WO2024106539A1 - Ligand conjugate substance, nucleic acid containing same, and use thereof - Google Patents

Ligand conjugate substance, nucleic acid containing same, and use thereof Download PDF

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WO2024106539A1
WO2024106539A1 PCT/JP2023/041480 JP2023041480W WO2024106539A1 WO 2024106539 A1 WO2024106539 A1 WO 2024106539A1 JP 2023041480 W JP2023041480 W JP 2023041480W WO 2024106539 A1 WO2024106539 A1 WO 2024106539A1
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optionally substituted
hydrogen atom
alkyl group
substituted alkyl
group
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PCT/JP2023/041480
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French (fr)
Japanese (ja)
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忠明 大木
友亮 阿部
智佐登 江村
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株式会社ボナック
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to novel compounds having an iminodiacetic acid group, and in particular to the field of therapeutic delivery having ligand conjugates via iminodiacetic acid derivatives.
  • pharmaceutical compositions comprising these conjugates advantageous for in vivo delivery, as well as pharmaceutical compositions suitable for in vivo therapeutic use, are provided.
  • the present invention relates to methods of using these compositions to introduce pharmaceutical compositions into cells for application in the treatment of various disease conditions.
  • Nucleic acid therapeutics are required to have serum stability, delivery to appropriate organs or cells, and transmembrane delivery.
  • protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, calcium phosphate-mediated transformation, etc. are utilized.
  • electroporation direct injection, cell fusion, viral vectors, calcium phosphate-mediated transformation, etc. are utilized.
  • most of these techniques are limited in the types of cells that can be transmembrane transported and the conditions required to achieve such transport.
  • the aim is to suppress disease-related proteins expressed in the liver, and intracellular transport via the asialoglycoprotein receptor (ASGPR) on the surface of the liver membrane using N-acetylgalactosamine (GalNAc) as a ligand to be conjugated thereto has become popular. Furthermore, with the aim of transporting the drug to organs other than the liver, development has begun on intracellular uptake via fatty acid receptors using lipids as ligands.
  • ASGPR asialoglycoprotein receptor
  • GalNAc N-acetylgalactosamine
  • the object of the present invention is to provide a therapeutic and/or prophylactic agent that is serum stable, delivers to the appropriate organ or cell, and enables transmembrane delivery, and further provides high functionality at low conjugate production costs.
  • a ligand-conjugated nucleic acid made of an iminodicarboxylic acid derivative enables serum stability, delivery to appropriate organs or cells, and transmembrane delivery, and thus completed the present invention.
  • A is a nucleic acid molecule
  • X represents -(CR 1 R 2 )n-R 3 - (wherein R 1 and R 2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group
  • R 3 represents -NR 4 - (wherein R 4 is a hydrogen atom or an optionally substituted alkyl group) or -S-heterocycle-CR 5 R 6 -NR 7 - (wherein R 5 , R 6 and R 7 are each independently a hydrogen atom or an optionally substituted alkyl group); and
  • n represents an integer of 1 to 10
  • B 1 , B 2 , B 3 and B 4 each independently represent a hydrogen atom or any of the following formulae: -( W2 ) n2 -CO-NH- X1 - Y1 , -( W3 ) n3 -CO-NH- X2 - Y2 , -(
  • nucleic acid molecule of A is an antisense nucleic acid for a target gene.
  • nucleic acid molecule of A is a single-stranded nucleic acid molecule capable of forming a duplex in which a guide strand for a target gene and a passenger strand complementary thereto are linked via a linker.
  • the nucleic acid molecule of A is represented by the following general formula (a):
  • X, Y, X 1 , Y 1 , X 2 , and Y 2 are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue;
  • Z is a linker connecting the 2'- or 5'-position of the sugar moiety of (X) to the 2'- or 3'-position of the sugar moiety of (Y), or a linker connecting the base moiety of (X) to the base moiety of (Y);
  • Sequence T is a nucleotide sequence containing an expression control sequence (T) of a target gene
  • sequence Q is a nucleotide sequence containing a sequence (Q) complementary to the expression control sequence T;
  • m1 and m2 are each independently an integer from 0 to 5; and
  • n1 and n2 are each independently an integer from 0 to 5.
  • Z is an amino protecting group
  • B 1 , B 2 , B 3 and B 4 are the same or different and each represents a hydrogen atom or any one of the following formulae: -( W2 ) n2 -CO-NH- X1 - Y1 , -( W3 ) n3 -CO-NH- X2 - Y2 , -( W5 ) n5 -CO-NH- X3 - Y3 , -( W6 ) n6 -CO-NH- X4 - Y4 ,
  • W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
  • W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally
  • Spacers X 1 , X 2 , X 3 and X 4 each independently represent -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); and n' represents an integer from 1 to 10).
  • the ligand-conjugated nucleic acid consisting of the iminodicarboxylic acid derivative, which is the compound of the present invention is capable of serum stability, delivery to appropriate organs or cells, and transmembrane delivery, and can provide a therapeutic and/or prophylactic agent that provides high functionality at low production costs.
  • 1 is an RP-HPLC chart of the compound prepared in Example 1-1.
  • 1 is an RP-HPLC chart of the compound prepared in Example 1-2.
  • 1 is a RP-HPLC chart of the compound prepared in Example 1-3.
  • 1 is a RP-HPLC chart of the compound prepared in Example 1-4.
  • 1 is a RP-HPLC chart of the compounds prepared in Examples 1-5.
  • 1 is a RP-HPLC chart of the compounds prepared in Examples 1-6.
  • RP-HPLC charts of the compounds prepared in Examples 1-7 RP-HPLC charts of the compounds prepared in Examples 1-8.
  • 1 is an RP-HPLC chart of the compound prepared in Example 2-1.
  • 1 is an RP-HPLC chart of the compound prepared in Example 2-2. 1 is an RP-HPLC chart of the compound prepared in Example 2-3. 1 is an RP-HPLC chart of the compound prepared in Example 2-4. 1 is an RP-HPLC chart of the compound prepared in Example 3-1. 1 is an RP-HPLC chart of the compound prepared in Example 3-2. 1 is an RP-HPLC chart of the compound prepared in Example 3-3. 1 is an RP-HPLC chart of the compound prepared in Example 4-1. 1 is an RP-HPLC chart of the compound prepared in Example 4-2. This is a graph showing the results of comparing the blood Factor VII concentrations of mice that were intravenously administered HDO or GalNAc-modified HDO.
  • amino group protecting groups include amide-based groups such as acetyl, trichloroacetyl, trifluoroacetyl, benzoyl, and N-phthalimide, and carbamate-based groups such as 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and t-butoxycarbonyl.
  • hydroxyl-protecting group refers to a general hydroxyl-protecting group known to those skilled in the art that is introduced to prevent reactions of the hydroxyl group, such as the protecting groups described in Protective Groups in Organic Synthesis, published by John Wiley and Sons (1980). Specific examples include acyl-based protecting groups such as acetyl and benzoyl, alkyl-based protecting groups such as trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl and benzyl, and silyl-based protecting groups such as trimethylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.
  • acyl-based protecting groups such as acetyl and benzoyl
  • alkyl-based protecting groups such as trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl and benzyl
  • silyl-based protecting groups such as trimethyl
  • electron-withdrawing group refers to a group that is more likely to attract electrons from the bonding atom than a hydrogen atom, and specific examples include cyano, nitro, alkylsulfonyl (e.g., methylsulfonyl, ethylsulfonyl), halogen (fluorine atom, chlorine atom, bromine atom, or iodine atom), arylsulfonyl (e.g., phenylsulfonyl, naphthylsulfonyl), trihalomethyl (e.g., trichloromethyl, trifluoromethyl), etc.
  • alkylsulfonyl e.g., methylsulfonyl, ethylsulfonyl
  • halogen fluorine atom, chlorine atom, bromine atom, or iodine atom
  • arylsulfonyl e.g.,
  • Halogen includes fluorine, chlorine, bromine and iodine atoms.
  • heterocycle refers to a 5-7 membered ring that contains at least one of a nitrogen atom, an oxygen atom, and a sulfur atom, and is substituted or unsubstituted, saturated or unsaturated. These may be monocyclic or may form a condensed ring together with another aryl ring or heterocycle.
  • heterocycles include a furan ring, a dihydrofuran ring, a tetrahydrofuran ring, a pyran ring, a dihydropyran ring, a tetrahydropyran ring, a benzofuran ring, an isobenzofuran ring, a chromene ring, a chroman ring, an isochroman ring, a thiophene ring, a benzothiophene ring, a pyrrole ring, a pyrroline ring, a pyrrolidine ring, an imidazole ring, an imidazoline ring, an imidazolidine ring, a pyrazole ring, a pyrazoline ring, a pyrazolidine ring, a triazole ring, a tetrazole ring, a pyridine ring, a pyridine oxide ring, a piperidine ring,
  • alkyl group refers to a straight-chain or branched-chain alkyl group having 1 to 30 carbon atoms, preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 4 carbon atoms, and specific examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and icosyl.
  • Preferred examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl.
  • alkyl group having 1 to 10 carbon atoms examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like, and preferably include a C1-6 alkyl group (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl).
  • a C1-6 alkyl group e.g
  • alkenyl group (alkenyl) means a straight-chain or branched-chain alkenyl group having 2 to 30 carbon atoms, preferably 2 to 12, and more preferably 2 to 8 carbon atoms, and examples of the alkyl group include those having one or more double bonds. Specific examples include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, and 3-methyl-2-butenyl.
  • Alkynyl group (alkynyl) means a straight-chain or branched-chain alkynyl group having 2 to 30 carbon atoms, preferably 2 to 12, and more preferably 2 to 8 carbon atoms, and examples of the alkyl group include those having one or more triple bonds. Specific examples include ethynyl, propynyl, propargyl, butynyl, pentynyl, and hexynyl. The alkynyl group may further have one or more double bonds.
  • Alkoxy group means a straight-chain or branched-chain alkoxy group having 1 to 30 carbon atoms, preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 4 carbon atoms, and specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, neopentyloxy, 2-pentyloxy, 3-pentyloxy, n-hexyloxy, 2-hexyloxy, etc.
  • Aryl group refers to an aryl group having 6 to 24 carbon atoms, preferably 6 to 10 carbon atoms, and examples of such groups include monocyclic aromatic hydrocarbon groups such as phenyl, and polycyclic aromatic hydrocarbon groups such as 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, and 9-phenanthryl.
  • Aryl groups having 6 to 10 carbon atoms include the above aryl groups having 6 to 10 carbon atoms, and specific examples include phenyl, naphthyl, etc.
  • arylene group refers to a divalent substituent formed by removing one hydrogen from the aryl group exemplified above.
  • heterocycloalkyl group refers to a heterocycloalkyl group having 6 to 24 carbon atoms, preferably 6 to 10 carbon atoms.
  • heterocycloalkyl groups include those in which one or more carbon atoms forming the cyclic structure of the cycloalkyl group described below are substituted with a nitrogen atom, an oxygen atom, a sulfur atom, or the like.
  • Specific examples include [1,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.
  • heterocycloalkyl groups having 4 to 10 carbon atoms include the above heterocycloalkyl groups having 4 to 10 carbon atoms, specifically pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl, thioxetanyl, tetrahydrothienyl, and tetrahydrothiopyranyl groups.
  • heterocycloalkylene group refers to a divalent substituent formed by removing one hydrogen from the heterocycloalkyl groups listed above.
  • Alkyl group means an aralkyl group having 7 to 30 carbon atoms, preferably 7 to 11 carbon atoms, and specific examples include benzyl, 2-phenethyl, and naphthalenylmethyl.
  • Cycloalkyl group means a cycloalkyl group having 3 to 24 carbon atoms, preferably 3 to 15 carbon atoms, and specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bridged cyclic hydrocarbon groups, spiro hydrocarbon groups, etc., and preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bridged cyclic hydrocarbon groups, etc.
  • bridged cyclic hydrocarbon groups include bicyclo[2.1.0]pentyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, tricyclo[2.2.1.0]heptyl, bicyclo[3.3.1]nonyl, 1-adamantyl, 2-adamantyl, etc.
  • spiro hydrocarbon groups include spiro[3.4]octyl, etc.
  • cycloalkyl groups having 4 to 10 carbon atoms include the above cycloalkyl groups having 4 to 10 carbon atoms, such as cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Cycloalkylene group refers to a divalent substituent formed by removing one hydrogen from the cycloalkyl groups listed above.
  • Cycloalkenyl group means a cycloalkenyl group having 3 to 24 carbon atoms, preferably 3 to 7 carbon atoms, containing at least one, preferably one or two double bonds, and specifically includes cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, etc.
  • the cycloalkenyl group also includes bridged cyclic hydrocarbon groups and spiro hydrocarbon groups having an unsaturated bond in the ring.
  • bridged cyclic hydrocarbon groups having an unsaturated bond in the ring include bicyclo[2.2.2]octenyl, bicyclo[3.2.1]octenyl, tricyclo[2.2.1.0]heptenyl, etc.
  • spiro hydrocarbon groups having an unsaturated bond in the ring include spiro[3.4]octenyl, etc.
  • Cycloalkylalkyl group refers to an alkyl group (described above) substituted with the cycloalkyl group, preferably a cycloalkylalkyl group having 4 to 30 carbon atoms, more preferably 4 to 11 carbon atoms. Specific examples include cyclopropylmethyl, 2-cyclobutylethyl, cyclopentylmethyl, 3-cyclopentylpropyl, cyclohexylmethyl, 2-cyclohexylethyl, cycloheptylmethyl, etc.
  • Alkoxyalkyl group refers to an alkyl group (described above) substituted with the above alkoxy group, and preferably refers to a straight-chain or branched-chain alkoxyalkyl group having 2 to 30 carbon atoms, more preferably 2 to 12 carbon atoms. Specific examples include methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, and t-butoxymethyl.
  • alkylene group (alkylene chain) means a straight-chain or branched-chain alkylene group having 1 to 30 carbon atoms, preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 4 carbon atoms, and specific examples include methylene, ethylene, and propylene.
  • heteroaryl group includes, for example, a monocyclic aromatic heterocyclic group and a fused aromatic heterocyclic group.
  • the heteroaryl includes, for example, furyl (e.g., 2-furyl, 3-furyl), thienyl (e.g., 2-thienyl, 3-thienyl), pyrrolyl (e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (e.g., 1-imidazolyl, 2-imidazolyl, 4-imidazolyl), pyrazolyl (e.g., 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl), triazolyl (e.g., 1,2,4-triazol-1-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-4-yl), tetrazolyl (e.g., 1-tetrazolyl, 2-tetrazolyl, 5-tetrazol
  • heteroaryl group having 2 to 10 carbon atoms examples include the above-mentioned heteroaryl groups having 2 to 10 carbon atoms, and specific examples thereof include a furyl group, a thienyl group, a pyrrolyl group, an oxazolyl group, a triazolyl group, a pyridyl group, and a quinolinyl group.
  • heteroarylene group refers to a divalent substituent formed by removing one hydrogen from the heteroaryl group exemplified above.
  • Substituent Group A (1) a halogen (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom); (2) an alkyl group (described above); (3) an alkoxy group (described above); (4) an alkenyl group (described above); (5) an alkynyl group (described above); (6) haloalkyl groups (e.g., chloromethyl, fluoromethyl, dichloromethyl, difluoromethyl, dichlorofluoromethyl, trifluoromethyl, pentafluoroethyl, etc.); (7) an aryl group (described above); (8) heteroaryl groups (described above); (9) an aralkyl group (described above); (10) a cycloalkyl
  • the present invention provides a compound represented by the following general formula (I):
  • the compound is a compound in which a nucleic acid molecule and a ligand are linked via a group consisting of an iminodicarboxylic acid derivative, and is also referred to as a ligand -conjugated nucleic acid in this specification.
  • A is a nucleic acid molecule
  • X represents -(CR 1 R 2 )n-R 3 - (wherein R 1 and R 2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group
  • R 3 represents -NR 4 - (wherein R 4 is a hydrogen atom or an optionally substituted alkyl group) or -S-heterocycle-CR 5 R 6 -NR 7 - (wherein R 5 , R 6 and R 7 are each independently a hydrogen atom or an optionally substituted alkyl group); and
  • n represents an integer of 1 to 10
  • B 1 , B 2 , B 3 and B 4 each independently represent a hydrogen atom or any of the following formulae: -( W2 ) n2 -CO-NH- X1 - Y1 , -( W3 ) n3 -CO-NH- X2 - Y2 , -(
  • the ligand-conjugated nucleic acid of the present invention represented by formula (I) is preferably a compound represented by the following formula (I-1):
  • X is preferably -(CH 2 ) 6 -NH- or -(CH 2 ) 6 -S-heterocycle-(CH 2 ) 2 -NH-.
  • X 0 is preferably a spacer represented by any one of the following formulas (1) to (4), preferably a spacer represented by the following formula (1), more preferably the spacer of X 0 is represented by -CO-(CH 2 )n L1 -CO- (wherein n L1 represents an integer of 1 to 10). n L1 is preferably 6.
  • L1 is (i) -(CR 1L1 R 2L1 ) n L1 (wherein R 1L1 and R 2L1 are the same or different and each represents a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; n L1 represents an integer of 1 to 10), (ii) -(CH 2 ) n L1 -Cy-(CH 2 ) m L1 - (wherein Cy represents an optionally substituted arylene group, an optionally substituted heteroarylene group, an optionally substituted cycloalkylene group, or an optionally substituted heterocycloalkylene group; n L1 and m L1 may be the same or different and represent an integer of 1 to 10), (iii) -NR 8 - (wherein R 8 represents a hydrogen atom or an optionally substituted alkyl group), (2) -L2 -CO- L3 -CO- (wherein L2
  • the spacers X 1 to X 4 are each independently -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); n' represents an integer of 1 to 10), and more preferably, they are each independently -(CH 2 ) 6 -O- or -(CH 2 ) 5 -NH-.
  • W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are —(CH 2 )—, and n 1 to n 6 are 1.
  • the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a sugar (preferably GalNAc) or a lipid.
  • the nucleic acid molecule represented by the symbol A contains a sequence that controls the expression of a gene (hereinafter also referred to as a target gene) whose expression is desired to be controlled (e.g., inhibited), and that is complementary to a specific site in the mRNA of the target gene (hereinafter also referred to as an expression control sequence).
  • a target gene whose expression is desired to be controlled (e.g., inhibited)
  • an expression control sequence a specific site in the mRNA of the target gene
  • the specific site is appropriately selected depending on the target gene, and may be a known sequence or a newly identified sequence.
  • sequences include expression inhibition sequences such as HPRT1 (Hypoxanthine Phosphoribosyltransferase 1), Factor VII (coagulation factor VII), NEK6 (NIMA related kinase 6), and TTR (transthyretin), which are used in the examples described below.
  • HPRT1 Hydropoxanthine Phosphoribosyltransferase 1
  • Factor VII coagulation factor VII
  • NEK6 NIMA related kinase 6
  • TTR transthyretin
  • the expression control sequence is a sequence complementary to the nucleotide sequence of a specific site of the target gene.
  • the "complementary sequence” may be not only a sequence that is completely complementary to the target sequence (i.e., hybridizes without mismatches), but also a sequence that contains a mismatch of one to several nucleotides, preferably one or two nucleotides, so long as it can specifically hybridize to the target sequence under physiological conditions of mammalian cells.
  • it may be a sequence that has an identity of 90% or more, preferably 95% or more, 97% or more, 98% or more, or 99% or more to the complementary strand sequence of the target nucleotide sequence in the target sequence.
  • a "complementary nucleotide sequence” is a nucleotide sequence that hybridizes with a target sequence under stringent conditions.
  • stringent conditions include, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, such as hybridization at 6x SSC (sodium chloride/sodium citrate)/45°C, followed by one or more washes at 0.2x SSC/0.1% SDS/50-65°C, but a person skilled in the art can appropriately select hybridization conditions that provide equivalent stringency.
  • the expression control sequence may be complementary to the entire target sequence or to a portion of the target sequence, but is usually preferably complementary to a sequence of 15 or more consecutive nucleotides in each target sequence.
  • the length of the nucleotide sequence targeted by the expression control sequence is a continuous partial nucleotide sequence of the target gene of, for example, 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less, and even more preferably 25 nucleotides or less. Therefore, the length of the nucleotide sequence targeted by the expression control sequence may be a partial nucleotide sequence of preferably 15 to 30 consecutive nucleotides, more preferably 15 to 25 consecutive nucleotides.
  • the nucleic acid molecule of the present invention may be RNA, DNA, or a DNA/RNA chimera, so long as it is capable of controlling the expression of a target gene.
  • the nucleic acid molecule of the present invention may be a double-stranded nucleic acid or a single-stranded nucleic acid, so long as it is capable of controlling the expression of a target gene.
  • a double-stranded nucleic acid it may be any of double-stranded DNA, double-stranded RNA, a DNA:RNA hybrid, and a hybrid of a DNA/RNA chimera with DNA, RNA, or a DNA/RNA chimera.
  • the nucleic acid molecule of the present invention when it is a single-stranded nucleic acid, it may have only a guide strand for the target gene, or the guide strand and passenger strand may be linked via an optional linker, and a sequence that suppresses gene expression within the molecule may hybridize with a complementary sequence to form a double strand.
  • examples of the constituent units of a nucleic acid molecule include ribonucleotide residues and deoxyribonucleotide residues. These nucleotide residues may be, for example, modified or unmodified.
  • the nucleic acid molecule of the present invention may have improved nuclease resistance and improved stability by, for example, including modified nucleotide residues.
  • the nucleic acid molecule of the present invention may further include non-nucleotide residues in addition to the nucleotide residues.
  • the constituent units of the regions other than the linker are preferably nucleotide residues.
  • Each region is composed of, for example, the following residues (1) to (3).
  • Modified nucleotide residues (3) Unmodified and modified nucleotide residues
  • the nucleic acid molecule of the present invention may be labeled with, for example, a labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes, etc.
  • the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, etc.
  • the dye include Alexa dyes such as Alexa488, etc.
  • the isotope include stable isotopes and radioisotopes. Stable isotopes, for example, have low risk of exposure and do not require dedicated facilities, making them easy to handle and reducing costs.
  • stable isotopes for example, do not cause changes in the physical properties of the labeled compound, and have excellent properties as a tracer.
  • stable isotopes include 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S, and 36 S.
  • Nucleotide residues contain sugar, base and phosphate as building blocks. Ribonucleotide residues have a ribose residue as the sugar and adenine (A), guanine (G), cytosine (C) and uracil (U) (which can also be replaced by thymine (T)) as bases, while deoxyribonucleotide residues have a deoxyribose residue as the sugar and adenine (dA), guanine (dG), cytosine (dC) and thymine (dT) (which can also be replaced by uracil (dU)) as bases.
  • a modified nucleotide residue may be one in which any of the components of the nucleotide residue has been modified.
  • “modification” may be, for example, the substitution, addition and/or removal of the components, or the substitution, addition and/or removal of atoms and/or functional groups in the components.
  • a modified nucleotide residue may be, for example, a naturally occurring modified nucleotide residue or an artificially modified nucleotide residue. For examples of naturally occurring modified nucleotide residues, see Limbach et al. (1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196).
  • modifications of nucleotide residues include modifications of the ribose-phosphate backbone (hereinafter, ribophosphate backbone).
  • the ribose residue can be modified.
  • the ribose residue can be modified, for example, at the 2' carbon, and specifically, for example, the hydroxyl group bonded to the 2' carbon can be substituted with an atom or group selected from the group consisting of a hydrogen atom, a halogen atom such as fluorine, or an -O-alkyl group (e.g., -O-Me group), an -O-acyl group (e.g., -O-COMe group), and an amino group, preferably an atom or group selected from the group consisting of a hydrogen atom, a methoxy group, and a fluorine atom.
  • the ribose residue can be substituted with deoxyribose.
  • the ribose residue can be substituted with a stereoisomer, for example, with an arabinose residue.
  • the ribophosphate backbone may be replaced with a non-ribophosphate backbone having, for example, a non-ribose residue and/or a non-phosphate.
  • the non-ribophosphate backbone may be, for example, an uncharged ribophosphate backbone.
  • substitutes for the nucleotide substituted with the non-ribophosphate backbone include, for example, morpholino, cyclobutyl, pyrrolidine, etc.
  • Other examples of the substitute include, for example, artificial nucleic acid monomer residues. Specific examples include, for example, PNA (peptide nucleic acid), LNA (locked nucleic acid), ENA (2'-O, 4'-C-ethylene bridged nucleic acid), etc., with PNA being preferred.
  • the phosphate group can also be modified.
  • the phosphate group closest to the sugar residue is called the ⁇ -phosphate group.
  • the ⁇ -phosphate group is negatively charged, and the charge is evenly distributed over the two oxygen atoms that are not bonded to the sugar residue.
  • the two oxygen atoms that are not bonded to the sugar residue in the phosphodiester bond between nucleotide residues are also referred to as "non-linking oxygens" below.
  • linking oxygens the two oxygen atoms that are bonded to the sugar residue in the phosphodiester bond between nucleotide residues are referred to as "linking oxygens" below. It is preferable to modify the ⁇ -phosphate group, for example, so that it becomes uncharged, or so that the charge distribution in the non-linking oxygen becomes asymmetric.
  • the phosphate group may, for example, replace the non-bonded oxygen.
  • the non-bonded oxygen can be replaced with any of the following atoms: S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl or aryl group), and is preferably replaced with S. It is preferable that both non-bonded oxygens are replaced, and more preferably, both are replaced with S.
  • modified phosphate groups include phosphorothioates, phosphorodithioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters, and among these, phosphorodithioates in which both of the two non-bonded oxygens are replaced with S are preferred.
  • the phosphate group may, for example, replace the bonded oxygen.
  • the bonded oxygen may, for example, be replaced by any of the atoms S (sulfur), C (carbon) and N (nitrogen).
  • Examples of such modified phosphate groups include bridged phosphoramidates substituted with N, bridged phosphorothioates substituted with S, and bridged methylene phosphonates substituted with C.
  • the bonded oxygen is preferably replaced, for example, at least one of the 5'-terminal nucleotide residue and the 3'-terminal nucleotide residue of the nucleic acid molecule of the present invention. In the case of the 5'-side, replacement with C is preferred, and in the case of the 3'-side, replacement with N is preferred.
  • the phosphate group may be replaced with, for example, a phosphorus-free linker.
  • linkers include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methylenecarbonylamino, and methyleneoxymethylimino, and preferably, methylenecarbonylamino and methylenemethylimino.
  • the nucleic acid molecule of the present invention may be modified, for example, at least one of the 3'-end and 5'-end nucleotide residues.
  • the modification is as described above, and is preferably performed on the terminal phosphate group.
  • the entire phosphate group may be modified, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.
  • Modifications of the terminal nucleotide residue include, for example, the addition of other molecules.
  • other molecules include functional molecules such as labeling substances and protecting groups.
  • protecting groups include S (sulfur), Si (silicon), B (boron), and ester-containing groups.
  • the functional molecules such as labeling substances can be used, for example, for detecting the nucleic acid molecule of the present invention.
  • Other molecules may be added to the phosphate group of a nucleotide residue, or may be added to the phosphate group or sugar residue via a spacer.
  • the terminal atom of the spacer in the nucleic acid molecule of the present invention can be added to or substituted for, for example, the bonded oxygen of the phosphate group, or the O, N, S, or C of the sugar residue.
  • the binding site of the sugar residue is preferably, for example, the 3' C or 5' C, or an atom bonded to these.
  • the spacer can also be added to or substituted for, for example, the terminal atom of a nucleotide substitute such as the PNA.
  • the spacer in the nucleic acid molecule of the present invention is not particularly limited, and examples thereof include -( CH2 ) n- , -( CH2 ) nN- , -(CH2) nO- , - ( CH2 ) nS- , O( CH2CH2O ) nCH2CH2OH , abasic sugar, amide, carboxy , amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, and morpholino, as well as biotin reagent and fluorescein reagent, etc.
  • the molecules added to the ends may include, for example, dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl) Examples of suitable glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O3
  • the nucleic acid molecules of the present invention may be modified at the 5' end with, for example, a phosphate group or a phosphate group analog.
  • the phosphate group may be, for example, a 5' monophosphate ((HO) 2 (O)PO-5'), a 5' diphosphate ((HO) 2 (O)POP(HO)(O)-O-5'), a 5' triphosphate ((HO) 2 (O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-guanosine cap (7-methylated or unmethylated, 7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-adenosine cap (Appp), any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5' monothio
  • the base is not particularly limited.
  • the base may be a natural base or a non-natural base.
  • common bases and their modified analogs can be used.
  • bases examples include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil, and thymine.
  • pyrimidine bases such as cytosine, uracil, and thymine.
  • Other examples of the base include inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, and tubercidine.
  • bases examples include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-azocytosine, and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, and 5-aminopropyluracil.
  • Purines and pyrimidines also include those disclosed, for example, in U.S. Pat. No. 3,687,808; in Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, edited by Kroschwitz J.I., John Wiley & Sons, 1990; and in Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613.
  • modified nucleotide residues may include, for example, residues lacking a base, i.e., an abasic ribophosphate backbone.
  • Modified nucleotide residues may also include, for example, residues described in U.S. Provisional Application No. 60/465,665 (filed April 25, 2003) and International Application No. PCT/US04/07070 (filed March 8, 2004: International Publication WO2004/080406), and the present invention may incorporate these documents.
  • the method for synthesizing the nucleic acid molecule of the present invention is not particularly limited, and a conventionally known method can be used.
  • the synthesis method include synthesis methods using genetic engineering techniques and chemical synthesis methods.
  • the genetic engineering techniques include in vitro transcription synthesis methods, methods using vectors, and methods using PCR cassettes.
  • the vector is not particularly limited, and examples include non-viral vectors such as plasmids, and viral vectors.
  • the chemical synthesis method is not particularly limited, and examples include the phosphoramidite method and the H-phosphonate method.
  • a commercially available automated nucleic acid synthesizer can be used for the chemical synthesis method. In general, amidites are used for the chemical synthesis method.
  • amidites are not particularly limited, and examples of commercially available amidites include RNA Phosphoramidites (2'-O-TBDMSi, product name, Sanzenri Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, and TEM amidite.
  • nucleic acid molecule of the present invention examples include siRNA and antisense nucleic acids against target genes.
  • examples of the nucleic acid molecule of the present invention include single-stranded nucleic acid molecules that can form a duplex in which a guide strand and a complementary passenger strand are linked via a linker.
  • siRNA for a target gene is a double-stranded oligoRNA consisting of a guide strand containing a sequence complementary to all or part of a target sequence of 25 or less consecutive nucleotides in the nucleotide sequence of the target gene, and a passenger strand containing a sequence complementary thereto, which is incorporated into the RISC complex, and the sequence complementary to the mRNA encoded by the target gene in the guide strand forms a double strand with the target sequence in the mRNA, thereby cleaving the mRNA and suppressing gene expression.
  • the "complementary sequence" has the same meaning as above.
  • the length of the siRNA is not particularly limited as long as the guide strand contains a sequence complementary to all or part of a target sequence of 25 or less consecutive nucleotides in the nucleotide sequence of the target gene and can suppress gene expression, but the nucleotide sequence targeted by the siRNA can be, in principle, 15 to 50 nucleotides, preferably 19 to 30 nucleotides, more preferably 19 to 27 nucleotides, and particularly preferably 19 to 21 nucleotides.
  • the guide strand and passenger strand may also have additional nucleotides at the 5' or 3' end. The length of the additional nucleotide is usually about 2 to 4 nucleotides, and the total length of the siRNA is 19 nucleotides or more.
  • the additional nucleotide may be either DNA or RNA, but using DNA may improve the stability of the nucleic acid in some cases.
  • additional nucleotide sequences include, but are not limited to, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', and uuuuu-3'.
  • the siRNA may have a 3'-overhang on one or both strands.
  • the length of the overhang is not particularly limited, and the lower limit is, for example, 1 base length, the upper limit is, for example, 4 base length or 3 base length, and the range is, for example, 1 to 4 base length, 1 to 3 base length, or 1 to 2 base length.
  • the sequence of the overhang is not particularly limited and may be any of A, U, G, C, and T.
  • Examples of the overhang sequence from the 3' side include TT, UU, CU, GC, UA, AA, CC, UG, CG, and AU.
  • TT or UU resistance to RNase can be imparted.
  • the method for synthesizing siRNA is not particularly limited, and a conventional method for producing nucleic acids can be used.
  • the synthesis method include a method in which a nucleic acid containing the complementary sequence and a nucleic acid having a complementary sequence thereto are each synthesized using an automatic DNA/RNA synthesizer, denatured in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then annealed at about 30 to about 70°C for about 1 to about 8 hours.
  • siRNA can be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it using dicer.
  • the nucleotide residues constituting siRNA may also be modified in the same manner as above to improve stability, specific activity, etc.
  • An antisense nucleic acid for a target gene refers to a nucleic acid that contains a sequence complementary to all or part of a target sequence of 25 or less consecutive nucleotides in the nucleotide sequence of a target gene, preferably a nucleotide sequence of 15 or more consecutive nucleotides in the nucleotide sequence, and has the effect of suppressing gene expression by binding to the target sequence in the nucleotide sequence of the target gene by forming a specific double strand.
  • the term "complementary sequence" has the same meaning as above.
  • the length of the antisense nucleic acid is not particularly limited, but may be, for example, 15 to 40 nucleotides, and preferably 15 to 30 nucleotides.
  • the antisense nucleic acid may be of the gapmer type.
  • a gapmer type antisense nucleic acid is a nucleic acid that has DNA and nucleic acid with modifications or crosslinks introduced on both sides of the DNA.
  • the DNA strand serves as the backbone, and RNA complementary to the backbone forms a heteroduplex nucleic acid, and the RNA is degraded by RNAase H. O-methylation of the 2' position of the sugar moiety improves the stability of the antisense nucleic acid and increases the binding affinity to the target.
  • the nuclease resistance of the antisense nucleic acid is increased by replacing the phosphate bond with a phosphorothioate bond.
  • the method for synthesizing antisense nucleic acids is not particularly limited, and any conventionally known method for producing nucleic acids can be used.
  • Examples of the synthesis method include a method in which nucleic acids containing the complementary sequences are prepared by synthesizing them using an automatic DNA/RNA synthesizer.
  • Antisense nucleic acids containing the various modifications described above can also be chemically synthesized using conventionally known methods.
  • single-stranded nucleic acid molecule for a target gene refers to a nucleic acid molecule that suppresses the expression of a target gene, in which a guide strand sequence T including a sequence Ta that is complementary to all or part of a target sequence of 25 or less consecutive nucleotides in a region represented by a specific sequence in the target gene and a passenger strand sequence Q including a sequence Qa that is complementary to Ta are linked in the order of T-L-Q in the 5' to 3' direction or the 3' to 5' direction via a linker L, and in an orientation in which the sequence Ta and the sequence Qa can form a double strand within the molecule.
  • the "complementary sequence” has the same meaning as above.
  • the sequence Ta is not particularly limited as long as it contains a sequence complementary to all or part of a target sequence of 25 consecutive nucleotides or less in a region represented by a specific sequence of the target gene, but the nucleotide sequence targeted by Ta can be, in principle, 15 to 49 nucleotides, preferably 19 to 30 nucleotides, more preferably 19 to 27 nucleotides, and particularly preferably 19 to 21 nucleotides.
  • the single-stranded nucleic acid molecule for a target gene is a nucleic acid molecule that includes a guide strand sequence T that includes a sequence Ta, and a passenger strand sequence Q that includes a sequence Qa that is complementary to the sequence Ta.
  • the guide strand sequence T may, for example, consist of only the sequence Ta, or may further have an additional sequence Tb. In the latter case, the additional sequence Tb does not need to be complementary to the nucleotide sequence of the target gene.
  • the additional sequence Tb may be added to either the 5' end or the 3' end of Ta, or may be added to both ends (Tb and Tb'). It is preferably added to the end of Ta that is linked to the linker L.
  • the length of the sequence Tb (Tb') is, for example, 1 to 35 nucleotides, preferably 1 to 25 nucleotides, more preferably 1 to 11 nucleotides, and particularly preferably 1, 2, 3, 4, 5, or 6 nucleotides.
  • the passenger strand sequence Q is not particularly limited as long as it contains a sequence Qa complementary to the sequence Ta, and may, for example, consist of only the sequence Qa, or may further have an additional sequence Qb.
  • the additional sequence Qb does not need to be complementary to the additional sequence Tb, but it is preferable that they are complementary, and in particular, when the additional sequences Tb and Qb are added to the ends of Ta and Qa that are linked to the linker L, respectively, it is more preferable that Tb and Qb are complementary.
  • the additional sequence Qb may be added to either the 5' end or the 3' end of Qa, or may be added to both ends (Qb and Qb'). It is preferably added to the end of Qa that is linked to the linker L.
  • the length of the sequence Yb (Yb') is, for example, 1 to 35 nucleotides, preferably 1 to 25 nucleotides, more preferably 1 to 11 nucleotides, and particularly preferably 1, 2, 3, 4, 5, or 6 nucleotides.
  • the guide strand sequence T and the passenger strand sequence Q may further have an overhang at the end not linked to the linker L.
  • the overhang is preferably added to the 3' end of the guide strand sequence or the passenger strand sequence whose 5' end is linked to the linker L.
  • the length of the overhang is not particularly limited, and the lower limit is, for example, 1 nucleotide, the upper limit is, for example, 4 nucleotides or 3 nucleotides, and the range is, for example, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides.
  • the sequence of the overhang is not particularly limited and may be any of A, U, G, C, and T.
  • Examples of the overhang sequence from the 5' side include TT, UU, CU, GC, UA, AA, CC, UG, CG, and AU.
  • TT or UU resistance to RNase can be imparted.
  • a single-stranded nucleic acid molecule for a gene is formed by linking a nucleotide sequence T and a nucleotide sequence Q via a linker L in the 5' to 3' or 3' to 5' direction in the order T-L-Q, and in an orientation in which the sequence Ta and the sequence Qa can form a double strand within the molecule.
  • the linker L may be composed of, for example, nucleotide residues, non-nucleotide residues, or nucleotide residues and non-nucleotide residues. Examples of nucleotide residues include ribonucleotide residues and deoxyribonucleotide residues.
  • the linker L When the linker L is composed of nucleotide residues, the sense region and the antisense region form a stem structure within a single molecule by base pairing with each other, and at the same time, the nucleotide sequence of the linker L forms a loop structure, so that the molecule as a whole forms a hairpin-type stem-loop structure, and the single-stranded nucleic acid molecule for the target gene can also be called shRNA (small hairpin RNA or short hairpin RNA).
  • shRNA small hairpin RNA or short hairpin RNA
  • the length of the linker L is not particularly limited, but it is preferably a length that allows the sequence Ta and the sequence Qa to form a double strand within the molecule.
  • the number of bases in the linker L has a lower limit of, for example, 1 base, 2 bases, or 3 bases, and an upper limit of, for example, 100 bases, 80 bases, or 50 bases.
  • Specific examples of the number of bases in each linker region include, but are not limited to, 1 to 50 bases, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 1 to 7 bases, and 1 to 4 bases. It is preferable that the linker L has a structure that does not cause self-annealing.
  • the linker L composed of non-nucleotide residues or the linker L composed of nucleotide residues and non-nucleotide residues, is represented, for example, by the following formula (1).
  • R3 is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on ring A;
  • L 1 is an alkylene chain consisting of m L carbon atoms, wherein the hydrogen atoms on the alkylene carbon atoms may or may not be substituted with OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a , or L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom, with the proviso that when Y 1 is NH, O, or S, the atom of L 1 bonded to Y 1 is carbon, and the atom of L 1 bonded
  • Nucleotide sequence T and nucleotide sequence Q are each linked to the non-nucleotide structure via -OR 1 - or -OR 2 -, where R 1 and R 2 may be present or absent, and when present, R 1 and R 2 are each independently a nucleotide residue or a structure of formula (1).
  • Y 1 and Y 2 each independently represent a single bond, CH 2 , NH, O or S.
  • l in ring A is 1 or 2.
  • ring A is a 5-membered ring, for example a pyrrolidine skeleton.
  • pyrrolidine skeletons include proline skeletons and prolinol skeletons, and their divalent structures can be exemplified.
  • ring A is a 6-membered ring, for example a piperidine skeleton.
  • one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen, or sulfur.
  • Ring A may also contain a carbon-carbon double bond or a carbon-nitrogen double bond within ring A.
  • Ring A may be, for example, either L-type or D-type.
  • R 3 is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on ring A.
  • the substituent R 3 may be one or more, or may be absent, and when there are more than one, they may be the same or different.
  • R 4 and R 5 are, for example, each independently a substituent or a protective group, and may be the same or different.
  • the substituent may be any of those described in the above-mentioned substituent group A, such as halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl, heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl, etc.
  • the substituent R 3 may be any of the substituents listed above.
  • the protecting group is, for example, a functional group that converts a highly reactive functional group into an inactive one, and examples of the protecting group include known protecting groups.
  • the description in the literature J. F. W. McOmie, "Protecting Groups in Organic Chemistry” Prenum Press, London and New York, 1973) can be cited.
