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WO1999021874A1 - Oligodesoxyribonucleotides contenant un nucleoside modifie et autre - Google Patents

Oligodesoxyribonucleotides contenant un nucleoside modifie et autre Download PDF

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Publication number
WO1999021874A1
WO1999021874A1 PCT/JP1998/004863 JP9804863W WO9921874A1 WO 1999021874 A1 WO1999021874 A1 WO 1999021874A1 JP 9804863 W JP9804863 W JP 9804863W WO 9921874 A1 WO9921874 A1 WO 9921874A1
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Prior art keywords
compound
reaction
group
solvent
acetonitrile
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Japanese (ja)
Inventor
Makoto Koizumi
Masakatsu Kaneko
Toshinori Ohmine
Hidehiko Furukawa
Takashi Nishigaki
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Sankyo Co Ltd
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Sankyo Co Ltd
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Priority to AU96489/98A priority Critical patent/AU9648998A/en
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    • 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
    • C12N15/1131—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 against viruses
    • C12N15/1132—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 against viruses against retroviridae, e.g. HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/336—Modified G
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the present invention relates to a novel modified oligodeoxyribonucleotide having excellent anti-AIDS virus activity.
  • BACKGROUND ART Antisense oligodeoxyribonucleotides are approximately 15 to 30-mer synthetic nucleic acids or derivatives thereof having a base sequence complementary to a specific gene or mRNA. These antisense oligodeoxyribonucleotides selectively inhibit the transcription or translation of a gene by binding to a target gene or mRNA, so that it can be expected to be able to treat diseases caused by this gene.
  • the nucleotide In order for antisense oligodoxyliponutide to function as a drug, the nucleotide must have nuclease stability, be capable of translocating into the cell or nucleus, and have a salt with the target gene or mRNA. It is important to have a group-pairing ability (Uhlmann, E. and Peyman, A. Chem. Rev., 90, 543, (1990)). In order to meet these requirements, various antisense oligodeoxyribonucleotides have been chemically modified. For example, one non-bridging oxygen atom of a phosphoric acid group in the phosphodiester bond of the nucleotide is replaced with a sulfur atom, a methyl group or a substituted amino group.
  • the modified phosphoric acid modified form and the modified form obtained by converting the nucleotide into peptide nucleic acids having an amide bond and the like have been reported (Uhlmann, E. and Peyman, ⁇ . Chem. Rev. , 90, 543-584, (1990)).
  • oligodoxyribonucleotides that bind to proteins are called aptamers, and those that bind to thrombin, gpl20, a surface protein of HIV, etc. have been found to date.
  • protein-binding nucleic acids have been searched using a technique called in vitro selection that combines RT-PCR with DNA having a random base sequence (Osborne, SE and Ellington, AD Chem Rev., 97, 349-370, (1997)).
  • antisense oligodeoxyribonucleotide in order for antisense oligodeoxyribonucleotide to exert its effect, it must form a stable base pair with mRNA or the like which is a target in vivo, and this base pair formation For this purpose, it has been said that the antisense oligodeoxyribonucleotide needs to have a length of at least about 15 nucleotides or more.
  • the antisense oligodeoxyribonucleotides cannot be said to be sufficiently satisfactory in the activity of inhibiting virus replication or cell growth, and furthermore, a diester phosphate bond forms a non-bridging oxygen atom of one of the phosphate groups.
  • Phosphorothioate oligodeoxyribonucleotides substituted with sulfur atoms have relatively high toxicity to normal host cells.
  • the inventors have invented short-chain oligodoxyribonucleotides having various substituents at the 5 ′ end and 3 ′ end, It discloses that the oligonucleotide exhibits anti-HIV-1 activity (Japanese Patent Application Laid-Open No. 7-87982).
  • oligodeoxyribonucleotides having a short chain length suppress degradation of 3'-nuclease from the 3'-terminal side, and have considered oligodeoxyribonucleotides.
  • a derivative having a 3 ', 3'-phosphate diester bond introduced therein and a derivative containing a base-free pseudonucleotide unit were synthesized in consideration of suppressing degradation of various nucleases. .
  • a derivative was also synthesized in which a relatively simple substituent was introduced into the base or sugar moiety of the nucleoside in oligodeoxyribonucleotide.
  • these modified oligodeoxyribonucleotide derivatives have high anti-HIV-1 activity, low toxicity to normal host cells, and are relatively easy to synthesize and practical.
  • the present invention has been completed.
  • a 2-N-methyl-2′-deoxyguanosine derivative is a useful production intermediate in producing chemically modified oligodeoxyribonucleotides, and completed the present invention.
  • An object of the present invention is to provide an oligodeoxyribonucleic acid having high anti-HIV-1 activity.
  • Another object of the present invention is to provide a method for treating or preventing AIDS disease, which comprises administering a pharmacologically effective amount of the oligodeoxyribonucleotide to a warm-blooded animal.
  • still another object of the present invention is to provide a 2-N-methyl-12'-deoxyguanosine derivative which is a useful production intermediate for synthesizing a modified oligodeoxyribonucleotide. That is.
  • the present invention also includes a pharmacologically acceptable salt of the oligodeoxyribonucleotide having (1).
  • a pharmacologically acceptable salt of the oligodeoxyribonucleotide having (1) in the general formula (1),
  • the M, X, R, is K n, A, G, C or T
  • beta 2 and beta 3 are the same or different
  • m represents an integer of 0 to 7
  • z represents an integer of 0 to 9.
  • 3 1 and 3 2 are the same or different and each represents a hydrogen atom, an alkyl group of 1 to 4 carbon atoms, shows the number of 1-4 alkoxy groups or halogen atoms carbon.
  • B 2 may be the same or different at each repetition of m
  • a, g, c, t, M, X, R, Kn , A, G, C and T are
  • n is an integer of 2 to 6.
  • the present invention is also a medicament, particularly a composition for treating or preventing an AIDS disease, comprising the compound (1) or a pharmacologically acceptable salt thereof. Furthermore, the present invention relates to the use of the above compound (1) or a pharmaceutically acceptable salt thereof for producing a medicament, particularly an anti-AIDS agent.
  • the present invention is a method for treating or preventing AIDS disease, which comprises administering a pharmacologically effective amount of the compound (1) or a pharmacologically acceptable salt thereof to a warm-blooded animal.
  • examples of the alkyl group having 1 to 4 carbon atoms of S and S 2 include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group and an s-butyl group.
  • a tert-butyl group and is preferably a methyl group.
  • the alkoxy group having 1 to 4 carbon atoms of Si and S 2 is a group in which the aforementioned “alkyl group having 1 to 4 carbon atoms” is bonded to an oxygen atom.
  • Examples include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, an s-butoxy group, and a tert-butoxy group, and a methoxy group is preferable.
  • the halogen atom of Si and S 2 is a fluorine atom or a salt.
  • examples include a fluorine atom, a bromine atom and an iodine atom, and a fluorine atom and a chlorine atom are preferred.
  • T, R or kappa eta is K 3 or the kappa 6, and most preferably a 3 T or kappa.
  • Beta 2 is preferably, A, a G, g, M, X or kappa beta, more preferably, a G, g, or M,
  • B 3 is preferably G, g or M, more preferably g, m is preferably 2 to 4, more preferably 2 to 3, most preferably Preferably 3,
  • z is preferably from 0 to 4, more preferably 0 or 1, most preferably 0,
  • ⁇ 1 and 3 2 is preferably a hydrogen atom
  • the sequence consisting of B 1 NB 2 and B 3 is preferably TGG gg, TGGG g, K 3 GGG, K 3 MMM or K 3 GGGG, and more preferably TGGG g.
  • ⁇ 2 is preferably A, G, M or X, more preferably A, G or M;
  • B 3 is preferably G, M or X, more preferably G or M
  • m is preferably from 0 to 4, more preferably from 2 to 4, and most preferably 4.
  • z is preferably 0 or 1, and more preferably, 0, ⁇ 1 and 3 2 is preferably a hydrogen atom,
  • the number of A in the sequence consisting of B or B 2 and B 3 is preferably 0 to 1, more preferably 1,
  • TMM 13 is a I B 2 and consisting of B 3 sequence, TMMM, TMGGAG, TGMGAG, TGGMAG , TGGGAM, a suitable TMMMAG or TXX XX, more preferably, TMGGAG, TGMGAG, T GGMAG, TGGG AM or TMMMAG It is.
  • the following compounds are preferred as compounds having the general formula (1).
  • B is T, R or K n (n is 2 to 6), B 2 is A, G, g, M, X or K Volunteer( ⁇ is 2 to 6), and B ⁇ ⁇ G, g or M, m is 2-4 and z is 0-4,
  • B is T, R or K n (n is 3 or 6), B 2 is G, g or M, B 3 is g, m is 2 to 3, z Is 0 or 1, and S i and S 2 are hydrogen atoms.
