WO1999019474A1 - Oligodesoxyribonucleotides modifies comportant la sequence tggg - Google Patents

Oligodesoxyribonucleotides modifies comportant la sequence tggg Download PDF

Info

Publication number
WO1999019474A1
WO1999019474A1 PCT/JP1998/004625 JP9804625W WO9919474A1 WO 1999019474 A1 WO1999019474 A1 WO 1999019474A1 JP 9804625 W JP9804625 W JP 9804625W WO 9919474 A1 WO9919474 A1 WO 9919474A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
compound
ch2ch2
meoph
dbp
Prior art date
Application number
PCT/JP1998/004625
Other languages
English (en)
Japanese (ja)
Inventor
Makoto Koizumi
Masakatsu Kaneko
Toshinori Ohmine
Hidehiko Furukawa
Takashi Nishigaki
Original Assignee
Sankyo Company, Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sankyo Company, Limited filed Critical Sankyo Company, Limited
Priority to AU94608/98A priority Critical patent/AU9460898A/en
Publication of WO1999019474A1 publication Critical patent/WO1999019474A1/fr

Links

Classifications

    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/311—Phosphotriesters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/314—Phosphoramidates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/314—Phosphoramidates
    • C12N2310/3145—Phosphoramidates with the nitrogen in 3' or 5'-position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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 oligodeoxyribonucleotide having an excellent anti-AIDS virus action.
  • BACKGROUND ART Oligodeoxyribonucleotides having a sequence complementary to a certain gene (hereinafter referred to as antisense oligodeoxyribonucleotide) are known to inhibit the functional expression of the gene. It has also been reported that antisense oligodeoxyribonucleotides of the viral gene or oncogene can inhibit viral replication or cell growth, respectively, by inhibiting the function of that gene. Natl. Acad. Sci. USA, Vol. 75, No. 1, page 280 (1977), and P. Zamecnik, J. Goodchild, Y. Taguchi, Sarin, Proc (PC Zamecnik, ML Stephenson, Proc. Natl. Natl. Acad. Sci. USA, Vol. 83, No. 6, 4143 (1986)).
  • the antisense oligodeoxyribonucleotide In order for the antisense oligodeoxyribonucleotide to exhibit the above-mentioned inhibitory action, it must form a stable hybrid with the target RNA or DNA in vivo. In order to form this hybrid, it is said that the antisense oligodeoxyribonucleotide needs to have a length of at least about 15 nucleotides or more. Had been.
  • the present inventors have proposed various nucleotide sequences and 5′-terminal and 3′-terminal oligo-nucleotide ribonucleotides, which have been considered to have no specific inhibitory activity.
  • modified oligodeoxyribonucleotides having a constant nucleotide sequence have a remarkably high anti-AIDS virus activity, and Found that the compound has low toxicity to normal cells, was relatively easy to synthesize, and was practically useful, and disclosed the compound described in Japanese Patent Laid-Open Publication No. 7-87982.
  • the present invention also includes a pharmacologically acceptable salt thereof.
  • Q represents a RiRsRsZ group (wherein, R, R 2 and R 3 may be the same or different and may have a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a substituent.
  • Z represents C or Si; or R 2 , R 3 and Z together form a fluorenyl group or a xanthyl group.
  • R 4 has a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a substituent, an aryl group or a substituent which may have a substituent.
  • ODN oligodeoxyribonucleotide
  • B the hydroxyl groups at the 5 'end and 3' end of each oligodeoxyribonucleotide are not included. The left end of each sequence is the 5 'end, and the right end is the 3' end.
  • R 2 , R 3 and R 4 are each a hydrogen atom
  • X is an ethylene group.
  • Y or ⁇ 2 , ⁇ 3 and ⁇ 4 are each an oxygen atom, ⁇ is a carbon atom, m is 1 to 3, and n is 1.
  • B is TGGGAG, is a 3,41- (dibenzyloxy) phenyl group
  • R 2 , R 3 and R 4 are each a hydrogen atom
  • X is an ethylene group
  • ⁇ 2 Upsilon 3 and Upsilon 4 are each an oxygen atom
  • m is 2 to 3
  • n is 1.
  • the present invention also relates to a drug, particularly an anti-AIDS agent, comprising the compound (1) or a pharmacologically acceptable salt thereof as an active ingredient.
  • examples of the anthraquinonyl group which may have a substituent of R 2 and R 3 in the Q group include, for example, 9,10-anthraquinone-11-yl, 9, Unsubstituted anthraquinonyl groups such as 10-anthraquinone-2-yl; such as 9,10-anthraquinone-1-4-methyl-1-yl, 9,10-anthraquinone-1-6-ethyl-2-yl An alkyl-substituted anthraquinonyl group having 1 to 4 carbon atoms; 1 to 4 carbon atoms such as 9,10-anthraquinone-5-methoxy-11-yl, 9,10-anthraquinone-18-ethoxy-2-yl 9 alkoxy-substituted anthraquinol groups; 9,10-anthraquinone-7-chloro-1-yl, 9,10-anthraquinone-18-flu
  • examples of the alkyl group having 1 to 4 carbon atoms which may have a substituent of R 4 include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, Unsubstituted alkyl groups such as isobutyl, s-butyl, and t-butyl; amino-substituted alkyl groups such as 2-aminoethyl and 3-aminopropyl; carbons such as 2-methoxyethoxy and 3-methoxypropyl Examples thereof include an alkoxy-substituted alkyl group having 1 to 4 alkoxy groups, preferably, methyl, ethyl, n-propyl, 2-aminoethyl, and 2-methoxethyl groups.
  • examples of the aryl group which may have a substituent of R 4 include, for example, phenyl group; alkyl having 1 to 4 carbon atoms such as 2-methylphenyl and 3-ethylphenyl. Substituted phenyl group; 2-phenylphenol, 2-phenylphenyl, 4-chlorophenyl, 2-bromophenyl, 2-phenylphenyl, etc.
  • Gen-substituted phenyl group A 2-substituted phenyl group such as 2-nitrophenyl, 4-nitrophenol; an alkoxy-substituted phenyl group such as 4-methoxyphenyl or 4-ethoxyphenyl; An alkylthio-substituted phenyl group having 1 to 4 carbon atoms such as 4-methylthiophene and 4-ethylthiophenyl; a naphthyl group; a phenanthrenyl group; an anthracenyl group; and a pyrenyl group. Examples include phenyl, halogen-substituted phenyl, and nitrogen-substituted phenyl groups.