  • the protecting group is not particularly limited, and examples thereof include tert-butyldimethylsilyl group (TBDMS), bis(2-acetoxyethyloxy)methyl group (ACE), triisopropylsilyloxymethyl group (TOM), 1-(2-cyanoethoxy)ethyl group (CEE), 2-cyanoethoxymethyl group (CEM), tolylsulfonylethoxymethyl group (TEM), dimethoxytrityl group (DMTr), and the like.
  • TBDMS group tert-butyldimethylsilyl group
  • ACE bis(2-acetoxyethyloxy)methyl group
  • TOM triisopropylsilyloxymethyl group
  • CEE 1-(2-cyanoethoxy)ethyl group
  • CEM 2-cyanoethoxymethyl group
  • TEM dimethoxytrityl group
  • DMTr dimethoxytrityl group
  • R 3 is OR 4
  • the protecting group is not particularly
  • L 1 is an alkylene chain consisting of m L carbon atoms.
  • the hydrogen atoms on the alkylene carbon atoms may be substituted with, for example, OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a , or may not be substituted.
  • L 1 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms.
  • the polyether chain is, for example, polyethylene glycol.
  • Y 1 is NH, O, or S
  • the atom of L 1 bonded to Y 1 is carbon
  • the atom of L 1 bonded to OR 1 is carbon
  • the oxygen atoms are not adjacent to each other. That is, for example, when Y 1 is O, the oxygen atom and the oxygen atom of L 1 are not adjacent, and the oxygen atom of OR 1 and the oxygen atom of L 1 are not adjacent.
  • L 2 is an alkylene chain consisting of nL carbon atoms.
  • the hydrogen atoms on the alkylene carbon atoms may be substituted with, for example, OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c , or may not be substituted.
  • L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms.
  • Y 2 is NH, O or S
  • the atom of L 2 bonded to Y 2 is carbon
  • the atom of L 2 bonded to OR 2 is carbon
  • the oxygen atoms are not adjacent to each other. That is, for example, when Y 2 is O, the oxygen atom and the oxygen atom of L 2 are not adjacent, and the oxygen atom of OR 2 and the oxygen atom of L 2 are not adjacent.
  • n L and m L of L1 and n L of L2 are not particularly limited, and the lower limit is, for example, 0, and the upper limit is also not particularly limited.
  • n L and m L can be appropriately set, for example, according to the desired length of the non-nucleotide structure.
  • n L and m L are each preferably 0 to 30, more preferably 0 to 20, and even more preferably 0 to 15.
  • n L +m L is, for example, 0 to 30, preferably 0 to 20, and more preferably 0 to 15.
  • R a , R b , R c and R d are, for example, each independently a substituent or a protecting group.
  • the substituent and protecting group are, for example, the same as those described above.
  • the hydrogen atoms may be, for example, each independently substituted with a halogen such as Cl, Br, F, or I.
  • the nucleotide sequence T and the nucleotide sequence Q are linked to the non-nucleotide structure, for example, via -OR 1 - or -OR 2 -, respectively.
  • R 1 and R 2 may be present or absent.
  • R 1 and R 2 are each independently a nucleotide residue or a structure of formula (1).
  • the structure of the linker L is formed, for example, from a non-nucleotide residue consisting of the structure of formula (1) excluding the nucleotide residues R 1 and/or R 2 , and a nucleotide residue.
  • the non-nucleotide structure is, for example, a structure in which two or more non-nucleotide residues consisting of the structure of formula (1) are linked.
  • the structure of formula (1) may include, for example, one, two, three, or four. In this way, when a plurality of structures of formula (1) are included, the structures of formula (1) may be, for example, directly linked or linked via a nucleotide residue.
  • the non-nucleotide structure is formed solely from non-nucleotide residues having the structure of, for example, formula (1).
  • the combination of bonds between the nucleotide sequence T and the nucleotide sequence Q and -OR 1 - and -OR 2 - is not particularly limited, and examples thereof include any of the following conditions.
  • Condition 1 Nucleotide sequence T is linked to the structure of formula (1) via --OR 2 --, and nucleotide sequence Q is linked to the structure of formula (1) via --OR 1 --.
  • Condition 2 Nucleotide sequence T is linked to the structure of formula (1) via --OR 1 --, and nucleotide sequence Q is linked to the structure of formula (1) via --OR 2 --.
  • formula (1) can be exemplified by the following formulae (1-1) to (1-9), in which nL and mL are the same as those in formula (1).
  • q is an integer of 0 to 10.
  • Single-stranded nucleic acid molecules for genes include not only hairpin-type nucleic acid molecules represented by T-L-Q, but also single-stranded nucleic acid molecules with a dumbbell-shaped structure in which linkers are added to both ends of a guide strand containing an expression control sequence, and a nucleotide sequence complementary to a portion of the guide strand is bound to a nucleotide sequence complementary to the remaining portion of the guide strand via each linker (e.g., Patent Nos. 4968811 and 4965745, etc.).
  • the single-stranded nucleic acid molecule for a gene may be a nucleic acid molecule having an L-type structure (L-type single-stranded nucleic acid molecule).
  • L-type single-stranded nucleic acid molecules include (i) L-type single-stranded nucleic acid molecules obtained by crosslinking the 2'-3' or 2'-5' of the sugar moiety of a nucleoside between the antisense strand and the sense strand, (ii) L-type single-stranded nucleic acid molecules using an alkyl chain having an internal amide bond as a linker, (iii) L-type single-stranded nucleic acid molecules in which the bases of nucleosides are directly linked, and (iv) L-type single-stranded nucleic acid molecules in which 1' of the sugar moiety of a nucleoside is replaced with H and the antisense strand and the sense strand are crosslinked between the 2'-2' of the sugar moiety of
  • T and Q are a sequence consisting of 14 to 30 contiguous ribonucleotide residues which are complementary to a target sequence and a ribonucleotide sequence complementary thereto (if one is a sequence complementary to a target nucleic acid sequence, the other is a sequence complementary thereto),
  • Z is a linker connecting the 2'- or 5'-position of the sugar moiety of (X) to the 2'- or 3'-position of the sugar moiety of (Y), or a linker connecting the base moiety of (X) to the base moiety of (Y);
  • m1 and m2 each independently represent an integer of 0 to 5; n1 and n2 each independently represent an integer from 0 to 5.
  • the nucleic acid molecule is represented by: In a preferred embodiment
  • a 1 and A 1 ' are each independently -O-, -NR 1a -, -S-, or -CR 1a R 1b - (wherein R 1a and R 1b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
  • a 2 and A 2 ′ each independently represent —CR 2a R 2b —, —CO—, an alkynyl group, an alkenyl group, or a single bond (wherein R 2a and R 2b each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
  • a 3 and A 3 ′ each independently represent —O—, —NR 3a —, —S—, —CR 3a R 3b —, or a single bond (wherein R 3a and R 3b each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
  • a 4 and A 4 ' are each independently -(CR 4a
  • E 2 and E 2 ' are each independently -CR 2a R 2b -, -CO-, an alkynyl group, an alkenyl group, or a single bond (wherein R 2a and R 2b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms); E 3 and E 3 ' are each independently -O-, -NR 3a -, -S-, -CR 3a R 3b - or a single bond (wherein R 3a and R 3b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms); E 4 and E 4 ' are each independently -(CR 4a R 4b ) n -, -(CR 4a R 4b ) n-ring D- (wherein ring D is an aryl group having 6 to 10 carbon atoms, a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or
  • the "alkyl group having 1 to 10 carbon atoms", the “aryl group having 6 to 10 carbon atoms", the “heteroaryl group having 2 to 10 carbon atoms”, the “cycloalkyl group having 4 to 10 carbon atoms” and the “heterocycloalkyl group having 4 to 10 carbon atoms” may be substituted at any substitutable position, and examples of the substituent include those described in the above-mentioned substituent group A.
  • the method for synthesizing a single-stranded nucleic acid molecule for a gene is not particularly limited, and a conventionally known method for producing nucleic acids can be used.
  • Examples of the synthesis method include synthesis using genetic engineering techniques and chemical synthesis.
  • Examples of the genetic engineering techniques include in vitro transcription synthesis, methods using vectors, and methods using PCR cassettes.
  • the vector is not particularly limited, and examples include non-viral vectors such as plasmids and viral vectors.
  • the chemical synthesis method is not particularly limited, and examples include the phosphoramidite method and the H-phosphonate method.
  • a commercially available automatic nucleic acid synthesizer can be used.
  • amidites are generally used.
  • the amidite is not particularly limited, and examples of commercially available amidites include RNA Phosphoramidites (2'-O-TBDMS amidite, ChemGenes), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc.
  • AEM amidite (WO2022/045224) can be used as the amidite for bridging the 2' position.
  • the single-stranded nucleic acid molecule for a gene When the single-stranded nucleic acid molecule for a gene is composed only of natural unmodified ribonucleotide residues, it can be provided as a precursor of the nucleic acid molecule in the form of a vector that encodes the nucleic acid molecule in an expressible state.
  • the expression vector is characterized by containing DNA encoding the single-stranded nucleic acid molecule for the target gene under the control of a promoter that is functional in the target cell, and other configurations are not limited in any way.
  • the vector into which the DNA is inserted is not particularly limited, and general vectors can be used, such as viral vectors and non-viral vectors.
  • An example of a non-viral vector is a plasmid vector.
  • Ligand conjugate substance The present invention provides a compound represented by the following general formula (II): This compound is a compound in which a ligand is bound to an iminodicarboxylic acid derivative, and is also referred to as a ligand conjugate substance in this specification.
  • Z is an amino protecting group
  • B 1 , B 2 , B 3 and B 4 are the same or different and each represents a hydrogen atom or any one of the following formulae: -( W2 ) n2 -CO-NH- X1 - Y1 , -( W3 ) n3 -CO-NH- X2 - Y2 , -( W5 ) n5 -CO-NH- X3 - Y3 , -( W6 ) n6 -CO-NH- X4 - Y4 ,
  • W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
  • W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally
  • the ligand conjugate substance of the present invention represented by formula (II) is preferably a compound represented by the following formula (II-1):
  • Z is an amino-protecting group, preferably benzyloxycarbonyl.
  • the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or combine together to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); n' represents an integer from 1 to 10), and more preferably, they are each independently -(CH 2 ) 6 -O- or -(CH 2 ) 5 -NH-.
  • W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are —(CH 2 )—, and n 1 to n 6 are 1.
  • the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a sugar (preferably GalNAc) or a lipid.
  • the present invention provides a pharmaceutical composition comprising the ligand-conjugated nucleic acid of the present invention.
  • the ligand corresponds to Y 1 to Y 4 in the above-mentioned general formula (I) or general formula (I-1).
  • the term "ligand” refers to a substance that forms a complex with a biomolecule to achieve a biological purpose.
  • the ligand is appropriately selected depending on the target biomolecule, and examples thereof include protein substances such as antibodies, enzymes, peptide aptamers, and receptors, nucleic acids such as DNA, RNA, or derivatives thereof, proteinaceous or non-proteinaceous bioactive substances such as antibiotics, and sugars that can bind to lectins (for example, lactose, sialyllactose, globotriose, lacto-N-neotetraose, oligomannose, and other sugar chains known to bind to lectins present in specific tissues in the body, and may be any sugar chain such as monosaccharides and oligosaccharides), lipids, etc.
  • protein substances such as antibodies, enzymes, peptide aptamers, and receptors
  • nucleic acids such as DNA, RNA, or derivatives thereof
  • proteinaceous or non-proteinaceous bioactive substances such as antibiotics
  • sugars that can bind to lectins for example, lactos
  • GalNAc N-acetylgalactosamine
  • a type of sugar chain attached to it.
  • GalNAc binds strongly to the asialoglycoprotein receptor specifically expressed on the cell surface of hepatocytes and is endocytosed. This receptor is actively recycled through endocytosis and exocytosis, so that the nucleic acid molecule is efficiently drawn into the hepatocytes.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally to mammals (e.g., humans, cats, ferrets, mink, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, and monkeys), but is preferably administered parenterally.
  • mammals e.g., humans, cats, ferrets, mink, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, and monkeys
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.
  • the formulations can be sealed in unit doses or multiple doses in containers such as ampoules and vials.
  • the active ingredient and a pharma- ceutical acceptable carrier can be freeze-dried and stored in a state in which it is sufficient to dissolve or suspend them in a suitable sterile vehicle immediately before use.
  • suitable formulations for parenteral administration include aerosols.
  • the content of the ligand-conjugated nucleic acid of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
  • the dosage of the pharmaceutical composition of the present invention varies depending on the purpose of administration, the method of administration, the type and severity of the target disease, and the condition of the recipient (sex, age, body weight, etc.).
  • a single dose of the ligand-conjugated nucleic acid of the present invention is usually preferably 2 nmol/kg or more and 50 nmol/kg or less, and when administered locally, 1 pmol/kg or more and 10 nmol/kg or less. It is desirable to administer such a dosage 1 to 10 times, more preferably 5 to 10 times.
  • the pharmaceutical composition of the present invention may contain two or more types of ligand-conjugated nucleic acids. It may also be used in combination with other drugs that have been reported to have therapeutic effects on genetic diseases for which the nucleic acid molecules contained in the pharmaceutical composition of the present invention are administered.
  • These concomitant drugs may be formulated together with the pharmaceutical composition of the present invention and administered as a single formulation, or may be formulated separately from the pharmaceutical composition of the present invention and administered simultaneously or at a later time via the same or a different route as the pharmaceutical composition of the present invention.
  • the dosage of these concomitant drugs may be the amount normally used when the drug is administered alone, or may be reduced from the amount normally used.
  • the pharmaceutical composition of the present invention may use an effective amount of the ligand-conjugated nucleic acid of the present invention alone, or may be formulated as a pharmaceutical composition together with any carrier, such as a pharma- ceutical acceptable carrier.
  • Examples of pharma- ceutically acceptable carriers include, but are not limited to, excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, flavorings such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, dispersing agents such as surfactants, diluents such as water and saline, base waxes, etc.
  • excipients such as sucrose and starch
  • binders such as cellulose and methylcellulose
  • disintegrants such as starch and carboxymethylcellulose
  • lubricants such as magnesium stearate and aerosil
  • flavorings such as citric acid and menthol
  • preservatives such as sodium benzoate and sodium bisulfit
  • the pharmaceutical composition of the present invention may further contain a nucleic acid introduction reagent.
  • the nucleic acid introduction reagent include atelocollagen; liposomes; nanoparticles; lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, and cationic lipids such as poly(ethyleneimine) (PEI).
  • the pharmaceutical composition of the present invention may be a pharmaceutical composition in which the ligand-conjugated nucleic acid of the present invention is encapsulated in a liposome.
  • a liposome is a fine closed vesicle having an internal phase surrounded by one or more lipid bilayers, and can usually hold a water-soluble substance in the internal phase and a fat-soluble substance in the lipid bilayer.
  • the term "encapsulation" refers to the ligand-conjugated nucleic acid of the present invention being held in the liposomal internal phase or in the lipid bilayer.
  • the liposome used in the present invention may be a single-layer membrane or a multi-layer membrane, and the particle size can be appropriately selected, for example, within the range of 10 to 1000 nm, preferably 50 to 300 nm. In consideration of the delivery to the target tissue, the particle size can be, for example, 200 nm or less, preferably 100 nm or less.
  • Methods for encapsulating water-soluble compounds such as nucleic acids into liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method, and any known method can be appropriately selected.
  • RNA is written in uppercase (ACGU).
  • ⁇ DNA is written in lower case (acgt). Unless otherwise specified, the sequences written vertically are 5' ⁇ 3' for the upper line and 3' ⁇ 5' for the lower line.
  • 2'-OMe modified nucleic acids Am, Cm, Gm, Um
  • 2'-F-modified nucleic acids Af, Cf, Gf, Uf LNA: AL, CL, GL, TL
  • Sulfurization Ns (sulfurization of the phosphate on the 3' side) 2'-OAEM-modified nucleic acid: Ae, Ce, Ge, Ue
  • a 1 M aqueous hydrochloric acid solution was added to the aqueous layer, and the mixture was extracted with dichloromethane. The organic layer was washed with water and then dried over magnesium sulfate. The solution was concentrated under reduced pressure to obtain 4.2 g of the target compound 3 as a colorless oily substance.
  • the organic solvent was concentrated under reduced pressure, and then water and a saturated aqueous sodium chloride solution were added, followed by extraction with chloroform.
  • the organic layer was dried over magnesium sulfate, and the solution was concentrated under reduced pressure to obtain 811 mg of the target compound 12 as a white solid.
  • the reaction solution was purified by HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 ⁇ m, 10 mm ⁇ 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN).
  • the purified product was precipitated with ethanol, and the resulting precipitate was dissolved in distilled water for injection to obtain the target compound 26.
  • Mass spectrometry 13998.6 (calculated value: 13998.0).
  • the reaction solution was purified by HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 ⁇ m, 10 mm ⁇ 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN).
  • the purified product was precipitated with ethanol, and the resulting precipitate was dissolved in distilled water for injection to obtain compound 29 (Example 1-1 compound) with a purity of 98.0%.
  • Mass analysis 15717.4 (calculated value: 15716.8).
  • the RP-HPLC chart is shown in Figure 1.
  • Examples 1-2 to 1-9 These compounds (nucleic acid molecules) were synthesized by the method of Example 1-1 or a combination of the method of Example 1-1 and an existing method. Their structural formulas are shown in Table 1. X in Table 1 has the same meaning as that in Example 1-1. Furthermore, RP-HPLC charts after purification of each of the nucleic acid molecules of Examples 1-2 to 1-9 are shown in Figures 2 to 9.
  • the line connecting the upper and lower bases represents the moiety corresponding to -Z- in formula (a), and means that the bases are linked at that site.
  • X represents the following structure.
  • reaction solution was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection (300.0 ⁇ L) and acetonitrile (100.0 ⁇ L), and purified by HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 ⁇ m, 10 mm ⁇ 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 90% CH 3 CN).
  • Examples 2-2 and 2-3 These compounds were synthesized by the method of Example 2-1 or a combination of the method of Example 2-1 and an existing method. Their structural formulas are shown in Table 2. In addition, RP-HPLC charts after purification of each of the nucleic acid molecules of Examples 2-2 and 2-3 are shown in Figures 11 and 12.
  • the reaction solution was subjected to ethanol precipitation and purified by RP-HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 ⁇ m, 10 mm ⁇ 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C.; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN), and the peak of the target substance was separated. The separated fraction was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain compound 37. Mass spectrometry: 20003.9 (calculated value: 20003.5). The RP-HPLC chart is shown in Figure 13.
  • the reaction solution was purified (column: XBridge Oligonucleotide, BEH C18, 2.5 ⁇ m, 10 mm ⁇ 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C.; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN), and the peak of the target product was separated. The collected fraction was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain SEQ ID NO:27-GalNAc4 with a purity of 99.6%.
  • Mass spectrometry 8689.9 (calculated value: 8689.3).
  • the RP-HPLC chart of SEQ ID NO:27-GalNAc4 is shown in FIG.
  • the sense strand SEQ ID NO:27-GalNAc4 obtained above and SEQ ID NO:28 were annealed in a PBS buffer solution of pH 7.4 to obtain the compound of Example 3-1.
  • Examples 3-2, 3-3, 4-1, and 4-2 These compounds were synthesized by synthesizing siRNA using the phosphoramidite method and the method of Example 3-1 or a combination of the method of Example 3-1 and an existing method.
  • the structural formulas of these nucleic acid molecules are shown in Table 3.
  • the RP-HPLC charts after purification of each of the nucleic acid molecules 3-2, 3-3, 4-1, and 4-2 are shown in Figures 15 to 18, respectively.
  • Y and Z have the following structures.
  • HDO DNA/RNA heteroduplex
  • GalNAc-modified HDO was intravenously administered at 1.5 mg/kg (5 mL/kg) to C57BL/6J mice (Charles River Japan) to measure blood Factor VII.
  • Two groups were set up, one each for
  • Test Example 1 Evaluation of Factor VII in the Blood of Mice Intravenously Administered HDO To measure Factor VII in the blood, each sample was evaluated using the BIOPHEN TM FVII kit. Plasma from mice administered HDO or GalNAc-modified HDO was used as a sample, and each sample was diluted 2000-fold with Tris-BSA buffer (R4) included in the kit. 30 ⁇ L of the diluted sample, 30 ⁇ L of Thromboplastin Calcium (R2) preincubated at 37° C., and 60 ⁇ L of Factor X (human) (R3) were added to a 96-well microplate, and incubated at 37° C. for 7 minutes after stirring. SXa-11 (R3) preincubated at 37° C.
  • Tris-BSA buffer R4 included in the kit.
  • mice administered GalNAc-modified HDO decreased by about 0.26 from 1 day to 3 days after administration and from 3 days to 7 days after administration.
  • the HDO used in this evaluation is a nucleic acid that suppresses the expression of Factor VII, and Factor VII in the blood is derived from the liver.
  • Test Example 2 Evaluation of Factor VII in the Blood of siRNA-Administered Mice
  • each sample was evaluated using the BIOPHEN TM FVII kit.
  • mice subcutaneously administered with GalNAc-modified siRNA and mice pulmonary administered with GalNAc-modified siRNA serum collected 7 days before administration was used as a control sample.
  • Each sample was diluted 2000-fold with Tris-BSA buffer (R4) included in the kit.
  • 60 ⁇ L of Factor X (human) (R3) were added to a 96-well microplate, and incubated at 37° C. for 7 minutes after stirring.
  • the ligand-conjugated nucleic acid comprising the iminodicarboxylic acid derivative, which is the compound of the present invention can provide a therapeutic and/or prophylactic agent that is serum stable, capable of being delivered to an appropriate organ or cell, and capable of being delivered through a membrane, and further provides high functionality at low production costs.
  • This application is based on Patent Application No. 2022-185308 filed in Japan (filing date: November 18, 2022), the contents of which are incorporated in their entirety herein.

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Abstract

This ligand conjugate nucleic acid represented by general formula (I) [In the formula (I), the symbols are as defined in the specification.] provides a therapeutic and/or prophylactic agent which enables serum stability, delivery to an appropriate organ or cell, and transmembrane delivery, and which can impart high functionality at low production cost.

Description

リガンドコンジュゲート物質及びそれを含む核酸並びにその用途Ligand conjugate substance, nucleic acid containing same, and use thereof
　本発明は、イミノジ酢酸基を有する新規な化合物に関し、具体的にはイミノジ酢酸誘導体を介したリガンドコンジュゲートを有する治療剤送達の分野に関する。特に、インビボ送達に有利なこれらのコンジュゲートを含む医薬組成物、ならびにインビボでの治療的使用に好適な医薬組成物を提供する。さらに、本発明は、これらの組成物をさまざまな疾患状態の治療のために適用すべく、これらを用いて医薬組成物を細胞に導入する方法に関する。 The present invention relates to novel compounds having an iminodiacetic acid group, and in particular to the field of therapeutic delivery having ligand conjugates via iminodiacetic acid derivatives. In particular, pharmaceutical compositions comprising these conjugates advantageous for in vivo delivery, as well as pharmaceutical compositions suitable for in vivo therapeutic use, are provided. Furthermore, the present invention relates to methods of using these compositions to introduce pharmaceutical compositions into cells for application in the treatment of various disease conditions.
　核酸の治療薬は、血清安定性、適切な臓器または細胞への送達、および膜貫通送達を有することが求められている。核酸の膜貫通送達の改善においては、タンパク質担体、抗体担体、リポソーム性送達系、エレクトロポレーション、直接注射、細胞融合、ウイルスベクター、およびリン酸カルシウム等を介する形質転換等が利用されている。しかしながら、これらの技術の多くは、膜貫通輸送が可能な細胞の種類と、このような輸送を達成するのに必要な条件が限定される。
　また、近年では、治療薬の観点から、肝臓内で発現する疾患関連タンパクの抑制を目指し、それにコンジュゲートするリガンドとして、Ｎ－アセチルガラクトサミン（ＧａｌＮＡｃ）を用いた、肝臓の膜表面にあるアシアロ糖タンパク質受容体（ＡＳＧＰＲ）を介した細胞内輸送も盛んである。
　さらには、肝臓以外の臓器への輸送を目指し、リガンドとして、脂質を利用した、脂肪酸受容体を介した細胞内取り込みの開発も始まっている。 Nucleic acid therapeutics are required to have serum stability, delivery to appropriate organs or cells, and transmembrane delivery. For improving the transmembrane delivery of nucleic acids, protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, calcium phosphate-mediated transformation, etc. are utilized. However, most of these techniques are limited in the types of cells that can be transmembrane transported and the conditions required to achieve such transport.
Moreover, in recent years, from the viewpoint of therapeutic drugs, the aim is to suppress disease-related proteins expressed in the liver, and intracellular transport via the asialoglycoprotein receptor (ASGPR) on the surface of the liver membrane using N-acetylgalactosamine (GalNAc) as a ligand to be conjugated thereto has become popular.
Furthermore, with the aim of transporting the drug to organs other than the liver, development has begun on intracellular uptake via fatty acid receptors using lipids as ligands.
　本発明の目的は、血清安定性、適切な臓器または細胞への送達、および膜貫通送達を可能とし、更に低いコンジュゲート製造コストで高い機能性を付与する、治療及び／又は予防剤を提供することである。 The object of the present invention is to provide a therapeutic and/or prophylactic agent that is serum stable, delivers to the appropriate organ or cell, and enables transmembrane delivery, and further provides high functionality at low conjugate production costs.
　本発明者らは、上記の目的を達成すべく鋭意研究を重ねた結果、イミノジカルボン酸誘導体からなるリガンドコンジュゲート核酸が、血清安定性、適切な臓器または細胞への送達、および膜貫通送達を可能とすることを見出し、本発明を完成するに至った。 As a result of extensive research aimed at achieving the above object, the inventors discovered that a ligand-conjugated nucleic acid made of an iminodicarboxylic acid derivative enables serum stability, delivery to appropriate organs or cells, and transmembrane delivery, and thus completed the present invention.
　即ち、本発明は以下のとおりである。
［１］下記一般式（Ｉ）： That is, the present invention is as follows.
[1] The following general formula (I):
［式中、
Ａは、核酸分子であり、
Ｘは、－（ＣＲ１Ｒ２）ｎ－Ｒ３－（式中、Ｒ１及びＲ２は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ３は－ＮＲ４－（式中、Ｒ４は、水素原子又は置換されていてもよいアルキル基である）又は－Ｓ－ヘテロ環－ＣＲ５Ｒ６－ＮＲ７－（式中、Ｒ５、Ｒ６、Ｒ７は、それぞれ独立して、水素原子又は置換されていてもよいアルキル基である）を表す；ｎは１～１０の整数を表す）を表し、
Ｂ１、Ｂ２、Ｂ３及びＢ４は、それぞれ独立して、水素原子又は下記式のいずれかを表し、
－（Ｗ２）ｎ２－ＣＯ－ＮＨ－Ｘ１－Ｙ１、
－（Ｗ３）ｎ３－ＣＯ－ＮＨ－Ｘ２－Ｙ２、
－（Ｗ５）ｎ５－ＣＯ－ＮＨ－Ｘ３－Ｙ３、
－（Ｗ６）ｎ６－ＣＯ－ＮＨ－Ｘ４－Ｙ４、
Ｗ２は、－（ＣＲ１Ｗ２Ｒ２Ｗ２）－（式中、Ｒ１Ｗ２及びＲ２Ｗ２は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ３は、－（ＣＲ１Ｗ３Ｒ２Ｗ３）－（式中、Ｒ１Ｗ３及びＲ２Ｗ３は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ５は、－（ＣＲ１Ｗ５Ｒ２Ｗ５）－（式中、Ｒ１Ｗ５及びＲ２Ｗ５は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ６は、－（ＣＲ１Ｗ６Ｒ２Ｗ６）－（式中、Ｒ１Ｗ６及びＲ２Ｗ６は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し[但し、Ｂ１～Ｂ４は、同時に水素原子ではない]、
ｎ２、ｎ３、ｎ５及びｎ６は、それぞれ独立して、１～５の整数であり、
ｎＢ１～ｎＢ４は、それぞれ独立して、１～３の整数を表し、
Ｘ０、Ｘ１、Ｘ２、Ｘ３及びＸ４は、それぞれ独立して、置換されても良いスペーサーを表し、
Ｙ１、Ｙ２、Ｙ３及びＹ４は、それぞれ独立して、リガンドを表し、
Ｗ１は、－（ＣＲ１Ｗ１Ｒ２Ｗ１）－（式中、Ｒ１Ｗ１及びＲ２Ｗ１は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ４は、－（ＣＲ１Ｗ４Ｒ２Ｗ４）－（式中、Ｒ１Ｗ４及びＲ２Ｗ４は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
ｎ１及びｎ４は、それぞれ独立して、１～５の整数である］
で表される化合物。
［２］下記一般式（Ｉ－１）： [Wherein,
A is a nucleic acid molecule,
X represents -(CR 1 R 2 )n-R 3 - (wherein R 1 and R 2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 3 represents -NR 4 - (wherein R 4 is a hydrogen atom or an optionally substituted alkyl group) or -S-heterocycle-CR 5 R 6 -NR 7 - (wherein R 5 , R 6 and R 7 are each independently a hydrogen atom or an optionally substituted alkyl group); and n represents an integer of 1 to 10);
B 1 , B 2 , B 3 and B 4 each independently represent a hydrogen atom or any of the following formulae:
-( W2 ) n2 -CO-NH- X1 - Y1 ,
-( W3 ) n3 -CO-NH- X2 - Y2 ,
-( W5 ) n5 -CO-NH- X3 - Y3 ,
-( W6 ) n6 -CO-NH- X4 - Y4 ,
W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W5 represents -( CR1W5R2W5 )- (wherein R1W5 and R2W5 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W 6 represents -(CR 1W6 R 2W6 )- (wherein R 1W6 and R 2W6 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group) (provided that B 1 to B 4 are not simultaneously hydrogen atoms);
n 2 , n 3 , n 5 and n 6 each independently represent an integer from 1 to 5;
n B1 to n B4 each independently represent an integer of 1 to 3;
X 0 , X 1 , X 2 , X 3 and X 4 each independently represent a spacer which may be substituted;
Y 1 , Y 2 , Y 3 and Y 4 each independently represent a ligand;
W 1 represents -(CR 1W1 R 2W1 )- (wherein R 1W1 and R 2W1 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group);
W 4 represents -(CR 1W4 R 2W4 )- (wherein R 1W4 and R 2W4 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
n1 and n4 each independently represent an integer from 1 to 5.
A compound represented by the formula:
[2] The following general formula (I-1):
（式中、各記号の定義は上記［１］と同義である）
で表される、上記［１］記載の化合物。
［３］Ｘ０のスペーサーが、下記式（１）～（４）のいずれかを表す、上記［１］又は［２］記載の化合物：
（１）－ＣＯ－Ｌ１－ＣＯ－
（式中、Ｌ１は
(i)－（ＣＲ１Ｌ１Ｒ２Ｌ１）ｎＬ１（式中、Ｒ１Ｌ１及びＲ２Ｌ１は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ１は１～１０の整数を表す）、
(ii)－（ＣＨ２）ｎＬ１－Ｃｙ－（ＣＨ２）ｍＬ１－（式中、Ｃｙは置換されていてもよいアリーレン基、置換されていてもよいヘテロアリーレン基、置換されていてもよいシクロアルキレン基、又は置換されていてもよいヘテロシクロアルキレン基を表し；ｎＬ１及びｍＬ１は同一又は異なって、１～１０の整数を表す）、
(iii)－ＮＲ８－（式中、Ｒ８は水素原子又は置換されていてもよいアルキル基を表す）、
（２）－Ｌ２－ＣＯ－Ｌ３－ＣＯ－
（式中、Ｌ２は、－（ＣＲ１Ｌ２Ｒ２Ｌ２）ｎＬ２－（式中、Ｒ１Ｌ２及びＲ２Ｌ２は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ２は０～６の整数を表す）を表し；Ｌ３は、－（ＣＲ１Ｌ３Ｒ２Ｌ３）ｎＬ３－（式中、Ｒ１Ｌ３及びＲ２Ｌ３は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ３は０～６の整数を表す、但し、ｎＬ２及びｎＬ３は同時に０ではない）を表す）、
（３）－ＣＯ－Ｌ４－ＣＯ－Ｌ５－ＣＯ－Ｌ６－ＣＯ－
（式中、Ｌ４は、－（ＣＲ１Ｌ４Ｒ２Ｌ４）ｎＬ４（式中、Ｒ１Ｌ４及びＲ２Ｌ４は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ４は１～６の整数を表す）を表し；Ｌ５は、－（ＯＣ２Ｈ４）ｎＬ５（式中、ｎＬ５は１～６の整数を表す）を表し；Ｌ６は、－（ＣＲ１Ｌ６Ｒ２Ｌ６）ｎＬ６（式中、Ｒ１Ｌ６及びＲ２Ｌ６は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ６は１～６の整数を表す）を表す）、
（４）－ＣＯ－Ｌ７－ＣＨ２ＣＯ－
（式中、Ｌ７は、－（ＯＣ２Ｈ４）ｎＬ７（式中、ｎＬ７は１～６の整数を表す）を表す）。
［４］Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＲ９Ｒ１０）ｎ’－Ｒ１１（式中、Ｒ９及びＲ１０は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ１１は－Ｏ－又は－ＮＲ１２－（式中、Ｒ１２は水素原子又は置換されていてもよいアルキル基である）を表す；ｎ’は１～１０の整数を表す）で表される上記［１］～［３］のいずれかに記載の化合物。
［５］Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５及びＷ６が、－（ＣＨ２）－で、ｎ１～ｎ６が１である上記［１］～［４］のいずれかに記載の化合物。
［６］Ｘ０のスペーサーが、－ＣＯ－（ＣＨ２）ｎＬ１－ＣＯ－（式中、ｎＬ１は１～１０の整数を表す）である、上記［１］～［５］のいずれかに記載の化合物。
［７］ｎＬ１が６である、上記［１］～［６］のいずれかに記載の化合物。
［８］Ｘが、－（ＣＨ２）６－ＮＨ－である、上記［１］～［７］のいずれかに記載の化合物。
［９］Ｘが、－（ＣＨ２）６－Ｓ－ヘテロ環－（ＣＨ２）２－ＮＨ－である、上記［１］～［７］のいずれかに記載の化合物。
［１０］Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）６－Ｏ－である、上記［１］～［９］のいずれかに記載の化合物。
［１１］Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）５－ＮＨ－である、上記［１］～［９］のいずれかに記載の化合物。
［１２］Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、糖である、上記［１］～［１１］のいずれかに記載の化合物。
［１３］糖がＧａｌＮＡｃである、上記［１２］記載の化合物。
［１４］Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、脂質である、上記［１］～［１１］のいずれかに記載の化合物。
［１５］Ａの核酸分子が、標的遺伝子に対するｓｉＲＮＡである、上記［１］～［１４］のいずれかに記載の化合物。
［１６］Ａの核酸分子が、標的遺伝子に対するアンチセンス核酸である、上記［１］～［１４］のいずれかに記載の化合物。
［１７］Ａの核酸分子が、標的遺伝子に対するガイド鎖とそれに相補的なパッセンジャー鎖とが、リンカーを介して連結された二重鎖を形成し得る一本鎖核酸分子である、上記［１］～［１４］のいずれかに記載の化合物。
［１８］Ａの核酸分子が、下記一般式（ａ）： (In the formula, the definitions of each symbol are the same as those in [1] above.)
The compound according to the above-mentioned [1], represented by the following formula:
[3] The compound according to the above [1] or [2], wherein the spacer of X 0 represents any one of the following formulas (1) to (4):
(1) -CO- L1 -CO-
(Wherein, L1 is
(i) -(CR 1L1 R 2L1 ) n L1 (wherein R 1L1 and R 2L1 are the same or different and each represents a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; n L1 represents an integer of 1 to 10),
(ii) -(CH 2 ) n L1 -Cy-(CH 2 ) m L1 - (wherein Cy represents an optionally substituted arylene group, an optionally substituted heteroarylene group, an optionally substituted cycloalkylene group, or an optionally substituted heterocycloalkylene group; n L1 and m L1 may be the same or different and represent an integer of 1 to 10),
(iii) -NR 8 - (wherein R 8 represents a hydrogen atom or an optionally substituted alkyl group),
(2) -L2 -CO- L3 -CO-
(wherein L2 represents -( CR1L2R2L2 ) nL2- (wherein R1L2 and R2L2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; nL2 represents an integer of 0 to 6); L3 represents -( CR1L3R2L3 ) nL3- (wherein R1L3 and R2L3 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; nL3 represents an integer of 0 to 6, with the proviso that nL2 and nL3 are not simultaneously 0));
(3) -CO- L4 -CO- L5 -CO- L6 -CO-
(wherein L 4 represents -(CR 1L4 R 2L4 )n L4 (wherein R 1L4 and R 2L4 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or taken together to form an alkylene group; n L4 represents an integer of 1 to 6); L 5 represents -(OC 2 H 4 )n L5 (wherein n L5 represents an integer of 1 to 6); L 6 represents -(CR 1L6 R 2L6 )n L6 (wherein R 1L6 and R 2L6 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or taken together to form an alkylene group; n L6 represents an integer of 1 to 6)).
(4) -CO-L 7 -CH 2 CO-
(wherein L7 represents -(OC 2 H 4 ) n L7 (wherein n L7 represents an integer of 1 to 6)).
[4] The compound according to any one of the above [1] to [3], wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently represented by -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); and n' represents an integer of 1 to 10).
[5] The compound according to any one of the above [1] to [4], wherein W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are —(CH 2 )—, and n 1 to n 6 are 1.
[6] The compound according to any one of the above [1] to [5], wherein the spacer of X 0 is -CO-(CH 2 ) n L1 -CO- (wherein n L1 represents an integer of 1 to 10).
[7] The compound according to any one of the above [1] to [6], wherein n L1 is 6.
[8] The compound according to any one of the above [1] to [7], wherein X is -(CH 2 ) 6 -NH-.
[9] The compound according to any one of the above [1] to [7], wherein X is -(CH 2 ) 6 -S-heterocycle-(CH 2 ) 2 -NH-.
[10] The compound according to any one of the above [1] to [9], wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 6 -O-.
[11] The compound according to any one of the above [1] to [9], wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 5 -NH-.
[12] The compound according to any one of the above [1] to [11], wherein the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a sugar.
[13] The compound according to the above-mentioned [12], wherein the sugar is GalNAc.
[14] The compound according to any one of the above [1] to [11], wherein the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a lipid.
[15] The compound according to any one of the above-mentioned [1] to [14], wherein the nucleic acid molecule of A is an siRNA for a target gene.
[16] The compound according to any one of the above [1] to [14], wherein the nucleic acid molecule of A is an antisense nucleic acid for a target gene.
[17] The compound according to any one of the above [1] to [14], wherein the nucleic acid molecule of A is a single-stranded nucleic acid molecule capable of forming a duplex in which a guide strand for a target gene and a passenger strand complementary thereto are linked via a linker.
[18] The nucleic acid molecule of A is represented by the following general formula (a):
［式中、Ｘ、Ｙ、Ｘ１、Ｙ１、Ｘ２、Ｙ２は、それぞれ独立して、修飾されていてもよいリボヌクレオチド残基又は修飾されていてもよいデオキシリボヌクレオチド残基であり；Ｚは（Ｘ）の糖部分の２’位若しくは５’位と（Ｙ）の糖部分の２’位若しくは３’位とを連結するリンカーであり、又は（Ｘ）の塩基部分と（Ｙ）の塩基部分とを連結するリンカーであり；
配列Ｔは標的遺伝子の発現制御配列（Ｔ）を含むヌクレオチド配列であり、配列Ｑは該発現制御配列Ｔに相補的な配列（Ｑ）を含むヌクレオチド配列であり；
ｍ１及びｍ２は、それぞれ独立して、０～５の整数であり；及び
ｎ１及びｎ２は、それぞれ独立して、０～５の整数である］
で表される、上記［１］～［１４］のいずれかに記載の化合物。
［１９］下記一般式（ＩＩ） [wherein X, Y, X 1 , Y 1 , X 2 , and Y 2 are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue; Z is a linker connecting the 2'- or 5'-position of the sugar moiety of (X) to the 2'- or 3'-position of the sugar moiety of (Y), or a linker connecting the base moiety of (X) to the base moiety of (Y);
Sequence T is a nucleotide sequence containing an expression control sequence (T) of a target gene, and sequence Q is a nucleotide sequence containing a sequence (Q) complementary to the expression control sequence T;
m1 and m2 are each independently an integer from 0 to 5; and n1 and n2 are each independently an integer from 0 to 5.
