the following. formula: (Where: R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, R 5 is hydrogen; R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy, Here, R 5 and R 6 may be combined to form a crosslinked structure, R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl) A group represented by:
  • R 6 examples include F, OCH 3 (OMe), OCH 2 CH 2 OCH 3 (MOE), and the like.
  • R 5 and R 6 may form a cross-linked structure together” includes, for example, a cross-linked structure between the 4′-position and the 2′-position of the following sugar.
  • 4 ′-(CR 8 R 9 ) mO-2 ′, 4 ′-(CR 8 R 9 ) m-S-2 ′, 4 ′-(CR 8 R 9 ) m—O—C ( ⁇ O) -2 ', 4 ′-(CR 8 R 9 ) m—NR 1 —O— (CR 8 R 9 ) m 1 ⁇ 2 ′, 4 ′-(CR 8 R 9 ) m 1 —C ( ⁇ O) —NR 1 ⁇ 2 '4'-(CR 8 R 9 ) m 2 -C ( O) -NR 1 -X 3 -2 ', 4'-(CR 8 R 9 ) m 1 -SO 2 -NR 1 -2 ', Or And here, X 3 is O, S, NH or CH 2 , R 1 is
  • R 1 is preferably a hydrogen atom, alkyl, alkenyl, alkynyl, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocycle It is alkyl, non-aromatic carbocyclic alkyl, aromatic heterocyclic alkyl or non-aromatic heterocyclic alkyl, and may have one or more arbitrary substituents selected from the ⁇ group.
  • the ⁇ group is a hydroxyl group, alkyl, alkyloxy, mercapto, alkylthio, amino, alkylamino, or halogen.
  • R 8 and R 9 are preferably a hydrogen atom.
  • the cross-linked structure is preferably 4 ′-(CR 8 R 9 ) m—O-2 ′ or 4 ′-(CR 8 R 9 ) m 1 —C ( ⁇ O) —NR 1 ⁇ 2 ′ (AmNA, Bridged nucleic acid) here
  • R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl
  • Each R 8 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl
  • Each R 9 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl
  • m is an integer
  • the cross-linked structure is particularly preferably 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′-C ( ⁇ O) —NR 1 -2 ′ (R 1 Is a hydrogen atom or alkyl).
  • 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4)
  • 4′—CH 2 —O-2 ′ (LNA, Locked Nucleic Acid) is particularly preferable.
  • Specific examples and preparation methods thereof are described in International Publication No. 98/39352, International Publication No. 2003/066875, International Publication No. 2005/021570, and the like.
  • 4′-C ( ⁇ O) —NR 1 ⁇ 2 ′ (R 1 is a hydrogen atom or alkyl)
  • 4′—C ( ⁇ O) —NCH 3 ⁇ 2 ′ is particularly preferable. Specific examples and preparation methods thereof are described in International Publication No. 2011/052436.
  • a group wherein at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is methyl, A group wherein R 13 or R 14 is F; A group wherein R 13 is OCH 3 (OMe) and R 14 is hydrogen; Examples include a group in which R 13 is OCH 2 CH 2 OCH 3 (MOE) and R 14 is hydrogen.
  • benzyl triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl and the like.
  • Halogen includes fluorine atom, chlorine atom, bromine atom and iodine atom. In particular, a fluorine atom and a chlorine atom are preferable.
  • Alkyl includes straight or branched hydrocarbon groups having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. To do. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl , Isooctyl, n-nonyl, n-decyl and the like.
  • alkyl examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and n-pentyl. Further preferred examples include methyl, ethyl, n-propyl, isopropyl and tert-butyl.
  • Alkenyl has 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms, having one or more double bonds at any position. These linear or branched hydrocarbon groups are included.
  • alkenyl include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, decenyl, tridecenyl, decenyl Etc.
  • alkenyl include vinyl, allyl, propenyl, isopropenyl and butenyl.
  • Alkynyl has 2 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, having one or more triple bonds at any position. Includes straight chain or branched hydrocarbon groups. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl and the like. These may further have a double bond at an arbitrary position. Preferred embodiments of “alkynyl” include ethynyl, propynyl, butynyl and pentynyl.
  • Alkylene is a straight or branched divalent hydrocarbon having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. Includes groups. Examples include methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene and the like.
  • Haloalkyl means a group in which one or more of the “halogen” is bonded to the “alkyl”. For example, monofluoromethyl, monofluoroethyl, monofluoropropyl, 2,2,3,3,3-pentafluoropropyl, monochloromethyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2, Examples include 2,2-trichloroethyl, 1,2-dibromoethyl, 1,1,1-trifluoropropan-2-yl and the like. Preferable embodiments of “haloalkyl” include trifluoromethyl and trichloromethyl.
  • Alkyloxy means a group in which the above “alkyl” is bonded to an oxygen atom. Examples thereof include methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy, tert-butyloxy, isobutyloxy, sec-butyloxy, pentyloxy, isopentyloxy, hexyloxy and the like. Preferable embodiments of “alkyloxy” include methoxy, ethoxy, n-propyloxy, isopropyloxy, tert-butyloxy.
  • Alkylthio means a group in which the above “alkyl” is bonded to a sulfur atom.
  • Alkylamino includes monoalkylamino and dialkylamino.
  • “Monoalkylamino” means a group in which the above “alkyl” is replaced with one hydrogen atom bonded to the nitrogen atom of the amino group.
  • methylamino, ethylamino, isopropylamino and the like can be mentioned.
  • methylamino and ethylamino are used.
  • “Dialkylamino” means a group in which the above “alkyl” is replaced with two hydrogen atoms bonded to the nitrogen atom of the amino group. The two alkyls may be the same or different.
  • Examples include dimethylamino, diethylamino, N, N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-ethylamino and the like. Preferable examples include dimethylamino and diethylamino.
  • Alkylcarbonylamino means a group in which alkylcarbonyl is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the amino group. In the case of two, each alkylcarbonyl group may be the same or different. For example, methylcarbonylamino, ethylcarbonylamino, propylcarbonylamino, isopropylcarbonylamino, tert-butylcarbonylamino, isobutylcarbonylamino, sec-butylcarbonylamino, dimethylcarbonylamino, diethylcarbonylamino, N, N-diisopropylcarbonylamino, etc. Is mentioned. Preferable embodiments of “alkylcarbonylamino” include methylcarbonylamino and ethylcarbonylamino.
  • Alkenylcarbonylamino means a group in which alkenylcarbonyl is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the amino group. In the case of two, each alkenylcarbonyl group may be the same or different. For example, vinylcarbonylamino, propenylcarbonylamino and the like can be mentioned.
  • Alkynylcarbonylamino means a group in which alkynylcarbonyl is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the amino group. In the case of two, each alkynylcarbonyl group may be the same or different. For example, ethynylcarbonylamino, propynylcarbonylamino and the like can be mentioned.
  • Alkylcarbamoyl means a group in which the above “alkyl” is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the carbamoyl group. In the case of two, each alkyl group may be the same or different. For example, methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl and the like can be mentioned.
  • Alkenylcarbamoyl means a group in which the above “alkenyl” is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the carbamoyl group. In the case of two, each alkenyl group may be the same or different. Examples thereof include vinyl carbamoyl, propenyl carbamoyl and the like.
  • Alkynylcarbamoyl means a group in which the above “alkynyl” is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the carbamoyl group. In the case of two, each alkynyl group may be the same or different. For example, ethynylcarbamoyl, propynylcarbamoyl and the like can be mentioned.
  • “Aromatic carbocyclic group” means a monocyclic or bicyclic or more cyclic aromatic hydrocarbon group. For example, phenyl, naphthyl, anthryl, phenanthryl and the like can be mentioned. A preferred embodiment of the “aromatic carbocyclic group” includes phenyl.
  • non-aromatic carbocyclic group means a cyclic saturated hydrocarbon group or a cyclic non-aromatic unsaturated hydrocarbon group having one or more rings.
  • the non-aromatic carbocyclic group having two or more rings includes a monocyclic ring or a non-aromatic carbocyclic group having two or more rings condensed with the ring in the above “aromatic carbocyclic group”.
  • the “non-aromatic carbocyclic group” includes a group which forms a bridge or a spiro ring as described below.
  • the monocyclic non-aromatic carbocyclic group preferably has 3 to 16 carbon atoms, more preferably 3 to 12 carbon atoms, and still more preferably 4 to 8 carbon atoms.
  • Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, and the like.
  • Examples of the two or more non-aromatic carbocyclic groups include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl, fluorenyl and the like.
  • “Aromatic heterocyclic group” means a monocyclic or bicyclic or more aromatic cyclic group having one or more heteroatoms arbitrarily selected from O, S and N in the ring. To do.
  • the aromatic heterocyclic group having two or more rings includes those obtained by condensing a ring in the above “aromatic carbocyclic group” to a monocyclic or two or more aromatic heterocyclic group.
  • the monocyclic aromatic heterocyclic group is preferably 5 to 8 members, more preferably 5 or 6 members.
  • Examples include pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl and the like.
  • bicyclic aromatic heterocyclic group examples include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzisoxazolyl, Oxazolyl, benzoxiadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyr Dazinyl, oxazolopyridyl, thiazolopyridyl and the like can be mentioned
  • aromatic heterocyclic group having 3 or more rings examples include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, dibenzofuryl and the like.
  • non-aromatic heterocyclic group is a monocyclic or bicyclic or more non-aromatic cyclic group having one or more of the same or different heteroatoms arbitrarily selected from O, S and N in the ring Means.
  • the non-aromatic heterocyclic group having two or more rings includes the above-mentioned “aromatic carbocyclic group”, “non-aromatic carbocyclic group”, and monocyclic or two or more non-aromatic heterocyclic groups, and Also included are those in which each ring in the “aromatic heterocyclic group” is condensed.
  • the “non-aromatic heterocyclic group” also includes a group that forms a bridge or a spiro ring as described below.
  • the monocyclic non-aromatic heterocyclic group is preferably 3 to 8 members, more preferably 5 or 6 members.
  • the carbon atom at any position may be bonded to one or more groups selected from the following substituents.
  • substituents halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkeny
  • substituent on the ring of “aromatic carbocycle” of “substituted or unsubstituted aromatic carbocyclic group” include the following substituents.
  • An atom at any position on the ring may be bonded to one or more groups selected from the following substituents.
  • Substituents halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkyloxyalkyl, alkylcarbonyl, alkenylcarbonyl, Alkynylcarbonyl, monoalkylamino, dialkylamin
  • the oxygen atom of “substituted or unsubstituted alkylene with O intervening” is present between carbon atoms of alkylene, or when present at the terminal, the oxygen atom is bonded to X 2 .
  • the oxygen atom of “substituted or unsubstituted alkylene intervening O” is not bonded to the oxygen atom described in the above formula (I).
  • “O-mediated substituted or unsubstituted alkylene” means, for example, Formula:-(CR 15 R 16 -CR 17 R 18 -O) n- (Where Each R 15 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl; Each R 16 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl; Each R 17 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl; Each R 18 is independently a hydrogen atom, halogen, cyano, substituted or un
  • X 1 is absent or is a substituted or unsubstituted alkylene which may be mediated by O, preferably absent or unsubstituted alkylene, more preferably absent or C1- C5 alkylene.
  • X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group, preferably substituted or unsubstituted aziridinyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, Substituted or unsubstituted piperidyl, substituted or unsubstituted piperidino, substituted or unsubstituted piperazinyl, substituted or unsubstituted piperazino, substituted or unsubstituted morpholinyl, substituted or unsubstituted morpholino, substituted or unsubstituted azepanyl or substituted Or
  • Oligonucleotide means nucleotides in which a plurality of identical or different nucleosides are bound via the same or different phosphate sites.
  • the oligonucleotide of the present invention is an oligonucleotide containing at least one bond represented by formula (I) at an arbitrary position.
  • the position and number of the bond represented by the formula (I) are not particularly limited, and can be appropriately designed according to the purpose.
  • the oligonucleotide of the present invention can be used as a nucleic acid pharmaceutical including siRNA, ribozyme, antigene, aptamer, decoy nucleic acid and the like.
  • the bond represented by formula (I) is a sequence that acts as an active ingredient of a nucleic acid pharmaceutical (for example, a sequence that binds to the mRNA of a target gene), a complementary strand of the sequence, and a sequence that can be included before and after the sequence and the complementary strand. (For example, a sequence that does not bind to the mRNA of the target gene, an abasic cation unit, etc.) may be contained.
  • the oligonucleotide of the present invention is particularly preferably used as a nucleic acid pharmaceutical comprising an antisense oligonucleotide.
  • the oligonucleotide of the present invention may be single-stranded or double-stranded. It may be a ribbon type in which both ends of the double strand are connected via a loop structure. In the case of a double strand, the bond represented by the formula (I) may be contained only in one chain, or the bond represented by the formula (I) may be contained in both chains. Examples of the case where the oligonucleotide of the present invention is single-stranded include antisense oligonucleotides, ribozymes, aptamers and the like.
  • the bond represented by the formula (I) is either a sequence that binds to the mRNA of the target gene or a sequence that can be included before or after the target gene. It may be contained.
  • the oligonucleotide of the present invention is double-stranded include siRNA, antigene, and decoy nucleic acid.
  • the double-stranded nucleic acid pharmaceutical containing the antisense oligonucleotide and the double stranded nucleic acid which consists of a nucleic acid complementary to this antisense oligonucleotide is mentioned.
  • Specific examples of the pharmaceutical composition and the preparation method thereof are described in Example 4 of the present specification, International Publication No. 2013/0889283 and the like.
  • the nucleic acid pharmaceutical may be bound with a ligand such as tocophenol (International Publication No. 2013/0889283) or GalNac derivative (PCT / JP2015 / 050083). Highly and efficiently delivered to the target organ, and the antisense oligonucleotide can effectively suppress the expression of the target gene.
  • the double-stranded oligonucleotide of the present invention is a double-stranded nucleic acid pharmaceutical comprising an antisense oligonucleotide and a double-stranded nucleic acid composed of a nucleic acid complementary to the antisense oligonucleotide, it is represented by the formula (I)
  • the bond may be contained in any of an antisense oligonucleotide (sequence that binds to the mRNA of the target gene), a complementary nucleic acid, a sequence that can be included before and after the antisense oligonucleotide and the complementary strand.
  • the bond represented by the formula (I) is contained in a sequence that can be contained before and after the antisense oligonucleotide and the complementary strand
  • the bond represented by the formula (I) is the 3′-terminal side of the complementary strand. May be contained in any of the sequences of 5′-terminal side, or may be contained in both of the terminal sequences.
  • the length of the oligonucleotide of the present invention is not particularly limited as long as it is pharmaceutically acceptable.
  • 8 to 25 bases 8 to 19 bases, 10 to 19 bases, 13 to 19 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases or 19 bases.
  • the oligonucleotide of the present invention contains at least one bond represented by the formula (I), it has a nucleotide modification known in the art even if the other part is the same as the natural nucleic acid. It may be.
  • the phosphate site other than the bond represented by the formula (I) in the oligonucleotide of the present invention include a phosphodiester bond, S-oligo (for example, phosphorothioate), M-oligo (for example, methylphosphonate) possessed by natural nucleic acids. ), Boranophosphate and the like.
  • the bond between nucleosides other than the bond represented by the formula (I) in the oligonucleotide of the present invention may be a bond having no phosphorus atom as long as it is a bond known in the art.
  • Examples include, but are not limited to, alkyl, non-aromatic carbocycle, haloalkyl, non-aromatic carbocycle substituted with halogen, and the like.
  • Examples include siloxane, sulfide, sulfoxide, sulfone, acetyl, acetyl formate, acetyl thioformate, acetyl methylene formate, acetyl thioformate, alkenyl, sulfamate, methylene imino, methylene hydrazino, sulfonate, sulfonamide, amide.
  • nucleobase portion of the oligonucleotide of the present invention is the same as the “nucleobase portion” described above. *
  • the oligonucleotide of the present invention may contain an abasic cation unit lacking the nucleobase portion of the nucleoside.
  • the position and number of abasic cation units are not particularly limited and can be appropriately designed according to the purpose. At that time, a hydrogen atom is bonded to the “nucleobase moiety”.
  • the sugar moiety in the oligonucleotide of the present invention is the same as the “sugar moiety” described above.
  • the oligonucleotide of the present invention includes both the sugar and its analogs contained in natural nucleic acids.
  • Examples of the pattern of sugars and analogs contained in natural nucleic acids include gapmers and mixmers.
  • a gapmer includes a central region (“gap”) and regions on both sides of the central region, wings (“5 ′ wing” on the 5 ′ side or “3 ′ wing” on the 3 ′ side), and each wing has at least An oligonucleotide containing one analog is meant.
  • “5 ′ wing” and / or “3 ′ wing” contains one or more analogs, preferably 1 to 5, more preferably 2 to 3.
  • the type, number and position of analogs in one wing may be the same or different from the type, number and position of analogs in the other wing.
  • a mixmer means an oligonucleotide containing analogs at random.
  • the oligonucleotide of the present invention is an antisense oligonucleotide, it is particularly preferably a gapmer.
  • Preferred examples of the sugar analogue used in the oligonucleotide of the present invention include the above-mentioned “modified sugar containing a 5-membered ring”.
  • a saccharide having a cross-linked structure between the 4 ′ position and the 2 ′ position can be mentioned.
  • the cross-linked structure is particularly preferably 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′-C ( ⁇ O) —NR 1 -2 ′ (R 1 is , A hydrogen atom or alkyl).
  • oligonucleotides of the present invention are not limited to a particular isomer, but all possible isomers (eg, keto-enol isomer, imine-enamine isomer, diastereoisomer, optical isomer, rotational isomerism) Body, etc.), racemate or a mixture thereof.
  • One or more hydrogen, carbon and / or other atoms of the oligonucleotides of the invention may be replaced with isotopes of hydrogen, carbon and / or other atoms, respectively.
  • isotopes are 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 123 I and Like 36 Cl, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine are included.
  • the oligonucleotides of the present invention also include oligonucleotides substituted with such isotopes.
  • Oligonucleotides substituted with the isotope are useful as pharmaceuticals and include all radiolabeled oligonucleotides of the invention.
  • a “radiolabeling method” for producing the “radiolabeled product” is also encompassed in the present invention, and is useful as a metabolic pharmacokinetic study, a study in a binding assay, and / or a diagnostic tool.
  • the radiolabeled oligonucleotide of the present invention can be prepared by methods well known in the art.
  • the oligonucleotide of the present invention labeled with tritium can be prepared by introducing tritium into the oligonucleotide of the present invention by catalytic dehalogenation reaction using tritium.
  • This method involves reacting a suitable halogen-substituted precursor of the oligonucleotide of the invention with tritium gas in the presence of a suitable catalyst such as Pd / C, in the presence or absence of a base.
  • Suitable methods for preparing other tritium labeled compounds include the document Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987).
  • the 14 C-labeled compound can be prepared by using a raw material having 14 C carbon.
  • the 3 ′ end and / or the 5 ′ end of the oligonucleotide of the present invention may be modified.
  • modifying groups known in the art to enable tracking of the oligonucleotide, to improve the pharmacokinetics or pharmacodynamics of the oligonucleotide, or to improve the stability or binding affinity of the oligonucleotide. Can do. Examples thereof include a hydroxyl protecting group, a reporter molecule, cholesterol, phospholipid, a dye, and a fluorescent molecule.
  • 3 'terminal and / or 5' terminal of the oligonucleotide of this invention may contain the phosphate ester part.
  • Phosphate ester moiety means a terminal phosphate group, including phosphate esters as well as modified phosphate esters.
  • the phosphate ester moiety may be located at either end, but is preferably a 5′-terminal nucleoside. Specifically, it is a group represented by the formula: —OP ( ⁇ O) (OH) OH or a modifying group thereof. That is, one or more of O and OH is substituted with H, O, S, N (R x ), or alkyl (where R x is H, an amino protecting group, or substituted or unsubstituted alkyl). It may be.
  • the 5 ′ and / or 3′-end groups may each independently contain 1 to 3 phosphate ester moieties that are substituted or unsubstituted.
  • the present invention contains a pharmaceutically acceptable salt of the oligonucleotide of the present invention.
  • the salt include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), metal salts (aluminum salt, iron salt, zinc salt, copper salt).
  • amine salt t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt , Diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) )amino Tan salts, etc.), inorganic acid salts (hydrofluoric acid salts, hydrochlorides, hydrobromides, hydroiodide salts such as hydroiodide, nitrates, perchlorates, sulfates, phosphorus Acid
  • the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof may form a solvate (for example, hydrate etc.) and / or a crystal polymorph, and the present invention provides such various solvates. And crystalline polymorphs.
  • the “solvate” may be coordinated with any number of solvent molecules (for example, water molecules) with respect to the oligonucleotide of the present invention.
  • solvent molecules for example, water molecules
  • the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof When the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof is left in the air, it may absorb moisture and adsorbed water may adhere or form a hydrate.
  • the crystalline polymorphism may be formed by recrystallizing the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof.
  • the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof may form a prodrug, and the present invention includes such various prodrugs.
  • a prodrug is a derivative of a compound of the present invention having a group that can be chemically or metabolically degraded and becomes a pharmaceutically active oligonucleotide of the present invention in vivo by solvolysis or under physiological conditions .
  • a prodrug is converted into an oligonucleotide of the present invention by hydrolysis with a compound that is enzymatically oxidized, reduced, hydrolyzed, etc. under physiological conditions in vivo to be converted into the oligonucleotide of the present invention, stomach acid, etc. And the like. Methods for selecting and producing suitable prodrug derivatives are described, for example, in Design of Prodrugs, Elsevier, Amsterdam 1985. Prodrugs may themselves have activity.
  • the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof has a hydroxyl group, for example, a compound having a hydroxyl group and an appropriate acyl halide, an appropriate acid anhydride, an appropriate sulfonyl chloride, an appropriate sulfonyl anhydride And prodrugs such as acyloxy derivatives and sulfonyloxy derivatives produced by reacting with mixed anhydride or by using a condensing agent.
  • the oligonucleotide of the present invention can be synthesized by a conventional method using an appropriate amidite such as the amidite of the present invention.
  • an appropriate amidite such as the amidite of the present invention.
  • it can be easily synthesized by a commercially available automatic nucleic acid synthesizer (for example, Applied Biosystems, Dainippon Seiki Co., Ltd.).
  • the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate. For example, it is disclosed in Tetrahedron Letters 22, 1859-1862 (1981), International Publication No. 2011/052436, and the like.
  • the substituent is preferably not protected with a protecting group.
  • a protecting group for example, the group shown below is mentioned. Therefore, when the nucleobase portion of an amidite such as the amidite of the present invention has a substituent protected by a protecting group, deprotection is performed during oligonucleotide synthesis.
  • the nucleic acid drug using the oligonucleotide of the present invention can be administered by various methods depending on whether local or systemic treatment is desired or on the region to be treated.
  • the administration method may be, for example, topical (including eye drops, intravaginal, rectal, intranasal, transdermal), oral, or parenteral.
  • Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by inhalation or inhalation, intradural administration, intraventricular administration, and the like.
  • compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets and the like.
  • compositions for parenteral, subdural space, or intraventricular administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.
  • Nucleic acid pharmaceuticals using the oligonucleotides of the present invention include various pharmaceutical additives such as excipients, binders, wetting agents, disintegrating agents, lubricants, diluents, etc. suitable for the dosage form in an effective amount of nucleic acids. It can be obtained by mixing as required. In the case of an injection, it may be sterilized with an appropriate carrier to form a preparation.
  • Excipients include lactose, sucrose, glucose, starch, calcium carbonate or crystalline cellulose.
  • binder include methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, and polyvinyl pyrrolidone.
  • disintegrant include carboxymethyl cellulose, sodium carboxymethyl cellulose, starch, sodium alginate, agar powder, or sodium lauryl sulfate.
  • lubricant include talc, magnesium stearate or macrogol. As a suppository base, cocoa butter, macrogol, methylcellulose, or the like can be used.
  • solubilizers when preparing as liquid or emulsion or suspension injections, commonly used solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, isotonic agents, etc. are added as appropriate. You may do it. In the case of oral administration, flavoring agents, fragrances and the like may be added.
  • the optimal dosing schedule can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill in the art can determine optimum dosages, dosing methodologies and repetition rates.
  • the optimal dose will vary depending on the relative potency of the individual nucleic acid drug, but can generally be calculated based on the IC50 or EC50 in in vitro and in vivo animal experiments. For example, given the molecular weight of a nucleic acid (derived from the nucleic acid sequence and chemical structure) and an effective dose (derived experimentally) such as IC50, the dose expressed in mg / kg is typically Calculated according to
  • R 2 is, for example, Formula: —NR 19 R 20 (Where Each R 19 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl; Each R 20 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl) Group etc. which are shown by these are mentioned.
  • R 2 is diisopropylamino.
  • Nucleoside structure means a structure composed of the above-mentioned “nucleobase moiety” and “sugar moiety”.
  • the “nucleoside structure” in the present specification also includes an abasic cation unit lacking a nucleobase moiety.
  • the “nucleoside structure” is preferably a nucleoside structure in which the “sugar moiety” is “a modified sugar containing a 5-membered ring”.
  • the formula (III) (Where: Bx is a hydrogen or nucleobase moiety; Z is a hydrogen atom or a hydroxyl protecting group, R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, R 5 is hydrogen; R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy, Here, R 5 and R 6 may be combined to form a crosslinked structure, R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or un
  • Bx is hydrogen or the above-mentioned “nucleobase moiety”. More preferred is hydrogen, substituted or unsubstituted purin-9-yl or substituted or unsubstituted 2-oxo-pyrimidin-1-yl.
  • Z is a hydrogen atom, a hydroxyl protecting group or a reactive phosphorus group.
  • a hydrogen atom or a hydroxyl protecting group is preferred. More preferably, a hydrogen atom, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2 , 4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, levulinoyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tri Phenylsilyl, triisopropylsily
  • R 3 to R 7 have the same meanings as the symbols in the above-mentioned “modified sugar containing a 5-membered ring”.
  • amidites of the present invention are not limited to specific isomers, but all possible isomers (eg keto-enol isomers, imine-enamine isomers, diastereoisomers, optical isomers, rotational isomers) Etc.), racemates or mixtures thereof.
  • One or more hydrogen, carbon and / or other atoms of the amidites of the present invention may be replaced with isotopes of hydrogen, carbon and / or other atoms, respectively.
  • isotopes are 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 123 I and Like 36 Cl, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine are included.
  • the amidites of the present invention also include compounds substituted with such isotopes.
  • the compound substituted with the isotope is also useful as a pharmaceutical, and includes all radiolabels of the amidite of the present invention.
  • a “radiolabeling method” for producing the “radiolabeled product” is also encompassed in the present invention, and is useful as a metabolic pharmacokinetic study, a study in a binding assay, and / or a diagnostic tool.
  • the radiolabeled amidite of the present invention can be prepared by methods well known in the art.
  • the tritium-labeled compound of the amidite of the present invention can be prepared by introducing tritium into the amidite of the present invention by, for example, catalytic dehalogenation reaction using tritium. This method involves reacting a tritium gas with a suitably halogen-substituted precursor of an amidite of the present invention in the presence of a suitable catalyst such as Pd / C, in the presence or absence of a base. .
  • suitable methods for preparing other tritium labeled compounds include the document Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987).
  • the 14 C-labeled compound can be prepared by using a raw material having 14 C carbon.
  • the present invention contains a salt capable of forming the amidite of the present invention.
  • the salt include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), metal salts (aluminum salt, iron salt, zinc salt, copper salt).
  • amine salt t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt , Diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) )amino Tan salts, etc.), inorganic acid salts (hydrofluoric acid salts, hydrochlorides, hydrobromides, hydroiodides, etc., halogen atom hydrohalates, nitrates, perchlorates, sulfates, etc.), inorganic acid salts (hydrofluoric acid salts
  • the amidite of the present invention or a salt thereof may form a solvate (for example, hydrate etc.) and / or a crystal polymorph, and the present invention also includes such various solvates and crystal polymorphs.
  • the “solvate” may be coordinated with an arbitrary number of solvent molecules (for example, water molecules) to the amidite of the present invention.
  • solvent molecules for example, water molecules
  • the amidite of the present invention or a pharmaceutically acceptable salt thereof When the amidite of the present invention or a pharmaceutically acceptable salt thereof is left in the air, it may absorb moisture and adsorbed water may adhere or form a hydrate.
  • those polymorphs may be formed by recrystallizing the amidite of the present invention or a pharmaceutically acceptable salt thereof.
  • the amidite of the present invention can be synthesized while taking into account techniques known in the art. For example, it can be produced by the synthesis method shown in Example 1. Extraction, purification, and the like may be performed in a normal organic chemistry experiment.
  • the oligonucleotide of the present invention has a higher target gene expression inhibitory activity than an oligonucleotide having only a phosphorothioate at the phosphate site.
  • the oligonucleotide of the present invention has improved plasma retention compared to an oligonucleotide whose phosphoric acid moiety is only phosphorothioate by devising the modification position and the number of modifications, for example, organs such as liver or lung Therefore, it can be efficiently delivered to the target organ and the expression of the target gene or target protein can be effectively suppressed. Therefore, the amidite of the present invention is very useful as a material for synthesizing nucleic acid pharmaceuticals containing antisense oligonucleotides and the like.
  • N, N-diisopropylethylamine (7.7 mL, 44 mmol) was added to a dichloromethane solution (100 mL) of compound 1 (20 g, 37 mmol), and the mixture was ice-cooled.
  • a dichloromethane solution 80 mL of bis (diisopropylamino) chlorophosphine (11.3 g, 42 mmol) was added dropwise over 60 minutes, and then the temperature was raised to room temperature over 30 minutes.
  • reaction solution was ice-cooled again, 1H-tetrazole (1.54 g, 22 mmol) in acetonitrile (49 mL) and quinuclidin-3-ol (5.6 g, 44 mmol) were sequentially added, and the mixture was warmed to room temperature. Stir for 2.5 hours.
  • a saturated aqueous sodium hydrogen carbonate solution 500 mL was added to the reaction mixture, and the mixture was extracted with ethyl acetate (600 mL). The obtained organic layer was washed with saturated brine, and then dried over anhydrous magnesium sulfate.