  • examples of the aralkyl group which may have a substituent for R 4 include, for example, a benzyl group; an alkyl-substituted benzyl group having 1 to 4 carbon atoms such as methylbenzyl and ethylbenzyl; Methoxyben Alkoxy-substituted benzinole groups having 1 to 4 carbon atoms, such as ethoxybenzyl; halogen-substituted benzinole groups, such as phenoleno benzone, chlorobenzenole, and bulomobenzinole; chloronaphthylmethyl Halogen-substituted naphthylmethyl group such as phenol; indulmethyl group; phenanthrenylmethyl group; anthracenylmethyl group; diphenylmethyl group; triphenylmethyl group; 1-phenylene, 2-phenethyl, 2,2-diphenylethylenol, Mono-
  • examples of the alkylene having 1 to 4 carbon atoms of Y 2 include methylene, ethylene, propylene, tetramethylene, and pentamethylene. And methylene, preferably methylene.
  • each of Y, Y 3 and Y 4 is preferably an oxygen atom.
  • ⁇ 2 is preferably an oxygen or sulfur atom.
  • ⁇ in the Q group is preferably a carbon atom.
  • examples of the linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a hydroxyl group of X include, for example, methylene, methylmethylene, ethylene, Propylene, tetramethylene, methinoleethylene, ⁇ -methinoletrimethylene, 2-methinoletrimethylene, 2-methyltetramethylene, 3-methyltrimethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, Examples thereof include nonamethylene, decamethylene, 2-hydroxytrimethylene, and 2-hydroxytramethylene, and preferably methylene, methylmethylene, ethylene, propylene, and methylethylene.
  • m is preferably 0 to 6, and more preferably 0 to 4.
  • m is preferably 1 to 3
  • B is TGGGAG
  • m is preferably 2 to 3.
  • n is preferably 0 to 6, and more preferably 1.
  • the sequence of B TG GG GT, TGG GGTT, TG GG GGT, TGG G CG, T GG GT G, T GG GG, TG GG GG or TG GGAG is selected from the group consisting of oligodeoxyribonucleotides. Preferred among them are the sequences TGGGGT, TGGGGGTT, TGGGGGT, TGGGGG, TGGGGG or TGGGGG.
  • A is adenine deoxyribonucleotide
  • G is guanine deoxyribonucleotide
  • C is cytosine deoxyribonucleotide
  • T is thymine deoxyribonucleotide. Shows Reotide.
  • the left end is the 5 'end and the right end is the 3' end. However, the hydroxyl groups at the 5 'end and 3' end of each oligodeoxylyl nucleotide are not included.
  • preferred groups at the 5 ′ end (Ri Rs Rs Z—Yi) obtained by combining R 2 , R 3 , and Z include triphenylmethyloxy, 3, 4- (dibenzinoleoxy) benzoyloxy, 3, 5- (dibenzyloxy) benzyloxy, t-butylinolethiophenolenosilyloxy, phenylenoleoleoni / reoxy, and phenylxanthenyloxy, More preferably, they are triphenylmethinoleoxy, 3,4- (dibenzyloxy) benzyloxy, 3,5- (dibenzyloxy) benzyloxy group or 3,5-bis [3,5- (dibenzyloxy) benzyloxy] benzyloxy group. Is a 3,4- (dibenzyloxy) benzyloxy group.
  • a suitable group at the 3 ′ terminal obtained by a combination of R 4 , X, Y 2 , ⁇ 3 , ⁇ 4 , m and ⁇ ([P (O) (Y 2 R 4 ) I Y 3 — (X- ⁇ 4 ) n ] m ⁇ ) includes hydrogen ( ⁇ ), methyl phosphoryl [P (0) (0CH 3 ) 0 H], 2 — phenylphosphoryl [ P (0) (0-2-C1- Ph) 0H], (O- methyl) Chiohosuhoriru [P (0) (0CH 3 ) SH], Mechiruhosu Honiru [P (0) (CH 3 ) 0H], main switch Luthiophosphonyl [P (0) (CH 3 ) SH], Phenylphosphonyl [P (0) (Ph) OH], 2 — Hydroxicetyl phosphoryl [P (0) (0H) 0CH 2 CH 2 0H] , [O— (2-hydroxy
  • preferred compounds include
  • B is an oligodeoxyribonucleotide selected from the sequence TGGGGT, TGGGGGTT, TGGGGGT, TGGGGG, TGGGGG or TGGGGG;
  • the 5 ′ terminal group (R i Rs Rg Z—Y i) is a triphenylmethyloxy, 3,4- (dibenzyloxy) benzyloxy, 3,5- (dibenzyloxy) benzyloxy group, and R 4 , X, Y 2 3 ′ terminal group ([P (0) (Y 2 R 4 ) — — 3 — (X—Y 4 ) n ] m H) force 'hydrogen obtained by the combination of, Y 3 , Y 4 , m and ⁇ , Methinole phosphoryl, 2-black mouth phenole phosphorinole,
  • B is an oligodoxyribonucleotide selected from the sequence TGGG GT, TG GGGTT or TGG GGGT Compound,
  • B represents oligodeoxyribonucleotide represented by the sequence TGGGG or TGGGGG, and m represents 1, 2 or 3.
  • G is guanine deoxyribonucleotide
  • T represents thymine deoxyribonucleotide.
  • the hydroxyl groups at the 5 'end and 3' end of each oligodeoxyribonucleotide are not included. The left end of each sequence is the 5 'end, and the right end is the 3' end.
  • B represents oligodeoxyribonucleotide represented by the sequence TGGGGAG, and m represents 2 or 3.
  • A indicates adenine deoxyribonucleotide
  • G indicates guaendeoxy ribonucleotide
  • T indicates thymine deoxyribonucleotide.
  • the hydroxyl groups at the 5 'end and the 3' end of the oligodeoxyribonucleotide are not included. The left end of the sequence is the 5 'end and the right end is the 3' end.
  • the compound having the general formula (1) of the present invention can be used in the form of a “pharmaceutically acceptable salt”.
  • a “pharmaceutically acceptable salt” examples include sodium and potassium salts.
  • Inorganic or organic salts such as alkaline metals such as calcium; alkaline earth metals such as calcium; ammonia; basic amino acids such as lysine and arginine; alkylamines such as triethylamine;
  • it is an alkali metal salt such as sodium or 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.
  • the present invention also includes hydrates and hydrates. 99/19474
  • Me is methyl
  • Et is ethyl
  • Pr is propyl
  • Bu is butyl
  • tBu is t-butyl
  • 3,4-DBP is 3,4-mono (dibenzyloxy) fuel
  • 3, 5-BDBBP is 3,5-bis ⁇ 3,5- (dibenzyloxy) benzyloxy ⁇ phenyl
  • 3,5-DBP is 3,5- (dibenzyloxy) phenyl
  • 1-PYR is pyrene-1-yl
  • 2-NAP is naphthalene-1-yl
  • 4-PhPh is 4- (phenyl) phenyl
  • 2-PhPh is 2- (phenyl) phenyl
  • 9-ANT is anthracene-9-yl
  • 2-ANT is anthracene-1-yl
  • 1-NAP is naphthalene-1-yl
  • 2-ANQ is anthraquinone-12-yl
  • 4-PHE is phenant
  • PhE is 1,4-phenylene
  • 2-NH2Et is 2-aminoethyl
  • 2-MeOEt is 2-methoxethyl
  • 1-Me-PrE is 1-methylpropyl.
  • Pyrene 2-Me-BuE is 2-methylbutylene
  • 2-0H-PrE is 2-hydroxypropylene
  • 1 is TGGGGT
  • 2 is TGGG GTT
  • 4 is TGGGCG
  • 5 is TGGGTG
  • 6 indicates TGGGG
  • 7 indicates TGGGGG
  • 8 indicates TGGG AG.