  • B is T or K n (n is 3 or 6), B 2 is G, g or M, B 3 is g, m is 2 to 3, and z force 0
  • S and S 2 are hydrogen atoms
  • (A- 4) BB 2 and consisting of B 3 sequence, TGG gg, TGGG g, K 3 GGG, K 3 MMM or K 3 compound is GGGG,
  • (A-5) a compound consisting of B or B 2 and B 3 is TGGG g, m is 2 to 3, z is 0, and S and S 2 are hydrogen atoms,
  • B- 2 is B i is T, B 2 is A, G or M, B 3 is G or M, m is 2 to 4, z is 0, 3 1 ⁇ Pi 3 2 is a hydrogen atom, B p B 2 and the number is 0 to 1 Kodea Ru compound of a in the sequence consisting of B 3,
  • B is T, 13 2 is a G or M, 8 3 a is ⁇ or 1 ⁇ , m is 4, z is 0, S, and S 2 is a hydrogen atom
  • B is a compound in which the number of A in the sequence consisting of B 2 and B 3 is 1,
  • (B- 4) B have B 2 and B 3 consisting of sequences, TMMM, a TMGGA G, TGMGAG, TGGMAG, TGGGAM , TMMMAG or T XX XX, m is 4, z is 0, 3 1
  • the compound having the general formula (1) of the present invention can be used in the form of a “pharmaceutically acceptable salt”.
  • examples of such salts include alkali metals such as sodium and potassium; alkaline earth metals such as calcium; ammonia; basic amino acids such as lysine and arginine; and triethylamine.
  • Inorganic salts or organic salts such as a suitable alkylamine; and preferred are alkali metal salts such as sodium and potassium.
  • the compound having the general formula (1) and a pharmacologically acceptable salt thereof may form a solvate in the production process or a hydrate during storage. And hydrates are also included in the present invention.
  • the 2-N-methyl-2′-deoxyguanosine derivative which is a useful production intermediate for producing oligodoxyribonucleotide of the present invention, comprises:
  • preferred compounds include 3, 4, 143, 160, 167, 239, 303, 304, 305, 306, 3 1 3 and 5 3 5 compounds,
  • Examples of suitable compounds include compounds of 4,303,304,305,306,313.
  • Examples of the compound having the general formula (2) of the present invention include the compounds described in Table 2.
  • 4,4'-DMT indicates 4,4'-dimethoxytrityl group
  • 4-band T indicates 4-monomethoxytrityl group.
  • suitable compounds include compounds 2-4 to 2-9.
  • suitable compounds include
  • 2-cyanoethyl 2- ⁇ -methyl-6-0-diphenylcarbamyl-2'-doxy-5'-0- (4-monomethoxytrityl) guanosine -3'-0-yl-1 ⁇ , ⁇ -Diisopropyl propyl phosphoramidite (Example number 2-8) and
  • Methinole 2— ⁇ —Methinole— 6—0—Dipheninorecanoreno kuminore— 2′—Deoxy—5′—0— (4-Monomethoxytrityl) Guanosine-3 '-0 -yl-1 ⁇ , ⁇ - Compounds of diisopropyl phosphoramidite (exemplification number 2-9) can be mentioned.
  • the most preferred compounds include 2-5 compounds.
  • the compounds of the general formula (1) of the present invention are prepared by the following methods (1), (2), (1), (3), (4) or (5), (6), (7) ),
  • nucleotide a compound prepared by the following method C on a DN ⁇ synthesizer using a commercially available nucleotide reagent for DNA synthesis (hereinafter referred to as “nucleotide”) as a raw material.
  • 10a), (1Ob) or (10c) de-dimethyloxytrityl (DMT) compound and a compound (A) prepared by the following A-1, A-2 or A-3 method ( 3) or (4) can be produced by bonding by the following D-I, D-2, D-3, D-4, D_5 or D-6 method.
  • the compound of the general formula (2) of the present invention can be produced by the B-5 method. A left
  • the 47th step is 0- (CH 2 ) n -0 -P-0- V
  • B i B 2 , B 3 , K n, m, n, z, S i S 2 , and R 2 are as defined above,
  • Tr trityl
  • MMT 4-monomethoxytrityl
  • DMT 4,4'-dimethoxytrityl
  • a 2 is a tri-substituted silyl group such as a tertiary butyldimethylsilyl (TB DMS) group or a triisopropylsilinole (TIPS) group; a trihalogenoethoxycarbonyl group such as a trichloroethoxycarbonyl (Troc) group; -Represents an aralkyloxycarbonyl group such as a (Z) group,
  • a 3 represents an alkyldisiloxane group such as a tetraisopropyldisiloxane (TIPDS) group generally used for protecting a hydroxyl group,
  • TIPDS tetraisopropyldisiloxane
  • D ' represents hydrogen, thymine, or adene, guanine or cytosine in which the amino group is protected by an acetyl group (eg, an acetyl group);
  • H a 1 represents a halogen atom (preferably a chlorine or bromine atom),
  • V is a protecting group for the phosphoric acid moiety (particularly, a lower alkyloxy group such as a methoxy group, a cyanoalkyloxy group such as a cyanoethyloxy group, and in the step 41 or 47, in particular, orthochlorophenyl).
  • a protecting group for the phosphoric acid moiety particularly, a lower alkyloxy group such as a methoxy group, a cyanoalkyloxy group such as a cyanoethyloxy group, and in the step 41 or 47, in particular, orthochlorophenyl).
  • U is an amino group of an amidite portion (particularly, a dialkylamino group such as a dimethylamino group or a diisopropylamino group; a heterocyclic group having one or two oxygen atoms and a Z or nitrogen atom in a ring such as a morpholino group)
  • Ph represents a phenyl group
  • DMT represents a 4,4 'dimethoxytrityl group
  • ⁇ 2 ′ and ⁇ 3 ′ respectively represent those in which the amino and phosphoric acid groups of the above ⁇ 2 and ⁇ 3 are protected by a protecting group
  • W, W 2 and W 3 are the portions of the compounds (10-7), (10-9) and (10-10) obtained by the method C which do not contain the hydrogen atom of the terminal hydroxyl group, respectively. Is shown.
  • Methods B-1, B-2, B-3, B-4 and B-5 produce compounds (5), (6), (7), (8) or (9) used in Method C Is the way.
  • the amidating reagent (5-4) is reacted with the hydroxyl group of the compound (10-2) obtained by converting one hydroxyl group of the diol compound (10-1) to DMT, and the compound (10-1) is reacted. — 3), and the compound (10-2) described above is bonded to CPG via a dicarboxylic acid in the same manner as in the method B-3 to form a CPG carrier (10-6) ), And using the CPG carrier (10-7) obtained by removing the DMT group as a raw material, using a nucleotide synthesizer as described above, and sequentially using the nucleotide unit described above, in the usual manner,
  • This is a method for producing a compound (10a) in which the compound (10a) has been extended to the position before the oligonucleotide by one less than the desired oligonucleotide, and further, a CPG carrier (10-7) is prepared on a DNA synthesizer.
  • the compound (3) or (4) is reacted with a phosphating agent to prepare a 3′-phosphorous acid derivative (11) or (14), and Of the compound (10a), (10b) or (10c) from which the DMT group has been removed, respectively, are condensed on a DN ⁇ synthesizer, and then oxidized.
  • the method further comprises cleaving the bond with CPG and, finally, removing the protecting group to produce the compound (1) of the present invention.
  • the D-2 and D-5 methods are based on the 3'-phosphate derivative (12) or (15) prepared by reacting a compound (3) or (4) with a phosphorylating agent, and the method C
  • the synthesized compound (10a;), (10b) or the corresponding compound from which (10c) from which the DMT group has been removed is condensed on a DNA synthesizer to break the bond with CPG.
  • a method for producing the compound (1) of the present invention by removing the protecting group.
  • the D-3 and D-6 methods are based on the compound (13) or (16) in which a phosphonic acid group is introduced at the 3 ′ position of the compound (3) or (4), and the compound (10) synthesized by the method C. a), (10b) or the corresponding compound from which (10c) the DMT group has been removed, respectively, is condensed and then oxidized to form a phosphodiester bond, and In this method, the bond is cleaved, and finally, the protecting group is removed to obtain the compound (1) of the present invention.
  • each step in the methods A-1 to D-6 will be described in detail. Steps that can be performed in the same manner will be described with the earliest step as a representative.
  • This step includes, in an inert solvent, the step of protecting the existing amino group for acylation in the case where the base moiety is A, G or C in the presence of the compound (3-1) or the like.
  • the protection step can be easily carried out by a known method (J. Am. Chem. Soc., 104, 1316, (1982)).
  • a lower aliphatic acyl or an aromatic acyl is generally used.
  • Examples of the lower aliphatic acyl used include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, bivaloyl, valeryl, and isovaleryl groups.
  • Examples of the aromatic acyl used include, for example, Benzyl, 4-acetoxybenzoyl, 4-methoxybenzoyl, 4-methylbenzoyl, 1-naphthyl and the like are preferred.
  • a benzoyl group when the base moiety is A or C
  • ⁇ 3> is a step of reacting a hydroxyl-protecting reagent to selectively produce a compound (3-2) or the like in which only the 5′-hydroxyl group is protected.
  • the solvent used is preferably an aromatic hydrocarbon such as benzene, toluene or xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, dichlorobenzene or the like.
  • Halogenated hydrocarbons esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisopyl pill ether, tetrahydrofuran, dioxane, dimethyloxetane, and dimethylene glycol Ethers such as dimethyl ether; acetone, Ketones such as methinoolethinoroketone, methylisobutinoleketone, isophorone and cyclohexanone; two-mouth compounds such as nitrotroethane and nitrobenzene; acetonitrile, isobuchi mouth-tolyl Such ethrils; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, and hexamethyl phosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane; Aliphatic tert
  • the protecting reagent to be used is not particularly limited as long as it can selectively protect only the 5′-position and can be removed under acidic and neutral conditions.
