WO2016017422A1 - Nucléoside et nucléotide de réticulation - Google Patents

Nucléoside et nucléotide de réticulation Download PDF

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WO2016017422A1
WO2016017422A1 PCT/JP2015/070201 JP2015070201W WO2016017422A1 WO 2016017422 A1 WO2016017422 A1 WO 2016017422A1 JP 2015070201 W JP2015070201 W JP 2015070201W WO 2016017422 A1 WO2016017422 A1 WO 2016017422A1
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group
carbon atoms
amino
nucleic acid
compound
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PCT/JP2015/070201
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Japanese (ja)
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功幸 張
聡 小比賀
昂志 大澤
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国立大学法人大阪大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals

Definitions

  • the present invention relates to a bridged nucleoside and a nucleotide. More particularly, it relates to bridged nucleosides and nucleotides having high binding affinity for single-stranded RNA and double-stranded DNA, and high resistance to nucleases.
  • anti-sense method anti-gene method, aptamer, siRNA and the like as treatment methods for diseases by nucleic acid medicine.
  • the antisense method introduces an oligonucleotide (antisense strand) complementary to the mRNA involved in the disease from the outside to form a double strand, thereby inhibiting the translation process of the pathogenic RNA and treating the disease.
  • siRNA is similar to this, and translation from mRNA to protein is inhibited by double-stranded RNA administered to a living body.
  • the antigene method suppresses transcription from DNA to RNA by introducing from the outside a triplex-forming oligonucleotide corresponding to a DNA site that transcribes pathogenic RNA.
  • aptamers are short nucleic acid molecules (oligonucleotides), they function by binding to biological components such as proteins that cause disease.
  • S-oligo phosphorothioate
  • 2 ′, 4′-BNA bridged nucleic acid
  • LNA locked nucleic acid
  • S-oligo is already marketed in the United States as an antisense drug against cytomegalovirus. Although this has high nuclease resistance, it has a drawback that its binding affinity to the target nucleic acid chain is low, and needs to be improved.
  • 2 ′, 4′-BNA / LNA developed so far has the highest binding affinity to the target nucleic acid chain as a 2 ′, 4′-bridged artificial nucleoside, and is the most useful material for nucleic acid medicine in the future. It is an expected molecule. However, resistance to nucleases is not sufficient, and there is room for improvement in terms of stability in vivo.
  • nucleoside based on the structure as shown in the above formula (a) is considered to have, for example, a very complicated and multi-step process for its synthesis, and there is room for improvement in resistance to nuclease. However, it was still not satisfactory. Further, it has been pointed out that the nucleoside serving as the base of the structure as shown in the formula (b) has a large seven-membered ring cross-linked structure, and thus it is considered that the binding affinity with the target nucleic acid chain is not satisfactory. It was. For this reason, it is desired to develop an oligonucleotide having performance equivalent to or higher than that of such an oligonucleotide and further excellent in industrial production efficiency.
  • the present invention solves the above-mentioned problems, and the object of the present invention is to prevent degradation by nucleases in vivo, have high binding affinity and specificity for target mRNA, and express a specific gene. It is an object of the present invention to provide a novel molecule for antisense methods and nucleic acid pharmaceuticals, which can efficiently control the above-described molecules, and has excellent productivity.
  • the present invention relates to a compound represented by the following formula (I) or (I ′) or a salt thereof:
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the ⁇ group, where The ⁇ group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis.
  • R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring
  • Chain or branch represents a chain alkylamino group];
  • R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring.
  • X is an oxygen atom or a sulfur atom).
  • X is an oxygen atom.
  • the Base is a 6-aminopurin-9-yl group, a 2,6-diaminopurin-9-yl group, a 2-amino-6- Chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group, 6-amino-2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurine-9 -Yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group, 4-amino- 2-oxo-5-fluoro-1,
  • the Base is a 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group.
  • the present invention also provides an oligonucleotide containing at least one nucleoside structure represented by the following formula (II) or (II ′) or a pharmacologically acceptable salt thereof:
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more optional substituents selected from the ⁇ group, where The ⁇ group is protected with a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group with 1 to 6 carbon atoms, a linear alkoxy group with 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis.
  • R 6 and R 7 are each independently a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring;
  • a group selected from the group consisting of: an alkoxy group of 7; an amino group; and an amino group protected with a protecting group for nucleic acid synthesis; or R 6 and R 7 taken together are ⁇ C (R 10 ) R 11 [wherein R 10 and R 11 are each independently a hydrogen atom, a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a mercapto group protected with a protecting group for nucleic acid
  • Chain or branch represents a chain alkylamino group];
  • R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring.
  • X is an oxygen atom or a sulfur atom).
  • X is an oxygen atom.
  • novel 2 ', 4'-bridged 6-membered ring nucleosides and nucleotides having a hetero atom at the 6'-position are provided.
  • This oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide has a binding affinity for single-stranded RNA and single-stranded DNA comparable to conventional oligonucleotides containing 2 ′, 4′-bridged artificial nucleotide.
  • the oligonucleotide of the present invention is expected to be applied to, for example, nucleic acid medicine.
  • the 2 ′, 4′-bridged nucleosides and nucleotides of the present invention can also introduce a heteroatom at the 6 ′ position in a 6-membered ring bridge structure, and through a simpler reaction process compared to the conventional one. Can be manufactured. For this reason, it is also possible to further increase industrial production efficiency.
  • linear alkyl group having 1 to 6 carbon atoms refers to any linear alkyl group having 1 to 6 carbon atoms, specifically a methyl group, an ethyl group, an n-propyl group, An n-butyl group, an n-pentyl group, or an n-hexyl group.