  • R p is acetyl, benzoyl or phenoxyacetyl
  • R q is isobutyryl, acetyl, benzoyl or phenoxyacetyl
  • Example 2 Synthesis of oligonucleotide of the present invention (an oligonucleotide containing one or more bonds represented by formula (I)) (1) Preparation of single-stranded oligonucleotide Oligo prepared from amidite obtained in Example 1 As a control and an oligonucleotide (0), (3-0), (4-0) or (5-0), in which all internucleoside linkages are phosphorothioate linkages, nS-8 (manufactured by Gene Design) was synthesized on a 0.2 ⁇ mol scale. The synthesized oligonucleotides are shown in Table 2. “S1 to s4” and “o1 to o4” mean bonds in which the symbols in formula (I) are as follows.
  • the amidite was used as a 0.1M acetonitrile solution.
  • the coupling time between the amidite and the hydroxyl group at the 5′-terminal was 2 minutes, and they were linked by two successive coupling steps.
  • the obtained crude product solution was neutralized with 1% aqueous citric acid solution under ice cooling.
  • a sample obtained by adding a 0.1 M triethylamine acetate aqueous solution (2 mL) to the neutralized sample was concentrated by a centrifugal evaporator (45 ° C., 1 hour).
  • the concentrated sample was purified by Sep-Pak C18 Plus Short Cartridge (manufactured by Waters), lyophilized, and then prepared into a 0.1 M oligonucleotide aqueous solution, which was used for the tests of Example 3 and later.
  • the purity and structure of the synthesized oligonucleotide were confirmed by LC-MS.
  • the results are shown in Table 4.
  • the measurement conditions for LC-MS are as follows.
  • Oligonucleotide (7-1) was purchased from Gene Design Co., Ltd. (Osaka, Japan). A method for synthesizing the single-stranded oligonucleotides (7-2) to (7-3) is shown below.
  • Single-stranded oligonucleotides synthesized by the same oligo synthesis method as in (1) are excised from the resin and subjected to HPLC purification, then the dimethoxytrityl group is removed, and HPLC purification is performed again, followed by salt exchange. Go to the final sample. The purity and structure of the synthesized oligonucleotide were confirmed by LC-MS in the same manner as in (1).
  • double-stranded oligonucleotides (7-4) and (7-5) were prepared as follows. After equimolar amounts of each oligonucleotide were mixed, distilled water was added to make a 0.5 mmol / L solution. Thereafter, the mixture was allowed to stand at 60 ° C. for 10 minutes, and then naturally cooled to room temperature to obtain a double-stranded nucleic acid. Double strand formation was confirmed by confirming that a normal Tm curve was drawn using UV-1800 made by Shimadzu, thereby forming a double chain. The synthesized oligonucleotides are shown in Table 6.
  • Example 3 Biological Activity Test (Complementary mRNA Cleavage Activity Test) of the Oligonucleotide of the Present Invention (Single-Stranded Oligonucleotide Containing One or More Bindings Represented by Formula (I)) (1)
  • HaCaT Human keratinocyte-derived cell line HaCaT was cultured in DMEM Low Glucose (Sigma) + 10% fetal bovine serum (FBS) + Penicillin (100 units / mL) + Streptomycin (100 ug / mL).
  • Hepa1c1c7 The mouse liver cancer-derived cell line Hepa1c1c7 was cultured in ⁇ -MEM (Gibco) + 10% FBS + Antibiotic Anticolytic Solution (10 mL / L).
  • Quantitative PCR was performed with the PLUS RT-PCR Kit (Takara). RPLP0 or GAPDH was used as an endogenous control.
  • the primer sequence used to measure the expression level of mouse Acsl1 is: Fw primer: AGGTGCTTCAGCCCACATC (SEQ ID NO: 5); Rv primer: AAAGTCCAACAGCCATCGCTTC (SEQ ID NO: 6) was used.
  • the primer sequence used to measure the expression level of mouse RPLP0 is: Fw primer: ATCAACGGGTCAAACGAGTC (SEQ ID NO: 7); Rv primer: CAGATGGATCAGCCCAAGAAGG (SEQ ID NO: 8) was used.
  • the primer sequence used to measure the expression level of mouse Gapdh is: Fw primer: TGTGTCCGTCGTGGATCTGA (SEQ ID NO: 9); Rv primer: TTGCTGTTTGAAGTCGCAGAG (SEQ ID NO: 10) was used.
  • the primer sequence used to measure the expression level of human Acsl1 is Fw primer: GCAGCGGGCATCATCAGAAAC (SEQ ID NO: 11); Rv primer: TGTCCATCATAGCCCGACTC (SEQ ID NO: 12) was used.
  • the primer sequence used to measure the expression level of human RPLP0 is Fw primer: ATCAACGGGTCAAACGAGTC (SEQ ID NO: 13); Rv primer: CAGATGGATCAGCCCAAGAAGG (SEQ ID NO: 14) was used.
  • Tables 7-12 Tables 7 to 10 and Table 12 show the amount of decrease in Acsl1 mRNA normalized by RPLP0 as a percentage of untreated cells as knockdown efficiency.
  • Table 11 shows the amount of Acsl1 mRNA decrease normalized with GAPDH as the knockdown efficiency as a percentage of untreated cells.
  • the oligonucleotides (1-1) to (1-4) and (2-1) to (2-4) of the present invention are compared with the oligonucleotide (0) having no bond represented by the formula (I),
  • the knockdown activity for the target mRNA (human Acsl1) was improved.
  • the oligonucleotides (2-1) to (2-4) in which the substituent Q of formula (I) is O are the same as the oligonucleotides (1-1) to (1-4) in which the substituent Q is S. In comparison, the knockdown activity was further improved.
  • the knockdown activity against the target mRNA was improved in the oligonucleotide (1-1) of the present invention compared to the oligonucleotide (0) having no bond represented by the formula (I). Since similar results were obtained in the human keratinocyte-derived cell line HaCaT in Test 1 and the mouse liver cancer-derived cell line Hepa1c1c7 in Test 2, the oligonucleotide of the present invention has a knockdown activity against the target mRNA regardless of the cell type. It was suggested to improve.
  • Oligonucleotides (3-0) and (3-1) are mismatched with respect to the target mRNA at one base at the 5 'end (cytidine).
  • the knockdown activity of the oligonucleotide (3-1) of the present invention was higher than that of the oligonucleotide (3-0) having no bond represented by the formula (I).
  • the oligonucleotide (3-1) of the present invention has a higher knockdown activity than the oligonucleotide (0) which is a sequence having no mismatch at the 5 ′ terminal base. Indicated. Therefore, it was suggested that the oligonucleotide of the present invention improves the knockdown activity against the target mRNA by having the bond represented by the formula (I) even when the nucleotide base is mismatched.
  • the oligonucleotide (4-1) of the present invention has an improved knockdown activity on the target mRNA (human Acsl1) compared to the oligonucleotide (4-0) having no bond represented by the formula (I). Similar results were obtained with the sugar site-modified nucleotide AmNA (Compound 1001) of Tests 1 to 3 and the like and the sugar site-modified nucleotide LNA (Compound 1002) of Test 4 and therefore, regardless of the type of sugar modification. It was suggested that the oligonucleotide improves the knockdown activity against the target mRNA.
  • Oligonucleotides (5-1-1) and (5-1-2) have a bond represented by the formula (I) in the central region (gap region) of the gapmer.
  • the oligonucleotides (5-1-1) and (5-1-2) of the present invention are more effective against the target mRNA (mouse Acsl1) than the oligonucleotide (5-0) having no bond represented by the formula (I).
  • Knockdown activity was improved. Similar results were obtained not only in the phosphate site modification of the 5 'terminal base as in Tests 1 to 4 but also in the case where the phosphate site modification was not at the 5' terminal as in Test 5. Therefore, it was suggested that the oligonucleotide of the present invention improves the knockdown activity for the target mRNA regardless of the modification position in the oligonucleotide.
  • the oligonucleotide (6-1-1) has one abasic cation unit (compound 1003) at the 5 ′ end, and the oligonucleotide (6-1-2) has two abasic cation units (compound 1003).
  • the oligonucleotides (6-1-1) and (6-1-2) of the present invention are knocked down against the target mRNA (human Acsl1) as compared with the oligonucleotide (0) having no binding represented by the formula (I) Activity improved. Therefore, it was suggested that the oligonucleotide of the present invention improves the knockdown activity against the target mRNA by having the bond represented by the formula (I) even when the nucleotide base is partially abasic.
  • Example 4 Evaluation of in vivo activity of oligonucleotide of the present invention (double-stranded oligonucleotide containing one or more bonds represented by formula (I)) Double-stranded oligo having no bond represented by formula (I)
  • the knockdown activity of the nucleotide (7-4) and the double-stranded oligonucleotide (7-5) of the present invention was evaluated by changing the expression level of Hprt1 mRNA in the mouse liver.
  • oligonucleotide (7-5) solution dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) was administered per mouse to C57BL / 6J mice (male, 10 weeks old, CLEA Japan). Intravenous administration was performed so that the amount was 0.5 mg / kg in terms of the amount of antisense oligo.
  • a solution of oligonucleotide (7-4) was administered so that the dose per mouse individual was 0.5 mg / kg in terms of the amount of antisense oligo. Seven days after administration, liver tissue was collected under isoflurane anesthesia.
  • RNA extraction from the liver was performed according to the manufacturer's recommended protocol using RNeasy Mini Kit (Qiagen). 100 ng of the obtained RNA was subjected to quantitative PCR using One Step SYBR PrimeScript PLUS RT-PCR Kit (manufactured by Takara Bio Inc.).
  • the primer sequence used to measure the expression level of mouse Hprt1 is Fw primer: TTGTTGTTGGATAGCCCCTTGACTA (SEQ ID NO: 18)
  • the primer sequence used for measuring the expression level of mouse Gapdh was the one shown in Example 3 (2) above.
  • the knockdown efficiency was expressed as a percentage of the physiological saline administration group with respect to the amount of Hprt1 mRNA decreased normalized by Gapdh.
  • the results are shown in FIG.
  • the double-stranded oligonucleotide (7-5) of the present invention was knocked in the liver as compared with the double-stranded antisense oligonucleotide (7-4) having no bond represented by the formula (I).
  • the down efficiency tended to increase.
  • the oligonucleotide of the present invention prepared from the amidite of the present invention exhibits excellent target gene expression suppression activity.
  • the oligonucleotide of the present invention improves plasma retention by devising the modification position and the number of modifications, and is efficiently delivered to a target organ, for example, by increasing accumulation in an organ such as the liver or lung. It has been suggested that it is possible to effectively suppress the expression of the target gene or target protein. Therefore, the oligonucleotide of this invention is very useful as a material for manufacturing the nucleic acid pharmaceutical containing an antisense oligonucleotide etc.

Abstract

　Provided are: a oligonucleotide having at least one bond represented by formula (I): (in the formula, Q is S or O, X1 does not exist, or is a substituted or unsubstituted alkylene in which O may be interposed, X2 is a substituted or unsubstituted monocyclic or duocyclic nitrogen-containing non-aromatic heterocyclic group) or a pharmaceutically acceptable salt thereof, and a novel amidite which can be used as a material for synthesizing said oligonucleotide. The oligonucleotide is a novel phosphate site modification which can be used as a nucleic acid pharmaceutical.

Description

含窒素非芳香族複素環を含む核酸のリン酸部位修飾Phosphate site modification of nucleic acids containing nitrogen-containing non-aromatic heterocycles
　本発明は、新規のリン酸部位修飾を有する核酸に関する。より詳細には、単環又は２環の含窒素非芳香族複素環を含むリン酸部位修飾を有するオリゴヌクレオチドに関する。さらに、該オリゴヌクレオチドを調製するために利用するアミダイトに関する。 The present invention relates to a nucleic acid having a novel phosphate site modification. More specifically, the present invention relates to an oligonucleotide having a phosphate site modification containing a monocyclic or bicyclic nitrogen-containing non-aromatic heterocycle. Further, the present invention relates to an amidite used for preparing the oligonucleotide.
　核酸医薬品による疾患の治療法として、アンチセンスオリゴヌクレオチド、ｓｉＲＮＡ、リボザイム、アンチジーン、アプタマー、デコイ核酸等を用いる方法がある。 There are methods using antisense oligonucleotides, siRNAs, ribozymes, antigenes, aptamers, decoy nucleic acids, etc. as methods for treating diseases caused by nucleic acid pharmaceuticals.
　アンチセンスオリゴヌクレオチドは、標的遺伝子のｍＲＮＡ、ｍＲＮＡ前駆体又はリボソームＲＮＡ、転移ＲＮＡ、ｍｉＲＮＡ等のｎｃＲＮＡ（ノンコーディングＲＮＡ）に対して相補的なオリゴヌクレオチドであり、約８～３０塩基からなる一本鎖のＤＮＡ、ＲＮＡ及び／又はそれらの構造類似体である。該アンチセンスオリゴヌクレオチドが標的とするｍＲＮＡ、ｍＲＮＡ前駆体、又はｎｃＲＮＡと二本鎖を形成することによりｍＲＮＡ、ｍＲＮＡ前駆体又はｎｃＲＮＡの働きを抑制する。 The antisense oligonucleotide is an oligonucleotide complementary to ncRNA (non-coding RNA) such as mRNA, mRNA precursor or ribosomal RNA, transfer RNA, miRNA and the like of a target gene, and consists of about 8 to 30 bases. Strand DNA, RNA and / or structural analogs thereof. The function of mRNA, mRNA precursor or ncRNA is suppressed by forming a double strand with the mRNA, mRNA precursor or ncRNA targeted by the antisense oligonucleotide.
　ｓｉＲＮＡは、標的遺伝子と相同な約１９～２５塩基対からなる低分子二本鎖ＲＮＡである。ＲＮＡ干渉と呼ばれる現象に関与しており、塩基配列特異的にｍＲＮＡを分解することによって、遺伝子の発現を抑制する。 SiRNA is a small double-stranded RNA consisting of about 19 to 25 base pairs homologous to the target gene. It is involved in a phenomenon called RNA interference and suppresses gene expression by degrading mRNA in a base sequence-specific manner.
　リボザイムは、核酸を切断する酵素活性を持つＲＮＡである。標的遺伝子のｍＲＮＡと二重鎖を形成し、特異的に該ｍＲＮＡを切断する。
　アンチジーンは、標的遺伝子の二重鎖ＤＮＡ部位に対応するオリゴヌクレオチドである。該ＤＮＡ部位とオリゴヌクレオチドで三重鎖を形成させることにより、ＤＮＡからｍＲＮＡへの転写を抑制する。
　アプタマーは、特定の分子と特異的に結合するＤＮＡ、ＲＮＡ及び／又はそれらの構造類似体である。標的タンパク質と結合することにより該タンパク質の機能を阻害する。
　デコイ核酸は、特定の転写調節因子の結合部位と同じ配列を含む短いＤＮＡである。該転写調節因子と遺伝子の結合を阻害し、該転写調節因子によって活性化される標的遺伝子の発現を抑制する。 Ribozymes are RNAs that have enzymatic activity to cleave nucleic acids. It forms a double strand with the mRNA of the target gene and specifically cleaves the mRNA.
Antigenes are oligonucleotides that correspond to the double stranded DNA site of the target gene. Transcription from DNA to mRNA is suppressed by forming a triplex with the DNA site and oligonucleotide.
Aptamers are DNA, RNA and / or structural analogs thereof that specifically bind to a specific molecule. By binding to the target protein, the function of the protein is inhibited.
Decoy nucleic acids are short DNAs that contain the same sequence as the binding site for a particular transcriptional regulator. The binding between the transcriptional regulatory factor and the gene is inhibited, and the expression of a target gene activated by the transcriptional regulatory factor is suppressed.
　しかし、これらの核酸医薬品は、生体内のヌクレアーゼにより分解されやすく、標的細胞への取り込み効率が低いため、実用化が難しい。２つの大きな問題点を克服するために、有効成分である核酸の塩基部分、糖部位及びリン酸部位の化学修飾の研究が長年行われている。修飾を行うことにより、標的遺伝子の発現抑制活性が上昇する、つまり、核酸医薬品の活性成分として利用可能な修飾も存在する。 However, these nucleic acid drugs are difficult to put to practical use because they are easily degraded by nucleases in living bodies and the efficiency of incorporation into target cells is low. In order to overcome two major problems, chemical modification of the base part, sugar part and phosphate part of nucleic acid, which is an active ingredient, has been studied for many years. By performing the modification, the target gene expression suppression activity increases, that is, there is a modification that can be used as an active ingredient of a nucleic acid pharmaceutical.
　核酸のリン酸部位の化学修飾としては、Ｓ－オリゴ（例えば、ホスホロチオエート）、Ｍ－オリゴ（例えば、メチルホスホネート）、ボラノホスホネート、特許文献１又は特許文献２記載の修飾等が知られている。しかし、単環又は２環の含窒素非芳香族複素環を含む修飾は知られていない。 Known chemical modifications of the phosphate site of nucleic acids include S-oligo (eg, phosphorothioate), M-oligo (eg, methylphosphonate), boranophosphonate, and the modifications described in Patent Document 1 or Patent Document 2. . However, there are no known modifications involving monocyclic or bicyclic nitrogen-containing non-aromatic heterocycles.
　特許文献３には含窒素鎖を含む核酸のリン酸部位修飾を有するオリゴヌクレオチドが記載されている。しかし、該オリゴヌクレオチドは、核酸医薬品のプロドラッグとして用いられており、体内で代謝される際に、該修飾は脱離する。
　非特許文献１には、フェノチアジンを有するリン酸保護基により、末端のリン酸部位が置換されている核酸が記載されている。該保護基は標識として用いられている。
　また、核酸を調製するために利用するアミダイトとしては、リン酸部位にピロリジンを含む保護基を有するアミダイト（特許文献４）やピリジンを含む保護基を有するアミダイト（非特許文献２）が知られている。該保護基は、オリゴヌクレオチドの合成時の安定性向上等のための修飾であり、オリゴヌクレオチドを担体より切り離す際に保護基は脱離する。
　よって、これらのリン酸部位修飾に、核酸医薬品の活性成分としての働き、つまり、標的遺伝子の発現抑制活性又は標的タンパク質の機能阻害活性を上昇する働きは存在しない。 Patent Document 3 describes an oligonucleotide having a phosphate site modification of a nucleic acid containing a nitrogen-containing chain. However, the oligonucleotide is used as a prodrug of a nucleic acid pharmaceutical, and the modification is eliminated when metabolized in the body.
Non-Patent Document 1 describes a nucleic acid in which a terminal phosphate site is substituted with a phosphate protecting group having phenothiazine. The protecting group is used as a label.
As amidites used for preparing nucleic acids, amidites having a protecting group containing pyrrolidine at the phosphate site (Patent Document 4) and amidites having a protecting group containing pyridine (Non-Patent Document 2) are known. Yes. The protecting group is a modification for improving stability during synthesis of the oligonucleotide, and the protecting group is removed when the oligonucleotide is separated from the carrier.
Therefore, these phosphate site modifications do not have a function as an active ingredient of a nucleic acid pharmaceutical, that is, a function of increasing the target gene expression suppressing activity or the target protein function inhibiting activity.
米国特許第６０３３９０９号明細書US Pat. No. 6,033,909 国際公開第２０１３／０２２９６６号International Publication No. 2013/022966 国際公開第２０１１／００５７６１号International Publication No. 2011/005761 米国特許出願公開第２００４／００８２７７４号明細書US Patent Application Publication No. 2004/0082774
　本発明の目的は、アンチセンスオリゴヌクレオチド、ｓｉＲＮＡ、リボザイム、アンチジーン、アプタマー、デコイ核酸等を含む核酸医薬品として利用可能な新規のリン酸部位修飾を有するオリゴヌクレオチド及び該オリゴヌクレオチドを合成するための材料として利用可能な新規のアミダイトを提供することにある。 An object of the present invention is to synthesize an oligonucleotide having a novel phosphate site modification that can be used as a nucleic acid drug including an antisense oligonucleotide, siRNA, ribozyme, antigene, aptamer, decoy nucleic acid, and the like, and the oligonucleotide. It is to provide a novel amidite that can be used as a material.
　本発明者らは、鋭意研究の結果、単環又は２環の含窒素非芳香族複素環を含むリン酸部位修飾、つまり、下記式（Ｉ）で示される結合を１以上含有するオリゴヌクレオチド（以下、「本発明のオリゴヌクレオチド」）が、全てのリン酸部位がホスホロチオエートであるオリゴヌクレオチドと比較して、標的遺伝子の発現抑制活性が向上することを見出した。
　また、本発明者らは、本発明のオリゴヌクレオチドは修飾位置や修飾数を工夫することで、全てのリン酸部位がホスホロチオエートであるオリゴヌクレオチドと比較して、マウスを用いた生物実験において血漿中滞留性が向上し、臓器（肝臓及び肺）分布が大きく変化すること、つまり、体内動態が大きく変化することを見出した。具体的には、全てのリン酸部位がホスホロチオエートであるオリゴヌクレオチドでは臓器（肝臓及び肺）中にほとんど集積されない場合でも、同じ配列であるが下記式（Ｉ）で示される結合を含むオリゴヌクレオチドは、下記式（Ｉ）で示される結合を多く含む程、マウス臓器（肝臓及び肺）への集積が増加した。本発明のオリゴヌクレオチド、つまり、下記式（Ｉ）で示される結合を利用することにより、もともとの配列が持っている臓器集積性と異なる結果が得られることから、効率的に標的臓器に送達され、標的遺伝子又は標的タンパク質の発現を効果的に抑制することを可能とすることが示唆された。
　よって、本発明のオリゴヌクレオチドは、アンチセンスオリゴヌクレオチド等を含む核酸医薬品として非常に有用である。
　また、本発明のオリゴヌクレオチドは、下記式（ＩＩ）で示される基及びヌクレオシド構造を含有する化合物（以下、「本発明のアミダイト」）を用いて、一般的な合成方法で、副産物無く、安定して合成することができる。本発明のアミダイトは、アンチセンスオリゴヌクレオチド等を含む核酸医薬品を合成するための材料として非常に有用である。 As a result of diligent research, the inventors of the present invention have made phosphate site modifications including monocyclic or bicyclic nitrogen-containing non-aromatic heterocycles, that is, oligonucleotides containing one or more bonds represented by the following formula (I) ( Hereinafter, the “oligonucleotide of the present invention”) has been found to have improved target gene expression-suppressing activity as compared to an oligonucleotide in which all phosphate sites are phosphorothioates.
In addition, the present inventors have devised the modification position and the number of modifications of the oligonucleotide of the present invention, so that compared to oligonucleotides in which all phosphate sites are phosphorothioates, in the biological experiment using mice, It was found that the retention was improved and the organ (liver and lung) distribution changed greatly, that is, the pharmacokinetics changed significantly. Specifically, even when oligonucleotides in which all phosphate sites are phosphorothioates are hardly accumulated in organs (liver and lung), oligonucleotides having the same sequence but containing a bond represented by the following formula (I) are: As the amount of the bond represented by the following formula (I) increases, accumulation in mouse organs (liver and lung) increases. By utilizing the oligonucleotide of the present invention, that is, the bond represented by the following formula (I), a result different from the organ accumulation property of the original sequence can be obtained, so that it can be efficiently delivered to the target organ. It was suggested that the expression of the target gene or target protein can be effectively suppressed.
Therefore, the oligonucleotide of this invention is very useful as a nucleic acid pharmaceutical containing an antisense oligonucleotide etc.
In addition, the oligonucleotide of the present invention is stable without any by-product by a general synthesis method using a compound containing a group represented by the following formula (II) and a nucleoside structure (hereinafter, “amidite of the present invention”). And can be synthesized. The amidite of the present invention is very useful as a material for synthesizing nucleic acid pharmaceuticals containing antisense oligonucleotides and the like.
　すなわち、本発明は、以下に関する。
（１）式（Ｉ）：
Figure JPOXMLDOC01-appb-C000005
（式中、
ＱはＳ又はＯであり、
Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、
Ｘ２は置換又は非置換の単環若しくは２環の含窒素非芳香族複素環式基である）
で示される結合を１以上含有するオリゴヌクレオチド又はその製薬上許容される塩。
（２）Ｘ２が、置換若しくは非置換のアジリジニル、置換若しくは非置換のアゼチジニル、置換若しくは非置換のピロリジニル、置換若しくは非置換のピペリジル、置換若しくは非置換のピペリジノ、置換若しくは非置換のピペラジニル、置換若しくは非置換のピペラジノ、置換若しくは非置換のモルホリニル、置換若しくは非置換のモルホリノ、置換若しくは非置換のアゼパニル又は置換若しくは非置換のキヌクリジニルである、（１）記載のオリゴヌクレオチド又はその製薬上許容される塩。
（３）Ｘ１は存在しないか、又は非置換のアルキレンである、（１）又は（２）記載のオリゴヌクレオチド又はその製薬上許容される塩。
（４）該オリゴヌクレオチドの長さが８～２５塩基である、（１）～（３）いずれかに記載のオリゴヌクレオチド又はその製薬上許容される塩。
（５）糖の２’位に置換基を有する糖修飾ヌクレオシド又は
糖の４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、
（１）～（４）いずれかに記載のオリゴヌクレオチド又はその製薬上許容される塩。
（６）該置換基が、F、ＯＣＨ３又はＯＣＨ２ＣＨ２ＯＣＨ３である、（５）記載のオリゴヌクレオチド又はその製薬上許容される塩。
（７）該架橋構造が、４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）又は４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、（５）記載のオリゴヌクレオチド又はその製薬上許容される塩。
（８）式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000006
（式中：
Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、
Ｘ２は置換又は非置換の単環若しくは２環の含窒素非芳香族複素環式基であり、
Ｒ２は置換若しくは非置換のアミノである）
で示される基
（但し、
Figure JPOXMLDOC01-appb-C000007
で示される基を除く）
及びヌクレオシド構造を含有する化合物、又はその塩。
（９）該ヌクレオシド構造が、式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000008
（式中：
Ｂｘは水素又は核酸塩基部分であり、
Ｚは水素原子又は水酸基保護基であり、
Ｒ３及びＲ４はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ５は水素であり、
Ｒ６は水素原子、ハロゲン、ヒドロキシ又は置換若しくは非置換のアルキルオキシであり、
ここでＲ５及びＲ６は、一緒になって架橋構造を形成していてもよく、
Ｒ７は水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルである）
で示される構造である、（８）記載の化合物、又はその塩。
（１０）Ｒ６が、Ｆ、ＯＣＨ３又はＯＣＨ２ＣＨ２ＯＣＨ３である、又は、
Ｒ５とＲ６が、一緒になって架橋構造を形成している、
（９）記載の化合物、又はその塩。
（１１）該架橋構造が、４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）又は４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、（１０）記載の化合物、又はその塩。
（１２）該水酸基保護基が、アセチル、ｔ－ブチル、ｔ－ブトキシメチル、メトキシメチル、テトラヒドロピラニル、１－エトキシエチル、１－（２－クロロエトキシ）エチル、２－トリメチルシリルエチル、ｐ－クロロフェニル、２，４－ジニトロフェニル、ベンジル、ベンゾイル、ｐ－フェニルベンゾイル、２，６－ジクロロベンジル、レブリノイル、ジフェニルメチル、ｐ－ニトロベンジル、トリメチルシリル、トリエチルシリル、ｔ－ブチルジメチルシリル、ｔ－ブチルジフェニルシリル、トリフェニルシリル、トリイソプロピルシリル、ギ酸ベンゾイル、クロロアセチル、トリクロロアセチル、トリフルオロアセチル、ピバロイル、イソブチリル、９－フルオレニルメチルオキシカルボニル、メタンスルホニル、ｐ－トルエンスルホニル、トリフルオロメタンスルホニル、トリチル、モノメトキシトリチル、ジメトキシトリチル、トリメトキシトリチル、９－フェニルキサンチン－９－イル又は９－（ｐ－メトキシフェニル）キサンチン－９－イルである、（９）～（１１）いずれかに記載の化合物、又はその塩。 That is, the present invention relates to the following.
(1) Formula (I):
Figure JPOXMLDOC01-appb-C000005
(Where
Q is S or O,
X 1 is a substituted or unsubstituted alkylene which is absent or O may be interposed,
X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group)
Or an pharmaceutically acceptable salt thereof.
(2) X 2 is substituted or unsubstituted aziridinyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted piperidyl, substituted or unsubstituted piperidino, substituted or unsubstituted piperazinyl, substituted Or the substituted piperazino, substituted or unsubstituted morpholinyl, substituted or unsubstituted morpholino, substituted or unsubstituted azepanyl, or substituted or unsubstituted quinuclidinyl, or the pharmaceutically acceptable oligonucleotide thereof (1) salt.
(3) The oligonucleotide or pharmaceutically acceptable salt thereof according to (1) or (2), wherein X 1 is absent or unsubstituted alkylene.
(4) The oligonucleotide according to any one of (1) to (3) or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide has a length of 8 to 25 bases.
(5) containing one or more sugar-modified nucleosides having a substituent at the 2′-position of the sugar or a sugar-modified nucleoside having a cross-linked structure between the 4′-position and the 2′-position of the sugar;
(1) to the oligonucleotide according to any one of (4) or a pharmaceutically acceptable salt thereof.
(6) The oligonucleotide or pharmaceutically acceptable salt thereof according to (5), wherein the substituent is F, OCH 3 or OCH 2 CH 2 OCH 3 .
(7) The crosslinked structure is 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′—C (═O) —NR 1 -2 ′ (R 1 is The oligonucleotide or pharmaceutically acceptable salt thereof according to (5), which is a hydrogen atom or alkyl).
(8) Formula (II):
Figure JPOXMLDOC01-appb-C000006
(Where:
X 1 is a substituted or unsubstituted alkylene which is absent or O may be interposed,
X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group,
R 2 is substituted or unsubstituted amino)
A group represented by (however,
Figure JPOXMLDOC01-appb-C000007
Except the group indicated by
And a compound containing a nucleoside structure, or a salt thereof.
(9) The nucleoside structure has the formula (III):
Figure JPOXMLDOC01-appb-C000008
(Where:
Bx is a hydrogen or nucleobase moiety;
Z is a hydrogen atom or a hydroxyl protecting group,
R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl,
R 5 is hydrogen;
R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy,
Here, R 5 and R 6 may be combined to form a crosslinked structure,
R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl)
The compound or a salt thereof according to (8), which has a structure represented by:
(10) R 6 is F, OCH 3 or OCH 2 CH 2 OCH 3 , or
R 5 and R 6 together form a crosslinked structure,
(9) The compound or salt thereof according to (9).