  • These are trimethyl halides such as lid, monomethoxytrityl chloride, and dimethoxytrityl chloride.
  • a base is usually used.
  • examples of the base used include heterocyclic amines such as pyridine, dimethylaminopyridine and pyrrolidinopyridine, and aliphatic tertiary amines such as trimethylamine and triethylamine.
  • heterocyclic amines such as pyridine, dimethylaminopyridine and pyrrolidinopyridine.
  • aliphatic tertiary amines such as trimethylamine and triethylamine.
  • Preferred are pyridine, dimethylamino pyridine and pyrrolidino pyridine.
  • the base When a liquid base is used as the solvent, the base itself acts as a deoxidizing agent, so that it is not necessary to add a base again.
  • the reaction temperature is usually from 0 to 150 ° C, preferably from 20 to 100 ° C, depending on the starting materials, reagents, solvents and the like used.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like to be used, but is usually 1 to 100 hours, preferably 2 to 24 hours.
  • the reaction solution is poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, etc., and the solvent is distilled off from the extract to obtain the desired compound (3-2). And so on, and usually used in the next step as it is. If desired, it can be isolated and purified by various chromatography or recrystallization methods.
  • This step is a step of producing a compound (3-3) or the like by reacting a compound for protecting a hydroxyl group with the compound (3-2) or the like in an inert solvent.
  • aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, carbon form, carbon tetrachloride, dichloroethane, carbon benzene and dichlorobenzene such as dichlorobenzene are preferably used.
  • esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisopyl propyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether Ethers such as acetone; ketones such as aceton, methinoolethinoleketone, methinolay soptino oketone, isophorone, cyclohexanone; and nitro compounds such as nitrotroethane, nitrobenzene; Toe Toril, Isobuchilo Nitritols such as nitril; amides such as formamide, dimethylformamide, dimethylacetamide and hexamethylphosphorotriamide; and sulfoxides such as dimethylsulfoxide and sulfolane. More preferably, polyesters (
  • the protecting reagent used is usually removed separately from the protecting group at the 5'-position.
  • silyl halides such as t-butyldimethylsilyl chloride or triisopropylsilyl chloride, or haloalkoxys such as trichloroethoxycarbyl chloride.
  • Examples include carbonyl halides and aralkyloxycarbonyl halides such as benzyloxycarboerk P-lide.
  • a base is usually used.
  • the base to be used is preferably an organic base (especially triethylamine, pyridine, N-methylmorpholine, DBU and imidazole).
  • the reaction temperature is usually from ⁇ 20 to 150 ° C., and preferably from ⁇ 10 to 50 ° C., depending on the reagent, raw material, solvent and the like used.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like to be used, but is usually 1 to 100 hours, preferably 1 to 24 hours.
  • reaction solution is poured into water, extracted with a water-immiscible solvent such as benzene, ether, ethyl acetate, and the like.
  • Etc. are usually obtained and used for the next step. If desired, it can be isolated and purified by various chromatography or recrystallization methods.
  • the compound (3-4) is reacted with a deprotection reagent in an inert solvent to selectively remove the protecting group for the 5′-hydroxyl group. This is the manufacturing process.
  • the solvent used is not particularly limited as long as it does not inhibit the reaction.
  • aromatic hydrocarbons such as benzene, toluene and xylene
  • halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, and dichloromethane
  • formic acid Esters such as tyl, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate
  • ethers such as getyl ether, diisopropyl ether, tetrahydrofuran, furan, dioxane, dimethyloxetane, and diethylene glycol dimethyl ether
  • the deprotecting reagent to be used is not particularly limited as long as it is a commonly used deprotecting reagent.
  • the protecting group is a triarylmethyl group, for example, acetic acid, acetic acid at the mouth of the mouth, trifluoroacetic acid , Hydrochloric acid, and Lewis acids such as zinc bromide, and acetic acid, dichloroacetic acid, and trifluoroacetic acid are preferred.
  • the reaction temperature varies depending on the used reagents, raw materials, solvents and the like, but is usually from 110 to 100 ° C, preferably from 0 to 50 ° C.
  • the reaction time is Although it varies depending on the starting material, solvent, reaction temperature and the like used, it is usually 1 minute to 50 hours, preferably 1 minute to 24 hours.
  • the reaction solution is neutralized with a base such as pyridine, poured into water, and extracted with a water-immiscible solvent such as benzene, ether, or butyl acetate, and the solvent is distilled off from the extract.
  • a base such as pyridine
  • a water-immiscible solvent such as benzene, ether, or butyl acetate
  • the solvent is distilled off from the extract.
  • the desired product (314) is obtained, and usually used as it is in the next step. If desired, it can be isolated and purified by various chromatography or recrystallization methods.
  • Steps 32 and 35 the desired compounds (10-7) and (10-9) can be collected by filtration, washed with an organic solvent such as methylene chloride, and then subjected to the next step. Used in the step.
  • the compound (3-5) is reacted with the compound (3-4) in the presence of a base in an inert solvent to produce the compound (3-6) and the like.
  • Preferred solvents used are aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform, carbon tetrachloride, di, cycloethane, cyclobenzene, dibenzene.
  • Hydrogenated hydrocarbons such as chlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisopropinole pinoleate ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol Ethers such as dimethyl ether; ketones such as acetone, methinolethynoleketone, meth ⁇ / isobutynoleketone, isophorone and cyclohexanone; and nitro compounds such as nitroethane and nitrobenzene; Setoetril, isoptilonito Nitrils such as ril; amides such as formamide, dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide; dimethylsulfoxyl And sulfolane such as sulfo
  • ethers particularly, tetrahydrofuran
  • ketones particularly, acetone
  • halogenated hydrocarbons particularly, methylene chloride
  • amides especially dimethylformamide
  • aromatic amines especially pyridine
  • organic bases particularly, triethylamine, pyridine, N-methylmorpholine, DBU, etc.
  • alkali metal hydrides particularly, sodium hydride
  • alkali metal Carbonates particularly sodium carbonate and lithium carbonate.
  • the reaction temperature is not particularly limited, it is generally 0 ° C. to 100 ° C., and preferably 20 to 60 ° C.
  • the reaction time is usually from 5 minutes to 30 hours, but when the reaction is carried out at 50 ° C, the reaction is completed in 10 hours.
  • the reaction mixture is appropriately neutralized, and if there is any insoluble matter, it is removed by filtration. Then, an immiscible organic solvent such as water and ethyl acetate is added.
  • the target product (3-6) can be obtained by separating the organic layer containing, drying with anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • the compound (3-6) is reacted with a deprotecting agent in an inert solvent to produce the compound (3) and the like.
  • the method of this step differs depending on the type of the protecting group such as compound (3-6).
  • a fluoride ion such as tetrabutylammonium fluoride is usually generated. It is removed by treatment with the compound.
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction, but ethers such as tetrahydrofuran and dioxane are preferable.
  • the reaction temperature is not particularly limited, but usually is 1 0 0 ° C from a 3 0 ° C, preferably carried out at 0 ° C to 3 0 D C.
  • the reaction time is usually from 5 minutes to 30 hours, but when the reaction is carried out at 20 ° C, the reaction is completed in 10 hours.
  • the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, and an immiscible organic solvent such as water and ethyl acetate is added.
  • an immiscible organic solvent such as water and ethyl acetate is added.
  • the organic layer containing the compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off to obtain the desired product (3).
  • the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction, but acetic acid, alcohol, or a mixed solvent of these with water is preferred.
  • the reaction temperature is not particularly limited, but is usually 0 to 100 ° C., and preferably is carried out at room temperature.
  • the reaction time is usually 5 minutes to 30 hours, but when the reaction is carried out at room temperature, the reaction is completed in 10 hours.
  • the reaction mixture is appropriately neutralized, and if there is any insoluble matter, it is removed by filtration. Then, an immiscible organic solvent such as water and ethyl acetate is added. The organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate, etc., and then the solvent is distilled off to obtain the desired product (3). Is obtained.
  • the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • reaction can be carried out by catalytic reduction or oxidation.
  • the reduction catalyst used in the catalytic reduction is not particularly limited as long as it is generally used in the catalytic reduction reaction, but is preferably palladium carbon, Raney nickel, platinum oxide, Platinum black, rhodium-aluminum oxide, triphenylphosphine rhodium monochloride, and palladium monosulfate barrier are used.
  • the pressure is not particularly limited, but it is usually 1 to 10 atm.
  • reaction temperature and the reaction time vary depending on the starting material, the solvent, the type of the catalyst and the like, but are usually from 0 to 100 ° C for 5 minutes to 24 hours.
  • the solvent used in the removal by oxidation is not particularly limited as long as it does not participate in this reaction, but is preferably a water-containing organic solvent.
  • the organic solvent preferably, ketones such as acetone, methylene chloride, chloroform, halogenated hydrocarbons such as carbon tetrachloride, -triles such as acetonitrile, Ethers such as Jethyl ether, tetrahydrofuran, and dioxane; amides such as dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide; and sulfoxides such as dimethylsulfoxide. Can be.
  • the oxidizing agent to be used is not particularly limited as long as it is a compound used for the oxidation.
  • potassium persulfate, sodium persulfate, ammonium cell nitrate (CAN), 2 , 3-Dicopento-5,6-dicyano-p-benzoquinone (DDQ) is used.