  • preferred compounds include 1 to 33, 32 27 to 39, 65 3 to 68, 979 to 1011, and 1089 to
  • the compounds of 121, 119, 1205, 121 and 122 to 123 can be cited.
  • preferred compounds include 1 to 11, 1, 327 to 337, 653 to 663, 979 to 989, 1089 to 1099, 1 Compounds of 199, 125, 1221, 122, and 123 to 123 can be mentioned.
  • the compound of the general formula (1) according to the present invention comprises the following compound (2) produced by the following methods A-1 and A_2
  • DMT-o-F-i w 4a (6a) DMT-o-F-w 5b (7b) de-DMT (4,4'-dimethoxytrityl) is used as a raw material, and the following C-1, It can be manufactured by the methods shown in C1-2 and C-3.
  • T r is a trityl group (T r), which is generally used to specifically protect the primary hydroxyl group of a nucleoside, a 4-monomethoxytrityl (MMT) group, and a 4,4′-dimethoxytrityl (DMT) group.
  • MMT 4-monomethoxytrityl
  • DMT 4,4′-dimethoxytrityl
  • a 2 is tert-butyldimethylsilyl (TB DMS) groups and triisopropylate Rushiriru (TIPS) good UNA trisubstituted silyl group group, Application Benefits Kuroroe Toki Shikaruboeru (T roc) Application Benefits Nono Rogenoe Tokishikarubo two Le groups such as An aralkyloxycarbonyl group such as a benzyloxycarbonyl (Z) group;
  • D ' represents a thymine base or a protected thymine base
  • D ′′ represents the base moiety of the nucleotide at the 3′-terminal where the amino group is protected with the acryl group
  • D "" indicates the base of any nucleotides used in DNA synthesis, and indicates a base selected from adene, guanine, cytosine and thymine or a corresponding protected base;
  • F is the oligonucleotide moiety excluding the nucleotide at the 5 'end of the desired nucleotide sequence, which is commonly used in DNA synthesis, and is protected by a protecting group on the phosphoric acid moiety. Indicates the oligonucleotide (including the 5'-terminal hydroxyl group of the 5'-terminal nucleoside and the 3'-position hydroxyl group of the 3'-terminal nucleoside);
  • 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 Step 39, particularly, an orthochlorophenoxy group).
  • Group); U is an amino group in the amidite portion (particularly a complex having one or two oxygen atoms and / or nitrogen atoms in the ring, such as a dialkylamino group such as a dimethylamino group or a diisopropylamino group, and a morpholino group).
  • Wi, W 2a , W 2 , W 3 , W, W 4 , W 5 a or W 5 b are obtained by converting the oligonucleotide F from the CPG of the final target compound in the method shown in B-1 to B-5. '3' of the terminal part up to the oxygen atom of the hydroxyl group;
  • E represents a hydrogen atom or an optionally protected hydroxyl or amino group
  • K represents an oxygen or sulfur atom
  • DMT represents 4, 4'-dimethoxytrityl group
  • H a 1 is (preferably, a chlorine or bromine atom) a halogen atom indicates;
  • R 5 is methyl, Echiru, propyl, butyl, phenyl, main butoxy, E butoxy, Purobokishi, butoxy, is Xia Roh ethyl O alkoxy or substituted Phenyloxy group (preferably unsubstituted).
  • Methods A-1 and A-2 are methods for producing compound (2), which is an important intermediate, for producing compound (1).
  • Method B-1 is based on a nucleotide having a protecting group at the 5 'terminal
  • the 3 'terminal nucleoside is linked to a controlled' pore glass' (CPG) via a linker as a starting material, and is a commercially available nucleoside reagent for DNA synthesis on a DNA synthesizer.
  • CPG controlled' pore glass'
  • DMT group a trimethyl group
  • One of the hydroxyl groups is condensed with a dicarboxylic anhydride to give succinic acid half ester (indicated by succinic acid as a representative of dicarboxylic acid in the scheme), and the carboxyl group of the ester and CPG
  • the CPG carrier (416) obtained by binding the amino group and removing the terminal DMT group is used to produce nucleotides on a DNA synthesizer using a nucleotide unit in the usual manner.
  • a nucleotide reagent capable of forming a phosphorothioate bond is reacted, and the nucleotide is then sequentially extended using a nucleotide unit in the usual manner. It is also the desired Origonuku Reochido Yori one less O Li Gonuku Reochido compound bound before (4 one b) Method of manufacturing.
  • a commercially available compound (5-2) is obtained from a CPG carrier (5-1) (that is, (4-1-6)) in which an alkylene diol and CPG are bonded via a dicarboxylic acid as a raw material.
  • a CPG carrier (5-1) that is, (4-1-6)
  • an alkylene diol and CPG are bonded via a dicarboxylic acid as a raw material.
  • one hydroxyl group of the diol compound (6-1) is converted to a compound (6-2) obtained by DMT, and then the other hydroxyl group is mixed with an amidating reagent (6-3 '). Reacting the compound (6-3) to obtain the compound (6-3).
  • the compound (6-2) is bound to CPG via dicarboxylic acid, and the CPG carrier is obtained. (6-6), and using the CPG carrier (6-7) obtained by removing the DMT group as a raw material.
  • the nucleotides are sequentially extended in a usual manner, and the compound is bound to a position before the desired oligonucleotide, which is one less than the desired oligonucleotide ( 6).
  • a ) is produced by reacting the compound (6-3) with the CPG carrier (6-7) once, twice or three times on a DNA synthesizer.
  • the product (6-9), (6-10) or (6-11) is a raw material, the nucleotide is sequentially extended by a usual method using a nucleotide unit to obtain a desired product. This is a method for producing a compound (6b), (6c) or (6d), which is bound to one oligonucleotide less than the oligonucleotide.
  • the B-5 method is based on the removal of the DMT group of a commercially available protected 2'-deoxynucleoside-linked CPG (hereinafter referred to as D '' '-CPG) by the linker (7- (Tetrahedron Letters, 27, 475 (19986))) (2-cyanoethoxy) one [2— (2'—O one) 4,4'-Dimethoxytrityloxetylsulfonyl) ethoxy]-(N, N-diisopropylamino) phosphine (7-1) to produce compound (7-3) Then, a compound obtained by removing the DMT group of the compound (7-3) and a compound separately synthesized by a conventional method