The compound according to any one of the above [1] to [14],
[19] The following general formula (II):
［式中、
Ｚはアミノ基保護基であり、
Ｂ１、Ｂ２、Ｂ３及びＢ４は、同一又は異なって水素原子又は下記式のいずれかを表し、
－（Ｗ２）ｎ２－ＣＯ－ＮＨ－Ｘ１－Ｙ１、
－（Ｗ３）ｎ３－ＣＯ－ＮＨ－Ｘ２－Ｙ２、
－（Ｗ５）ｎ５－ＣＯ－ＮＨ－Ｘ３－Ｙ３、
－（Ｗ６）ｎ６－ＣＯ－ＮＨ－Ｘ４－Ｙ４、
Ｗ２は、－（ＣＲ１Ｗ２Ｒ２Ｗ２）－（式中、Ｒ１Ｗ２及びＲ２Ｗ２は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ３は、－（ＣＲ１Ｗ３Ｒ２Ｗ３）－（式中、Ｒ１Ｗ３及びＲ２Ｗ３は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ５は、－（ＣＲ１Ｗ５Ｒ２Ｗ５）－（式中、Ｒ１Ｗ５及びＲ２Ｗ５は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ６は、－（ＣＲ１Ｗ６Ｒ２Ｗ６）－（式中、Ｒ１Ｗ６及びＲ２Ｗ６は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
ｎ２、ｎ３、ｎ５及びｎ６は、それぞれ独立して、１～５の整数であり、
ｎＢ１’～ｎＢ４’は、それぞれ独立して、０～３の整数（但し、同時に０でない）を表し、
Ｘ１、Ｘ２、Ｘ３及びＸ４は、それぞれ独立して、置換されても良いスペーサーであり、Ｙ１、Ｙ２、Ｙ３及びＹ４は、それぞれ独立して、リガンドであり、
Ｗ１は、－（ＣＲ１Ｗ１Ｒ２Ｗ１）－（式中、Ｒ１Ｗ１及びＲ２Ｗ１は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ４は、－（ＣＲ１Ｗ４Ｒ２Ｗ４）－（式中、Ｒ１Ｗ４及びＲ２Ｗ４は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
ｎ１及びｎ４は、それぞれ独立して、１～５の整数である］
で表される化合物。
［２０］下記一般式（ＩＩ－１）： [Wherein,
Z is an amino protecting group;
B 1 , B 2 , B 3 and B 4 are the same or different and each represents a hydrogen atom or any one of the following formulae:
-( W2 ) n2 -CO-NH- X1 - Y1 ,
-( W3 ) n3 -CO-NH- X2 - Y2 ,
-( W5 ) n5 -CO-NH- X3 - Y3 ,
-( W6 ) n6 -CO-NH- X4 - Y4 ,
W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W5 represents -( CR1W5R2W5 )- (wherein R1W5 and R2W5 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W6 represents -( CR1W6R2W6 )- (wherein R1W6 and R2W6 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
n 2 , n 3 , n 5 and n 6 each independently represent an integer from 1 to 5;
n B1 ' to n B4 ' each independently represent an integer of 0 to 3 (but are not all 0 at the same time);
X 1 , X 2 , X 3 and X 4 each independently represent a spacer which may be substituted; Y 1 , Y 2 , Y 3 and Y 4 each independently represent a ligand;
W 1 represents -(CR 1W1 R 2W1 )- (wherein R 1W1 and R 2W1 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group);
W 4 represents -(CR 1W4 R 2W4 )- (wherein R 1W4 and R 2W4 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
n1 and n4 each independently represent an integer from 1 to 5.
A compound represented by the formula:
[20] The following general formula (II-1):
（式中、各記号の定義は上記［１９］と同義である）
で表される、上記［１９］記載の化合物。
［２１］Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＲ９Ｒ１０）ｎ’－Ｒ１１（式中、Ｒ９及びＲ１０は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ１１は－Ｏ－又は－ＮＲ１２－（式中、Ｒ１２は水素原子又は置換されていてもよいアルキル基である）を表す；ｎ’は１～１０の整数を表す）
で表される上記［１９］又は［２０］に記載の化合物。
［２２］Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５及びＷ６が、－（ＣＨ２）－で、ｎ１～ｎ６が１である上記［１９］～［２１］のいずれかに記載の化合物。
［２３］Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）６－Ｏ－である、上記［１９］～［２２］のいずれかに記載の化合物。
［２４］Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）５－ＮＨ－である、上記［１９］～［２２］のいずれかに記載の化合物。
［２５］Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、タンパク質性物質、核酸、生体内活性物質、糖及び脂質からなる群より選択される少なくとも１種である、上記［１９］～［２４］のいずれかに記載の化合物。
［２６］Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、糖である、上記［１９］～［２５］のいずれかに記載の化合物。
［２７］糖がＧａｌＮＡｃである、上記［２６］記載の化合物。
［２８］Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、脂質である、上記［１９］～［２５］のいずれかに記載の化合物。 (In the formula, the definitions of each symbol are the same as those in [19] above.)
The compound according to the above-mentioned [19], represented by the formula:
[21] Spacers X 1 , X 2 , X 3 and X 4 each independently represent -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); and n' represents an integer from 1 to 10).
The compound according to the above [19] or [20],
[22] The compound according to any one of the above [19] to [21], wherein W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are —(CH 2 )—, and n 1 to n 6 are 1.
[23] The compound according to any one of the above [19] to [22], wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 6 -O-.
[24] The compound according to any one of the above [19] to [22], wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 5 -NH-.
[25] The compound according to any one of the above [19] to [24], wherein the ligands of Y 1 , Y 2 , Y 3 and Y 4 are each independently at least one selected from the group consisting of a proteinaceous substance, a nucleic acid, a biologically active substance, a sugar and a lipid.
[26] The compound according to any one of the above [19] to [25], wherein the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a sugar.
[27] The compound according to the above-mentioned [26], wherein the sugar is GalNAc.
[28] The compound according to any one of the above-mentioned [19] to [25], wherein the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a lipid.
　本発明の化合物であるイミノジカルボン酸誘導体からなるリガンドコンジュゲート核酸は、血清安定性、適切な臓器または細胞への送達、および膜貫通送達を可能とし、更に低い製造コストで高い機能性を付与する、治療及び／又は予防剤を提供することができた。 The ligand-conjugated nucleic acid consisting of the iminodicarboxylic acid derivative, which is the compound of the present invention, is capable of serum stability, delivery to appropriate organs or cells, and transmembrane delivery, and can provide a therapeutic and/or prophylactic agent that provides high functionality at low production costs.
実施例１－１で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 1-1. 実施例１－２で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 1-2. 実施例１－３で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is a RP-HPLC chart of the compound prepared in Example 1-3. 実施例１－４で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is a RP-HPLC chart of the compound prepared in Example 1-4. 実施例１－５で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is a RP-HPLC chart of the compounds prepared in Examples 1-5. 実施例１－６で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is a RP-HPLC chart of the compounds prepared in Examples 1-6. 実施例１－７で製造した化合物のＲＰ－ＨＰＬＣチャートである。RP-HPLC charts of the compounds prepared in Examples 1-7. 実施例１－８で製造した化合物のＲＰ－ＨＰＬＣチャートである。RP-HPLC charts of the compounds prepared in Examples 1-8. 実施例１－９で製造した化合物のＲＰ－ＨＰＬＣチャートである。RP-HPLC charts of the compounds prepared in Examples 1-9. 実施例２－１で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 2-1. 実施例２－２で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 2-2. 実施例２－３で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 2-3. 実施例２－４で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 2-4. 実施例３－１で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 3-1. 実施例３－２で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 3-2. 実施例３－３で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 3-3. 実施例４－１で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 4-1. 実施例４－２で製造した化合物のＲＰ－ＨＰＬＣチャートである。1 is an RP-HPLC chart of the compound prepared in Example 4-2. ＨＤＯあるいはＧａｌＮＡｃ修飾ＨＤＯを静脈内投与したマウスの血中Ｆａｃｔｏｒ　ＶＩＩ濃度を比較した結果を示すグラフである。This is a graph showing the results of comparing the blood Factor VII concentrations of mice that were intravenously administered HDO or GalNAc-modified HDO. ｓｉＲＮＡあるいはＧａｌＮＡｃ修飾したｓｉＲＮＡを投与（皮下投与）したマウスの血中Ｆａｃｔｏｒ　ＶＩＩ濃度を比較した結果を示すグラフである。This is a graph showing the results of comparing the blood Factor VII concentration of mice administered siRNA or GalNAc-modified siRNA (subcutaneous administration). ｓｉＲＮＡあるいはＧａｌＮＡｃ修飾したｓｉＲＮＡを投与（経肺投与）したマウスの血中Ｆａｃｔｏｒ　ＶＩＩ濃度を比較した結果を示すグラフである。This is a graph showing the results of comparing the blood Factor VII concentration of mice administered siRNA or GalNAc-modified siRNA (pulmonary administration).
　本明細書で使用する用語は、特に言及しない限り、当該技術分野で通常用いられる意味で用いることができる。
　以下に本発明で使用する用語および記号を定義する。 Terms used in this specification can be used in the same manner as commonly used in the art unless otherwise specified.
The terms and symbols used in the present invention are defined below.
　「アミノ基保護基」とは、具体的には、アセチル、トリクロロアセチル、トリフルオロアセチル、ベンゾイル、Ｎ－フタルイミド等のアミド系、及び９－フルオレニルメトキシカルボニル、ベンジルオキシカルボニル、ｔ－ブトキシカルボニル等のカルバメート系が挙げられる。 Specific examples of "amino group protecting groups" include amide-based groups such as acetyl, trichloroacetyl, trifluoroacetyl, benzoyl, and N-phthalimide, and carbamate-based groups such as 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and t-butoxycarbonyl.
　「水酸基保護基」とは、水酸基の反応を防ぐために導入される、当業者に公知の一般的な水酸基の保護基を意味し、例えば、Protective Groups in Organic Synthesis, published by John Wiley and Sons (1980)に記載の保護基等であり、具体的には、アセチル、ベンゾイル等のアシル系保護基、トリチル、４－メトキシトリチル、４，４’－ジメトキシトリチル、ベンジル等のアルキル系保護基、トリメチルシリル、ｔｅｒｔ－ブチルジメチルシリル、ｔｅｒｔ－ブチルジフェニルシリル等のシリル系保護基が挙げられる。 The term "hydroxyl-protecting group" refers to a general hydroxyl-protecting group known to those skilled in the art that is introduced to prevent reactions of the hydroxyl group, such as the protecting groups described in Protective Groups in Organic Synthesis, published by John Wiley and Sons (1980). Specific examples include acyl-based protecting groups such as acetyl and benzoyl, alkyl-based protecting groups such as trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl and benzyl, and silyl-based protecting groups such as trimethylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl.
　「電子求引基」とは、水素原子と比べて、結合原子側から電子をひきつけやすい基を示し、具体的には、シアノ、ニトロ、アルキルスルホニル（例、メチルスルホニル、エチルスルホニル）、ハロゲン（フッ素原子、塩素原子、臭素原子またはヨウ素原子）、アリールスルホニル（例、フェニルスルホニル、ナフチルスルホニル）、トリハロメチル（例、トリクロロメチル、トリフルオロメチル）等が挙げられる。 The term "electron-withdrawing group" refers to a group that is more likely to attract electrons from the bonding atom than a hydrogen atom, and specific examples include cyano, nitro, alkylsulfonyl (e.g., methylsulfonyl, ethylsulfonyl), halogen (fluorine atom, chlorine atom, bromine atom, or iodine atom), arylsulfonyl (e.g., phenylsulfonyl, naphthylsulfonyl), trihalomethyl (e.g., trichloromethyl, trifluoromethyl), etc.
　「ハロゲン」としてはフッ素原子、塩素原子、臭素原子及びヨウ素原子が挙げられる。 "Halogen" includes fluorine, chlorine, bromine and iodine atoms.
　「ヘテロ環」とは、窒素原子、酸素原子、硫黄原子のうち少なくとも一つを含む５～７員環の、置換もしくは無置換の、飽和もしくは不飽和のヘテロ環である。これらは単環であっても良いし、更に他のアリール環もしくはヘテロ環と共に縮合環を形成しても良い。ヘテロ環としては、例えば、フラン環、ジヒドロフラン環、テトラヒドロフラン環、ピラン環、ジヒドロピラン環、テトラヒドロピラン環、ベンゾフラン環、イソベンゾフラン環、クロメン環、クロマン環、イソクロマン環、チオフェン環、ベンゾチオフェン環、ピロール環、ピロリン環、ピロリジン環、イミダゾール環、イミダゾリン環、イミダゾリジン環、ピラゾール環、ピラゾリン環、ピラゾリジン環、トリアゾール環、テトラゾール環、ピリジン環、ピリジンオキシド環、ピペリジン環、ピラジン環、ピペラジン環、ピリミジン環、ピリダジン環、インドリジン環、インドール環、インドリン環、イソインドール環、イソインドリン環、インダゾール環、ベンゾイミダゾール環、プリン環、キノリジン環、キノリン環、フタラジン環、ナフチリジン環、キノキサリン環、キナゾリン環、シンノリン環、プテリジン環、オキサゾール環、オキサゾリジン環、イソキサゾール環、イソキサゾリジン環、チアゾール環、ベンゾチアゾール環、チアジリジン環、イソチアゾール環、イソチアゾリジン環、ジオキサン環、ジチアン環、モルホリン環、チオモルホリン環、フタルイミド環等を挙げることができる。 The term "heterocycle" refers to a 5-7 membered ring that contains at least one of a nitrogen atom, an oxygen atom, and a sulfur atom, and is substituted or unsubstituted, saturated or unsaturated. These may be monocyclic or may form a condensed ring together with another aryl ring or heterocycle. Examples of heterocycles include a furan ring, a dihydrofuran ring, a tetrahydrofuran ring, a pyran ring, a dihydropyran ring, a tetrahydropyran ring, a benzofuran ring, an isobenzofuran ring, a chromene ring, a chroman ring, an isochroman ring, a thiophene ring, a benzothiophene ring, a pyrrole ring, a pyrroline ring, a pyrrolidine ring, an imidazole ring, an imidazoline ring, an imidazolidine ring, a pyrazole ring, a pyrazoline ring, a pyrazolidine ring, a triazole ring, a tetrazole ring, a pyridine ring, a pyridine oxide ring, a piperidine ring, a pyrazine ring, a piperazine ring, a pyrimidine ring, a pyridazine ring, Examples of the ring include an indolizine ring, an indole ring, an indoline ring, an isoindole ring, an isoindoline ring, an indazole ring, a benzimidazole ring, a purine ring, a quinolizine ring, a quinoline ring, a phthalazine ring, a naphthyridine ring, a quinoxaline ring, a quinazoline ring, a cinnoline ring, a pteridine ring, an oxazole ring, an oxazolidine ring, an isoxazole ring, an isoxazolidine ring, a thiazole ring, a benzothiazole ring, a thiaziridine ring, an isothiazole ring, an isothiazolidine ring, a dioxane ring, a dithiane ring, a morpholine ring, a thiomorpholine ring, and a phthalimide ring.
　「アルキル基」（アルキル、アルキル鎖）とは、炭素数１～３０、好ましくは１～１２、より好ましくは１～６、特に好ましくは１～４の直鎖又は分岐鎖アルキル基を意味し、具体的には、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－ブチルおよびｔｅｒｔ－ブチル、ペンチル、ヘキシル、ヘプチル、オクチル、ノニル、デシル、ウンデシル、ドデシル、トリデシル、テトラデシル、ペンタデシル、ヘキサデシル、ヘプタデシル、オクタデシル、ノナデシル、イコシル等が挙げられる。好ましくは、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－ぺンチル、イソペンチル、ネオペンチル、ｎ－ヘキシル、イソヘキシル等が挙げられる。「炭素数１～１０のアルキル基」としては、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－ブチルおよびｔｅｒｔ－ブチル、ペンチル、ヘキシル、ヘプチル、オクチル、ノニル、デシル等が挙げられ、好ましくはＣ１－６アルキル基（例、メチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ペンチル、イソペンチル、ネオペンチル、１－エチルプロピル、ヘキシル、イソヘキシル、１，１－ジメチルブチル、２，２－ジメチルブチル、３，３－ジメチルブチル、２－エチルブチル）が挙げられる。 The term "alkyl group" (alkyl, alkyl chain) refers to a straight-chain or branched-chain alkyl group having 1 to 30 carbon atoms, preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 4 carbon atoms, and specific examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and icosyl. Preferred examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl. Examples of the "alkyl group having 1 to 10 carbon atoms" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like, and preferably include a C1-6 alkyl group (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl).
　「アルケニル基」（アルケニル）とは、炭素数２～３０、好ましくは２～１２、より好ましく２～８の直鎖又は分岐鎖アルケニル基を意味し、前記アルキル基において、１個又は複数の二重結合を有するもの等が挙げられる。具体的には、ビニル、１－プロペニル、２－プロペニル、１－ブテニル、２－ブテニル、３－ブテニル、１，３－ブタジエニル、３－メチル－２－ブテニル等が挙げられる。 "Alkenyl group" (alkenyl) means a straight-chain or branched-chain alkenyl group having 2 to 30 carbon atoms, preferably 2 to 12, and more preferably 2 to 8 carbon atoms, and examples of the alkyl group include those having one or more double bonds. Specific examples include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, and 3-methyl-2-butenyl.
　「アルキニル基」（アルキニル）とは、炭素数２～３０、好ましくは２～１２、より好ましく２～８の直鎖又は分岐鎖アルキニル基を意味し、前記アルキル基において、１個又は複数の三重結合を有するもの等が挙げられる。具体的には、エチニル、プロピニル、プロパルギル、ブチニル、ペンチニル、ヘキシニル等が挙げられる。該アルキニル基は、さらに１個又は複数の二重結合を有していてもよい。 "Alkynyl group" (alkynyl) means a straight-chain or branched-chain alkynyl group having 2 to 30 carbon atoms, preferably 2 to 12, and more preferably 2 to 8 carbon atoms, and examples of the alkyl group include those having one or more triple bonds. Specific examples include ethynyl, propynyl, propargyl, butynyl, pentynyl, and hexynyl. The alkynyl group may further have one or more double bonds.
　「アルコキシ基」（アルコキシ）とは、炭素数１～３０、好ましくは１～１２、より好ましくは１～６、特に好ましくは１～４の直鎖又は分岐鎖アルコキシ基を意味し、具体的には、メトキシ、エトキシ、ｎ－プロポキシ、イソプロポキシ、ｎ－ブトキシ、イソブトキシ、ｓｅｃ－ブトキシ、ｔｅｒｔ－ブトキシ、ｎ－ペンチルオキシ、イソペンチルオキシ、ｔｅｒｔ－ペンチルオキシ、ネオペンチルオキシ、２－ペンチルオキシ、３－ペンチルオキシ、ｎ－ヘキシルオキシ、２－ヘキシルオキシ等が挙げられる。 "Alkoxy group" (alkoxy) means a straight-chain or branched-chain alkoxy group having 1 to 30 carbon atoms, preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 4 carbon atoms, and specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, neopentyloxy, 2-pentyloxy, 3-pentyloxy, n-hexyloxy, 2-hexyloxy, etc.
　「アリール基」とは、炭素数６～２４、好ましくは６～１０のアリール基を意味し、フェニル等の単環芳香族炭化水素基、１－ナフチル、２－ナフチル、１－アントリル、２－アントリル、９－アントリル、１－フェナントリル、２－フェナントリル、３－フェナントリル、４－フェナントリル、９－フェナントリル等の多環芳香族炭化水素基が挙げられる。「炭素数６～１０のアリール基」としては、上記アリール基のうち、炭素数が６～１０のものが挙げられ、具体的にはフェニル、ナフチル等が挙げられる。 "Aryl group" refers to an aryl group having 6 to 24 carbon atoms, preferably 6 to 10 carbon atoms, and examples of such groups include monocyclic aromatic hydrocarbon groups such as phenyl, and polycyclic aromatic hydrocarbon groups such as 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, and 9-phenanthryl. "Aryl groups having 6 to 10 carbon atoms" include the above aryl groups having 6 to 10 carbon atoms, and specific examples include phenyl, naphthyl, etc.
　「アリーレン基」とは、上記で例示したアリール基から水素を１つ除去した２価の置換基を表す。 An "arylene group" refers to a divalent substituent formed by removing one hydrogen from the aryl group exemplified above.
　「ヘテロシクロアルキル基」とは、炭素数６～２４、好ましくは６～１０のヘシクロアルキル基を意味する。ヘテロシクロアルキル基としては、後述のシクロアルキル基の環状構造を形成する１個若しくはそれ以上の炭素原子が、窒素原子、酸素原子、硫黄原子などで置換されたものが挙げられる。具体的には、［１，３］ジオキソラニル、ピロリジニル、ピラゾリニル、ピラゾリジニル、イミダゾリニル、イミダゾリジニル、ピペリジニル、ピペラジニル、オキサゾリジニル、イソオキサゾリジニル、モルホリニル、チアゾリジニル、イソチアゾリジニル、及びテトラヒドロフリル等が挙げられる。「炭素数４～１０のヘテロシクロアルキル基」としては上記ヘテロシクロアルキル基のうち、炭素数が４～１０のものが挙げられ、具体的にはピロリジニル、ピペリジニル、ピペラジニル、モルホリニル、オキセタニル、テトラヒドロフリル、テトラヒドロピラニル、チオキセタニル、テトラヒドロチエニル、及びテトラヒドロチオピラニル基等が挙げられる。 The term "heterocycloalkyl group" refers to a heterocycloalkyl group having 6 to 24 carbon atoms, preferably 6 to 10 carbon atoms. Examples of heterocycloalkyl groups include those in which one or more carbon atoms forming the cyclic structure of the cycloalkyl group described below are substituted with a nitrogen atom, an oxygen atom, a sulfur atom, or the like. Specific examples include [1,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Examples of "heterocycloalkyl groups having 4 to 10 carbon atoms" include the above heterocycloalkyl groups having 4 to 10 carbon atoms, specifically pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl, thioxetanyl, tetrahydrothienyl, and tetrahydrothiopyranyl groups.
　「ヘテロシクロアルキレン基」とは、上記で例示したヘテロシクロアルキル基から水素を１つ除去した２価の置換基を表す。 The term "heterocycloalkylene group" refers to a divalent substituent formed by removing one hydrogen from the heterocycloalkyl groups listed above.
　「アラルキル基」とは、炭素数７～３０、好ましくは７～１１のアラルキル基を意味し、具体的には、ベンジル、２－フェネチル、およびナフタレニルメチル等が挙げられる。 "Aralkyl group" means an aralkyl group having 7 to 30 carbon atoms, preferably 7 to 11 carbon atoms, and specific examples include benzyl, 2-phenethyl, and naphthalenylmethyl.
　「シクロアルキル基」とは、炭素数３～２４、好ましくは３～１５のシクロアルキル基を意味し、具体的には、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、橋かけ環式炭化水素基、スピロ炭化水素基等が挙げられ、好ましくは、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、橋かけ環式炭化水素基等が挙げられる。「橋かけ環式炭化水素基」としては、ビシクロ［２．１．０］ペンチル、ビシクロ［２．２．１］ヘプチル、ビシクロ［２．２．２］オクチル、ビシクロ［３．２．１］オクチル、トリシクロ［２．２．１．０］ヘプチル、ビシクロ［３．３．１］ノニル、１－アダマンチル、２－アダマンチル等が挙げられる。「スピロ炭化水素基」としては、スピロ［３．４］オクチル等が挙げられる。「炭素数４～１０のシクロアルキル基」としては、上記シクロアルキル基のうち、炭素数が４～１０のものが挙げられ、具体的にはシクロブチル、シクロペンチル、シクロヘキシル、シクロヘプチル、シクロオクチル等が挙げられる。 "Cycloalkyl group" means a cycloalkyl group having 3 to 24 carbon atoms, preferably 3 to 15 carbon atoms, and specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bridged cyclic hydrocarbon groups, spiro hydrocarbon groups, etc., and preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bridged cyclic hydrocarbon groups, etc. Examples of "bridged cyclic hydrocarbon groups" include bicyclo[2.1.0]pentyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, tricyclo[2.2.1.0]heptyl, bicyclo[3.3.1]nonyl, 1-adamantyl, 2-adamantyl, etc. Examples of "spiro hydrocarbon groups" include spiro[3.4]octyl, etc. Examples of "cycloalkyl groups having 4 to 10 carbon atoms" include the above cycloalkyl groups having 4 to 10 carbon atoms, such as cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
　「シクロアルキレン基」とは、上記で例示したシクロアルキル基から水素を１つ除去した２価の置換基を表す。 "Cycloalkylene group" refers to a divalent substituent formed by removing one hydrogen from the cycloalkyl groups listed above.
　「シクロアルケニル基」とは、少なくとも１個、好ましくは１または２個の二重結合を含む炭素数３～２４、好ましくは３～７のシクロアルケニル基を意味し、具体的には、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、シクロヘプテニル等が挙げられる。前記シクロアルケニル基は、環中に不飽和結合を有する橋かけ環式炭化水素基およびスピロ炭化水素基も含む。「環中に不飽和結合を有する橋かけ環式炭化水素基」としては、ビシクロ［２．２．２］オクテニル、ビシクロ［３．２．１］オクテニル、トリシクロ［２．２．１．０］ヘプテニル等が挙げられる。「環中に不飽和結合を有するスピロ炭化水素基」としては、スピロ［３．４］オクテニル等が挙げられる。 "Cycloalkenyl group" means a cycloalkenyl group having 3 to 24 carbon atoms, preferably 3 to 7 carbon atoms, containing at least one, preferably one or two double bonds, and specifically includes cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, etc. The cycloalkenyl group also includes bridged cyclic hydrocarbon groups and spiro hydrocarbon groups having an unsaturated bond in the ring. Examples of "bridged cyclic hydrocarbon groups having an unsaturated bond in the ring" include bicyclo[2.2.2]octenyl, bicyclo[3.2.1]octenyl, tricyclo[2.2.1.0]heptenyl, etc. Examples of "spiro hydrocarbon groups having an unsaturated bond in the ring" include spiro[3.4]octenyl, etc.
　「シクロアルキルアルキル基」とは、前記シクロアルキル基で置換されたアルキル基（上述）を意味し、好ましくは炭素数４～３０、より好ましくは４～１１のシクロアルキルアルキル基を意味する。具体的には、シクロプロピルメチル、２－シクロブチルエチル、シクロペンチルメチル、３－シクロペンチルプロピル、シクロヘキシルメチル、２－シクロヘキシルエチル、シクロヘプチルメチル等が挙げられる。 "Cycloalkylalkyl group" refers to an alkyl group (described above) substituted with the cycloalkyl group, preferably a cycloalkylalkyl group having 4 to 30 carbon atoms, more preferably 4 to 11 carbon atoms. Specific examples include cyclopropylmethyl, 2-cyclobutylethyl, cyclopentylmethyl, 3-cyclopentylpropyl, cyclohexylmethyl, 2-cyclohexylethyl, cycloheptylmethyl, etc.
　「アルコキシアルキル基」とは、前記アルコキシ基で置換されたアルキル基（上述）を意味し、好ましくは炭素数２～３０、より好ましくは２～１２の直鎖又は分岐鎖アルコキシアルキル基を意味する。具体的には、メトキシメチル、メトキシエチル、エトキシメチル、エトキシエチルおよびｔ－ブトキシメチル等が挙げられる。 "Alkoxyalkyl group" refers to an alkyl group (described above) substituted with the above alkoxy group, and preferably refers to a straight-chain or branched-chain alkoxyalkyl group having 2 to 30 carbon atoms, more preferably 2 to 12 carbon atoms. Specific examples include methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, and t-butoxymethyl.
　「アルキレン基」（アルキレン鎖）とは、炭素数１～３０、好ましくは１～１２、より好ましくは１～６、特に好ましくは１～４の直鎖又は分岐鎖アルキレン基を意味し、具体的には、メチレン、エチレン、およびプロピレン等が挙げられる。「一緒になってアルキレン基を形成する」とは２つの置換基が一緒になって＝ＣＨ２（メチリデン基ともいう）を形成することを意味する。 The term "alkylene group" (alkylene chain) means a straight-chain or branched-chain alkylene group having 1 to 30 carbon atoms, preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 4 carbon atoms, and specific examples include methylene, ethylene, and propylene. The term "together to form an alkylene group" means that two substituents together form =CH 2 (also called a methylidene group).
　「ヘテロアリール基」は、例えば、単環芳香族複素環式基および縮合芳香族複素環式基を含む。前記ヘテロアリールは、例えば、フリル（例：２－フリル、３－フリル）、チエニル（例：２－チエニル、３－チエニル）、ピロリル（例：１－ピロリル、２－ピロリル、３－ピロリル）、イミダゾリル（例：１－イミダゾリル、２－イミダゾリル、４－イミダゾリル）、ピラゾリル（例：１－ピラゾリル、３－ピラゾリル、４－ピラゾリル）、トリアゾリル（例：１，２，４－トリアゾール－１－イル、１，２，４－トリアゾール－３－イル、１，２，４－トリアゾール－４－イル）、テトラゾリル（例：１－テトラゾリル、２－テトラゾリル、５－テトラゾリル）、オキサゾリル（例：２－オキサゾリル、４－オキサゾリル、５－オキサゾリル）、イソキサゾリル（例：３－イソキサゾリル、４－イソキサゾリル、５－イソキサゾリル）、チアゾリル（例：２－チアゾリル、４－チアゾリル、５－チアゾリル）、チアジアゾリル、イソチアゾリル（例：３－イソチアゾリル、４－イソチアゾリル、５－イソチアゾリル）、ピリジル（例：２－ピリジル、３－ピリジル、４－ピリジル）、ピリダジニル（例：３－ピリダジニル、４－ピリダジニル）、ピリミジニル（例：２－ピリミジニル、４－ピリミジニル、５－ピリミジニル）、フラザニル（例：３－フラザニル）、ピラジニル（例：２－ピラジニル）、オキサジアゾリル（例：１，３，４－オキサジアゾール－２－イル）、ベンゾフリル（例：２－ベンゾ［ｂ］フリル、３－ベンゾ［ｂ］フリル、４－ベンゾ［ｂ］フリル、５－ベンゾ［ｂ］フリル、６－ベンゾ［ｂ］フリル、７－ベンゾ［ｂ］フリル）、ベンゾチエニル（例：２－ベンゾ［ｂ］チエニル、３－ベンゾ［ｂ］チエニル、４－ベンゾ［ｂ］チエニル、５－ベンゾ［ｂ］チエニル、６－ベンゾ［ｂ］チエニル、７－ベンゾ［ｂ］チエニル）、ベンズイミダゾリル（例：１－ベンズイミダゾリル、２－ベンズイミダゾリル、４－ベンズイミダゾリル、５－ベンゾイミダゾリル）、ジベンゾフリル、ベンゾオキサゾリル、ベンゾチアゾリル、キノキサリニル（例：２－キノキサリニル、５－キノキサリニル、６－キノキサリニル）、シンノリニル（例：３－シンノリニル、４－シンノリニル、５－シンノリニル、６－シンノリニル、７－シンノリニル、８－シンノリニル）、キナゾリニル（例：２－キナゾリニル、４－キナゾリニル、５－キナゾリニル、６－キナゾリニル、７－キナゾリニル、８－キナゾリニル）、キノリル（例：２－キノリル、３－キノリル、４－キノリル、５－キノリル、６－キノリル、７－キノリル、８－キノリル）、フタラジニル（例：１－フタラジニル、５－フタラジニル、６－フタラジニル）、イソキノリル（例：１－イソキノリル、３－イソキノリル、４－イソキノリル、５－イソキノリル、６－イソキノリル、７－イソキノリル、８－イソキノリル）、プリル、プテリジニル（例：２－プテリジニル、４－プテリジニル、６－プテリジニル、７－プテリジニル）、カルバゾリル、フェナントリジニル、アクリジニル（例：１－アクリジニル、２－アクリジニル、３－アクリジニル、４－アクリジニル、９－アクリジニル）、インドリル（例：１－インドリル、２－インドリル、３－インドリル、４－インドリル、５－インドリル、６－インドリル、７－インドリル）、イソインドリル、フェナジニル（例：１－フェナジニル、２－フェナジニル）またはフェノチアジニル（例：１－フェノチアジニル、２－フェノチアジニル、３－フェノチアジニル、４－フェノチアジニル）等が挙げられる。
　「炭素数２～１０のヘテロアリール基」としては上記ヘテロアリール基のうち、炭素数が２～１０のものが挙げられ、具体的にはフリル基、チエニル基、ピロリル基、オキサゾリル基、トリアゾリル基、ピリジル基、キノリニル基等が挙げられる。 The "heteroaryl group" includes, for example, a monocyclic aromatic heterocyclic group and a fused aromatic heterocyclic group. The heteroaryl includes, for example, furyl (e.g., 2-furyl, 3-furyl), thienyl (e.g., 2-thienyl, 3-thienyl), pyrrolyl (e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (e.g., 1-imidazolyl, 2-imidazolyl, 4-imidazolyl), pyrazolyl (e.g., 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl), triazolyl (e.g., 1,2,4-triazol-1-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-4-yl), tetrazolyl (e.g., 1-tetrazolyl, 2-tetrazolyl, 5-tetrazolyl), oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), isoxazolyl (e.g., 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), thiazolyl (e.g., 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), thiadiazolyl, isothiazolyl (e.g., 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl), pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl), pyridazinyl (e.g., 3-pyridazinyl, 4-pyridazinyl), pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), furazanyl (e.g., 3-furazanyl), pyrazinyl (e.g., 2-pyrazinyl), oxy sadiazolyl (e.g., 1,3,4-oxadiazol-2-yl), benzofuryl (e.g., 2-benzo[b]furyl, 3-benzo[b]furyl, 4-benzo[b]furyl, 5-benzo[b]furyl, 6-benzo[b]furyl, 7-benzo[b]furyl), benzothienyl (e.g., 2-benzo[b]thienyl, 3-benzo[b]thienyl, 4-benzo[b]thienyl, 5-benzo[b]thienyl, 6-benzo[b]thienyl, 7-benzo[b]furyl), enyl, 7-benzo[b]thienyl), benzimidazolyl (e.g., 1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl), dibenzofuryl, benzoxazolyl, benzothiazolyl, quinoxalinyl (e.g., 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl), cinnolinyl (e.g., 3-cinnolinyl, 4-cinnolinyl, 5-cinnolinyl), , 6-cinnolinyl, 7-cinnolinyl, 8-cinnolinyl), quinazolinyl (e.g., 2-quinazolinyl, 4-quinazolinyl, 5-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl, 8-quinazolinyl), quinolyl (e.g., 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), phthalazinyl (e.g., 1-phthalazinyl, 5-phthalazinyl, 6-phthalazinyl, nyl), isoquinolyl (e.g., 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), pryl, pteridinyl (e.g., 2-pteridinyl, 4-pteridinyl, 6-pteridinyl, 7-pteridinyl), carbazolyl, phenanthridinyl, acridinyl (e.g., 1-acridinyl, 2-acridinyl, 3-acridinyl, 4- acridinyl, 9-acridinyl), indolyl (e.g., 1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), isoindolyl, phenazinyl (e.g., 1-phenazinyl, 2-phenazinyl), or phenothiazinyl (e.g., 1-phenothiazinyl, 2-phenothiazinyl, 3-phenothiazinyl, 4-phenothiazinyl), and the like.
Examples of the "heteroaryl group having 2 to 10 carbon atoms" include the above-mentioned heteroaryl groups having 2 to 10 carbon atoms, and specific examples thereof include a furyl group, a thienyl group, a pyrrolyl group, an oxazolyl group, a triazolyl group, a pyridyl group, and a quinolinyl group.
　「ヘテロアリーレン基」とは、上記で例示したヘテロアリール基から水素を１つ除去した２価の置換基を表す。 The term "heteroarylene group" refers to a divalent substituent formed by removing one hydrogen from the heteroaryl group exemplified above.
　本発明において「置換されていてもよい」とは「置換基を有していてもよい」と同義であるが、「置換基」としては、下記置換基群Ａに記載のものが挙げられる。
置換基群Ａ
（１）ハロゲン（フッ素原子、塩素原子、臭素原子またはヨウ素原子）；
（２）アルキル基（上述）；
（３）アルコキシ基（上述）；
（４）アルケニル基（上述）；
（５）アルキニル基（上述）；
（６）ハロアルキル基（例、クロロメチル、フルオロメチル、ジクロロメチル、ジフルオロメチル、ジクロロフルオロメチル、トリフルオロメチル、ペンタフルオロエチル等）；
（７）アリール基（上述）；
（８）ヘテロアリール基（上述）；
（９）アラルキル基（上述）；
（１０）シクロアルキル基（上述）；
（１１）シクロアルケニル基（上述）；
（１２）シクロアルキルアルキル基（上述）；
（１３）シクロアルケニルアルキル基（例、シクロペンテニルエチル、シクロヘキセニルエチル、シクロヘキセニルブチル等）；
（１４）ヒドロキシアルキル基（例、ヒドロキシメチル、ヒドロキシエチル、ヒドロキシプロピル、ヒドロキシブチル等）；
（１５）アルコキシアルキル基（上述）；
（１６）アミノアルキル基（例、アミノメチル、アミノエチル、アミノプロピル等）；
（１７）ヘテロシクリル基（例、１－ピロリニル、２－ピロリニル、３－ピロリニル、１－ピロリジニル、２－ピロリジニル、３－ピロリジニル、ピロリジノニル、１－イミダゾリニル、２－イミダゾリニル、４－イミダゾリニル、１－イミダゾリジニル、２－イミダゾリジニル、４－イミダゾリジニル、イミダゾリジノニル、１－ピラゾリニル、３－ピラゾリニル、４－ピラゾリニル、１－ピラゾリジニル、３－ピラゾリジニル、４－ピラゾリジニル、ピペリジノニル、ピペリジノ、２－ピペリジニル、３－ピペリジニル、４－ピペリジニル、１－ピペラジニル、２－ピペラジニル、ピペラジノニル、２－モルホリニル、３－モルホリニル、モルホリノ、テトラヒドロピラニル、テトラヒドロフラニル等）；
（１８）ヘテロシクリルアルケニル基（例、２－ピペリジニルエテニル等）；
（１９）ヘテロシクリルアルキル基（例、ピペリジニルメチル、ピペラジニルメチル等）；
（２０）ヘテロアリールアルキル基（例、ピリジルメチル、キノリン－３－イルメチル等）；
（２１）シリル基；
（２２）シリルオキシアルキル基（例、シリルオキシメチル、シリルオキシエチル等）；
（２３）モノ・ジもしくはトリアルキルシリル基（例、メチルシリル、エチルシリル等）；及び
（２４）モノ・ジもしくはトリアルキルシリルオキシアルキル基（例、トリメチルシリルオキシメチル等）。 In the present invention, the term "optionally substituted" has the same meaning as "optionally having a substituent", and examples of the "substituent" include those described in the following Substituent Group A.
Substituent Group A
(1) a halogen (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom);
(2) an alkyl group (described above);
(3) an alkoxy group (described above);
(4) an alkenyl group (described above);
(5) an alkynyl group (described above);
(6) haloalkyl groups (e.g., chloromethyl, fluoromethyl, dichloromethyl, difluoromethyl, dichlorofluoromethyl, trifluoromethyl, pentafluoroethyl, etc.);
(7) an aryl group (described above);
(8) heteroaryl groups (described above);
(9) an aralkyl group (described above);
(10) a cycloalkyl group (described above);
(11) a cycloalkenyl group (described above);
(12) cycloalkylalkyl group (described above);
(13) cycloalkenylalkyl groups (e.g., cyclopentenylethyl, cyclohexenylethyl, cyclohexenylbutyl, etc.);
(14) hydroxyalkyl groups (e.g., hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, etc.);
(15) alkoxyalkyl groups (described above);
(16) aminoalkyl groups (e.g., aminomethyl, aminoethyl, aminopropyl, etc.);
(17) heterocyclyl groups (e.g., 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, pyrrolidinonyl, 1-imidazolinyl, 2-imidazolinyl, 4-imidazolinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, imidazolidinonyl, 1-pyrazolinyl, 3-pyridazolinyl, pyrazolinyl, 4-pyrazolinyl, 1-pyrazolidinyl, 3-pyrazolidinyl, 4-pyrazolidinyl, piperidinonyl, piperidino, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 1-piperazinyl, 2-piperazinyl, piperazinonyl, 2-morpholinyl, 3-morpholinyl, morpholino, tetrahydropyranyl, tetrahydrofuranyl, etc.);
(18) heterocyclylalkenyl groups (e.g., 2-piperidinylethenyl, etc.);
(19) heterocyclylalkyl groups (e.g., piperidinylmethyl, piperazinylmethyl, etc.);
(20) heteroarylalkyl groups (e.g., pyridylmethyl, quinolin-3-ylmethyl, etc.);
(21) a silyl group;
(22) silyloxyalkyl groups (e.g., silyloxymethyl, silyloxyethyl, etc.);
(23) mono-, di- or trialkylsilyl groups (eg, methylsilyl, ethylsilyl, etc.); and (24) mono-, di- or trialkylsilyloxyalkyl groups (eg, trimethylsilyloxymethyl, etc.).
リガンドコンジュゲート核酸
　本発明は、下記一般式（Ｉ）で表される化合物を提供する。当該化合物は、イミノジカルボン酸誘導体からなる基を介して核酸分子とリガンドとを連結させた化合物であり、本明細書中、リガンドコンジュゲート核酸とも称する。 The present invention provides a compound represented by the following general formula (I): The compound is a compound in which a nucleic acid molecule and a ligand are linked via a group consisting of an iminodicarboxylic acid derivative, and is also referred to as a ligand -conjugated nucleic acid in this specification.