  • reaction temperature and reaction time vary depending on the starting material, solvent, type of catalyst, etc. However, it is usually carried out at 0 to 150 ° C. for 10 minutes to 24 hours.
  • an alkali metal such as lithium metal or metal sodium is allowed to act at 178 to 120 ° C. Can also be removed.
  • an alkylsilyl halide such as aluminum chloride-sodium iodide or trimethylsilyl iodide in an inert solvent.
  • the solvent to be used is not particularly limited as long as it does not participate in the reaction, but preferably, nitriles such as acetonitrile, methylene chloride, and chloroform are preferred. Halogenated hydrocarbons such as form or mixed solvents thereof are used.
  • reaction temperature and reaction time vary depending on the starting material, solvent and the like, but are usually carried out at 0 to 50 ° C for 5 minutes to 3 hours.
  • reaction substrate has a sulfur atom
  • aluminum chloride sodium iodide is preferably used.
  • reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration. Then, an immiscible organic solvent such as water and ethyl acetate is added. The organic layer containing is separated, dried with magnesium sulfate anhydride and the like, and the solvent is distilled off to obtain the desired product (3).
  • the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • a compound (5-3) or the like is reacted with a phosphite (5-4) in an inert solvent in the presence of a deoxidizing agent to convert the 3,3 phosphite derivative (5) or the like. This is the manufacturing process.
  • phosphorylating agent (5-4) morpholine morpholinomethoxyphosphine, chloromonorenolynosinoetoxyphosphine, chlorodimethinoleamine methoxyphosphine, chlorodimethylaminosinoetoxyphosphine, chlorodisopropylamino methoxyphosphine
  • phosphines such as black diisopropylaminoamino ethoxyphosphine, and preferably black monorenoline methoxyphosphine, black monorenoline ethoxy phosphine, chlorodisopropylamino methoxy phosphine, and chlorophyll. It is oral dipropylamino cyanoethoxyphosphine.
  • the solvent to be used is not particularly limited as long as it does not affect the reaction, but is preferably an ether such as tetrahydrofuran, getyl ether, or dioxane.
  • deoxidizing agent used examples include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine and diisopyrupyrethylamine. Amides (especially diisopropylethylamine).
  • the reaction temperature is not particularly limited, but is usually from 150 to 50 ° C., preferably room temperature.
  • the reaction time varies depending on the starting materials, reagents, temperature and the like to be used, but is usually 5 minutes to 30 hours, preferably 30 minutes when the reaction is carried out at room temperature.
  • the target compound is neutralized, for example, by appropriately neutralizing the reaction mixture. If there is an insoluble substance, it is removed by filtration, and then an immiscible organic solvent such as water and ethyl acetate is added, followed by washing with water. Thereafter, the organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off.
  • the obtained target compound can be obtained by a conventional method, for example, recrystallization, It can be further purified by reprecipitation or by mouth chromatography.
  • a compound (3-1) is reacted with a hydroxyl-protecting reagent in an inert solvent to produce a compound (6-1) in which the 5′- and 3′-hydroxyl groups are protected. It is.
  • the solvent used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; or a hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, or dichlorobenzene.
  • aromatic hydrocarbon such as benzene, toluene, or xylene
  • hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, or dichlorobenzene.
  • Esters such as ethyl ethyl formate, ethyl ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisoprop, pirate ether, tetrahydrofuran, dioxane, dimethoxetane, diethylene glycol Ethers such as ethyldimethylether; ketones such as acetone, methinoleetinoleketone, methionine / raysobutinoleketone, isophorone, cyclohexanone; and nitrobenzene and nitrobenzene Acetonitrile compounds; Nitrils such as butyronitrile; amides such as formamide, dimethylformamide (DMF), dimethylacetamide and hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane Aliphatic tertiary
  • the protecting reagent to be used is not particularly limited as long as it can protect the 5′-position and 3′-position hydroxyl groups and then selectively remove only the 5′-position hydroxyl groups in the 16th step.
  • trityl chloride are triaryl methyl halides, such as lithium-rich mouth lids and 4,4'-dimethoxytrityl chloride.
  • Examples of the base used include heterocyclic amines such as pyridine, dimethylaminopyridine and pyrrolidinopyridine, and aliphatic tertiary amines such as trimethylamine and triethylamine.
  • organic bases especially Pyridine, dimethylaminopyridine, pyrrolidinopyridine.
  • the reaction temperature is usually from 0 to 150 ° C, preferably from 20 to 100 ° C, depending on the starting materials, reagents, solvents and the like used.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like to be used, but it requires a longer time than the first step, and is usually 1 to 100 hours, preferably 2 to 24 hours.
  • reaction solution is usually poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, and the like. Use it in the next step as it is. If desired, it can be isolated and purified by various chromatography or recrystallization methods.
  • a water-immiscible solvent for example, benzene, ether, ethyl acetate, and the like.
  • the compound (6-1) is reacted with a deprotecting reagent in an inert solvent to selectively remove the 5′-hydroxyl protecting group (AJ) to give the compound (612). This is the manufacturing process.
  • the solvent to be used preferably, methylene chloride, Halogenated hydrocarbons such as carbon tetrachloride, dichloroethane, cyclobenzene, dichlorobenzene; esters such as ethyl ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; methanol, ethanol, n -Alcohols such as propanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, methinoleserosolonolev, etc .; Ketones such as tyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; Nitro compounds such as nitroethane and nitrobenzen
  • Examples of the deprotecting reagent used include Lewis acids such as zinc bromide, and zinc bromide is preferred.
  • the reaction temperature varies depending on the reagents, raw materials, solvents and the like used, but is usually from 110 to 100 ° C, preferably from 0 to 50 ° C.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like used, but is usually 1 minute to 50 hours, preferably 1 minute to 24 hours.
  • the mixture obtained by pouring into water, extracting with a water-immiscible solvent such as benzene, ether, or ethyl acetate, and distilling off the solvent from the extract is usually used as is in the next step. Used for the process. If desired, it can be isolated and purified by various chromatographic or recrystallization methods.
  • Examples of the protecting group include silyl halides.
  • Examples of protecting groups capable of protecting the 3′- and 5′-hydroxyl groups with a single protecting group include triisopropylsilyl-capped lidodichlorotetrisopropyldiloxane and the like. And t-butyldimethylsilyl chloride as a protecting group for protecting the 3′- and 5′-position hydroxyl groups, respectively.
  • Preferred solvents used are aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, and dichlorobenzene.
  • Novel hydrogenated hydrocarbons esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisop ore pirueter, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol.
  • Ethers such as coal dimethyl ether; ketones such as acetone, methinoleetinoleketone, methinolay sobutinoleketone, isophorone and cyclohexanone; Compounds; acetonitrile, isobutyro Nitrils such as tolyl; amides such as formamide, dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide; and sulfoxides such as dimethylsulfoxide and sulfolane. More preferred are ethers (especially tetrahydrofuran), halogenated hydrocarbons (especially methylene chloride), aromatic hydrocarbons (especially toluene), and imides (especially DMF).
  • the base used is preferably an organic base (especially triethylamine, pyridine, N-methylmorpholine, DBU and imidazole).
  • the reaction temperature is usually from 120 to 150 ° C, preferably from 110 to 50 ° C, depending on the reagents, raw materials, solvents and the like used.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like to be used, but is usually 1 to 100 hours, preferably 1 to 24 hours.
  • reaction solution is usually poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, and the like. Use it in the next step as it is. If desired, it can be isolated and purified by various chromatography or recrystallization methods.
  • a water-immiscible solvent for example, benzene, ether, ethyl acetate, and the like.
  • the compound (9-12) is added with an acid, formaldehyde and an alkyl-substituted fuerthiol in a solvent and reacted to obtain a 2-N-alkyl-substituted phenylthiomethyl compound, which is then reduced and subjected to 2-N —This is a step of producing the compound (9-13) which is a methyl form.
  • the solvent used for the alkyl-substituted phenylthiomethylation is not particularly limited as long as it does not hinder the reaction and dissolves the starting material to some extent, but aromatic hydrocarbons such as benzene, toluene, and xylene can be used.
  • Alcohols such as ethanol, n-butanol, isobutanol and isoamyl alcohol; diluent acids such as sulfuric acid water; water; acetone; ketones such as methylethylketone; heterocyclic amines such as pyridine or ⁇ Se Toyu bets such as Lil - tolyl acids can be mentioned, preferably, methanol, Etanonore, n- Purono ⁇ 0 Nonore, isoproterenol Bruno, 0 Bruno one Honoré, n- Butanonore, iso butanol Ichiru, isoamyl Alcohols such as alcohol.
  • the acid used for the alkyl-substituted phenylthiomethylation is not particularly limited as long as it does not hinder the reaction and dissolves the starting material to some extent.
  • Alkyl carboxylic acids are preferred, and acetic acid is preferred.
  • the alkyl-substituted phenolthiol used for the alkyl-substituted phenylthiomethylation is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, and examples thereof include thiophenol-toluenethiol. And preferably toluene thiol.
  • Substituted Hue - reagent reaction temperature used in Ruchiomechiru reduction raw materials, usually by and solvent - a 2 0 to 1 5 0 ° C, preferably an 1 0 ⁇ optimum 5 0 e C.