  • linear alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methyloxy group, an ethyloxy group, and an n-propyloxy group.
  • a linear or branched alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having an arbitrary linear or branched alkyl group having 1 to 6 carbon atoms.
  • Examples thereof include a methyloxy group, an ethyloxy group, an n-propyloxy group, an isopropyloxy group, an n-butyloxy group, an isobutyloxy group, a tert-butyloxy group, an n-pentyloxy group, and an isopentyloxy group.
  • linear alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methylthio group, an ethylthio group, and an n-propylthio group.
  • a linear or branched alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having an arbitrary linear or branched alkyl group having 1 to 6 carbon atoms.
  • Examples include a methylthio group, an ethylthio group, an n-propylthio group, an isopropylthio group, an n-butylthio group, an isobutylthio group, a tert-butylthio group, an n-pentylthio group, and an isopentylthio group.
  • C1-C6 cyanoalkoxy group refers to a group in which at least one hydrogen atom constituting the straight-chain alkoxy group having 1 to 6 carbon atoms is substituted with a cyano group.
  • linear alkylamino group having 1 to 6 carbon atoms means a group in which one or two hydrogen atoms constituting the amino group are substituted with a linear alkyl group having 1 to 6 carbon atoms. Is included. Examples thereof include a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, and a diethylamino group.
  • a linear or branched alkylamino group having 1 to 6 carbon atoms means any linear or branched group in which one or two hydrogen atoms constituting the amino group are 1 to 6 carbon atoms. Includes groups substituted with chain alkyl groups.
  • Examples include methylamino group, dimethylamino group, ethylamino group, methylethylamino group, diethylamino group, n-propylamino group, di-n-propylamino group, isopropylamino group, diisopropylamino group and the like.
  • an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring means any linear alkyl group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. Includes a chain alkyl group and any cyclic alkyl group having 3 to 7 carbon atoms. It may be simply referred to as “lower alkyl group”.
  • arbitrary linear alkyl groups having 1 to 7 carbon atoms include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, and n-heptyl group.
  • Examples of the branched alkyl group having 3 to 7 carbon atoms include isopropyl group, isobutyl group, tert-butyl group, isopentyl group and the like, and optional cyclic alkyl group having 3 to 7 carbon atoms include A cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. are mentioned.
  • an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring means any linear alkenyl group having 2 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. Chain alkenyl groups, and any cyclic alkenyl group having 3 to 7 carbon atoms are included. It may be simply referred to as “lower alkenyl group”.
  • Examples of the branched alkenyl group having 3 to 7 carbon atoms include isopropenyl group, 1-methyl-1-propenyl group, 1-methyl -2-propenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-methyl-2-butenyl group, etc., and any cyclic alkenyl group having 3 to 7 carbon atoms includes a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and the like.
  • an alkoxy group having 1 to 7 carbon atoms which may form a branch or a ring means any linear alkoxy group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. It includes a chain alkoxy group and any cyclic alkoxy group having 3 to 7 carbon atoms. It may be simply referred to as “lower alkoxy group”.
  • any linear alkoxy group having 1 to 7 carbon atoms includes a methoxy group, an ethoxy group, an n-propoxy group, an n-butoxy, an n-pentyloxy group, an n-hexyloxy group, and an n-heptyloxy group.
  • Examples of the branched alkoxy group having 3 to 7 carbon atoms include isopropoxy group, isobutoxy group, tert-butoxy group, isopentyloxy group, etc., and any cyclic group having 3 to 7 carbon atoms
  • Examples of the alkoxy group include a cyclobutoxy group, a cyclopentyloxy group, and a cyclohexyloxy group.
  • an aryl group having 3 to 12 carbon atoms that may contain a heteroatom refers to any aryl group having 6 to 12 carbon atoms, which is composed of only hydrocarbons, and the aryl group. Including any heteroaryl group having 3 to 12 carbon atoms in which at least one carbon atom constituting the ring structure is substituted with a heteroatom (eg, a nitrogen atom, an oxygen atom, and a sulfur atom, and combinations thereof) To do.
  • a heteroatom eg, a nitrogen atom, an oxygen atom, and a sulfur atom, and combinations thereof
  • Examples of the aryl group having 6 to 12 carbon atoms include a phenyl group, a naphthyl group, an indenyl group, and an azulenyl group, and examples of the heteroaryl group having 3 to 12 carbon atoms include a pyridyl group, a pyrrolyl group, A quinolyl group, an indolyl group, an imidazolyl group, a furyl group, a thienyl group, and the like can be given.
  • aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom examples include a benzyl group, a phenethyl group, a naphthylmethyl group, a 3-phenylpropyl group, -Phenylpropyl, 4-phenylbutyl, 2-phenylbutyl, pyridylmethyl, indolylmethyl, furylmethyl, thienylmethyl, pyrrolylmethyl, 2-pyridylethyl, 1-pyridylethyl, 3 -Thienylpropyl group and the like.
  • examples of the term “acyl group” include aliphatic acyl groups and aromatic acyl groups.