  • the compound (2) is reacted with a phosphating agent to prepare a 3′-phosphorous acid derivative (8), and the compound (3) synthesized by the methods B-1 to B-5 is used. ), (4—a), (4—b), (5), (6a), (6b),
  • the C-12 method is based on the compound (2), which is prepared by reacting a phosphorylating agent with a 3, monophosphate derivative (9), and the compounds (3), (4) synthesized by the methods B-1 to B-5.
  • a), (4-1b), (5), (6a), (6b), (6c), (6d), (7a), or (7b) with the DMT group removed This is a method for producing the compound (1) of the present invention by condensing the compound and on a DNA synthesizer, cleaving the bond with CPG, and finally removing the protecting group.
  • the C-13 method comprises a compound (10) in which a phosphonic acid group is introduced at the 3′-position of the compound (2), and a compound (3), (4—a) synthesized by the methods B-1 to B-5. ), (4-b), (5), (6a), (6b), (6c), (6d), (7a), and the corresponding compound from which the DMT group has been removed.
  • a compound (10) in which a phosphonic acid group is introduced at the 3′-position of the compound (2) and a compound (3), (4—a) synthesized by the methods B-1 to B-5.
  • (4-b), (5), (6a), (6b), (6c), (6d), (7a) are condensed and then oxidized to form a phosphodiester bond, further cleave the bond with CPG, and finally, remove the protecting group to obtain the compound (1) of the present invention. Is the way.
  • This step includes, in an inert solvent, the step of protecting compound (2-1) ⁇ wherein the base moiety is A, G or C in the presence of A, G or C, for protecting an existing amino group from acylation.
  • the protection step can be easily performed by a known method (J. Am. Chetn. Soc., 104, 1316, (1982)).
  • a lower aliphatic acyl or an aromatic acyl is generally used.
  • the lower aliphatic acyl used includes, for example, formyl, acetinole, propiole, butyryl, isobutylinole, pentanoyl, pivaloyl, valeryl, isovaleryl, and the like.
  • the benzyl include benzoyl, 4-acetoxybenzoyl, 4-methoxybenzoyl, 4-methylbenzoyl, and 11-naphthyl.
  • the base moiety is A or C
  • a compound (2-2) in which only the hydroxyl group at the 5′-position is selectively protected is produced by reacting a hydroxyl-protecting reagent. '
  • the solvent used is preferably an aromatic hydrocarbon such as benzene, toluene or xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, or cyclobenzene. And halogenated hydrocarbons such as dichlorobenzene; esters such as engineered formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisopropyl propylether, tetrahydrofuran, dioxane, and the like.
  • aromatic hydrocarbon such as benzene, toluene or xylene
  • halogenated hydrocarbons such as dichlorobenzene
  • esters such as engineered formate, ethyl acetate, propyl acetate
  • Ethers such as dimethoxetane and diethylene glycol dimethyl ether; ketones such as acetone, methylethylketone, methylisobutylketone, isofolone and cyclohexanone; nitrotroethane, nitrite Nitro compounds such as benzene; acetonitrile, a Buchiroyu Tritoles such as trinole; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, hexamethylphosphorotriamide; dimethylsulfoxide Sulfolane, sulfolane; aliphatic tertiary amines such as trimethylamine, triethylamine, N-methylmorpholine; aromatic amines, such as pyridine and picolin. More preferred are halogenated hydrocarbons (particularly methylene chloride) and amides (particularly DMF).
  • the protecting reagent used is not particularly limited as long as it can selectively protect only at the 5'-position and can be removed under acidic and neutral conditions.
  • Trimethyl chlorides such as lithichloride, monomethoxytrityl chloride, and dimethoxytrityl chloride.
  • a base is usually used.
  • the base to be used may be a heterocyclic amine such as pyridine, dimethylaminopyridine, pyrrolidinopyridine, or an aliphatic tertiary amine such as trimethylamine or triethylamine.
  • Amines are preferred, and pyridine, dimethylaminopyridine and pyrrolidinopyridine are preferred.
  • 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.
  • reaction solution is poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, and the like, and the solvent is distilled off from the extract to obtain the desired product (2-2). And usually the next Used in the step. 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
  • compound (2-2) is reacted with a hydroxyl-protecting reagent in an inert solvent to produce compound (2-3).
  • the solvent used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, or dichloromethane.
  • Halogenated hydrocarbons such as chlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; getyl ether, diisopropyl ether, tetrahydrofuran, dioxane, and dimethoxetane
  • Ethenes such as diethylene glycol dimethinoleone; ketones such as acetone, methylethylketone, methylisobutylketone, isofolone and cyclohexanone; nitroethane, nitrobenzene
  • Nitro compounds such as: acetonitrile, Butyramides such as soptilonitrile; amides such as formamide, dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide; and sulfoxides such as dimethylsulfoxide and sulfolane. More preferably, ethers (
  • the protecting reagent to be used is not particularly limited as long as it can be deprotected in distinction from the protecting group at the 5'-position.
  • t-butyldimethylsilyl chloride is used.
  • Certain compounds include silyl halides such as triisoprovir silyl chloride, haloalkoxycarbon halides such as triethoxy ethoxycarbonyl chloride, and benzyloxy canoleles. Examples include aralkyloxycarbonyl halides such as bojyl chloride.
  • a base is usually used.
  • the base used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.).
  • the reaction temperature is usually from 120 to 150 ° C, preferably from ⁇ 10 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 poured into water, extracted with a water-immiscible solvent, for example, benzene, ether, ethyl acetate, or the like. 3) 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.