  • the reaction time of the alkyl-substituted thiothiomethylation varies depending on the starting materials used, the solvent, the reaction temperature and the like, but is usually 1 to 100 hours, and preferably 1 to 24 hours.
  • the reaction solution is poured into water, extracted with a water-immiscible solvent such as benzene, ether, or ethyl acetate, and the solvent is distilled off from the extract.
  • a water-immiscible solvent such as benzene, ether, or ethyl acetate
  • the product obtained is usually used as it is in the next step.
  • Various chromatographs as desired Alternatively, it can be isolated and purified by a recrystallization method.
  • the solvent used in the subsequent reduction reaction is not particularly limited as long as it does not hinder the reaction and dissolves the starting materials to some extent; however, aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride Halogenated hydrocarbons such as alcohol, tetrahydrofuran, dioxane, and dimethoxetane; dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide Sulphoxides such as dimethylsulphoxide; methanol, ethanol, n-propanol and isoprono ⁇ .
  • aromatic hydrocarbons such as benzene, toluene and xylene
  • Halogenated hydrocarbons such as alcohol, tetrahydrofuran, dioxane, and dimethoxetane
  • dimethylformamide dimethylacetamide
  • hexamethylphosphorotriamide Sulphoxides such as dimethylsulphoxide
  • Tolyls can be mentioned, and preferred are methano mono, ethano nor, n-prono nor and isov.
  • the reducing reagent is not particularly limited as long as it does not inhibit the reaction, and examples thereof include sodium borohydride and nickel nickel raney, and more preferably, sodium borohydride.
  • the reaction temperature is usually from 120 to 150 ° C., and preferably from ⁇ 10 to 50 ° C., depending on the reagent, raw material, solvent and the like used. C.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like to be used, but is usually 1 to 100 hours, preferably 1 to 24 hours.
  • reaction solution is usually poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, and the like. Use it in the next step as it is. If desired, isolate and purify by various chromatographic or recrystallization methods. You can also. (Step 24)
  • a water-immiscible solvent for example, benzene, ether, ethyl acetate, and the like.
  • This step is a step of reacting compound (9-3) with diphenylcarbamyl chloride to produce compound (914).
  • the solvent used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; or a hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, or dichlorobenzene.
  • Esters such as ethylen formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; tert-hydrofuran, dioxane, dimethoxetane, and dimethylene glycol.
  • Ethers such as dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as trotroethane and nitrobenzene; acetonitrile, Issobutyroni Tril Trilinoles; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, hexamethylphosphorotriamide; snorreoxides such as dimethyl snolefoxide and snoreholane; trimethinorea Aliphatic tertiary amines such as amine, triethylamine and N-methylmorpholine; aromatic amines such as pyridine and picoline; more preferably, halogenated hydrocarbons (particularly methylene chloride). And amides (especially DMF).
  • a base is usually used for the protection reaction.
  • the base used include heterocyclic amines such as pyridine, dimethylaminopyridine and pyrrolidinopyridine, and aliphatic tertiary amines such as trimethylamine and triethylamine. (Especially pyridine, dimethylaminopyri) Gin, pyrrolidinopyridine).
  • the organic amine When an organic amine is used as a solvent, the organic amine itself functions as a deoxidizing agent, so that it is not necessary to add another deoxidizing agent.
  • the reaction temperature is usually from 0 to 150 ° C, preferably from 20 to 100 ° C, depending on the starting materials, reagents, solvents and the like used.
  • the reaction time varies depending on the starting material, solvent, reaction temperature and the like to be used, but is usually 1 to 100 hours, preferably 2 to 24 hours.
  • reaction solution is usually poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, and the like. Use it in the next step as it is. If desired, it can be isolated and purified by various chromatography or recrystallization methods.
  • a water-immiscible solvent for example, benzene, ether, ethyl acetate, and the like.
  • the compound (10-2) is reacted with a dicarboxylic acid anhydride in the presence of a basic catalyst to produce a dicarboxylic acid half ester (10-4).
  • succinic acid is described as a representative, but the same can be applied to other dicarboxylic acids.
  • the dicarboxylic acid used is not particularly limited, but preferably has 2 to 10 carbon atoms, most preferably succinic acid or glutaric acid.
  • Examples of the basic catalyst used include aminopyridines such as dimethylaminopyridine and pyrrolidinoviridine, tertiary amines such as trimethylamine / triethylamine, sodium hydrogen carbonate, and potassium carbonate. Preferred are alkali metal carbonates, but dimethylamino pyridine, Pyrrolidino pyridine is most preferred.
  • the solvent used is not particularly limited as long as it does not hinder the reaction and dissolves the starting material to some extent, but is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; Halogenated hydrocarbons such as lids and chloroforms; ethers such as ethers, tetrahydrofuran, dioxane, and dimethoxetane; dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide Amides; sulfoxides such as dimethyl sulfoxide; methanol, ethanol, n-prono II. Nonore, I Soprono.
  • Alcohols such as ethanol, n-butanol, isobutanol, and isoamyl alcohol; diluent acids such as sulfuric acid; diluent bases such as sodium hydroxide; water; acetone; Ketones include heterocyclic amines such as pyridine and nitriles such as acetonitrile, and preferably, nitriles (especially acetonitrile), ethers ( Especially, tetrahydrofuran) and halogenated hydrocarbons (especially methylene chloride).
  • diluent acids such as sulfuric acid
  • diluent bases such as sodium hydroxide
  • water acetone
  • Ketones include heterocyclic amines such as pyridine and nitriles such as acetonitrile, and preferably, nitriles (especially acetonitrile), ethers ( Especially, tetrahydrofuran) and halogenated hydrocarbons (especially methylene chlor
  • the reaction is carried out at a temperature of 150 to 100 ° C., and the reaction time is usually 30 minutes to 15 hours, although it depends mainly on the reaction temperature, the starting compound or the type of the solvent used.
  • the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, and water and an immiscible organic solvent such as ethyl acetate are added.
  • the target compound (10-4) can be obtained by separating the organic layer containing the target compound, drying over an anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • the dicarboxylic acid obtained in Step 30 is treated in the presence of a condensing agent.
  • Phenol (10-4) is reacted with phenols such as pentachloronorenophenol to form an active ester, and then, in the presence of a base, the active ester and amino-CPG (10-5) ) To obtain the desired product (10-6).
  • phenols used are not particularly limited as long as they react with a carboxylic acid to form an active ester, but pentachlorophenol and 4-ethylphenol are preferred. .
  • the base to be used is not particularly limited as long as it is used as a base in a usual reaction, but is preferably triethylamine, tributylamine or diisopropylamine.
  • the solvent used is not particularly limited as long as it does not hinder the reaction and dissolves the starting material to some extent.
  • the solvent include dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide.
  • they are amides such as dimethylformamide.
  • the base to be used is not particularly limited as long as it is used as a base in a usual reaction.
  • Organic bases such as 2] octane (DAB CO), 1,8-diazabicyclo [5,4,0] pandec-7-ene (DBU), and preferably, triethylamine, pyridine, N— Methylmorpholine, DBU.
  • the reaction temperature is from 150 to 100 ° C, and the reaction time depends mainly on the reaction temperature, the type of the starting compounds and the type of the solvent used. 0 to 50 hours.
  • the target substance is collected, washed with an organic solvent such as methylene chloride, and used as it is in the next step.
  • a nucleoside-supporting CPG (10-7) which is a nucleotide at the 3 'end of the target oligonucleotide, is used as a raw material, and the DNA is usually extended on a DNA synthesizer.
  • the procedure used in the reaction is repeated to extend the oligonucleotide to the oligonucleotide from which the nucleotides other than the 5 'end of the target oligonucleotide have been removed, and to obtain the oligodeoxyribonucleotide (10a) etc. while being supported on CPG. is there.
  • the phosphoramidite method will be described for the elongation reaction of the DNA chain on the DNA synthesizer, but is not particularly limited to this method.
  • the CPG carrier (10-7) and the like are removed on a DNA synthesizer using a DMT-removing reagent to remove DMT, and triphosphite formed by condensing nucleotide units.
  • the ester bond is oxidized to a phosphoric acid triester using an oxidizing agent.
  • oligonucleotide is extended to an oligonucleotide excluding nucleotides other than the 5 'end of the desired oligonucleotide, and the oligodeoxynucleotide (1) supported on a CPG having a DMT group at the 5' end. 0a) (hereinafter, oligodoxynucleotide is abbreviated as ODN).
  • a CPG carrying an ODN of the desired nucleotide sequence protected at the 5 'end with a DMT group can be synthesized on a DNA synthesizer (for example, Applied Biosystems' phosphoramidite model 380B or Milligen®).
  • the compound can be synthesized by the method described in Nucleic Acids Res, 124539 (19894) or a modified method thereof using the cycloamidite method of the phosphoramidite method of Z Biosearch.
  • the base of the nucleotide unit used in the synthesis of the oligonucleotide one protected with an aliphatic or aromatic acyl group is used.
  • the base is A or C
  • a benzoyl group is used
  • the base is G
  • an isobutyryl group is suitably used.
  • the solvent used in the condensation reaction in this step is not particularly limited as long as it does not inhibit the reaction.
  • Acetonitrile and tetrahydrofuran are suitable, and are used as catalysts.
  • As an acidic substance, tetrazole is preferable.
  • the reaction temperature may be anywhere from 130 to 50 ° C., but is usually carried out at room temperature.