  • examples of the aliphatic acyl group include formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group, isovaleryl group, octanoyl group, nonanoyl group, decanoyl group, 3-methylnonanoyl group, 8-methylnonanoyl group, 3-ethyloctanoyl group, 3,7-dimethyloctanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, 1-methylpentadecanoyl group, 14-methylpentadecanoyl group, 13,13-
  • aromatic acyl group examples include arylcarbonyl groups such as benzoyl group, ⁇ -naphthoyl group and ⁇ -naphthoyl group; halogenoarylcarbonyl groups such as 2-bromobenzoyl group and 4-chlorobenzoyl group; 2 , 4,6-trimethylbenzoyl group, lower alkylated arylcarbonyl group such as 4-toluoyl group; lower alkoxylated arylcarbonyl group such as 4-anisoyl group; 2-carboxybenzoyl group, 3-carboxybenzoyl group, 4 A carboxylated arylcarbonyl group such as a carboxybenzoyl group; a nitrated arylcarbonyl group such as a 4-nitrobenzoyl group or a 2-nitrobenzoyl group; a lower alkoxycarbonylated arylcarbonyl such as a 2- (methoxycarbonyl) benzoyl
  • sil group examples include trimethylsilyl group, triethylsilyl group, isopropyldimethylsilyl group, t-butyldimethylsilyl group, methyldiisopropylsilyl group, methyldi-t-butylsilyl group, and triisopropylsilyl group.
  • a tri-lower alkylsilyl group such as diphenylmethylsilyl group, butyldiphenylbutylsilyl group, diphenylisopropylsilyl group, tri-lower alkylsilyl group substituted with 1 to 2 aryl groups such as phenyldiisopropylsilyl group, etc.
  • a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, and a t-butyldiphenylsilyl group are preferable, and a trimethylsilyl group is more preferable.
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferable is a fluorine atom or a chlorine atom.
  • protecting group for amino group for nucleic acid synthesis is capable of stably protecting an amino group, a hydroxyl group, a phosphate group or a mercapto group during nucleic acid synthesis. If it is, it will not be restrict
  • protecting group that is stable under acidic or neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis.
  • protecting groups include lower alkyl groups, lower alkenyl groups, acyl groups, tetrahydropyranyl or tetrahydrothiopyranyl groups, tetrahydrofuranyl or tetrahydrothiofuranyl groups, silyl groups, lower alkoxymethyl groups, lower alkoxy groups.
  • examples of the tetrahydropyranyl group or tetrahydrothiopyranyl group include a tetrahydropyran-2-yl group, a 3-bromotetrahydropyran-2-yl group, a 4-methoxytetrahydropyran-4-yl group, a tetrahydro Examples include a thiopyran-4-yl group and a 4-methoxytetrahydrothiopyran-4-yl group.
  • examples of the tetrahydrofuranyl group or the tetrahydrothiofuranyl group include a tetrahydrofuran-2-yl group and a tetrahydrothiofuran-2-yl group.
  • Examples of the lower alkoxymethyl group include a methoxymethyl group, a 1,1-dimethyl-1-methoxymethyl group, an ethoxymethyl group, a propoxymethyl group, an isopropoxymethyl group, a butoxymethyl group, and a t-butoxymethyl group.
  • Examples of the lower alkoxylated lower alkoxymethyl group include 2-methoxyethoxymethyl group.
  • Examples of the halogeno lower alkoxymethyl group include 2,2,2-trichloroethoxymethyl group and bis (2-chloroethoxy) methyl group.
  • Examples of the lower alkoxylated ethyl group include 1-ethoxyethyl group and 1- (isopropoxy) ethyl group.
  • Examples of the halogenated ethyl group include 2,2,2-trichloroethyl group.
  • Examples of the methyl group substituted with 1 to 3 aryl groups include benzyl group, ⁇ -naphthylmethyl group, ⁇ -naphthylmethyl group, diphenylmethyl group, triphenylmethyl group, ⁇ -naphthyldiphenylmethyl group, 9-anne.
  • Examples include a thrylmethyl group.
  • Examples of the “methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a lower alkyl group, lower alkoxy group, halogen atom or cyano group” include 4-methylbenzyl group, 2,4,6- Trimethylbenzyl group, 3,4,5-trimethylbenzyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, 4,4'-dimethoxytriphenylmethyl group, 2-nitrobenzyl group, 4-nitrobenzyl group 4-chlorobenzyl group, 4-bromobenzyl group, 4-cyanobenzyl group and the like.
  • Examples of the lower alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl group, and an isobutoxycarbonyl group.
  • Examples of the “aryl group substituted with a halogen atom, lower alkoxy group or nitro group” include 4-chlorophenyl group, 2-fluorophenyl group, 4-methoxyphenyl group, 4-nitrophenyl group, 2,4-dinitrophenyl group Etc.
  • Examples of the “lower alkoxycarbonyl group substituted with a halogen atom or tri-lower alkylsilyl group” include 2,2,2-trichloroethoxycarbonyl group, 2-trimethylsilylethoxycarbonyl group and the like.
  • Examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group and an aryloxycarbonyl group.
  • Examples of the “aralkyloxycarbonyl group whose aryl ring may be substituted with a lower alkoxy or nitro group” include benzyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 3,4-dimethoxybenzyloxycarbonyl group, 2-nitro Examples include benzyloxycarbonyl group, 4-nitrobenzyloxycarbonyl group and the like.
  • the “hydroxyl-protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, “lower alkyl, lower alkoxy, halogen, cyano” A methyl group substituted with 1 to 3 aryl groups substituted with an aryl ring by a group ”, or a silyl group, and more preferably an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzoyl group, a dimethoxy group A trityl group, a monomethoxytrityl group or a tert-butyldiphenylsilyl group;
  • Preferred examples of the protecting group for the “hydroxyl group protected with a protecting group for nucleic acid synthesis” include aliphatic acyl groups, aromatic acyl groups, “methyl groups substituted with 1 to 3 aryl groups
  • the “protecting group for the amino group for nucleic acid synthesis” is preferably an acyl group, more preferably a benzoyl group.
  • the “protecting group” of the “phosphate group protected with a protecting group for nucleic acid synthesis” is preferably a lower alkyl group, a lower alkyl group substituted with a cyano group, an aralkyl group, a “nitro group or a halogen atom”.