［式中、
Ａは、核酸分子であり、
Ｘは、－（ＣＲ１Ｒ２）ｎ－Ｒ３－（式中、Ｒ１及びＲ２は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ３は－ＮＲ４－（式中、Ｒ４は、水素原子又は置換されていてもよいアルキル基である）又は－Ｓ－ヘテロ環－ＣＲ５Ｒ６－ＮＲ７－（式中、Ｒ５、Ｒ６、Ｒ７は、それぞれ独立して、水素原子又は置換されていてもよいアルキル基である）を表す；ｎは１～１０の整数を表す）を表し、
Ｂ１、Ｂ２、Ｂ３及びＢ４は、それぞれ独立して、水素原子又は下記式のいずれかを表し、
－（Ｗ２）ｎ２－ＣＯ－ＮＨ－Ｘ１－Ｙ１、
－（Ｗ３）ｎ３－ＣＯ－ＮＨ－Ｘ２－Ｙ２、
－（Ｗ５）ｎ５－ＣＯ－ＮＨ－Ｘ３－Ｙ３、
－（Ｗ６）ｎ６－ＣＯ－ＮＨ－Ｘ４－Ｙ４、
Ｗ２は、－（ＣＲ１Ｗ２Ｒ２Ｗ２）－（式中、Ｒ１Ｗ２及びＲ２Ｗ２は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ３は、－（ＣＲ１Ｗ３Ｒ２Ｗ３）－（式中、Ｒ１Ｗ３及びＲ２Ｗ３は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ５は、－（ＣＲ１Ｗ５Ｒ２Ｗ５）－（式中、Ｒ１Ｗ５及びＲ２Ｗ５は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ６は、－（ＣＲ１Ｗ６Ｒ２Ｗ６）－（式中、Ｒ１Ｗ６及びＲ２Ｗ６は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し[但し、Ｂ１～Ｂ４は、同時に水素原子ではない] 、
ｎ２、ｎ３、ｎ５及びｎ６は、それぞれ独立して、１～５の整数であり、
ｎＢ１～ｎＢ４は、それぞれ独立して、1～３の整数を表し、
Ｘ０、Ｘ１、Ｘ２、Ｘ３及びＸ４は、それぞれ独立して、置換されても良いスペーサーを表し、
Ｙ１、Ｙ２、Ｙ３及びＹ４は、それぞれ独立して、リガンドを表し、
Ｗ１は、－（ＣＲ１Ｗ１Ｒ２Ｗ１）－（式中、Ｒ１Ｗ１及びＲ２Ｗ１は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ４は、－（ＣＲ１Ｗ４Ｒ２Ｗ４）－（式中、Ｒ１Ｗ４及びＲ２Ｗ４は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
ｎ１及びｎ４は、それぞれ独立して、１～５の整数である］。 [Wherein,
A is a nucleic acid molecule,
X represents -(CR 1 R 2 )n-R 3 - (wherein R 1 and R 2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 3 represents -NR 4 - (wherein R 4 is a hydrogen atom or an optionally substituted alkyl group) or -S-heterocycle-CR 5 R 6 -NR 7 - (wherein R 5 , R 6 and R 7 are each independently a hydrogen atom or an optionally substituted alkyl group); and n represents an integer of 1 to 10);
B 1 , B 2 , B 3 and B 4 each independently represent a hydrogen atom or any of the following formulae:
-( W2 ) n2 -CO-NH- X1 - Y1 ,
-( W3 ) n3 -CO-NH- X2 - Y2 ,
-( W5 ) n5 -CO-NH- X3 - Y3 ,
-( W6 ) n6 -CO-NH- X4 - Y4 ,
W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W5 represents -( CR1W5R2W5 )- (wherein R1W5 and R2W5 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W 6 represents -(CR 1W6 R 2W6 )- (wherein R 1W6 and R 2W6 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group), provided that B 1 to B 4 are not simultaneously hydrogen atoms;
n 2 , n 3 , n 5 and n 6 each independently represent an integer from 1 to 5;
n B1 to n B4 each independently represent an integer of 1 to 3;
X 0 , X 1 , X 2 , X 3 and X 4 each independently represent a spacer which may be substituted;
Y 1 , Y 2 , Y 3 and Y 4 each independently represent a ligand;
W 1 represents -(CR 1W1 R 2W1 )- (wherein R 1W1 and R 2W1 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group);
W 4 represents -(CR 1W4 R 2W4 )- (wherein R 1W4 and R 2W4 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
n1 and n4 are each independently an integer from 1 to 5.
　式（Ｉ）で表される本発明のリガンドコンジュゲート核酸は、好ましくは下記式（Ｉ－１）で表される化合物である。 The ligand-conjugated nucleic acid of the present invention represented by formula (I) is preferably a compound represented by the following formula (I-1):
（式中、各記号の定義は式（Ｉ）と同義である）
　式（Ｉ）及び式（Ｉ－１）中、Ｘは、好ましくは、－（ＣＨ２）６－ＮＨ－又は－（ＣＨ２）６－Ｓ－ヘテロ環－（ＣＨ２）２－ＮＨ－である。
　式（Ｉ）及び式（Ｉ－１）中、Ｘ０は、好ましくは、下記式（１）～（４）のいずれかで表されるスペーサー、好ましくは下記式（１）で表されるスペーサー、より好ましくは、Ｘ０のスペーサーは、－ＣＯ－（ＣＨ２）ｎＬ１－ＣＯ－（式中、ｎＬ１は１～１０の整数を表す）で表される。ｎＬ１は好ましくは６である。
（１）－ＣＯ－Ｌ１－ＣＯ－
（式中、Ｌ１は
(i)－（ＣＲ１Ｌ１Ｒ２Ｌ１）ｎＬ１（式中、Ｒ１Ｌ１及びＲ２Ｌ１は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ１は１～１０の整数を表す）、
(ii)－（ＣＨ２）ｎＬ１－Ｃｙ－（ＣＨ２）ｍＬ１－（式中、Ｃｙは置換されていてもよいアリーレン基、置換されていてもよいヘテロアリーレン基、置換されていてもよいシクロアルキレン基、又は置換されていてもよいヘテロシクロアルキレン基を表し；ｎＬ１及びｍＬ１は同一又は異なって、１～１０の整数を表す）、
(iii)－ＮＲ８－（式中、Ｒ８は水素原子又は置換されていてもよいアルキル基を表す）、
（２）－Ｌ２－ＣＯ－Ｌ３－ＣＯ－
（式中、Ｌ２は、－（ＣＲ１Ｌ２Ｒ２Ｌ２）ｎＬ２－（式中、Ｒ１Ｌ２及びＲ２Ｌ２は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ２は０～６の整数を表す）を表し；Ｌ３は、－（ＣＲ１Ｌ３Ｒ２Ｌ３）ｎＬ３－（式中、Ｒ１Ｌ３及びＲ２Ｌ３は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ３は０～６の整数を表す、但し、ｎＬ２及びｎＬ３は同時に０ではない）を表す）、
（３）－ＣＯ－Ｌ４－ＣＯ－Ｌ５－ＣＯ－Ｌ６－ＣＯ－
（式中、Ｌ４は、－（ＣＲ１Ｌ４Ｒ２Ｌ４）ｎＬ４（式中、Ｒ１Ｌ４及びＲ２Ｌ４は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ４は１～６の整数を表す）を表し；Ｌ５は、－（ＯＣ２Ｈ４）ｎＬ５（式中、ｎＬ５は１～６の整数を表す）を表し；Ｌ６は、－（ＣＲ１Ｌ６Ｒ２Ｌ６）ｎＬ６（式中、Ｒ１Ｌ６及びＲ２Ｌ６は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ６は１～６の整数を表す）を表す）、
（４）－ＣＯ－Ｌ７－ＣＨ２ＣＯ－
（式中、Ｌ７は、－（ＯＣ２Ｈ４）ｎＬ７（式中、ｎＬ７は１～６の整数を表す）。 (In the formula, the definitions of each symbol are the same as those in formula (I)).
In formula (I) and formula (I-1), X is preferably -(CH 2 ) 6 -NH- or -(CH 2 ) 6 -S-heterocycle-(CH 2 ) 2 -NH-.
In formula (I) and formula (I-1), X 0 is preferably a spacer represented by any one of the following formulas (1) to (4), preferably a spacer represented by the following formula (1), more preferably the spacer of X 0 is represented by -CO-(CH 2 )n L1 -CO- (wherein n L1 represents an integer of 1 to 10). n L1 is preferably 6.
(1) -CO- L1 -CO-
(Wherein, L1 is
(i) -(CR 1L1 R 2L1 ) n L1 (wherein R 1L1 and R 2L1 are the same or different and each represents a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; n L1 represents an integer of 1 to 10),
(ii) -(CH 2 ) n L1 -Cy-(CH 2 ) m L1 - (wherein Cy represents an optionally substituted arylene group, an optionally substituted heteroarylene group, an optionally substituted cycloalkylene group, or an optionally substituted heterocycloalkylene group; n L1 and m L1 may be the same or different and represent an integer of 1 to 10),
(iii) -NR 8 - (wherein R 8 represents a hydrogen atom or an optionally substituted alkyl group),
(2) -L2 -CO- L3 -CO-
(wherein L2 represents -( CR1L2R2L2 ) nL2- (wherein R1L2 and R2L2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; nL2 represents an integer of 0 to 6); L3 represents -( CR1L3R2L3 ) nL3- (wherein R1L3 and R2L3 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; nL3 represents an integer of 0 to 6, with the proviso that nL2 and nL3 are not simultaneously 0));
(3) -CO- L4 -CO- L5 -CO- L6 -CO-
(wherein L 4 represents -(CR 1L4 R 2L4 )n L4 (wherein R 1L4 and R 2L4 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or taken together to form an alkylene group; n L4 represents an integer of 1 to 6); L 5 represents -(OC 2 H 4 )n L5 (wherein n L5 represents an integer of 1 to 6); L 6 represents -(CR 1L6 R 2L6 )n L6 (wherein R 1L6 and R 2L6 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or taken together to form an alkylene group; n L6 represents an integer of 1 to 6)).
(4) -CO-L 7 -CH 2 CO-
(In the formula, L7 is --(OC 2 H 4 )n L7 (In the formula, n L7 represents an integer of 1 to 6).
　式（Ｉ）及び式（Ｉ－１）中、好ましくは、Ｘ１～Ｘ４のスペーサーは、それぞれ独立して、－（ＣＲ９Ｒ１０）ｎ’－Ｒ１１（式中、Ｒ９及びＲ１０は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ１１は－Ｏ－又は－ＮＲ１２－（式中、Ｒ１２は水素原子又は置換されていてもよいアルキル基である）を表す；ｎ’は１～１０の整数を表す）であり、より好ましくは、それぞれ独立して、－（ＣＨ２）６－Ｏ－であるか、－（ＣＨ２）５－ＮＨ－である。 In formula (I) and formula (I-1), preferably, the spacers X 1 to X 4 are each independently -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); n' represents an integer of 1 to 10), and more preferably, they are each independently -(CH 2 ) 6 -O- or -(CH 2 ) 5 -NH-.
　式（Ｉ）及び式（Ｉ－１）中、好ましくは、Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５及びＷ６は、－（ＣＨ２）－で、ｎ１～ｎ６が１である。 In formula (I) and formula (I-1), preferably, W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are —(CH 2 )—, and n 1 to n 6 are 1.
　式（Ｉ）及び式（Ｉ－１）中、好ましくは、Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドは、それぞれ独立して、糖（好ましくはＧａｌＮＡｃ）であるか脂質である。 In formula (I) and formula (I-1), preferably, the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a sugar (preferably GalNAc) or a lipid.
　式（Ｉ）及び式（Ｉ－１）中、記号Ａで表される核酸分子（本明細書中では、本発明の核酸分子と称する場合がある）は、その発現を制御する（例、抑制する）ことが所望される遺伝子（以下、標的遺伝子とも称する）の発現を制御する配列（以下、発現制御配列とも称する）として、該標的遺伝子のｍＲＮＡの特定の部位と相補的な配列を含む。該特定の部位は標的遺伝子によって適宜選択され、既知配列であっても、新たに特定されたものであってもよい。例えば、後述の実施例で用いた、HPRT1 (Hypoxanthine Phosphoribosyltransferase 1)、FactorVII (coagulation factor VII)、NEK6 (NIMA related kinase 6)、TTR (transthyretin)等の発現抑制配列等が挙げられる。 In formula (I) and formula (I-1), the nucleic acid molecule represented by the symbol A (sometimes referred to herein as the nucleic acid molecule of the present invention) contains a sequence that controls the expression of a gene (hereinafter also referred to as a target gene) whose expression is desired to be controlled (e.g., inhibited), and that is complementary to a specific site in the mRNA of the target gene (hereinafter also referred to as an expression control sequence). The specific site is appropriately selected depending on the target gene, and may be a known sequence or a newly identified sequence. Examples of such sequences include expression inhibition sequences such as HPRT1 (Hypoxanthine Phosphoribosyltransferase 1), Factor VII (coagulation factor VII), NEK6 (NIMA related kinase 6), and TTR (transthyretin), which are used in the examples described below.
　発現制御配列は、標的遺伝子の特定の部位のヌクレオチド配列と相補的な配列である。ここで、「相補的な配列」とは、標的配列に対して完全相補的な（即ち、ミスマッチなくハイブリダイズする）配列だけでなく、哺乳動物細胞の生理的条件下で標的配列と特異的にハイブリダイズし得る限り、１ないし数ヌクレオチド、好ましくは、１又は２ヌクレオチドのミスマッチを含む配列であってもよい。例えば、標的配列中の標的ヌクレオチド配列の相補鎖配列に対して、９０％以上、好ましくは９５％以上、９７％以上、９８％以上、９９％以上の同一性を有する配列が挙げられる。本発明における「ヌクレオチド配列の同一性」は、相同性計算アルゴリズムＮＣＢＩ　ＢＬＡＳＴ(National Center for Biotechnology Information Basic Local Alignment Search Tool)を用い、以下の条件（期待値＝１０；ギャップを許す；フィルタリング＝ＯＮ；マッチスコア＝１；ミスマッチスコア＝－３）にて計算することができる。また、個々の塩基における相補性は、対象となる塩基とワトソン・クリック型塩基対を形成することに限定されるものではなく、フーグスティーン型塩基対やゆらぎ塩基対(Wobble base pair)を形成することも含む。 The expression control sequence is a sequence complementary to the nucleotide sequence of a specific site of the target gene. Here, the "complementary sequence" may be not only a sequence that is completely complementary to the target sequence (i.e., hybridizes without mismatches), but also a sequence that contains a mismatch of one to several nucleotides, preferably one or two nucleotides, so long as it can specifically hybridize to the target sequence under physiological conditions of mammalian cells. For example, it may be a sequence that has an identity of 90% or more, preferably 95% or more, 97% or more, 98% or more, or 99% or more to the complementary strand sequence of the target nucleotide sequence in the target sequence. The "identity of nucleotide sequences" in the present invention can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). Furthermore, the complementarity of individual bases is not limited to forming Watson-Crick base pairs with the target base, but also includes forming Hoogsteen base pairs and wobble base pairs.
　あるいは、「相補的なヌクレオチド配列」とは、標的配列とストリンジェントな条件下でハイブリダイズするヌクレオチド配列である。ここで「ストリンジェントな条件」とは、例えば、Current Protocols in Molecular Biology, John Wiley & Sons,6.3.1-6.3.6,1999に記載される条件、例えば、６×ＳＳＣ（ｓｏｄｉｕｍ　ｃｈｌｏｒｉｄｅ／ｓｏｄｉｕｍ　ｃｉｔｒａｔｅ）／４５℃でのハイブリダイゼーション、次いで０．２×ＳＳＣ／０．１％　ＳＤＳ／５０～６５℃での一回以上の洗浄等が挙げられるが、当業者であれば、これと同等のストリンジェンシーを与えるハイブリダイゼーションの条件を適宜選択することができる。 Alternatively, a "complementary nucleotide sequence" is a nucleotide sequence that hybridizes with a target sequence under stringent conditions. Here, "stringent conditions" include, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, such as hybridization at 6x SSC (sodium chloride/sodium citrate)/45°C, followed by one or more washes at 0.2x SSC/0.1% SDS/50-65°C, but a person skilled in the art can appropriately select hybridization conditions that provide equivalent stringency.
　発現制御配列は、各標的配列の全部に相補的であってもよいし、該標的配列中の一部に対して相補的であってもよいが、通常、各標的配列中の連続する１５ヌクレオチド以上の配列に対して相補的であることが好ましい。発現制御配列が標的とするヌクレオチド配列の長さの上限は特に制限はないが、合成の容易さ等を考慮すれば、例えば１００ヌクレオチド以下、好ましくは５０ヌクレオチド以下、より好ましくは３０ヌクレオチド以下、さらに好ましくは２５ヌクレオチド以下の、標的遺伝子の連続する部分ヌクレオチド配列である。従って、発現制御配列が標的とするヌクレオチド配列の長さは、好ましくは、連続する１５～３０ヌクレオチド、より好ましくは、連続する１５～２５ヌクレオチドの部分ヌクレオチド配列であり得る。 The expression control sequence may be complementary to the entire target sequence or to a portion of the target sequence, but is usually preferably complementary to a sequence of 15 or more consecutive nucleotides in each target sequence. There is no particular upper limit to the length of the nucleotide sequence targeted by the expression control sequence, but taking into consideration ease of synthesis, etc., it is a continuous partial nucleotide sequence of the target gene of, for example, 100 nucleotides or less, preferably 50 nucleotides or less, more preferably 30 nucleotides or less, and even more preferably 25 nucleotides or less. Therefore, the length of the nucleotide sequence targeted by the expression control sequence may be a partial nucleotide sequence of preferably 15 to 30 consecutive nucleotides, more preferably 15 to 25 consecutive nucleotides.
　本発明の核酸分子は、標的遺伝子の発現を制御し得る限りＲＮＡであっても、ＤＮＡであってもよく、ＤＮＡ／ＲＮＡキメラであってもよい。また、本発明の核酸分子は、標的遺伝子の発現を制御し得る限り、二本鎖核酸であっても、一本鎖核酸であってもよい。二本鎖核酸の場合、二本鎖ＤＮＡ、二本鎖ＲＮＡ、ＤＮＡ：ＲＮＡハイブリッド、ＤＮＡ／ＲＮＡキメラとＤＮＡ、ＲＮＡ又はＤＮＡ／ＲＮＡキメラとのハイブリッドのいずれであってもよい。 The nucleic acid molecule of the present invention may be RNA, DNA, or a DNA/RNA chimera, so long as it is capable of controlling the expression of a target gene. Furthermore, the nucleic acid molecule of the present invention may be a double-stranded nucleic acid or a single-stranded nucleic acid, so long as it is capable of controlling the expression of a target gene. In the case of a double-stranded nucleic acid, it may be any of double-stranded DNA, double-stranded RNA, a DNA:RNA hybrid, and a hybrid of a DNA/RNA chimera with DNA, RNA, or a DNA/RNA chimera.
　本発明の核酸分子が一本鎖核酸の場合、標的遺伝子に対するガイド鎖のみを有する場合と、ガイド鎖とパッセンジャー鎖とが任意のリンカーを介して連結され、分子内で遺伝子の発現を抑制する配列とそれに相補的な配列とがハイブリダイズして二重鎖を形成し得る場合とがある。 When the nucleic acid molecule of the present invention is a single-stranded nucleic acid, it may have only a guide strand for the target gene, or the guide strand and passenger strand may be linked via an optional linker, and a sequence that suppresses gene expression within the molecule may hybridize with a complementary sequence to form a double strand.
　本発明において、核酸分子の構成単位としては、例えば、リボヌクレオチド残基およびデオキシリボヌクレオチド残基があげられる。これらのヌクレオチド残基は、例えば、修飾されていても非修飾であってもよい。本発明の核酸分子は、例えば、修飾ヌクレオチド残基を含むことによって、ヌクレアーゼ耐性が向上し、安定性の改善が可能である。また、本発明の核酸分子は、例えば、前記ヌクレオチド残基の他に、さらに、非ヌクレオチド残基を含んでもよい。 In the present invention, examples of the constituent units of a nucleic acid molecule include ribonucleotide residues and deoxyribonucleotide residues. These nucleotide residues may be, for example, modified or unmodified. The nucleic acid molecule of the present invention may have improved nuclease resistance and improved stability by, for example, including modified nucleotide residues. Furthermore, the nucleic acid molecule of the present invention may further include non-nucleotide residues in addition to the nucleotide residues.
　本発明の核酸分子において、リンカー以外の領域（ガイド鎖やパッセンジャー鎖）の構成単位は、ヌクレオチド残基であることが好ましい。各領域は、例えば、下記（１）～（３）の残基で構成される。
（１）非修飾ヌクレオチド残基
（２）修飾ヌクレオチド残基
（３）非修飾ヌクレオチド残基および修飾ヌクレオチド残基 In the nucleic acid molecule of the present invention, the constituent units of the regions other than the linker (guide strand and passenger strand) are preferably nucleotide residues. Each region is composed of, for example, the following residues (1) to (3).
(1) Unmodified nucleotide residues (2) Modified nucleotide residues (3) Unmodified and modified nucleotide residues
　本発明の核酸分子は、例えば、標識物質で標識化されてもよい。標識物質は、特に制限されず、例えば、蛍光物質、色素、同位体等があげられる。標識物質は、例えば、ピレン、ＴＡＭＲＡ、フルオレセイン、Ｃｙ３色素、Ｃｙ５色素等の蛍光団があげられ、色素としては、例えば、Ａｌｅｘａ４８８等のＡｌｅｘａ色素等があげられる。同位体としては、例えば、安定同位体および放射性同位体があげられる。安定同位体は、例えば、被曝の危険性が少なく、専用の施設も不要であることから取り扱い性に優れ、また、コストも低減できる。また、安定同位体は、例えば、標識した化合物の物性変化がなく、トレーサーとしての性質にも優れる。安定同位体としては、例えば、２Ｈ、１３Ｃ、１５Ｎ、１７Ｏ、１８Ｏ、３３Ｓ、３４Ｓおよび３６Ｓがあげられる。 The nucleic acid molecule of the present invention may be labeled with, for example, a labeling substance. The labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes, etc. Examples of the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, etc., and examples of the dye include Alexa dyes such as Alexa488, etc. Examples of the isotope include stable isotopes and radioisotopes. Stable isotopes, for example, have low risk of exposure and do not require dedicated facilities, making them easy to handle and reducing costs. In addition, stable isotopes, for example, do not cause changes in the physical properties of the labeled compound, and have excellent properties as a tracer. Examples of stable isotopes include 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S, and 36 S.
　ヌクレオチド残基は、構成要素として、糖、塩基およびリン酸を含む。リボヌクレオチド残基は、糖としてリボース残基を有し、塩基として、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）およびウラシル（Ｕ）（チミン（Ｔ）に置き換えることもできる）を有し、デオキシリボヌクレオチド残基は、糖としてデオキシリボース残基を有し、塩基として、アデニン（ｄＡ）、グアニン（ｄＧ）、シトシン（ｄＣ）およびチミン（ｄＴ）（ウラシル（ｄＵ）に置き換えることもできる）を有する。 Nucleotide residues contain sugar, base and phosphate as building blocks. Ribonucleotide residues have a ribose residue as the sugar and adenine (A), guanine (G), cytosine (C) and uracil (U) (which can also be replaced by thymine (T)) as bases, while deoxyribonucleotide residues have a deoxyribose residue as the sugar and adenine (dA), guanine (dG), cytosine (dC) and thymine (dT) (which can also be replaced by uracil (dU)) as bases.
　修飾ヌクレオチド残基は、ヌクレオチド残基の構成要素のいずれが修飾されていてもよい。本発明において、「修飾」は、例えば、前記構成要素の置換、付加および／または脱離、前記構成要素における原子および／または官能基の置換、付加および／または脱離であり得る。修飾ヌクレオチド残基は、例えば、天然に存在する修飾ヌクレオチド残基であっても、人工的に修飾したヌクレオチド残基であってもよい。天然由来の修飾ヌクレオチド残基としては、例えば、リンバックら(Limbach et al.、1994、Summary:the modified nucleosides of RNA、Nucleic Acids Res.22:2183-2196)を参照できる。 A modified nucleotide residue may be one in which any of the components of the nucleotide residue has been modified. In the present invention, "modification" may be, for example, the substitution, addition and/or removal of the components, or the substitution, addition and/or removal of atoms and/or functional groups in the components. A modified nucleotide residue may be, for example, a naturally occurring modified nucleotide residue or an artificially modified nucleotide residue. For examples of naturally occurring modified nucleotide residues, see Limbach et al. (1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196).
　ヌクレオチド残基の修飾としては、例えば、リボース－リン酸骨格（以下、リボリン酸骨格）の修飾があげられる。 Examples of modifications of nucleotide residues include modifications of the ribose-phosphate backbone (hereinafter, ribophosphate backbone).
　前記リボリン酸骨格において、例えば、リボース残基を修飾できる。前記リボース残基は、例えば、２’位炭素を修飾でき、具体的には、例えば、２’位炭素に結合する水酸基を、水素原子、フッ素等のハロゲン原子又は－Ｏ－アルキル基（例、－Ｏ－Ｍｅ基）、－Ｏ－アシル基（例、－Ｏ－ＣＯＭｅ基）及びアミノ基からなる群より選ばれる原子又は基、好ましくは、水素原子、メトキシ基及びフッ素原子からなる群より選ばれる原子又は基に置換できる。前記２’位炭素の水酸基を水素に置換することで、リボース残基をデオキシリボースに置換できる。前記リボース残基は、例えば、立体異性体に置換でき、例えば、アラビノース残基に置換してもよい。 In the ribophosphate skeleton, for example, the ribose residue can be modified. The ribose residue can be modified, for example, at the 2' carbon, and specifically, for example, the hydroxyl group bonded to the 2' carbon can be substituted with an atom or group selected from the group consisting of a hydrogen atom, a halogen atom such as fluorine, or an -O-alkyl group (e.g., -O-Me group), an -O-acyl group (e.g., -O-COMe group), and an amino group, preferably an atom or group selected from the group consisting of a hydrogen atom, a methoxy group, and a fluorine atom. By substituting the hydroxyl group of the 2' carbon with hydrogen, the ribose residue can be substituted with deoxyribose. The ribose residue can be substituted with a stereoisomer, for example, with an arabinose residue.
　リボリン酸骨格は、例えば、非リボース残基および／または非リン酸を有する非リボリン酸骨格に置換してもよい。非リボリン酸骨格は、例えば、リボリン酸骨格の非荷電体があげられる。非リボリン酸骨格に置換されたヌクレオチドの代替物としては、例えば、モルホリノ、シクロブチル、ピロリジン等があげられる。前記代替物は、この他に、例えば、人工核酸モノマー残基があげられる。具体例として、例えば、PNA（ペプチド核酸）、LNA（Locked Nucleic Acid）、ENA（2’-O，4’-C-Ethylene bridged Nucleic Acid）等があげられ、好ましくはPNAである。 The ribophosphate backbone may be replaced with a non-ribophosphate backbone having, for example, a non-ribose residue and/or a non-phosphate. The non-ribophosphate backbone may be, for example, an uncharged ribophosphate backbone. Examples of substitutes for the nucleotide substituted with the non-ribophosphate backbone include, for example, morpholino, cyclobutyl, pyrrolidine, etc. Other examples of the substitute include, for example, artificial nucleic acid monomer residues. Specific examples include, for example, PNA (peptide nucleic acid), LNA (locked nucleic acid), ENA (2'-O, 4'-C-ethylene bridged nucleic acid), etc., with PNA being preferred.
　リボリン酸骨格において、リン酸基を修飾することもできる。リボリン酸骨格において、糖残基に最も隣接するリン酸基は、αリン酸基と呼ばれる。αリン酸基は、負に荷電し、その電荷は、糖残基に非結合の2つの酸素原子にわたって、均一に分布している。αリン酸基における4つの酸素原子のうち、ヌクレオチド残基間のホスホジエステル結合において、糖残基と非結合である2つの酸素原子は、以下、「非結合（non-linking）酸素」ともいう。他方、ヌクレオチド残基間のホスホジエステル結合において、糖残基と結合している2つの酸素原子は、以下、「結合（linking）酸素」という。αリン酸基は、例えば、非荷電となる修飾、または、非結合酸素における電荷分布が非対称型となる修飾を行うことが好ましい。 In the ribophosphate backbone, the phosphate group can also be modified. In the ribophosphate backbone, the phosphate group closest to the sugar residue is called the α-phosphate group. The α-phosphate group is negatively charged, and the charge is evenly distributed over the two oxygen atoms that are not bonded to the sugar residue. Of the four oxygen atoms in the α-phosphate group, the two oxygen atoms that are not bonded to the sugar residue in the phosphodiester bond between nucleotide residues are also referred to as "non-linking oxygens" below. On the other hand, the two oxygen atoms that are bonded to the sugar residue in the phosphodiester bond between nucleotide residues are referred to as "linking oxygens" below. It is preferable to modify the α-phosphate group, for example, so that it becomes uncharged, or so that the charge distribution in the non-linking oxygen becomes asymmetric.
　リン酸基は、例えば、非結合酸素を置換してもよい。非結合酸素は、例えば、S（硫黄）、Se（セレン）、B（ホウ素）、C（炭素）、H（水素）、N（窒素）およびOR（Rは、アルキル基またはアリール基）のいずれかの原子で置換でき、好ましくは、Sで置換される。非結合酸素は、例えば、両方が置換されていることが好ましく、より好ましくは、両方がSで置換される。このような修飾リン酸基としては、例えば、ホスホロチオエート、ホスホロジチオエート、ホスホロセレネート、ボラノホスフェート、ボラノホスフェートエステル、ホスホネート水素、ホスホロアミデート、アルキルまたはアリールホスホネート、およびホスホトリエステル等があげられ、中でも、前記2つの非結合酸素が両方ともSで置換されているホスホロジチオエートが好ましい。 The phosphate group may, for example, replace the non-bonded oxygen. The non-bonded oxygen can be replaced with any of the following atoms: S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl or aryl group), and is preferably replaced with S. It is preferable that both non-bonded oxygens are replaced, and more preferably, both are replaced with S. Examples of such modified phosphate groups include phosphorothioates, phosphorodithioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters, and among these, phosphorodithioates in which both of the two non-bonded oxygens are replaced with S are preferred.
　リン酸基は、例えば、結合酸素を置換してもよい。結合酸素は、例えば、S（硫黄）、C（炭素）およびN（窒素）のいずれかの原子で置換でき、このような修飾リン酸基としては、例えば、Nで置換した架橋ホスホロアミデート、Sで置換した架橋ホスホロチオエート、およびCで置換した架橋メチレンホスホネート等があげられる。結合酸素の置換は、例えば、本発明の核酸分子の5’末端ヌクレオチド残基および3’末端ヌクレオチド残基の少なくとも一方において行うことが好ましく、5'側の場合、Cによる置換が好ましく、3’側の場合、Nによる置換が好ましい。 The phosphate group may, for example, replace the bonded oxygen. The bonded oxygen may, for example, be replaced by any of the atoms S (sulfur), C (carbon) and N (nitrogen). Examples of such modified phosphate groups include bridged phosphoramidates substituted with N, bridged phosphorothioates substituted with S, and bridged methylene phosphonates substituted with C. The bonded oxygen is preferably replaced, for example, at least one of the 5'-terminal nucleotide residue and the 3'-terminal nucleotide residue of the nucleic acid molecule of the present invention. In the case of the 5'-side, replacement with C is preferred, and in the case of the 3'-side, replacement with N is preferred.
　リン酸基は、例えば、リン非含有のリンカーに置換してもよい。リンカーとしては、例えば、シロキサン、カーボネート、カルボキシメチル、カルバメート、アミド、チオエーテル、エチレンオキサイドリンカー、スルホネート、スルホンアミド、チオホルムアセタール、ホルムアセタール、オキシム、メチレンイミノ、メチレンメチルイミノ、メチレンヒドラゾ、メチレンジメチルヒドラゾ、メチレンカルボニルアミノおよびメチレンオキシメチルイミノ等があげられ、好ましくは、メチレンカルボニルアミノおよびメチレンメチルイミノが挙げられる。 The phosphate group may be replaced with, for example, a phosphorus-free linker. Examples of linkers include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, methylenecarbonylamino, and methyleneoxymethylimino, and preferably, methylenecarbonylamino and methylenemethylimino.
　本発明の核酸分子は、例えば、3’末端および5’末端の少なくとも一方のヌクレオチド残基が修飾されてもよい。当該修飾は前述のとおりであり、好ましくは、末端のリン酸基に行うことが好ましい。リン酸基は全体を修飾してもよいし、リン酸基における1つ以上の原子を修飾してもよい。前者の場合、例えば、リン酸基全体の置換でもよいし、欠失でもよい。 The nucleic acid molecule of the present invention may be modified, for example, at least one of the 3'-end and 5'-end nucleotide residues. The modification is as described above, and is preferably performed on the terminal phosphate group. The entire phosphate group may be modified, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.
　末端のヌクレオチド残基の修飾としては、例えば、他の分子の付加があげられる。他の分子としては、例えば、標識物質、保護基等の機能性分子があげられる。保護基としては、例えば、S（硫黄）、Si（ケイ素）、B（ホウ素）、エステル含有基等があげられる。前記標識物質等の機能性分子は、例えば、本発明の核酸分子の検出等に利用できる。 Modifications of the terminal nucleotide residue include, for example, the addition of other molecules. Examples of other molecules include functional molecules such as labeling substances and protecting groups. Examples of protecting groups include S (sulfur), Si (silicon), B (boron), and ester-containing groups. The functional molecules such as labeling substances can be used, for example, for detecting the nucleic acid molecule of the present invention.
　他の分子は、ヌクレオチド残基のリン酸基に付加してもよいし、スペーサーを介して、リン酸基または糖残基に付加してもよい。本発明の核酸分子におけるスペーサーの末端原子は、例えば、リン酸基の結合酸素、または、糖残基のO、N、SもしくはCに、付加または置換できる。糖残基の結合部位は、例えば、3’位のCもしくは5’位のC、またはこれらに結合する原子が好ましい。スペーサーは、例えば、前記PNA等のヌクレオチド代替物の末端原子に、付加または置換することもできる。 Other molecules may be added to the phosphate group of a nucleotide residue, or may be added to the phosphate group or sugar residue via a spacer. The terminal atom of the spacer in the nucleic acid molecule of the present invention can be added to or substituted for, for example, the bonded oxygen of the phosphate group, or the O, N, S, or C of the sugar residue. The binding site of the sugar residue is preferably, for example, the 3' C or 5' C, or an atom bonded to these. The spacer can also be added to or substituted for, for example, the terminal atom of a nucleotide substitute such as the PNA.
　本発明の核酸分子におけるスペーサーは特に制限されず、例えば、-(CH2)n-、-(CH2)nN-、-(CH2)nO-、-(CH2)nS-、O(CH2CH2O)nCH2CH2OH、無塩基糖、アミド、カルボキシ、アミン、オキシアミン、オキシイミン、チオエーテル、ジスルフィド、チオ尿素、スルホンアミド、およびモルホリノ等、ならびに、ビオチン試薬およびフルオレセイン試薬等が挙げられる。前記式において、nは、正の整数であり、n＝3または6が好ましい。 The spacer in the nucleic acid molecule of the present invention is not particularly limited, and examples thereof include -( CH2 ) n- , -( CH2 ) nN- , -(CH2) nO- , - ( CH2 ) nS- , O( CH2CH2O ) nCH2CH2OH , abasic sugar, amide, carboxy , amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, and morpholino, as well as biotin reagent and fluorescein reagent, etc. In the above formula, n is a positive integer, and n=3 or 6 is preferred.
　末端に付加する分子は、これらの他に、例えば、色素、インターカレート剤（例えば、アクリジン）、架橋剤（例えば、ソラレン、マイトマイシンC）、ポルフィリン（TPPC4、テキサフィリン、サッフィリン）、多環式芳香族炭化水素（例えば、フェナジン、ジヒドロフェナジン）、人工エンドヌクレアーゼ（例えば、EDTA）、親油性担体（例えば、コレステロール、コール酸、アダマンタン酢酸、1-ピレン酪酸、ジヒドロテストステロン、1，3-ビス-O（ヘキサデシル）グリセロール、ゲラニルオキシヘキシル基、ヘキサデシルグリセロール、ボルネオール、メントール、1，3-プロパンジオール、ヘプタデシル基、パルミチン酸、ミリスチン酸、O3-（オレオイル）リトコール酸、O3-(オレオイル）コール酸、ジメトキシトリチル、またはフェノキサジン）およびペプチド複合体（例えば、アンテナペディアペプチド、Tatペプチド）、アルキル化剤、リン酸、アミノ、メルカプト、PEG（例えば、PEG-40K）、MPEG、［MPEG］2、ポリアミノ、アルキル、置換アルキル、放射線標識マーカー、酵素、ハプテン（例えば、ビオチン）、輸送／吸収促進剤（例えば、アスピリン、ビタミンE、葉酸）、合成リボヌクレアーゼ（例えば、イミダゾール、ビスイミダゾール、ヒスタミン、イミダゾールクラスター、アクリジン－イミダゾール複合体、テトラアザマクロ環のEu3+複合体）等があげられる。 In addition to these, the molecules added to the ends may include, for example, dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl) Examples of suitable glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, and Eu3 + complexes of tetraazamacrocycles).
　本発明の核酸分子は、5’末端が、例えば、リン酸基またはリン酸基アナログで修飾されてもよい。リン酸基は、例えば、5’一リン酸（(HO)2(O)P-O-5’）、5’二リン酸（(HO)2(O)P-O-P(HO)(O)-O-5’）、5’三リン酸（(HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’）、5’－グアノシンキャップ（7-メチル化または非メチル化、7m-G-O-5’-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’）、5’－アデノシンキャップ（Appp）、任意の修飾または非修飾ヌクレオチドキャップ構造（N-O-5’-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’）、5’一チオリン酸（ホスホロチオエート：(HO)2(S)P-O-5’）、5’ジチオリン酸（ホスホロジチオエート：(HO)(HS)(S)P-O-5’）、5’-ホスホロチオール酸（(HO)2(O)P-S-5’）、硫黄置換の一リン酸、二リン酸および三リン酸（例えば、5’-α-チオ三リン酸、5’-γ-チオ三リン酸等）、5’-ホスホルアミデート（(HO)2(O)P-NH-5’、(HO)(NH2)(O)P-O-5’）、5’－アルキルホスホン酸（例えば、RP(OH)(O)-O-5’、(OH)2(O)P-5’-CH2、Rはアルキル（例えば、メチル、エチル、イソプロピル、プロピル等））、5’-アルキルエーテルホスホン酸（例えば、RP(OH)(O)-O-5’、Rはアルキルエーテル（例えば、メトキシメチル、エトキシメチル等））等があげられる。 The nucleic acid molecules of the present invention may be modified at the 5' end with, for example, a phosphate group or a phosphate group analog. The phosphate group may be, for example, a 5' monophosphate ((HO) 2 (O)PO-5'), a 5' diphosphate ((HO) 2 (O)POP(HO)(O)-O-5'), a 5' triphosphate ((HO) 2 (O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-guanosine cap (7-methylated or unmethylated, 7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-adenosine cap (Appp), any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5' monothiophosphate (phosphorothioate: (HO) 2 (S)PO-5'), 5'-dithiophosphate (phosphorodithioate: (HO)(HS)(S)PO-5'), 5'-phosphorothiolic acid ((HO) 2 (O)PS-5'), sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)PO-5'), 5'-alkylphosphonic acids (e.g., RP(OH)(O)-O-5', (OH) 2 (O)P-5'-CH 2 , where R is alkyl (e.g., methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkyl ether phosphonic acids (e.g., RP(OH)(O)-O-5', where R is alkyl ether (e.g., methoxymethyl, ethoxymethyl, etc.)), and the like.
　ヌクレオチド残基において、塩基は特に制限されない。塩基は、天然の塩基でもよいし、非天然の塩基でもよい。例えば、一般的な塩基、その修飾アナログ等が使用できる。 In the nucleotide residue, the base is not particularly limited. The base may be a natural base or a non-natural base. For example, common bases and their modified analogs can be used.