  • the reaction time varies depending on the reaction temperature from 1 minute to 20 hours. When the reaction is carried out at room temperature, the reaction is completed in 10 minutes.
  • the solvent used in the oxidation reaction in this step is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent.
  • the heterocyclic amine particularly, Gin
  • nitriles especially acetonitrile
  • ethers especially tetrahydrofuran
  • halogenated hydrocarbons especially methylene chloride
  • peracids such as m-chloroperbenzoic acid
  • peroxides such as t-butyl hydroperoxide
  • iodine-pyridine Is water water.
  • the reaction is carried out at a temperature of from 150 to 100 ° C, and the reaction time is usually from 30 minutes to 15 hours, although it depends mainly on the reaction temperature, the type of the starting compound or the type of the solvent used.
  • the desired product (10a) and the like are collected by filtration, washed with an organic solvent such as methylene chloride, and used as they are in the next step.
  • the steps 36 and 38 can be performed in the same manner as described above.
  • This step is performed in the same manner as in step 33 except that the compound (10-3) is reacted in place of the nucleoside phosphoramidite, which is a nucleotide unit for a DNA synthesizer, in the step 33.
  • This is a step of producing a CPG carrier (10-8) having an ester bond.
  • the CPG carrier (10-9) obtained in the step 35 is repeated by repeating the steps 34 and 35 Z -1 times ( Z has the same meaning as described above).
  • the compound (11) obtained in the step 39, etc., and the compound obtained by removing the DMT group at the 5′-position, such as the compound (10a), are reacted with the presence of an acid catalyst.
  • This is a step of condensing, then oxidizing, further cleaving the CPG, and finally removing the protecting group to obtain the target compound (1) of the present invention through a purification operation.
  • the acid catalyst used is preferably tetrazole.
  • the oxidizing agent to be used is not particularly limited as long as it is usually used in the oxidation reaction, but is preferably a peracid such as m-chloroperbenzoic acid. Peroxides such as t-butyl hydroperoxide; iodine-pyridine-water.
  • the solvent used is not particularly limited as long as it does not hinder the reaction and dissolves the starting material to some extent, but is preferably a heterocyclic amine (particularly, pyridine) or nitrile. (Especially acetonitrile), ethers (especially tetrahydrofuran), and halogenated hydrocarbons (especially methylene chloride).
  • the reaction is carried out at a temperature of 150 to 100 ° C, and the reaction time varies depending on the reaction temperature, the type of the starting compound or the solvent used, but is usually 30 minutes to 15 hours. is there.
  • the reaction is accelerated by adding a layer transfer catalyst such as triethylbenzylammonium chloride or tributylbenzylammonium bromide.
  • a layer transfer catalyst such as triethylbenzylammonium chloride or tributylbenzylammonium bromide.
  • CPG cleavage and removal of the protecting group in the final step can be performed by a known method (J. Am. Chem. Soc., 103, 3185, (1981)).
  • the reaction mixture containing the compound of the general formula (1) thus obtained is subjected to various chromatographies such as reversed-phase and ion-exchange chromatography (including high-performance liquid chromatography).
  • the target compound can be obtained by purification by a procedure usually performed for oligonucleotide purification.
  • the compound (3) or the like is treated with a phosphorylating reagent (for example, V-substituted phosphoropis triazolide (where V is 2-cyclophenyl group) in an inert solvent.
  • a phosphorylating reagent for example, V-substituted phosphoropis triazolide (where V is 2-cyclophenyl group) in an inert solvent.
  • this is the step of reacting 2-chlorophenylphosphorobistriazolide) to obtain an intermediate, such as mononucleotide (12).
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction. Usually, an aromatic amine such as pyridine is used.
  • the reaction temperature is not particularly limited up to 120 to 100 ° C., but it is usually carried out at room temperature.
  • the reaction time varies depending on the solvent and the reaction temperature, but is 1 hour when pyridine is used as the reaction solvent at room temperature.
  • the target compound is neutralized, for example, by appropriately neutralizing the reaction mixture. If insolubles are present, they are removed by filtration, and an immiscible organic solvent such as water and ethyl acetate is added. It can be obtained by separating the organic layer containing the target compound, drying over anhydrous magnesium sulfate or the like, and distilling off the solvent.
  • the obtained target compound can be further purified, if necessary, by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction, but an aromatic amine such as pyridine is preferably used.
  • Examples of the condensing agent used for the condensation include disuccinic carbodiimide (DCC), mesitylene sulfonic acid chloride (Ms—C 1), triisopropylbenzene sulfonic acid chloride, and mesitylene sulfonic acid triazolide.
  • DCC disuccinic carbodiimide
  • Ms—C 1 mesitylene sulfonic acid chloride
  • Ms—C 1 mesitylene sulfonic acid chloride
  • mesitylene sulfonic acid triazolide examples include disuccinic carbodiimide (DCC), mesitylene sulfonic acid chloride (Ms—C 1), triisopropylbenzene sulfonic acid chloride, and mesitylene sulfonic acid triazolide.
  • MS T mesitylenesulfonic acid-3-nitrotriazolide
  • MS NT mesitylenesulfonic acid-3-nitrotriazolide
  • TPS-Te mesitylenesulfonic acid tetrazolide
  • TPS-NI triisopropyl benzene sulfonic acid nitroimidazolide
  • MS NT and TPS-Te and TPS-NI are used.
  • the reaction temperature is not particularly limited, and is usually from room temperature to 110 ° C to 1 ° C.
  • the reaction time varies depending on the solvent used and the reaction temperature. However, when pyridine is used as the reaction solvent and the reaction is carried out at room temperature, the reaction time is 30 minutes.
  • CPG cleavage and removal of the protecting group in the final step can be performed by a known method (J. Am. Chem. Soc., 103, 3185, (1981)).
  • the thus obtained reaction mixture containing the compound of the general formula (1) is subjected to ordinary chromatography using various chromatographies such as reversed-phase and ion-exchange chromatography (including high-performance liquid chromatography).
  • the target compound can be obtained by purification by the operation performed in oligonucleotide purification.
  • compound (3) is added to compound (3) in an inert solvent, for example, according to literature (BC Freohler, PG Ng and MD. Matteucci, Nucleic Acids Res., 145399 (1986)).
  • an inert solvent for example, according to literature (BC Freohler, PG Ng and MD. Matteucci, Nucleic Acids Res., 145399 (1986)).
  • tris- (1,2,4-triazolyl) phosphite prepared from 2,4-triazole is reacted to obtain nucleoside 3'H-phosphonate (13).
  • the solvent used is not particularly limited as long as it does not inhibit the reaction, but is preferably a halogenated hydrocarbon such as methylene chloride.
  • the reaction temperature ranges from 120 to 100 ° C., although there is no particular limitation. Apply at
  • reaction time varies depending on the solvent and the reaction time, but the reaction time is 30 minutes in methylene chloride at room temperature.
  • the target compound is neutralized, for example, by appropriately neutralizing the reaction mixture.- If insolubles are present, remove them by filtration, then add an immiscible organic solvent such as water and ethyl acetate, and wash with water. Thereafter, the organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off.
  • an immiscible organic solvent such as water and ethyl acetate
  • the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • the compound (13) obtained in the step 43 etc. is combined with the compound (10a), (10b) or (10c) obtained by removing the DMT group at the 5 'end of the compound (10a).
  • the compound is condensed in the presence of a condensing agent and a deoxidizing agent, and then oxidized.
  • the CPG is cleaved under basic conditions, and at the same time, the protecting group is removed to obtain the compound (1) of the present invention. This is the step of obtaining
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction.
  • anhydrous acetonitril is used.
  • the condensing agent to be used carboxylic acid or acid chloride of phosphoric acid is used, and pivaloyl chloride is preferably used.
  • the oxidizing agent to be used is not particularly limited as long as it is usually used in an oxidation reaction, but is preferably a peracid such as m-mouth perbenzoic acid; or a t-butyl hydroperoxide.
  • Major peroxides iodine-pyridin-water.
  • the deoxidizing agents used include pyridine and dimethylaminopyridine.
  • Such heterocyclic amines, and aliphatic amines such as trimethylamine, triethylamine, and diisopropylethylamine, and preferably aliphatic amines (particularly, diisopropylethylamine).
  • the reaction temperature is not particularly limited, it is generally 50 to 50 ° C., and preferably room temperature.
  • the reaction time varies depending on the starting materials, reagents, temperature and the like to be used, but is usually 5 minutes to 30 hours, and preferably 30 minutes when reacted at room temperature.
  • CPG cleavage and removal of protecting groups in the final step can be carried out by a known method (J. Am. Chem. Soc., 103, 3185, (1981)).
  • the reaction mixture containing the compound of the general formula (1) thus obtained is subjected to ordinary nucleic acid chromatography, such as reversed-phase and ion-exchange chromatography (including high-performance liquid chromatography).
  • the compound having the general formula (1) can be obtained by purification by the same operation as used for the purification of The compound (1) of the present invention and a pharmaceutically acceptable salt thereof exhibit excellent anti-IV-1 activity, and when the compound (1) and a pharmaceutically acceptable salt thereof are used as an anti-AIDS agent, Mixed with excipients, diluents, etc., or as appropriate pharmacologically acceptable, orally by tablets, capsules, granules, powders or syrups, or parenterally by injections, etc. Can be administered.