  • the protecting group constituting the “phosphate group protected with a protecting group for nucleic acid synthesis” may be one or more.
  • the “protecting group” of the “mercapto group protected with a protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group or an aromatic acyl group, and more preferably a benzoyl group.
  • —P (R 4 ) R 5 wherein R 4 and R 5 are each independently a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a protecting group for nucleic acid synthesis.
  • the groups represented by the formula (1) represents a linear or branched alkylamino group of 1 to 6, the group that R 4 can represent as OR 4a and R 5 as NR 5a can be referred to as a “phosphoramidite group”. .
  • the phosphoramidite group is preferably a group represented by the formula —P (OC 2 H 4 CN) (N (iPr) 2 ) or a formula —P (OCH 3 ) (N (iPr) 2 ). And the group represented.
  • iPr represents an isopropyl group.
  • nucleoside and nucleoside analog mean an unnatural type of “nucleoside” in which a purine or pyrimidine base and a sugar are bonded, and an aromatic heterocycle other than purine and pyrimidine. And an aromatic hydrocarbon ring capable of substituting with a purine or pyrimidine base and having a sugar bound thereto.
  • the terms “artificial oligonucleotide” and “oligonucleotide analog” refer to an “oligonucleotide” in which 2 to 50 identical or different “nucleosides” or “nucleoside analogs” are linked by phosphodiester bonds.
  • Such analogs preferably include a sugar derivative having a modified sugar moiety; a thioate derivative in which the phosphodiester moiety is thioated; an ester form in which the terminal phosphate moiety is esterified; Examples include amides in which the amino group is amidated, and more preferable examples include sugar derivatives in which the sugar moiety is modified.
  • a salt thereof refers to a salt of a compound represented by the formula (I) or (I ′) of the present invention.
  • examples of such salts include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phene
  • the term “pharmacologically acceptable salt thereof” refers to a salt of an oligonucleotide analog containing at least one nucleoside structure represented by the formula (II) or (II ′) of the present invention.
  • salts include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (Hydroxy Amine salts such as organic salts such as
  • the compound of the present invention or a salt thereof is 2 ', 4'-bridged nucleoside and nucleotide or a salt thereof.
  • the compound of the present invention or a salt thereof has the following formula (I) or (I ′):
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group optionally having one or more arbitrary substituents selected from ⁇ group
  • the ⁇ group is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, or a protecting group for nucleic acid synthesis.
  • R 2 and R 3 each independently form a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, a branch or a ring
  • Chain or branch represents a chain alkylamino group];
  • R 8 and R 9 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, or a 1 to 7 carbon atom that may form a branch or a ring.
  • X is an oxygen atom or a sulfur atom).
  • Base is a purine base (ie, purin-9-yl group) or a pyrimidine base (ie, 2-oxo-1,2-dihydropyrimidin-1-yl group) ).
  • bases are a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and 1 to carbon atoms. It may have one or more arbitrary substituents selected from the ⁇ group consisting of 6 linear alkylamino groups and halogen atoms.
  • Base examples include 6-aminopurin-9-yl group (adenylyl group), 2,6-diaminopurine-9-yl group, 2-amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group (guaninyl group), 6-amino- 2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6-dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (cytosynyl group), 4-amino- 2-oxo-5-
  • Base has the following structural formula from the viewpoint of introduction into nucleic acid medicine:
  • 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group), 2-oxo-4-amino-1,2-dihydropyrimidine-1 -Yl group (cytosynyl group), 6-aminopurin-9-yl group (adenylyl group), 2-amino-6-hydroxypurin-9-yl group (guaninyl group), 4-amino-5-methyl-2-
  • the oxo-1,2-dihydropyrimidin-1-yl group and the 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group are preferred, and in particular, the 2-oxo-4 A -hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group (thyminyl group) is preferred.
  • the hydroxyl group and amino group are protected by a protecting group.
  • the 2 ′, 4′-bridged nucleoside of the present invention has a hetero atom such as an oxygen atom or a sulfur atom introduced into the 6 ′ position of the conventional 2 ′, 4′-bridge structure, which will be described later. Enzyme resistance performance of the oligonucleotide to be improved. In addition, such a heteroatom in the 2 ', 4'-crosslinked structure directly affects the conformation of the sugar moiety. For this reason, the 2 ', 4'-bridged nucleoside of the present invention can further improve the binding affinity of the resulting oligonucleotide to single-stranded RNA (ssRNA) due to the influence.
  • ssRNA single-stranded RNA
  • the 2 ′, 4′-bridged nucleoside of the present invention can be synthesized in a single step by introducing a heteroatom at the 6 ′ position by using a 4′-exoolefin compound.
  • a radical cyclization reaction By utilizing a radical cyclization reaction, a crosslinked structure having a hetero atom at the 6′-position can be produced through very few steps. Therefore, the 2 ′, 4′-bridged nucleoside of the present invention can be synthesized more efficiently through fewer steps than conventional ENA or EoNA in which a hetero atom is introduced into the bridge structure. .
  • Isomers (i) and R-form (ii) and S-form (iii) with controlled stereochemistry can be synthesized separately.
  • oligonucleotides (2 ', 4'-bridged artificial nucleotides) can be prepared using such 2', 4'-bridged nucleosides.
  • triphosphorylation can be easily performed according to the method described in Non-Patent Document 5.
  • the oligonucleotide of the present invention or a pharmacologically acceptable salt thereof includes at least one nucleoside structure represented by the following formula (II) or (II ′):
  • the oligonucleotide of the present invention has at least one nucleoside structure at any position.
  • the position and number are not particularly limited, and can be appropriately designed according to the purpose.