　塩基としては、例えば、アデニンおよびグアニン等のプリン塩基、シトシン、ウラシルおよびチミン等のピリミジン塩基があげられる。前記塩基は、この他に、イノシン、チミン、キサンチン、ヒポキサンチン、ヌバラリン（nubularine）、イソグアニシン（isoguanisine）、ツベルシジン（tubercidine）等があげられる。塩基は、例えば、2-アミノアデニン、6-メチル化プリン等のアルキル誘導体；2-プロピル化プリン等のアルキル誘導体；5-ハロウラシルおよび5-ハロシトシン；5-プロピニルウラシルおよび5-プロピニルシトシン；6-アゾウラシル、6-アゾシトシンおよび6-アゾチミン；5-ウラシル（プソイドウラシル）、4-チオウラシル、5-ハロウラシル、5-（2-アミノプロピル）ウラシル、5-アミノアリルウラシル；8-ハロ化、アミノ化、チオール化、チオアルキル化、ヒドロキシル化および他の8-置換プリン；5-トリフルオロメチル化および他の5-置換ピリミジン；7-メチルグアニン；5-置換ピリミジン；6-アザピリミジン；N-2、N-6、およびO-6置換プリン（2-アミノプロピルアデニンを含む）；5-プロピニルウラシルおよび5-プロピニルシトシン；ジヒドロウラシル；3-デアザ－5-アザシトシン；2-アミノプリン；5-アルキルウラシル；7-アルキルグアニン；5-アルキルシトシン；7-デアザアデニン；N6，N6-ジメチルアデニン；2，6-ジアミノプリン；5-アミノ－アリル－ウラシル；N3-メチルウラシル；置換1，2，4-トリアゾール；2-ピリジノン；5-ニトロインドール；3-ニトロピロール；5-メトキシウラシル；ウラシル－5-オキシ酢酸；5-メトキシカルボニルメチルウラシル；5-メチル-2-チオウラシル；5-メトキシカルボニルメチル-2-チオウラシル；5-メチルアミノメチル-2-チオウラシル；3-（3-アミノ-3-カルボキシプロピル）ウラシル；3-メチルシトシン；5-メチルシトシン；N4-アセチルシトシン；2-チオシトシン；N6-メチルアデニン；N6-イソペンチルアデニン；2-メチルチオ-N6-イソペンテニルアデニン；N-メチルグアニン；O-アルキル化塩基等であり得る。また、プリンおよびピリミジンには、例えば、米国特許第3,687,808号、「Concise Encyclopedia Of Polymer Science And Engineering」、858～859頁、クロシュビッツ ジェー アイ（Kroschwitz J.I.）編、John Wiley & Sons、1990、およびイングリッシュら（Englischら）、Angewandte Chemie、International Edition、1991、30巻、p.613に開示されるものが含まれる。 Examples of bases include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil, and thymine. Other examples of the base include inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, and tubercidine. Examples of bases include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-azocytosine, and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, and 5-aminopropyluracil. 8-halo, aminated, thiolated, thioalkylated, hydroxylated and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine); 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2-azapyrimidines; minopurine; 5-alkyluracil; 7-alkylguanine; 5-alkylcytosine; 7-deazaadenine; N6,N6-dimethyladenine; 2,6-diaminopurine; 5-amino-allyl-uracil; N3-methyluracil; substituted 1,2,4-triazoles; 2-pyridinone; 5-nitroindole; 3-nitropyrrole; 5-methoxyuracil; uracil-5-oxyacetic acid; 5-methoxycarbonylmethyluracil; 5-methyl- It may be 2-thiouracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-methylaminomethyl-2-thiouracil; 3-(3-amino-3-carboxypropyl)uracil; 3-methylcytosine; 5-methylcytosine; N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6-isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; O-alkylated bases, and the like. Purines and pyrimidines also include those disclosed, for example, in U.S. Pat. No. 3,687,808; in Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, edited by Kroschwitz J.I., John Wiley & Sons, 1990; and in Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613.
　修飾ヌクレオチド残基は、これらの他に、例えば、塩基を欠失する残基、すなわち、無塩基のリボリン酸骨格を含んでもよい。また、修飾ヌクレオチド残基は、例えば、米国仮出願第60/465,665号（出願日：2003年4月25日）、および国際出願第PCT/US04/07070号（出願日：2004年3月8日：国際公開WO2004/080406）に記載される残基が使用でき、本発明は、これらの文献を援用できる。 In addition to the above, modified nucleotide residues may include, for example, residues lacking a base, i.e., an abasic ribophosphate backbone. Modified nucleotide residues may also include, for example, residues described in U.S. Provisional Application No. 60/465,665 (filed April 25, 2003) and International Application No. PCT/US04/07070 (filed March 8, 2004: International Publication WO2004/080406), and the present invention may incorporate these documents.
　本発明の核酸分子の合成方法は、特に制限されず、従来公知の方法が採用できる。前記合成方法は、例えば、遺伝子工学的手法による合成法、化学合成法等があげられる。遺伝子工学的手法は、例えば、インビトロ転写合成法、ベクターを用いる方法、ＰＣＲカセットによる方法があげられる。前記ベクターは、特に制限されず、プラスミド等の非ウイルスベクター、ウイルスベクター等があげられる。前記化学合成法は、特に制限されず、例えば、ホスホロアミダイト法およびＨ－ホスホネート法等があげられる。前記化学合成法は、例えば、市販の自動核酸合成機を使用可能である。前記化学合成法は、一般に、アミダイトが使用される。前記アミダイトは、特に制限されず、市販のアミダイトとして、例えば、ＲＮＡ　Ｐｈｏｓｐｈｏｒａｍｉｄｉｔｅｓ（２’－Ｏ－ＴＢＤＭＳｉ、商品名、三千里製薬）、ＡＣＥアミダイトおよびＴＯＭアミダイト、ＣＥＥアミダイト、ＣＥＭアミダイト、ＴＥＭアミダイト等があげられる。 The method for synthesizing the nucleic acid molecule of the present invention is not particularly limited, and a conventionally known method can be used. Examples of the synthesis method include synthesis methods using genetic engineering techniques and chemical synthesis methods. Examples of the genetic engineering techniques include in vitro transcription synthesis methods, methods using vectors, and methods using PCR cassettes. The vector is not particularly limited, and examples include non-viral vectors such as plasmids, and viral vectors. The chemical synthesis method is not particularly limited, and examples include the phosphoramidite method and the H-phosphonate method. For example, a commercially available automated nucleic acid synthesizer can be used for the chemical synthesis method. In general, amidites are used for the chemical synthesis method. The amidites are not particularly limited, and examples of commercially available amidites include RNA Phosphoramidites (2'-O-TBDMSi, product name, Sanzenri Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, and TEM amidite.
　本発明の核酸分子としては、例えば、標的遺伝子に対するｓｉＲＮＡ、アンチセンス核酸等が挙げられる。また、本発明の核酸分子としては、ガイド鎖とそれに相補的なパッセンジャー鎖とが、リンカーを介して連結された二重鎖を形成し得る一本鎖核酸分子を挙げることができる。 Examples of the nucleic acid molecule of the present invention include siRNA and antisense nucleic acids against target genes. In addition, examples of the nucleic acid molecule of the present invention include single-stranded nucleic acid molecules that can form a duplex in which a guide strand and a complementary passenger strand are linked via a linker.
（ｉ）ｓｉＲＮＡ
　標的遺伝子に対するｓｉＲＮＡとは、標的遺伝子のヌクレオチド配列中の連続する２５ヌクレオチド以下の標的配列の全部もしくは一部と相補的な配列を含むガイド鎖と、それに相補的な配列を含むパッセンジャー鎖とからなる二本鎖オリゴＲＮＡであって、ＲＩＳＣ複合体に取り込まれ、ガイド鎖中の標的遺伝子にコードされるｍＲＮＡに相補的な配列が当該ｍＲＮＡ中の標的配列と二重鎖を形成することで、当該ｍＲＮＡを切断し、遺伝子発現を抑制する核酸をいう。ここで「相補的な配列」とは、前記と同義である。 (i) siRNA
The siRNA for a target gene is a double-stranded oligoRNA consisting of a guide strand containing a sequence complementary to all or part of a target sequence of 25 or less consecutive nucleotides in the nucleotide sequence of the target gene, and a passenger strand containing a sequence complementary thereto, which is incorporated into the RISC complex, and the sequence complementary to the mRNA encoded by the target gene in the guide strand forms a double strand with the target sequence in the mRNA, thereby cleaving the mRNA and suppressing gene expression. Here, the "complementary sequence" has the same meaning as above.
　ｓｉＲＮＡの長さは、ガイド鎖中に、標的遺伝子のヌクレオチド配列中の連続する２５ヌクレオチド以下の標的配列の全部もしくは一部と相補的な配列を含み、遺伝子発現を抑制し得る限り特に限定されないが、ｓｉＲＮＡが標的とするヌクレオチド配列は、原則的には１５～５０ヌクレオチド、好ましくは１９～３０ヌクレオチド、更に好ましくは１９～２７ヌクレオチド、特に好ましくは１９～２１ヌクレオチドであり得る。また、ガイド鎖及びパッセンジャー鎖は、５’または３’末端に、付加的なヌクレオチドを有していてもよい。該付加的ヌクレオチドの長さは、通常２～４ヌクレオチド程度であり、ｓｉＲＮＡの全長として１９ヌクレオチド以上である。該付加的ヌクレオチドは、ＤＮＡでもＲＮＡでもよいが、ＤＮＡを用いると核酸の安定性を向上させることができる場合がある。このような付加的ヌクレオチドの配列としては、例えばｕｇ－３’、ｕｕ－３’、ｔｇ－３’、ｔｔ－３’、ｇｇｇ－３’、ｇｕｕｕ－３’、ｇｔｔｔ－３’、ｔｔｔｔｔ－３’、ｕｕｕｕｕ－３’などの配列が挙げられるが、これらに限定されるものではない。 The length of the siRNA is not particularly limited as long as the guide strand contains a sequence complementary to all or part of a target sequence of 25 or less consecutive nucleotides in the nucleotide sequence of the target gene and can suppress gene expression, but the nucleotide sequence targeted by the siRNA can be, in principle, 15 to 50 nucleotides, preferably 19 to 30 nucleotides, more preferably 19 to 27 nucleotides, and particularly preferably 19 to 21 nucleotides. The guide strand and passenger strand may also have additional nucleotides at the 5' or 3' end. The length of the additional nucleotide is usually about 2 to 4 nucleotides, and the total length of the siRNA is 19 nucleotides or more. The additional nucleotide may be either DNA or RNA, but using DNA may improve the stability of the nucleic acid in some cases. Examples of such additional nucleotide sequences include, but are not limited to, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', and uuuuu-3'.
　ｓｉＲＮＡは、一方もしくは両方の鎖に３’－オーバーハングを有していてもよい。オーバーハングを有する場合、オーバーハングの長さは、特に限定されず、下限が、例えば、１塩基長であり、上限が、例えば、４塩基長、３塩基長であり、範囲が、例えば、１～４塩基長、１～３塩基長、１～２塩基長である。 The siRNA may have a 3'-overhang on one or both strands. When an overhang is present, the length of the overhang is not particularly limited, and the lower limit is, for example, 1 base length, the upper limit is, for example, 4 base length or 3 base length, and the range is, for example, 1 to 4 base length, 1 to 3 base length, or 1 to 2 base length.
　オーバーハングの配列は、特に限定されず、Ａ、Ｕ、Ｇ、Ｃ、Ｔのいずれであってもよい。オーバーハングの配列は、例えば、３’側から、ＴＴ、ＵＵ、ＣＵ、ＧＣ、ＵＡ、ＡＡ、ＣＣ、ＵＧ、ＣＧ、ＡＵ等が例示できる。前記オーバーハングは、例えば、ＴＴ、ＵＵとすることで、ＲＮＡ分解酵素に対する耐性を付加できる。 The sequence of the overhang is not particularly limited and may be any of A, U, G, C, and T. Examples of the overhang sequence from the 3' side include TT, UU, CU, GC, UA, AA, CC, UG, CG, and AU. By making the overhang, for example, TT or UU, resistance to RNase can be imparted.
　ｓｉＲＮＡの合成方法は、特に限定されず、従来公知の核酸の製造方法が採用できる。合成方法としては、例えば、前記相補的な配列を含む核酸およびそれに相補的な配列の核酸をＤＮＡ／ＲＮＡ自動合成機でそれぞれ合成し、適当なアニーリング緩衝液中、約９０～約９５℃で約１分程度変性させた後、約３０～約７０℃で約１～約８時間アニーリングさせることにより調製する方法等が挙げられる。また、ｓｉＲＮＡの前駆体となるｓｈＲＮＡを合成し、ダイサー（ｄｉｃｅｒ）を用いてこれを切断することにより調製することもできる。ｓｉＲＮＡを構成するヌクレオチド残基もまた、安定性、比活性などを向上させるために、上記と同様の修飾を受けていてよい。 The method for synthesizing siRNA is not particularly limited, and a conventional method for producing nucleic acids can be used. Examples of the synthesis method include a method in which a nucleic acid containing the complementary sequence and a nucleic acid having a complementary sequence thereto are each synthesized using an automatic DNA/RNA synthesizer, denatured in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then annealed at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, siRNA can be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it using dicer. The nucleotide residues constituting siRNA may also be modified in the same manner as above to improve stability, specific activity, etc.
（ｉｉ）標的遺伝子に対するアンチセンス核酸
　標的遺伝子に対するアンチセンス核酸とは、標的遺伝子のヌクレオチド配列中の連続する２５ヌクレオチド以下の標的配列の全部もしくは一部、好ましくは該ヌクレオチド配列中、連続する１５ヌクレオチド以上のヌクレオチド配列と相補的な配列を含み、標的遺伝子のヌクレオチド配列中の標的配列と特異的な二重鎖を形成して結合することにより、遺伝子発現を抑制する作用を有する核酸をいう。ここで「相補的な配列」とは、前記と同義である。 (ii) Antisense Nucleic Acid for a Target Gene An antisense nucleic acid for a target gene refers to a nucleic acid that contains a sequence complementary to all or part of a target sequence of 25 or less consecutive nucleotides in the nucleotide sequence of a target gene, preferably a nucleotide sequence of 15 or more consecutive nucleotides in the nucleotide sequence, and has the effect of suppressing gene expression by binding to the target sequence in the nucleotide sequence of the target gene by forming a specific double strand. Here, the term "complementary sequence" has the same meaning as above.
　アンチセンス核酸の長さは特に限定されないが、例えば、１５～４０ヌクレオチドであり、好ましくは１５～３０ヌクレオチドであり得る。 The length of the antisense nucleic acid is not particularly limited, but may be, for example, 15 to 40 nucleotides, and preferably 15 to 30 nucleotides.
　アンチセンス核酸は、ギャップマー型であってもよい。ギャップマー型のアンチセンス核酸とは、ＤＮＡと、その両側に、修飾や架橋が導入された核酸とを有する核酸である。ＤＮＡ鎖を主鎖として、主鎖に相補的なＲＮＡがヘテロ２本鎖核酸を形成し、ＲＮＡは、ＲＮＡａｓｅ　Ｈにより分解される。糖部の２’位のＯ－メチル化により、アンチセンス核酸の安定性が向上し、ターゲットへの結合親和性が増大する。また、リン酸結合をホスホロチオエート結合に置換することにより、アンチセンス核酸のヌクレアーゼ耐性が高まる。 The antisense nucleic acid may be of the gapmer type. A gapmer type antisense nucleic acid is a nucleic acid that has DNA and nucleic acid with modifications or crosslinks introduced on both sides of the DNA. The DNA strand serves as the backbone, and RNA complementary to the backbone forms a heteroduplex nucleic acid, and the RNA is degraded by RNAase H. O-methylation of the 2' position of the sugar moiety improves the stability of the antisense nucleic acid and increases the binding affinity to the target. In addition, the nuclease resistance of the antisense nucleic acid is increased by replacing the phosphate bond with a phosphorothioate bond.
　アンチセンス核酸の合成方法は、特に限定されず、従来公知の核酸の製造方法が採用できる。合成方法としては、例えば、前記相補的な配列を含む核酸をＤＮＡ／ＲＮＡ自動合成機でそれぞれ合成することにより調製する方法等が挙げられる。また、上記した各種修飾を含むアンチセンス核酸も、従来公知の手法により、化学的に合成することができる。 The method for synthesizing antisense nucleic acids is not particularly limited, and any conventionally known method for producing nucleic acids can be used. Examples of the synthesis method include a method in which nucleic acids containing the complementary sequences are prepared by synthesizing them using an automatic DNA/RNA synthesizer. Antisense nucleic acids containing the various modifications described above can also be chemically synthesized using conventionally known methods.
（ｉｉｉ）標的遺伝子に対する一本鎖核酸分子
　本発明において、「標的遺伝子に対する一本鎖核酸分子」とは、標的遺伝子における特定配列で示される領域中の連続する２５ヌクレオチド以下の標的配列の全部もしくは一部と相補的な配列Ｔａを含むガイド鎖配列Ｔと、Ｔａに相補的な配列Ｑａを含むパッセンジャー鎖配列Ｑとが、リンカーＬを介して、５’から３’方向又は３’から５’方向にＴ－Ｌ－Ｑの順序で、かつ配列Ｔａと配列Ｑａとが分子内で二重鎖を形成し得る配向で連結された、標的遺伝子の発現を抑制する核酸分子をいう。ここで「相補的な配列」とは、前記と同義である。 (iii) Single-stranded nucleic acid molecule for a target gene In the present invention, the term "single-stranded nucleic acid molecule for a target gene" refers to a nucleic acid molecule that suppresses the expression of a target gene, in which a guide strand sequence T including a sequence Ta that is complementary to all or part of a target sequence of 25 or less consecutive nucleotides in a region represented by a specific sequence in the target gene and a passenger strand sequence Q including a sequence Qa that is complementary to Ta are linked in the order of T-L-Q in the 5' to 3' direction or the 3' to 5' direction via a linker L, and in an orientation in which the sequence Ta and the sequence Qa can form a double strand within the molecule. Here, the "complementary sequence" has the same meaning as above.
　配列Ｔａは、標的遺伝子の特定配列で示される領域中の連続する２５ヌクレオチド以下の標的配列の全部又は一部と相補的な配列を含む限り特に限定されないが、Ｔａが標的とするヌクレオチド配列は、原則的には１５～４９ヌクレオチド、好ましくは１９～３０ヌクレオチド、更に好ましくは１９～２７ヌクレオチド、特に好ましくは１９～２１ヌクレオチドであり得る。 The sequence Ta is not particularly limited as long as it contains a sequence complementary to all or part of a target sequence of 25 consecutive nucleotides or less in a region represented by a specific sequence of the target gene, but the nucleotide sequence targeted by Ta can be, in principle, 15 to 49 nucleotides, preferably 19 to 30 nucleotides, more preferably 19 to 27 nucleotides, and particularly preferably 19 to 21 nucleotides.
　標的遺伝子に対する一本鎖核酸分子は、好ましい一実施態様においては、配列Ｔａを含むガイド鎖配列Ｔと、配列Ｔａに相補的な配列Ｑａを含むパッセンジャー鎖配列Ｑとを含む核酸分子等が挙げられる。 In a preferred embodiment, the single-stranded nucleic acid molecule for a target gene is a nucleic acid molecule that includes a guide strand sequence T that includes a sequence Ta, and a passenger strand sequence Q that includes a sequence Qa that is complementary to the sequence Ta.
　ガイド鎖配列Ｔは、例えば、配列Ｔａのみからなってもよいし、さらに付加配列Ｔｂを有してもよい。後者の場合、付加配列Ｔｂは標的遺伝子のヌクレオチド配列と相補的であることを要しない。付加配列Ｔｂは、Ｔａの５’末端もしくは３’末端のいずれに付加されてもよく、両端に付加されてもよい（Ｔｂ及びＴｂ’）。好ましくは、ＴａのリンカーＬと連結される側の末端に付加される。配列Ｔｂ（Ｔｂ’）の長さは、例えば、１～３５ヌクレオチドであり、好ましくは、１～２５ヌクレオチドであり、より好ましくは、１～１１ヌクレオチドであり、特に好ましくは、１、２、３、４、５又は６ヌクレオチドである。 The guide strand sequence T may, for example, consist of only the sequence Ta, or may further have an additional sequence Tb. In the latter case, the additional sequence Tb does not need to be complementary to the nucleotide sequence of the target gene. The additional sequence Tb may be added to either the 5' end or the 3' end of Ta, or may be added to both ends (Tb and Tb'). It is preferably added to the end of Ta that is linked to the linker L. The length of the sequence Tb (Tb') is, for example, 1 to 35 nucleotides, preferably 1 to 25 nucleotides, more preferably 1 to 11 nucleotides, and particularly preferably 1, 2, 3, 4, 5, or 6 nucleotides.
　パッセンジャー鎖配列Ｑは、配列Ｔａに相補的な配列Ｑａを含む限り、特に限定されず、例えば、配列Ｑａのみからなってもよいし、さらに付加配列Ｑｂを有してもよい。後者の場合、付加配列Ｑｂは付加配列Ｔｂと相補的であることを要しないが、相補的であることが望ましく、特に、付加配列Ｔｂ及びＱｂが、それぞれＴａ及びＱａのリンカーＬと連結される側の末端に付加される場合、ＴｂとＱｂとは相補的であることがより望ましい。付加配列Ｑｂは、Ｑａの５’末端もしくは３’末端のいずれに付加されてもよく、両端に付加されてもよい（Ｑｂ及びＱｂ’）。好ましくは、ＱａのリンカーＬと連結される側の末端に付加される。配列Ｙｂ（Ｙｂ’）の長さは、例えば、１～３５ヌクレオチドであり、好ましくは、１～２５ヌクレオチドであり、より好ましくは、１～１１ヌクレオチドであり、特に好ましくは、１、２、３、４、５又は６ヌクレオチドである。 The passenger strand sequence Q is not particularly limited as long as it contains a sequence Qa complementary to the sequence Ta, and may, for example, consist of only the sequence Qa, or may further have an additional sequence Qb. In the latter case, the additional sequence Qb does not need to be complementary to the additional sequence Tb, but it is preferable that they are complementary, and in particular, when the additional sequences Tb and Qb are added to the ends of Ta and Qa that are linked to the linker L, respectively, it is more preferable that Tb and Qb are complementary. The additional sequence Qb may be added to either the 5' end or the 3' end of Qa, or may be added to both ends (Qb and Qb'). It is preferably added to the end of Qa that is linked to the linker L. The length of the sequence Yb (Yb') is, for example, 1 to 35 nucleotides, preferably 1 to 25 nucleotides, more preferably 1 to 11 nucleotides, and particularly preferably 1, 2, 3, 4, 5, or 6 nucleotides.
　ガイド鎖配列Ｔ及びパッセンジャー鎖配列Ｑは、リンカーＬに連結しない側の末端に、さらにオーバーハングを有してもよい。オーバーハングは、好ましくは、ガイド鎖配列及びパッセンジャー鎖配列のうち、その５’末端がリンカーＬに連結される方の配列の３’末端に付加される。 The guide strand sequence T and the passenger strand sequence Q may further have an overhang at the end not linked to the linker L. The overhang is preferably added to the 3' end of the guide strand sequence or the passenger strand sequence whose 5' end is linked to the linker L.
　オーバーハングの長さは、特に限定されず、下限が、例えば、１ヌクレオチド長であり、上限が、例えば、４ヌクレオチド長、３ヌクレオチド長であり、範囲が、例えば、１～４ヌクレオチド長、１～３ヌクレオチド長、１～２ヌクレオチド長である。 The length of the overhang is not particularly limited, and the lower limit is, for example, 1 nucleotide, the upper limit is, for example, 4 nucleotides or 3 nucleotides, and the range is, for example, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides.
　オーバーハングの配列は、特に限定されず、Ａ、Ｕ、Ｇ、Ｃ、Ｔのいずれであってもよい。オーバーハングの配列は、例えば、５’側から、ＴＴ、ＵＵ、ＣＵ、ＧＣ、ＵＡ、ＡＡ、ＣＣ、ＵＧ、ＣＧ、ＡＵ等が例示できる。オーバーハングは、例えば、ＴＴ、ＵＵとすることで、ＲＮＡ分解酵素に対する耐性を付加できる。 The sequence of the overhang is not particularly limited and may be any of A, U, G, C, and T. Examples of the overhang sequence from the 5' side include TT, UU, CU, GC, UA, AA, CC, UG, CG, and AU. By making the overhang, for example, TT or UU, resistance to RNase can be imparted.
　好ましい一実施態様において、遺伝子に対する一本鎖核酸分子は、ヌクレオチド配列Ｔと、ヌクレオチド配列Ｑとが、リンカーＬを介して、５’から３’方向又は３’から５’方向にＴ－Ｌ－Ｑの順序で、かつ配列Ｔａと配列Ｑａとが分子内で二重鎖を形成し得る配向で連結される。リンカーＬは、例えば、ヌクレオチド残基から構成されてもよいし、非ヌクレオチド残基から構成されてもよく、ヌクレオチド残基および非ヌクレオチド残基から構成されてもよい。ヌクレオチド残基としては、リボヌクレオチド残基およびデオキシリボヌクレオチド残基があげられる。 In a preferred embodiment, a single-stranded nucleic acid molecule for a gene is formed by linking a nucleotide sequence T and a nucleotide sequence Q via a linker L in the 5' to 3' or 3' to 5' direction in the order T-L-Q, and in an orientation in which the sequence Ta and the sequence Qa can form a double strand within the molecule. The linker L may be composed of, for example, nucleotide residues, non-nucleotide residues, or nucleotide residues and non-nucleotide residues. Examples of nucleotide residues include ribonucleotide residues and deoxyribonucleotide residues.
　リンカーＬがヌクレオチド残基から構成されている場合、一分子内でセンス領域とアンチセンス領域が互いに塩基対合してステム構造を形成し、同時にリンカーＬのヌクレオチド配列がループ構造を形成することによって、分子全体としてヘアピン型のステム－ループ構造を形成しており、標的遺伝子に対する一本鎖核酸分子は、ｓｈＲＮＡ(small hairpin RNAまたはshort hairpin RNA)とも言える。リンカーＬの長さは、特に限定されないが、例えば、配列Ｔａと配列Ｑａとが分子内で二重鎖を形成可能な長さであることが好ましい。リンカーＬの塩基数は、その下限が、例えば、１塩基、２塩基、３塩基であり、その上限が、例えば、１００塩基、８０塩基、５０塩基である。各リンカー領域の塩基数は、具体例として、例えば、１～５０塩基、１～３０塩基、１～２０塩基、１～１０塩基、１～７塩基、１～４塩基等が例示できるが、これには限定されない。リンカーＬは、自己アニーリングを生じない構造であることが好ましい。 When the linker L is composed of nucleotide residues, the sense region and the antisense region form a stem structure within a single molecule by base pairing with each other, and at the same time, the nucleotide sequence of the linker L forms a loop structure, so that the molecule as a whole forms a hairpin-type stem-loop structure, and the single-stranded nucleic acid molecule for the target gene can also be called shRNA (small hairpin RNA or short hairpin RNA). The length of the linker L is not particularly limited, but it is preferably a length that allows the sequence Ta and the sequence Qa to form a double strand within the molecule. The number of bases in the linker L has a lower limit of, for example, 1 base, 2 bases, or 3 bases, and an upper limit of, for example, 100 bases, 80 bases, or 50 bases. Specific examples of the number of bases in each linker region include, but are not limited to, 1 to 50 bases, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 1 to 7 bases, and 1 to 4 bases. It is preferable that the linker L has a structure that does not cause self-annealing.
　非ヌクレオチド残基から構成されているリンカーＬ、又はヌクレオチド残基および非ヌクレオチド残基から構成されているリンカーＬは、例えば、下記式（１）で表わされる。 The linker L composed of non-nucleotide residues, or the linker L composed of nucleotide residues and non-nucleotide residues, is represented, for example, by the following formula (1).
　式（１）中、例えば、Ｘ１ａおよびＸ１ｂは、共に水素原子であるか、一緒になって＝Ｏ、＝Ｓまたは＝ＮＨを形成し；Ｘ２ａおよびＸ２ｂは、共に水素原子であるか、一緒になって＝Ｏ、＝Ｓまたは＝ＮＨを形成し；
Ｒ３は、環Ａ上のＣ－３、Ｃ－４、Ｃ－５またはＣ－６に結合する水素原子または置換基であり；
Ｌ１は、ｍＬ個の炭素原子からなるアルキレン鎖であり、ここで、アルキレン炭素原子上の水素原子は、ＯＨ、ＯＲａ、ＮＨ２、ＮＨＲａ、ＮＲａＲｂ、ＳＨ、もしくはＳＲａで置換されても置換されていなくてもよく、または、Ｌ１は、アルキレン鎖の一つ以上の炭素原子が、酸素原子で置換されたポリエーテル鎖であり、ただし、Ｙ１が、ＮＨ、ＯまたはＳの場合、Ｙ１に結合するＬ１の原子は炭素であり、ＯＲ１に結合するＬ１の原子は炭素であり、酸素原子同士は隣接せず；
Ｌ２は、ｎＬ個の炭素原子からなるアルキレン鎖であり、ここで、アルキレン炭素原子上の水素原子は、ＯＨ、ＯＲｃ、ＮＨ２、ＮＨＲｃ、ＮＲｃＲｄ、ＳＨもしくはＳＲｃで置換されても置換されていなくてもよく、または、Ｌ２は、アルキレン鎖の一つ以上の炭素原子が、酸素原子で置換されたポリエーテル鎖であり、ただし、Ｙ２が、ＮＨ、ＯまたはＳの場合、Ｙ２に結合するＬ２の原子は炭素であり、ＯＲ２に結合するＬ２の原子は炭素であり、酸素原子同士は隣接せず；
Ｒａ、Ｒｂ、ＲｃおよびＲｄは、それぞれ独立して、置換基または保護基であり；
ｌは、１または２であり；
ｍＬは、０～３０の範囲の整数であり；
ｎＬは、０～３０の範囲の整数であり；
環Ａは、環Ａ上のＣ－２以外の１個の炭素原子が、窒素、酸素、硫黄で置換されてもよく；
環Ａ内に、炭素－炭素二重結合または炭素－窒素二重結合を含んでもよい。
ヌクレオチド配列Ｔおよびヌクレオチド配列Ｑは、それぞれ、－ＯＲ１－または－ＯＲ２－を介して、非ヌクレオチド構造に結合し、ここで、Ｒ１およびＲ２は、存在しても存在しなくてもよく、存在する場合、Ｒ１およびＲ２は、それぞれ独立して、ヌクレオチド残基または式（１）の構造である。 In formula (1), for example, X 1a and X 1b are both hydrogen atoms or are taken together to form =O, =S or =NH; X 2a and X 2b are both hydrogen atoms or are taken together to form =O, =S or =NH;
R3 is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on ring A;
L 1 is an alkylene chain consisting of m L carbon atoms, wherein the hydrogen atoms on the alkylene carbon atoms may or may not be substituted with OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a , or L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom, with the proviso that when Y 1 is NH, O, or S, the atom of L 1 bonded to Y 1 is carbon, and the atom of L 1 bonded to OR 1 is carbon, and the oxygen atoms are not adjacent to each other;
L2 is an alkylene chain consisting of nL carbon atoms, wherein the hydrogen atoms on the alkylene carbon atoms may or may not be substituted with OH, ORc , NH2 , NHRc , NRcRd , SH or SRc , or L2 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom, with the proviso that when Y2 is NH, O or S, the atom of L2 bonded to Y2 is carbon, the atom of L2 bonded to OR2 is carbon, and the oxygen atoms are not adjacent to each other;
R a , R b , R c and R d are each independently a substituent or a protecting group;
l is 1 or 2;
m L is an integer ranging from 0 to 30;
n L is an integer ranging from 0 to 30;
In ring A, one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen, or sulfur;
Ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond.
Nucleotide sequence T and nucleotide sequence Q are each linked to the non-nucleotide structure via -OR 1 - or -OR 2 -, where R 1 and R 2 may be present or absent, and when present, R 1 and R 2 are each independently a nucleotide residue or a structure of formula (1).
　式（１）中、Ｘ１ａおよびＸ１ｂは、例えば、共に水素原子であるか、一緒になって＝Ｏ、＝Ｓまたは＝ＮＨを形成する。Ｘ２ａおよびＸ２ｂについても同様である。 In formula (1), X 1a and X 1b are, for example, both hydrogen atoms or are combined to form =O, =S or =NH. The same applies to X 2a and X 2b .
　式（１）中、Ｙ１およびＹ２は、それぞれ独立して、単結合、ＣＨ２、ＮＨ、ＯまたはＳである。 In formula (1), Y 1 and Y 2 each independently represent a single bond, CH 2 , NH, O or S.
　式（１）中、環Ａにおいて、ｌは、１または２である。ｌ＝１の場合、環Ａは、５員環であり、例えば、ピロリジン骨格である。ピロリジン骨格は、例えば、プロリン骨格、プロリノール骨格等があげられ、これらの二価の構造が例示できる。ｌ＝２の場合、環Ａは、６員環であり、例えば、ピペリジン骨格である。環Ａは、環Ａ上のＣ－２以外の１個の炭素原子が、窒素、酸素または硫黄で置換されてもよい。また、環Ａは、環Ａ内に、炭素－炭素二重結合または炭素－窒素二重結合を含んでもよい。環Ａは、例えば、Ｌ型およびＤ型のいずれでもよい。 In formula (1), l in ring A is 1 or 2. When l=1, ring A is a 5-membered ring, for example a pyrrolidine skeleton. Examples of pyrrolidine skeletons include proline skeletons and prolinol skeletons, and their divalent structures can be exemplified. When l=2, ring A is a 6-membered ring, for example a piperidine skeleton. In ring A, one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen, or sulfur. Ring A may also contain a carbon-carbon double bond or a carbon-nitrogen double bond within ring A. Ring A may be, for example, either L-type or D-type.
　式（１）中、Ｒ３は、環Ａ上のＣ－３、Ｃ－４、Ｃ－５またはＣ－６に結合する水素原子または置換基である。Ｒ３が置換基の場合、置換基Ｒ３は、１個でも複数でも、存在しなくてもよく、複数の場合、同一でも異なってもよい。 In formula (1), R 3 is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on ring A. When R 3 is a substituent, the substituent R 3 may be one or more, or may be absent, and when there are more than one, they may be the same or different.
　置換基Ｒ３は、例えば、ハロゲン、ＯＨ、ＯＲ４、ＮＨ２、ＮＨＲ４、ＮＲ４Ｒ５、ＳＨ、ＳＲ４またはオキソ基（＝Ｏ）等である。 The substituent R3 is, for example, a halogen, OH, OR4 , NH2 , NHR4 , NR4R5 , SH, SR4 or an oxo group (=O).
　Ｒ４およびＲ５は、例えば、それぞれ独立して、置換基または保護基であり、同一でも異なってもよい。置換基は、上記置換基群Ａに記載のものが挙げられ、例えば、ハロゲン、アルキル、アルケニル、アルキニル、ハロアルキル、アリール、ヘテロアリール、アリールアルキル、シクロアルキル、シクロアルケニル、シクロアルキルアルキル、シクロアルケニルアルキル、ヒドロキシアルキル、アルコキシアルキル、アミノアルキル、ヘテロシクリルアルケニル、ヘテロシクリルアルキル、ヘテロアリールアルキル、シリル、シリルオキシアルキル等があげられる。以下、同様である。置換基Ｒ３は、これらの列挙する置換基であってもよい。 R 4 and R 5 are, for example, each independently a substituent or a protective group, and may be the same or different. The substituent may be any of those described in the above-mentioned substituent group A, such as halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl, heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl, etc. The same applies below. The substituent R 3 may be any of the substituents listed above.
　保護基は、例えば、反応性の高い官能基を不活性に変換する官能基であり、公知の保護基等があげられる。保護基は、例えば、文献(J. F. W. McOmie, 「Protecting Groups in Organic Chemistry」 PrenumPress, London and New York, 1973)の記載を援用できる。保護基は、特に制限されず、例えば、ｔｅｒｔ－ブチルジメチルシリル基（ＴＢＤＭＳ）、ビス（２－アセトキシエチルオキシ）メチル基（ＡＣＥ）、トリイソプロピルシリルオキシメチル基（ＴＯＭ）、１－（２－シアノエトキシ）エチル基（ＣＥＥ）、２－シアノエトキシメチル基（ＣＥＭ）およびトリルスルフォニルエトキシメチル基（ＴＥＭ）、ジメトキシトリチル基（ＤＭＴｒ）等があげられる。Ｒ３がＯＲ４の場合、保護基は、特に制限されず、例えば、ＴＢＤＭＳ基、ＡＣＥ基、ＴＯＭ基、ＣＥＥ基、ＣＥＭ基およびＴＥＭ基等があげられる。この他にも、シリル含有基もあげられる。以下、同様である。 The protecting group is, for example, a functional group that converts a highly reactive functional group into an inactive one, and examples of the protecting group include known protecting groups. For example, the description in the literature (J. F. W. McOmie, "Protecting Groups in Organic Chemistry" Prenum Press, London and New York, 1973) can be cited. The protecting group is not particularly limited, and examples thereof include tert-butyldimethylsilyl group (TBDMS), bis(2-acetoxyethyloxy)methyl group (ACE), triisopropylsilyloxymethyl group (TOM), 1-(2-cyanoethoxy)ethyl group (CEE), 2-cyanoethoxymethyl group (CEM), tolylsulfonylethoxymethyl group (TEM), dimethoxytrityl group (DMTr), and the like. When R 3 is OR 4 , the protecting group is not particularly limited, and examples thereof include TBDMS group, ACE group, TOM group, CEE group, CEM group, and TEM group. Other examples include silyl-containing groups. The same applies below.
　式（１）中、Ｌ１は、ｍＬ個の炭素原子からなるアルキレン鎖である。アルキレン炭素原子上の水素原子は、例えば、ＯＨ、ＯＲａ、ＮＨ２、ＮＨＲａ、ＮＲａＲｂ、ＳＨ、もしくはＳＲａで置換されてもよいし、置換されていなくてもよい。または、Ｌ１は、アルキレン鎖の１つ以上の炭素原子が酸素原子で置換されたポリエーテル鎖でもよい。ポリエーテル鎖は、例えば、ポリエチレングリコールである。なお、Ｙ１が、ＮＨ、ＯまたはＳの場合、Ｙ１に結合するＬ１の原子は炭素であり、ＯＲ１に結合するＬ１の原子は炭素であり、酸素原子同士は隣接しない。つまり、例えば、Ｙ１がＯの場合、その酸素原子とＬ１の酸素原子は隣接せず、ＯＲ１の酸素原子とＬ１の酸素原子は隣接しない。 In formula (1), L 1 is an alkylene chain consisting of m L carbon atoms. The hydrogen atoms on the alkylene carbon atoms may be substituted with, for example, OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a , or may not be substituted. Alternatively, L 1 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms. The polyether chain is, for example, polyethylene glycol. Note that, when Y 1 is NH, O, or S, the atom of L 1 bonded to Y 1 is carbon, the atom of L 1 bonded to OR 1 is carbon, and the oxygen atoms are not adjacent to each other. That is, for example, when Y 1 is O, the oxygen atom and the oxygen atom of L 1 are not adjacent, and the oxygen atom of OR 1 and the oxygen atom of L 1 are not adjacent.
　式（１）中、Ｌ２は、ｎＬ個の炭素原子からなるアルキレン鎖である。アルキレン炭素原子上の水素原子は、例えば、ＯＨ、ＯＲｃ、ＮＨ２、ＮＨＲｃ、ＮＲｃＲｄ、ＳＨもしくはＳＲｃで置換されてもよいし、置換されていなくてもよい。または、Ｌ２は、アルキレン鎖の１つ以上の炭素原子が酸素原子で置換されたポリエーテル鎖でもよい。なお、Ｙ２が、ＮＨ、ＯまたはＳの場合、Ｙ２に結合するＬ２の原子は炭素であり、ＯＲ２に結合するＬ２の原子は炭素であり、酸素原子同士は隣接しない。つまり、例えば、Ｙ２がＯの場合、その酸素原子とＬ２の酸素原子は隣接せず、ＯＲ２の酸素原子とＬ２の酸素原子は隣接しない。 In formula (1), L 2 is an alkylene chain consisting of nL carbon atoms. The hydrogen atoms on the alkylene carbon atoms may be substituted with, for example, OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c , or may not be substituted. Alternatively, L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms. Note that, when Y 2 is NH, O or S, the atom of L 2 bonded to Y 2 is carbon, the atom of L 2 bonded to OR 2 is carbon, and the oxygen atoms are not adjacent to each other. That is, for example, when Y 2 is O, the oxygen atom and the oxygen atom of L 2 are not adjacent, and the oxygen atom of OR 2 and the oxygen atom of L 2 are not adjacent.
　Ｌ１のｍＬおよびＬ２のｎＬは、特に制限されず、それぞれ、下限は、例えば、０であり、上限も、特に制限されない。ｎＬおよびｍＬは、例えば、非ヌクレオチド構造の所望の長さに応じて、適宜設定できる。ｎＬおよびｍＬは、例えば、製造コストおよび収率等の点から、それぞれ、０～３０が好ましく、より好ましくは０～２０であり、さらに好ましくは０～１５である。ｎＬとｍＬは、同じでもよいし（ｎＬ＝ｍＬ）、異なってもよい。ｎＬ＋ｍＬは、例えば、０～３０であり、好ましくは０～２０であり、より好ましくは０～１５である。 m L of L1 and n L of L2 are not particularly limited, and the lower limit is, for example, 0, and the upper limit is also not particularly limited. n L and m L can be appropriately set, for example, according to the desired length of the non-nucleotide structure. For example, in terms of production cost and yield, n L and m L are each preferably 0 to 30, more preferably 0 to 20, and even more preferably 0 to 15. n L and m L may be the same (n L =m L ), or may be different. n L +m L is, for example, 0 to 30, preferably 0 to 20, and more preferably 0 to 15.
　Ｒａ、Ｒｂ、ＲｃおよびＲｄは、例えば、それぞれ独立して、置換基または保護基である。置換基および保護基は、例えば、前述と同様である。 R a , R b , R c and R d are, for example, each independently a substituent or a protecting group. The substituent and protecting group are, for example, the same as those described above.
　式（１）において、水素原子は、例えば、それぞれ独立して、Ｃｌ、Ｂｒ、ＦおよびＩ等のハロゲンに置換されてもよい。 In formula (1), the hydrogen atoms may be, for example, each independently substituted with a halogen such as Cl, Br, F, or I.