  • These preparations may be excipients (eg, lactose, sucrose, glucose, mannite, saccharides such as sorbite; corn starch, potato starch, starch such as ⁇ -starch, dextrin, carboxymethyl starch).
  • excipients eg, lactose, sucrose, glucose, mannite, saccharides such as sorbite; corn starch, potato starch, starch such as ⁇ -starch, dextrin, carboxymethyl starch.
  • microcrystalline cellulose, low-substituted hydroxypropylcellulose, hydroxypropinolemethinoresenorelose, canoleboxymethinoresenolle Cellulose derivatives such as oral, carboxymethylcellulose calcium, internal cross-linking power, norboxoxymethylcellulose sodium; arabiagome; dextran; pullulan; light anhydrous silicic acid, synthetic aluminum silicate, magnesium metasilicate aluminum Silicates; Phosphates such as calcium phosphate; Carbonates such as calcium carbonate; Sulfates such as calcium sulfate; binders (eg, the above-mentioned excipients; gelatin; polybutylpyrrolidone; Disintegrants (eg, the above excipients; croscarmellose sodium, carboxymethyl starch sodium, crosslinked polyvinylpyrrolidone, starch, cellulose derivatives, etc.) ), Lubricants (eg, talc; Metal salts of stearic acid such
  • a turbidity agent for example, polysorbate 80, sodium carboxymethylcellulose
  • a diluent for example, water, ethanol, glycerin, etc.
  • 1 to 200 Omg preferably 10 to 100 Omg
  • 1 to 200 Omg per day preferably, 10 to 100 Omg
  • It can also be administered continuously to maintain the concentration of compound (1) required for effective treatment.
  • the base sequence for example, Tgggg
  • the salt form is a triethylamine salt.
  • Example 1 (Exemplified compound 1 in Table 1)
  • the supplied synthesis reagents were connected to a PerkinElmer 392 DNA / RNA synthesizer, and ⁇ / zmol DNA synthesis was used as a program.
  • Example 12b of Japanese Patent Application Laid-Open No. 7-87982 was weighed for 7 ⁇ 0 1 minute, and packed in an empty column for ⁇ ⁇ ⁇ ⁇ .
  • the oligonucleotide protected on the CPG is activated.
  • CPG was removed by filtration, and washed twice with 1 Om of water.
  • reverse phase silica gel column chromatography (Cosmosil 75C18-0PN, 1.515 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; 10 — 50% B; linear gradient; 254 nm) was used.
  • the same post-treatment as in Example 1 was performed to obtain 39 OD (260 nm) of the target compound in an amorphous state.
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetonitrile, 0.1 MTE AA, pH 7.0, B: acetonitrile; 10-60 % B / 20 min; linear gradient; 1 ml / min; 260 nm) eluted at 13.98 min.
  • the sequence was changed to the base sequence of 5GGG6T, and was synthesized in the same manner as in Example 1 using 7 ⁇ mol of modified CPG.
  • the same post-treatment as in Example 1 was performed to obtain an amorphous target compound (173 nm, 260 nm).
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetonitrile, 0.1 MTEAA, pH 7.0, B: acetonitrine); — 60% Bz 20 min; linear gradient; 1 ml / min; 260 nm) eluted at 14.0 1 min.
  • the compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetonitrile, 0.1 MTE AA, pH 7.0, B: acetonitrile; 10- When analyzed by 60% B / 20 min; linear gradient; 1 ml / min; 260 nm), it was eluted at 15.29 min.
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 150 mm; A: 5% acetonitrile, 0.1 M TEAA, pH 7.0, B: acetonitrile; 10— When analyzed by 60% BZ 20 min; linear gradient; 1 ml / min; 260 nm), it was eluted at 13.80 min.
  • the resulting precipitate was collected by filtration, and the precipitate was dissolved in chloroform (400 ml). After washing with water, the organic layer was filtered using IPS filter paper (manufactured by Wattman), and the solvent was distilled off under reduced pressure. . The residue was dissolved in pyridine, evaporated and dried. Pyridine (50 ml) was added and dissolved therein, and diisopropylethinoreamin 2.4 m 1 (13 mmol) and dipheninolone-forced milk milk mouth lid 4.17 g (18 mmol) were added. For 2 hours. The solvent was distilled off under reduced pressure, and methylene chloride (400 ml) was added for dissolution.
  • IPS filter paper manufactured by Wattman
  • Example 7d Approximately 0.1 M solution of the compound of Example 7d in acetonitrile in bottle # 7
  • the nucleotide sequence was changed to 57GGAGT and input, and synthesized in the same manner as in Example 1.
  • reverse-phase silica gel column chromatography Preparative C18, Waters, 1.5 x 11 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; 50% B; linear gradient; 254 nm.
  • the same post-treatment as in Example 1 was carried out to obtain a target compound in the form of amorphous (269 OD, 260 nm).
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate trilue, 0.1 M TEAA, pH 7.0, B: acetate nitrile; 10 — 60% B at 20 min; linear gradient; 1 ml / min; 260 nm) eluted at 14.39 min.
  • Example 8 (Exemplified compound 304 in Table 1) Using a 0.1 M solution of the compound of Example 7d in acetonitrile in bottle # 7, the nucleotide sequence was changed to 5G7GAGT, and the mixture was synthesized in the same manner as in Example 1. However, in the purification, reverse-phase silica gel column chromatography (Preparative C18, Waters, 1.511 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; 50% B; linear gradient; 254 nm). The same post-treatment as in Example 1 was carried out to obtain a target compound in the form of amorphous (275 OD (260 nm)).
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 150 mm; A: 5% acetate nitrile, 0.1 MT EAA, pH 7.0, B: acetate nitrile; 1 It was eluted at 14.49 min when analyzed by 0—60% By 20 min; linear gradient; 1 ml / min; 260 nm).
  • Example 7d Using a 0.1 M solution of the compound of Example 7d in acetonitrile in a bottle # 7, the nucleotide sequence was changed to 5GG7AGT, and input.
  • the synthesis was carried out in the same manner as in Example 1. However, in the purification, reverse-phase silica gel column chromatography (Preparative C18, Waters, 1.5 x 11 cm; A: 50 mM TEAB, pH 7.5, B: acetone nitrile); 50% B; linear gradient; 254 nm).
  • the same post-treatment as in Example 1 was carried out to obtain a target compound in the form of amorphous (225 OD (260 nm)).
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate ditriol, 0.1 MTE AA, pH 7.0, B: acetate nitrile; 10 - 6 0% B / / 2 0 min; 1 4. eluted in 5 2 min and analyzed by 2 6 0 nm); linear gradient ; 1 ml / min.
  • Example 7d Using a 0.1 M solution of the compound of Example 7d in acetonitrile in bottle # 7, the nucleotide sequence was changed to 5GGGA7T, and input.
  • the synthesis was carried out in the same manner as in Example 1. However, in the purification, reverse phase silica gel column chromatography (Preparative C18, Waters, 1.5 x 11 cm; A: 50 m MTEAB, pH 7.5, B: acetonitrile; 15-50% B; linear gradient; 254 nm).
  • the same post-treatment as in Example 1 was carried out to obtain a target compound in the form of amorphous (288 OD (260 nm)).
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 150 mm; A: 5% acetate nitrile, 0.1 M TEAA, pH 7.0, B: acetate nitrile; 10— When analyzed by 60% B / 2Omin; linear gradient; 1 ml / min; 260 nm), it was eluted at 14.52 min.
  • Example 7d Using a 0.1 M acetonitrile solution of the compound of Example 7d in bottle # 7, the nucleotide sequence was changed to 5777AGT, and input.
  • the synthesis was performed in the same manner as in Example 1. However, in the purification, reverse phase silica gel column chromatography (Preparative C18, Waters, 1.5 ⁇ 11 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; 15-50% B; linear gradient; 254 nm) was used.
  • the same post-treatment as in Example 1 was carried out to obtain an amorphous target compound at 1966 OD (260 nm).
  • the compound I is a reversed phase HPLC (Wakosil WS_DNA, 4.6 150 mm; A: 5% acetoethanol, 0.1 M TEAA, pH 7.0, B: acetonitrine; 10—60 When analyzed by B / 2 0 min; linear gradient; 1 ml / min; 260 nm), it was eluted at 14.39 min.
  • Example 12b Using a 0.1 M solution of the acetonitrile solution of the compound of Example 12b in Bottonore # 5, the sequence was changed to 5GGGAGT, and input was performed in the same manner as in Example 1. And synthesized. However, in the purification, reverse-phase silica gel column chromatography (Preparative C18, Waters, 1.5 x 11 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; 15-50 % B; linear gradient; 254 nm). The same post-treatment as in Example 1 was carried out to obtain a target compound in the form of amorphous (251 OD (260 nm)).
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate etryl, 0.1% TEAA, pH 7.0, B: acetone nitrile; 10-6 When eluted with 0% B / 20 min; linear gradient; 1 ml / min; 260 nm;
  • the solvent was distilled off under reduced pressure, the residue was dissolved in 200 mL of ethylene chloride, washed twice with 150 mL of 0.01 N aqueous ammonium chloride solution, and then dried over anhydrous magnesium sulfate.
  • the product was obtained and purified again with the same column (15 g, same solution) to obtain 0.19 g (10%) of the target compound.