  • Oligonucleotide analogs containing such a nucleoside structure have a dramatic improvement in enzyme resistance compared to, for example, conventional EoNA. Moreover, it has a single-stranded RNA (ssRNA) binding affinity that exceeds the EoNA.
  • ssRNA single-stranded RNA
  • oligonucleotide analogues synthesized using the 2 ′, 4′-bridged nucleosides of the present invention inhibit the action of specific genes including antitumor agents and antiviral agents.
  • both binding affinity to complementary sense strand RNA and resistance to in vivo DNA-degrading enzyme are required.
  • the structure of the sugar part is constantly fluctuating between a shape close to a DNA duplex and a shape close to a DNA-RNA duplex or an RNA duplex. It has been known. When a single-stranded nucleic acid forms a double strand with a complementary RNA strand, its sugar structure is fixed.
  • the sugar portion is fixed in the state in which a double strand is formed in advance, so that it is easy to form a double strand with a target RNA strand and is stable. Can be present. It is also known that nucleic acid duplexes are stabilized by hydrated water connected like a chain of water molecules.
  • the oligonucleotide analog of the present invention contains, for example, adjuvants commonly used in the pharmaceutical formulation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and flavoring agents.
  • adjuvants commonly used in the pharmaceutical formulation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and flavoring agents.
  • it can be a parenteral preparation or a liposome preparation.
  • a topical preparation such as a solution, cream, ointment, etc. can be prepared by blending a pharmaceutical carrier usually used in the art.
  • anhydrous dimethylformamide solution 100 mL of compound 1 (9.30 g, 38.7 mmol) synthesized from 5-methyluridine by the method described in Non-Patent Document 8 was added at 0 ° C. to 1,4 Add diazabicyclo [2.2.2] octane (21.7 g, 194 mmol), tert-butyldimethylchlorosilane (7.00 g, 46.5 mmol), silver nitrate (7.90 g, 46.5 mmol) and add 12 at room temperature. Stir for hours. After completion of the reaction, the reaction solution was filtered through celite. Water was added to the filtrate and extracted with diethyl ether.
  • Table 5 shows the physical property data of the obtained compound 5.
  • Table 6 shows the physical property data of the obtained compound 6.
  • Table 7 shows the physical property data of the obtained compound 7.
  • N, N-diisopropylethylamine (0.21 mL, 1.18 mmol) was added to an anhydrous dichloromethane solution (3.0 mL) of the compound 7 (230 mg, 0.393 mmol) obtained above, and the mixture was brought to 0 ° C.
  • 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.11 mL, 0.472 mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium hydrogen carbonate at 0 ° C., and extracted with ethyl acetate.
  • Table 10 shows the physical property data of the obtained compound 10.
  • N, N-diisopropylethylamine (0.21 mL, 1.15 mmol) was added to an anhydrous dichloromethane solution (3.0 mL) of the compound 10 obtained above (230 mg, 0.383 mmol), and 0 ° C. was added dropwise 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.10 mL, 0.460 mmol) and stirred at room temperature for 6 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
  • Table 11 shows the physical property data of the obtained compound 11.
  • Table 13 shows the physical property data of the obtained compound 13.
  • Table 14 shows the physical property data of the obtained compound 14.
  • Table 15 shows the physical property data of the obtained compound 15.
  • N, N-diisopropylethylamine (0.55 mL, 3.10 mmol) was added to an anhydrous dichloromethane solution (5.0 mL) of the compound 15 obtained above (372 mg, 0.619 mmol), and 0 ° C.
  • 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.21 mL, 0.929 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
  • Table 16 shows the physical property data of the obtained compound 16.
  • Example 4 Synthesis and purification of oligonucleotide (1) Compound 1.7 (Exomethylene), Compound 11 ((R) -methyl), Compound 16 ((S) obtained in Example 1.7, Example 2.3 and Example 3.5, and Comparative Example 1 ) - methyl body) and EoNA, used respectively as amidites blocks, these and d m C (Ac) and T phosphoramidites (both sigma - prepared Aldrich) and anhydrous acetonitrile 0.1M of Each of the oligonucleotides shown in Table 17 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where X is Corresponds to the structure derived from compound 8 (exomethylene) obtained in Example 1.7.
  • nS-8 Oligonucleotides Synthesizer an oligonucle
  • Y corresponds to the structure derived from the compound 11 ((R) -methyl form) obtained in Example 2.3
  • Z represents the compound 16 ((S) -methyl form obtained in Example 3.5.
  • W corresponds to the structure derived from EoNA obtained in Comparative Example 1).
  • the synthesis scale in the synthesis was 0.2 ⁇ mol, and was performed under trityl-on conditions.
  • As the activator 5- [3,5-bis (trifluoromethyl) phenyl] -1H-tetrazole (0.25 M anhydrous acetonitrile solution, Activator 42, Proligo (registered trademark)) was used.
  • the condensation time is 10 minutes for the amidite blocks X, Y and Z obtained in Example 1.7, Example 2.3 and Example 3.5 and Comparative Example 1, and EoNA, and the natural amidite The block was 32 seconds.
  • the mixture was treated with 28% aqueous ammonia at room temperature for 1.5 hours to cut out from the column carrier and to deprotect the base part and the phosphoric diester part. Subsequently, purification was performed by a simple gel filtration column (Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters), and further purified by reverse phase HPLC.
  • a simple gel filtration column Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters
  • Example 5 Measurement of melting temperature ( Tm ) (1) Sample solution containing 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 ⁇ M oligonucleotide obtained in Example 4 and 4 ⁇ M single-stranded RNA or single-stranded DNA shown in Table 17 (130 ⁇ L) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
  • Table 17 shows the results when single-stranded RNA was used as the target strand and the results when single-stranded DNA was used as the target strand.