　ヌクレオチド配列Ｔおよびヌクレオチド配列Ｑは、例えば、それぞれ、－ＯＲ１－または－ＯＲ２－を介して、非ヌクレオチド構造に結合する。ここで、Ｒ１およびＲ２は、存在しても存在しなくてもよい。Ｒ１およびＲ２が存在する場合、Ｒ１およびＲ２は、それぞれ独立して、ヌクレオチド残基または式（１）の構造である。Ｒ１および／またはＲ２がヌクレオチド残基の場合、リンカーＬの構造は、例えば、ヌクレオチド残基Ｒ１および／またはＲ２を除く式（１）の構造からなる非ヌクレオチド残基と、ヌクレオチド残基とから形成される。Ｒ１および／またはＲ２が式（１）の構造の場合、非ヌクレオチド構造は、例えば、式（１）の構造からなる非ヌクレオチド残基が、２つ以上連結された構造となる。式（１）の構造は、例えば、１個、２個、３個または４個含んでもよい。このように、複数含む場合、式（１）の構造は、例えば、直接連結されてもよいし、ヌクレオチド残基を介して結合してもよい。他方、Ｒ１およびＲ２が存在しない場合、非ヌクレオチド構造は、例えば、式（１）の構造からなる非ヌクレオチド残基のみから形成される。 The nucleotide sequence T and the nucleotide sequence Q are linked to the non-nucleotide structure, for example, via -OR 1 - or -OR 2 -, respectively. Here, R 1 and R 2 may be present or absent. When R 1 and R 2 are present, R 1 and R 2 are each independently a nucleotide residue or a structure of formula (1). When R 1 and/or R 2 are nucleotide residues, the structure of the linker L is formed, for example, from a non-nucleotide residue consisting of the structure of formula (1) excluding the nucleotide residues R 1 and/or R 2 , and a nucleotide residue. When R 1 and/or R 2 are the structure of formula (1), the non-nucleotide structure is, for example, a structure in which two or more non-nucleotide residues consisting of the structure of formula (1) are linked. The structure of formula (1) may include, for example, one, two, three, or four. In this way, when a plurality of structures of formula (1) are included, the structures of formula (1) may be, for example, directly linked or linked via a nucleotide residue. On the other hand, when R 1 and R 2 are not present, the non-nucleotide structure is formed solely from non-nucleotide residues having the structure of, for example, formula (1).
　ヌクレオチド配列Ｔおよびヌクレオチド配列Ｑと、－ＯＲ１－および－ＯＲ２－との結合の組合せは、特に制限されず、例えば、以下のいずれかの条件があげられる。
条件１
　ヌクレオチド配列Ｔは、－ＯＲ２－を介して、ヌクレオチド配列Ｑは、－ＯＲ１－を介して、式（１）の構造と結合する。
条件２
　ヌクレオチド配列Ｔは、－ＯＲ１－を介して、ヌクレオチド配列Ｑは、－ＯＲ２－を介して、式（１）の構造と結合する。 The combination of bonds between the nucleotide sequence T and the nucleotide sequence Q and -OR 1 - and -OR 2 - is not particularly limited, and examples thereof include any of the following conditions.
Condition 1
Nucleotide sequence T is linked to the structure of formula (1) via --OR 2 --, and nucleotide sequence Q is linked to the structure of formula (1) via --OR 1 --.
Condition 2
Nucleotide sequence T is linked to the structure of formula (1) via --OR 1 --, and nucleotide sequence Q is linked to the structure of formula (1) via --OR 2 --.
　式（１）の構造は、例えば、下記式（１－１）～式（１－９）が例示でき、下記式において、ｎＬおよびｍＬは、式（１）と同じである。下記式において、ｑは、０～１０の整数である。 The structure of formula (1) can be exemplified by the following formulae (1-1) to (1-9), in which nL and mL are the same as those in formula (1). In the following formulae, q is an integer of 0 to 10.
　式（１－１）～（１－９）において、ｎＬ、ｍＬおよびｑは、特に制限されず、前述の通りである。
　具体例として、式（１－１）において、ｎＬ＝８、式（１－２）において、ｎＬ＝３、式（１－３）において、ｎＬ＝４または８、式（１－４）において、ｎＬ＝７または８、式（１－５）において、ｎＬ＝３およびｍＬ＝４、式（１－６）において、ｎＬ＝８およびｍＬ＝４、式（１－７）において、ｎＬ＝８およびｍＬ＝４、式（１－８）において、ｎＬ＝５およびｍＬ＝４、式（１－９）において、ｑ＝１およびｍＬ＝４があげられる。式（１－４）の一例（ｎＬ＝８）を、下記式（１－４ａ）に、式（１－８）の一例（ｎＬ＝５、ｍＬ＝４）を、下記式（１－８ａ）に示す。 In the formulas (1-1) to (1-9), n L , m L and q are not particularly limited and are as defined above.
Specific examples include: in formula (1-1), n L =8, in formula (1-2), n L =3, in formula (1-3), n L =4 or 8, in formula (1-4), n L =7 or 8, in formula (1-5), n L =3 and m L =4, in formula (1-6), n L =8 and m L =4, in formula (1-7), n L =8 and m L =4, in formula (1-8), n L =5 and m L =4, and in formula (1-9), q =1 and m L =4. An example of formula (1-4) (n L =8) is shown in formula (1-4a) below, and an example of formula (1-8) (n L =5, m L =4) is shown in formula (1-8a) below.
　また、式（１－８）の一例（ｎＬ＝５、ｍＬ＝４）である、下記式で表されるプロリン誘導体リンカーを以下に示す。 Moreover, a proline derivative linker represented by the following formula, which is an example of formula (1-8) (n L =5, m L =4), is shown below.
　遺伝子に対する一本鎖核酸分子として、Ｔ－Ｌ－Ｑで表されるヘアピン型核酸分子だけでなく、発現制御配列を含むガイド鎖の両端にリンカーが付加され、各リンカーを介してガイド鎖の一部に相補的なヌクレオチド配列と、ガイド鎖の残りの部分に相補的なヌクレオチド配列とが結合した、ダンベル型の構造を有する一本鎖核酸分子（例えば、特許第４９６８８１１号、特許第４９６５７４５号等）も包含される。 Single-stranded nucleic acid molecules for genes include not only hairpin-type nucleic acid molecules represented by T-L-Q, but also single-stranded nucleic acid molecules with a dumbbell-shaped structure in which linkers are added to both ends of a guide strand containing an expression control sequence, and a nucleotide sequence complementary to a portion of the guide strand is bound to a nucleotide sequence complementary to the remaining portion of the guide strand via each linker (e.g., Patent Nos. 4968811 and 4965745, etc.).
　さらに、遺伝子に対する一本鎖核酸分子はＬ型構造を有する核酸分子であってもよい（Ｌ型一本鎖核酸分子）。Ｌ型一本鎖核酸分子としては、（ｉ）アンチセンス鎖及びセンス鎖の連結がヌクレオシドの糖部の２’，３’間又は２’，５’間をクロスリンクして得られたＬ型一本鎖核酸分子、（ｉｉ）リンカーとして、内部にアミド結合を有するアルキル鎖を用いたＬ型一本鎖核酸分子、（ｉｉｉ）ヌクレオシドの塩基間を直接連結したＬ型一本鎖核酸分子、及び（ｉｖ）ヌクレオシドの糖部の１’がＨで置換され且つ、アンチセンス鎖及びセンス鎖の連結がヌクレオシドの糖部の２’，２’間をクロスリンクして得られたＬ型一本鎖核酸分子が挙げられる。
　Ｌ型一本鎖核酸分子としては、下式の一般式： Furthermore, the single-stranded nucleic acid molecule for a gene may be a nucleic acid molecule having an L-type structure (L-type single-stranded nucleic acid molecule). Examples of L-type single-stranded nucleic acid molecules include (i) L-type single-stranded nucleic acid molecules obtained by crosslinking the 2'-3' or 2'-5' of the sugar moiety of a nucleoside between the antisense strand and the sense strand, (ii) L-type single-stranded nucleic acid molecules using an alkyl chain having an internal amide bond as a linker, (iii) L-type single-stranded nucleic acid molecules in which the bases of nucleosides are directly linked, and (iv) L-type single-stranded nucleic acid molecules in which 1' of the sugar moiety of a nucleoside is replaced with H and the antisense strand and the sense strand are crosslinked between the 2'-2' of the sugar moiety of a nucleoside.
The L-type single-stranded nucleic acid molecule has the following general formula:
［式中、Ｘ、Ｙ、Ｘ１、Ｙ１、Ｘ２、Ｙ２は、それぞれ独立して、修飾されていてもよいリボヌクレオチド残基又は修飾されていてもよいデオキシリボヌクレオチド残基であり、Ｔ及びＱは、標的配列に対して相補的な、連続する１４～３０個の修飾されていてもよいリボヌクレオチド残基からなる配列とそれに相補的なリボヌクレオチド配列であり（一方が標的核酸配列に対して相補的な配列であれば、他方はそれに相補的な配列である）、
Ｚは（Ｘ）の糖部分の２’位若しくは５’位と（Ｙ）の糖部分の２’位若しくは３’位を連結するリンカーであり、又は（Ｘ）の塩基部分と（Ｙ）の塩基部分とを連結するリンカーであり、
ｍ１及びｍ２は、それぞれ独立して、０～５の整数であり；
ｎ１及びｎ２は、それぞれ独立して、０～５の整数である］
で示される核酸分子、が挙げられる。
　好適な態様において一般式（ａ）で表される核酸分子における [wherein X, Y, X1 , Y1 , X2 , and Y2 are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue, T and Q are a sequence consisting of 14 to 30 contiguous ribonucleotide residues which are complementary to a target sequence and a ribonucleotide sequence complementary thereto (if one is a sequence complementary to a target nucleic acid sequence, the other is a sequence complementary thereto),
Z is a linker connecting the 2'- or 5'-position of the sugar moiety of (X) to the 2'- or 3'-position of the sugar moiety of (Y), or a linker connecting the base moiety of (X) to the base moiety of (Y);
m1 and m2 each independently represent an integer of 0 to 5;
n1 and n2 each independently represent an integer from 0 to 5.
The nucleic acid molecule is represented by:
In a preferred embodiment, the nucleic acid molecule represented by the general formula (a)
の構造は、 The structure of is
又は or
である。
　上記［式１］～［式４］において、
Ａ１およびＡ１’は、それぞれ独立して－Ｏ－、－ＮＲ１ａ－、－Ｓ－又は－ＣＲ１ａＲ１ｂ－であり（ここで、Ｒ１ａ及びＲ１ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基である）、
Ａ２およびＡ２’は、それぞれ独立して－ＣＲ２ａＲ２ｂ－、－ＣＯ－、アルキニル基、アルケニル基又は単結合であり（ここで、Ｒ２ａ及びＲ２ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基である）、
Ａ３およびＡ３’は、それぞれ独立して－Ｏ－又は－ＮＲ３ａ－、－Ｓ－、－ＣＲ３ａＲ３ｂ－又は単結合であり（ここで、Ｒ３ａ及びＲ３ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基である）、
Ａ４およびＡ４’は、それぞれ独立して－（ＣＲ４ａＲ４ｂ）ｎ－、－（ＣＲ４ａＲ４ｂ）ｎ－環Ｄ－（ここで、環Ｄは、炭素数６～１０のアリール基、炭素数２～１０のヘテロアリール基、炭素数４～１０のシクロアルキル基又は炭素数４～１０のヘテロシクロアルキル基であり、Ｒ４ａ及びＲ４ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基であり；ｎは１～６の整数である）又は単結合であり、
Ａ５およびＡ５’は、それぞれ独立して－ＮＲ５ａ－又は単結合であり（ここで、Ｒ５ａは水素原子又は炭素数１～１０のアルキル基である）、
Ａ６およびＡ６’は、それぞれ独立して－（ＣＲ６ａＲ６ｂ）ｎ－又は単結合であり（ここで、Ｒ６ａ及びＲ６ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基であり；ｎは１～６の整数である）、
Ｗ１は、－（ＣＲ１Ｒ２）ｎ－、－ＣＯ－、－（ＣＲ１Ｒ２）ｎ－ＣＯＯ－（ＣＲ１Ｒ２）ｎ－ＣＯＯ－（ＣＲ１Ｒ２）ｎ、－（ＣＲ１Ｒ２）ｎ－Ｏ－（ＣＲ１Ｒ２ＣＲ１Ｒ２Ｏ）ｎ－ＣＨ２－、－（ＣＲ１Ｒ２）ｎ－環Ｄ－（ＣＲ１Ｒ２）ｎ－又は－（ＣＲ１Ｒ２）ｎ－ＳＳ－（ＣＲ１Ｒ２）ｎ－であり（ここで、環Ｄは、炭素数６～１０のアリール基、炭素数２～１０のヘテロアリール基、炭素数４～１０のシクロアルキル基又は炭素数４～１０のヘテロシクロアルキル基であり；Ｒ１とＲ２は、それぞれ独立して水素原子又は炭素数１～１０のアルキル基でありｎは１～６の整数である）、
Ｅ２およびＥ２’は、それぞれ独立して－ＣＲ２ａＲ２ｂ－、－ＣＯ－、アルキニル基、アルケニル基又は単結合であり（ここで、Ｒ２ａ及びＲ２ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基である）、
Ｅ３およびＥ３’は、それぞれ独立して－Ｏ－又は－ＮＲ３ａ－、－Ｓ－、－ＣＲ３ａＲ３ｂ－又は単結合であり（ここで、Ｒ３ａ及びＲ３ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基である）、
Ｅ４およびＥ４’は、それぞれ独立して－（ＣＲ４ａＲ４ｂ）ｎ－、－（ＣＲ４ａＲ４ｂ）ｎ－環Ｄ－（ここで、環Ｄは、炭素数６～１０のアリール基、炭素数２～１０のヘテロアリール基、炭素数４～１０のシクロアルキル基又は炭素数４～１０のヘテロシクロアルキル基であり；Ｒ４ａ及びＲ４ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基であり；ｎは１～６の整数である）又は単結合であり、
Ｅ５およびＥ５’は、それぞれ独立して－ＮＲ５ａ－又は単結合であり（ここで、Ｒ５ａは水素原子又は炭素数１～１０のアルキル基である）、
Ｅ６およびＥ６’は、それぞれ独立して－（ＣＲ６ａＲ６ｂ）ｎ－又は単結合である（ここで、Ｒ６ａ及びＲ６ｂは、それぞれ独立して水素原子又は炭素数１～１０のアルキル基であり：ｎは１～６の整数である）。 It is.
In the above formulas 1 to 4,
A 1 and A 1 ' are each independently -O-, -NR 1a -, -S-, or -CR 1a R 1b - (wherein R 1a and R 1b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
A 2 and A 2 ′ each independently represent —CR 2a R 2b —, —CO—, an alkynyl group, an alkenyl group, or a single bond (wherein R 2a and R 2b each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
A 3 and A 3 ′ each independently represent —O—, —NR 3a —, —S—, —CR 3a R 3b —, or a single bond (wherein R 3a and R 3b each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
A 4 and A 4 ' are each independently -(CR 4a R 4b ) n -, -(CR 4a R 4b ) n-ring D- (wherein ring D is an aryl group having 6 to 10 carbon atoms, a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or a heterocycloalkyl group having 4 to 10 carbon atoms, R 4a and R 4b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; n is an integer of 1 to 6) or a single bond;
A 5 and A 5 ' each independently represent -NR 5a - or a single bond (wherein R 5a represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
A 6 and A 6 ′ each independently represent —(CR 6a R 6b )n- or a single bond (wherein R 6a and R 6b each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; n is an integer of 1 to 6);
W 1 is -(CR 1 R 2 )n-, -CO-, -(CR 1 R 2 )n-COO-(CR 1 R 2 )n-COO-(CR 1 R 2 )n, -(CR 1 R 2 )n-O-(CR 1 R 2 CR 1 R 2 O)n-CH 2 -, -(CR 1 R 2 )n-ring D-(CR 1 R 2 )n- or -(CR 1 R 2 )n-SS-(CR 1 R 2 )n- (wherein ring D is an aryl group having 6 to 10 carbon atoms, a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms or a heterocycloalkyl group having 4 to 10 carbon atoms; Each of 2 is independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and n is an integer of 1 to 6.
E 2 and E 2 ' are each independently -CR 2a R 2b -, -CO-, an alkynyl group, an alkenyl group, or a single bond (wherein R 2a and R 2b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
E 3 and E 3 ' are each independently -O-, -NR 3a -, -S-, -CR 3a R 3b - or a single bond (wherein R 3a and R 3b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
E 4 and E 4 ' are each independently -(CR 4a R 4b ) n -, -(CR 4a R 4b ) n-ring D- (wherein ring D is an aryl group having 6 to 10 carbon atoms, a heteroaryl group having 2 to 10 carbon atoms, a cycloalkyl group having 4 to 10 carbon atoms, or a heterocycloalkyl group having 4 to 10 carbon atoms; R 4a and R 4b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; and n is an integer of 1 to 6) or a single bond;
E 5 and E 5 ' each independently represent -NR 5a - or a single bond (wherein R 5a represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms);
E 6 and E 6 ' are each independently -(CR 6a R 6b )n- or a single bond (wherein R 6a and R 6b are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; n is an integer of 1 to 6).
　上記式において、「炭素数１～１０のアルキル基」、「炭素数６～１０のアリール基」、「炭素数２～１０のヘテロアリール基」、「炭素数４～１０のシクロアルキル基」及び「炭素数４～１０のヘテロシクロアルキル基」は置換可能な位置で置換されていてもよく、置換基としては前述の置換基群Ａに記載のものが挙げられる。 In the above formula, the "alkyl group having 1 to 10 carbon atoms", the "aryl group having 6 to 10 carbon atoms", the "heteroaryl group having 2 to 10 carbon atoms", the "cycloalkyl group having 4 to 10 carbon atoms" and the "heterocycloalkyl group having 4 to 10 carbon atoms" may be substituted at any substitutable position, and examples of the substituent include those described in the above-mentioned substituent group A.
　遺伝子に対する一本鎖核酸分子の合成方法は、特に限定されず、従来公知の核酸の製造方法が採用できる。合成方法としては、例えば、遺伝子工学的手法による合成法、化学合成法等があげられる。遺伝子工学的手法は、例えば、インビトロ転写合成法、ベクターを用いる方法、ＰＣＲカセットによる方法があげられる。ベクターは、特に制限されず、プラスミド等の非ウイルスベクター、ウイルスベクター等があげられる。化学合成法は、特に制限されず、例えば、ホスホロアミダイト法およびＨ－ホスホネート法等があげられる。化学合成法は、例えば、市販の自動核酸合成機を使用可能である。化学合成法は、一般に、アミダイトが使用される。アミダイトは、特に制限されず、市販のアミダイトとして、例えば、ＲＮＡ　Ｐｈｏｓｐｈｏｒａｍｉｄｉｔｅｓ（２’－Ｏ－ＴＢＤＭＳアミダイト、ＣｈｅｍＧｅｎｅｓ）、ＡＣＥアミダイトおよびＴＯＭアミダイト、ＣＥＥアミダイト、ＣＥＭアミダイト、ＴＥＭアミダイト等があげられる。また、２’位架橋用アミダイトとしてＡＥＭアミダイト（ＷＯ２０２２／０４５２２４）を用いることができる。 The method for synthesizing a single-stranded nucleic acid molecule for a gene is not particularly limited, and a conventionally known method for producing nucleic acids can be used. Examples of the synthesis method include synthesis using genetic engineering techniques and chemical synthesis. Examples of the genetic engineering techniques include in vitro transcription synthesis, methods using vectors, and methods using PCR cassettes. The vector is not particularly limited, and examples include non-viral vectors such as plasmids and viral vectors. The chemical synthesis method is not particularly limited, and examples include the phosphoramidite method and the H-phosphonate method. For the chemical synthesis method, for example, a commercially available automatic nucleic acid synthesizer can be used. For the chemical synthesis method, amidites are generally used. The amidite is not particularly limited, and examples of commercially available amidites include RNA Phosphoramidites (2'-O-TBDMS amidite, ChemGenes), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc. In addition, AEM amidite (WO2022/045224) can be used as the amidite for bridging the 2' position.
　遺伝子に対する一本鎖核酸分子が、天然の非修飾リボヌクレオチド残基のみで構成される場合、該核酸分子の前駆体として、該核酸分子を発現可能な状態でコードするベクターの形態で提供することもできる。該発現ベクターは、標的遺伝子に対する一本鎖核酸分子をコードするＤＮＡを標的細胞内で機能的なプロモーターの制御下に含むことを特徴とし、その他の構成は何ら制限されない。前記ＤＮＡを挿入するベクターは特に制限されず、一般的なベクターを使用することができ、例えば、ウイルスベクターおよび非ウイルスベクター等があげられる。非ウイルスベクターとしては、例えば、プラスミドベクターがあげられる。該発現ベクターを、自体公知の遺伝子導入法を用いて、標的細胞（標的遺伝子を発現し得る哺乳動物細胞）に導入することにより、該細胞内での標的遺伝子の発現を制御することができる。 When the single-stranded nucleic acid molecule for a gene is composed only of natural unmodified ribonucleotide residues, it can be provided as a precursor of the nucleic acid molecule in the form of a vector that encodes the nucleic acid molecule in an expressible state. The expression vector is characterized by containing DNA encoding the single-stranded nucleic acid molecule for the target gene under the control of a promoter that is functional in the target cell, and other configurations are not limited in any way. The vector into which the DNA is inserted is not particularly limited, and general vectors can be used, such as viral vectors and non-viral vectors. An example of a non-viral vector is a plasmid vector. By introducing the expression vector into a target cell (a mammalian cell capable of expressing a target gene) using a gene transfer method known per se, it is possible to control the expression of the target gene in the cell.
リガンドコンジュゲート物質
　本発明は、下記一般式（ＩＩ）で表される化合物を提供する。当該化合物は、イミノジカルボン酸誘導体にリガンドが結合した化合物であり、本明細書中、リガンドコンジュゲート物質とも称する。 Ligand conjugate substance The present invention provides a compound represented by the following general formula (II): This compound is a compound in which a ligand is bound to an iminodicarboxylic acid derivative, and is also referred to as a ligand conjugate substance in this specification.
［式中、
Ｚはアミノ基保護基であり、
Ｂ１、Ｂ２、Ｂ３及びＢ４は、同一又は異なって水素原子又は下記式のいずれかを表し、
－（Ｗ２）ｎ２－ＣＯ－ＮＨ－Ｘ１－Ｙ１、
－（Ｗ３）ｎ３－ＣＯ－ＮＨ－Ｘ２－Ｙ２、
－（Ｗ５）ｎ５－ＣＯ－ＮＨ－Ｘ３－Ｙ３、
－（Ｗ６）ｎ６－ＣＯ－ＮＨ－Ｘ４－Ｙ４、
Ｗ２は、－（ＣＲ１Ｗ２Ｒ２Ｗ２）－（式中、Ｒ１Ｗ２及びＲ２Ｗ２は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ３は、－（ＣＲ１Ｗ３Ｒ２Ｗ３）－（式中、Ｒ１Ｗ３及びＲ２Ｗ３は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ５は、－（ＣＲ１Ｗ５Ｒ２Ｗ５）－（式中、Ｒ１Ｗ５及びＲ２Ｗ５は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ６は、－（ＣＲ１Ｗ６Ｒ２Ｗ６）－（式中、Ｒ１Ｗ６及びＲ２Ｗ６は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
ｎ２、ｎ３、ｎ５及びｎ６は、それぞれ独立して、１～５の整数であり、
ｎＢ１’～ｎＢ４’は、０～３の整数（但し、同時に０でない）を表し、
Ｘ１、Ｘ２、Ｘ３及びＸ４は、それぞれ独立して、置換されても良いスペーサーであり、Ｙ１、Ｙ２、Ｙ３及びＹ４は、それぞれ独立して、リガンドであり、
Ｗ１は、－（ＣＲ１Ｗ１Ｒ２Ｗ１）－（式中、Ｒ１Ｗ１及びＲ２Ｗ１は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
Ｗ４は、－（ＣＲ１Ｗ４Ｒ２Ｗ４）－（式中、Ｒ１Ｗ４及びＲ２Ｗ４は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
ｎ１及びｎ４は、それぞれ独立して、１～５の整数である］
で表される化合物。 [Wherein,
Z is an amino protecting group;
B 1 , B 2 , B 3 and B 4 are the same or different and each represents a hydrogen atom or any one of the following formulae:
-( W2 ) n2 -CO-NH- X1 - Y1 ,
-( W3 ) n3 -CO-NH- X2 - Y2 ,
-( W5 ) n5 -CO-NH- X3 - Y3 ,
-( W6 ) n6 -CO-NH- X4 - Y4 ,
W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W5 represents -( CR1W5R2W5 )- (wherein R1W5 and R2W5 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
W6 represents -( CR1W6R2W6 )- (wherein R1W6 and R2W6 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
n 2 , n 3 , n 5 and n 6 each independently represent an integer from 1 to 5;
n B1 ' to n B4 ' each represent an integer of 0 to 3 (but are not all 0);
X 1 , X 2 , X 3 and X 4 each independently represent a spacer which may be substituted; Y 1 , Y 2 , Y 3 and Y 4 each independently represent a ligand;
W 1 represents -(CR 1W1 R 2W1 )- (wherein R 1W1 and R 2W1 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group);
W 4 represents -(CR 1W4 R 2W4 )- (wherein R 1W4 and R 2W4 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
n1 and n4 each independently represent an integer from 1 to 5.
A compound represented by the formula:
　式（ＩＩ）で表される本発明のリガンドコンジュゲート物質は、好ましくは下記式（ＩＩ－１）で表される化合物である。 The ligand conjugate substance of the present invention represented by formula (II) is preferably a compound represented by the following formula (II-1):
（式中、各記号の定義は上記［ＩＩ］と同義である）
　式（ＩＩ）及び式（ＩＩ－１）中、Ｚはアミノ基保護基である。好ましくは、ベンジルオキシカルボニル等が挙げられる。 (In the formula, the definitions of each symbol are the same as those in [II] above.)
In formula (II) and formula (II-1), Z is an amino-protecting group, preferably benzyloxycarbonyl.
　式（ＩＩ）及び式（ＩＩ－１）中、好ましくは、Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーは、それぞれ独立して、－（ＣＲ９Ｒ１０）ｎ’－Ｒ１１（式中、Ｒ９及びＲ１０は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ１１は－Ｏ－又は－ＮＲ１２－（式中、Ｒ１２は水素原子又は置換されていてもよいアルキル基である）を表す；ｎ’は１～１０の整数を表す）であり、より好ましくは、それぞれ独立して、－（ＣＨ２）６－Ｏ－であるか、－（ＣＨ２）５－ＮＨ－である。 In formula (II) and formula (II-1), preferably, the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or combine together to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); n' represents an integer from 1 to 10), and more preferably, they are each independently -(CH 2 ) 6 -O- or -(CH 2 ) 5 -NH-.
　式（ＩＩ）及び式（ＩＩ－１）中、好ましくは、Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５及びＷ６は、－（ＣＨ２）－で、ｎ１～ｎ６が１である。 In formula (II) and formula (II-1), preferably, W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are —(CH 2 )—, and n 1 to n 6 are 1.
　式（ＩＩ）及び式（ＩＩ－１）中、好ましくは、Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドは、それぞれ独立して、糖（好ましくはＧａｌＮＡｃ）であるか脂質である。 In formula (II) and formula (II-1), preferably, the ligands Y 1 , Y 2 , Y 3 and Y 4 are each independently a sugar (preferably GalNAc) or a lipid.
医薬組成物
　本発明は、本発明のリガンドコンジュゲート核酸を含む医薬組成物を提供する。ここで、リガンドは、上記した一般式（Ｉ）又は一般式（Ｉ－１）におけるＹ１～Ｙ４に相当する。「リガンド」とは、生体分子と複合体を形成して生物学的な目的を果たす物質を意味する。リガンドとしては、標的とする生体分子によって適宜選択されるが、例えば、抗体、酵素、ペプチドアプタマー、受容体などのタンパク質性物質、ＤＮＡ、ＲＮＡ又はこれらの誘導体などの核酸、抗生物質などのタンパク質性もしくは非タンパク質性生体内活性物質、あるいは、レクチンなどに結合し得る糖（例えば、ラクトース、シアリルラクトース、グロボトリオース、ラクト-Ｎ-ネオテトラオース、オリゴマンノースなどの他、生体内の特定の組織等に存在するレクチンと結合することが既知の糖鎖などであり、単糖、オリゴ糖などいかなる糖鎖であってもよい）、脂質などを挙げることができる。
　一例として、糖鎖の一種であるＧａｌＮＡｃ（Ｎ－アセチルガラクトサミン）が付加された核酸分子が挙げられる。ＧａｌＮＡｃは肝実質細胞の細胞表面に特異的に発現するアシアロ糖タンパク質受容体と強く結合し、エンドサイトーシスされるが、この受容体はエンドサイトーシスとエキソサイトーシスのリサイクリングが活発に行われるため、核酸分子が効率よく肝実質細胞に引き込まれる。 Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising the ligand-conjugated nucleic acid of the present invention. Here, the ligand corresponds to Y 1 to Y 4 in the above-mentioned general formula (I) or general formula (I-1). The term "ligand" refers to a substance that forms a complex with a biomolecule to achieve a biological purpose. The ligand is appropriately selected depending on the target biomolecule, and examples thereof include protein substances such as antibodies, enzymes, peptide aptamers, and receptors, nucleic acids such as DNA, RNA, or derivatives thereof, proteinaceous or non-proteinaceous bioactive substances such as antibiotics, and sugars that can bind to lectins (for example, lactose, sialyllactose, globotriose, lacto-N-neotetraose, oligomannose, and other sugar chains known to bind to lectins present in specific tissues in the body, and may be any sugar chain such as monosaccharides and oligosaccharides), lipids, etc.
One example is a nucleic acid molecule with GalNAc (N-acetylgalactosamine), a type of sugar chain, attached to it. GalNAc binds strongly to the asialoglycoprotein receptor specifically expressed on the cell surface of hepatocytes and is endocytosed. This receptor is actively recycled through endocytosis and exocytosis, so that the nucleic acid molecule is efficiently drawn into the hepatocytes.
　本発明の医薬組成物は、経口的にまたは非経口的に、哺乳動物（例、ヒト、ネコ、フェレット、ミンク、ラット、マウス、モルモット、ウサギ、ヒツジ、ウマ、ブタ、ウシ、サル）に対して投与することが可能であるが、非経口的に投与するのが望ましい。 The pharmaceutical composition of the present invention can be administered orally or parenterally to mammals (e.g., humans, cats, ferrets, mink, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, and monkeys), but is preferably administered parenterally.
　非経口的な投与（例えば、静脈内注射、皮下注射、筋肉注射、局所注入、腹腔内投与など）に好適な製剤としては、水性および非水性の等張な無菌の注射液剤があり、これには抗酸化剤、緩衝液、制菌剤、等張化剤等が含まれていてもよい。また、水性および非水性の無菌の懸濁液剤が挙げられ、これには懸濁剤、可溶化剤、増粘剤、安定化剤、防腐剤等が含まれていてもよい。当該製剤は、アンプルやバイアルのように単位投与量あるいは複数回投与量ずつ容器に封入することができる。また、有効成分および医薬上許容される担体を凍結乾燥し、使用直前に適当な無菌のビヒクルに溶解または懸濁すればよい状態で保存することもできる。
　非経口的な投与に好適な別の製剤としては、噴霧剤等を挙げることが出来る。 Suitable formulations for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The formulations can be sealed in unit doses or multiple doses in containers such as ampoules and vials. Also, the active ingredient and a pharma- ceutical acceptable carrier can be freeze-dried and stored in a state in which it is sufficient to dissolve or suspend them in a suitable sterile vehicle immediately before use.
Other suitable formulations for parenteral administration include aerosols.
　医薬組成物中の本発明のリガンドコンジュゲート核酸の含有量は、例えば、医薬組成物全体の約０．１ないし１００重量％である。 The content of the ligand-conjugated nucleic acid of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
　本発明の医薬組成物の投与量は、投与の目的、投与方法、対象疾患の種類、重篤度、投与対象の状況（性別、年齢、体重など）によって異なるが、例えば、成人に全身投与する場合、通常、本発明のリガンドコンジュゲート核酸の一回投与量として２ｎｍｏｌ／ｋｇ以上５０ｎｍｏｌ／ｋｇ以下、局所投与する場合、１ｐｍｏｌ／ｋｇ以上１０ｎｍｏｌ／ｋｇ以下が望ましい。かかる投与量を１～１０回、より好ましくは５～１０回投与することが望ましい。 The dosage of the pharmaceutical composition of the present invention varies depending on the purpose of administration, the method of administration, the type and severity of the target disease, and the condition of the recipient (sex, age, body weight, etc.). For example, when administered systemically to an adult, a single dose of the ligand-conjugated nucleic acid of the present invention is usually preferably 2 nmol/kg or more and 50 nmol/kg or less, and when administered locally, 1 pmol/kg or more and 10 nmol/kg or less. It is desirable to administer such a dosage 1 to 10 times, more preferably 5 to 10 times.
　本発明の医薬組成物は、２種以上のリガンドコンジュゲート核酸を含んでいてもよい。また、本発明の医薬組成物に含まれる核酸分子の投与対象となる遺伝子疾患に対する治療効果が報告されている他の医薬と組み合わせて用いることができる。これらの併用薬剤は、本発明の医薬組成物とともに製剤化して単一の製剤として投与することもできるし、あるいは、本発明の医薬組成物とは別個に製剤化して、本発明の医薬組成物と同一もしくは別ルートで、同時もしくは時間差をおいて投与することもできる。また、これらの併用薬剤の投与量は、該薬剤を単独投与する場合に通常用いられる量であってよく、あるいは通常用いられる量より減量することもできる。 The pharmaceutical composition of the present invention may contain two or more types of ligand-conjugated nucleic acids. It may also be used in combination with other drugs that have been reported to have therapeutic effects on genetic diseases for which the nucleic acid molecules contained in the pharmaceutical composition of the present invention are administered. These concomitant drugs may be formulated together with the pharmaceutical composition of the present invention and administered as a single formulation, or may be formulated separately from the pharmaceutical composition of the present invention and administered simultaneously or at a later time via the same or a different route as the pharmaceutical composition of the present invention. The dosage of these concomitant drugs may be the amount normally used when the drug is administered alone, or may be reduced from the amount normally used.
　本発明の医薬組成物は、有効量の本発明のリガンドコンジュゲート核酸を単独で用いてもよいし、任意の担体、例えば医薬上許容される担体とともに、医薬組成物として製剤化することもできる。 The pharmaceutical composition of the present invention may use an effective amount of the ligand-conjugated nucleic acid of the present invention alone, or may be formulated as a pharmaceutical composition together with any carrier, such as a pharma- ceutical acceptable carrier.
　医薬上許容される担体としては、例えば、ショ糖、デンプン等の賦形剤、セルロース、メチルセルロース等の結合剤、デンプン、カルボキシメチルセルロース等の崩壊剤、ステアリン酸マグネシウム、エアロジル等の滑剤、クエン酸、メントール等の芳香剤、安息香酸ナトリウム、亜硫酸水素ナトリウム等の保存剤、クエン酸、クエン酸ナトリウム等の安定剤、メチルセルロース、ポリビニルピロリドン等の懸濁剤、界面活性剤等の分散剤、水、生理食塩水等の希釈剤、ベースワックス等が挙げられるが、それらに限定されるものではない。 Examples of pharma- ceutically acceptable carriers include, but are not limited to, excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, flavorings such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, dispersing agents such as surfactants, diluents such as water and saline, base waxes, etc.
　本発明のリガンドコンジュゲート核酸の標的細胞内への導入を促進するために、本発明の医薬組成物は更に核酸導入用試薬を含むことができる。該核酸導入用試薬としては、アテロコラーゲン；リポソーム；ナノパーティクル；リポフェクチン、リプフェクタミン（ｌｉｐｏｆｅｃｔａｍｉｎｅ）、ＤＯＧＳ（トランスフェクタム）、ＤＯＰＥ、ＤＯＴＡＰ、ＤＤＡＢ、ＤＨＤＥＡＢ、ＨＤＥＡＢ、ポリブレン、あるいはポリ（エチレンイミン）（ＰＥＩ）等の陽イオン性脂質等を用いることが出来る。 In order to promote the introduction of the ligand-conjugated nucleic acid of the present invention into target cells, the pharmaceutical composition of the present invention may further contain a nucleic acid introduction reagent. Examples of the nucleic acid introduction reagent include atelocollagen; liposomes; nanoparticles; lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, and cationic lipids such as poly(ethyleneimine) (PEI).
　好ましい一実施態様において、本発明の医薬組成物は、本発明のリガンドコンジュゲート核酸がリポソームに封入されてなる医薬組成物であり得る。リポソームは、１以上の脂質二重層により包囲された内相を有する微細閉鎖小胞であり、通常は水溶性物質を内相に、脂溶性物質を脂質二重層内に保持することができる。本明細書において「封入」という場合には、本発明のリガンドコンジュゲート核酸はリポソーム内相に保持されてもよいし、脂質二重層内に保持されてもよい。本発明に用いられるリポソームは単層膜であっても多層膜であってもよく、また、粒子径は、例えば１０～１０００ｎｍ、好ましくは５０～３００ｎｍの範囲で適宜選択できる。標的組織への送達性を考慮すると、粒子径は、例えば２００ｎｍ以下、好ましくは１００ｎｍ以下であり得る。 In a preferred embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition in which the ligand-conjugated nucleic acid of the present invention is encapsulated in a liposome. A liposome is a fine closed vesicle having an internal phase surrounded by one or more lipid bilayers, and can usually hold a water-soluble substance in the internal phase and a fat-soluble substance in the lipid bilayer. In the present specification, the term "encapsulation" refers to the ligand-conjugated nucleic acid of the present invention being held in the liposomal internal phase or in the lipid bilayer. The liposome used in the present invention may be a single-layer membrane or a multi-layer membrane, and the particle size can be appropriately selected, for example, within the range of 10 to 1000 nm, preferably 50 to 300 nm. In consideration of the delivery to the target tissue, the particle size can be, for example, 200 nm or less, preferably 100 nm or less.
　核酸のような水溶性化合物のリポソームへの封入法としては、リピドフィルム法（ボルテックス法）、逆相蒸発法、界面活性剤除去法、凍結融解法、リモートローディング法等が挙げられるが、これらに限定されず、任意の公知の方法を適宜選択することができる。 Methods for encapsulating water-soluble compounds such as nucleic acids into liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method, and any known method can be appropriately selected.
　以下に実施例を示して本発明をさらに具体的に説明するが、本発明はこれらの実施例に何ら制限されない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.
本実施例で使用した各定義についての説明
１）配列について
・ＲＮＡは大文字で表記した（ＡＣＧＵ）。
・ＤＮＡは小文字で表記した（ａｃｇｔ）。
・上下に記載した配列は、特に指定のない場合には、上段は５’→３’、下段は３’→５’とした。
２）修飾核酸の記載
・２’－ＯＭｅ化した核酸：Ａｍ，Ｃｍ，Ｇｍ，Ｕｍ
・２’－Ｆ化した核酸：Ａｆ，Ｃｆ，Ｇｆ，Ｕｆ
・ＬＮＡ：ＡＬ，ＣＬ，ＧＬ，ＴＬ
・Ｓ化：Ｎｓ（３’側のリン酸を硫化）
・２’－ＯＡＥＭ化した核酸：Ａｅ，Ｃｅ，Ｇｅ，Ｕｅ Explanation of each definition used in this example: 1) Regarding sequences: RNA is written in uppercase (ACGU).
・DNA is written in lower case (acgt).
Unless otherwise specified, the sequences written vertically are 5'→3' for the upper line and 3'→5' for the lower line.
2) Description of modified nucleic acids 2'-OMe modified nucleic acids: Am, Cm, Gm, Um
2'-F-modified nucleic acids: Af, Cf, Gf, Uf
LNA: AL, CL, GL, TL
Sulfurization: Ns (sulfurization of the phosphate on the 3' side)
2'-OAEM-modified nucleic acid: Ae, Ce, Ge, Ue
製造例１　（ＧａｌＮＡｃ）×４個＋リンカーの合成
１）化合物４の合成 Production Example 1: Synthesis of 4 (GalNAc) + Linker 1) Synthesis of Compound 4
（１）化合物２の合成
　化合物１（３．０ｇ，１５．９ｍｍｏｌ）のテトラヒドロフラン（３０ｍＬ）溶液に、アルゴン雰囲気下、ジイソプロピルエチルアミン（３．３ｍＬ，１．２ｅｑ．）、クロロ蟻酸ベンジル（２．７ｍＬ，１．２ｅｑ．）を加え、室温にて終夜撹拌した。反応混合物に、水、飽和炭酸水素ナトリウム水溶液を加え、ジクロロメタンにて抽出し、有機層を硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン：酢酸エチル＝５０：５０～３０：７０）にて精製し、目的化合物２を無色油状物質として５．２ｇ得た。
ESI-Mass:346.13[M+Na]+
（２）化合物３の合成
　化合物２（５．２ｇ）のエタノール（８０ｍＬ）溶液に、アルゴン雰囲気下、１Ｍ水酸化ナトリウム水溶液（４８．０ｍＬ，３．０ｅｑ．）を加え、室温にて３時間撹拌した。反応混合物に、水を加え、ジクロロメタンにて洗浄した。水層に１Ｍ塩酸水溶液を加え、ジクロロメタンにて抽出した。有機層を水で洗浄した後、硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮し、目的化合物３を無色油状物質として４．２ｇ得た。
ESI-Mass:266.07[M-H]-
（３）化合物４の合成
　化合物３（３．６ｇ，１３．３ｍｍｏｌ）のジクロロメタン（１００ｍＬ）溶液に、アルゴン雰囲気下、ペンタフルオロフェノール（６．１ｇ，２．５ｅｑ．）、１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（６．４ｇ，２．５ｅｑ．）、４－ジメチルアミノピリジン（１６２ｍｇ，０．１ｅｑ．）を加え、室温にて３時間撹拌した。減圧下、溶液を３分の１以下に濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（ジクロロメタン）にて精製し、目的化合物４を淡黄色油状物質として５．８ｇ得た。
ESI-Mass:622.04[M+Na]+ (1) Synthesis of Compound 2 Diisopropylethylamine (3.3 mL, 1.2 eq.) and benzyl chloroformate (2.7 mL, 1.2 eq.) were added to a solution of Compound 1 (3.0 g, 15.9 mmol) in tetrahydrofuran (30 mL) under an argon atmosphere, and the mixture was stirred overnight at room temperature. Water and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, which was extracted with dichloromethane, and the organic layer was dried over magnesium sulfate. After concentrating the solution under reduced pressure, the resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=50:50 to 30:70) to obtain 5.2 g of the target compound 2 as a colorless oily substance.