(11) The crosslinked structure is 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′—C (═O) —NR 1 -2 ′ (R 1 is The compound according to (10), which is a hydrogen atom or alkyl, or a salt thereof.
(12) The hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl. 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, levulinoyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl , Triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl, 9-fluorenylmethyloxycarbonyl, methanesulfonyl, p-toluenesulfur Nyl, trifluoromethanesulfonyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl or 9- (p-methoxyphenyl) xanthin-9-yl, (9) to (11 ) Any one of the compounds or salts thereof.
　本発明オリゴヌクレオチドは、
標的遺伝子に対してより優れた発現抑制活性を示すこと、また、
本発明のオリゴヌクレオチドは、修飾位置や修飾数を工夫することで、血漿中滞留性が向上し、例えば、肝臓又は肺等の臓器への集積を増加させることができ、効率的に標的臓器に送達され、標的遺伝子又は標的タンパク質の発現を効果的に抑制することを可能であること
から、核酸医薬品への応用が期待される。 The oligonucleotide of the present invention comprises
Exhibit superior expression suppression activity against the target gene, and
The oligonucleotide of the present invention improves plasma retention by devising the modification position and the number of modifications, for example, can increase accumulation in organs such as the liver or lung, and efficiently target the target organ Since it is delivered and it is possible to effectively suppress the expression of the target gene or target protein, application to nucleic acid pharmaceuticals is expected.
カチオン修飾オリゴヌクレオチドのノックダウン評価Knockdown evaluation of cation-modified oligonucleotides
　本明細書において使用される用語は、特に言及する場合を除いて、当該分野で通常用いられる意味で用いられる。
　本発明においては、当該分野で公知の遺伝子操作方法の使用が可能である。例えば、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，　Ｔｈｉｒｄ　Ｅｄｉｔｉｏｎ，　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（２００１）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，　Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ　（２００３）に記載された方法等が挙げられる。 The terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.
In the present invention, a genetic manipulation method known in the art can be used. For example, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Current Protocols in Molecular Biology, Jon.
　以下に本明細書中で使用する各用語を説明する。なお、本明細書中、各用語は単独で使用されている場合も、又は他の用語と一緒になって使用されている場合も、特に記載の無い限り、同一の意義を有する。 The terms used in this specification are explained below. In addition, in this specification, each term has the same meaning, whether used alone or in combination with other terms, unless otherwise specified.
　「核酸」とは、核酸塩基部分と糖部位とが共有結合した化合物であるヌクレオシドの糖分子にリン酸部位が結合した化合物であるヌクレオチドを構成単位とした高分子物質である。 “Nucleic acid” is a high-molecular substance composed of a nucleotide, which is a compound in which a phosphate moiety is bonded to a sugar molecule of a nucleoside, which is a compound in which a nucleobase moiety and a sugar moiety are covalently bonded.
　「核酸塩基部分」とは、核酸塩基又はその類縁体を含む置換基を意味する。天然の核酸塩基としては、アデニン（Ａ）、グアニン（Ｇ）、チミン（Ｔ）、シトシン（Ｃ）又はウラシル（Ｕ）が挙げられる。本発明の核酸塩基は、それらに限定されず、他の人工又は天然の核酸塩基も含まれる。例えば、５－メチルシトシン（５－ｍｅ－Ｃ）、５－ヒドロキシメチルシトシン、キサンチン、ヒポキサンチン、２－アミノアデニン、７－デアザ－アデニン、７－デアザグアノシン、２－アミノピリジン、２－ピリドン等が挙げられる。
　つまり、本発明における「核酸塩基部分」とは、核酸（ＤＮＡ、ＲＮＡ）の塩基部分を構成する置換若しくは非置換の複素環式基又は置換若しくは非置換の炭素環式基である。
　該複素環は、Ｏ、Ｓ及びＮから任意に選択される同一又は異なるヘテロ原子を環内に１以上有する単環又は縮合環を含む。例えば、プリン、ピリミジン、チオフェン、チアントレン、フラン、ピラン、イソベンゾフラン、クロメン、キサンテン、フェノキサチイン、ピロール、イミダゾール、ピラゾール、イソチアゾール、イソキサゾール、ピリダジン、インドリジン、インドール、イソインドール、イソキノリン、キノリン、ナフチリジン、キノキサリン、キナゾリン、プテリジン、カルバゾール、フェナントリジン、アクリジン、ペリミジン、フェナジン、フェナルサジン、フェノチアジン、フラザン、フェノキサジン、ピロリジン、ピロリン、イミダゾリジン、イミダゾリン、ピラゾリジン等が挙げられる。好ましくはプリン又はピリミジンである。
　該炭素環は、単環又は縮合環の炭化水素環を含む。例えば、ベンゼン、ナフタレン、アントラセン、フェナントレン、インダン、インデン、テトラヒドロナフチレン、ビフェニレン等が挙げられる。好ましくは、ベンゼン又はナフタレンである。
　該複素環式基又は炭素環式基の置換基としては、置換基群αに含まれる置換基が挙げられる。任意の位置の炭素原子が置換基群αから選択される１以上の置換基と結合していてもよい。
　置換基群α：ハロゲン、ヒドロキシ、核酸合成に用いられる保護基で保護された水酸基、アルキル、アルキルオキシ、アルキルチオ、アルキルアミノ、アルケニル、アルキニル、メルカプト、核酸合成に用いられる保護基で保護されたメルカプト、アミノ、核酸合成に用いられる保護基で保護されたアミノ。 “Nucleobase moiety” means a substituent comprising a nucleobase or an analog thereof. Natural nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) or uracil (U). The nucleobases of the present invention are not limited to these, but also include other artificial or natural nucleobases. For example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone Etc.
That is, the “nucleobase moiety” in the present invention is a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted carbocyclic group that constitutes the base part of a nucleic acid (DNA, RNA).
The heterocyclic ring includes a single ring or a condensed ring having one or more of the same or different heteroatoms arbitrarily selected from O, S and N in the ring. For example, purine, pyrimidine, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, Examples include naphthyridine, quinoxaline, quinazoline, pteridine, carbazole, phenanthridine, acridine, perimidine, phenazine, phenalsazine, phenothiazine, furazane, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, and pyrazolidine. Preferred is purine or pyrimidine.
The carbocycle includes a monocyclic or condensed hydrocarbon ring. Examples thereof include benzene, naphthalene, anthracene, phenanthrene, indane, indene, tetrahydronaphthylene, biphenylene and the like. Preferred is benzene or naphthalene.
Examples of the substituent of the heterocyclic group or carbocyclic group include substituents included in the substituent group α. The carbon atom at any position may be bonded to one or more substituents selected from the substituent group α.
Substituent group α: halogen, hydroxy, hydroxyl group protected with a protecting group used for nucleic acid synthesis, alkyl, alkyloxy, alkylthio, alkylamino, alkenyl, alkynyl, mercapto, mercapto protected with a protecting group used for nucleic acid synthesis , Amino, amino protected with a protecting group used in nucleic acid synthesis.
　「核酸合成に用いられる保護基で保護された水酸基」の保護基とは、核酸合成の際に安定して水酸基を保護し得るものであれば、特に限定されない。具体的には、酸性又は中性条件で安定であり、加水素分解、加水分解、電気分解及び光分解のような化学的方法により開裂し得る保護基である。例えば、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル、ホルミル又は以下の保護基が挙げられる。
脂肪族アシル：アルキルカルボニル（例えば、アセチル、プロピオニル、ブチリル、イソブチリル、ペンタノイル、ピバロイル、バレリル、イソバレリル、オクタノイル、ノナノイル、デカノイル、３－メチルノナノイル、８－メチルノナノイル、３－エチルオクタノイル、３，７－ジメチルオクタノイル、ウンデカノイル、ドデカノイル、トリデカノイル、テトラデカノイル、ペンタデカノイル、ヘキサデカノイル、１－メチルペンタデカノイル、１４－メチルペンタデカノイル、１３，１３－ジメチルテトラデカノイル、ヘプタデカノイル、１５－メチルヘキサデカノイル、オクタデカノイル、１－メチルヘプタデカノイル、ノナデカノイル、アイコサノイル、ヘナイコサノイル等）、カルボキシアルキルカルボニル（例えば、スクシノイル、グルタロイル、アジポイル等）、ハロアルキルカルボニル（例えば、クロロアセチル、ジクロロアセチル、トリクロロアセチル、トリフルオロアセチル等）、アルキルオキシアルキルカルボニル（例えば、メトキシアセチル等）、不飽和アルキルカルボニル（例えば、（Ｅ）－２－メチル－２－ブテノイル等）等。
芳香族アシル：芳香族炭素環式基カルボニル（例えば、ベンゾイル、α－ナフトイル、β－ナフトイル等）、ハロゲンで置換された芳香族炭素環式基カルボニル（例えば、２－ブロモベンゾイル、４－クロロベンゾイル等）、アルキルで置換された芳香族炭素環式基カルボニル（例えば、２，４，６－トリメチルベンゾイル、４－トルオイル等）、アルキルオキシで置換された芳香族炭素環式基カルボニル（例えば、４－アニソイル等）、カルボキシで置換された芳香族炭素環式基カルボニル（２－カルボキシベンゾイル、３－カルボキシベンゾイル、４－カルボキシベンゾイル等）、ニトロで置換された芳香族炭素環式基カルボニル（４－ニトロベンゾイル、２－ニトロベンゾイル等）アルキルオキシカルボニルで置換された芳香族炭素環式基カルボニル（２－（メトキシカルボニル）ベンゾイル等）、芳香族炭素環式基で置換された芳香族炭素環式基カルボニル（４－フェニルベンゾイル等）等。
テトラヒドロピラニル：テトラヒドロピラン－２－イル、３－ブロモテトラヒドロピラン－２－イル、４－メトキシテトラヒドロピラン－４－イル等。
テトラヒドロチオピラニル：テトラヒドロチオピラン－２－イル、４－メトキシテトラヒドロチオピラン－４－イル等。
テトラヒドロフラニル：テトラヒドロフラン－２－イル等。
テトラヒドロチオフラニル：テトラヒドロチオフラン－２－イル等。
シリル：トリアルキルシリル（トリメチルシリル、トリエチルシリル、イソプロピルジメチルシリル、ｔ－ブチルジメチルシリル、メチルジイソプロピルシリル、メチルジ－ｔ－ブチルシリル、トリイソプロピルシリル等）、１～２個の芳香族炭素環式基で置換されたトリアルキルシリル（ジフェニルメチルシリル、ジフェニルブチルシリル、ジフェニルイソプロピルシリル、フェニルジイソプロピルシリル等）等。
アルキルオキシメチル：メトキシメチル、１，１－ジメチル－１－メトキシメチル、エトキシメチル、プロポキシメチル、イソプロポキシメチル、ブトキシメチル、ｔ－ブトキシメチル等。
アルキルオキシ化アルキルオキシメチル：２－メトキシエトキシメチル等。
ハロゲノアルキルオキシメチル：２，２，２－トリクロロエトキシメチル、ビス（２－クロロエトキシ）メチル等。
アルキルオキシ化エチル：１－エトキシエチル、１－（イソプロポキシ）エチル等。
ハロゲン化エチル：２，２，２－トリクロロエチル等。
１～３個の芳香族炭素環式基で置換されたメチル：ベンジル、α－ナフチルメチル、β－ナフチルメチル、ジフェニルメチル、トリフェニルメチル、α－ナフチルジフェニルメチル、９－アンスリルメチル等。
アルキル、アルキルオキシ、ハロゲン又はシアノで芳香族炭素環が置換された１～３個の芳香族炭素環式基で置換されたメチル：４－メチルベンジル、２，４，６－トリメチルベンジル、３，４，５－トリメチルベンジル、４－メトキシベンジル、４－メトキシフェニルジフェニルメチル、４，４’－ジメトキシトリフェニルメチル、２－ニトロベンジル、４－ニトロベンジル、４－クロロベンジル、４－ブロモベンジル、４－シアノベンジル等。
アルキルオキシカルボニル：メトキシカルボニル、エトキシカルボニル、ｔ－ブトキシカルボニル、イソブトキシカルボニル等。
ハロゲン、アルキルオキシ又はニトロで置換された芳香族炭素環式基：４－クロロフェニル、２－フロロフェニル、４－メトキシフェニル、４－ニトロフェニル、２，４－ジニトロフェニル等。
ハロゲン又はトリアルキルシリル基で置換されたアルキルオキシカルボニル：２，２，２－トリクロロエトキシカルボニル、２－トリメチルシリルエトキシカルボニル等。
アルケニルオキシカルボニル：ビニルオキシカルボニル、芳香族炭素環式基オキシカルボニル等。
１～２個のアルキルオキシ又はニトロで芳香族炭素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：ベンジルオキシカルボニル、４－メトキシベンジルオキシカルボニル、３，４－ジメトキシベンジルオキシカルボニル、２－ニトロベンジルオキシカルボニル、４－ニトロベンジルオキシカルボニル等。
　好ましい保護基としては、アルキル、アルケニル、「脂肪族アシル」、「芳香族アシル」、「１～３個の芳香族炭素環式基で置換されたメチル」、「ハロゲン、アルキルオキシ又はニトロで置換された芳香族炭素環式基」等が挙げられる。さらに好ましくは、ベンゾイル、ベンジル、２－クロロフェニル、４－クロロフェニル、２－プロペニル等が挙げられる。 The protecting group of “hydroxyl group protected by a protecting group used for nucleic acid synthesis” is not particularly limited as long as it can stably protect a hydroxyl group during nucleic acid synthesis. Specifically, it is 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 include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, formyl, and the following protecting groups.
Aliphatic acyl: alkylcarbonyl (eg, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyl Octanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexayl Decanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, icosanoyl, heinacosanoyl, etc.), carboxyalkylcarbonyl (eg, succino , Glutaroyl, adipoyl etc.), haloalkylcarbonyl (eg chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl etc.), alkyloxyalkylcarbonyl (eg methoxyacetyl etc.), unsaturated alkylcarbonyl (eg (E) -2-methyl-2-butenoyl and the like.
Aromatic acyl: Aromatic carbocyclic group carbonyl (eg, benzoyl, α-naphthoyl, β-naphthoyl, etc.), aromatic carbocyclic group carbonyl substituted with halogen (eg, 2-bromobenzoyl, 4-chlorobenzoyl) Etc.), an aromatic carbocyclic group carbonyl substituted with alkyl (eg 2,4,6-trimethylbenzoyl, 4-toluoyl etc.), an aromatic carbocyclic group carbonyl substituted with alkyloxy (eg 4 -Anisoyl etc.), carboxy substituted aromatic carbocyclic groups carbonyl (2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl etc.), nitro substituted aromatic carbocyclic groups carbonyl (4- Nitrobenzoyl, 2-nitrobenzoyl, etc.) Aromatic carbocyclic substituted with alkyloxycarbonyl A group carbonyl (such as 2- (methoxycarbonyl) benzoyl), an aromatic carbocyclic group carbonyl (such as 4-phenylbenzoyl) substituted with an aromatic carbocyclic group, and the like.
Tetrahydropyranyl: tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl and the like.
Tetrahydrothiopyranyl: tetrahydrothiopyran-2-yl, 4-methoxytetrahydrothiopyran-4-yl and the like.
Tetrahydrofuranyl: tetrahydrofuran-2-yl and the like.
Tetrahydrothiofuranyl: tetrahydrothiofuran-2-yl and the like.
Silyl: Trialkylsilyl (trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, etc.) substituted with 1 to 2 aromatic carbocyclic groups Trialkylsilyl (diphenylmethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl, etc.) and the like.
Alkyloxymethyl: methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl and the like.
Alkyloxylated alkyloxymethyl: 2-methoxyethoxymethyl and the like.
Halogenoalkyloxymethyl: 2,2,2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl and the like.
Alkyloxylated ethyl: 1-ethoxyethyl, 1- (isopropoxy) ethyl and the like.
Ethyl halide: 2,2,2-trichloroethyl and the like.
Methyl substituted with 1 to 3 aromatic carbocyclic groups: benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl and the like.
Methyl substituted with 1 to 3 aromatic carbocyclic groups in which the aromatic carbocycle is substituted with alkyl, alkyloxy, halogen or cyano: 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3, 4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4 -Cyanobenzyl and the like.
Alkyloxycarbonyl: methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl and the like.
Aromatic carbocyclic groups substituted with halogen, alkyloxy or nitro: 4-chlorophenyl, 2-fluorophenyl, 4-methoxyphenyl, 4-nitrophenyl, 2,4-dinitrophenyl and the like.
Alkyloxycarbonyl substituted with a halogen or trialkylsilyl group: 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl and the like.
Alkenyloxycarbonyl: vinyloxycarbonyl, aromatic carbocyclic group oxycarbonyl and the like.
Aralkyloxycarbonyl optionally substituted with 1 to 2 alkyloxy or nitro aromatic carbocycles: benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2-nitrobenzyloxy Carbonyl, 4-nitrobenzyloxycarbonyl and the like.
Preferred protecting groups include alkyl, alkenyl, “aliphatic acyl”, “aromatic acyl”, “methyl substituted with 1 to 3 aromatic carbocyclic groups”, “substituted with halogen, alkyloxy or nitro” Aromatic carbocyclic group "and the like. More preferred are benzoyl, benzyl, 2-chlorophenyl, 4-chlorophenyl, 2-propenyl and the like.
　「核酸合成に用いられる保護基で保護されたメルカプト」の保護基としては、核酸合成の際に安定してメルカプトを保護し得るものであれば、特に限定されない。具体的には、酸性又は中性条件で安定であり、加水素分解、加水分解、電気分解及び光分解のような化学的方法により開裂し得る保護基である。
　例えば、上記水酸基の保護基として挙げられたものの他、以下も含まれる。
ジスルフィドを形成する基：アルキルチオ（メチルチオ、エチルチオ、ｔｅｒｔ－ブチルチオ等）、芳香族炭素環式基チオ（ベンジルチオ等）等。
　好ましい保護基としては、「脂肪族アシル」、「芳香族アシル」等が挙げられる。さらに好ましくは、ベンゾイル等が挙げられる。 The protecting group of “mercapto protected with a protecting group used for nucleic acid synthesis” is not particularly limited as long as it can stably protect mercapto during nucleic acid synthesis. Specifically, it is 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.
For example, in addition to those mentioned as the protective group for the hydroxyl group, the following are also included.
Groups that form disulfides: alkylthio (methylthio, ethylthio, tert-butylthio, etc.), aromatic carbocyclic groups thio (benzylthio, etc.), etc.
Preferable protecting groups include “aliphatic acyl”, “aromatic acyl” and the like. More preferably, benzoyl etc. are mentioned.
　「核酸合成に用いられる保護基で保護されたアミノ」の保護基としては、核酸合成の際に安定してアミノを保護し得るものであれば、特に限定されない。具体的には、酸性又は中性条件で安定であり、加水素分解、加水分解、電気分解及び光分解のような化学的方法により開裂し得る保護基である。
　例えば、ホルミル、上記水酸基の保護基として挙げられた「脂肪族アシル」、「芳香族アシル」、「アルキルオキシカルボニル」、「ハロゲン又はトリアルキルシリル基で置換されたアルキルオキシカルボニル」、「アルケニルオキシカルボニル」、「１～２個のアルキルオキシ又はニトロで芳香族炭素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」が挙げられる。
　好ましい保護基としては、「脂肪族アシル」、「芳香族アシル」等が挙げられる。さらに好ましくは、ベンゾイル等が挙げられる。 The protecting group of “amino protected with a protecting group used for nucleic acid synthesis” is not particularly limited as long as it can stably protect amino during nucleic acid synthesis. Specifically, it is 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.
For example, formyl, “aliphatic acyl”, “aromatic acyl”, “alkyloxycarbonyl”, “alkyloxycarbonyl substituted with a halogen or trialkylsilyl group”, “alkenyloxy” mentioned as the protective group for the above hydroxyl group Carbonyl "," aralkyloxycarbonyl optionally substituted with 1 to 2 alkyloxy or nitro aromatic carbocycle ".
Preferable protecting groups include “aliphatic acyl”, “aromatic acyl” and the like. More preferably, benzoyl etc. are mentioned.
　「核酸塩基部分」として、好ましくは、置換若しくは非置換のプリン－９－イル、又は、置換若しくは非置換の２－オキソ－ピリミジン－１－イル等が挙げられる。核酸塩基部分に含まれる環の置換基としては、上記置換基群αに含まれる置換基が挙げられる。任意の位置の炭素原子が置換基群αから選択される１以上の置換基と結合していてもよい。さらに好ましくは、上記置換基群αから選択される１以上の置換基で置換された、プリン－９－イル又は２－オキソ－ピリミジン－１－イルである。特に好ましくは、上記置換基群αから選択される１又は２の置換基で置換された、プリン－９－イル又は２－オキソ－ピリミジン－１－イルである。
　例えば、６－アミノプリン－９－イル（即ち、アデニニル）、アミノが核酸合成に用いられる保護基で保護された６－アミノプリン－９－イル、２，６－ジアミノプリン－９－イル、アミノが核酸合成に用いられる保護基で保護された２，６－ジアミノプリン－９－イル、６－クロロプリン－９－イル、２－アミノ－６－クロロプリン－９－イル、アミノが核酸合成に用いられる保護基で保護された２－アミノ－６－クロロプリン－９－イル、６－フルオロプリン－９－イル、２－アミノ－６－フルオロプリン－９－イル、アミノが核酸合成に用いられる保護基で保護された２－アミノ－６－フルオロプリン－９－イル、６－ブロモプリン－９－イル、２－アミノ－６－ブロモプリン－９－イル、アミノが核酸合成に用いられる保護基で保護された２－アミノ－６－ブロモプリン－９－イル、２－アミノ－６－ヒドロキシプリン－９－イル（即ち、グアニニル）、アミノが核酸合成に用いられる保護基で保護された２－アミノ－６－ヒドロキシプリン－９－イル、６－アミノ－２－メトキシプリン－９－イル、６－アミノ－２－クロロプリン－９－イル、６－アミノ－２－フルオロプリン－９－イル、２，６－ジメトキシプリン－９－イル、２，６－ジクロロプリン－９－イル、６－メルカプトプリン－９－イル、２－オキソ－４－アミノ－１，２－ジヒドロピリミジン－１－イル（即ち、シトシニル）、アミノが核酸合成に用いられる保護基で保護された２－オキソ－４－アミノ－１，２－ジヒドロピリミジン－１－イル、２－オキソ－４－アミノ－５－フルオロ－１，２－ジヒドロピリミジン－１－イル、アミノが核酸合成に用いられる保護基で保護された２－オキソ－４－アミノ－５－フルオロ－１，２－ジヒドロピリミジン－１－イル、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、２－オキソ－４－メトキシ－１，２－ジヒドロピリミジン－１－イル、２－オキソ－４－メルカプト－１，２－ジヒドロピリミジン－１－イル、２－オキソ－４－ヒドロキシ－１，２－ジヒドロピリミジン－１－イル（即ち、ウラシニル）、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（即ち、チミニル）、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（即ち、５－メチルシトシニル）、又は、アミノが核酸合成に用いられる保護基で保護された４－アミノ－５－メチル－２－オキソ－１，２－ジヒドロピリミジン－１－イル等が挙げられる。 The “nucleobase moiety” is preferably substituted or unsubstituted purin-9-yl, substituted or unsubstituted 2-oxo-pyrimidin-1-yl, and the like. Examples of the ring substituent contained in the nucleobase moiety include those contained in the above substituent group α. The carbon atom at any position may be bonded to one or more substituents selected from the substituent group α. More preferred is purin-9-yl or 2-oxo-pyrimidin-1-yl substituted with one or more substituents selected from the above substituent group α. Particularly preferred is purin-9-yl or 2-oxo-pyrimidin-1-yl substituted with one or two substituents selected from the above substituent group α.
For example, 6-aminopurin-9-yl (ie, adeninyl), 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, amino protected with a protecting group used in nucleic acid synthesis, amino 2,6-diaminopurin-9-yl, 6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, amino protected with a protecting group used for nucleic acid synthesis 2-Amino-6-chloropurin-9-yl, 6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl, amino protected with the protecting groups used are used for nucleic acid synthesis Protecting groups protected with 2-amino-6-fluoropurin-9-yl, 6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, amino protected with a protecting group Protected by 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (ie, guaninyl), amino protected with a protecting group used in nucleic acid synthesis 6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2, 6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (ie, Cytosynyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, amino protected with a protecting group used for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2, -Jihi Ropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, amino protected with a protecting group used for nucleic acid synthesis, 4-amino-2-oxo- 5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1 -Yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (ie uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl ( Ie, thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl), or amino is used in nucleic acid synthesis. And 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl protected with a protecting group.
　さらに具体的には、以下に記載の式：(Ｂ－１)～(Ｂ－４)で示される基が挙げられる。
　式（Ｂ－１）：
Figure JPOXMLDOC01-appb-C000009
（式中、Ｒａは、水素原子又はアルキルであり、
Ｒｂは、水素原子又はアルキルである。）
で示される基。
　Ｒａは、好ましくは、水素原子又はＣ１－Ｃ５アルキルである。さらに好ましくは水素原子又はメチルである。
Ｒｂは、好ましくは、水素原子である。 More specifically, groups represented by the following formulas: (B-1) to (B-4) can be mentioned.
Formula (B-1):
Figure JPOXMLDOC01-appb-C000009
(Wherein R a is a hydrogen atom or alkyl,
R b is a hydrogen atom or alkyl. )
A group represented by
R a is preferably a hydrogen atom or C1-C5 alkyl. More preferably, they are a hydrogen atom or methyl.
R b is preferably a hydrogen atom.
　式（Ｂ－２）：
Figure JPOXMLDOC01-appb-C000010
（式中、
Ｒｃは、水素原子、ハロゲン又はアルキルであり、
Ｒｄは、アミノ、メルカプト、アルキルオキシ、ＮＨＣＯＲｅ、ＮＨＣＯＣＨ２ＯＲｅ又はＮ＝ＮＲｆであり、
Ｒｅは、置換若しくは非置換のアルキル又は置換若しくは非置換の芳香族炭素環式基であり、
Ｒｆは、水素原子又はアルキルである）
で示される基。
　Ｒｃは、好ましくは、水素原子又はＣ１－Ｃ５アルキルである。さらに好ましくは水素原子又はメチルである。
　Ｒｄは、好ましくは、ＮＨＣＯＰｈ、ＮＨＣＯＣＨ３、ＮＨＣＯＣＨ２ＯＰｈ、ＮＨＣＯＣＨ２Ｏ－（４－ｔＢｕ）Ｐｈである。 Formula (B-2):
Figure JPOXMLDOC01-appb-C000010
(Where
R c is a hydrogen atom, halogen or alkyl,
R d is amino, mercapto, alkyloxy, NHCOR e , NHCOCH 2 OR e or N═NR f ;
R e is a substituted or unsubstituted alkyl or a substituted or unsubstituted aromatic carbocyclic group;
R f is a hydrogen atom or alkyl)
A group represented by
R c is preferably a hydrogen atom or C1-C5 alkyl. More preferably, they are a hydrogen atom or methyl.
R d is preferably NHCOPh, NHCOCH 3 , NHCOCH 2 OPh, NHCOCH 2 O— (4-tBu) Ph.
　式（Ｂ－３）：
Figure JPOXMLDOC01-appb-C000011
（式中、
Ｒｇは、ハロゲン、アミノ、メルカプト、アルキルオキシ、ＮＨＣＯＲｉ、ＮＨＣＯＣＨ２ＯＲｉ又はＮ＝ＮＲｊであり、
Ｒｈは、水素原子、ハロゲン、アミノ又はアルキルオキシであり、
Ｒｉは、置換若しくは非置換のアルキル又は置換若しくは非置換の芳香族炭素環式基であり、
Ｒｊは、水素原子又はアルキルである）
で示される基。
　Ｒｇは、好ましくは、ＮＨＣＯＰｈ、ＮＨＣＯＣＨ３、ＮＨＣＯＣＨ２ＯＰｈ、ＮＨＣＯＣＨ２Ｏ－（４－ｔＢｕ）Ｐｈである。
　Ｒｈは、好ましくは、水素原子である。 Formula (B-3):
Figure JPOXMLDOC01-appb-C000011
(Where
R g is halogen, amino, mercapto, alkyloxy, NHCOR i , NHCOCH 2 OR i or N = NR j ;
R h is a hydrogen atom, halogen, amino or alkyloxy,
R i is a substituted or unsubstituted alkyl or a substituted or unsubstituted aromatic carbocyclic group;
R j is a hydrogen atom or alkyl)
A group represented by
R g is preferably NHCOPh, NHCOCH 3 , NHCOCH 2 OPh, NHCOCH 2 O— (4-tBu) Ph.
R h is preferably a hydrogen atom.
　式（Ｂ－４）：
Figure JPOXMLDOC01-appb-C000012
（式中、
Ｒｋは、アミノ、ＮＨＣＯＲｍ、ＮＨＣＯＣＨ２ＯＲｍ又はＮ＝ＮＲｎであり、
Ｒｍは、置換若しくは非置換のアルキル又は置換若しくは非置換の芳香族炭素環式基であり、
Ｒｎは、水素原子又はアルキルである）
で示される基。
　Ｒｋは、好ましくは、ＮＨＣＯＰｈ、ＮＨＣＯＣＨ３、ＮＨＣＯＣＨ（ＣＨ３）２、ＮＨＣＯＣＨ２ＯＰｈ、ＮＨＣＯＣＨ２Ｏ－（４－ｔＢｕ）Ｐｈである。 Formula (B-4):
Figure JPOXMLDOC01-appb-C000012
(Where
R k is amino, NHCOR m , NHCOCH 2 OR m or N═NR n ;
R m is a substituted or unsubstituted alkyl or a substituted or unsubstituted aromatic carbocyclic group;
R n is a hydrogen atom or alkyl)
A group represented by
R k is preferably NHCOPh, NHCOCH 3 , NHCOCH (CH 3 ) 2 , NHCOCH 2 OPh, NHCOCH 2 O— (4-tBu) Ph.