  • Example 6 Measurement of melting temperature (T m ) (2) (Evaluation of triple chain forming ability)
  • the final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 ⁇ M of the oligonucleotide obtained in Example 4, and 1.5 ⁇ M of the double-stranded DNA shown in Table 18.
  • the containing sample solution (130 ⁇ L) was bathed in boiling water and slowly cooled to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C.
  • Example 1.7 the compounds 8 (exomethylene) and 16 ((S) -methyl) obtained in Example 1.7, Example 2.3 and Example 3.5 were amidite.
  • the oligonucleotide synthesized using the block showed a higher Tm value for double-stranded DNA and higher binding affinity than the oligonucleotide synthesized using EoNA as the amidite block. Recognize.
  • Example 7 Oligonucleotide Synthesis and Purification (2) Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl), thymidine (naturally occurring), LNA and ENA were used as amidite blocks, respectively, in the same manner as in Example 4 according to the phosphoramidite method known in the art. Nucleotide synthesis was performed.
  • Example 8 Enzyme resistance experiment (1) The final concentrations were respectively Tris-HCl buffer (pH 8.0) 50 mM, magnesium chloride 10 mM, each oligonucleotide 7.5 ⁇ M obtained in Example 7, and 3′-exonuclease (Crotalus Admanteus Venom Phosphoesterase: CAVP, Pharmacia Biotech) The sample solution (100 ⁇ L) adjusted to 1.5 ⁇ g / mL was kept at 37 ° C. for reaction. A part of the reaction solution (20 ⁇ L) was collected over time, heated at 90 ° C. for 2 minutes to inactivate the enzyme, and the remaining amount of oligonucleotide was determined by HPLC (LC-20AT, SPD-20A, CTO manufactured by SHIMADZU). -20A, CBM-20A).
  • compound 8 (exomethylene) obtained in Example 1.7, compound 11 ((R) -methyl) obtained in Example 2.3, or Example 3.5
  • the oligonucleotide synthesized using the compound 16 ((S) -methyl derivative) obtained in (1) as an amidite block has a residual amount of unreacted oligonucleotide as compared with those using natural thymidine or LNA. It can be seen that the result was extremely high. Further, even when compared with the oligonucleotides using ENA, the oligonucleotides using the exomethylene, (R) -methyl, or (S) -methyl are not left unreacted with time. It can be seen that the amount was not reduced, and had better enzyme resistance.
  • Table 19 shows the physical property data of the obtained compound 17.
  • 1,8-diazabicycloundecene (62 ⁇ L, 0.416 mmol) was added dropwise to an anhydrous tetrahydrofuran solution (2.0 mL) of the obtained crude product under a nitrogen stream, and iodine (43 0.7 mg, 0.166 mmol) was added and stirred at room temperature for 2 hours.
  • the reaction solution was quenched by adding a saturated aqueous sodium thiosulfate solution at 0 ° C., and extracted with ethyl acetate. The organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (70.0 mg).
  • N, N-diisopropylethylamine (0.23 mL, 1.28 mmol) was added to an anhydrous dichloromethane solution (5.0 mL) of the compound 19 (251 mg, 0.428 mmol) obtained above, 2-Cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.11 mL, 0.513 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
  • the organic layer was washed once with water and once with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (3.49 g).
  • Table 27 shows the physical property data of the obtained compound 25.
  • N, N-diisopropylethylamine (0.61 mL, 3.42 mmol) was added to a solution of compound 25 (700 mg, 1.14 mmol) obtained in Example 10.5 in anhydrous dichloromethane (10 mL).
  • 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.31 mL, 1.37 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
  • Table 33 shows the physical property data of the obtained compound 31.
  • N, N-diisopropylethylamine (0.35 mL, 1.96 mmol) was added to a solution of compound 31 (400 mg, 0.653 mmol) obtained in Example 11.5 in anhydrous dichloromethane (10 mL).
  • 2-cyanoethyl-N, N-diisopropylchlorophosphoramidide (0.17 mL, 0.783 mmol) was added dropwise at 0 ° C., and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was quenched by adding saturated aqueous sodium bicarbonate at 0 ° C., and extracted with dichloromethane.
  • Example 12 Synthesis and purification of oligonucleotide (3) Compound 9.4 (unsubstituted product), Compound 26 ((R) -methyl-exomethylene compound), and Compound 32 ((S)) obtained in Example 9.4, Example 10.6 and Example 11.6 -Methyl-exomethylene) were used as amidite blocks, respectively, to prepare 0.1M anhydrous acetonitrile solution of these with d m C (Ac) and T phosphoramidites (both from Sigma-Aldrich).
  • Each of the oligonucleotides shown in Table 35 was synthesized according to a phosphoramidite method known in the art using an nS-8 Oligonucleotides Synthesizer (an oligonucleotide synthesizer manufactured by Gene Design Co., Ltd.) (where Q is This corresponds to the structure derived from compound 20 (unsubstituted product) obtained in Example 9.4, and R Corresponds to the structure derived from compound 26 ((R) -methyl-exomethylene) obtained in Example 10.6. S represents compound 32 ((S) -methyl obtained in Example 11.6. -Corresponds to a structure derived from an exomethylene).
  • the synthesis scale in the synthesis was 0.2 ⁇ mol, and was performed under trityl-on conditions.
  • As the activator 5- [3,5-bis (trifluoromethyl) phenyl] -1H-tetrazole (0.25 M anhydrous acetonitrile solution, Activator 42, Proligo (registered trademark)) was used.
  • the condensation time was 10 minutes for the amidite blocks Q, R, and S obtained in Example 9.4, Example 10.6, and Example 11.6, and 32 seconds for the natural amidite block.