ESI-Mass: 346.13 [M+Na] +
(2) Synthesis of Compound 3 To a solution of Compound 2 (5.2 g) in ethanol (80 mL), 1 M aqueous sodium hydroxide solution (48.0 mL, 3.0 eq.) was added under an argon atmosphere, and the mixture was stirred at room temperature for 3 hours. Water was added to the reaction mixture, and the mixture was washed with dichloromethane. A 1 M aqueous hydrochloric acid solution was added to the aqueous layer, and the mixture was extracted with dichloromethane. The organic layer was washed with water and then dried over magnesium sulfate. The solution was concentrated under reduced pressure to obtain 4.2 g of the target compound 3 as a colorless oily substance.
ESI-Mass:266.07[MH] -
(3) Synthesis of Compound 4 To a solution of Compound 3 (3.6 g, 13.3 mmol) in dichloromethane (100 mL), pentafluorophenol (6.1 g, 2.5 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.4 g, 2.5 eq.), and 4-dimethylaminopyridine (162 mg, 0.1 eq.) were added under an argon atmosphere, and the mixture was stirred at room temperature for 3 hours. The solution was concentrated to one-third or less under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane) to obtain 5.8 g of the target compound 4 as a pale yellow oily substance.
ESI-Mass: 622.04 [M+Na] +
２）化合物６の合成 2) Synthesis of compound 6
（１）化合物６の合成
　既知の方法で得られた化合物５（３．０ｇ，５．２ｍｍｏｌ）のメタノール（３０ｍＬ）溶液に、酢酸（５滴）、１０ｗｔ％５５％ｗｅｔパラジウム－活性炭素（５００ｍｇ，０．０５ｅｑ．）を加え、水素雰囲気下、室温にて４時間撹拌した。反応混合物をセライトろ過した後、トリフルオロ酢酸（４７５μＬ，１．２ｅｑ．）を加え、減圧下、溶液を濃縮し、目的化合物６を白色泡状物質として得た。全量を次の反応に使用した。 (1) Synthesis of Compound 6 To a solution of compound 5 (3.0 g, 5.2 mmol) obtained by a known method in methanol (30 mL), acetic acid (5 drops) and 10 wt % 55% wet palladium-activated carbon (500 mg, 0.05 eq.) were added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 4 hours. The reaction mixture was filtered through Celite, and then trifluoroacetic acid (475 μL, 1.2 eq.) was added, and the solution was concentrated under reduced pressure to obtain the target compound 6 as a white foamy substance. The entire amount was used in the next reaction.
３）化合物８の合成 3) Synthesis of compound 8
（１）化合物７の合成
　化合物４（１．６ｇ，２．６ｍｍｏｌ）のジメトキシエタン（２０ｍＬ）溶液に、アルゴン雰囲気下、化合物６（５．２ｍｍｏｌ，２．０ｅｑ．）、ジイソプロピルエチルアミン（１．４ｍＬ，３．０ｅｑ．）を加え、室温にて４時間撹拌した。反応混合物に、水、飽和炭酸水素ナトリウム水溶液を加え、ジクロロメタンにて抽出した。有機層を０．１Ｍ塩酸水溶液で洗浄した後、水で洗浄し、硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（クロロホルム：メタノール＝１００：０～９０：１０）にて精製し、目的化合物７を白色泡状物質として２．５ｇ得た。
ESI-Mass:1146.49[M+Na]+
（２）化合物８の合成
　化合物７（１．７ｇ，１．５ｍｍｏｌ）のメタノール（２５ｍＬ）溶液に、１０ｗｔ％５５％ｗｅｔパラジウム－活性炭素（２８９ｍｇ，０．１ｅｑ．）を加え、水素雰囲気下、室温にて終夜撹拌した。反応混合物をセライトろ過した後、減圧下、溶液を濃縮し、目的化合物８を白色泡状物質として１．５ｇ得た。 (1) Synthesis of Compound 7 Compound 6 (5.2 mmol, 2.0 eq.) and diisopropylethylamine (1.4 mL, 3.0 eq.) were added to a solution of compound 4 (1.6 g, 2.6 mmol) in dimethoxyethane (20 mL) under an argon atmosphere, and the mixture was stirred at room temperature for 4 hours. Water and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a 0.1 M aqueous solution of hydrochloric acid, then washed with water, and dried over magnesium sulfate. The solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform:methanol = 100:0 to 90:10) to obtain 2.5 g of the target compound 7 as a white foamy substance.
ESI-Mass: 1146.49 [M+Na] +
(2) Synthesis of Compound 8 To a solution of Compound 7 (1.7 g, 1.5 mmol) in methanol (25 mL), 10 wt % 55% wet palladium-activated carbon (289 mg, 0.1 eq.) was added, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. The reaction mixture was filtered through Celite, and the solution was concentrated under reduced pressure to obtain 1.5 g of the target compound 8 as a white foam.
４）化合物１１の合成 4) Synthesis of compound 11
（１）化合物１０の合成
　化合物９（５．３ｇ，２１．６ｍｍｏｌ）及びスベリン酸モノメチル（４．９ｇ，１．２ｅｑ．）のジクロロメタン（５０ｍＬ）溶液に、アルゴン雰囲気下、１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（５．０ｇ，１．２ｅｑ．）を加え、室温にて終夜撹拌した。反応混合物に、水、飽和炭酸水素ナトリウム水溶液を加え、ジクロロメタンにて抽出し、有機層を硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（ｎ－ヘキサン：酢酸エチル＝３０：７０～０：１００）にて精製し、目的化合物１０を無色油状物質として９．４ｇ得た。
ESI-Mass:438.25[M+Na]+
（２）化合物１１の合成
　化合物１０（９．４ｇ，２２．６ｍｍｏｌ）のジクロロメタン（８０ｍＬ）溶液に、アルゴン雰囲気下、トリフルオロ酢酸（１７．３ｍＬ，１０ｅｑ．）を加え、室温にて３時間撹拌した。減圧下、溶液を濃縮し、目的化合物１１を淡黄色油状物質として８．３ｇ得た。
ESI-Mass:304.10[M+H]+ (1) Synthesis of Compound 10 To a solution of compound 9 (5.3 g, 21.6 mmol) and monomethyl suberate (4.9 g, 1.2 eq.) in dichloromethane (50 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.0 g, 1.2 eq.) was added under an argon atmosphere, and the mixture was stirred overnight at room temperature. Water and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, which was extracted with dichloromethane, and the organic layer was dried over magnesium sulfate. After concentrating the solution under reduced pressure, the resulting residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=30:70 to 0:100) to obtain 9.4 g of the target compound 10 as a colorless oily substance.
ESI-Mass: 438.25 [M+Na] +
(2) Synthesis of Compound 11 To a solution of Compound 10 (9.4 g, 22.6 mmol) in dichloromethane (80 mL), trifluoroacetic acid (17.3 mL, 10 eq.) was added under an argon atmosphere, and the mixture was stirred at room temperature for 3 hours. The solution was concentrated under reduced pressure to obtain 8.3 g of the target compound 11 as a pale yellow oily substance.
ESI-Mass: 304.10 [M+H] +
５）化合物１３の合成 5) Synthesis of compound 13
（１）化合物１２の合成
　化合物１１（２０２ｍｇ，６６８μｍｏｌ）及び化合物８（１．５ｇ，２．２ｅｑ．）のジクロロメタン（３０ｍＬ）溶液に、アルゴン雰囲気下、１－［ビス（ジメチルアミノ）メチレン］－１Ｈ－１，２，３－トリアゾロ［４，５－ｂ］ピリジニウム３－オキシドヘキサフルオロホスファート（１．０ｇ，４．０ｅｑ．）、ジイソプロピルエチルアミン（８１４μＬ，７．０ｅｑ．）を加え、室温にて６時間撹拌した。反応混合物に、水を加え、クロロホルムにて抽出し、有機層を硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣を逆相シリカゲルカラムクロマトグラフィー（水：アセトニトリル＝９０：１０～０：１００）にて精製した。減圧下、有機溶媒を濃縮した後、水、飽和塩化ナトリウム水溶液を加え、クロロホルムにて抽出した。有機層を硫酸マグネシウムにて乾燥した後、減圧下、溶液を濃縮し、目的化合物１２を白色固体として８１１ｍｇ得た。
ESI-Mass:2269.04[M+Na]+
（２）化合物１３の合成
　化合物１２（７１６ｍｇ，３１９μｍｏｌ）のメタノール（５１ｍＬ）溶液に、アルゴン雰囲気下、１Ｍ水酸化ナトリウム水溶液（１２．７ｍＬ，４０ｅｑ．）を加え、室温にて終夜撹拌した。反応混合物に、アンバーライトＩＲＣ７６を加え、ｐＨ６とした。ろ過後、減圧下、溶液を濃縮した後、得られた残渣を逆相シリカゲルカラムクロマトグラフィー（水：アセトニトリル＝９０：１０～７０：３０）にて精製した。精製物をメタノール（１ｍＬ）に溶解した後、撹拌下、室温にて酢酸エチル（３０ｍＬ）に滴下した。析出固体をろ取、減圧下乾燥し、目的化合物１３を白色固体として３５５ｍｇ得た。
ESI-Mass:1750.90[M+Na]+ (1) Synthesis of Compound 12 To a dichloromethane (30 mL) solution of Compound 11 (202 mg, 668 μmol) and Compound 8 (1.5 g, 2.2 eq.), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (1.0 g, 4.0 eq.) and diisopropylethylamine (814 μL, 7.0 eq.) were added under an argon atmosphere, and the mixture was stirred at room temperature for 6 hours. Water was added to the reaction mixture, which was extracted with chloroform, and the organic layer was dried over magnesium sulfate. The solution was concentrated under reduced pressure, and the resulting residue was purified by reversed-phase silica gel column chromatography (water:acetonitrile = 90:10 to 0:100). The organic solvent was concentrated under reduced pressure, and then water and a saturated aqueous sodium chloride solution were added, followed by extraction with chloroform. The organic layer was dried over magnesium sulfate, and the solution was concentrated under reduced pressure to obtain 811 mg of the target compound 12 as a white solid.
ESI-Mass: 2269.04 [M+Na] +
(2) Synthesis of Compound 13 To a solution of compound 12 (716 mg, 319 μmol) in methanol (51 mL), 1M aqueous sodium hydroxide solution (12.7 mL, 40 eq.) was added under an argon atmosphere, and the mixture was stirred overnight at room temperature. Amberlite IRC76 was added to the reaction mixture to adjust the pH to 6. After filtration, the solution was concentrated under reduced pressure, and the resulting residue was purified by reversed-phase silica gel column chromatography (water:acetonitrile = 90:10 to 70:30). The purified product was dissolved in methanol (1 mL), and then dropped into ethyl acetate (30 mL) at room temperature under stirring. The precipitated solid was collected by filtration and dried under reduced pressure to obtain 355 mg of the target compound 13 as a white solid.
ESI-Mass: 1750.90 [M+Na] +
６）化合物１４の合成 6) Synthesis of compound 14
（１）化合物１４の合成
　化合物１３（４０ｍｇ，２３μｍｏｌ）のジメチルホルムアミド（０．５ｍＬ）溶液に、Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチル－Ｏ－（Ｎ－スクシンイミジル）ウロニウムテトラフルオロボラート（８．７ｍｇ，１．３ｅｑ．）のジメチルホルムアミド（０．２ｍＬ）溶液、ジイソプロピルエチルアミン（４．８μＬ，１．２ｅｑ．）のジメチルホルムアミド（０．２ｍＬ）溶液を加え、氷冷下にて３０分間撹拌した。反応混合物を、ジエチルエーテル（６．０ｍＬ）に滴下した後、４℃にて１０分間、遠心分離した。上澄み液を除去した後、減圧下乾燥し、目的化合物１４を無色油状物質として得た。収率１００％として、次の反応に使用した。
ESI-Mass:1825.93[M＋H]+ (1) Synthesis of Compound 14 To a solution of compound 13 (40 mg, 23 μmol) in dimethylformamide (0.5 mL), a solution of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (8.7 mg, 1.3 eq.) in dimethylformamide (0.2 mL) and a solution of diisopropylethylamine (4.8 μL, 1.2 eq.) in dimethylformamide (0.2 mL) were added and stirred for 30 minutes under ice cooling. The reaction mixture was dropped into diethyl ether (6.0 mL) and then centrifuged at 4° C. for 10 minutes. The supernatant was removed and dried under reduced pressure to obtain the target compound 14 as a colorless oily substance. The yield was 100% and used in the next reaction.
ESI-Mass: 1825.93 [M+H] +
製造例２　（ＧａｌＮＡｃ）×２個＋リンカーの合成
１）化合物１５の合成 Production Example 2 Synthesis of (GalNAc) x 2 + Linker 1) Synthesis of Compound 15
　化合物１１（３．０ｇ，９．９ｍｍｏｌ）のジクロロメタン（８０ｍＬ）溶液に、アルゴン雰囲気下、ペンタフルオロフェノール（４．６ｇ，２．５ｅｑ．）、１－（３－ジメチルアミノプロピル）－３－エチルカルボジイミド塩酸塩（４．７ｇ，２．５ｅｑ．）、４－ジメチルアミノピリジン（１２１ｍｇ，０．１ｅｑ．）を加え、室温にて終夜撹拌した。減圧下、溶液を３分の１以下に濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（ジクロロメタン：酢酸エチル＝１００：０～８５：１５）にて精製し、目的化合物１５を淡黄色油状物質として５．６ｇ得た。
ESI-Mass:658.09[M+Na]+ To a solution of compound 11 (3.0 g, 9.9 mmol) in dichloromethane (80 mL), pentafluorophenol (4.6 g, 2.5 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.7 g, 2.5 eq.), and 4-dimethylaminopyridine (121 mg, 0.1 eq.) were added under an argon atmosphere, and the mixture was stirred overnight at room temperature. The solution was concentrated to one-third or less under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate=100:0 to 85:15) to obtain 5.6 g of the target compound 15 as a pale yellow oily substance.
ESI-Mass: 658.09 [M+Na] +
２）化合物１６の合成 2) Synthesis of compound 16
　化合物１５（１．０ｇ，１．６ｍｍｏｌ）のジメトキシエタン（１５ｍＬ）溶液に、アルゴン雰囲気下、化合物６（１．８ｇ，２．０ｅｑ．）、ジイソプロピルエチルアミン（１．６ｍＬ，６．０ｅｑ．）を加え、室温にて終夜撹拌した。反応混合物に、水、飽和炭酸水素ナトリウム水溶液を加え、ジクロロメタンにて抽出した。有機層を０．１Ｍ塩酸水溶液で洗浄した後、水で洗浄し、硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１００：０～９０：１０）にて精製し、目的化合物１６を白色固体として１．１ｇ得た。
ESI-Mass:1182.54[M+Na]+ Compound 6 (1.8 g, 2.0 eq.) and diisopropylethylamine (1.6 mL, 6.0 eq.) were added to a solution of compound 15 (1.0 g, 1.6 mmol) in dimethoxyethane (15 mL) under an argon atmosphere, and the mixture was stirred overnight at room temperature. Water and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with a 0.1 M aqueous solution of hydrochloric acid, then washed with water, and dried over magnesium sulfate. The solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane:methanol = 100:0 to 90:10) to obtain 1.1 g of the target compound 16 as a white solid.
ESI-Mass: 1182.54 [M+Na] +
３）化合物１８の合成 3) Synthesis of compound 18
（１）化合物１７の合成
　化合物１６（１．１ｇ，１．０ｍｍｏｌ）のメタノール（７６ｍＬ）溶液に、アルゴン雰囲気下、１Ｍ水酸化ナトリウム水溶液（１９．０ｍＬ，２０ｅｑ．）を加え、室温にて４時間撹拌した。反応混合物に、１Ｍ塩酸水溶液（１９．０ｍＬ，２０ｅｑ．）を加えた後、減圧下、溶液を濃縮した。得られた残渣を逆相シリカゲルカラムクロマトグラフィー（水：アセトニトリル＝９０：１０～７０：３０）にて精製し、目的化合物１７を白色固体として４２６ｍｇ得た。
ESI-Mass:916.47[M+Na]+
（２）化合物１８の合成
　化合物１７（１０ｍｇ，１１．２μｍｏｌ）のジメチルホルムアミド（０．５ｍＬ）溶液に、氷冷下、Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチル－Ｏ－（Ｎ－スクシンイミジル）ウロニウムテトラフルオロボラート（４．２ｍｇ，１．３ｅｑ．）のジメチルホルムアミド（０．２ｍＬ）溶液、ジイソプロピルエチルアミン（２．３μＬ，１．２ｅｑ．）のジメチルホルムアミド（０．２ｍＬ）溶液を加え、氷冷下にて３０分間撹拌した。反応混合物を、ジエチルエーテル（６．０ｍＬ）に滴下した後、４℃にて１０分間、遠心分離した。上澄み液を除去した後、減圧下乾燥し、目的化合物１８を白色固体として得た。収率１００％として、次の反応に使用した。
ESI-Mass:991.48[M+H]+ (1) Synthesis of Compound 17 To a solution of compound 16 (1.1 g, 1.0 mmol) in methanol (76 mL), 1 M aqueous sodium hydroxide solution (19.0 mL, 20 eq.) was added under an argon atmosphere, and the mixture was stirred at room temperature for 4 hours. After adding 1 M aqueous hydrochloric acid solution (19.0 mL, 20 eq.) to the reaction mixture, the solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase silica gel column chromatography (water:acetonitrile = 90:10 to 70:30), and 426 mg of the target compound 17 was obtained as a white solid.
ESI-Mass: 916.47 [M+Na] +
(2) Synthesis of Compound 18 To a solution of Compound 17 (10 mg, 11.2 μmol) in dimethylformamide (0.5 mL), a solution of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (4.2 mg, 1.3 eq.) in dimethylformamide (0.2 mL) and a solution of diisopropylethylamine (2.3 μL, 1.2 eq.) in dimethylformamide (0.2 mL) were added under ice cooling, and the mixture was stirred for 30 minutes under ice cooling. The reaction mixture was dropped into diethyl ether (6.0 mL), and then centrifuged at 4° C. for 10 minutes. After removing the supernatant, the mixture was dried under reduced pressure to obtain the target compound 18 as a white solid. The yield was 100%, and the product was used in the next reaction.
ESI-Mass: 991.48[M+H] +
製造例３　脂肪酸＋リンカーの合成
１）化合物２３の合成 Preparation Example 3 Synthesis of fatty acid + linker 1) Synthesis of compound 23
（１）化合物１９の合成
　化合物１５（７６２ｍｇ，１．２ｍｍｏｌ）のジメトキシエタン（１０ｍＬ）溶液に、アルゴン雰囲気下、Ｎ－（ｔｅｒｔ－ブトキシカルボニル）－１，５－ジアミノペンタン（５３４ｍｇ，２．２ｅｑ．）、ジイソプロピルエチルアミン（６２７μＬ，３．０ｅｑ．）を加え、室温にて終夜撹拌した。反応混合物に、水、飽和炭酸水素ナトリウム水溶液を加え、クロロホルムにて抽出し、有機層を硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（クロロホルム：メタノール＝１００：０～９４：６）にて精製し、目的化合物１９を無色油状物質として９６８ｍｇ得た。
ESI-Mass:694.43[M+Na]+
（２）化合物２０の合成
　化合物１９（１．２ｇ，１．８ｍｍｏｌ）のジオキサン（１０ｍＬ）溶液に、アルゴン雰囲気下、４Ｍ塩酸－ジオキサン（１０ｍＬ）を加え、室温にて終夜撹拌した。反応混合物に、メタノール（１０ｍＬ）を加え、減圧下、溶液を濃縮し、目的化合物２０を橙色油状物質として１．１ｇ得た。
ESI-Mass:472.35[M+H]+
（３）化合物２１の合成
　化合物２０（３００ｍｇ，５５１μｍｏｌ）のジクロロメタン（１０ｍＬ）溶液に、アルゴン雰囲気下、ジイソプロピルエチルアミン（５７６μＬ，６．０ｅｑ．）、パルミチン酸クロリド（３６６μＬ，２．２ｅｑ．）を加え、室温にて１時間撹拌した。反応混合物に、水、飽和炭酸水素ナトリウム水溶液を加え、クロロホルムにて抽出し、有機層を硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮した後、得られた残渣をシリカゲルカラムクロマトグラフィー（クロロホルム：メタノール＝１００：０～９０：１０）にて精製し、目的化合物２１を３２４ｍｇ得た。
ESI-Mass:970.78[M+Na]+
（４）化合物２２の合成
　化合物２１（３００ｍｇ，３１６μｍｏｌ）のメタノール（１０ｍＬ）、テトラヒドロフラン（５ｍＬ）、クロロホルム（５ｍＬ）溶液に、アルゴン雰囲気下、１Ｍ水酸化ナトリウム水溶液（３．２ｍＬ，１０．０ｅｑ．）を加え、室温にて１日間撹拌した。反応混合物に、水、１２Ｍ塩酸水溶液（１ｍＬ）を加え、クロロホルムにて抽出し、有機層を硫酸マグネシウムにて乾燥した。減圧下、溶液を濃縮し、目的化合物２２を白色固体として３０１ｍｇ得た。
ESI-Mass:956.78[M+Na]+
（５）化合物２２の精製
　化合物２２（２８１ｍｇ，３０１μｍｏｌ）をシリカゲルカラムクロマトグラフィー（クロロホルム：メタノール＝１００：０～９０：１０）にて精製し、目的化合物２２を白色固体として２４０ｍｇ得た。
（６）化合物２３の合成
　化合物２２（４．１ｍｇ，４．４μｍｏｌ）のピリジン（０．２ｍＬ）溶液に、ジイソプロピルエチルアミン（２．３μＬ，３．０ｅｑ．）のピリジン（０．１ｍＬ）溶液、Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチル－Ｏ－（Ｎ－スクシンイミジル）ウロニウムテトラフルオロボラート（２．０ｍｇ，１．５ｅｑ．）のジメチルホルムアミド（０．１ｍＬ）溶液を加え、室温にて１時間撹拌した。反応混合物を、水（４．０ｍＬ）に滴下した後、４℃にて１５分間、遠心分離した。上澄み液を除去し、酢酸エチル（４．０ｍＬ）を加え振とうした後、４℃にて１５分間、遠心分離した。上澄み液を除去した後、減圧下乾燥し、目的化合物２３を得た。収率１００％として、次の反応に使用した。
ESI-Mass:1053.78[M+H]+ (1) Synthesis of Compound 19 To a solution of compound 15 (762 mg, 1.2 mmol) in dimethoxyethane (10 mL), N-(tert-butoxycarbonyl)-1,5-diaminopentane (534 mg, 2.2 eq.) and diisopropylethylamine (627 μL, 3.0 eq.) were added under an argon atmosphere, and the mixture was stirred overnight at room temperature. Water and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, which was extracted with chloroform, and the organic layer was dried over magnesium sulfate. After concentrating the solution under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform:methanol=100:0 to 94:6) to obtain 968 mg of the target compound 19 as a colorless oily substance.
ESI-Mass: 694.43 [M+Na] +
(2) Synthesis of Compound 20 To a solution of Compound 19 (1.2 g, 1.8 mmol) in dioxane (10 mL), 4 M hydrochloric acid-dioxane (10 mL) was added under an argon atmosphere, and the mixture was stirred overnight at room temperature. Methanol (10 mL) was added to the reaction mixture, and the solution was concentrated under reduced pressure to obtain 1.1 g of the target compound 20 as an orange oily substance.
ESI-Mass: 472.35 [M+H] +
(3) Synthesis of Compound 21 Diisopropylethylamine (576 μL, 6.0 eq.) and palmitic acid chloride (366 μL, 2.2 eq.) were added to a dichloromethane (10 mL) solution of compound 20 (300 mg, 551 μmol) under an argon atmosphere, and the mixture was stirred at room temperature for 1 hour. Water and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, which was extracted with chloroform, and the organic layer was dried over magnesium sulfate. After concentrating the solution under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform:methanol=100:0 to 90:10) to obtain 324 mg of the target compound 21.
ESI-Mass: 970.78 [M+Na] +
(4) Synthesis of Compound 22 Compound 21 (300 mg, 316 μmol) was dissolved in methanol (10 mL), tetrahydrofuran (5 mL), and chloroform (5 mL) and 1M aqueous sodium hydroxide solution (3.2 mL, 10.0 eq.) was added under an argon atmosphere and stirred at room temperature for 1 day. Water and 12M aqueous hydrochloric acid solution (1 mL) were added to the reaction mixture, which was extracted with chloroform and the organic layer was dried over magnesium sulfate. The solution was concentrated under reduced pressure to obtain 301 mg of the target compound 22 as a white solid.
ESI-Mass: 956.78 [M+Na] +
(5) Purification of Compound 22 Compound 22 (281 mg, 301 μmol) was purified by silica gel column chromatography (chloroform:methanol=100:0 to 90:10) to obtain 240 mg of the target compound 22 as a white solid.
(6) Synthesis of Compound 23 A pyridine (0.2 mL) solution of compound 22 (4.1 mg, 4.4 μmol) was added with a pyridine (0.1 mL) solution of diisopropylethylamine (2.3 μL, 3.0 eq.) and a dimethylformamide (0.1 mL) solution of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (2.0 mg, 1.5 eq.), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was dropped into water (4.0 mL), and then centrifuged at 4° C. for 15 minutes. The supernatant was removed, and ethyl acetate (4.0 mL) was added and shaken, and then centrifuged at 4° C. for 15 minutes. After removing the supernatant, the mixture was dried under reduced pressure to obtain the target compound 23. The yield was 100%, and the mixture was used in the next reaction.
ESI-Mass: 1053.78[M+H] +
実施例１－１ Example 1-1
　化合物２４及び化合物２５を反応させて核酸分子である化合物２６～２９を合成した。
１）化合物２６（Ｘ＝Ｘ１）の合成
　０．５０ｍｍｏｌ／Ｌの化合物２４の水溶液（８０．０μＬ）、０．４５ｍｍｏｌ／Ｌの化合物２５の水溶液（８９．９μＬ）、注射用蒸留水（４６．１μＬ）、及びｐＨ８．５の１ｍｏｌ／Ｌリン酸緩衝液（６０μＬ）の混合物に、室温にて、５０ｍｍｏｌ／ＬのＢＳ３水溶液（２４μＬ）を加え、２５℃にて４時間撹拌した。反応液を、ＨＰＬＣにて精製（カラム：ＸＢｒｉｄｇｅ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ，ＢＥＨ　Ｃ１８，２．５μｍ，１０ｍｍ×５０ｍｍ；流速：４．７ｍＬ／ｍｉｎ；検出：ＵＶ２６０ｎｍ；カラムオーブン：６０℃；Ｂｕｆｆｅｒ　Ａ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５％ＣＨ３ＣＮ；Ｂｕｆｆｅｒ　Ｂ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５０％ＣＨ３ＣＮ）した。精製物のエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、目的化合物２６を得た。質量分析：１３９９８．６（計算値：１３９９８．０）。 Compound 24 and compound 25 were reacted to synthesize nucleic acid molecules, compounds 26 to 29.
1) Synthesis of Compound 26 (X = X1) To a mixture of a 0.50 mmol/L aqueous solution of compound 24 (80.0 μL), a 0.45 mmol/L aqueous solution of compound 25 (89.9 μL), distilled water for injection (46.1 μL), and a 1 mol/L phosphate buffer solution of pH 8.5 (60 μL) was added a 50 mmol/L aqueous solution of BS3 (24 μL) at room temperature, and the mixture was stirred at 25° C. for 4 hours. The reaction solution was purified by HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 μm, 10 mm × 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN). The purified product was precipitated with ethanol, and the resulting precipitate was dissolved in distilled water for injection to obtain the target compound 26. Mass spectrometry: 13998.6 (calculated value: 13998.0).
２）化合物２７（Ｘ＝Ｘ２）の合成
　５６．６μｍｏｌ／Ｌの化合物２６の水溶液（２５０．０μＬ）、ｐＨ８．０の１ｍｏｌ／Ｌリン酸緩衝液（５０μＬ）の混合物に、室温にて、１６２ｍｍｏｌ／Ｌのジチオトレイトール水溶液（８．５μＬ）を加え、３５℃にて２日間撹拌した。反応液にエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、目的化合物２７を得た。質量分析：１３８６６．３（計算値：１３８６５．８）。 2) Synthesis of Compound 27 (X = X2) A mixture of 56.6 μmol/L aqueous solution of Compound 26 (250.0 μL) and 1 mol/L phosphate buffer (50 μL) at pH 8.0 was added with 162 mmol/L aqueous solution of dithiothreitol (8.5 μL) at room temperature, and stirred at 35°C for 2 days. The reaction solution was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain the target compound 27. Mass spectrometry: 13866.3 (calculated value: 13865.8).
３）化合物２８（Ｘ＝Ｘ３）の合成
　８８．４μｍｏｌ／Ｌの化合物２７の水溶液（１６０．０μＬ）、ｐＨ７．０の１ｍｏｌ／Ｌリン酸緩衝液（４０μＬ）の混合物に、室温にて、９８．４ｍｍｏｌ／ＬのＮ－（２－アミノエチル）マレイミド　トリフルオロ酢酸塩の水溶液（７．０μＬ）を加え、２５℃にて３０分間撹拌した。反応液にエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、目的化合物２８を得た。質量分析：１４００６．５（計算値：１４００５．９）。 3) Synthesis of Compound 28 (X = X3) To a mixture of an 88.4 μmol/L aqueous solution of Compound 27 (160.0 μL) and a 1 mol/L phosphate buffer solution (40 μL) of pH 7.0, an aqueous solution of N-(2-aminoethyl)maleimide trifluoroacetate (7.0 μL) of 98.4 mmol/L was added at room temperature, and the mixture was stirred at 25°C for 30 minutes. The reaction solution was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain the target compound 28. Mass spectrometry: 14006.5 (calculated value: 14005.9).
４）化合物２９（Ｘ＝Ｘ；実施例１－１）の合成
　７０．７μｍｏｌ／Ｌの化合物２８の水溶液（１４５．０μＬ）、注射用蒸留水（２５．０μＬ）、及びｐＨ８．５の１ｍｏｌ／Ｌリン酸緩衝液（５０μＬ）の混合物に、室温にて、１０ｍｍｏｌ／Ｌの化合物１４の水溶液（５０．０μＬ）を加え、２５℃にて終夜撹拌した。反応液を、ＨＰＬＣにて精製（カラム：ＸＢｒｉｄｇｅ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ，ＢＥＨ　Ｃ１８，２．５μｍ，１０ｍｍ×５０ｍｍ；流速：４．７ｍＬ／ｍｉｎ；検出：ＵＶ２６０ｎｍ；カラムオーブン：６０℃；Ｂｕｆｆｅｒ　Ａ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５％ＣＨ３ＣＮ；Ｂｕｆｆｅｒ　Ｂ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５０％ＣＨ３ＣＮ）した。精製物のエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、純度９８．０％の化合物２９（実施例１－１化合物）を得た。質量分析：１５７１７．４（計算値：１５７１６．８）。ＲＰ－ＨＰＬＣチャートを図１に示す。 4) Synthesis of Compound 29 (X = X ; Example 1-1) To a mixture of a 70.7 μmol/L aqueous solution of compound 28 (145.0 μL), distilled water for injection (25.0 μL), and a 1 mol/L phosphate buffer solution having a pH of 8.5 (50 μL) was added a 10 mmol/L aqueous solution of compound 14 (50.0 μL) at room temperature, and the mixture was stirred at 25°C overnight. The reaction solution was purified by HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 μm, 10 mm × 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN). The purified product was precipitated with ethanol, and the resulting precipitate was dissolved in distilled water for injection to obtain compound 29 (Example 1-1 compound) with a purity of 98.0%. Mass analysis: 15717.4 (calculated value: 15716.8). The RP-HPLC chart is shown in Figure 1.
実施例１－２～１－９
　これらの化合物（核酸分子）は実施例１－１の方法または実施例１－１の方法と既存方法の組合せによって合成した。それらの構造式を表１に示す。表１中のＸは実施例１－１におけるそれと同義である。また各々の核酸分子である実施例１－２～１－９の精製後のＲＰ－ＨＰＬＣチャートを図２～図９に示した。 Examples 1-2 to 1-9
These compounds (nucleic acid molecules) were synthesized by the method of Example 1-1 or a combination of the method of Example 1-1 and an existing method. Their structural formulas are shown in Table 1. X in Table 1 has the same meaning as that in Example 1-1. Furthermore, RP-HPLC charts after purification of each of the nucleic acid molecules of Examples 1-2 to 1-9 are shown in Figures 2 to 9.
　表中、上段と下段の塩基同士を結ぶ線は、式（ａ）の－Ｚ－に相当する部分であり、該部位で連結していることを意味する。
　表中、Ｘは下記構造であることを意味する。 In the table, the line connecting the upper and lower bases represents the moiety corresponding to -Z- in formula (a), and means that the bases are linked at that site.
In the table, X represents the following structure.
実施例２－１ Example 2-1
　５００．０μｍｏｌ／Ｌの化合物３０の水溶液（２０．０μＬ）、ピリジン（１８．０μＬ）、及びジイソプロピルエチルアミン（２．０μＬ）の混合物に、室温にて、１４．７ｍｍｏｌ／Ｌの化合物２３のピリジン溶液（２０．０μＬ）を加え、２５℃にて１時間撹拌した。反応液のエタノール沈殿を行い、生じた沈殿を注射用蒸留水（３００．０μＬ）、アセトニトリル（１００．０μＬ）に溶解させ、ＨＰＬＣにて精製（カラム：ＸＢｒｉｄｇｅ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ，ＢＥＨ　Ｃ１８，２．５μｍ，１０ｍｍ×５０ｍｍ；流速：４．７ｍＬ／ｍｉｎ；検出：ＵＶ２６０ｎｍ；カラムオーブン：６０℃；Ｂｕｆｆｅｒ　Ａ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５％ＣＨ３ＣＮ；Ｂｕｆｆｅｒ　Ｂ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、９０％ＣＨ３ＣＮ）した。精製物のエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、純度９６．５％の化合物３１（実施例２－１）を得た。質量分析：１５４４７．２（計算値：１５４４６．４）。ＲＰ－ＨＰＬＣチャートを図１０に示す。 To a mixture of a 500.0 μmol/L aqueous solution of compound 30 (20.0 μL), pyridine (18.0 μL), and diisopropylethylamine (2.0 μL) was added a 14.7 mmol/L pyridine solution of compound 23 (20.0 μL) at room temperature, and the mixture was stirred at 25° C. for 1 hour. The reaction solution was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection (300.0 μL) and acetonitrile (100.0 μL), and purified by HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 μm, 10 mm × 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 90% CH 3 CN). The purified product was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain compound 31 (Example 2-1) with a purity of 96.5%. Mass spectrometry: 15447.2 (calculated value: 15446.4). The RP-HPLC chart is shown in FIG.
実施例２－２、２－３
　これらの化合物は実施例２－１の方法または実施例２－１方法と既存方法の組合せによって合成した。それらの構造式を表２に示す。また各々の核酸分子である実施例２－２、及び２－３の精製後のＲＰ－ＨＰＬＣチャートを図１１及び図１２に示した。 Examples 2-2 and 2-3
These compounds were synthesized by the method of Example 2-1 or a combination of the method of Example 2-1 and an existing method. Their structural formulas are shown in Table 2. In addition, RP-HPLC charts after purification of each of the nucleic acid molecules of Examples 2-2 and 2-3 are shown in Figures 11 and 12.
実施例２－４ Example 2-4
１）化合物３３（Ｗ＝Ｗ２，Ｘ＝Ｘ１）の合成
　既知の方法で得られた２１８μｍｏｌ／Ｌの化合物３２の水溶液（９２μＬ）、注射用蒸留水（８４μＬ）、及びｐＨ８．５の１ｍｏｌ／Ｌリン酸緩衝液（６４μＬ）の混合物に、室温にて、１０ｍｍｏｌ／ＬのＤＢＣＯ－ＮＨＳのＤＭＦの溶液（９０μＬ；４５ｅｑ．）を加え、３５℃にて３時間撹拌した。反応溶液のエタノール沈殿を行い、沈殿物として化合物３３を得た。質量分析：１６４６１．５（計算値：１６４６１．５）。 1) Synthesis of Compound 33 (W = W2, X = X1) A mixture of a 218 μmol/L aqueous solution (92 μL) of Compound 32 obtained by a known method, distilled water for injection (84 μL), and 1 mol/L phosphate buffer solution (64 μL) of pH 8.5 was added with a 10 mmol/L DMF solution (90 μL; 45 eq.) of DBCO-NHS at room temperature, and stirred at 35° C. for 3 hours. The reaction solution was precipitated with ethanol to obtain Compound 33 as a precipitate. Mass spectrometry: 16461.5 (calculated value: 16461.5).
２）化合物３４（Ｗ＝Ｗ２，Ｘ＝Ｘ２）の合成
　１５０μｍｏｌ／Ｌの化合物３３の水溶液（１００μＬ）、及びｐＨ８．０の１ｍｏｌ／Ｌリン酸緩衝液（４０μＬ）の混合物に、２０ｍｇ／ｍＬに調製したジチオトレイトール水溶液（６０μＬ）を加え、２５℃にて１時間撹拌した。反応溶液のエタノール沈殿を行い、沈殿物として化合物３４を得た。 2) Synthesis of Compound 34 (W = W2, X = X2) A 20 mg/mL dithiothreitol aqueous solution (60 μL) was added to a mixture of a 150 μmol/L aqueous solution of Compound 33 (100 μL) and a 1 mol/L phosphate buffer solution (40 μL) at pH 8.0, and the mixture was stirred for 1 hour at 25° C. Ethanol precipitation of the reaction solution was performed to obtain Compound 34 as a precipitate.
３）化合物３５（Ｗ＝Ｗ２，Ｘ＝Ｘ３）の合成
　１１０μｍｏｌ／Ｌの化合物３４の水溶液（１３５μＬ）、及びｐＨ７．０の１ｍｏｌ／Ｌリン酸緩衝液（２０μＬ）の混合物に、１０ｍｍｏｌ／Ｌに調製したＮ－（２－アミノエチル）マレイミド　トリフルオロ酢酸塩のＤＭＦ溶液（４５μＬ；３０ｅｑ．）を加え、室温にて１時間撹拌した。反応溶液のエタノール沈殿を行い、沈殿物として化合物３５を得た。 3) Synthesis of Compound 35 (W = W2, X = X3) A 10 mmol/L solution of N-(2-aminoethyl)maleimide trifluoroacetate in DMF (45 μL; 30 eq.) was added to a mixture of a 110 μmol/L aqueous solution of Compound 34 (135 μL) and a 1 mol/L phosphate buffer solution (20 μL) of pH 7.0, and the mixture was stirred at room temperature for 1 hour. The reaction solution was precipitated with ethanol to obtain Compound 35 as a precipitate.
４）化合物３６（Ｗ＝Ｗ２，Ｘ＝Ｘ）の合成
　１３０μｍｏｌ／Ｌの化合物３５の水溶液（１１５μＬ）、及びｐＨ８．５の１ｍｏｌ／Ｌリン酸緩衝液（４０μＬ）の混合物に、室温にて、１０ｍｍｏｌ／Ｌに調製した化合物１４のＤＭＦ溶液（６０μＬ；４０ｅｑ．）を加え、２５℃にて１．５時間撹拌した。反応溶液のエタノール沈殿を行い沈殿物として化合物３６を得た。質量分析：１８１８０．７（計算値：１８１８０．４）。 4) Synthesis of Compound 36 (W = W2, X = X ) A mixture of a 130 μmol/L aqueous solution of Compound 35 (115 μL) and a 1 mol/L phosphate buffer solution (40 μL) at pH 8.5 was added with a 10 mmol/L DMF solution of Compound 14 (60 μL; 40 eq.) at room temperature, and stirred at 25°C for 1.5 hours. The reaction solution was precipitated with ethanol to obtain Compound 36 as a precipitate. Mass spectrometry: 18180.7 (calculated value: 18180.4).