　例えば、以下が挙げられる。
Figure JPOXMLDOC01-appb-C000013
（式中、Ｒ’は、水素原子又は核酸合成に用いられるアミノの保護基である。例えば、イソブチル、アセチル、ベンゾイル、フェノキシアセチル等が挙げられる） Examples include the following.
Figure JPOXMLDOC01-appb-C000013
(In the formula, R ′ is a hydrogen atom or an amino protecting group used for nucleic acid synthesis. Examples thereof include isobutyl, acetyl, benzoyl, phenoxyacetyl, etc.)
　「糖部位」とは、天然の核酸に含まれる糖又はその類縁体を含む置換基を意味する。
　「天然の核酸に含まれる糖の置換基」としては、以下のＤＮＡの糖部位
Figure JPOXMLDOC01-appb-C000014
及び以下のＲＮＡの糖部位
Figure JPOXMLDOC01-appb-C000015
が挙げられる。 “Sugar moiety” means a substituent containing a sugar or analog thereof contained in a natural nucleic acid.
“Substituents of sugars contained in natural nucleic acids” include the following sugar sites of DNA
Figure JPOXMLDOC01-appb-C000014
And the following sugar sites of RNA:
Figure JPOXMLDOC01-appb-C000015
Is mentioned.
　「糖の類縁体を含む置換基」としては、天然の核酸に含まれる５員環を含む糖の修飾体、テトラヒドロピラン等の６員環を含む糖の修飾体、その他当該分野で用いられる２環又は３環を含む糖の修飾体（例えば、Ｂｉｏｏｒｇａｎｉｃ　＆　Ｍｅｄｉｃｉｎａｌ　Ｃｈｅｍｉｓｔｒｙ，２００２、１０、８４１－８５４参照）等が挙げられる。 Examples of the “substituent containing a sugar analogue” include a modified sugar containing a 5-membered ring, a modified sugar containing a 6-membered ring such as tetrahydropyran, and the like used in this field. Modified examples of sugars containing a ring or 3 rings (see, for example, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
　「５員環を含む糖の修飾体」としては、例えば、以下が挙げられる。
式：
Figure JPOXMLDOC01-appb-C000016
（式中：
Ｒ３及びＲ４はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ５は水素であり、
Ｒ６は水素原子、ハロゲン、ヒドロキシ又は置換若しくは非置換のアルキルオキシであり、
ここでＲ５及びＲ６は、一緒になって架橋構造を形成していてもよく、
Ｒ７は水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルである）
で示される基。 Examples of “modified sugar containing a 5-membered ring” include the following.
formula:
Figure JPOXMLDOC01-appb-C000016
(Where:
R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl,
R 5 is hydrogen;
R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy,
Here, R 5 and R 6 may be combined to form a crosslinked structure,
R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl)
A group represented by
　Ｒ６は、例えば、Ｆ、ＯＣＨ３（ＯＭｅ）、ＯＣＨ２ＣＨ２ＯＣＨ３（ＭＯＥ）等が挙げられる。 Examples of R 6 include F, OCH 3 (OMe), OCH 2 CH 2 OCH 3 (MOE), and the like.
　「Ｒ５及びＲ６は、一緒になって架橋構造を形成していてもよい」とは、例えば、以下の糖の４’位と２’位との間の架橋構造が挙げられる。
　４’－（ＣＲ８Ｒ９）ｍ－Ｏ－２’、４’－（ＣＲ８Ｒ９）ｍ－Ｓ－２’、４’－（ＣＲ８Ｒ９）ｍ－Ｏ－Ｃ（＝Ｏ）－２’、
４’－（ＣＲ８Ｒ９）ｍ－ＮＲ１－Ｏ－（ＣＲ８Ｒ９）ｍ１－２’、４’－（ＣＲ８Ｒ９）ｍ１-Ｃ（＝Ｏ）－ＮＲ１－２’、４’－（ＣＲ８Ｒ９）ｍ２-Ｃ（＝Ｏ）－ＮＲ１－Ｘ３－２’、４’－（ＣＲ８Ｒ９）ｍ１－ＳＯ２－ＮＲ１－２’、又は
Figure JPOXMLDOC01-appb-C000017
であり、
ここで、
Ｘ３は、Ｏ、Ｓ、ＮＨ又はＣＨ２であり、
Ｒ１は、水素原子、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル、置換若しくは非置換のアルキニル、置換若しくは非置換の芳香族炭素環式基、置換若しくは非置換の非芳香族炭素環式基、置換若しくは非置換の芳香族複素環式基、置換若しくは非置換の非芳香族複素環式基、置換若しくは非置換の芳香族炭素環アルキル、置換若しくは非置換の非芳香族炭素環アルキル、置換若しくは非置換の芳香族複素環アルキル又は置換若しくは非置換の非芳香族複素環アルキルであり、
Ｒ８はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ９はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｙ１はＣＲ１０又はＮであり、
Ｙ２はＣＲ１１又はＮであり、
Ｙ３はＣＲ１２又はＮであり、
Ｒ１０、Ｒ１１及びＲ１２はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル、置換若しくは非置換のアルキニル、置換若しくは非置換のアミノ、置換若しくは非置換のアルキルオキシ、置換若しくは非置換のアルキルカルボニルアミノ、置換若しくは非置換のアルケニルカルボニルアミノ、置換若しくは非置換のアルキニルカルボニルアミノ、置換若しくは非置換のアルキルカルバモイル、置換若しくは非置換のアルケニルカルバモイル又は置換若しくは非置換のアルキニルカルバモイルであり、
ｍは、１～４の整数であり、
ｍ１は、０～３の整数であり、
ｍ２は、０又は１である。 “R 5 and R 6 may form a cross-linked structure together” includes, for example, a cross-linked structure between the 4′-position and the 2′-position of the following sugar.
4 ′-(CR 8 R 9 ) mO-2 ′, 4 ′-(CR 8 R 9 ) m-S-2 ′, 4 ′-(CR 8 R 9 ) m—O—C (═O) -2 ',
4 ′-(CR 8 R 9 ) m—NR 1 —O— (CR 8 R 9 ) m 1 −2 ′, 4 ′-(CR 8 R 9 ) m 1 —C (═O) —NR 1 −2 '4'-(CR 8 R 9 ) m 2 -C (= O) -NR 1 -X 3 -2 ', 4'-(CR 8 R 9 ) m 1 -SO 2 -NR 1 -2 ', Or
Figure JPOXMLDOC01-appb-C000017
And
here,
X 3 is O, S, NH or CH 2 ,
R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aromatic carbocyclic group, substituted or unsubstituted non-aromatic carbocycle Group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted non-aromatic heterocyclic group, substituted or unsubstituted aromatic carbocyclic alkyl, substituted or unsubstituted non-aromatic carbocyclic alkyl Substituted or unsubstituted aromatic heterocyclic alkyl or substituted or unsubstituted non-aromatic heterocyclic alkyl,
Each R 8 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 9 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Y 1 is CR 10 or N;
Y 2 is CR 11 or N;
Y 3 is CR 12 or N;
R 10 , R 11 and R 12 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amino, substituted Or unsubstituted alkyloxy, substituted or unsubstituted alkylcarbonylamino, substituted or unsubstituted alkenylcarbonylamino, substituted or unsubstituted alkynylcarbonylamino, substituted or unsubstituted alkylcarbamoyl, substituted or unsubstituted alkenylcarbamoyl, or Substituted or unsubstituted alkynylcarbamoyl,
m is an integer of 1 to 4,
m 1 is an integer from 0 to 3,
m 2 is 0 or 1.
　Ｒ１は、好ましくは、水素原子、アルキル、アルケニル、アルキニル、芳香族炭素環式基、非芳香族炭素環式基、芳香族複素環式基、非芳香族複素環式基、芳香族炭素環アルキル、非芳香族炭素環アルキル、芳香族複素環アルキル又は非芳香族複素環アルキルであり、β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。
　β群は、水酸基、アルキル、アルキルオキシ、メルカプト、アルキルチオ、アミノ、アルキルアミノ又はハロゲンである。 R 1 is preferably a hydrogen atom, alkyl, alkenyl, alkynyl, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocycle It is alkyl, non-aromatic carbocyclic alkyl, aromatic heterocyclic alkyl or non-aromatic heterocyclic alkyl, and may have one or more arbitrary substituents selected from the β group.
The β group is a hydroxyl group, alkyl, alkyloxy, mercapto, alkylthio, amino, alkylamino, or halogen.
　Ｒ８及びＲ９は、好ましくは、水素原子である。 R 8 and R 9 are preferably a hydrogen atom.
　該架橋構造として、好ましくは、４’－（ＣＲ８Ｒ９）ｍ－Ｏ－２’又は４’－（ＣＲ８Ｒ９）ｍ１-Ｃ（＝Ｏ）－ＮＲ１－２’（ＡｍＮＡ、Ｂｒｉｄｇｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ）であり、
ここで、
Ｒ１は、水素原子、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ８はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ９はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
ｍは、１～４の整数であり、
ｍ１は、０～２の整数である。 The cross-linked structure is preferably 4 ′-(CR 8 R 9 ) m—O-2 ′ or 4 ′-(CR 8 R 9 ) m 1 —C (═O) —NR 1 −2 ′ (AmNA, Bridged nucleic acid)
here,
R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl,
Each R 8 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 9 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
m is an integer of 1 to 4,
m 1 is an integer of 0-2.
　該架橋構造として、特に好ましくは、４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）又は、４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である。
　４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）の中で、特に好ましくは４’－ＣＨ２－Ｏ－２’（ＬＮＡ、Ｌｏｃｋｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ）である。具体例及びその調製方法は、国際公開第９８／３９３５２号、国際公開第２００３／０６８７９５号、国際公開第２００５／０２１５７０号等に記載されている。
　４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）の中で、特に好ましくは４’－Ｃ（＝Ｏ）－ＮＣＨ３－２’である。具体例及びその調製方法は、国際公開第２０１１／０５２４３６号に記載されている。 The cross-linked structure is particularly preferably 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′-C (═O) —NR 1 -2 ′ (R 1 Is a hydrogen atom or alkyl).
Among 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4), 4′—CH 2 —O-2 ′ (LNA, Locked Nucleic Acid) is particularly preferable. Specific examples and preparation methods thereof are described in International Publication No. 98/39352, International Publication No. 2003/066875, International Publication No. 2005/021570, and the like.
Among 4′-C (═O) —NR 1 −2 ′ (R 1 is a hydrogen atom or alkyl), 4′—C (═O) —NCH 3 −2 ′ is particularly preferable. Specific examples and preparation methods thereof are described in International Publication No. 2011/052436.
　「６員環を含む糖の修飾体」としては、例えば、以下が挙げられる。
式：
Figure JPOXMLDOC01-appb-C000018
（式中：
ｑ１、ｑ２、ｑ３、ｑ４、ｑ５、ｑ６及びｑ７はそれぞれ独立して、水素、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル、置換若しくは非置換のアルケニルであり、
Ｒ１３及びＲ１４は、それぞれ独立して、水素、ハロゲン、置換若しくは非置換のアルキルオキシ、ＮＪ１Ｊ２、ＳＪ１、Ｎ３、ＯＣ（＝Ｘ４）Ｊ１、ＯＣ（＝Ｘ４）ＮＪ１Ｊ２、ＮＪ３Ｃ（＝Ｘ４）ＮＪ１Ｊ２又はＣＮであり、
Ｘ４はＯ、Ｓ又はＮＪ１であり、
Ｊ１、Ｊ２及びＪ３はそれぞれ独立して、水素又はアルキルである）
で示される基。 Examples of the “modified sugar containing a 6-membered ring” include the following.
formula:
Figure JPOXMLDOC01-appb-C000018
(Where:
q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkenyl. ,
R 13 and R 14 are each independently hydrogen, halogen, substituted or unsubstituted alkyloxy, NJ 1 J 2 , SJ 1 , N 3 , OC (= X 4 ) J 1 , OC (= X 4 ). NJ 1 J 2 , NJ 3 C (= X 4 ) NJ 1 J 2 or CN,
X 4 is O, S or NJ 1 ,
J 1 , J 2 and J 3 are each independently hydrogen or alkyl)
A group represented by
　例えば、
ｑ１、ｑ２、ｑ３、ｑ４、ｑ５、ｑ６及びｑ７のうち少なくとも１つがメチルである基、
Ｒ１３又はＲ１４がＦである基、
Ｒ１３がＯＣＨ３（ＯＭｅ）であり、かつ、Ｒ１４が水素である基、
Ｒ１３がＯＣＨ２ＣＨ２ＯＣＨ３（ＭＯＥ）であり、かつ、Ｒ１４が水素である基
等が挙げられる。 For example,
a group wherein at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is methyl,
A group wherein R 13 or R 14 is F;
A group wherein R 13 is OCH 3 (OMe) and R 14 is hydrogen;
Examples include a group in which R 13 is OCH 2 CH 2 OCH 3 (MOE) and R 14 is hydrogen.
　上記当該分野で公知の核酸塩基部分や糖部位の修飾及び修飾方法については、例えば、以下の特許文献にも開示されている。
国際公開第９８／３９３５２号、国際公開第９９／０１４２２６号、国際公開２０００／０５６７４８号、国際公開第２００５／０２１５７０号、国際公開第２００３／０６８７９５号、国際公開第２０１１／０５２４３６号、国際公開第２００４／０１６７４９号、国際公開第２００５／０８３１２４号、国際公開２００７／１４３３１５号、国際公開第２００９／０７１６８０号、国際公開２０１４／１１２４６３号、国際公開２０１４／１２６２２９号等。 The modification and modification method of the nucleobase moiety and sugar moiety known in the art are also disclosed in, for example, the following patent documents.
International Publication No. 98/39352, International Publication No. 99/014226, International Publication No. 2000/056748, International Publication No. 2005/021570, International Publication No. 2003/066875, International Publication No. 2011/052436, International Publication No. 2004/016749, International Publication No. 2005/083124, International Publication No. 2007/143315, International Publication No. 2009/071680, International Publication No. 2014/112463, International Publication No. 2014/126229, and the like.
　式（ＩＩＩ）のＺの「水酸基保護基」としては、上記「核酸合成に用いられる保護基で保護された水酸基」の保護基として挙げた保護基が挙げられる。好ましくは、アルキル、アルケニル、「脂肪族アシル」、「芳香族アシル」等が挙げられる。
　さらに好ましくは、アセチル、ｔ－ブチル、ｔ－ブトキシメチル、メトキシメチル、テトラヒドロピラニル、１－エトキシエチル、１－（２－クロロエトキシ）エチル、２－トリメチルシリルエチル、ｐ－クロロフェニル、２，４－ジニトロフェニル、ベンジル、ベンゾイル、ｐ－フェニルベンゾイル、２，６－ジクロロベンジル、レブリノイル、ジフェニルメチル、ｐ－ニトロベンジル、トリメチルシリル、トリエチルシリル、ｔ－ブチルジメチルシリル、ｔ－ブチルジフェニルシリル、トリフェニルシリル、トリイソプロピルシリル、ギ酸ベンゾイル、クロロアセチル、トリクロロアセチル、トリフルオロアセチル、ピバロイル、イソブチリル、９－フルオレニルメチルオキシカルボニル、メタンスルホニル、ｐ－トルエンスルホニル、トリフルオロメタンスルホニル、トリフェニルメチル（トリチル）、モノメトキシトリチル、ジメトキシトリチル（ＤＭＴｒ）、トリメトキシトリチル、９－フェニルキサンチン－９－イル（Ｐｉｘｙｌ）又は９－（ｐ－メトキシフェニル）キサンチン－９－イル（ＭＯＸ）である。特に好ましくは、ベンジル、トリエチルシリル、ｔ－ブチルジメチルシリル、ｔ－ブチルジフェニルシリル、トリイソプロピルシリル、トリチル、モノメトキシトリチル、ジメトキシトリチル、トリメトキシトリチル等が挙げられる。 Examples of the “hydroxyl protecting group” for Z in the formula (III) include the protecting groups mentioned as the protecting group for the above “hydroxyl group protected with a protecting group used for nucleic acid synthesis”. Preferred are alkyl, alkenyl, “aliphatic acyl”, “aromatic acyl” and the like.
More preferably, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4- Dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, levulinoyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, Triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl, 9-fluorenylmethyloxycarbonyl, methanesulfonyl, p-toluenesulfonyl, Trifluoromethanesulfonyl, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) or 9- (p-methoxyphenyl) xanthine-9- Il (MOX). Particularly preferred are benzyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl and the like.
　「ハロゲン」とは、フッ素原子、塩素原子、臭素原子、及びヨウ素原子を包含する。特にフッ素原子、及び塩素原子が好ましい。 “Halogen” includes fluorine atom, chlorine atom, bromine atom and iodine atom. In particular, a fluorine atom and a chlorine atom are preferable.
　「アルキル」とは、炭素数１～１５、好ましくは炭素数１～１０、より好ましくは炭素数１～６、さらに好ましくは炭素数１～４の直鎖又は分枝状の炭化水素基を包含する。例えば、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、ネオペンチル、ｎ－ヘキシル、イソヘキシル、ｎ－へプチル、イソヘプチル、ｎ－オクチル、イソオクチル、ｎ－ノニル、ｎ－デシル等が挙げられる。
　「アルキル」の好ましい態様として、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチルが挙げられる。さらに好ましい態様として、メチル、エチル、ｎ－プロピル、イソプロピル、ｔｅｒｔ－ブチルが挙げられる。 “Alkyl” includes straight or branched hydrocarbon groups having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. To do. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl , Isooctyl, n-nonyl, n-decyl and the like.
Preferred embodiments of “alkyl” include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and n-pentyl. Further preferred examples include methyl, ethyl, n-propyl, isopropyl and tert-butyl.
　「アルケニル」とは、任意の位置に１以上の二重結合を有する、炭素数２～１５、好ましくは炭素数２～１０、より好ましくは炭素数２～６、さらに好ましくは炭素数２～４の直鎖又は分枝状の炭化水素基を包含する。例えば、ビニル、アリル、プロペニル、イソプロペニル、ブテニル、イソブテニル、プレニル、ブタジエニル、ペンテニル、イソペンテニル、ペンタジエニル、ヘキセニル、イソヘキセニル、ヘキサジエニル、ヘプテニル、オクテニル、ノネニル、デセニル、ウンデセニル、ドデセニル、トリデセニル、テトラデセニル、ペンタデセニル等が挙げられる。
　「アルケニル」の好ましい態様として、ビニル、アリル、プロペニル、イソプロペニル、ブテニルが挙げられる。 “Alkenyl” has 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms, having one or more double bonds at any position. These linear or branched hydrocarbon groups are included. For example, vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, decenyl, tridecenyl, decenyl Etc.
Preferred embodiments of “alkenyl” include vinyl, allyl, propenyl, isopropenyl and butenyl.
　「アルキニル」とは、任意の位置に１以上の三重結合を有する、炭素数２～１０、好ましくは炭素数２～８、さらに好ましくは炭素数２～６、さらに好ましくは炭素数２～４の直鎖又は分枝状の炭化水素基を包含する。例えば、エチニル、プロピニル、ブチニル、ペンチニル、ヘキシニル、ヘプチニル、オクチニル、ノニニル、デシニル等を包含する。これらはさらに任意の位置に二重結合を有していてもよい。
　「アルキニル」の好ましい態様として、エチニル、プロピニル、ブチニル、ペンチニルが挙げられる。 “Alkynyl” has 2 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, having one or more triple bonds at any position. Includes straight chain or branched hydrocarbon groups. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl and the like. These may further have a double bond at an arbitrary position.
Preferred embodiments of “alkynyl” include ethynyl, propynyl, butynyl and pentynyl.
　「アルキレン」とは、炭素数１～１５、好ましくは炭素数１～１０、より好ましくは炭素数１～６、さらに好ましくは炭素数１～４の直鎖又は分枝状の２価の炭化水素基を包含する。例えば、メチレン、エチレン、トリメチレン、プロピレン、テトラメチレン、ペンタメチレン、ヘキサメチレン等が挙げられる。 “Alkylene” is a straight or branched divalent hydrocarbon having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. Includes groups. Examples include methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene and the like.
　「ハロアルキル」とは、１以上の上記「ハロゲン」が上記「アルキル」に結合した基を意味する。例えば、モノフルオロメチル、モノフルオロエチル、モノフルオロプロピル、２，２，３，３，３－ペンタフルオロプロピル、モノクロロメチル、トリフルオロメチル、トリクロロメチル、２，２，２－トリフルオロエチル、２，２，２－トリクロロエチル、１，２－ジブロモエチル、１，１，１－トリフルオロプロパン－２－イル等が挙げられる。
　「ハロアルキル」の好ましい態様として、トリフルオロメチル、トリクロロメチルが挙げられる。 “Haloalkyl” means a group in which one or more of the “halogen” is bonded to the “alkyl”. For example, monofluoromethyl, monofluoroethyl, monofluoropropyl, 2,2,3,3,3-pentafluoropropyl, monochloromethyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2, Examples include 2,2-trichloroethyl, 1,2-dibromoethyl, 1,1,1-trifluoropropan-2-yl and the like.
Preferable embodiments of “haloalkyl” include trifluoromethyl and trichloromethyl.
　「アルキルオキシ」とは、上記「アルキル」が酸素原子に結合した基を意味する。例えば、メトキシ、エトキシ、ｎ－プロピルオキシ、イソプロピルオキシ、ｎ－ブチルオキシ、ｔｅｒｔ－ブチルオキシ、イソブチルオキシ、ｓｅｃ－ブチルオキシ、ペンチルオキシ、イソペンチルオキシ、へキシルオキシ等が挙げられる。
　「アルキルオキシ」の好ましい態様として、メトキシ、エトキシ、ｎ－プロピルオキシ、イソプロピルオキシ、ｔｅｒｔ－ブチルオキシが挙げられる。 “Alkyloxy” means a group in which the above “alkyl” is bonded to an oxygen atom. Examples thereof include methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy, tert-butyloxy, isobutyloxy, sec-butyloxy, pentyloxy, isopentyloxy, hexyloxy and the like.
Preferable embodiments of “alkyloxy” include methoxy, ethoxy, n-propyloxy, isopropyloxy, tert-butyloxy.
　「アルキルチオ」とは、上記「アルキル」が硫黄原子に結合した基を意味する。 “Alkylthio” means a group in which the above “alkyl” is bonded to a sulfur atom.
　「アルキルアミノ」には、モノアルキルアミノとジアルキルアミノが含まれる。
　「モノアルキルアミノ」とは、上記「アルキル」がアミノ基の窒素原子と結合している水素原子１個と置き換わった基を意味する。例えば、メチルアミノ、エチルアミノ、イソプロピルアミノ等が挙げられる。好ましくは、メチルアミノ、エチルアミノが挙げられる。
　「ジアルキルアミノ」とは、上記「アルキル」がアミノ基の窒素原子と結合している水素原子２個と置き換わった基を意味する。２個のアルキルは、同一でも異なっていてもよい。例えば、ジメチルアミノ、ジエチルアミノ、Ｎ，Ｎ－ジイソプロピルアミノ、Ｎ－メチル－Ｎ－エチルアミノ、Ｎ－イソプロピル－Ｎ－エチルアミノ等が挙げられる。好ましくは、ジメチルアミノ、ジエチルアミノが挙げられる。 “Alkylamino” includes monoalkylamino and dialkylamino.
“Monoalkylamino” means a group in which the above “alkyl” is replaced with one hydrogen atom bonded to the nitrogen atom of the amino group. For example, methylamino, ethylamino, isopropylamino and the like can be mentioned. Preferably, methylamino and ethylamino are used.
“Dialkylamino” means a group in which the above “alkyl” is replaced with two hydrogen atoms bonded to the nitrogen atom of the amino group. The two alkyls may be the same or different. Examples include dimethylamino, diethylamino, N, N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-ethylamino and the like. Preferable examples include dimethylamino and diethylamino.
　「アルキルカルボニルアミノ」とは、アルキルカルボニルがアミノ基の窒素原子と結合している水素原子１個又は２個と置き換わった基を意味する。２個の場合、それぞれのアルキルカルボニル基は、同一でも異なっていてもよい。例えば、メチルカルボニルアミノ、エチルカルボニルアミノ、プロピルカルボニルアミノ、イソプロピルカルボニルアミノ、ｔｅｒｔ－ブチルカルボニルアミノ、イソブチルカルボニルアミノ、ｓｅｃ－ブチルカルボニルアミノ、ジメチルカルボニルアミノ、ジエチルカルボニルアミノ、Ｎ，Ｎ－ジイソプロピルカルボニルアミノ等が挙げられる。
　「アルキルカルボニルアミノ」の好ましい態様としては、メチルカルボニルアミノ、エチルカルボニルアミノが挙げられる。 “Alkylcarbonylamino” means a group in which alkylcarbonyl is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the amino group. In the case of two, each alkylcarbonyl group may be the same or different. For example, methylcarbonylamino, ethylcarbonylamino, propylcarbonylamino, isopropylcarbonylamino, tert-butylcarbonylamino, isobutylcarbonylamino, sec-butylcarbonylamino, dimethylcarbonylamino, diethylcarbonylamino, N, N-diisopropylcarbonylamino, etc. Is mentioned.
Preferable embodiments of “alkylcarbonylamino” include methylcarbonylamino and ethylcarbonylamino.
　「アルケニルカルボニルアミノ」とは、アルケニルカルボニルがアミノ基の窒素原子と結合している水素原子１個又は２個と置き換わった基を意味する。２個の場合、それぞれのアルケニルカルボニル基は、同一でも異なっていてもよい。例えば、ビニルカルボニルアミノ、プロペニルカルボニルアミノ等が挙げられる。 “Alkenylcarbonylamino” means a group in which alkenylcarbonyl is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the amino group. In the case of two, each alkenylcarbonyl group may be the same or different. For example, vinylcarbonylamino, propenylcarbonylamino and the like can be mentioned.
　「アルキニルカルボニルアミノ」とは、アルキニルカルボニルがアミノ基の窒素原子と結合している水素原子１個又は２個と置き換わった基を意味する。２個の場合、それぞれのアルキニルカルボニル基は、同一でも異なっていてもよい。例えば、エチニルカルボニルアミノ、プロピニルカルボニルアミノ等が挙げられる。 “Alkynylcarbonylamino” means a group in which alkynylcarbonyl is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the amino group. In the case of two, each alkynylcarbonyl group may be the same or different. For example, ethynylcarbonylamino, propynylcarbonylamino and the like can be mentioned.
　「アルキルカルバモイル」とは、上記「アルキル」がカルバモイル基の窒素原子と結合している水素原子１個又は２個と置き換わった基を意味する。２個の場合、それぞれのアルキル基は、同一でも異なっていてもよい。例えば、メチルカルバモイル、エチルカルバモイル、ジメチルカルバモイル、ジエチルカルバモイル等が挙げられる。 “Alkylcarbamoyl” means a group in which the above “alkyl” is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the carbamoyl group. In the case of two, each alkyl group may be the same or different. For example, methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl and the like can be mentioned.
　「アルケニルカルバモイル」とは、上記「アルケニル」がカルバモイル基の窒素原子と結合している水素原子１個又は２個と置き換わった基を意味する。２個の場合、それぞれのアルケニル基は、同一でも異なっていてもよい。例えば、ビニルカルバモイル、プロペニルカルバモイル等が挙げられる。 “Alkenylcarbamoyl” means a group in which the above “alkenyl” is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the carbamoyl group. In the case of two, each alkenyl group may be the same or different. Examples thereof include vinyl carbamoyl, propenyl carbamoyl and the like.
　「アルキニルカルバモイル」とは、上記「アルキニル」がカルバモイル基の窒素原子と結合している水素原子１個又は２個と置き換わった基を意味する。２個の場合、それぞれのアルキニル基は、同一でも異なっていてもよい。例えば、エチニルカルバモイル、プロピニルカルバモイル等が挙げられる。 “Alkynylcarbamoyl” means a group in which the above “alkynyl” is replaced with one or two hydrogen atoms bonded to the nitrogen atom of the carbamoyl group. In the case of two, each alkynyl group may be the same or different. For example, ethynylcarbamoyl, propynylcarbamoyl and the like can be mentioned.
　「芳香族炭素環式基」とは、単環又は２環以上の、環状芳香族炭化水素基を意味する。例えば、フェニル、ナフチル、アントリル、フェナントリル等が挙げられる。
　「芳香族炭素環式基」の好ましい態様として、フェニルが挙げられる。 “Aromatic carbocyclic group” means a monocyclic or bicyclic or more cyclic aromatic hydrocarbon group. For example, phenyl, naphthyl, anthryl, phenanthryl and the like can be mentioned.
A preferred embodiment of the “aromatic carbocyclic group” includes phenyl.
　「非芳香族炭素環式基」とは、単環又は２環以上の、環状飽和炭化水素基又は環状非芳香族不飽和炭化水素基を意味する。２環以上の非芳香族炭素環式基は、単環又は２環以上の非芳香族炭素環式基に、上記「芳香族炭素環式基」における環が縮合したものも包含する。
　さらに、「非芳香族炭素環式基」は、以下のように架橋している基、又はスピロ環を形成する基も包含する。
Figure JPOXMLDOC01-appb-C000019
　単環の非芳香族炭素環式基としては、炭素数３～１６が好ましく、より好ましくは炭素数３～１２、さらに好ましくは炭素数４～８である。例えば、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、シクロヘプチル、シクロオクチル、シクロノニル、シクロデシル、シクロプロペニル、シクロブテニル、シクロペンテニル、シクロヘキセニル、シクロヘプテニル、シクロヘキサジエニル等が挙げられる。
　２環以上の非芳香族炭素環式基としては、例えば、インダニル、インデニル、アセナフチル、テトラヒドロナフチル、フルオレニル等が挙げられる。 The “non-aromatic carbocyclic group” means a cyclic saturated hydrocarbon group or a cyclic non-aromatic unsaturated hydrocarbon group having one or more rings. The non-aromatic carbocyclic group having two or more rings includes a monocyclic ring or a non-aromatic carbocyclic group having two or more rings condensed with the ring in the above “aromatic carbocyclic group”.
Furthermore, the “non-aromatic carbocyclic group” includes a group which forms a bridge or a spiro ring as described below.