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 150 mm; A: 5% acetate ditriol, 0.1 MT EAA, pH 7.0, B: acetate etyr; 10— When analyzed by 60% B / 20 min; linear gradient; 1 ml / min; 260 nm), it was eluted at 17.03 min.
  • the aqueous layer was back-extracted with a black hole form, the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure.
  • Example 14a Compound 1.Dissolve 1 l (4 mmol) in 8 m of tetrahydrofuran, add 230 mg (8 mmol) of 60% Nali under nitrogen atmosphere, and stir at 60 ° C for 2 hours. did. After returning to room temperature, a solution of 1.69 g (5 mmol) of 3,4- (dibenzyloxy) benzyl chloride in 2.5 mL of tetrahydrofuran was added dropwise, and 374 mg of sodium iodide (2.5 mmol) was added. ) was added and the mixture was stirred at room temperature.
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetateton) Ril, 0.1 MT EAA, pH 7.0, B: Acetonitrile; 10-60% B '20 min; linear gradient; 1 ml / min; 260 nm) It was eluted at 14.92 min.
  • Example 12b Using a 0.1 M solution of the acetonitrile solution of the compound of Example 12b in bottle # 5, the nucleotide sequence was changed to 5GGGGT and synthesized in the same manner as in Example 1. However, in the purification, reverse phase silica gel column chromatography (Preparative C18, Waters, 1.511 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; 50% B; linear gradient; 254 nm). The same post-treatment as in Example 1 was carried out to obtain the desired compound in an amorphous state at 248 OD (260 nm).
  • This compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate nitrile, 0.1 MT EAA, pH 7.0, B: acetate nitrile; 10- When analyzed by 60% B / 20 rn in; linear gradient; 1 ml / min; 260 nm), it was eluted at 15.59 min.
  • Acetonitrile, 0.1 MTE AA, pH 7.0, B Acetonitrile; When analyzed by 1 0 — 60% B / 20 min; linear gradient; 1 ml / min; 260 nm), it was eluted at 15.43 min.
  • the compound was analyzed by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate ditriol, 0.1 M EEA, pH 7.0, B: acetate ethl; When analyzed by 1 0—D 0% B / 20 m 1 n; linear gradient; 1 ml / min; 260 nm), it was eluted at 15.41 min.
  • Example 18 (Example compound 4 4 1 in Table 1) 05 Approximately 0.1 M acetonitrile solution of the compound of Example 12b in bottle # 5, approximately 0.1 M in bottle # 6, approximately 0.1 M of the compound of Example 16a in bottle # 7, approximately 0.1 M in bottle # 7
  • the compound was synthesized in the same manner as in Example 1 except that the base sequence was changed to 57776GT using an acetonitrile solution of the compound of Example 7d.
  • reverse phase silica gel column chromatography Preparative 18, Waters, 1.511 cm; A: 50 mM MT EAB, pH 7.5, B: acetonitrile 15-50% B; 1 inear gradient; 254 nm
  • Example 2 The same post-treatment as in Example 1 was carried out to obtain a target compound in the form of amorphous (170 OD (260 nm)).
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate nitrile, 0.1 MT EAA, pH 7.0, B: acetate nitrile; 10 — 60% B / 20 min; 1 inear gradient; 1 ml / min; 260 nm) eluted at 15.45 min.
  • This compound was obtained by reverse-phase HPLC (Wakosil WS-DNA, 4.6 x 150 mm; A: 5% acetate nitrile, 0.1 MTE AA, pH 7.0, B: acetate nitrile; 10 When analyzed by -60% B / 20 min; linear gradient; 1 ml / rain; 260 nm), it was eluted at 15.81 min.
  • Example 2 The same procedure as in Example 1 was carried out except that the base sequence of 5GGGT was changed to about 0.1 M of an acetonitrile solution of the compound of Example 12b in bottle # 5.
  • reverse-phase silica gel column chromatography Preparative C18, Waters, 1.5 x 11 cm; A: 50 mM TEAB, pI-17.5, B: acetonitrile
  • Ril 15-50% B; linear gradient; 254 nm
  • the same post-treatment as in Example 1 was performed to obtain 210 OD (26 Onm) of the amorphous target compound.
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 x 0 mm; A: 5% acetate nitrile, 0.1 MT EAA, pH 7.0, B: acetate nitrile; 10 — 60% BZ 20 min; linear gradient; 1 ral / min; 260 nm) It was eluted at 15.76 minutes.
  • Example 7d Using a 0.1 M solution of the compound of Example 7d in acetonitrile in bottle # 7, the nucleotide sequence was changed to 5777T, and synthesis was performed in the same manner as in Example 1. However, in the purification, reverse-phase silica gel column chromatography (Preparative C18, Waters, 1.5 x 11 cm; A: 50 mM TEAB, pH 7.5, B: acetonitrile; — 50% B; linear gradient; 254 nm) was used. The same post-treatment as in Example 1 was performed to obtain 98 OD (260 nm) of the target compound in an amorphous state.
  • This compound was obtained by reversed-phase HPLC (Wakosil WS-DNA, 4.6 ⁇ 150 mm; A: 5 ⁇ acetonitrile, 0.1 MTE AA, pH 7.0, B: acetonitrile) When analyzed with 10-60% B / 20 min; linear gradient; 1 ml / min; 260 nm; it was eluted at 15.20 min.
  • Unit capsules were prepared by filling each of the standard bisected hard gelatin capsules with 100 mg of the powdered Example 11 compound, 150 mg lactose, 50 mg cellulose and 6 mg magnesium stearate. Manufacture, wash and dry. (Formulation Example 5) Soft capsule
  • Anti-HIV-1 activity was measured by the method of Pawel et al. (R. Pauel et al., J. Virology Methods 20, 309-321 (1988)). That is, the MT- 4 cells in the logarithmic growth phase were centrifuged for 5 min at 0.99 X g, a concentration of the resulting After the cell pellets were suspended in a medium HIV- 1 ( ⁇ ⁇ type) to 10 CCID 5 Q Infected for 1 hour at 37 ° C. Thereafter, HIV-1 infected MT-4 cells were obtained by centrifugation and washing with RPMI-1640 medium containing 10% fetal calf serum (hereinafter referred to as "serum medium").
  • HIV-1 infected MT-4 cells and HIV-1 uninfected MT-4 cells were each suspended in a serum medium so as to be 4 ⁇ 10 5 cells / ml.
  • a serum medium so as to be 4 ⁇ 10 5 cells / ml.
  • Into a 96-well plastic microtiter plate add 100 ⁇ l of the test compound solution (dissolved in serum medium) serially diluted in advance to each well, and then add 100 ⁇ l of the above cell suspension to each well. The mixture was added, and culturing was carried out for 6 days in the presence of 5% carbon dioxide.
  • HIV-1 infected ⁇ -4 cells supplemented with the test compound and no test compound
  • the added ⁇ IV-1 non-infected MT-4 cells were cultured.
  • the number of viable cells was measured based on the MTT (3- (4,5-dimethylthiazole-2-yl) -2, 5-diphenyltetrazolium bromide) method (LM Green et al., J. Immunol. Methods , 70, 257-268 (1984)), to determine the cytotoxic activity of HIV-1.
  • the cytotoxic activity of HIV-1 infected MT-4 cells without sample compound addition is defined as 100%, and the cytotoxic activity of HIV-1 (IIIB) uninfected MT-4 cells without sample compound is defined as 0%.
  • the concentration (IC 50 ) of a sample capable of suppressing the cytotoxic activity of MT-4 cells infected with HIV-1 by 50 % was determined.
  • a concentration (CC 5 ) that inhibits the proliferation of HIV-1 non-infected MT-4 cells by 50% was determined. The results of these measurements are shown in Table 3 or Table 4.
  • the 2-N-methyl-2'-deoxyguanosine derivative represented by the general formula (2) of the present invention is useful for synthesizing a 2-N-methylguanosine-containing nucleotide.

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Abstract

Cette invention concerne de nouveaux oligodesoxynucléotides modifiés présentant une excellente activité contre le virus de l'immunodéficience humaine. De manière plus spécifique cette invention concerne des composés représentés par la formule (1) ou des sels pharmacologiquement accceptables de ces derniers. Dans la formule B1, B2, et B3 sont identiques ou différents et chacun représente A, G, C, T, a, g, c, t, M, X, etc.; m représente un entier compris entre 0 et 7; S1 et S2 représentent chacun hydrogène, alkyle, alcoxy ou halogéno; z représente un entier compris entre 0 et 9; dans les m répétitions de B2, les B2 peuvent être identiques ou différents; a, g, c et t représentent respectivement A, G, C et T qui sont chacun liés au niveau de l'extrémité 3'; M représente 2-N-méthyle-G; et X représente 2'-méthoxy-G.
PCT/JP1998/004863 1997-10-27 1998-10-27 Oligodesoxyribonucleotides contenant un nucleoside modifie et autre Ceased WO1999021874A1 (fr)

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JP9/293821 1997-10-27
JP29382197 1997-10-27

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Publication number Priority date Publication date Assignee Title
WO2006093157A1 (fr) * 2005-02-28 2006-09-08 Tokyo Institute Of Technology Derive oligonucleotidique, sonde de detection du gene, et puce a adn
US7851157B1 (en) 2005-02-28 2010-12-14 Tokyo Institute Of Technology Oligonucleotide derivative, probe for detection of gene, and DNA chip

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JPH11199597A (ja) 1999-07-27

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