  • the mixture was treated with 28% aqueous ammonia at room temperature for 1.5 hours to cut out from the column carrier and to deprotect the base part and the phosphoric diester part. Subsequently, purification was performed by a simple gel filtration column (Sep-Pak (registered trademark) Plus C18 Environmental Cartridges manufactured by Waters), and further purified by reverse phase HPLC.
  • Tm melting temperature
  • the final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 4 ⁇ M of the oligonucleotide obtained in Example 12, and a sample solution containing 4 ⁇ M of single-stranded RNA or single-stranded DNA shown in Table 35. (130 ⁇ L) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C. using SHIMADZU UV-1650PC and SHIMADZU UV-1800 spectrometers (manufactured by Shimadzu Corporation) and plotted did. All Tm values were calculated by the midline method and used as the average of three independent measurement results.
  • Table 35 shows the results when single-stranded RNA was used as the target strand and the results when single-stranded DNA was used as the target strand.
  • Example 14 Measurement of melting temperature ( Tm ) (4) (Evaluation of triple chain forming ability) The final concentration was 10 mM sodium cacodylate buffer (pH 7.2), 140 mM potassium chloride, 5 mM magnesium chloride, 1.5 ⁇ M oligonucleotide obtained in Example 12, and 1.5 ⁇ M hairpin double-stranded DNA shown in Table 36.
  • the sample solution containing (130 ⁇ L) was bathed in boiling water and cooled slowly to room temperature, and then each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 90 ° C. at a rate of 0.5 ° C.
  • Table 36 shows the results when double-stranded DNA was used as the target strand.
  • Example 15 Oligonucleotide Synthesis and Purification (4)
  • Compound 8 (Exomethylene) obtained in Example 1.7, Compound 11 ((R) -methyl) obtained in Example 2.3, Compound 16 obtained in Example 3.5 (( S) -methyl form), compound 20 obtained in Example 9.4 (unsubstituted product), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene form), Example 11
  • compound 32 ((S) -methyl-exomethylene), thymidine (natural product), and ENA obtained in .6 were used as amidite blocks, respectively.
  • the following oligonucleotides were synthesized according to a known phosphoramidite method.
  • Example 16 Enzyme Resistance Experiment (2) The final concentrations were Tris-HCl buffer (pH 8.0) 50 mM, magnesium chloride 10 mM, each oligonucleotide 7.5 ⁇ M obtained in Example 15, 3′-exonuclease (Crotalus Admanteus Venophophophosterase: CAVP, Pharmacia Biotech) (Product) 2.5 ⁇ g / mL sample solution (100 ⁇ L) was kept at 37 ° C. for reaction. A part of the reaction solution (20 ⁇ L) was collected over time, heated at 90 ° C. for 2 minutes to inactivate the enzyme, and the remaining amount of oligonucleotide was determined by HPLC (LC-20AT, SPD-20A, CTO manufactured by SHIMADZU). -20A, CBM-20A).
  • Example 2 As shown in FIG. 2, in a system in which the concentration of 3′-exonuclease was higher than that in the experimental system described in Example 8, compound 8 (exomethylene) obtained in Example 1.7, Example 2 was obtained. Compound 11 ((R) -methyl compound) obtained in .3, Compound 16 ((S) -methyl compound) obtained in Example 3.5, and Compound 20 obtained in Example 9.4 (none) Substituent), compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene), or compound 32 obtained in Example 11.6 ((S) -methyl-exomethylene) It can be seen that the oligonucleotide synthesized using amidite block showed a significantly higher residual amount of unreacted oligonucleotide compared to natural thymidine and ENA whose residual amount decreased over time.
  • the remaining amount of the unreacted oligonucleotide of the oligonucleotide using (R) -methyl or (R) -methyl-exomethylene is less decreased with time. It can be seen that the enzyme had better enzyme resistance.
  • Example 17 Oligonucleotide Synthesis and Purification (5)
  • one of the following artificial nucleic acids was incorporated into an oligonucleotide composed of four types of bases (A, T, G and C).
  • Compound 8 Exomethylene
  • Compound 11 ((R) -methyl) obtained in Example 2.3
  • Compound 16 obtained in Example 3.5
  • compound 20 obtained in Example 9.4 (unsubstituted product)
  • compound 26 obtained in Example 10.6 ((R) -methyl-exomethylene form)
  • Example 11 6 except that Compound 32 ((S) -methyl-exomethylene), thymidine (natural product), LNA and ENA obtained in .6 were used as amidite blocks, respectively.
  • oligonucleotides were synthesized according to the known phosphoramidite method.
  • sequence of the oligonucleotide is 5′-d (TTCAGCATTGGTATTC) -3 ′ (SEQ ID NO: 5).
  • Example 18 Measurement of melting temperature (Tm) (4)
  • each oligonucleotide obtained in Example 17 was double-stranded with a single-stranded oligo RNA of 5′-r (GAAUACCAAUGCUGAA) -3 ′ (SEQ ID NO: 6) as a target strand, and the RNA The melting temperature was measured.
  • the final concentration is 10 mM sodium phosphate buffer (pH 7.2), 100 mM sodium chloride, 4 ⁇ M of the oligonucleotide obtained in Example 17, and the sample solution (130 ⁇ L) containing the single-stranded oligo RNA 4 ⁇ M is bathed in boiling water. Then, after slowly cooling to room temperature, each sample solution was cooled to 15 ° C. and measurement of the melting temperature (T m ) was started. The temperature was raised to 95 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured at intervals of 0.5 ° C.
  • any artificial oligonucleic acid can have sufficient binding ability to the target single-stranded RNA. Indicated.