Figure JPOXMLDOC01-appb-C000019
The monocyclic non-aromatic carbocyclic group preferably has 3 to 16 carbon atoms, more preferably 3 to 12 carbon atoms, and still more preferably 4 to 8 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, and the like.
Examples of the two or more non-aromatic carbocyclic groups include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl, fluorenyl and the like.
　「芳香族複素環式基」とは、Ｏ、Ｓ及びＮから任意に選択される同一又は異なるヘテロ原子を環内に１以上有する、単環又は２環以上の、芳香族環式基を意味する。
　２環以上の芳香族複素環式基は、単環又は２環以上の芳香族複素環式基に、上記「芳香族炭素環式基」における環が縮合したものも包含する。
　単環の芳香族複素環式基としては、５～８員が好ましく、より好ましくは５員又は６員である。例えば、ピロリル、イミダゾリル、ピラゾリル、ピリジル、ピリダジニル、ピリミジニル、ピラジニル、トリアゾリル、トリアジニル、テトラゾリル、フリル、チエニル、イソオキサゾリル、オキサゾリル、オキサジアゾリル、イソチアゾリル、チアゾリル、チアジアゾリル等が挙げられる。
　２環の芳香族複素環式基としては、例えば、インドリル、イソインドリル、インダゾリル、インドリジニル、キノリニル、イソキノリニル、シンノリニル、フタラジニル、キナゾリニル、ナフチリジニル、キノキサリニル、プリニル、プテリジニル、ベンズイミダゾリル、ベンズイソオキサゾリル、ベンズオキサゾリル、ベンズオキサジアゾリル、ベンズイソチアゾリル、ベンゾチアゾリル、ベンゾチアジアゾリル、ベンゾフリル、イソベンゾフリル、ベンゾチエニル、ベンゾトリアゾリル、イミダゾピリジル、トリアゾロピリジル、イミダゾチアゾリル、ピラジノピリダジニル、オキサゾロピリジル、チアゾロピリジル等が挙げられる。
　３環以上の芳香族複素環式基としては、例えば、カルバゾリル、アクリジニル、キサンテニル、フェノチアジニル、フェノキサチイニル、フェノキサジニル、ジベンゾフリル等が挙げられる。 “Aromatic heterocyclic group” means a monocyclic or bicyclic or more aromatic cyclic group having one or more heteroatoms arbitrarily selected from O, S and N in the ring. To do.
The aromatic heterocyclic group having two or more rings includes those obtained by condensing a ring in the above “aromatic carbocyclic group” to a monocyclic or two or more aromatic heterocyclic group.
The monocyclic aromatic heterocyclic group is preferably 5 to 8 members, more preferably 5 or 6 members. Examples include pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl and the like.
Examples of the bicyclic aromatic heterocyclic group include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzisoxazolyl, Oxazolyl, benzoxiadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyr Dazinyl, oxazolopyridyl, thiazolopyridyl and the like can be mentioned.
Examples of the aromatic heterocyclic group having 3 or more rings include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, dibenzofuryl and the like.
　「非芳香族複素環式基」とは、Ｏ、Ｓ及びＮから任意に選択される同一又は異なるヘテロ原子を環内に１以上有する、単環又は２環以上の、非芳香族環式基を意味する。
　２環以上の非芳香族複素環式基は、単環又は２環以上の非芳香族複素環式基に、上記「芳香族炭素環式基」、「非芳香族炭素環式基」、及び／又は「芳香族複素環式基」におけるそれぞれの環が縮合したものも包含する。
　さらに、「非芳香族複素環式基」は、以下のように架橋している基、又はスピロ環を形成する基も包含する。
Figure JPOXMLDOC01-appb-C000020
　単環の非芳香族複素環式基としては、３～８員が好ましく、より好ましくは５員又は６員である。例えば、ジオキサニル、チイラニル、オキシラニル、オキセタニル、オキサチオラニル、アゼチジニル、チアニル、チアゾリジニル、ピロリジニル、ピロリニル、イミダゾリジニル、イミダゾリニル、ピラゾリジニル、ピラゾリニル、ピペリジル、ピペラジニル、モルホリニル、モルホリノ、チオモルホリニル、チオモルホリノ、ジヒドロピリジル、テトラヒドロピリジル、テトラヒドロフリル、テトラヒドロピラニル、ジヒドロチアゾリル、テトラヒドロチアゾリル、テトラヒドロイソチアゾリル、ジヒドロオキサジニル、ヘキサヒドロアゼピニル、テトラヒドロジアゼピニル、テトラヒドロピリダジニル、ヘキサヒドロピリミジニル、ジオキソラニル、ジオキサジニル、アジリジニル、ジオキソリニル、オキセパニル、チオラニル、チイニル、チアジニル等が挙げられる。
　２環以上の非芳香族複素環式基としては、例えば、インドリニル、イソインドリニル、クロマニル、イソクロマニル等が挙げられる。 The “non-aromatic heterocyclic group” is a monocyclic or bicyclic or more non-aromatic cyclic group having one or more of the same or different heteroatoms arbitrarily selected from O, S and N in the ring Means.
The non-aromatic heterocyclic group having two or more rings includes the above-mentioned “aromatic carbocyclic group”, “non-aromatic carbocyclic group”, and monocyclic or two or more non-aromatic heterocyclic groups, and Also included are those in which each ring in the “aromatic heterocyclic group” is condensed.
Furthermore, the “non-aromatic heterocyclic group” also includes a group that forms a bridge or a spiro ring as described below.
Figure JPOXMLDOC01-appb-C000020
The monocyclic non-aromatic heterocyclic group is preferably 3 to 8 members, more preferably 5 or 6 members. For example, dioxanyl, thiranyl, oxiranyl, oxetanyl, oxathiolanyl, azetidinyl, thianyl, thiazolidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, morpholinyl, morpholino, thiomorpholinyl, morpholino, thiomorpholinyl, morpholino, thiomorpholinyl Furyl, tetrahydropyranyl, dihydrothiazolyl, tetrahydrothiazolyl, tetrahydroisothiazolyl, dihydrooxazinyl, hexahydroazepinyl, tetrahydrodiazepinyl, tetrahydropyridazinyl, hexahydropyrimidinyl, dioxolanyl, dioxazinyl Aziridinyl, dioxolinyl, oxepanyl, thiolanyl, thii Le, triazinyl, and the like.
Examples of the non-aromatic heterocyclic group having two or more rings include indolinyl, isoindolinyl, chromanyl, isochromanyl and the like.
　「置換若しくは非置換のアミノ」、「置換若しくは非置換のアルキル」、「置換若しくは非置換のアルケニル」、「置換若しくは非置換のアルキニル」、「置換若しくは非置換のアルキレン」、「置換若しくは非置換のアルキルオキシ」「置換若しくは非置換のアルキルカルボニルアミノ」、「置換若しくは非置換のアルケニルカルボニルアミノ」、「置換若しくは非置換のアルキニルカルボニルアミノ」、「置換若しくは非置換のアルキルカルバモイル」、「置換若しくは非置換のアルケニルカルバモイル」、「置換若しくは非置換のアルキニルカルバモイル」の置換基としては、次の置換基が挙げられる。任意の位置の炭素原子が次の置換基から選択される１以上の基と結合していてもよい。
　置換基：ハロゲン、ヒドロキシ、カルボキシ、アミノ、イミノ、ヒドロキシアミノ、ヒドロキシイミノ、ホルミル、ホルミルオキシ、カルバモイル、スルファモイル、スルファニル、スルフィノ、スルホ、チオホルミル、チオカルボキシ、ジチオカルボキシ、チオカルバモイル、シアノ、ニトロ、ニトロソ、アジド、ヒドラジノ、ウレイド、アミジノ、グアニジノ、トリアルキルシリル、アルキルオキシ、アルケニルオキシ、アルキニルオキシ、ハロアルキルオキシ、アルキルカルボニル、アルケニルカルボニル、アルキニルカルボニル、モノアルキルアミノ、ジアルキルアミノ、アルキルスルホニル、アルケニルスルホニル、アルキニルスルホニル、モノアルキルカルボニルアミノ、ジアルキルカルボニルアミノ、モノアルキルスルホニルアミノ、ジアルキルスルホニルアミノ、アルキルイミノ、アルケニルイミノ、アルキニルイミノ、アルキルカルボニルイミノ、アルケニルカルボニルイミノ、アルキニルカルボニルイミノ、アルキルオキシイミノ、アルケニルオキシイミノ、アルキニルオキシイミノ、アルキルカルボニルオキシ、アルケニルカルボニルオキシ、アルキニルカルボニルオキシ、アルキルオキシカルボニル、アルケニルオキシカルボニル、アルキニルオキシカルボニル、アルキルスルファニル、アルケニルスルファニル、アルキニルスルファニル、アルキルスルフィニル、アルケニルスルフィニル、アルキニルスルフィニル、モノアルキルカルバモイル、ジアルキルカルバモイル、モノアルキルスルファモイル、ジアルキルスルファモイル、芳香族炭素環式基、非芳香族炭素環式基、芳香族複素環式基、非芳香族複素環式基、芳香族炭素環オキシ、非芳香族炭素環オキシ、芳香族複素環オキシ、非芳香族複素環オキシ、芳香族炭素環カルボニル、非芳香族炭素環カルボニル、芳香族複素環カルボニル、非芳香族複素環カルボニル、芳香族炭素環オキシカルボニル、非芳香族炭素環オキシカルボニル、芳香族複素環オキシカルボニル、非芳香族複素環オキシカルボニル、芳香族炭素環アルキルオキシ、非芳香族炭素環アルキルオキシ、芳香族複素環アルキルオキシ、非芳香族複素環アルキルオキシ、芳香族炭素環アルキルオキシカルボニル、非芳香族炭素環アルキルオキシカルボニル、芳香族複素環アルキルオキシカルボニル、非芳香族複素環アルキルオキシカルボニル、芳香族炭素環アルキルアミノ、非芳香族炭素環アルキルアミノ、芳香族複素環アルキルアミノ、非芳香族複素環アルキルアミノ、芳香族炭素環スルファニル、非芳香族炭素環スルファニル、芳香族複素環スルファニル、非芳香族複素環スルファニル、非芳香族炭素環スルホニル、芳香族炭素環スルホニル、芳香族複素環スルホニル、及び非芳香族複素環スルホニル。 "Substituted or unsubstituted amino", "Substituted or unsubstituted alkyl", "Substituted or unsubstituted alkenyl", "Substituted or unsubstituted alkynyl", "Substituted or unsubstituted alkylene", "Substituted or unsubstituted Alkyloxy ”,“ substituted or unsubstituted alkylcarbonylamino ”,“ substituted or unsubstituted alkenylcarbonylamino ”,“ substituted or unsubstituted alkynylcarbonylamino ”,“ substituted or unsubstituted alkylcarbamoyl ”,“ substituted or Examples of the substituent of “unsubstituted alkenylcarbamoyl” and “substituted or unsubstituted alkynylcarbamoyl” include the following substituents. The carbon atom at any position may be bonded to one or more groups selected from the following substituents.
Substituents: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, alkynyl Sulfonyl, monoalkylcarbonylamino, dialkylcarbonylamino, monoalkylsulfonyl Mino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino, alkenylcarbonylimino, alkynylcarbonylimino, alkyloxyimino, alkenyloxyimino, alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy Alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylsulfanyl, alkenylsulfanyl, alkynylsulfanyl, alkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, monoalkylcarbamoyl, dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic Group carbocyclic group, Aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocyclic oxy, non-aromatic carbocyclic oxy, aromatic heterocyclic oxy, non-aromatic heterocyclic oxy, aromatic Carbocyclic carbonyl, non-aromatic carbocyclic carbonyl, aromatic heterocyclic carbonyl, non-aromatic heterocyclic carbonyl, aromatic carbocyclic oxycarbonyl, non-aromatic carbocyclic oxycarbonyl, aromatic heterocyclic oxycarbonyl, non-aromatic hetero Ring oxycarbonyl, aromatic carbocyclic alkyloxy, non-aromatic carbocyclic alkyloxy, aromatic heterocyclic alkyloxy, non-aromatic heterocyclic alkyloxy, aromatic carbocyclic alkyloxycarbonyl, non-aromatic carbocyclic alkyloxycarbonyl , Aromatic heterocyclic alkyloxycarbonyl, non-aromatic heterocyclic alkyloxycarbonyl, aromatic carbocyclic alkylamino, non-aromatic Aromatic carbocyclic alkylamino, aromatic heterocyclic alkylamino, non-aromatic heterocyclic alkylamino, aromatic carbocyclic sulfanyl, non-aromatic carbocyclic sulfanyl, aromatic heterocyclic sulfanyl, non-aromatic heterocyclic sulfanyl, non-aromatic Aromatic carbocyclic sulfonyl, aromatic carbocyclic sulfonyl, aromatic heterocyclic sulfonyl, and non-aromatic heterocyclic sulfonyl.
　「置換若しくは非置換の芳香族炭素環式基」の「芳香族炭素環」の環上の置換基としては、次の置換基が挙げられる。環上の任意の位置の原子が次の置換基から選択される１以上の基と結合していてもよい。
　置換基：ハロゲン、ヒドロキシ、カルボキシ、アミノ、イミノ、ヒドロキシアミノ、ヒドロキシイミノ、ホルミル、ホルミルオキシ、カルバモイル、スルファモイル、スルファニル、スルフィノ、スルホ、チオホルミル、チオカルボキシ、ジチオカルボキシ、チオカルバモイル、シアノ、ニトロ、ニトロソ、アジド、ヒドラジノ、ウレイド、アミジノ、グアニジノ、トリアルキルシリル、アルキル、アルケニル、アルキニル、ハロアルキル、アルキルオキシ、アルケニルオキシ、アルキニルオキシ、ハロアルキルオキシ、アルキルオキシアルキル、アルキルオキシアルキルオキシ、アルキルカルボニル、アルケニルカルボニル、アルキニルカルボニル、モノアルキルアミノ、ジアルキルアミノ、アルキルスルホニル、アルケニルスルホニル、アルキニルスルホニル、モノアルキルカルボニルアミノ、ジアルキルカルボニルアミノ、モノアルキルスルホニルアミノ、ジアルキルスルホニルアミノ、アルキルイミノ、アルケニルイミノ、アルキニルイミノ、アルキルカルボニルイミノ、アルケニルカルボニルイミノ、アルキニルカルボニルイミノ、アルキルオキシイミノ、アルケニルオキシイミノ、アルキニルオキシイミノ、アルキルカルボニルオキシ、アルケニルカルボニルオキシ、アルキニルカルボニルオキシ、アルキルオキシカルボニル、アルケニルオキシカルボニル、アルキニルオキシカルボニル、アルキルスルファニル、アルケニルスルファニル、アルキニルスルファニル、アルキルスルフィニル、アルケニルスルフィニル、アルキニルスルフィニル、モノアルキルカルバモイル、ジアルキルカルバモイル、モノアルキルスルファモイル、ジアルキルスルファモイル、芳香族炭素環式基、非芳香族炭素環式基、芳香族複素環式基、非芳香族複素環式基、芳香族炭素環オキシ、非芳香族炭素環オキシ、芳香族複素環オキシ、非芳香族複素環オキシ、芳香族炭素環カルボニル、非芳香族炭素環カルボニル、芳香族複素環カルボニル、非芳香族複素環カルボニル、芳香族炭素環オキシカルボニル、非芳香族炭素環オキシカルボニル、芳香族複素環オキシカルボニル、非芳香族複素環オキシカルボニル、芳香族炭素環アルキル、非芳香族炭素環アルキル、芳香族複素環アルキル、非芳香族複素環アルキル、芳香族炭素環アルキルオキシ、非芳香族炭素環アルキルオキシ、芳香族複素環アルキルオキシ、非芳香族複素環アルキルオキシ、芳香族炭素環アルキルオキシカルボニル、非芳香族炭素環アルキルオキシカルボニル、芳香族複素環アルキルオキシカルボニル、非芳香族複素環アルキルオキシカルボニル、芳香族炭素環アルキルオキシアルキル、非芳香族炭素環アルキルオキシアルキル、芳香族複素環アルキルオキシアルキル、非芳香族複素環アルキルオキシアルキル、芳香族炭素環アルキルアミノ、非芳香族炭素環アルキルアミノ、芳香族複素環アルキルアミノ、非芳香族複素環アルキルアミノ、芳香族炭素環スルファニル、非芳香族炭素環スルファニル、芳香族複素環スルファニル、非芳香族複素環スルファニル、非芳香族炭素環スルホニル、芳香族炭素環スルホニル、芳香族複素環スルホニル、及び非芳香族複素環スルホニル。 Examples of the substituent on the ring of “aromatic carbocycle” of “substituted or unsubstituted aromatic carbocyclic group” include the following substituents. An atom at any position on the ring may be bonded to one or more groups selected from the following substituents.
Substituents: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkyloxyalkyl, alkyloxyalkyloxy, alkylcarbonyl, alkenylcarbonyl, Alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, Lucinylsulfonyl, monoalkylcarbonylamino, dialkylcarbonylamino, monoalkylsulfonylamino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino, alkenylcarbonylimino, alkynylcarbonylimino, alkyloxyimino, alkenyloxyimino Alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylsulfanyl, alkenylsulfanyl, alkynylsulfanyl, alkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, monoalkylcarba Mo , Dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocycle Oxy, non-aromatic carbocyclic oxy, aromatic heterocyclic oxy, non-aromatic heterocyclic oxy, aromatic carbocyclic carbonyl, non-aromatic carbocyclic carbonyl, aromatic heterocyclic carbonyl, non-aromatic heterocyclic carbonyl, aromatic Carbocyclic oxycarbonyl, non-aromatic carbocyclic oxycarbonyl, aromatic heterocyclic oxycarbonyl, non-aromatic heterocyclic oxycarbonyl, aromatic carbocyclic alkyl, non-aromatic carbocyclic alkyl, aromatic heterocyclic alkyl, non-aromatic Heterocyclic alkyl, aromatic carbocyclic alkyloxy, non-aromatic carbocyclic alkyloxy, aromatic heterocyclic alkyloxy, non-aromatic heterocyclic alkyl Oxy, aromatic carbocyclic alkyloxycarbonyl, non-aromatic carbocyclic alkyloxycarbonyl, aromatic heterocyclic alkyloxycarbonyl, non-aromatic heterocyclic alkyloxycarbonyl, aromatic carbocyclic alkyloxyalkyl, non-aromatic carbocyclic alkyl Oxyalkyl, aromatic heterocyclic alkyloxyalkyl, non-aromatic heterocyclic alkyloxyalkyl, aromatic carbocyclic alkylamino, non-aromatic carbocyclic alkylamino, aromatic heterocyclic alkylamino, non-aromatic heterocyclic alkylamino, Aromatic carbocyclic sulfanyl, non-aromatic carbocyclic sulfanyl, aromatic heterocyclic sulfanyl, non-aromatic heterocyclic sulfanyl, non-aromatic carbocyclic sulfonyl, aromatic carbocyclic sulfonyl, aromatic heterocyclic sulfonyl, and non-aromatic hetero Ring sulfonyl.
　以下に、本発明のオリゴヌクレオチド又はその製薬上許容される塩の好ましい様態を説明する。 Hereinafter, preferred embodiments of the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof will be described.
式（Ｉ）：
Figure JPOXMLDOC01-appb-C000021
（式中、
ＱはＳ又はＯであり、
Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、
Ｘ２は置換又非置換の単環若しくは２環の含窒素非芳香族複素環式基である）
で示される結合を１以上含有するオリゴヌクレオチド又はその製薬上許容される塩。
　「Ｏが介在している置換若しくは非置換のアルキレン」の酸素原子はアルキレンの炭素原子間に存在するか、末端に存在する場合は該酸素原子はＸ２に結合する。「Ｏが介在している置換若しくは非置換のアルキレン」の酸素原子が上記式（Ｉ）に記載の酸素原子に結合することは無い。 Formula (I):
Figure JPOXMLDOC01-appb-C000021
(Where
Q is S or O,
X 1 is a substituted or unsubstituted alkylene which is absent or O may be interposed,
X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group)
Or an pharmaceutically acceptable salt thereof.
The oxygen atom of “substituted or unsubstituted alkylene with O intervening” is present between carbon atoms of alkylene, or when present at the terminal, the oxygen atom is bonded to X 2 . The oxygen atom of “substituted or unsubstituted alkylene intervening O” is not bonded to the oxygen atom described in the above formula (I).
　「Ｏが介在している置換若しくは非置換のアルキレン」とは、例えば、
式：－（ＣＲ１５Ｒ１６－ＣＲ１７Ｒ１８－Ｏ）ｎ－
（式中、
Ｒ１５はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ１６はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ１７はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ１８はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
ｎは１～５の整数である）
で示される基等が挙げられる。なお、Ｘ１が上記式で表される場合、上記式中、右側（酸素原子）にＸ２が結合する。 “O-mediated substituted or unsubstituted alkylene” means, for example,
Formula:-(CR 15 R 16 -CR 17 R 18 -O) n-
(Where
Each R 15 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 16 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 17 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 18 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl;
n is an integer of 1 to 5)
Group etc. which are shown by these are mentioned. When X 1 is represented by the above formula, X 2 is bonded to the right side (oxygen atom) in the above formula.
　Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、好ましくは、存在しないか、又は非置換のアルキレンであり、さらに好ましくは、存在しないか、Ｃ１－Ｃ５アルキレンである。
　Ｘ２は置換又非置換の単環若しくは２環の含窒素非芳香族複素環式基であり、好ましくは、置換若しくは非置換のアジリジニル、置換若しくは非置換のアゼチジニル、置換若しくは非置換のピロリジニル、置換若しくは非置換のピペリジル、置換若しくは非置換のピペリジノ、置換若しくは非置換のピペラジニル、置換若しくは非置換のピペラジノ、置換若しくは非置換のモルホリニル、置換若しくは非置換のモルホリノ、置換若しくは非置換のアゼパニル又は置換若しくは非置換のキヌクリジニルである。特に好ましくは、置換若しくは非置換のピペリジル、置換若しくは非置換のモルホリノ又置換若しくは非置換のキヌクリジニルである。 X 1 is absent or is a substituted or unsubstituted alkylene which may be mediated by O, preferably absent or unsubstituted alkylene, more preferably absent or C1- C5 alkylene.
X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group, preferably substituted or unsubstituted aziridinyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, Substituted or unsubstituted piperidyl, substituted or unsubstituted piperidino, substituted or unsubstituted piperazinyl, substituted or unsubstituted piperazino, substituted or unsubstituted morpholinyl, substituted or unsubstituted morpholino, substituted or unsubstituted azepanyl or substituted Or unsubstituted quinuclidinyl. Particularly preferred are substituted or unsubstituted piperidyl, substituted or unsubstituted morpholino or substituted or unsubstituted quinuclidinyl.
　「オリゴヌクレオチド」とは、同一又は異なるヌクレオシドが、同一又は異なるリン酸部位を挟んで複数個結合したヌクレオチドを意味する。 “Oligonucleotide” means nucleotides in which a plurality of identical or different nucleosides are bound via the same or different phosphate sites.
　本発明のオリゴヌクレオチドは式（Ｉ）で示される結合を任意の位置に少なくとも１つ含有するオリゴヌクレオチドである。式（Ｉ）で示される結合の位置及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 is an oligonucleotide containing at least one bond represented by formula (I) at an arbitrary position. The position and number of the bond represented by the formula (I) are not particularly limited, and can be appropriately designed according to the purpose.
　本発明のオリゴヌクレオチドは、ｓｉＲＮＡ、リボザイム、アンチジーン、アプタマー、デコイ核酸等を含む核酸医薬品として利用可能である。式（Ｉ）で示される結合は、核酸医薬品の活性成分として働く配列（例えば、標的遺伝子のｍＲＮＡに結合する配列）、該配列の相補鎖、該配列及びその相補鎖の前後に含まれ得る配列（例えば、標的遺伝子のｍＲＮＡに結合しない配列、無塩基のカチオンユニット等）のいずれに含有されていてもよい。
　本発明のオリゴヌクレオチドは、特に好ましくは、アンチセンスオリゴヌクレオチドを含む核酸医薬品として利用される。 The oligonucleotide of the present invention can be used as a nucleic acid pharmaceutical including siRNA, ribozyme, antigene, aptamer, decoy nucleic acid and the like. The bond represented by formula (I) is a sequence that acts as an active ingredient of a nucleic acid pharmaceutical (for example, a sequence that binds to the mRNA of a target gene), a complementary strand of the sequence, and a sequence that can be included before and after the sequence and the complementary strand. (For example, a sequence that does not bind to the mRNA of the target gene, an abasic cation unit, etc.) may be contained.
The oligonucleotide of the present invention is particularly preferably used as a nucleic acid pharmaceutical comprising an antisense oligonucleotide.
　本発明のオリゴヌクレオチドは、一本鎖であっても二本鎖であってもよい。二本鎖の両端がそれぞれループ構造を介してつながっているリボン型であってもよい。二本鎖である場合、一方の鎖にのみ式（Ｉ）で示される結合が含有されていてもよいし、両方の鎖に式（Ｉ）で示される結合が含有されていてもよい。
　本発明のオリゴヌクレオチドが、一本鎖である場合とは、例えば、アンチセンスオリゴヌクレオチド、リボザイム、アプタマー等が挙げられる。
　本発明の一本鎖オリゴヌクレオチドが、アンチセンスオリゴヌクレオチドを含む核酸医薬品である場合、式（Ｉ）で示される結合は、標的遺伝子のｍＲＮＡに結合する配列又は前後に含まれ得る配列のいずれに含有されていてもよい。
　本発明のオリゴヌクレオチドが、二本鎖である場合とは、ｓｉＲＮＡ、アンチジーン、デコイ核酸等が挙げられる。また、アンチセンスオリゴヌクレオチド及び該アンチセンスオリゴヌクレオチドに相補的な核酸からなる二重鎖核酸を含む二本鎖の核酸医薬品が挙げられる。該医薬組成物の具体例及びその調製方法は、本明細書実施例４、国際公開第２０１３／０８９２８３号等に記載されている。また、該核酸医薬品は、トコフェノール（国際公開第２０１３／０８９２８３号）やＧａｌＮａｃ誘導体（ＰＣＴ／ＪＰ２０１５／０５００８３）等のリガンドが結合していてもよく、該リガンドにより、アンチセンスオリゴヌクレオチドを特異性高く効率的に標的臓器に送達し、かつ該アンチセンスオリゴヌクレオチドにより、標的遺伝子の発現を効果的に抑制することを可能とする。
　本発明の二本鎖オリゴヌクレオチドが、アンチセンスオリゴヌクレオチド及び該アンチセンスオリゴヌクレオチドに相補的な核酸からなる二重鎖核酸を含む二本鎖の核酸医薬品である場合、式（Ｉ）で示される結合は、アンチセンスオリゴヌクレオチド（標的遺伝子のｍＲＮＡに結合する配列）、相補的な核酸、該アンチセンスオリゴヌクレオチド及び相補鎖の前後に含まれ得る配列のいずれに含有されていてもよい。該式（Ｉ）で示される結合が該アンチセンスオリゴヌクレオチド及び相補鎖の前後に含まれ得る配列に含有される場合、該式（Ｉ）で示される結合は、相補鎖の３’‐末端側の配列、５’‐末端側の配列のいずれに含有されていてもよいし、両末端の配列に共に含有されていてもよい。 The oligonucleotide of the present invention may be single-stranded or double-stranded. It may be a ribbon type in which both ends of the double strand are connected via a loop structure. In the case of a double strand, the bond represented by the formula (I) may be contained only in one chain, or the bond represented by the formula (I) may be contained in both chains.
Examples of the case where the oligonucleotide of the present invention is single-stranded include antisense oligonucleotides, ribozymes, aptamers and the like.
When the single-stranded oligonucleotide of the present invention is a nucleic acid pharmaceutical containing an antisense oligonucleotide, the bond represented by the formula (I) is either a sequence that binds to the mRNA of the target gene or a sequence that can be included before or after the target gene. It may be contained.
Examples of the case where the oligonucleotide of the present invention is double-stranded include siRNA, antigene, and decoy nucleic acid. Moreover, the double-stranded nucleic acid pharmaceutical containing the antisense oligonucleotide and the double stranded nucleic acid which consists of a nucleic acid complementary to this antisense oligonucleotide is mentioned. Specific examples of the pharmaceutical composition and the preparation method thereof are described in Example 4 of the present specification, International Publication No. 2013/0889283 and the like. In addition, the nucleic acid pharmaceutical may be bound with a ligand such as tocophenol (International Publication No. 2013/0889283) or GalNac derivative (PCT / JP2015 / 050083). Highly and efficiently delivered to the target organ, and the antisense oligonucleotide can effectively suppress the expression of the target gene.
When the double-stranded oligonucleotide of the present invention is a double-stranded nucleic acid pharmaceutical comprising an antisense oligonucleotide and a double-stranded nucleic acid composed of a nucleic acid complementary to the antisense oligonucleotide, it is represented by the formula (I) The bond may be contained in any of an antisense oligonucleotide (sequence that binds to the mRNA of the target gene), a complementary nucleic acid, a sequence that can be included before and after the antisense oligonucleotide and the complementary strand. When the bond represented by the formula (I) is contained in a sequence that can be contained before and after the antisense oligonucleotide and the complementary strand, the bond represented by the formula (I) is the 3′-terminal side of the complementary strand. May be contained in any of the sequences of 5′-terminal side, or may be contained in both of the terminal sequences.
　本発明のオリゴヌクレオチドの長さは、製薬上許容される限り、特に限定されない。例えば、８～２５塩基、８～１９塩基、１０～１９塩基、１３～１９塩基、１３塩基、１４塩基、１５塩基、１６塩基、１７塩基、１８塩基又は１９塩基である。 The length of the oligonucleotide of the present invention is not particularly limited as long as it is pharmaceutically acceptable. For example, 8 to 25 bases, 8 to 19 bases, 10 to 19 bases, 13 to 19 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases or 19 bases.