  • a water-immiscible solvent for example, benzene, ether, ethyl acetate, or the like. 3
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction.
  • aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform and tetrachloride Halogenated hydrocarbons such as carbon, dichloroethane, cyclobenzene, and dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and ethyl carbonate Esters such as tyl; ethers such as getyl ether, disopropyl ether, tetrahydrofuran, dioxane, dimethoxetane, and methyl dimethyl ether; methanol, ethanol, n -Prono.
  • Alcohols such as ethanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, methinolace Ketones such as acetone, methylethylketone, methylisobutylketone, isophorone and cyclohexanone; two-mouth compounds such as nitroethane and nitrobenzene; acetonitrile, isobutyroni Ditolyls, such as tolyl; amides, such as formamide, dimethylformamide, dimethylacetamide, hexamethylphosphotriamide; sulfoxides, such as dimethylsulfoxide and sulfolane; More preferably, alcohols ( Methanol, ethanol) or if you methylene chloride and deprotection reagent using acetic acid is a mixture of acetic acid
  • the deprotecting reagent to be used is not particularly limited as long as it is a commonly used deprotecting reagent. If the protecting group is a triarylmethyl group, for example, acetic acid, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid And Lewis acids such as zinc bromide, preferably acetic acid, dichloroacetic acid and trifluoroacetic acid.
  • the reaction temperature varies depending on the used reagents, raw materials, solvents and the like, but is usually from 10 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 reaction solution is neutralized with a base such as pyridine, poured into water, extracted with a water-immiscible solvent such as benzene, ether, ethyl acetate, etc., and the solvent is distilled off from the extract.
  • a base such as pyridine
  • a water-immiscible solvent such as benzene, ether, ethyl acetate, etc.
  • the product (2-4) 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.
  • the desired compounds (4_6), (6-7) and (6-9) can be collected by filtration, and an organic solvent such as methylene chloride can be used. After washing with, use it as it is in the next step.
  • compound (2-5) is reacted with compound (2-1) or (2-4) in an inert solvent in the presence of a base to produce compound (2) or (216)
  • the solvent used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; a halogen such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, or dichlorobenzene.
  • aromatic hydrocarbon such as benzene, toluene, or xylene
  • a halogen such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, cyclobenzene, or dichlorobenzene.
  • Esters such as ethyl ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and getyl carbonate; dimethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethyl oxetane, diethylene glycol dimethynoate ether Ethers; ketones such as acetone, methylethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane, nitrobenzene; acetonitrile; Issobutyroni Tril Nitriles; amides such as formamide, dimethylformamide, dimethylacetamide and hexamethylphosphorotriamide; and sulfoxides such as dimethylsulfoxide and sulfolane.
  • Ethers especially tetrahydrofuran
  • ketones especially acetone
  • halogenated hydrocarbons especially methylene chloride
  • amides especially dimethylformamide
  • aromatic amines especially , Pyridine
  • the base used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, etc.), an alkali metal hydride (particularly, sodium hydride), and an alkali metal carbonate. Salts (particularly sodium carbonate and lithium carbonate) are preferred.
  • Z in the compound (2-5) is a silicon atom
  • an organic base such as imidazole is most preferred.
  • reaction temperature is not particularly limited, it is generally 0 to 100 ° C, 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.
  • 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 the compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off to obtain the desired product (2) or (2-6).
  • 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 (2) or (2-6).
  • the obtained target compound can be further purified, if necessary, by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • This step is a step of producing compound (2) by reacting compound (2-6) with a deprotecting agent in an inert solvent.
  • the method of this step differs depending on the type of the protecting group of compound (2-6).
  • the solvent used is not particularly limited as long as it does not inhibit the reaction, but ethers such as tetrahydrofuran and dioxane are preferred. Suitable.
  • the reaction temperature is not particularly limited, but is usually from 130 ° C to 100 ° C, and preferably from 0 ° C to 30 ° C.
  • the reaction time is usually 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. Then, an immiscible organic solvent such as water and ethyl acetate is added. After separating the organic layer containing, the organic layer is dried over anhydrous magnesium sulfate or the like, and the solvent is distilled off to obtain the desired product (2).
  • 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 solvent used is not particularly limited as long as it does not inhibit the reaction, but acetic acid, alcohol, or a mixed solvent of these and 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 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 the compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off to obtain the desired product (2).
  • 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 (2).
  • the obtained target compound can be further purified, if necessary, by a conventional method, for example, recrystallization, reprecipitation or chromatography. 3) When an aralkyloxycarbonyl group is used as the 3′-position deprotection, the 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 usually used in the catalytic reduction reaction, but is preferably palladium carbon, Raney nickel, platinum oxide, platinum black. , Rhodium aluminum monoxide, triphenylphosphine-rhodium chloride, and palladium monosulfate.