  • Example 19 Antisense evaluation in cell culture system Huh-7 (human hepatoma cell line: PS20) prepared to 4.51 ⁇ 10 5 cells / mL was seeded in a 96-well flat bottom microplate (IWAKI) and incubated at 37 ° C. For 24 hours under 5% CO 2 .
  • Lipofectamine 3000 manufactured by Life Technologies
  • Opti-MEM manufactured by Life Technologies were further added and mixed so that each antisense nucleic acid molecule had a final concentration of 100 nM or 400 nM and allowed to stand at room temperature for 15 minutes. Later, it was added to each well. Each oligonucleotide obtained in Example 17 was added, and cells were collected 24 hours later.
  • CDNA was synthesized from the collected total RNA using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.). After the obtained cDNA was appropriately diluted, real-time PCR was performed to quantify the amount of ApoB mRNA. In real-time PCR, the amount of GAPDH mRNA of the housekeeping gene was also quantified. Thus, the amount of ApoB mRNA relative to the amount of GAPDH mRNA was quantitatively evaluated. For real-time PCR, Fast SYBR (registered trademark) Green Master Mix (Applied Biosystems) was used.
  • Hs_ApoB_Fw GGCTCACCCTGAGAGAAGTG (SEQ ID NO: 8)
  • Hs_ApoB_Rv GCTGCTTTCTGGGAACCTCAC (SEQ ID NO: 8)
  • Hs_GAPDH_Fw GGCCTCCCAAGGAGTAAGACC (SEQ ID NO: 9)
  • Hs_GAPDH_Rv AGGGTCTCATAGGGCACTG (SEQ ID NO: 10)
  • EoDNA structure is an oligonucleotide comprising a structure derived from compound 20 (unsubstituted product) obtained in Example 9.4 among the oligonucleotides synthesized in Example 17;
  • MeEoDNA structure is an oligonucleotide comprising a structure derived from compound 11 ((R) -methyl) obtained in Example 2.3 among the oligonucleotides synthesized in Example 17;
  • S-MeEoDNA structure Is an oligonucleotide containing a structure derived from the compound 32 ((S) -methyl-exomethylene form) obtained in Example 11.6 among the oligonucleotides synthesized in Example 17;
  • Methylene EoDNA structure "Is derived from compound 8 (exomethylene) obtained in Example 1.7 among the oligonucleotides synthesized in Example 17.
  • R-Me-methylene structure is the same as the compound 26 ((R) -methyl-exomethylene form) obtained in Example 10.6 among the oligonucleotides synthesized in Example 17.
  • novel 2 ', 4'-bridged 6-membered ring nucleosides and nucleotides having a hetero atom at the 6'-position are provided.
  • This oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide has a binding affinity for single-stranded RNA and double-stranded DNA comparable to conventional oligonucleotide containing 2 ′, 4′-bridged artificial nucleotide.
  • the oligonucleotide of the present invention is useful as a material for nucleic acid medicine, for example.

Abstract

La présente invention concerne un nucléoside et un nucléotide de réticulation. Le nucléoside comporte une structure de réticulation en 2',4', et est représenté par la formule (I) ou (I'). Un oligonucléotide contenant le 2',4'-nucléotide artificiel de réticulation présente une affinité de liaison à un ARN monocaténaire comparable à celle d'oligonucléotides contenant des 2',4'-nucléotides artificiels de réticulation traditionnels. Un oligonucléotide selon la présente invention est utilisé, par exemple, en tant que matériau-médicament à base d'acide nucléique.
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WO2018007475A1 (fr) 2016-07-05 2018-01-11 Biomarin Technologies B.V. Oligonucléotides de commutation ou de modulation d'épissage de pré-arnm comprenant des fragments d'échafaudage bicycliques, présentant des caractéristiques améliorées pour le traitement des troubles d'origine génétique
WO2018091544A1 (fr) 2016-11-16 2018-05-24 Biomarin Pharmaceutical, Inc. Substances pour le ciblage de divers organes ou tissus sélectionnés
WO2018155451A1 (fr) 2017-02-21 2018-08-30 国立大学法人大阪大学 Composé d'acide nucléique et oligonucléotide
WO2018155450A1 (fr) 2017-02-21 2018-08-30 国立大学法人大阪大学 Acide oligonucléique anti-sens
WO2019009298A1 (fr) * 2017-07-05 2019-01-10 国立大学法人大阪大学 INHIBITEUR DE L'EXPRESSION DE L'α-SYNUCLÉINE
JP2019525916A (ja) * 2016-07-27 2019-09-12 ロシュ イノベーション センター コペンハーゲン エーエス ５’ｓ−ｌｎａヌクレオチドおよびオリゴヌクレオチド
WO2020050307A1 (fr) 2018-09-05 2020-03-12 国立大学法人大阪大学 Oligonucléotide antisens ciblant une molécule d'arl4c, et médicament à base d'acide nucléique utilisant un oligonucléotide antisens
WO2020089325A1 (fr) 2018-11-02 2020-05-07 Biomarin Technologies B.V. Oligonucléotides antisens bispécifiques pour le saut d'exon de la dystrophine
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
WO2021256297A1 (fr) 2020-06-15 2021-12-23 リードファーマ株式会社 Nucléoside et nucléotide pontés
WO2022069511A1 (fr) 2020-09-30 2022-04-07 Biomarin Technologies B.V. Oligonucléotide anti-sens ciblant l'exon 51 du gène de la dystrophine
WO2023192904A1 (fr) 2022-03-30 2023-10-05 Biomarin Pharmaceutical Inc. Oligonucléotides de saut d'exon de dystrophine

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WO2021256297A1 (fr) 2020-06-15 2021-12-23 リードファーマ株式会社 Nucléoside et nucléotide pontés
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