　本発明のオリゴヌクレオチドは、式（Ｉ）で示される結合を少なくとも１つ含有していれば、他の部分が天然の核酸と同じであっても、当該分野で公知のヌクレオチドの修飾を有していてもよい。
　本発明のオリゴヌクレオチド中の式（Ｉ）で示される結合以外のリン酸部位としては、天然の核酸が有するリン酸ジエステル結合、Ｓ－オリゴ（例えば、ホスホロチオエート）、Ｍ－オリゴ（例えば、メチルホスホネート）、ボラノホスフェート等が挙げられる。
　また、本発明のオリゴヌクレオチド中の式（Ｉ）で示される結合以外のヌクレオシド間の結合は、当該分野で公知の結合であれば、リン原子を有していない結合であってもよい。アルキル、非芳香族炭素環、ハロアルキル、ハロゲンで置換された非芳香族炭素環等を含むが、これらに限定されない。例えば、シロキサン、スルフィド、スルホキシド、スルホン、アセチル、ギ酸アセチル、チオギ酸アセチル、メチレンギ酸アセチル、チオギ酸アセチル、アルケニル、スルファマート、メチレンイミノ、メチレンヒドラジノ、スルホナート、スルホンアミド、アミドを含む。 As long as the oligonucleotide of the present invention contains at least one bond represented by the formula (I), it has a nucleotide modification known in the art even if the other part is the same as the natural nucleic acid. It may be.
Examples of the phosphate site other than the bond represented by the formula (I) in the oligonucleotide of the present invention include a phosphodiester bond, S-oligo (for example, phosphorothioate), M-oligo (for example, methylphosphonate) possessed by natural nucleic acids. ), Boranophosphate and the like.
In addition, the bond between nucleosides other than the bond represented by the formula (I) in the oligonucleotide of the present invention may be a bond having no phosphorus atom as long as it is a bond known in the art. Examples include, but are not limited to, alkyl, non-aromatic carbocycle, haloalkyl, non-aromatic carbocycle substituted with halogen, and the like. Examples include siloxane, sulfide, sulfoxide, sulfone, acetyl, acetyl formate, acetyl thioformate, acetyl methylene formate, acetyl thioformate, alkenyl, sulfamate, methylene imino, methylene hydrazino, sulfonate, sulfonamide, amide.
　本発明のオリゴヌクレオチドの核酸塩基部分は、上記「核酸塩基部分」と同じである。　 The nucleobase portion of the oligonucleotide of the present invention is the same as the “nucleobase portion” described above. *
　また、本発明のオリゴヌクレオチドは、ヌクレオシドの核酸塩基部分を欠く無塩基のカチオンユニットを含んでいてもよい。無塩基のカチオンユニットの位置及び数は特に限定されず、目的に応じて適宜設計され得る。その際、「核酸塩基部分」には水素原子が結合する。 The oligonucleotide of the present invention may contain an abasic cation unit lacking the nucleobase portion of the nucleoside. The position and number of abasic cation units are not particularly limited and can be appropriately designed according to the purpose. At that time, a hydrogen atom is bonded to the “nucleobase moiety”.
　本発明のオリゴヌクレオチド中の糖部位としては、上記「糖部位」と同じである。好ましくは、本発明のオリゴヌクレオチドは、天然の核酸に含まれる糖及びその類縁体の両方を含む。天然の核酸に含まれる糖とその類縁体のパターンとしては、例えば、ギャップマー又はミクスマーが挙げられる。
　ギャップマーとは、中心領域（“ギャップ”）と該中心領域の両側の領域、ウイング（５’側の“５’ウイング”又は３’側の“３’ウイング”）を含み、各ウイングに少なくとも１つの類縁体を含むオリゴヌクレオチドを意味する。例えば、“５’ウイング”及び ／又は“３’ウイング”に、類縁体を１以上、好ましくは、１～５、さらに好ましくは、２～３含有する。一方のウイング内の類縁体の種類、数、位置は他方のウイングにおける類縁体の種類、数、位置と同じであっても異なっていてもよい。
　ミクスマーとは、ランダムに類縁体を含むオリゴヌクレオチドを意味する。
　本発明のオリゴヌクレオチドがアンチセンスオリゴヌクレオチドである場合、特に好ましくは、ギャップマーである。 The sugar moiety in the oligonucleotide of the present invention is the same as the “sugar moiety” described above. Preferably, the oligonucleotide of the present invention includes both the sugar and its analogs contained in natural nucleic acids. Examples of the pattern of sugars and analogs contained in natural nucleic acids include gapmers and mixmers.
A gapmer includes a central region (“gap”) and regions on both sides of the central region, wings (“5 ′ wing” on the 5 ′ side or “3 ′ wing” on the 3 ′ side), and each wing has at least An oligonucleotide containing one analog is meant. For example, “5 ′ wing” and / or “3 ′ wing” contains one or more analogs, preferably 1 to 5, more preferably 2 to 3. The type, number and position of analogs in one wing may be the same or different from the type, number and position of analogs in the other wing.
A mixmer means an oligonucleotide containing analogs at random.
When the oligonucleotide of the present invention is an antisense oligonucleotide, it is particularly preferably a gapmer.
　本発明のオリゴヌクレオチドに用いられる糖の類縁体として、好ましくは、上記「５員環を含む糖の修飾体」が挙げられる。例えば、４’位と２’位との間に架橋構造を有する糖が挙げられる。該架橋構造として、特に好ましくは、４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）又は４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である。 Preferred examples of the sugar analogue used in the oligonucleotide of the present invention include the above-mentioned “modified sugar containing a 5-membered ring”. For example, a saccharide having a cross-linked structure between the 4 ′ position and the 2 ′ position can be mentioned. The cross-linked structure is particularly preferably 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′-C (═O) —NR 1 -2 ′ (R 1 is , A hydrogen atom or alkyl).
　本発明のオリゴヌクレオチドは、特定の異性体に限定するものではなく、全ての可能な異性体（例えば、ケト－エノール異性体、イミン－エナミン異性体、ジアステレオ異性体、光学異性体、回転異性体等）、ラセミ体又はそれらの混合物を含む。 The oligonucleotides of the present invention are not limited to a particular isomer, but all possible isomers (eg, keto-enol isomer, imine-enamine isomer, diastereoisomer, optical isomer, rotational isomerism) Body, etc.), racemate or a mixture thereof.
　本発明のオリゴヌクレオチドの一つ以上の水素、炭素及び／又は他の原子は、それぞれ水素、炭素及び／又は他の原子の同位体で置換され得る。そのような同位体の例としては、それぞれ２Ｈ、３Ｈ、１１Ｃ、１３Ｃ、１４Ｃ、１５Ｎ、１８Ｏ、１７Ｏ、３１Ｐ、３２Ｐ、３５Ｓ、１８Ｆ、123Ｉ及び３６Ｃｌのように、水素、炭素、窒素、酸素、リン、硫黄、フッ素、ヨウ素及び塩素が包含される。本発明のオリゴヌクレオチドは、そのような同位体で置換されたオリゴヌクレオチドも包含する。該同位体で置換されたオリゴヌクレオチドは、医薬品としても有用であり、本発明のオリゴヌクレオチドのすべての放射性標識体を包含する。また該「放射性標識体」を製造するための「放射性標識化方法」も本発明に包含され、代謝薬物動態研究、結合アッセイにおける研究及び／又は診断のツールとして有用である。 One or more hydrogen, carbon and / or other atoms of the oligonucleotides of the invention may be replaced with isotopes of hydrogen, carbon and / or other atoms, respectively. Examples of such isotopes are 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 123 I and Like 36 Cl, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine are included. The oligonucleotides of the present invention also include oligonucleotides substituted with such isotopes. Oligonucleotides substituted with the isotope are useful as pharmaceuticals and include all radiolabeled oligonucleotides of the invention. A “radiolabeling method” for producing the “radiolabeled product” is also encompassed in the present invention, and is useful as a metabolic pharmacokinetic study, a study in a binding assay, and / or a diagnostic tool.
　本発明のオリゴヌクレオチドの放射性標識体は、当該技術分野で周知の方法で調製できる。例えば、トリチウムで標識した本発明のオリゴヌクレオチドは、トリチウムを用いた触媒的脱ハロゲン化反応によって、本発明のオリゴヌクレオチドにトリチウムを導入することで調製できる。この方法は、適切な触媒、例えばＰｄ／Ｃの存在下、塩基の存在下又は非存在下で、本発明のオリゴヌクレオチドが適切にハロゲン置換された前駆体とトリチウムガスとを反応させることを包含する。他のトリチウム標識化合物を調製するための適切な方法としては、文書Ｉｓｏｔｏｐｅｓ　ｉｎ　ｔｈｅ　Ｐｈｙｓｉｃａｌ　ａｎｄ　Ｂｉｏｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅｓ，Ｖｏｌ．１，Ｌａｂｅｌｅｄ　Ｃｏｍｐｏｕｎｄｓ　（Ｐａｒｔ　Ａ），Ｃｈａｐｔｅｒ　６　（１９８７年）を参照にできる。１４Ｃ－標識化合物は、１４Ｃ炭素を有する原料を用いることによって調製できる。 The radiolabeled oligonucleotide of the present invention can be prepared by methods well known in the art. For example, the oligonucleotide of the present invention labeled with tritium can be prepared by introducing tritium into the oligonucleotide of the present invention by catalytic dehalogenation reaction using tritium. This method involves reacting a suitable halogen-substituted precursor of the oligonucleotide of the invention with tritium gas in the presence of a suitable catalyst such as Pd / C, in the presence or absence of a base. To do. Suitable methods for preparing other tritium labeled compounds include the document Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987). The 14 C-labeled compound can be prepared by using a raw material having 14 C carbon.
　本発明のオリゴヌクレオチドの３’末端及び／又は５’末端は修飾されていてもよい。オリゴヌクレオチドの追跡を可能にするため、オリゴヌクレオチドの薬物動態又は薬力学を改善するため、あるいはオリゴヌクレオチドの安定性又は結合親和性を向上させるために、当該分野で公知の修飾基を利用することができる。例えば、水酸基の保護基、レポーター分子、コレステロール、リン脂質、色素、蛍光分子等が挙げられる。
　また、本発明のオリゴヌクレオチドの３’末端及び／又は５’末端はリン酸エステル部分を含んでいてもよい。「リン酸エステル部分」とは、リン酸エステル並びに修飾リン酸エステルが含まれる、末端リン酸基を意味する。リン酸エステル部分は、いずれの末端に位置してもよいが、５’－末端ヌクレオシドであることが好ましい。具体的には、式：－Ｏ－Ｐ（＝Ｏ）（ＯＨ）ＯＨで示される基又はその修飾基である。つまり、Ｏ及びＯＨの１以上が、Ｈ、Ｏ、Ｓ、Ｎ（ＲX）、又はアルキル（ここでＲXは、Ｈ、アミノ保護基、又は置換若しくは非置換のアルキルである）で置換されていてもよい。５’及び／又は３’－末端基は、それぞれ独立して置換又は非置換の、１～３のリン酸エステル部分を含んでいてもよい。 The 3 ′ end and / or the 5 ′ end of the oligonucleotide of the present invention may be modified. Utilize modifying groups known in the art to enable tracking of the oligonucleotide, to improve the pharmacokinetics or pharmacodynamics of the oligonucleotide, or to improve the stability or binding affinity of the oligonucleotide. Can do. Examples thereof include a hydroxyl protecting group, a reporter molecule, cholesterol, phospholipid, a dye, and a fluorescent molecule.
Moreover, 3 'terminal and / or 5' terminal of the oligonucleotide of this invention may contain the phosphate ester part. "Phosphate ester moiety" means a terminal phosphate group, including phosphate esters as well as modified phosphate esters. The phosphate ester moiety may be located at either end, but is preferably a 5′-terminal nucleoside. Specifically, it is a group represented by the formula: —OP (═O) (OH) OH or a modifying group thereof. That is, one or more of O and OH is substituted with H, O, S, N (R x ), or alkyl (where R x is H, an amino protecting group, or substituted or unsubstituted alkyl). It may be. The 5 ′ and / or 3′-end groups may each independently contain 1 to 3 phosphate ester moieties that are substituted or unsubstituted.
　本発明は、本発明のオリゴヌクレオチドの製薬上許容される塩を含有する。該塩としては、例えば、アルカリ金属塩（ナトリウム塩、カリウム塩、リチウム塩等）、アルカリ土類金属塩（カルシウム塩、マグネシウム塩等）、金属塩（アルミニウム塩、鉄塩、亜鉛塩、銅塩、ニッケル塩、コバルト塩等）、アンモニウム塩、アミン塩（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）アミノメタン塩等）、無機酸塩（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）、アルカンスルホン酸塩（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）、芳香族炭素環式基スルホン酸塩（ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩等）、有機酸塩（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）、アミノ酸塩（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、アスパラギン酸塩等）等が挙げられる。これらの塩は、通常行われる方法によって形成させることができる。 The present invention contains a pharmaceutically acceptable salt of the oligonucleotide of the present invention. Examples of the salt include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), metal salts (aluminum salt, iron salt, zinc salt, copper salt). , Nickel salt, cobalt salt, etc.), ammonium salt, amine salt (t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt , Diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) )amino Tan salts, etc.), inorganic acid salts (hydrofluoric acid salts, hydrochlorides, hydrobromides, hydroiodide salts such as hydroiodide, nitrates, perchlorates, sulfates, phosphorus Acid salts), alkane sulfonates (methanesulfonate, trifluoromethanesulfonate, ethanesulfonate), aromatic carbocyclic sulfonates (benzenesulfonate, p-toluenesulfonate, etc.) , Organic acid salt (acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.), amino acid salt (glycine salt, lysine salt, arginine salt, ornithine) Salt, glutamate, aspartate, etc.). These salts can be formed by a commonly performed method.
　本発明のオリゴヌクレオチド又はその製薬上許容される塩は、溶媒和物（例えば、水和物等）及び／又は結晶多形を形成する場合があり、本発明はそのような各種の溶媒和物及び結晶多形も包含する。「溶媒和物」は、本発明のオリゴヌクレオチドに対し、任意の数の溶媒分子（例えば、水分子等）と配位していてもよい。本発明のオリゴヌクレオチド又はその製薬上許容される塩を、大気中に放置することにより、水分を吸収し、吸着水が付着する場合や、水和物を形成する場合がある。また、本発明のオリゴヌクレオチド又はその製薬上許容される塩を、再結晶することでそれらの結晶多形を形成する場合がある。 The oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof may form a solvate (for example, hydrate etc.) and / or a crystal polymorph, and the present invention provides such various solvates. And crystalline polymorphs. The “solvate” may be coordinated with any number of solvent molecules (for example, water molecules) with respect to the oligonucleotide of the present invention. When the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof is left in the air, it may absorb moisture and adsorbed water may adhere or form a hydrate. Moreover, the crystalline polymorphism may be formed by recrystallizing the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof.
　本発明のオリゴヌクレオチド又はその製薬上許容される塩は、プロドラッグを形成する場合があり、本発明はそのような各種のプロドラッグも包含する。プロドラッグは、化学的又は代謝的に分解できる基を有する本発明化合物の誘導体であり、加溶媒分解により又は生理学的条件下でインビボにおいて薬学的に活性な本発明のオリゴヌクレオチドとなる化合物である。プロドラッグは、生体内における生理条件下で酵素的に酸化、還元、加水分解等を受けて本発明のオリゴヌクレオチドに変換される化合物、胃酸等により加水分解されて本発明のオリゴヌクレオチドに変換される化合物等を包含する。適当なプロドラッグ誘導体を選択する方法及び製造する方法は、例えばＤｅｓｉｇｎ　ｏｆ　Ｐｒｏｄｒｕｇｓ，　Ｅｌｓｅｖｉｅｒ，　Ａｍｓｔｅｒｄａｍ　１９８５に記載されている。プロドラッグは、それ自身が活性を有する場合がある。 The oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof may form a prodrug, and the present invention includes such various prodrugs. A prodrug is a derivative of a compound of the present invention having a group that can be chemically or metabolically degraded and becomes a pharmaceutically active oligonucleotide of the present invention in vivo by solvolysis or under physiological conditions . A prodrug is converted into an oligonucleotide of the present invention by hydrolysis with a compound that is enzymatically oxidized, reduced, hydrolyzed, etc. under physiological conditions in vivo to be converted into the oligonucleotide of the present invention, stomach acid, etc. And the like. Methods for selecting and producing suitable prodrug derivatives are described, for example, in Design of Prodrugs, Elsevier, Amsterdam 1985. Prodrugs may themselves have activity.
　本発明のオリゴヌクレオチド又はその製薬上許容される塩がヒドロキシル基を有する場合は、例えば、ヒドロキシル基を有する化合物と適当なアシルハライド、適当な酸無水物、適当なスルホニルクロライド、適当なスルホニルアンハイドライド及びミックスドアンハイドライドとを反応させることにより或いは縮合剤を用いて反応させることにより製造されるアシルオキシ誘導体やスルホニルオキシ誘導体のようなプロドラッグが例示される。例えば、ＣＨ３ＣＯＯ－、Ｃ２Ｈ５ＣＯＯ－、ｔｅｒｔ－ＢｕＣＯＯ－、Ｃ１５Ｈ３１ＣＯＯ－、ＰｈＣＯＯ－、（ｍ－ＮａＯＯＣＰｈ）ＣＯＯ－、ＮａＯＯＣＣＨ２ＣＨ２ＣＯＯ－、ＣＨ３ＣＨ（ＮＨ２）ＣＯＯ－、ＣＨ２Ｎ（ＣＨ３）２ＣＯＯ－、ＣＨ３ＳＯ３－、ＣＨ３ＣＨ２ＳＯ３－、ＣＦ３ＳＯ３－、ＣＨ２ＦＳＯ３－、ＣＦ３ＣＨ２ＳＯ３－、ｐ－ＣＨ３Ｏ－ＰｈＳＯ３－、ＰｈＳＯ３－、ｐ－ＣＨ３ＰｈＳＯ３－が挙げられる。 When the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof has a hydroxyl group, for example, a compound having a hydroxyl group and an appropriate acyl halide, an appropriate acid anhydride, an appropriate sulfonyl chloride, an appropriate sulfonyl anhydride And prodrugs such as acyloxy derivatives and sulfonyloxy derivatives produced by reacting with mixed anhydride or by using a condensing agent. For example, CH 3 COO—, C 2 H 5 COO—, tert-BuCOO—, C 15 H 31 COO—, PhCOO—, (m-NaOOCPh) COO—, NaOOCCH 2 CH 2 COO—, CH 3 CH (NH 2 ) COO—, CH 2 N (CH 3 ) 2 COO—, CH 3 SO 3 —, CH 3 CH 2 SO 3 —, CF 3 SO 3 —, CH 2 FSO 3 —, CF 3 CH 2 SO 3 —, p -CH 3 O-PhSO 3- , PhSO 3- , p-CH 3 PhSO 3 -can be mentioned.
　本発明のオリゴヌクレオチドは、本発明のアミダイト等の適切なアミダイトを用いて、常法によって合成することができる。例えば、市販の核酸自動合成装置（例えば、ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ社製、（株）大日本精機製等）によって容易に合成することができる。合成法はホスホロアミダイトを用いた固相合成法、ハイドロジェンホスホネートを用いた固相合成法等がある。例えば、Ｔｅｔｒａｈｅｄｒｏｎ　Ｌｅｔｔｅｒｓ　２２，１８５９－１８６２（１９８１）、国際公開第２０１１／０５２４３６号等に開示されている。 The oligonucleotide of the present invention can be synthesized by a conventional method using an appropriate amidite such as the amidite of the present invention. For example, it can be easily synthesized by a commercially available automatic nucleic acid synthesizer (for example, Applied Biosystems, Dainippon Seiki Co., Ltd.). Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate. For example, it is disclosed in Tetrahedron Letters 22, 1859-1862 (1981), International Publication No. 2011/052436, and the like.
　本発明のオリゴヌクレオチド中の核酸塩基部分は、置換基が保護基で保護されていないことが好ましい。例えば、以下で示される基が挙げられる。
Figure JPOXMLDOC01-appb-C000022
　よって、本発明のアミダイト等のアミダイトの核酸塩基部分が、保護基で保護された置換基を有する場合、オリゴヌクレオチド合成の際に、脱保護を行う。 In the nucleobase moiety in the oligonucleotide of the present invention, the substituent is preferably not protected with a protecting group. For example, the group shown below is mentioned.
Figure JPOXMLDOC01-appb-C000022
Therefore, when the nucleobase portion of an amidite such as the amidite of the present invention has a substituent protected by a protecting group, deprotection is performed during oligonucleotide synthesis.
　本発明のオリゴヌクレオチドを利用した核酸医薬品は、局所的あるいは全身的な治療のいずれが望まれるのか、又は治療すべき領域に応じて、様々な方法により投与することができる。投与方法としては、例えば、局所的（点眼、膣内、直腸内、鼻腔内、経皮を含む）、経口的、又は、非経口的であってもよい。非経口的投与としては、静脈内注射若しくは点滴、皮下、腹腔内若しくは筋肉内注入、吸引若しくは吸入による肺投与、硬膜下腔内投与、脳室内投与等が挙げられる。 The nucleic acid drug using the oligonucleotide of the present invention can be administered by various methods depending on whether local or systemic treatment is desired or on the region to be treated. The administration method may be, for example, topical (including eye drops, intravaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by inhalation or inhalation, intradural administration, intraventricular administration, and the like.
　本発明のオリゴヌクレオチドを利用した核酸医薬品を局所投与する場合、経皮パッチ、軟膏、ローション、クリーム、ゲル、滴下剤、坐剤、噴霧剤、液剤、散剤等の製剤を用いることができる。
　経口投与用組成物としては、散剤、顆粒剤、水若しくは非水性媒体に溶解させた懸濁液又は溶液、カプセル、粉末剤、錠剤等が挙げられる。
　非経口、硬膜下腔、又は、脳室内投与用組成物としては、バッファー、希釈剤及びその他の適当な添加剤を含む無菌水溶液等が挙げられる。 When the nucleic acid pharmaceutical using the oligonucleotide of the present invention is locally administered, preparations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders and the like can be used.
Examples of compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets and the like.
Examples of compositions for parenteral, subdural space, or intraventricular administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.
　本発明のオリゴヌクレオチドを利用した核酸医薬品は、核酸の有効量にその剤型に適した賦形剤、結合剤、湿潤剤、崩壊剤、滑沢剤、希釈剤等の各種医薬用添加剤を必要に応じて混合して得ることができる。注射剤の場合には適当な担体と共に滅菌処理を行なって製剤とすればよい。 Nucleic acid pharmaceuticals using the oligonucleotides of the present invention include various pharmaceutical additives such as excipients, binders, wetting agents, disintegrating agents, lubricants, diluents, etc. suitable for the dosage form in an effective amount of nucleic acids. It can be obtained by mixing as required. In the case of an injection, it may be sterilized with an appropriate carrier to form a preparation.
　賦形剤としては乳糖、白糖、ブドウ糖、デンプン、炭酸カルシウム又は結晶セルロース等が挙げられる。結合剤としてはメチルセルロース、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ゼラチン又はポリビニルピロリドン等が挙げられる。崩壊剤としてはカルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、デンプン、アルギン酸ナトリウム、カンテン末又はラウリル硫酸ナトリウム等が挙げられる。滑沢剤としてはタルク、ステアリン酸マグネシウム又はマクロゴール等が挙げられる。坐剤の基剤としてはカカオ脂、マクロゴール又はメチルセルロース等を用いることができる。また、液剤又は乳濁性、懸濁性の注射剤として調製する場合には通常使用されている溶解補助剤、懸濁化剤、乳化剤、安定化剤、保存剤、等張剤等を適宜添加しても良い。経口投与の場合には嬌味剤、芳香剤等を加えても良い。 Excipients include lactose, sucrose, glucose, starch, calcium carbonate or crystalline cellulose. Examples of the binder include methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, and polyvinyl pyrrolidone. Examples of the disintegrant include carboxymethyl cellulose, sodium carboxymethyl cellulose, starch, sodium alginate, agar powder, or sodium lauryl sulfate. Examples of the lubricant include talc, magnesium stearate or macrogol. As a suppository base, cocoa butter, macrogol, methylcellulose, or the like can be used. In addition, when preparing as liquid or emulsion or suspension injections, commonly used solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, isotonic agents, etc. are added as appropriate. You may do it. In the case of oral administration, flavoring agents, fragrances and the like may be added.
　投与は、治療される病態の重度と反応度に依存し、治療コースは、数日から数ヶ月、あるいは、治癒が実現されるまで、又は、病状の減退が達成されるまで持続する。最適投与スケジュールは、生体における薬剤蓄積の測定から計算が可能である。当該分野の当業者であれば、最適用量、投与法、及び、繰り返し頻度を定めることができる。最適用量は、個々の核酸医薬品の相対的効力に応じて変動するが、一般に、インビトロ及びインビボの動物実験におけるＩＣ５０又はＥＣ５０に基づいて計算することが可能である。例えば、核酸の分子量（核酸配列及び化学構造から導かれる）と、例えば、ＩＣ５０のような効果的用量（実験的に導かれる）が与えられたならば、ｍｇ／ｋｇで表される用量が通例に従って計算される。 Administration depends on the severity and responsiveness of the condition being treated, and the course of treatment lasts from days to months, or until healing is achieved, or until disease status is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill in the art can determine optimum dosages, dosing methodologies and repetition rates. The optimal dose will vary depending on the relative potency of the individual nucleic acid drug, but can generally be calculated based on the IC50 or EC50 in in vitro and in vivo animal experiments. For example, given the molecular weight of a nucleic acid (derived from the nucleic acid sequence and chemical structure) and an effective dose (derived experimentally) such as IC50, the dose expressed in mg / kg is typically Calculated according to
　以下に、本発明のアミダイトの好ましい様態を説明する。 Hereinafter, preferred embodiments of the amidite of the present invention will be described.
式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000023
（式中：
Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、
Ｘ２は置換又非置換の単環若しくは２環の含窒素非芳香族複素環式基であり、
Ｒ２は置換若しくは非置換のアミノである）
で示される基
及びヌクレオシド構造を含有する化合物、又はその塩。 Formula (II):
Figure JPOXMLDOC01-appb-C000023
(Where:
X 1 is a substituted or unsubstituted alkylene which is absent or O may be interposed,
X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group,
R 2 is substituted or unsubstituted amino)
Or a salt thereof, which contains a group represented by the above formula and a nucleoside structure.
　式中のＸ１及びＸ２は式（Ｉ）で示される結合における各記号と同意義である。 X 1 and X 2 in the formula are the same as the symbols in the bond represented by the formula (I).
　Ｒ２は、例えば、
式：－ＮＲ１９Ｒ２０
（式中、
Ｒ１９はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ２０はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルである）
で示される基等が挙げられる。好ましくは、Ｒ２はジイソプロピルアミノである。 R 2 is, for example,
Formula: —NR 19 R 20
(Where
Each R 19 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl;
Each R 20 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl)
Group etc. which are shown by these are mentioned. Preferably R 2 is diisopropylamino.
　「ヌクレオシド構造」とは、上記「核酸塩基部分」と「糖部位」からなる構造を意味する。また、本明細書における「ヌクレオシド構造」には、核酸塩基部分を欠く無塩基のカチオンユニットも含まれる。 “Nucleoside structure” means a structure composed of the above-mentioned “nucleobase moiety” and “sugar moiety”. The “nucleoside structure” in the present specification also includes an abasic cation unit lacking a nucleobase moiety.
　「ヌクレオシド構造」として、好ましくは、「糖部位」が「５員環を含む糖の修飾体」であるヌクレオシド構造である。詳しくは、式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000024
（式中：
Ｂｘは水素又は核酸塩基部分であり、
Ｚは水素原子又は水酸基保護基であり、
Ｒ３及びＲ４はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ５は水素であり、
Ｒ６は水素原子、ハロゲン、ヒドロキシ又は置換若しくは非置換のアルキルオキシであり、
ここでＲ５及びＲ６は、一緒になって架橋構造を形成していてもよく、
Ｒ７は水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルである）
で示される構造である。 The “nucleoside structure” is preferably a nucleoside structure in which the “sugar moiety” is “a modified sugar containing a 5-membered ring”. Specifically, the formula (III):
Figure JPOXMLDOC01-appb-C000024
(Where:
Bx is a hydrogen or nucleobase moiety;
Z is a hydrogen atom or a hydroxyl protecting group,
R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl,
R 5 is hydrogen;
R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy,
Here, R 5 and R 6 may be combined to form a crosslinked structure,
R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl)
It is a structure shown by.
　Ｂｘは水素又は上記「核酸塩基部分」である。より好ましくは、水素、置換若しくは非置換のプリン－９－イル又は置換若しくは非置換の２－オキソ－ピリミジン－１－イルである。 Bx is hydrogen or the above-mentioned “nucleobase moiety”. More preferred is hydrogen, substituted or unsubstituted purin-9-yl or substituted or unsubstituted 2-oxo-pyrimidin-1-yl.
　Ｚは、水素原子、水酸基保護基又は反応性リン基である。好ましくは、水素原子又は水酸基保護基である。より好ましくは、水素原子、アセチル、ｔ－ブチル、ｔ－ブトキシメチル、メトキシメチル、テトラヒドロピラニル、１－エトキシエチル、１－（２－クロロエトキシ）エチル、２－トリメチルシリルエチル、ｐ－クロロフェニル、２，４－ジニトロフェニル、ベンジル、ベンゾイル、ｐ－フェニルベンゾイル、２，６－ジクロロベンジル、レブリノイル、ジフェニルメチル、ｐ－ニトロベンジル、トリメチルシリル、トリエチルシリル、ｔ－ブチルジメチルシリル、ｔ－ブチルジフェニルシリル、トリフェニルシリル、トリイソプロピルシリル、ギ酸ベンゾイル、クロロアセチル、トリクロロアセチル、トリフルオロアセチル、ピバロイル、イソブチリル、９－フルオレニルメチルオキシカルボニル、メタンスルホニル、ｐ－トルエンスルホニル、トリフルオロメタンスルホニル、トリフェニルメチル（トリチル）、モノメトキシトリチル、ジメトキシトリチル（ＤＭＴｒ）、トリメトキシトリチル、９－フェニルキサンチン－９－イル（Ｐｉｘｙｌ）又は９－（ｐ－メトキシフェニル）キサンチン－９－イル（ＭＯＸ）である。特に好ましくは、水素原子、ベンジル、トリエチルシリル、ｔ－ブチルジメチルシリル、ｔ－ブチルジフェニルシリル、トリイソプロピルシリル、トリチル、モノメトキシトリチル、ジメトキシトリチル、トリメトキシトリチル等である。 Z is a hydrogen atom, a hydroxyl protecting group or a reactive phosphorus group. A hydrogen atom or a hydroxyl protecting group is preferred. More preferably, a hydrogen atom, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2 , 4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, levulinoyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tri Phenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl, 9-fluorenylmethyloxycarbonyl, methanesulfonyl, p-toluenesulfur Nyl, trifluoromethanesulfonyl, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) or 9- (p-methoxyphenyl) xanthine-9 -Ile (MOX). Particularly preferred are a hydrogen atom, benzyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl and the like.