  • the pressure is not particularly limited, but it is usually 1 to 10 atm.
  • the reaction temperature and reaction time vary depending on the type of the starting material, the solvent and the catalyst, and the like.
  • 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, halogenated hydrocarbons such as methylene-based lid, chloroform-based form, carbon tetrachloride, and acetonitrile are preferred.
  • Nitriles such as Jethyl ether, tetrahydrofuran, dioxane, amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide, and dimethylsulfoxide Sulphoxides.
  • the oxidizing agent to be used is not particularly limited as long as it is a compound used for the oxidation, but preferably, potassium persulfate, sodium persulfate, ammonium permite nitrate (CAN), 2, 3-Zikuguchi-5, 6-Diciano-p-Venzoquinone (DDQ) is used.
  • 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 carried out at 0 to 150 ° C for 10 minutes to 24 hours.
  • liquid ammonia or in alcohols such as methanol and ethanol, -78 to -20 It can also be removed by the action of alkali metals such as tritium.
  • 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 this reaction, but preferably, -tolyls such as acetonitrile, methylene chloride, and chloroform Halogenated hydrocarbons such as form or a mixed solvent thereof are used.
  • reaction temperature and reaction time vary depending on the starting materials, the solvent, and the like, but are usually 0 to 50 ° C. for 5 minutes to 3B.
  • reaction substrate has a sulfur atom
  • sodium aluminum chloride and iodide are preferably used.
  • the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, and then 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 target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off to obtain the desired product (2).
  • the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.
  • CPG (3-1) carrying a nucleoside which is the nucleotide at the 3 'end of the target oligonucleotide, is used as a raw material and is usually used for a DNA chain elongation reaction on a DNA synthesizer.
  • This procedure is repeated to extend the oligonucleotide to the oligonucleotide excluding the nucleotide other than the 5 'end of the desired oligonucleotide, and the oligonucleotide is supported on cPG.
  • This is the step of obtaining oxyribonucleotide (3) and the like.
  • a CPG (3-1) carrying a nucleoside which is a nucleotide on the 3 ′ end side of the target oligonucleotide, which is commercially available or obtained by the method described above or below, is added to D.
  • the phosphite-triester bond formed by condensing the nucleotide unit is converted to phosphoric acid-triester using an oxidizing agent. Oxidize.
  • oligodeoxynucleotide is abbreviated as ODN.
  • a CPG carrying the ODN of the desired nucleotide sequence protected at the 5 ′ end with a DMT group can be synthesized using a DNA synthesizer (for example, Applied Biosystems' phosphoramidite model 380B, The compound can be synthesized by the method in Nucleic Acids Res, 124539 (1994) or a modified method thereof using a cycloamidite method by the phosphoramidite method of Z Biosearch.
  • a DNA synthesizer for example, Applied Biosystems' phosphoramidite model 380B
  • the compound can be synthesized by the method in Nucleic Acids Res, 124539 (1994) or a modified method thereof using a cycloamidite method by the phosphoramidite method of Z Biosearch.
  • nucleotide unit of the nucleotide unit used for the synthesis of an oligonucleotide a nucleotide unit protected with an aliphatic or aromatic acyl group is used.
  • base is A or C
  • a benzoyl group is used
  • 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. Hydrofuran is preferred, and tetrazole is preferred as the acidic substance used as the catalyst.
  • 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 of 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 amines particularly, pyridine
  • Nitriles especially, acetonitrile
  • ethers especially, tetrahydrofuran
  • nitrogenated hydrocarbons especially, methylene chloride
  • Oxides iodine-pyridine-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 reaction is accelerated by adding an interlayer transfer catalyst such as tributylbenzylammonium mouthride or tributylbenzylammonium bromide.
  • an interlayer transfer catalyst such as tributylbenzylammonium mouthride or tributylbenzylammonium bromide.
  • the desired product (3) is collected by filtration, washed with an organic solvent such as methylene chloride, and used as it is in the next step.
  • the 12th, 14th, 15th, 15th, 17th, 23rd, 26th, 28th, 30th, 33th, and 35th steps can be performed in the same manner as described above.
  • Steps 9 and 20 In this step, the compound (4-2) or (6-2) is reacted with an anhydride of a dicarboxylic acid in the presence of a basic catalyst to form a dicarboxylic acid half ester (4-3) or (6-4). This is the step of obtaining.
  • succinic acid is described as a representative, but the same can be performed for other dicarboxylic acids.
  • the dicarboxylic acid used is not particularly limited, but is preferably one having 2 to 10 carbon atoms, and most preferably succinic acid or glutaric acid.
  • Examples of the basic catalyst used include aminopyridines such as dimethylaminopyridine and pyrrolidinoviridine, tertiary amines such as trimethylaminetriethylamine, and hydrogencarbonate.
  • Alkali metal carbonates such as lithium and lithium carbonate are preferred, but dimethylaminopyridine and pyrrolidinopyridine are 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; methylene chloride Halogenated hydrocarbons such as lids and chloroform; ethers such as ether, tetrahydrofuran, dioxane, and dimethoxetane; dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide Amides such as amides; sulfoxides such as dimethyl sulfoxide; methanol, ethanol, n-propanol, and isoprono II.
  • aromatic hydrocarbon such as benzene, toluene or xylene
  • Halogenated hydrocarbons such as lids and chloroform