５）化合物３７（実施例２－４）（Ｗ＝Ｗ３，Ｘ＝Ｘ）の合成
　７１μｍｏｌ／Ｌの化合物３６の水溶液（７０μＬ）に、１０ｍｇ／ｍＬに調製したＳ１９－Ｎｔｅｒ－ＡｚｉｄｅのＤＭＳＯ溶液（１０μＬ；５．４ｅｑ．）を加えて２５℃にて５分間撹拌し、その後４℃にて終夜静置した。反応溶液のエタノール沈殿を行い、ＲＰ－ＨＰＬＣにて精製し（カラム：ＸＢｒｉｄｇｅ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ，ＢＥＨ　Ｃ１８，２．５μｍ，１０ｍｍ×５０ｍｍ；流速：４．７ｍＬ／ｍｉｎ；検出：ＵＶ２６０ｎｍ；カラムオーブン：６０℃；Ｂｕｆｆｅｒ　Ａ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５％ＣＨ３ＣＮ；Ｂｕｆｆｅｒ　Ｂ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５０％ＣＨ３ＣＮ）、目的物のピークを分取した。分取したフラクションのエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、化合物３７を取得した。質量分析：２０００３．９（計算値：２０００３．５）。ＲＰ－ＨＰＬＣチャートを図１３に示す。 5) Synthesis of Compound 37 (Example 2-4) (W=W3, X= X ) A DMSO solution of S19-Nter-Azide (10 μL; 5.4 eq.) adjusted to 10 mg/mL was added to an aqueous solution (70 μL) of 71 μmol/L of Compound 36, and the mixture was stirred at 25° C. for 5 minutes and then allowed to stand at 4° C. overnight. The reaction solution was subjected to ethanol precipitation and purified by RP-HPLC (column: XBridge Oligonucleotide, BEH C18, 2.5 μm, 10 mm × 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C.; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN), and the peak of the target substance was separated. The separated fraction was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain compound 37. Mass spectrometry: 20003.9 (calculated value: 20003.5). The RP-HPLC chart is shown in Figure 13.
実施例３－１ Example 3-1
　１ｍｍｏｌ／Ｌの配列番号２７の水溶液（１４３．０μＬ）、注射用蒸留水（１０７μＬ）、ｐＨ８．５の１ｍｏｌ／Ｌリン酸緩衝液（１００μＬ）の混合物に、室温にて、１９ｍｍｏｌ／Ｌの化合物１４の水溶液（１５０．０μＬ；２０ｅｑ．）を加え、３０℃にて２時間撹拌した。反応液を精製し（カラム：ＸＢｒｉｄｇｅ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ，ＢＥＨ　Ｃ１８，２．５μｍ，１０ｍｍ×５０ｍｍ；流速：４．７ｍＬ／ｍｉｎ；検出：ＵＶ２６０ｎｍ；カラムオーブン：６０℃；Ｂｕｆｆｅｒ　Ａ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５％ＣＨ３ＣＮ；Ｂｕｆｆｅｒ　Ｂ：５０ｍｍｏｌ／Ｌ　ＴＥＡＡ（ｐＨ７．３）、５０％ＣＨ３ＣＮ）、目的物のピークを分取した。分取したフラクションのエタノール沈殿を行い、生じた沈殿を注射用蒸留水に溶解させ、純度９９．６％の配列番号２７－ＧａｌＮＡｃ４を得た。質量分析：８６８９．９（計算値：８６８９．３）。配列番号２７－ＧａｌＮＡｃ４のＲＰ－ＨＰＬＣチャートを図１４に示す。
　上述にて取得したセンス鎖配列番号２７－ＧａｌＮＡｃ４と配列番号２８をｐＨ７．４のＰＢＳ緩衝液にてアニーリングし、実施例３－１の化合物を取得した。 A mixture of 1 mmol/L aqueous solution of SEQ ID NO: 27 (143.0 μL), distilled water for injection (107 μL), and 1 mol/L phosphate buffer solution (100 μL) of pH 8.5 was added with 19 mmol/L aqueous solution of compound 14 (150.0 μL; 20 eq.) at room temperature and stirred for 2 hours at 30 ° C. The reaction solution was purified (column: XBridge Oligonucleotide, BEH C18, 2.5 μm, 10 mm × 50 mm; flow rate: 4.7 mL / min; detection: UV 260 nm; column oven: 60 ° C.; Buffer A: 50 mmol / L TEAA (pH 7.3), 5% CH 3 CN; Buffer B: 50 mmol / L TEAA (pH 7.3), 50% CH 3 CN), and the peak of the target product was separated. The collected fraction was subjected to ethanol precipitation, and the resulting precipitate was dissolved in distilled water for injection to obtain SEQ ID NO:27-GalNAc4 with a purity of 99.6%. Mass spectrometry: 8689.9 (calculated value: 8689.3). The RP-HPLC chart of SEQ ID NO:27-GalNAc4 is shown in FIG.
The sense strand SEQ ID NO:27-GalNAc4 obtained above and SEQ ID NO:28 were annealed in a PBS buffer solution of pH 7.4 to obtain the compound of Example 3-1.
実施例３－２、３－３、４－１、４－２
　これらの化合物はｓｉＲＮＡをホスホロアミダイト法及び実施例３－１の方法または実施例３－１の方法と既存方法の組合せによって合成した。それらの核酸分子の構造式を表３に示した。また各々の核酸分子３－２、３－３、４－１、４－２の精製後のＲＰ－ＨＰＬＣチャートをそれぞれ図１５～１８に示した。 Examples 3-2, 3-3, 4-1, and 4-2
These compounds were synthesized by synthesizing siRNA using the phosphoramidite method and the method of Example 3-1 or a combination of the method of Example 3-1 and an existing method. The structural formulas of these nucleic acid molecules are shown in Table 3. Furthermore, the RP-HPLC charts after purification of each of the nucleic acid molecules 3-2, 3-3, 4-1, and 4-2 are shown in Figures 15 to 18, respectively.
　表中、Ｙ、Ｚは下記構造であることを意味する。 In the table, Y and Z have the following structures.
試験例
＜薬理評価方法＞
１．ＤＮＡ／ＲＮＡヘテロ二本鎖核酸（ＨＤＯ）静脈内投与マウスの血中Ｆａｃｔｏｒ　ＶＩＩアッセイ用サンプル採材
　血中Ｆａｃｔｏｒ　ＶＩＩ測定のため、Ｃ５７ＢＬ／６Ｊマウス（日本チャールス・リバー）にＨＤＯあるいはＧａｌＮＡｃ修飾ＨＤＯを１．５ｍｇ／ｋｇ（５ｍＬ／ｋｇ）で静脈内投与した。ＨＤＯあるいはＧａｌＮＡｃ修飾ＨＤＯを各１群、ｎ＝６の計２群を設定した。静脈内投与１及び３日後に頚静脈から経時採血と７日後に後大静脈採血を行った。また、無処置動物１例を投与翌日に後大静脈より採血を行った。分取した血漿を凍結保存し、評価試料とした。 Test Example <Pharmacological Evaluation Method>
1. Sample collection for blood Factor VII assay of mice intravenously administered DNA/RNA heteroduplex (HDO) HDO or GalNAc-modified HDO was intravenously administered at 1.5 mg/kg (5 mL/kg) to C57BL/6J mice (Charles River Japan) to measure blood Factor VII. Two groups were set up, one each for HDO and GalNAc-modified HDO, n=6 in total. Blood was collected over time from the jugular vein 1 and 3 days after intravenous administration, and blood was collected from the posterior vena cava 7 days later. In addition, blood was collected from one untreated animal the day after administration from the posterior vena cava. The collected plasma was frozen and stored as an evaluation sample.
２．ｓｉＲＮＡ皮下投与マウスの血中Ｆａｃｔｏｒ　ＶＩＩアッセイ用サンプル採材
　血中Ｆａｃｔｏｒ　ＶＩＩ測定のため、Ｃ５７ＢＬ／６Ｊマウス（日本チャールス・リバー）にＧａｌＮＡｃ修飾ｓｉＲＮＡを投与用量０．３ｍｇ／ｋｇ及び３．０ｍｇ／ｋｇで皮下投与した。投与用量に対し各１群、ｎ＝４の計２群を設定した。投与７日前、皮下投与１、３、７及び１４日後に尾静脈から経時採血と２１日後に後大静脈採血を行った。分取した血清を凍結保存し、評価試料とした。 2. Sample collection for blood Factor VII assay of mice subcutaneously administered siRNA GalNAc-modified siRNA was subcutaneously administered at doses of 0.3 mg/kg and 3.0 mg/kg to measure blood Factor VII in C57BL/6J mice (Charles River Japan). Two groups (n=4) were set for each dose. Blood was collected from the tail vein 7 days before administration, 1, 3, 7 and 14 days after subcutaneous administration, and from the posterior vena cava 21 days later. The collected serum was frozen and stored as an evaluation sample.
３．ｓｉＲＮＡ経肺投与マウスの血中Ｆａｃｔｏｒ　ＶＩＩアッセイ用サンプル採材
　血中Ｆａｃｔｏｒ　ＶＩＩ測定のため、Ｃ５７ＢＬ／６Ｊマウス（日本チャールス・リバー）にＧａｌＮＡｃ修飾ｓｉＲＮＡを投与用量０．３ｍｇ／ｋｇ及び３．０ｍｇ／ｋｇで経肺投与した。投与用量に対して各１群、ｎ＝４の計２群を設定した。投与７日前、皮下投与１、３、７及び１４日後に尾静脈から経時採血と２１日後に後大静脈採血を行った。分取した血清を凍結保存し、評価試料とした。 3. Sample collection for blood Factor VII assay of mice administered siRNA via the lungs GalNAc-modified siRNA was administered via the lungs at doses of 0.3 mg/kg and 3.0 mg/kg to measure blood Factor VII in C57BL/6J mice (Charles River Japan). Two groups were set up, one for each dose, with n=4 in total. Blood was collected from the tail vein 7 days before administration, and 1, 3, 7, and 14 days after subcutaneous administration, and blood was collected from the caudal vein 21 days later. The collected serum was frozen and stored as an evaluation sample.
試験例１．ＨＤＯ静脈内投与マウスの血中Ｆａｃｔｏｒ　ＶＩＩの評価
　血中Ｆａｃｔｏｒ　ＶＩＩを測定するため、各サンプルについてＢＩＯＰＨＥＮＴＭＦＶＩＩキットを用いて評価を実施した。ＨＤＯあるいはＧａｌＮＡｃ修飾ＨＤＯを投与したマウスの血漿をサンプルとし、各サンプルはキットに付属のＴｒｉｓ－ＢＳＡ　ｂｕｆｆｅｒ（Ｒ４）で２０００倍希釈した。９６ｗｅｌｌマイクロプレートに、希釈サンプル３０μＬ、３７℃でプレインキュベートしたＴｈｒｏｍｂｏｐｌａｓｔｉｎ　Ｃａｌｃｉｕｍ（Ｒ２）３０μＬ及びＦａｃｔｏｒ　Ｘ（ｈｕｍａｎ）（Ｒ３）６０μＬを加え、撹拌後３７℃で７分間インキュベートした。さらに３７℃でプレインキュベートしたＳＸａ－１１（Ｒ３）を添加し、撹拌後３７℃で５分間インキュベートした後に、２％クエン酸溶液で反応を停止した。反応停止後、マイクロプレートリーダーで４０５ｎｍの吸光度を測定し、ＨＤＯあるいはＧａｌＮＡｃ修飾ＨＤＯの経時的な吸光度変化を比較することで活性を評価した。
　結果を図１９に示す。
　ＨＤＯあるいはＧａｌＮＡｃ修飾ＨＤＯを静脈内に投与したマウスから経時的に採取した血漿のＦａｃｔｏｒＶＩＩ活性を４０５ｎｍの吸収を指標として比較した。その結果、ＨＤＯを投与したマウスでは投与１日後と投与３日後では４０５ｎｍの吸光度に差はなく、投与７日後に０．１３程度低下した。一方、ＧａｌＮＡｃ修飾ＨＤＯを投与したマウスでは投与１日後から投与３日後、投与３日後から投与７日後にそれぞれ０．２６程度低下した。
　本評価に用いたＨＤＯはＦａｃｔｏｒＶＩＩの発現を抑制する核酸であり、また血中ＦａｃｔｏｒＶＩＩは肝臓に由来している。この結果より、ＧａｌＮＡｃ修飾によってＨＤＯの肝臓への集積が向上していることを明らかとした。 Test Example 1. Evaluation of Factor VII in the Blood of Mice Intravenously Administered HDO To measure Factor VII in the blood, each sample was evaluated using the BIOPHEN ™ FVII kit. Plasma from mice administered HDO or GalNAc-modified HDO was used as a sample, and each sample was diluted 2000-fold with Tris-BSA buffer (R4) included in the kit. 30 μL of the diluted sample, 30 μL of Thromboplastin Calcium (R2) preincubated at 37° C., and 60 μL of Factor X (human) (R3) were added to a 96-well microplate, and incubated at 37° C. for 7 minutes after stirring. SXa-11 (R3) preincubated at 37° C. was further added, and incubated at 37° C. for 5 minutes after stirring, and the reaction was stopped with a 2% citric acid solution. After the reaction was stopped, the absorbance at 405 nm was measured using a microplate reader, and the activity was evaluated by comparing the change in absorbance over time for HDO or GalNAc-modified HDO.
The results are shown in Figure 19.
Factor VII activity of plasma collected over time from mice intravenously administered HDO or GalNAc-modified HDO was compared using absorbance at 405 nm as an index. As a result, in mice administered HDO, there was no difference in absorbance at 405 nm between 1 day and 3 days after administration, and it decreased by about 0.13 after 7 days. On the other hand, in mice administered GalNAc-modified HDO, it decreased by about 0.26 from 1 day to 3 days after administration and from 3 days to 7 days after administration.
The HDO used in this evaluation is a nucleic acid that suppresses the expression of Factor VII, and Factor VII in the blood is derived from the liver. These results demonstrated that the accumulation of HDO in the liver was improved by GalNAc modification.
試験例２．ｓｉＲＮＡ投与マウスの血中Ｆａｃｔｏｒ　ＶＩＩの評価
　血中Ｆａｃｔｏｒ　ＶＩＩを測定するため、各サンプルについてＢＩＯＰＨＥＮＴＭＦＶＩＩキットを用いて評価を実施した。ＧａｌＮＡｃ修飾ｓｉＲＮＡ皮下投与マウス及びＧａｌＮＡｃ修飾ｓｉＲＮＡ経肺投与マウスでは、投与７日前に採取した血清をコントロールサンプルとした。各サンプルはキットに付属のＴｒｉｓ－ＢＳＡ　ｂｕｆｆｅｒ（Ｒ４）で２０００倍希釈した。９６ｗｅｌｌマイクロプレートに、希釈サンプル３０μＬ、３７℃でプレインキュベートしたＴｈｒｏｍｂｏｐｌａｓｔｉｎ　Ｃａｌｃｉｕｍ（Ｒ２）３０μＬ及びＦａｃｔｏｒ　Ｘ（ｈｕｍａｎ）（Ｒ３）６０μＬを加え、撹拌後３７℃で７分間インキュベートした。さらに３７℃でプレインキュベートしたＳＸａ－１１（Ｒ３）を添加し、撹拌後３７℃で５分間インキュベートした後に、２％クエン酸溶液で反応を停止した。反応停止後、マイクロプレートリーダーで４０５ｎｍの吸光度を測定し、コントロールの吸光度に対する評価サンプルの吸光度の比を試算し、相対活性として評価した。
　結果を図２０（皮下投与）及び図２１（経肺投与）に示す。
　ＧａｌＮＡｃ修飾ｓｉＲＮＡの投与７日前に採取した血清のＦａｃｔｏｒＶＩＩ活性を基準として、各時間点で採取した血清のＦａｃｔｏｒＶＩＩ活性を評価したところ、皮下投与及び経肺投与共に、３ｍｇ／ｋｇ投与群で投与３日後から投与７日後にかけてＦａｃｔｏｒ　ＶＩＩ活性が低下し、その後投与２１日後までに活性が回復することを確認した。一方で、０．３ｍｇ投与群では、皮下投与では、投与３日後にかけて活性が上昇した後、投与７日後にかけて基準とする投与７日前の活性と同程度まで低下し、その後投与２１日後まで変化が認められなかった。また、経肺投与で投与１日後にわずかに活性が上昇した後、投与３日後に活性がわずかに低下し、投与２１日後までほとんど変化が認められなかった。
　本評価に用いたｓｉＲＮＡはＦａｃｔｏｒＶＩＩの発現を抑制する核酸であり、また血中ＦａｃｔｏｒＶＩＩは肝臓に由来している。これらの結果から、ＧａｌＮＡｃ修飾ｓｉＲＮＡを経肺又は皮下に３ｍｇ／ｋｇ投与することで、ｓｉＲＮＡが肝臓に集積し、血中のＦａｃｔｏｒＶＩＩ活性を低下させることを明らかにした。 Test Example 2. Evaluation of Factor VII in the Blood of siRNA-Administered Mice To measure Factor VII in the blood, each sample was evaluated using the BIOPHEN ™ FVII kit. For mice subcutaneously administered with GalNAc-modified siRNA and mice pulmonary administered with GalNAc-modified siRNA, serum collected 7 days before administration was used as a control sample. Each sample was diluted 2000-fold with Tris-BSA buffer (R4) included in the kit. 30 μL of the diluted sample, 30 μL of Thromboplastin Calcium (R2) preincubated at 37° C., and 60 μL of Factor X (human) (R3) were added to a 96-well microplate, and incubated at 37° C. for 7 minutes after stirring. Further, SXa-11 (R3) preincubated at 37° C. was added, and after stirring, the mixture was incubated at 37° C. for 5 minutes, and then the reaction was stopped with a 2% citric acid solution. After the reaction was stopped, the absorbance at 405 nm was measured with a microplate reader, and the ratio of the absorbance of the evaluation sample to the absorbance of the control was calculated and evaluated as the relative activity.
The results are shown in FIG. 20 (subcutaneous administration) and FIG. 21 (pulmonary administration).
The Factor VII activity of serum collected at each time point was evaluated based on the Factor VII activity of serum collected 7 days before administration of GalNAc-modified siRNA. In both subcutaneous and pulmonary administration, Factor VII activity decreased from 3 days after administration to 7 days after administration in the 3 mg/kg administration group, and activity was confirmed to recover by 21 days after administration. On the other hand, in the 0.3 mg administration group, activity increased by 3 days after administration in the subcutaneous administration, and then decreased to the same level as the activity before 7 days after administration as the standard by 7 days after administration, and no change was observed until 21 days after administration. In addition, activity increased slightly 1 day after administration in the pulmonary administration, and then activity decreased slightly 3 days after administration, and almost no change was observed until 21 days after administration.
The siRNA used in this evaluation is a nucleic acid that suppresses the expression of Factor VII, and Factor VII in the blood originates from the liver. These results demonstrated that the siRNA accumulates in the liver and reduces Factor VII activity in the blood by administering GalNAc-modified siRNA via the lungs or subcutaneously at 3 mg/kg.
　本発明の化合物であるイミノジカルボン酸誘導体からなるリガンドコンジュゲート核酸は、血清安定性、適切な臓器または細胞への送達、および膜貫通送達を可能とし、更に低い製造コストで高い機能性を付与する、治療及び／又は予防剤を提供することができる。
　本出願は、日本で出願された特願２０２２－１８５３０８（出願日：２０２２年１１月１８日）を基礎としておりその内容は本明細書に全て包含されるものである。 The ligand-conjugated nucleic acid comprising the iminodicarboxylic acid derivative, which is the compound of the present invention, can provide a therapeutic and/or prophylactic agent that is serum stable, capable of being delivered to an appropriate organ or cell, and capable of being delivered through a membrane, and further provides high functionality at low production costs.
This application is based on Patent Application No. 2022-185308 filed in Japan (filing date: November 18, 2022), the contents of which are incorporated in their entirety herein.

Claims (27)

  1. 　下記一般式（Ｉ）：
    ［式中、
    Ａは、核酸分子であり、
    Ｘは、－（ＣＲ１Ｒ２）ｎ－Ｒ３－（式中、Ｒ１及びＲ２は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ３は－ＮＲ４－（式中、Ｒ４は、水素原子又は置換されていてもよいアルキル基である）又は－Ｓ－ヘテロ環－ＣＲ５Ｒ６－ＮＲ７－（式中、Ｒ５、Ｒ６、Ｒ７は、それぞれ独立して、水素原子又は置換されていてもよいアルキル基である）を表す；ｎは１～１０の整数を表す）を表し、
    Ｂ１、Ｂ２、Ｂ３及びＢ４は、それぞれ独立して、水素原子又は下記式のいずれかを表し、
    －（Ｗ２）ｎ２－ＣＯ－ＮＨ－Ｘ１－Ｙ１、
    －（Ｗ３）ｎ３－ＣＯ－ＮＨ－Ｘ２－Ｙ２、
    －（Ｗ５）ｎ５－ＣＯ－ＮＨ－Ｘ３－Ｙ３、
    －（Ｗ６）ｎ６－ＣＯ－ＮＨ－Ｘ４－Ｙ４、
    Ｗ２は、－（ＣＲ１Ｗ２Ｒ２Ｗ２）－（式中、Ｒ１Ｗ２及びＲ２Ｗ２は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ３は、－（ＣＲ１Ｗ３Ｒ２Ｗ３）－（式中、Ｒ１Ｗ３及びＲ２Ｗ３は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ５は、－（ＣＲ１Ｗ５Ｒ２Ｗ５）－（式中、Ｒ１Ｗ５及びＲ２Ｗ５は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ６は、－（ＣＲ１Ｗ６Ｒ２Ｗ６）－（式中、Ｒ１Ｗ６及びＲ２Ｗ６は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し[但し、Ｂ１～Ｂ４は、同時に水素原子ではない]、
    ｎ２、ｎ３、ｎ５及びｎ６は、それぞれ独立して、１～５の整数であり、
    ｎＢ１～ｎＢ４は、それぞれ独立して、1～３の整数を表し、
    Ｘ０、Ｘ１、Ｘ２、Ｘ３及びＸ４は、それぞれ独立して、置換されても良いスペーサーを表し、
    Ｙ１、Ｙ２、Ｙ３及びＹ４は、それぞれ独立して、リガンドを表し、
    Ｗ１は、－（ＣＲ１Ｗ１Ｒ２Ｗ１）－（式中、Ｒ１Ｗ１及びＲ２Ｗ１は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ４は、－（ＣＲ１Ｗ４Ｒ２Ｗ４）－（式中、Ｒ１Ｗ４及びＲ２Ｗ４は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    ｎ１及びｎ４は、それぞれ独立して、１～５の整数である］
    で表される化合物。     The following general formula (I):
    [Wherein,
    A is a nucleic acid molecule,
    X represents -(CR 1 R 2 )n-R 3 - (wherein R 1 and R 2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 3 represents -NR 4 - (wherein R 4 is a hydrogen atom or an optionally substituted alkyl group) or -S-heterocycle-CR 5 R 6 -NR 7 - (wherein R 5 , R 6 and R 7 are each independently a hydrogen atom or an optionally substituted alkyl group); and n represents an integer of 1 to 10);
    B 1 , B 2 , B 3 and B 4 each independently represent a hydrogen atom or any of the following formulae:
    -( W2 ) n2 -CO-NH- X1 - Y1 ,
    -( W3 ) n3 -CO-NH- X2 - Y2 ,
    -( W5 ) n5 -CO-NH- X3 - Y3 ,
    -( W6 ) n6 -CO-NH- X4 - Y4 ,
    W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W5 represents -( CR1W5R2W5 )- (wherein R1W5 and R2W5 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W 6 represents -(CR 1W6 R 2W6 )- (wherein R 1W6 and R 2W6 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group) (provided that B 1 to B 4 are not simultaneously hydrogen atoms);
    n 2 , n 3 , n 5 and n 6 each independently represent an integer from 1 to 5;
    n B1 to n B4 each independently represent an integer of 1 to 3;
    X 0 , X 1 , X 2 , X 3 and X 4 each independently represent a spacer which may be substituted;
    Y 1 , Y 2 , Y 3 and Y 4 each independently represent a ligand;
    W 1 represents -(CR 1W1 R 2W1 )- (wherein R 1W1 and R 2W1 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W 4 represents -(CR 1W4 R 2W4 )- (wherein R 1W4 and R 2W4 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    n1 and n4 each independently represent an integer from 1 to 5.
    A compound represented by the formula:
  2. 　下記一般式（Ｉ－１）：
    （式中、各記号の定義は請求項１と同義である）
    で表される、請求項１記載の化合物。     The following general formula (I-1):
    (In the formula, the definitions of each symbol are the same as those in claim 1.)
    The compound according to claim 1, represented by the formula:
  3. 　Ｘ０のスペーサーが、下記式（１）～（４）のいずれかを表す、請求項１又は２記載の化合物：
    （１）－ＣＯ－Ｌ１－ＣＯ－
    （式中、Ｌ１は
    (i)－（ＣＲ１Ｌ１Ｒ２Ｌ１）ｎＬ１（式中、Ｒ１Ｌ１及びＲ２Ｌ１は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ１は１～１０の整数を表す）、
    (ii)－（ＣＨ２）ｎＬ１－Ｃｙ－（ＣＨ２）ｍＬ１－（式中、Ｃｙは置換されていてもよいアリーレン基、置換されていてもよいヘテロアリーレン基、置換されていてもよいシクロアルキレン基、又は置換されていてもよいヘテロシクロアルキレン基を表し；ｎＬ１及びｍＬ１は同一又は異なって、１～１０の整数を表す）、
    (iii)－ＮＲ８－（式中、Ｒ８は水素原子又は置換されていてもよいアルキル基を表す）、
    （２）－Ｌ２－ＣＯ－Ｌ３－ＣＯ－
    （式中、Ｌ２は、－（ＣＲ１Ｌ２Ｒ２Ｌ２）ｎＬ２－（式中、Ｒ１Ｌ２及びＲ２Ｌ２は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ２は０～６の整数を表す）を表し；Ｌ３は、－（ＣＲ１Ｌ３Ｒ２Ｌ３）ｎＬ３－（式中、Ｒ１Ｌ３及びＲ２Ｌ３は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ３は０～６の整数を表す、但し、ｎＬ２及びｎＬ３は同時に０ではない）を表す）、
    （３）－ＣＯ－Ｌ４－ＣＯ－Ｌ５－ＣＯ－Ｌ６－ＣＯ－
    （式中、Ｌ４は、－（ＣＲ１Ｌ４Ｒ２Ｌ４）ｎＬ４（式中、Ｒ１Ｌ４及びＲ２Ｌ４は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ４は１～６の整数を表す）を表し；Ｌ５は、－（ＯＣ２Ｈ４）ｎＬ５（式中、ｎＬ５は１～６の整数を表す）を表し；Ｌ６は、－（ＣＲ１Ｌ６Ｒ２Ｌ６）ｎＬ６（式中、Ｒ１Ｌ６及びＲ２Ｌ６は同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；ｎＬ６は１～６の整数を表す）を表す）、
    （４）－ＣＯ－Ｌ７－ＣＨ２ＣＯ－
    （式中、Ｌ７は、－（ＯＣ２Ｈ４）ｎＬ７（式中、ｎＬ７は１～６の整数を表す）を表す）。     The compound according to claim 1 or 2, wherein the spacer X 0 represents any one of the following formulas (1) to (4):
    (1) -CO- L1 -CO-
    (Wherein, L1 is
    (i) -(CR 1L1 R 2L1 ) n L1 (wherein R 1L1 and R 2L1 are the same or different and each represents a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; n L1 represents an integer of 1 to 10),
    (ii) -(CH 2 ) n L1 -Cy-(CH 2 ) m L1 - (wherein Cy represents an optionally substituted arylene group, an optionally substituted heteroarylene group, an optionally substituted cycloalkylene group, or an optionally substituted heterocycloalkylene group; n L1 and m L1 may be the same or different and represent an integer of 1 to 10),
    (iii) -NR 8 - (wherein R 8 represents a hydrogen atom or an optionally substituted alkyl group),
    (2) -L2 -CO- L3 -CO-
    (wherein L2 represents -( CR1L2R2L2 ) nL2- (wherein R1L2 and R2L2 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; nL2 represents an integer of 0 to 6); L3 represents -( CR1L3R2L3 ) nL3- (wherein R1L3 and R2L3 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or together form an alkylene group; nL3 represents an integer of 0 to 6, with the proviso that nL2 and nL3 are not simultaneously 0));
    (3) -CO- L4 -CO- L5 -CO- L6 -CO-
    (wherein L 4 represents -(CR 1L4 R 2L4 )n L4 (wherein R 1L4 and R 2L4 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or taken together to form an alkylene group; n L4 represents an integer of 1 to 6); L 5 represents -(OC 2 H 4 )n L5 (wherein n L5 represents an integer of 1 to 6); L 6 represents -(CR 1L6 R 2L6 )n L6 (wherein R 1L6 and R 2L6 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or taken together to form an alkylene group; n L6 represents an integer of 1 to 6)).
    (4) -CO-L 7 -CH 2 CO-
    (wherein L7 represents -(OC 2 H 4 ) n L7 (wherein n L7 represents an integer of 1 to 6)).
  4. 　Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＲ９Ｒ１０）ｎ’－Ｒ１１（式中、Ｒ９及びＲ１０は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ１１は－Ｏ－又は－ＮＲ１２－（式中、Ｒ１２は水素原子又は置換されていてもよいアルキル基である）を表す；ｎ’は１～１０の整数を表す）
    で表される請求項１又は２記載の化合物。     spacers X 1 , X 2 , X 3 and X 4 each independently represent -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); and n' represents an integer from 1 to 10).
    The compound according to claim 1 or 2, represented by the formula:
  5. 　Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５及びＷ６が、－（ＣＨ２）－で、ｎ１～ｎ６が１である請求項１又は２記載の化合物。 3. The compound according to claim 1 or 2, wherein W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are -(CH 2 )-, and n 1 to n 6 are 1.
  6. 　Ｘ０のスペーサーが、－ＣＯ－（ＣＨ２）ｎＬ１－ＣＯ－（式中、ｎＬ１は１～１０の整数を表す）である、請求項１又は２記載の化合物。 3. The compound according to claim 1 or 2, wherein the spacer X 0 is -CO-(CH 2 )n L1 -CO- (wherein n L1 represents an integer of 1 to 10).
  7. 　ｎＬ１が６である、請求項１又は２記載の化合物。 The compound according to claim 1 or 2, wherein n L1 is 6.
  8. 　Ｘが、－（ＣＨ２）６－ＮＨ－である、請求項１又は２記載の化合物。 The compound according to claim 1 or 2, wherein X is -(CH 2 ) 6 -NH-.
  9. 　Ｘが、－（ＣＨ２）６－Ｓ－ヘテロ環－（ＣＨ２）２－ＮＨ－である、請求項１又は２記載の化合物。 The compound of claim 1 or 2, wherein X is -(CH 2 ) 6 -S-heterocycle-(CH 2 ) 2 -NH-.
  10. 　Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）６－Ｏ－である、請求項１又は２記載の化合物。 3. The compound according to claim 1 or 2, wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 6 -O-.
  11. 　Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）５－ＮＨ－である、請求項１又は２記載の化合物。 3. The compound according to claim 1 or 2, wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 5 -NH-.
  12. 　Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、糖である、請求項１又は２記載の化合物。 3. The compound of claim 1 or 2, wherein the ligands Y1 , Y2 , Y3 and Y4 are each independently a sugar.
  13. 　糖がＧａｌＮＡｃである、請求項１２記載の化合物。 The compound of claim 12, wherein the sugar is GalNAc.
  14. 　Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、脂質である、請求項１又は２記載の化合物。 3. The compound of claim 1 or 2, wherein the ligands Y1 , Y2 , Y3 and Y4 are each independently a lipid.
  15. 　Ａの核酸分子が、標的遺伝子に対するｓｉＲＮＡである、請求項１又は２記載の化合物。 The compound according to claim 1 or 2, wherein the nucleic acid molecule A is an siRNA directed against a target gene.
  16. 　Ａの核酸分子が、標的遺伝子に対するアンチセンス核酸である、請求項１又は２記載の化合物。 The compound according to claim 1 or 2, wherein the nucleic acid molecule A is an antisense nucleic acid for a target gene.
  17. 　Ａの核酸分子が、標的遺伝子に対するガイド鎖とそれに相補的なパッセンジャー鎖とが、リンカーを介して連結された二重鎖を形成し得る一本鎖核酸分子である、請求項１又は２記載の化合物。 The compound according to claim 1 or 2, wherein the nucleic acid molecule A is a single-stranded nucleic acid molecule capable of forming a duplex in which a guide strand for a target gene and a passenger strand complementary thereto are linked via a linker.
  18. 　Ａの核酸分子が、下記一般式（ａ）：
    ［式中、Ｘ、Ｙ、Ｘ１、Ｙ１、Ｘ２、Ｙ２は、それぞれ独立して、修飾されていてもよいリボヌクレオチド残基又は修飾されていてもよいデオキシリボヌクレオチド残基であり；Ｚは（Ｘ）の糖部分の２’位若しくは５’位と（Ｙ）の糖部分の２’位若しくは３’位とを連結するリンカーであり、又は（Ｘ）の塩基部分と（Ｙ）の塩基部分とを連結するリンカーであり；
    配列Ｔは標的遺伝子の発現制御配列（Ｔ）を含むヌクレオチド配列であり、配列Ｑは該発現制御配列Ｔに相補的な配列（Ｑ）を含むヌクレオチド配列であり；
    ｍ１及びｍ２は、それぞれ独立して、０～５の整数であり；及び
    ｎ１及びｎ２は、それぞれ独立して、０～５の整数である］
    で表される、請求項１又は２記載の化合物。     The nucleic acid molecule of A has the following general formula (a):
    [wherein X, Y, X 1 , Y 1 , X 2 , and Y 2 are each independently an optionally modified ribonucleotide residue or an optionally modified deoxyribonucleotide residue; Z is a linker connecting the 2'- or 5'-position of the sugar moiety of (X) to the 2'- or 3'-position of the sugar moiety of (Y), or a linker connecting the base moiety of (X) to the base moiety of (Y);
    Sequence T is a nucleotide sequence containing an expression control sequence (T) of a target gene, and sequence Q is a nucleotide sequence containing a sequence (Q) complementary to the expression control sequence T;
    m1 and m2 are each independently an integer from 0 to 5; and n1 and n2 are each independently an integer from 0 to 5.
    The compound according to claim 1 or 2, represented by the formula:
  19. 　下記一般式（ＩＩ）
    ［式中、
    Ｚはアミノ基保護基であり、
    Ｂ１、Ｂ２、Ｂ３及びＢ４は、同一又は異なって水素原子又は下記式のいずれかを表し、
    －（Ｗ２）ｎ２－ＣＯ－ＮＨ－Ｘ１－Ｙ１、
    －（Ｗ３）ｎ３－ＣＯ－ＮＨ－Ｘ２－Ｙ２、
    －（Ｗ５）ｎ５－ＣＯ－ＮＨ－Ｘ３－Ｙ３、
    －（Ｗ６）ｎ６－ＣＯ－ＮＨ－Ｘ４－Ｙ４、
    Ｗ２は、－（ＣＲ１Ｗ２Ｒ２Ｗ２）－（式中、Ｒ１Ｗ２及びＲ２Ｗ２は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ３は、－（ＣＲ１Ｗ３Ｒ２Ｗ３）－（式中、Ｒ１Ｗ３及びＲ２Ｗ３は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ５は、－（ＣＲ１Ｗ５Ｒ２Ｗ５）－（式中、Ｒ１Ｗ５及びＲ２Ｗ５は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ６は、－（ＣＲ１Ｗ６Ｒ２Ｗ６）－（式中、Ｒ１Ｗ６及びＲ２Ｗ６は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    ｎ２、ｎ３、ｎ５及びｎ６は、それぞれ独立して、１～５の整数であり、
    ｎＢ１’～ｎＢ４’は、それぞれ独立して、０～３の整数（但し、同時に０でない）を表し、
    Ｘ１、Ｘ２、Ｘ３及びＸ４は、それぞれ独立して、置換されても良いスペーサーであり、Ｙ１、Ｙ２、Ｙ３及びＹ４は、それぞれ独立して、リガンドであり、
    Ｗ１は、－（ＣＲ１Ｗ１Ｒ２Ｗ１）－（式中、Ｒ１Ｗ１及びＲ２Ｗ１は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    Ｗ４は、－（ＣＲ１Ｗ４Ｒ２Ｗ４）－（式中、Ｒ１Ｗ４及びＲ２Ｗ４は、同一又は異なって水素原子又は置換されていてもよいアルキル基である）を表し、
    ｎ１及びｎ４は、それぞれ独立して、１～５の整数である］
    で表される化合物。     The following general formula (II)
    [Wherein,
    Z is an amino protecting group;
    B 1 , B 2 , B 3 and B 4 are the same or different and each represents a hydrogen atom or any one of the following formulae:
    -( W2 ) n2 -CO-NH- X1 - Y1 ,
    -( W3 ) n3 -CO-NH- X2 - Y2 ,
    -( W5 ) n5 -CO-NH- X3 - Y3 ,
    -( W6 ) n6 -CO-NH- X4 - Y4 ,
    W2 represents -( CR1W2R2W2 )- (wherein R1W2 and R2W2 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W3 represents -( CR1W3R2W3 )- (wherein R1W3 and R2W3 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W5 represents -( CR1W5R2W5 )- (wherein R1W5 and R2W5 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    W6 represents -( CR1W6R2W6 )- (wherein R1W6 and R2W6 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    n 2 , n 3 , n 5 and n 6 each independently represent an integer from 1 to 5;
    n B1 ' to n B4 ' each independently represent an integer of 0 to 3 (but are not all 0 at the same time);
    X 1 , X 2 , X 3 and X 4 each independently represent a spacer which may be substituted; Y 1 , Y 2 , Y 3 and Y 4 each independently represent a ligand;
    W 1 represents -(CR 1W1 R 2W1 )- (wherein R 1W1 and R 2W1 may be the same or different and represent a hydrogen atom or an optionally substituted alkyl group);
    W 4 represents -(CR 1W4 R 2W4 )- (wherein R 1W4 and R 2W4 may be the same or different and each represents a hydrogen atom or an optionally substituted alkyl group);
    n1 and n4 each independently represent an integer from 1 to 5.
    A compound represented by the formula:
  20. 　下記一般式（ＩＩ－１）：
    （式中、各記号の定義は請求項１９と同義である）
    で表される、請求項１９記載の化合物。     The following general formula (II-1):
    (In the formula, the definitions of each symbol are the same as those in claim 19.)
    The compound according to claim 19, represented by:
  21. 　Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＲ９Ｒ１０）ｎ’－Ｒ１１（式中、Ｒ９及びＲ１０は、同一又は異なって、水素原子、置換されていてもよいアルキル基を表すか、又は一緒になってアルキレン基を形成する；Ｒ１１は－Ｏ－又は－ＮＲ１２－（式中、Ｒ１２は水素原子又は置換されていてもよいアルキル基である）を表す；ｎ’は１～１０の整数を表す）
    で表される請求項１９又は２０記載の化合物。     spacers X 1 , X 2 , X 3 and X 4 each independently represent -(CR 9 R 10 )n'-R 11 (wherein R 9 and R 10 are the same or different and represent a hydrogen atom, an optionally substituted alkyl group, or are joined together to form an alkylene group; R 11 represents -O- or -NR 12 - (wherein R 12 is a hydrogen atom or an optionally substituted alkyl group); and n' represents an integer of 1 to 10).
    The compound according to claim 19 or 20, represented by the formula:
  22. 　Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５及びＷ６が、－（ＣＨ２）－で、ｎ１～ｎ６が１である請求項１９又は２０記載の化合物。 The compound according to claim 19 or 20, wherein W 1 , W 2 , W 3 , W 4 , W 5 and W 6 are -(CH 2 )-, and n 1 to n 6 are 1.
  23. 　Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）６－Ｏ－である、請求項１９又は２０記載の化合物。 The compound according to claim 19 or 20, wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 6 -O-.
  24. 　Ｘ１、Ｘ２、Ｘ３及びＸ４のスペーサーが、それぞれ独立して、－（ＣＨ２）５－ＮＨ－である、請求項１９又は２０記載の化合物。 The compound according to claim 19 or 20, wherein the spacers X 1 , X 2 , X 3 and X 4 are each independently -(CH 2 ) 5 -NH-.
  25. 　Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、糖である、請求項１９又は２０記載の化合物。 21. The compound of claim 19 or 20, wherein the ligands Y1 , Y2 , Y3 and Y4 are each independently a sugar.
  26. 　糖がＧａｌＮＡｃである、請求項２５記載の化合物。 The compound of claim 25, wherein the sugar is GalNAc.
  27. 　Ｙ１、Ｙ２、Ｙ３及びＹ４のリガンドが、それぞれ独立して、脂質である、請求項１９又は２０記載の化合物。 21. The compound of claim 19 or 20, wherein the ligands Y1 , Y2 , Y3 and Y4 are each independently a lipid.
PCT/JP2023/041480 2022-11-18 2023-11-17 Ligand conjugate substance, nucleic acid containing same, and use thereof WO2024106539A1 (en)

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