　Ｒ３～Ｒ７は上記「５員環を含む糖の修飾体」における各記号と同意義である。 R 3 to R 7 have the same meanings as the symbols in the above-mentioned “modified sugar containing a 5-membered ring”.
　本発明のアミダイトは、特定の異性体に限定するものではなく、全ての可能な異性体（例えば、ケト－エノール異性体、イミン－エナミン異性体、ジアステレオ異性体、光学異性体、回転異性体等）、ラセミ体又はそれらの混合物を含む。 The amidites of the present invention are not limited to specific isomers, but all possible isomers (eg keto-enol isomers, imine-enamine isomers, diastereoisomers, optical isomers, rotational isomers) Etc.), racemates or mixtures thereof.
　本発明のアミダイトの一つ以上の水素、炭素及び／又は他の原子は、それぞれ水素、炭素及び／又は他の原子の同位体で置換され得る。そのような同位体の例としては、それぞれ２Ｈ、３Ｈ、１１Ｃ、１３Ｃ、１４Ｃ、１５Ｎ、１８Ｏ、１７Ｏ、３１Ｐ、３２Ｐ、３５Ｓ、１８Ｆ、123Ｉ及び３６Ｃｌのように、水素、炭素、窒素、酸素、リン、硫黄、フッ素、ヨウ素及び塩素が包含される。本発明のアミダイトは、そのような同位体で置換された化合物も包含する。該同位体で置換された化合物は、医薬品としても有用であり、本発明のアミダイトのすべての放射性標識体を包含する。また該「放射性標識体」を製造するための「放射性標識化方法」も本発明に包含され、代謝薬物動態研究、結合アッセイにおける研究及び／又は診断のツールとして有用である。 One or more hydrogen, carbon and / or other atoms of the amidites of the present invention may be replaced with isotopes of hydrogen, carbon and / or other atoms, respectively. Examples of such isotopes are 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 123 I and Like 36 Cl, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine are included. The amidites of the present invention also include compounds substituted with such isotopes. The compound substituted with the isotope is also useful as a pharmaceutical, and includes all radiolabels of the amidite of the present invention. A “radiolabeling method” for producing the “radiolabeled product” is also encompassed in the present invention, and is useful as a metabolic pharmacokinetic study, a study in a binding assay, and / or a diagnostic tool.
　本発明のアミダイトの放射性標識体は、当該技術分野で周知の方法で調製できる。例えば、本発明のアミダイトのトリチウム標識化合物は、例えば、トリチウムを用いた触媒的脱ハロゲン化反応によって、本発明のアミダイトにトリチウムを導入することで調製できる。この方法は、適切な触媒、例えばＰｄ／Ｃの存在下、塩基の存在下又は非存在下で、本発明のアミダイトが適切にハロゲン置換された前駆体とトリチウムガスとを反応させることを包含する。他のトリチウム標識化合物を調製するための適切な方法としては、文書Ｉｓｏｔｏｐｅｓ　ｉｎ　ｔｈｅ　Ｐｈｙｓｉｃａｌ　ａｎｄ　Ｂｉｏｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅｓ，Ｖｏｌ．１，Ｌａｂｅｌｅｄ　Ｃｏｍｐｏｕｎｄｓ　（Ｐａｒｔ　Ａ），Ｃｈａｐｔｅｒ　６　（１９８７年）を参照にできる。１４Ｃ－標識化合物は、１４Ｃ炭素を有する原料を用いることによって調製できる。 The radiolabeled amidite of the present invention can be prepared by methods well known in the art. For example, the tritium-labeled compound of the amidite of the present invention can be prepared by introducing tritium into the amidite of the present invention by, for example, catalytic dehalogenation reaction using tritium. This method involves reacting a tritium gas with a suitably halogen-substituted precursor of an amidite of the present invention in the presence of a suitable catalyst such as Pd / C, in the presence or absence of a base. . Suitable methods for preparing other tritium labeled compounds include the document Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987). The 14 C-labeled compound can be prepared by using a raw material having 14 C carbon.
　本発明は、本発明のアミダイトの生成可能な塩を含有する。該塩としては、例えば、アルカリ金属塩（ナトリウム塩、カリウム塩、リチウム塩等）、アルカリ土類金属塩（カルシウム塩、マグネシウム塩等）、金属塩（アルミニウム塩、鉄塩、亜鉛塩、銅塩、ニッケル塩、コバルト塩等）、アンモニウム塩、アミン塩（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）アミノメタン塩等）、無機酸塩（フッ化水素酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩等の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）、アルカンスルホン酸塩（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等）、芳香族炭素環式基スルホン酸塩（ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩等）、有機酸塩（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）、アミノ酸塩（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、アスパラギン酸塩等）等が挙げられる。これらの塩は、通常行われる方法によって形成させることができる。 The present invention contains a salt capable of forming the amidite of the present invention. Examples of the salt include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), metal salts (aluminum salt, iron salt, zinc salt, copper salt). , Nickel salt, cobalt salt, etc.), ammonium salt, amine salt (t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt , Diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) )amino Tan salts, etc.), inorganic acid salts (hydrofluoric acid salts, hydrochlorides, hydrobromides, hydroiodides, etc., halogen atom hydrohalates, nitrates, perchlorates, sulfates, phosphoric acids, etc.) Salt), alkane sulfonate (methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, etc.), aromatic carbocyclic sulfonate (benzenesulfonate, p-toluenesulfonate, etc.) , Organic acid salt (acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.), amino acid salt (glycine salt, lysine salt, arginine salt, ornithine) Salt, glutamate, aspartate, etc.). These salts can be formed by a commonly performed method.
　本発明のアミダイト又はその塩は、溶媒和物（例えば、水和物等）及び／又は結晶多形を形成する場合があり、本発明はそのような各種の溶媒和物及び結晶多形も包含する。「溶媒和物」は、本発明のアミダイトに対し、任意の数の溶媒分子（例えば、水分子等）と配位していてもよい。本発明のアミダイト又はその製薬上許容される塩を、大気中に放置することにより、水分を吸収し、吸着水が付着する場合や、水和物を形成する場合がある。また、本発明のアミダイト又はその製薬上許容される塩を、再結晶することでそれらの結晶多形を形成する場合がある。 The amidite of the present invention or a salt thereof may form a solvate (for example, hydrate etc.) and / or a crystal polymorph, and the present invention also includes such various solvates and crystal polymorphs. To do. The “solvate” may be coordinated with an arbitrary number of solvent molecules (for example, water molecules) to the amidite of the present invention. When the amidite of the present invention or a pharmaceutically acceptable salt thereof is left in the air, it may absorb moisture and adsorbed water may adhere or form a hydrate. Moreover, those polymorphs may be formed by recrystallizing the amidite of the present invention or a pharmaceutically acceptable salt thereof.
　本発明のアミダイトは、当該分野において公知の手法を参酌しながら合成することができる。例えば、実施例１に示す合成法によって製造することができる。また、抽出、精製等は、通常の有機化学の実験で行う処理を行えばよい。 The amidite of the present invention can be synthesized while taking into account techniques known in the art. For example, it can be produced by the synthesis method shown in Example 1. Extraction, purification, and the like may be performed in a normal organic chemistry experiment.
　本発明のオリゴヌクレオチドは、リン酸部位がホスホロチオエートのみのオリゴヌクレオチドと比較して高い標的遺伝子の発現抑制活性を有する。また、本発明のオリゴヌクレオチドは、修飾位置や修飾数を工夫することで、リン酸部位がホスホロチオエートのみのオリゴヌクレオチドと比較して、血漿中滞留性が向上し、例えば、肝臓又は肺等の臓器への集積性が高いことから、効率的に標的臓器に送達され、標的遺伝子又は標的タンパク質の発現を効果的に抑制することを可能である。よって本発明のアミダイトは、アンチセンスオリゴヌクレオチド等を含む核酸医薬品を合成するための材料として非常に有用である。 The oligonucleotide of the present invention has a higher target gene expression inhibitory activity than an oligonucleotide having only a phosphorothioate at the phosphate site. In addition, the oligonucleotide of the present invention has improved plasma retention compared to an oligonucleotide whose phosphoric acid moiety is only phosphorothioate by devising the modification position and the number of modifications, for example, organs such as liver or lung Therefore, it can be efficiently delivered to the target organ and the expression of the target gene or target protein can be effectively suppressed. Therefore, the amidite of the present invention is very useful as a material for synthesizing nucleic acid pharmaceuticals containing antisense oligonucleotides and the like.
　なお、本明細書中で用いる略語は以下の意味を表す。
ＤＭＴｒ：ジメトキシトリチル
Ｐｈ：フェニル
ｔＢｕ：ｔｅｒｔ－ブチル In addition, the abbreviation used in this specification represents the following meaning.
DMTr: dimethoxytrityl Ph: phenyl tBu: tert-butyl
　以下に本発明の実施例及び参考例、ならびに試験例を挙げて本発明をさらに詳しく説明するが、本発明はこれらにより限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples, and Test Examples of the present invention, but the present invention is not limited thereto.
　実施例で得られた化合物のＮＭＲ分析は４００ＭＨｚで行い、ＣＤＣｌ３を用いて測定した。
　ＲＴとあるのは、ＬＣ／ＭＳ：液体クロマトグラフィー／質量分析でのリテンションタイムを表し以下の条件で測定した。
　測定条件：カラム：Ａｃｑｕｉｔｙ　ＵＰＬＣ（登録商標）ＢＥＨ　Ｃ１８（１．７μｍ、ｉ．ｄ．２．１ｘ５０ｍｍ）（Ｗａｔｅｒｓ）
流速：０．８ｍＬ／分
ＵＶ検出波長：２５４ｎｍ
移動相：［Ａ］は１０ｍＭ炭酸アンモニウム含有水溶液、［Ｂ］は１０ｍＭ炭酸アンモニウム含有アセトニトリル溶液
グラジエント：３．５分間で５％－１００％溶媒［Ｂ］のリニアグラジエントを行い、０．５分間、１００％溶媒［Ｂ］を維持した。 The NMR analysis of the compounds obtained in the examples was performed at 400 MHz and measured using CDCl 3 .
RT represents the retention time in LC / MS: liquid chromatography / mass spectrometry and was measured under the following conditions.
Measurement conditions: Column: Acquity UPLC (registered trademark) BEH C18 (1.7 μm, id 2.1 × 50 mm) (Waters)
Flow rate: 0.8 mL / min UV detection wavelength: 254 nm
Mobile phase: [A] is a 10 mM ammonium carbonate-containing aqueous solution, [B] is a 10 mM ammonium carbonate-containing acetonitrile solution Gradient: A linear gradient of 5% -100% solvent [B] is performed in 3.5 minutes, and 0.5 minutes. 100% solvent [B] was maintained.
実施例１　本発明のアミダイト（式（ＩＩ）で示される基及びヌクレオシド構造を含有する化合物）の合成
（１－１）化合物（ＩＩ－１－１）の合成
Figure JPOXMLDOC01-appb-C000025
 Example 1 Synthesis of Amidite of the Present Invention (Compound Containing Group Shown by Formula (II) and Nucleoside Structure) (1-1) Synthesis of Compound (II-1-1)
Figure JPOXMLDOC01-appb-C000025
　　窒素雰囲気下、化合物１（２０ｇ、３７ｍｍｏｌ）のジクロロメタン溶液（１００ｍＬ）にＮ，Ｎ－ジイソプロピルエチルアミン（７．７ｍＬ、４４ｍｍｏｌ）を加え、氷冷した。この原料溶液に対し、ビス（ジイソプロピルアミノ）クロロフォスフィン（１１．３ｇ、４２ｍｍｏｌ）のジクロロメタン溶液（８０ｍＬ）を、６０分間かけて滴下し、その後３０分かけて室温まで昇温した。その後、再び反応溶液を氷冷し、１Ｈ－テトラゾール（１．５４ｇ、２２ｍｍｏｌ）のアセトニトリル溶液（４９ｍＬ）とキヌクリジン－３－オール（５．６ｇ、４４ｍｍｏｌ）を順次加えた後に室温まで昇温し、２時間半撹拌した。反応液へ飽和炭酸水素ナトリウム水溶液（５００ｍＬ）を加えた後に、酢酸エチル（６００ｍＬ）で抽出した。得られた有機層を飽和食塩水で洗浄した後に、無水硫酸マグネシウムで乾燥した。ろ過の後に溶媒を除去し、得られた化合物（ＩＩ－１－１）の粗製物（３１．６ｇ）をシリカゲルカラムクロマトグラフィー（（０－２５分）酢酸エチル、２５－４５分）５％→１００％Ｂ溶媒　ｉｎ　Ａ溶媒、（４５－６０分）１００％Ｂ溶媒　ｉｎ　Ａ溶媒；Ａ溶媒　クロロホルム／メタノール／トリエチルアミン＝１００：０：１、Ｂ溶媒　クロロホルム／メタノール／トリエチルアミン＝７５：２５：１）で精製し、化合物（ＩＩ－１－１）（２０．７ｇ、７０％）を白色固体物質として得た。
31P NMR (CDCl3, 400 MHz) δP: 147.0, 146.8, 146.7, 146.3．
LC-ESI-SQ-MS: m/Z 801.55 [M+H]+, cald. 801.4 [M+H]+. 
LCMS Rt (min): 2.76, 2.88, 2.91, 2.98. Under a nitrogen atmosphere, N, N-diisopropylethylamine (7.7 mL, 44 mmol) was added to a dichloromethane solution (100 mL) of compound 1 (20 g, 37 mmol), and the mixture was ice-cooled. To this raw material solution, a dichloromethane solution (80 mL) of bis (diisopropylamino) chlorophosphine (11.3 g, 42 mmol) was added dropwise over 60 minutes, and then the temperature was raised to room temperature over 30 minutes. Thereafter, the reaction solution was ice-cooled again, 1H-tetrazole (1.54 g, 22 mmol) in acetonitrile (49 mL) and quinuclidin-3-ol (5.6 g, 44 mmol) were sequentially added, and the mixture was warmed to room temperature. Stir for 2.5 hours. A saturated aqueous sodium hydrogen carbonate solution (500 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (600 mL). The obtained organic layer was washed with saturated brine, and then dried over anhydrous magnesium sulfate. After filtration, the solvent was removed, and the resulting crude product (31.6 g) of compound (II-1-1) was subjected to silica gel column chromatography ((0-25 minutes) ethyl acetate, 25-45 minutes) 5% → 100% B solvent in A solvent, (45-60 minutes) 100% B solvent in A solvent; A solvent chloroform / methanol / triethylamine = 100: 0: 1, B solvent chloroform / methanol / triethylamine = 75: 25: 1) To give compound (II-1-1) (20.7 g, 70%) as a white solid substance.
31 P NMR (CDCl 3 , 400 MHz) δ P : 147.0, 146.8, 146.7, 146.3.
LC-ESI-SQ-MS: m / Z 801.55 [M + H] + , cald. 801.4 [M + H] + .
LCMS Rt (min): 2.76, 2.88, 2.91, 2.98.
（１－２）化合物（ＩＩ－１－２）の合成
Figure JPOXMLDOC01-appb-C000026
　Ｎ－ベンゾイル－５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－デオキシシチジン（５ｇ）及びキヌクリジン－３－オール（１．２ｇ）を用いて（１－１）と同様の方法で化合物（ＩＩ－１－２）（４．５ｇ、６４％）を淡黄色固体物質として得た。
31P NMR (CDCl3, 400 MHz) δP: 147.5, 147.0, 146.9, 146.7．
LC-ESI-SQ-MS: m/Z 890.54 [M+H]+, cald. 890.4 [M+H]+.  
LCMS Rt (min): 2.94, 3.11, 3.24. (1-2) Synthesis of compound (II-1-2)
Figure JPOXMLDOC01-appb-C000026
The same method as (1-1) using N-benzoyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxycytidine (5 g) and quinuclidin-3-ol (1.2 g) Gave compound (II-1-2) (4.5 g, 64%) as a pale yellow solid substance.
31 P NMR (CDCl 3 , 400 MHz) δ P : 147.5, 147.0, 146.9, 146.7.
LC-ESI-SQ-MS: m / Z 890.54 [M + H] + , cald. 890.4 [M + H] + .
LCMS Rt (min): 2.94, 3.11, 3.24.
（１－３）化合物（ＩＩ－１－３）の合成
Figure JPOXMLDOC01-appb-C000027
　５’－Ｏ－（４，４’－ジメトキシトリチル）－１，２－デオキシ－Ｄ－リボース（Ｂｅｒｒｙ　＆　Ａｓｓｏｃｉａｔｅｓ社製）（０．８ｇ）及びキヌクリジン－３－オール（０．２９ｇ）を用いて（１－１）と同様の方法で化合物（ＩＩ－１－３）（０．４７ｇ、３６％）を白色固体物質として得た。
31P NMR (CDCl3, 400 MHz) δP: 145.7, 145.6, 145.3, 144.9．
LC-ESI-SQ-MS: m/Z 677.49 [M+H]+, cald. 677.36 [M+H]+.  
LCMS Rt (min): 3.26, 3.44. (1-3) Synthesis of compound (II-1-3)
Figure JPOXMLDOC01-appb-C000027
Using 5′-O- (4,4′-dimethoxytrityl) -1,2-deoxy-D-ribose (Berry & Associates) (0.8 g) and quinuclidin-3-ol (0.29 g) Compound (II-1-3) (0.47 g, 36%) was obtained as a white solid substance by a method similar to (1-1).
31 P NMR (CDCl 3 , 400 MHz) δ P : 145.7, 145.6, 145.3, 144.9.
LC-ESI-SQ-MS: m / Z 677.49 [M + H] + , cald. 677.36 [M + H] + .
LCMS Rt (min): 3.26, 3.44.
（２－１）化合物（ＩＩ－２－１）の合成
Figure JPOXMLDOC01-appb-C000028
　５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－デオキシチミジン（２．５ｇ）及び２，２，６，６－テトラメチルピペリジン－４－オール（０．８７ｇ）を用いて（１－１）と同様の方法で化合物（ＩＩ－２－１）（０．５ｇ、１３％）を白色固体物質として得た。
31P NMR (CDCl3, 400 MHz) δP: 144.25．
LC-ESI-SQ-MS: m/Z 831.68 [M+H]+, cald. 832 [M+H]+.  
LCMS Rt (min): 3.1, 3.26. (2-1) Synthesis of compound (II-2-1)
Figure JPOXMLDOC01-appb-C000028
Using 5′-O- (4,4′-dimethoxytrityl) -2′-deoxythymidine (2.5 g) and 2,2,6,6-tetramethylpiperidin-4-ol (0.87 g) ( Compound (II-2-1) (0.5 g, 13%) was obtained as a white solid substance in the same manner as in 1-1).
31 P NMR (CDCl 3 , 400 MHz) δ P : 144.25.
LC-ESI-SQ-MS: m / Z 831.68 [M + H] + , cald. 832 [M + H] + .
LCMS Rt (min): 3.1, 3.26.
（３－１）化合物（ＩＩ－３－１）の合成
Figure JPOXMLDOC01-appb-C000029
　５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－デオキシチミジン（２ｇ）及び３－モルホリノプロパン－１－オール（０．５３ｇ）を用いて（１－１）と同様の方法で化合物（ＩＩ－３－１）（１．１ｇ、３７％）を白色固体物質として得た。
31P NMR (CDCl3, 400 MHz) δP: 147.3, 147.2, 146.9．
LC-ESI-SQ-MS: m/Z 819.59 [M+H]+, cald. 819.4 [M+H]+.  
LCMS Rt (min): 3.01, 3.08. (3-1) Synthesis of compound (II-3-1)
Figure JPOXMLDOC01-appb-C000029
Using 5′-O- (4,4′-dimethoxytrityl) -2′-deoxythymidine (2 g) and 3-morpholinopropan-1-ol (0.53 g) in the same manner as (1-1) Compound (II-3-1) (1.1 g, 37%) was obtained as a white solid material.
31 P NMR (CDCl 3 , 400 MHz) δ P : 147.3, 147.2, 146.9.
LC-ESI-SQ-MS: m / Z 819.59 [M + H] + , cald. 819.4 [M + H] + .
LCMS Rt (min): 3.01, 3.08.
（４－１）化合物（ＩＩ－４－１）
Figure JPOXMLDOC01-appb-C000030
　５’－Ｏ－（４，４’－ジメトキシトリチル）－２’－デオキシチミジン（２ｇ）及び（１－メチルピペリジン－４－イル）メタノール（０．４７ｇ）を用いて（１－１）と同様の方法で化合物（ＩＩ－４－１）（０．２９ｇ、９．８％）を白色固体物質として得た。
31P NMR (CDCl3, 400 MHz) δP: 147.5, 147.3．
LC-ESI-SQ-MS: m/Z 803.58 [M+H]+, cald. 803.41 [M+H]+.  
LCMS Rt (min): 3.07,  3.20. (4-1) Compound (II-4-1)
Figure JPOXMLDOC01-appb-C000030
Same as (1-1) using 5′-O- (4,4′-dimethoxytrityl) -2′-deoxythymidine (2 g) and (1-methylpiperidin-4-yl) methanol (0.47 g) The compound (II-4-1) (0.29 g, 9.8%) was obtained as a white solid substance by the above method.
31 P NMR (CDCl 3 , 400 MHz) δ P : 147.5, 147.3.
LC-ESI-SQ-MS: m / Z 803.58 [M + H] + , cald. 803.41 [M + H] + .
LCMS Rt (min): 3.07, 3.20.
　同様に、以下の化合物も合成することができる。
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000032
（式中、Ｒｐは、アセチル、ベンゾイル又はフェノキシアセチルであり、Ｒｑは、イソブチリル、アセチル、ベンゾイル又はフェノキシアセチルである）

Figure JPOXMLDOC01-appb-T000033
 Similarly, the following compounds can also be synthesized.
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000032
(Wherein R p is acetyl, benzoyl or phenoxyacetyl, and R q is isobutyryl, acetyl, benzoyl or phenoxyacetyl)

Figure JPOXMLDOC01-appb-T000033
実施例２　本発明のオリゴヌクレオチド（式（Ｉ）で示される結合を１以上含有するオリゴヌクレオチド）の合成
（１）一本鎖オリゴヌクレオチドの調製
　実施例１で得られたアミダイトから調製されるオリゴヌクレオチド、及びコントロールとして、全てのヌクレオシド間結合がホスホロチオエート結合であるオリゴヌクレオチド（０）、（３－０）、（４－０）若しくは（５－０）を、ｎＳ－８（ジーンデザイン社製）により０．２μｍｏｌスケールで合成した。合成したオリゴヌクレオチドを表２に示す。
Figure JPOXMLDOC01-appb-T000034
Figure JPOXMLDOC01-appb-C000035
“ｓ１～ｓ４”及び“ｏ１～ｏ４”は、式（Ｉ）における各記号が以下である、結合を意味する。
Figure JPOXMLDOC01-appb-T000036
 Example 2 Synthesis of oligonucleotide of the present invention (an oligonucleotide containing one or more bonds represented by formula (I)) (1) Preparation of single-stranded oligonucleotide Oligo prepared from amidite obtained in Example 1 As a control and an oligonucleotide (0), (3-0), (4-0) or (5-0), in which all internucleoside linkages are phosphorothioate linkages, nS-8 (manufactured by Gene Design) Was synthesized on a 0.2 μmol scale. The synthesized oligonucleotides are shown in Table 2.
Figure JPOXMLDOC01-appb-T000034
Figure JPOXMLDOC01-appb-C000035
“S1 to s4” and “o1 to o4” mean bonds in which the symbols in formula (I) are as follows.
Figure JPOXMLDOC01-appb-T000036
　アミダイトは、０．１Ｍアセトニトリル溶液として用いた。アミダイトと５’－末端の水酸基とのカップリング時間は２分とし、かつ連続する二度のカップリング工程により連結した。５’－末端がＤＭＴｒ基により保護され、かつ固相に支持されたオリゴヌクレオチドを０．１Ｍ水酸化ナトリウム溶液（メタノール／水＝４：１、０．５ｍＬ／サンプル、一晩）で処理した。得られた粗製物溶液を氷冷下、１％クエン酸水溶液で中和した。中和後のサンプルに０．１Ｍ　トリエチルアミン酢酸塩水溶液（２ｍＬ）を加えたものを遠心エバポレーターで濃縮した（４５℃、１時間）。濃縮後のサンプルをＳｅｐ－Ｐａｋ　Ｃ１８　Ｐｌｕｓ　Ｓｈｏｒｔ　Ｃａｒｔｒｉｄｇｅ（Ｗａｔｅｒｓ社製）により精製し、凍結乾燥した後に０．１Ｍオリゴヌクレオチド水溶液へと調製して、実施例３以降の試験へと供した。合成したオリゴヌクレオチドの純度と構造の確認は、ＬＣ－ＭＳにより確認した。結果を表４に示す。
　なお、ＬＣ-ＭＳの測定条件は以下の通りである。
カラム：Ａｃｑｕｉｔｙ　ＵＰＬＣ（登録商標）ＯＳＴ　Ｃ１８（１．７μｍ、ｉ．ｄ．２．１ｘ５０ｍｍ）
流速：０．８ｍＬ／分
ＵＶ検出波長：２６０ｎｍ
移動相：［Ａ］はメタノール、［Ｂ］は２００ｍＭ　ＨＦＩＰと８ｍＭ　ＴＥＡ含有水溶液（ｐＨ８．８）
グラジエント：１３分間で１０％－３０％溶媒［Ａ］のリニアグラジエントを行い、１００％溶媒［Ｂ］を維持した。
ＭＳはＥＳＩ－ＴＯＦを用い、ネガティブモードにて検出した。
Figure JPOXMLDOC01-appb-T000037
 The amidite was used as a 0.1M acetonitrile solution. The coupling time between the amidite and the hydroxyl group at the 5′-terminal was 2 minutes, and they were linked by two successive coupling steps. Oligonucleotides whose 5′-ends were protected by DMTr groups and supported on a solid phase were treated with 0.1 M sodium hydroxide solution (methanol / water = 4: 1, 0.5 mL / sample, overnight). The obtained crude product solution was neutralized with 1% aqueous citric acid solution under ice cooling. A sample obtained by adding a 0.1 M triethylamine acetate aqueous solution (2 mL) to the neutralized sample was concentrated by a centrifugal evaporator (45 ° C., 1 hour). The concentrated sample was purified by Sep-Pak C18 Plus Short Cartridge (manufactured by Waters), lyophilized, and then prepared into a 0.1 M oligonucleotide aqueous solution, which was used for the tests of Example 3 and later. The purity and structure of the synthesized oligonucleotide were confirmed by LC-MS. The results are shown in Table 4.
The measurement conditions for LC-MS are as follows.
Column: Acquity UPLC® OST C18 (1.7 μm, id 2.1 × 50 mm)
Flow rate: 0.8 mL / min UV detection wavelength: 260 nm
Mobile phase: [A] is methanol, [B] is an aqueous solution containing 200 mM HFIP and 8 mM TEA (pH 8.8)
Gradient: A linear gradient of 10% -30% solvent [A] was performed in 13 minutes to maintain 100% solvent [B].
MS was detected in negative mode using ESI-TOF.
Figure JPOXMLDOC01-appb-T000037
（２）二本鎖オリゴヌクレオチドの調製
　オリゴヌクレオチド（７－１）は株式会社ジーンデザイン社(大阪、日本)から購入した。
　一本鎖オリゴヌクレオチド（７－２）～（７－３）の合成法を以下に示す。（１）と同様のオリゴ合成法により合成した一本鎖オリゴヌクレオチドは、樹脂より切り出し反応を行った後にＨＰＬＣ精製を行い、その後ジメトキシトリチル基を除去し、もう一度ＨＰＬＣ精製を行った後に塩交換を行って最終サンプルとした。合成したオリゴヌクレオチドの純度と構造の確認は、（１）と同様の方法によりＬＣ－ＭＳにより確認した。結果を表５に示す。
Figure JPOXMLDOC01-appb-T000038
　さらに二本鎖オリゴヌクレオチド（７－４）及び（７－５）を以下のようにして調製した。各オリゴヌクレオチドを等モル量混合した後、蒸留水を加えて濃度０．５ｍｍｏｌ／Ｌ溶液とした。その後６０℃下で１０分間静置後、室温まで自然冷却させることで二重鎖核酸を得た。二重鎖形成の確認は、島津製ＵＶ－１８００を用いて正常なＴｍカーブを描くことを確認することで、二重鎖を形成しているものとした。
　合成したオリゴヌクレオチドを表６に示す。
Figure JPOXMLDOC01-appb-T000039
Figure JPOXMLDOC01-appb-C000040
 (2) Preparation of double-stranded oligonucleotide Oligonucleotide (7-1) was purchased from Gene Design Co., Ltd. (Osaka, Japan).
A method for synthesizing the single-stranded oligonucleotides (7-2) to (7-3) is shown below. Single-stranded oligonucleotides synthesized by the same oligo synthesis method as in (1) are excised from the resin and subjected to HPLC purification, then the dimethoxytrityl group is removed, and HPLC purification is performed again, followed by salt exchange. Go to the final sample. The purity and structure of the synthesized oligonucleotide were confirmed by LC-MS in the same manner as in (1). The results are shown in Table 5.
Figure JPOXMLDOC01-appb-T000038
Furthermore, double-stranded oligonucleotides (7-4) and (7-5) were prepared as follows. After equimolar amounts of each oligonucleotide were mixed, distilled water was added to make a 0.5 mmol / L solution. Thereafter, the mixture was allowed to stand at 60 ° C. for 10 minutes, and then naturally cooled to room temperature to obtain a double-stranded nucleic acid. Double strand formation was confirmed by confirming that a normal Tm curve was drawn using UV-1800 made by Shimadzu, thereby forming a double chain.
The synthesized oligonucleotides are shown in Table 6.