  • ethers such as ether, tetrahydrofuran, dioxane, and dimethoxetane
  • dimethylformamide dimethyl
  • Alcohols such as ethanol, n-butanol, isobutanol, and isoamino alcohol; dilute acids such as sulfuric acid water; dilute bases such as sodium hydroxide water; water; acetate; methylethylketone; Ketones; heterocyclic amides such as pyridine or nitriles such as acetonitrile
  • they are nitriles (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 is usually 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 reaction mixture is appropriately neutralized, and if there is any insoluble matter, it is removed by filtration. Then, a water-immiscible organic solvent such as water and ethyl acetate is added, and the mixture is washed 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, whereby the target product (4-1-3) or (6-4) can be obtained.
  • a water-immiscible organic solvent such as water and ethyl acetate
  • the phenol used is not particularly limited as long as it reacts with a carboxylic acid to form an active ester, but pentachlorophenol 412 trophenol is preferred.
  • the base to be used is not particularly limited as long as it is used as a base in a usual reaction, and is preferably triethylamine, tributylamine, or diisopropylamine.
  • the solvent used does not hinder the reaction and dissolves the starting materials to some extent.
  • amides such as dimethylformamide, dimethylacetamide and hexamethylphosphorotriamide
  • sulfoxides such as dimethylsulfoxide
  • acetate Ketones such as luethyl ketone
  • heterocyclic amides such as pyridine or nitriles such as acetonitrile
  • amides such as dimethylformamide. It is a kind.
  • 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, diisopropylamineethyl. , N-methylmorpholinepyridine, 4- (N, N-dimethylamino) pyridine, N, N-dimethylaniline, N, N-getinoleaniline, 1,5-diazabicyclo [4,3,0] ] Nona 1-5, 1,4 diazabicyclo [2,2,2] octane (DABCO), 1,8 diazabicyclo [5,4,0] Ndeku 7-Den (DBU) Examples thereof include organic bases, preferably, triethylamine, pyridine, N-methylmorpholine, and DBU.
  • the reaction is carried out at a temperature of 150 to 100 ° C, and the reaction time varies depending mainly on the reaction temperature, the type of the starting compound or the solvent used. 30 to 50 hours.
  • the target substance is collected by filtration, washed with an organic solvent such as methylene chloride, and used as it is in the next step.
  • the compound (416) obtained in the 1st step is reacted with a commercially available 5'-O-DMT-nucleoside-13'-phosphoramidite reagent on a DNA synthesizer. Then, the chelating reagent is reacted, This is a step of obtaining the desired product (4.7) which is a CPG carrier having a C bond.
  • the reagent for the formation of a cholate is not particularly limited as long as it can react with trivalent phosphorus to form a cholate. Tetraethylthiuram disulfide (TETD) or Beaucage reagent, which can be obtained from Miragen Z Bioresearch, is suitable.
  • the desired product (4-7) can be obtained by processing according to the method or a modification thereof.
  • the compound (7-3) is reacted with a 5'-O-DMT-nucleotide 3'-phosphoramidite reagent, and a phosphoric acid triester bond is formed by a usual treatment.
  • the desired product (7-7) can be obtained by reacting the thioating reagent with a thioating reagent having a certain force.
  • the desired product is collected by filtration, washed with an organic solvent such as methylene chloride, and used as it is in the next step.
  • the condensing agent to be used is not particularly limited as long as it forms a mixed acid anhydride with the phosphonic acid monoester, but is preferably adamanta.
  • 1-Carbonyl chloride and pivaloyl chloride are used.
  • the deoxidizing agent used is not particularly limited as long as it is a deoxidizing agent used for acylation using a fatty acid halide, but usually pyridin is used.
  • the solvent to be used is not particularly limited as long as it does not inhibit the reaction, but nitriles such as anhydrous acetonitrile are preferably used.
  • the reaction time is 5 to 60 minutes when the reaction is carried out at room temperature.
  • the target substance is collected by filtration, washed with an organic solvent such as methylene chloride, and used as it is in the next step.
  • This step is a step of reacting the compound (5-3) with a desired alkylamine and carbon tetrachloride to form a phosphoramidate bond.
  • reaction temperature is from 150 ° C. to 100 ° C., and is not particularly limited. In the case of room temperature, the reaction time is from 1 hour to 10 hours.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • AIDS & HIV (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention se rapporte à de nouveaux oligodésoxyribonucléotides modifiés possédant une activité excellente dirigée contre le virus de l'immunodéficience humaine (VIH-1). Ces composés sont représentés par la formule (1) ou en sont des sels pharmacologiquement acceptables. Dans la formule (1), Q est un groupe représenté par R1R2R3Z (où R1, R2 et R3 représente chacun H, un alkyle C1-4, un aryle éventuellement substitué ou un anthraquinonyle éventuellement substitué et Z représente C ou Si, ou bien R2, R3 et Z représentent conjointement un fluorényle ou un xanthényle); R4 est H, alkyle C1-4 éventuellement substitué, aryle éventuellement substitué ou aralkyle éventuellement substitué; Y1, Y3 et Y4 sont chacun O, S ou NH; Y2 est O, S, NH, alkylène C1-4, ou phénylène; Z est C ou Si; X est alkylène C1-10 éventuellement substitué par OH; m et n sont chacun un entier compris entre 0 et 10; et B est un oligodésoxyribonucléotide comportant une séquence représentée par TGGGGT, TGGGGTT, TGGGGGT, TGGGCG, TGGGTG, TGGGG, TGGGGG ou TGGGAG.
PCT/JP1998/004625 1997-10-14 1998-10-13 Oligodesoxyribonucleotides modifies comportant la sequence tggg WO1999019474A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU94608/98A AU9460898A (en) 1997-10-14 1998-10-13 Modified oligodeoxyribonucleotides having tggg sequence