Figure JPOXMLDOC01-appb-T000039
Figure JPOXMLDOC01-appb-C000040
実施例３　本発明のオリゴヌクレオチド（式（Ｉ）で示される結合を１以上含有する一本鎖のオリゴヌクレオチド）の生物活性試験（相補ｍＲＮＡ切断活性試験）
（１）インビトロモデル　細胞培養
　細胞を、以下に記載される適当な培地で培養し、３７℃、９５～９８％湿度及び５％
ＣＯ２で維持した。
　ＨａＣａＴ：ヒトケラチノサイト由来細胞株ＨａＣａＴは、ＤＭＥＭ　Ｌｏｗ　Ｇｌｕｃｏｓｅ（Ｓｉｇｍａ）＋１０％ウシ胎児血清（ＦＢＳ）＋Ｐｅｎｉｃｉｌｌｉｎ（１００ｕｎｉｔｓ／ｍＬ）＋Ｓｔｒｅｐｔｏｍｙｃｉｎ（１００ｕｇ／ｍＬ）で培養した。
　Ｈｅｐａ１ｃ１ｃ７：マウス肝ガン由来細胞株Ｈｅｐａ１ｃ１ｃ７は、α－ＭＥＭ（Ｇｉｂｃｏ）＋１０％　ＦＢＳ＋Ａｎｔｉｂｉｏｔｉｃ　Ａｎｔｉｍｙｃｏｔｉｃ　Ｓｏｌｕｔｉｏｎ（１０ｍＬ／Ｌ）で培養した。 Example 3 Biological Activity Test (Complementary mRNA Cleavage Activity Test) of the Oligonucleotide of the Present Invention (Single-Stranded Oligonucleotide Containing One or More Bindings Represented by Formula (I))
(1) In vitro model Cell culture Cells are cultured in an appropriate medium as described below, 37 ° C, 95-98% humidity and 5%
Maintained with CO 2 .
HaCaT: Human keratinocyte-derived cell line HaCaT was cultured in DMEM Low Glucose (Sigma) + 10% fetal bovine serum (FBS) + Penicillin (100 units / mL) + Streptomycin (100 ug / mL).
Hepa1c1c7: The mouse liver cancer-derived cell line Hepa1c1c7 was cultured in α-MEM (Gibco) + 10% FBS + Antibiotic Anticolytic Solution (10 mL / L).
（２）自然取り込みによる導入実験
　実施例２に記載の通り、設計・製造したオリゴヌクレオチドを用いて、ヒトＨａＣａＴ細胞及び、マウスＨｅｐａ１ｃ１ｃ７細胞でノックダウン実験を行った。すべての細胞において、試薬を用いずにオリゴヌクレオチドを細胞へ導入した。
　ヒトＨａＣａＴ細胞及びマウスＨｅｐａ１ｃ１ｃ７細胞に対して、各細胞培養液にアンチセンスヌクレオチドの最終濃度が５μＭになるように添加した。ヒトＨａＣａＴ細胞に対しては、導入２日後に、マウスＨｅｐａ１ｃ１ｃ７細胞に対しては、導入５日後にＦａｓｔｌａｎｅ(ＱＩＡＧＥＮ)、若しくは、ＣｅｌｌＡｍｐ　ＲＮＡ　Ｐｒｅｐ　Ｋｉｔ（Ｔａｋａｒａ）にて細胞を回収し、Ｏｎｅ　Ｓｔｅｐ　ＳＹＢＲ　ＰｒｉｍｅＳｃｒｉｐｔ　ＰＬＵＳ　ＲＴ－ＰＣＲ　Ｋｉｔ（Ｔａｋａｒａ）により定量的ＰＣＲを行った。内在性コントロールとしてＲＰＬＰ０又はＧＡＰＤＨを使用した。

　マウスＡｃｓｌ１の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＡＧＧＴＧＣＴＴＣＡＧＣＣＣＡＣＣＡＴＣ（配列番号５）；
Ｒｖプライマー：ＡＡＡＧＴＣＣＡＡＣＡＧＣＣＡＴＣＧＣＴＴＣ（配列番号６）
を用いた。
　マウスＲＰＬＰ０の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＡＴＣＡＡＣＧＧＧＴＡＣＡＡＡＣＧＡＧＴＣ（配列番号７）；
Ｒｖプライマー：ＣＡＧＡＴＧＧＡＴＣＡＧＣＣＡＡＧＡＡＧＧ（配列番号８）
を用いた。
　マウスＧａｐｄｈの発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＴＧＴＧＴＣＣＧＴＣＧＴＧＧＡＴＣＴＧＡ（配列番号９）；
Ｒｖプライマー：ＴＴＧＣＴＧＴＴＧＡＡＧＴＣＧＣＡＧＧＡＧ（配列番号１０）
を用いた。
　ヒトＡｃｓｌ１の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＡＧＣＧＧＣＡＴＣＡＴＣＡＧＡＡＡＣ（配列番号１１）；
Ｒｖプライマー：ＴＧＴＣＡＣＣＡＴＣＡＧＣＣＧＧＡＣＴＣ（配列番号１２）
を用いた。
　ヒトＲＰＬＰ０の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＡＴＣＡＡＣＧＧＧＴＡＣＡＡＡＣＧＡＧＴＣ（配列番号１３）；
Ｒｖプライマー：ＣＡＧＡＴＧＧＡＴＣＡＧＣＣＡＡＧＡＡＧＧ（配列番号１４）
を用いた。 (2) Introduction experiment by natural uptake As described in Example 2, using the designed and produced oligonucleotide, a knockdown experiment was conducted with human HaCaT cells and mouse Hepa1c1c7 cells. In all cells, oligonucleotides were introduced into the cells without using reagents.
To human HaCaT cells and mouse Hepa1c1c7 cells, each cell culture solution was added to a final concentration of antisense nucleotide of 5 μM. For human HaCaT cells, cells were collected with Fastlane (QIAGEN) or CellAmp RNA Prep Kit (Takara) 5 days after introduction for mouse Hepa1c1c7 cells 2 days after introduction, and One Step SYBR PrimeScript. Quantitative PCR was performed with the PLUS RT-PCR Kit (Takara). RPLP0 or GAPDH was used as an endogenous control.

The primer sequence used to measure the expression level of mouse Acsl1 is:
Fw primer: AGGTGCTTCAGCCCACATC (SEQ ID NO: 5);
Rv primer: AAAGTCCAACAGCCATCGCTTC (SEQ ID NO: 6)
Was used.
The primer sequence used to measure the expression level of mouse RPLP0 is:
Fw primer: ATCAACGGGTCAAACGAGTC (SEQ ID NO: 7);
Rv primer: CAGATGGATCAGCCCAAGAAGG (SEQ ID NO: 8)
Was used.
The primer sequence used to measure the expression level of mouse Gapdh is:
Fw primer: TGTGTCCGTCGTGGATCTGA (SEQ ID NO: 9);
Rv primer: TTGCTGTTTGAAGTCGCAGAG (SEQ ID NO: 10)
Was used.
The primer sequence used to measure the expression level of human Acsl1 is
Fw primer: GCAGCGGGCATCATCAGAAAC (SEQ ID NO: 11);
Rv primer: TGTCCATCATAGCCCGACTC (SEQ ID NO: 12)
Was used.
The primer sequence used to measure the expression level of human RPLP0 is
Fw primer: ATCAACGGGTCAAACGAGTC (SEQ ID NO: 13);
Rv primer: CAGATGGATCAGCCCAAGAAGG (SEQ ID NO: 14)
Was used.
　結果を表７～１２に示す。表７～１０及び表１２にはＲＰＬＰ０で正規化したＡｃｓｌ１のｍＲＮＡ減少量を、未処理細胞に対する割合をノックダウン効率として示した。表１１には、ＧＡＰＤＨで正規化したＡｃｓｌ１のｍＲＮＡ減少量を、未処理細胞に対する割合をノックダウン効率として示した。 Results are shown in Tables 7-12. Tables 7 to 10 and Table 12 show the amount of decrease in Acsl1 mRNA normalized by RPLP0 as a percentage of untreated cells as knockdown efficiency. Table 11 shows the amount of Acsl1 mRNA decrease normalized with GAPDH as the knockdown efficiency as a percentage of untreated cells.
（試験１：ＨａＣａＴ細胞、導入２日後）
Figure JPOXMLDOC01-appb-T000041
 (Test 1: HaCaT cells, 2 days after introduction)
Figure JPOXMLDOC01-appb-T000041
　本発明のオリゴヌクレオチド（１－１）～（１－４）及び（２－１）～（２－４）は、式（Ｉ）で示される結合を有さないオリゴヌクレオチド（０）と比べ、標的ｍＲＮＡ（ヒトＡｃｓｌ１）に対するノックダウン活性が向上した。また、式（Ｉ）の置換基ＱがＯであるオリゴヌクレオチド（２－１）～（２－４）は、置換基ＱがＳであるオリゴヌクレオチド（１－１）～（１－４）と比較して、さらにノックダウン活性が向上した。 The oligonucleotides (1-1) to (1-4) and (2-1) to (2-4) of the present invention are compared with the oligonucleotide (0) having no bond represented by the formula (I), The knockdown activity for the target mRNA (human Acsl1) was improved. Further, the oligonucleotides (2-1) to (2-4) in which the substituent Q of formula (I) is O are the same as the oligonucleotides (1-1) to (1-4) in which the substituent Q is S. In comparison, the knockdown activity was further improved.
（試験２：Ｈｅｐａ１ｃ１ｃ７細胞、導入５日後）
Figure JPOXMLDOC01-appb-T000042
 (Test 2: Hepa1c1c7 cells, 5 days after introduction)
Figure JPOXMLDOC01-appb-T000042
　本発明のオリゴヌクレオチド（１－１）は式（Ｉ）で示される結合を有さないオリゴヌクレオチド（０）と比べ、標的ｍＲＮＡ（マウスＡｃｓｌ１）に対するノックダウン活性が向上した。試験１のヒトケラチノサイト由来細胞株ＨａＣａＴ及び試験２のマウス肝ガン由来細胞株Ｈｅｐａ１ｃ１ｃ７において同様の結果が得られたことから、細胞種に関わらず、本発明のオリゴヌクレオチドは標的ｍＲＮＡに対するノックダウン活性を向上させることが示唆された。 The knockdown activity against the target mRNA (mouse Acsl1) was improved in the oligonucleotide (1-1) of the present invention compared to the oligonucleotide (0) having no bond represented by the formula (I). Since similar results were obtained in the human keratinocyte-derived cell line HaCaT in Test 1 and the mouse liver cancer-derived cell line Hepa1c1c7 in Test 2, the oligonucleotide of the present invention has a knockdown activity against the target mRNA regardless of the cell type. It was suggested to improve.
（試験３：ＨａＣａＴ細胞、導入２日後）
Figure JPOXMLDOC01-appb-T000043
 (Test 3: HaCaT cells, 2 days after introduction)
Figure JPOXMLDOC01-appb-T000043
　オリゴヌクレオチド（３－０）及び（３－１）は、５’末端の１塩基（シチジン）が標的ｍＲＮＡに対して、ミスマッチである。本発明のオリゴヌクレオチド（３－１）のノックダウン活性は、式（Ｉ）で示される結合を有さないオリゴヌクレオチド（３－０）よりも高い活性を示した。また、試験１の結果と比較すると明らかなように、５’末端塩基部にミスマッチを持たない配列であるオリゴヌクレオチド（０）よりも本発明のオリゴヌクレオチド（３－１）は高いノックダウン活性を示した。よって、本発明のオリゴヌクレオチドは、ヌクレオチドの塩基がミスマッチであっても、式（Ｉ）で示される結合を有することにより、標的ｍＲＮＡに対するノックダウン活性を向上させることが示唆された。 Oligonucleotides (3-0) and (3-1) are mismatched with respect to the target mRNA at one base at the 5 'end (cytidine). The knockdown activity of the oligonucleotide (3-1) of the present invention was higher than that of the oligonucleotide (3-0) having no bond represented by the formula (I). Further, as is clear from the results of Test 1, the oligonucleotide (3-1) of the present invention has a higher knockdown activity than the oligonucleotide (0) which is a sequence having no mismatch at the 5 ′ terminal base. Indicated. Therefore, it was suggested that the oligonucleotide of the present invention improves the knockdown activity against the target mRNA by having the bond represented by the formula (I) even when the nucleotide base is mismatched.
（試験４：ＨａＣａＴ細胞、導入２日後）
Figure JPOXMLDOC01-appb-T000044
 (Test 4: HaCaT cells, 2 days after introduction)
Figure JPOXMLDOC01-appb-T000044
　本発明のオリゴヌクレオチド（４－１）は式（Ｉ）で示される結合を有さないオリゴヌクレオチド（４－０）と比べ、標的ｍＲＮＡ（ヒトＡｃｓｌ１）に対するノックダウン活性が向上した。試験１～３等の糖部位修飾ヌクレオチドＡｍＮＡ（化合物１００１）及び試験４の糖部位修飾ヌクレオチドＬＮＡ（化合物１００２）において同様の結果が得られたことから、糖修飾の種類に関わらず、本発明のオリゴヌクレオチドは標的ｍＲＮＡに対するノックダウン活性を向上させることが示唆された。 The oligonucleotide (4-1) of the present invention has an improved knockdown activity on the target mRNA (human Acsl1) compared to the oligonucleotide (4-0) having no bond represented by the formula (I). Similar results were obtained with the sugar site-modified nucleotide AmNA (Compound 1001) of Tests 1 to 3 and the like and the sugar site-modified nucleotide LNA (Compound 1002) of Test 4 and therefore, regardless of the type of sugar modification. It was suggested that the oligonucleotide improves the knockdown activity against the target mRNA.
（試験５：Ｈｅｐａ１ｃ１ｃ７細胞、導入５日後）
Figure JPOXMLDOC01-appb-T000045
 (Test 5: Hepa1c1c7 cells, 5 days after introduction)
Figure JPOXMLDOC01-appb-T000045
　オリゴヌクレオチド（５－１－１）及び（５－１－２）は、ギャップマーの中心領域（ギャップ領域）に式（Ｉ）で示される結合を有する。本発明のオリゴヌクレオチド（５－１－１）及び（５－１－２）は式（Ｉ）で示される結合を有さないオリゴヌクレオチド（５－０）と比べ、標的ｍＲＮＡ（マウスＡｃｓｌ１）に対するノックダウン活性が向上した。試験１～４等のように５’末端塩基のリン酸部位修飾だけでなく、試験５のようにリン酸部位修飾が、５’末端でなくとも、同様の結果が得られた。よって、オリゴヌクレオチドにおける修飾位置に関わらず、本発明のオリゴヌクレオチドは標的ｍＲＮＡに対するノックダウン活性を向上させることが示唆された。 Oligonucleotides (5-1-1) and (5-1-2) have a bond represented by the formula (I) in the central region (gap region) of the gapmer. The oligonucleotides (5-1-1) and (5-1-2) of the present invention are more effective against the target mRNA (mouse Acsl1) than the oligonucleotide (5-0) having no bond represented by the formula (I). Knockdown activity was improved. Similar results were obtained not only in the phosphate site modification of the 5 'terminal base as in Tests 1 to 4 but also in the case where the phosphate site modification was not at the 5' terminal as in Test 5. Therefore, it was suggested that the oligonucleotide of the present invention improves the knockdown activity for the target mRNA regardless of the modification position in the oligonucleotide.
（試験６：ＨａＣａＴ細胞、導入２日後）
Figure JPOXMLDOC01-appb-T000046
 (Test 6: HaCaT cells, 2 days after introduction)
Figure JPOXMLDOC01-appb-T000046
　オリゴヌクレオチド（６－１－１）は５’末端に１個、オリゴヌクレオチド（６－１－２）は５’末端に２個の無塩基のカチオンユニット（化合物１００３）を有し、該カチオンユニットのリン酸部位に式（Ｉ）で示される結合を有する。本発明のオリゴヌクレオチド（６－１－１）及び（６－１－２）は式（Ｉ）で示される結合を有さないオリゴヌクレオチド（０）と比べ、標的ｍＲＮＡ（ヒトＡｃｓｌ１）に対するノックダウン活性が向上した。よって、本発明のオリゴヌクレオチドは、ヌクレオチドの塩基が一部無塩基であっても、式（Ｉ）で示される結合を有することにより、標的ｍＲＮＡに対するノックダウン活性を向上させることが示唆された。 The oligonucleotide (6-1-1) has one abasic cation unit (compound 1003) at the 5 ′ end, and the oligonucleotide (6-1-2) has two abasic cation units (compound 1003). Have a bond represented by formula (I). The oligonucleotides (6-1-1) and (6-1-2) of the present invention are knocked down against the target mRNA (human Acsl1) as compared with the oligonucleotide (0) having no binding represented by the formula (I) Activity improved. Therefore, it was suggested that the oligonucleotide of the present invention improves the knockdown activity against the target mRNA by having the bond represented by the formula (I) even when the nucleotide base is partially abasic.
実施例４　本発明のオリゴヌクレオチド（式（Ｉ）で示される結合を１以上含有する二本鎖のオリゴヌクレオチド）のｉｎ　ｖｉｖｏ活性評価
　式（Ｉ）で示される結合を有さない二本鎖オリゴヌクレオチド（７－４）と本発明の二本鎖オリゴヌクレオチド（７－５）のマウス肝臓におけるＨｐｒｔ１　ｍＲＮＡの発現量変化によるノックダウン活性評価を行った。Ｃ５７ＢＬ／６Ｊマウス（オス１０週齢、日本クレア社）に、生理食塩水（大塚生食注、大塚製薬工場）に溶解したオリゴヌクレオチド（７－５）の溶液　約０．１ｍＬを、マウス個体あたり投与量がアンチセンスオリゴ量換算で０．５ｍｇ／ｋｇとなるように静脈投与した。陽性対照として、オリゴヌクレオチド（７－４）の溶液を、マウス個体あたり投与量がアンチセンスオリゴ量換算で０．５ｍｇ／ｋｇとなるように投与した。投与７日後にイソフルラン麻酔下で肝臓組織を採取した。肝臓からのＲＮＡ抽出はＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（Ｑｉａｇｅｎ社製）を用いてメーカー推奨プロトコル通りに行った。得られたＲＮＡのうち１００ｎｇをＯｎｅ　Ｓｔｅｐ　ＳＹＢＲ　ＰｒｉｍｅＳｃｒｉｐｔ　ＰＬＵＳ　ＲＴ－ＰＣＲ　Ｋｉｔ（タカラバイオ社製）を用いて定量的ＰＣＲを行った。
　マウスＨｐｒｔ１の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＴＴＧＴＴＧＴＴＧＧＡＴＡＴＧＣＣＣＴＴＧＡＣＴＡ（配列番号：１８）
Ｒｖプライマー：ＡＧＧＣＡＧＡＴＧＧＣＣＡＣＡＧＧＡＣＴＡ（配列番号：１９）
を用い、
マウスＧａｐｄｈの発現量を測定するために使用したプライマー配列は、上記実施例３（２）で示したものを用いた。
　ノックダウン効率は、Ｇａｐｄｈで正規化したＨｐｒｔ１のｍＲＮＡ減少量について、生理食塩水投与群に対する割合として示した。結果を図１に示す。この結果、本発明の二本鎖オリゴヌクレオチド（７－５）は、式（Ｉ）で示される結合を有さない二本鎖アンチセンスオリゴヌクレオチド（７－４）と比較して肝臓でのノックダウン効率が上昇する傾向を示した。 Example 4 Evaluation of in vivo activity of oligonucleotide of the present invention (double-stranded oligonucleotide containing one or more bonds represented by formula (I)) Double-stranded oligo having no bond represented by formula (I) The knockdown activity of the nucleotide (7-4) and the double-stranded oligonucleotide (7-5) of the present invention was evaluated by changing the expression level of Hprt1 mRNA in the mouse liver. About 0.1 mL of oligonucleotide (7-5) solution dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) was administered per mouse to C57BL / 6J mice (male, 10 weeks old, CLEA Japan). Intravenous administration was performed so that the amount was 0.5 mg / kg in terms of the amount of antisense oligo. As a positive control, a solution of oligonucleotide (7-4) was administered so that the dose per mouse individual was 0.5 mg / kg in terms of the amount of antisense oligo. Seven days after administration, liver tissue was collected under isoflurane anesthesia. RNA extraction from the liver was performed according to the manufacturer's recommended protocol using RNeasy Mini Kit (Qiagen). 100 ng of the obtained RNA was subjected to quantitative PCR using One Step SYBR PrimeScript PLUS RT-PCR Kit (manufactured by Takara Bio Inc.).
The primer sequence used to measure the expression level of mouse Hprt1 is
Fw primer: TTGTTGTTGGATAGCCCCTTGACTA (SEQ ID NO: 18)
Rv primer: AGGCAGATGGCCACAGGAACTA (SEQ ID NO: 19)
Use
The primer sequence used for measuring the expression level of mouse Gapdh was the one shown in Example 3 (2) above.
The knockdown efficiency was expressed as a percentage of the physiological saline administration group with respect to the amount of Hprt1 mRNA decreased normalized by Gapdh. The results are shown in FIG. As a result, the double-stranded oligonucleotide (7-5) of the present invention was knocked in the liver as compared with the double-stranded antisense oligonucleotide (7-4) having no bond represented by the formula (I). The down efficiency tended to increase.
　以上の実施例から明らかなように、本発明のアミダイト等から調製される本発明のオリゴヌクレオチドは、優れた標的遺伝子の発現抑制活性を示す。また、本発明のオリゴヌクレオチドは、修飾位置や修飾数を工夫することで、血漿中滞留性が向上し、例えば、肝臓又は肺等の臓器への集積の増加により、効率的に標的臓器に送達され、標的遺伝子又は標的タンパク質の発現を効果的に抑制することを可能であることが示唆されている。従って、本発明のオリゴヌクレオチドは、アンチセンスオリゴヌクレオチド等を含む核酸医薬品を製造するための材料として非常に有用である。 As is clear from the above examples, the oligonucleotide of the present invention prepared from the amidite of the present invention exhibits excellent target gene expression suppression activity. In addition, the oligonucleotide of the present invention improves plasma retention by devising the modification position and the number of modifications, and is efficiently delivered to a target organ, for example, by increasing accumulation in an organ such as the liver or lung. It has been suggested that it is possible to effectively suppress the expression of the target gene or target protein. Therefore, the oligonucleotide of this invention is very useful as a material for manufacturing the nucleic acid pharmaceutical containing an antisense oligonucleotide etc.

Claims (12)

  1. 式（Ｉ）：
    Figure JPOXMLDOC01-appb-C000001
    （式中、
    ＱはＳ又はＯであり、
    Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、
    Ｘ２は置換又は非置換の単環若しくは２環の含窒素非芳香族複素環式基である）
    で示される結合を１以上含有するオリゴヌクレオチド又はその製薬上許容される塩。     Formula (I):
    Figure JPOXMLDOC01-appb-C000001
    (Where
    Q is S or O,
    X 1 is a substituted or unsubstituted alkylene which is absent or O may be interposed,
    X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group)
    Or an pharmaceutically acceptable salt thereof.
  2. Ｘ２が、置換若しくは非置換のアジリジニル、置換若しくは非置換のアゼチジニル、置換若しくは非置換のピロリジニル、置換若しくは非置換のピペリジル、置換若しくは非置換のピペリジノ、置換若しくは非置換のピペラジニル、置換若しくは非置換のピペラジノ、置換若しくは非置換のモルホリニル、置換若しくは非置換のモルホリノ、置換若しくは非置換のアゼパニル又は置換若しくは非置換のキヌクリジニルである、請求項１記載のオリゴヌクレオチド又はその製薬上許容される塩。 X 2 is substituted or unsubstituted aziridinyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted piperidyl, substituted or unsubstituted piperidino, substituted or unsubstituted piperazinyl, substituted or unsubstituted The oligonucleotide or pharmaceutically acceptable salt thereof according to claim 1, which is piperazino, substituted or unsubstituted morpholinyl, substituted or unsubstituted morpholino, substituted or unsubstituted azepanyl or substituted or unsubstituted quinuclidinyl.
  3. Ｘ１は存在しないか、又は非置換のアルキレンである、請求項１又は２記載のオリゴヌクレオチド又はその製薬上許容される塩。 Or X 1 is absent, is or unsubstituted alkylene, claim 1 or 2 oligonucleotide or a pharmaceutically acceptable salt thereof.
  4. 該オリゴヌクレオチドの長さが８～２５塩基である、請求項１～３いずれかに記載のオリゴヌクレオチド又はその製薬上許容される塩。 The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide has a length of 8 to 25 bases.
  5. 糖の２’位に置換基を有する糖修飾ヌクレオシド又は
    糖の４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシド
    を１以上含有する、請求項１～４いずれかに記載のオリゴヌクレオチド又はその製薬上許容される塩。     5. The sugar-modified nucleoside having a substituent at the 2′-position of the sugar or one or more sugar-modified nucleosides having a cross-linking structure between the 4′-position and the 2′-position of the sugar. Oligonucleotide or a pharmaceutically acceptable salt thereof.
  6. 該置換基が、F、ＯＣＨ３又はＯＣＨ２ＣＨ２ＯＣＨ３である、請求項５記載のオリゴヌクレオチド又はその製薬上許容される塩。 The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 5, wherein the substituent is F, OCH 3 or OCH 2 CH 2 OCH 3 .
  7. 該架橋構造が、４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）又は４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、請求項５記載のオリゴヌクレオチド又はその製薬上許容される塩。 The cross-linked structure is 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′—C (═O) —NR 1 -2 ′ (R 1 is a hydrogen atom or 6. The oligonucleotide according to claim 5 or a pharmaceutically acceptable salt thereof.
  8. 式（ＩＩ）：
    Figure JPOXMLDOC01-appb-C000002
    （式中：
    Ｘ１は存在しないか、又はＯが介在していてもよい置換若しくは非置換のアルキレンであり、
    Ｘ２は置換又は非置換の単環若しくは２環の含窒素非芳香族複素環式基であり、
    Ｒ２は置換若しくは非置換のアミノである）
    で示される基
    （但し、
    Figure JPOXMLDOC01-appb-C000003
    で示される基を除く）
    及びヌクレオシド構造を含有する化合物、又はその塩。     Formula (II):
    Figure JPOXMLDOC01-appb-C000002
    (Where:
    X 1 is a substituted or unsubstituted alkylene which is absent or O may be interposed,
    X 2 is a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing non-aromatic heterocyclic group,
    R 2 is substituted or unsubstituted amino)
    A group represented by (however,
    Figure JPOXMLDOC01-appb-C000003
    Except the group indicated by
    And a compound containing a nucleoside structure, or a salt thereof.
  9. 該ヌクレオシド構造が、式（ＩＩＩ）：
    Figure JPOXMLDOC01-appb-C000004
    （式中：
    Ｂｘは水素又は核酸塩基部分であり、
    Ｚは水素原子又は水酸基保護基であり、
    Ｒ３及びＲ４はそれぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
    Ｒ５は水素であり、
    Ｒ６は水素原子、ハロゲン、ヒドロキシ又は置換若しくは非置換のアルキルオキシであり、
    ここでＲ５及びＲ６は、一緒になって架橋構造を形成していてもよく、
    Ｒ７は水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルである）
    で示される構造である、請求項８記載の化合物、又はその塩。     The nucleoside structure has the formula (III):
    Figure JPOXMLDOC01-appb-C000004
    (Where:
    Bx is a hydrogen or nucleobase moiety;
    Z is a hydrogen atom or a hydroxyl protecting group,
    R 3 and R 4 are each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl,
    R 5 is hydrogen;
    R 6 is a hydrogen atom, halogen, hydroxy, or substituted or unsubstituted alkyloxy,
    Here, R 5 and R 6 may be combined to form a crosslinked structure,
    R 7 is a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl)
    The compound or its salt of Claim 8 which is a structure shown by these.
  10. Ｒ６が、Ｆ、ＯＣＨ３又はＯＣＨ２ＣＨ２ＯＣＨ３である、又は、
    Ｒ５とＲ６が、一緒になって架橋構造を形成している、
    請求項９記載の化合物、又はその塩。     R 6 is F, OCH 3 or OCH 2 CH 2 OCH 3 , or
    R 5 and R 6 together form a crosslinked structure,
    The compound of Claim 9, or its salt.
  11. 該架橋構造が、４’－（ＣＨ２）ｍ－Ｏ－２’（ｍは１～４の整数）又は４’－Ｃ（＝Ｏ）－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、請求項１０記載の化合物、又はその塩。 The cross-linked structure is 4 ′-(CH 2 ) m—O-2 ′ (m is an integer of 1 to 4) or 4′—C (═O) —NR 1 -2 ′ (R 1 is a hydrogen atom or 11. The compound according to claim 10, which is alkyl), or a salt thereof.
  12. 該水酸基保護基が、アセチル、ｔ－ブチル、ｔ－ブトキシメチル、メトキシメチル、テトラヒドロピラニル、１－エトキシエチル、１－（２－クロロエトキシ）エチル、２－トリメチルシリルエチル、ｐ－クロロフェニル、２，４－ジニトロフェニル、ベンジル、ベンゾイル、ｐ－フェニルベンゾイル、２，６－ジクロロベンジル、レブリノイル、ジフェニルメチル、ｐ－ニトロベンジル、トリメチルシリル、トリエチルシリル、ｔ－ブチルジメチルシリル、ｔ－ブチルジフェニルシリル、トリフェニルシリル、トリイソプロピルシリル、ギ酸ベンゾイル、クロロアセチル、トリクロロアセチル、トリフルオロアセチル、ピバロイル、イソブチリル、９－フルオレニルメチルオキシカルボニル、メタンスルホニル、ｐ－トルエンスルホニル、トリフルオロメタンスルホニル、トリチル、モノメトキシトリチル、ジメトキシトリチル、トリメトキシトリチル、９－フェニルキサンチン－９－イル又は９－（ｐ－メトキシフェニル）キサンチン－９－イルである、請求項９～１１いずれかに記載の化合物、又はその塩。 The hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2, 4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, levulinoyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenyl Silyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl, 9-fluorenylmethyloxycarbonyl, methanesulfonyl, p-toluenesulfonyl, The trifluoromethanesulfonyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl or 9- (p-methoxyphenyl) xanthin-9-yl Or a salt thereof.
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