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP28021097 1997-10-14
JP9/280210 1997-10-14
JP28641497 1997-10-20
JP9/286414 1997-10-20

Publications (1)

Publication Number Publication Date
WO1999019474A1 true WO1999019474A1 (fr) 1999-04-22

Family

ID=26553681

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/004625 WO1999019474A1 (fr) 1997-10-14 1998-10-13 Oligodesoxyribonucleotides modifies comportant la sequence tggg

Country Status (2)

Country Link
AU (1) AU9460898A (fr)
WO (1) WO1999019474A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766183A (zh) * 2011-05-05 2012-11-07 中国人民解放军军事医学科学院毒物药物研究所 具有抗hiv-1融合活性的修饰的核酸结构
WO2019222264A1 (fr) * 2018-05-15 2019-11-21 Illumina, Inc. Compositions, procédés de clivage chimique et procédés de déprotection d'oligonucléotides liés à une surface

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05310779A (ja) * 1992-01-22 1993-11-22 Hoechst Ag オリゴヌクレオチド類似体、その製造法および用途
JPH0787982A (ja) * 1993-01-29 1995-04-04 Sankyo Co Ltd 修飾オリゴデオキシリボヌクレオチド

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05310779A (ja) * 1992-01-22 1993-11-22 Hoechst Ag オリゴヌクレオチド類似体、その製造法および用途
JPH0787982A (ja) * 1993-01-29 1995-04-04 Sankyo Co Ltd 修飾オリゴデオキシリボヌクレオチド

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AGATSUMA T., ET AL.: "PROTECTION OF HU-PBL-SCID/BEIGE MICE FROM HIV-1 INFECTION BY A 6-MER MODIFIED OLIGONUCLEOTIDE, R-95288.", ANTIVIRAL RESEARCH, ELSEVIER BV, NL, vol. 34., 1 January 1997 (1997-01-01), NL, pages 121 - 130., XP002915796, ISSN: 0166-3542, DOI: 10.1016/S0166-3542(97)01032-2 *
HOTODA H., ET AL.: "BIOLOGICALLY ACTIVE OLIGODEOXYRIBONUCLEOTIDES - IV: ANTI-HIV-1 ACTIVITY OF TGGGAG HAVING HYDROPHOBIC SUBSTITUENT AT ITS 5'-END VIAPHOSPHODIESTER LINKAGE.", NUCLEOSIDES & NUCLEOTIDES, MARCEL DEKKER INC, US, vol. 15., no. 01/03., 1 January 1996 (1996-01-01), US, pages 531 - 538., XP002915797, ISSN: 0732-8311 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766183A (zh) * 2011-05-05 2012-11-07 中国人民解放军军事医学科学院毒物药物研究所 具有抗hiv-1融合活性的修饰的核酸结构
WO2012149906A1 (fr) * 2011-05-05 2012-11-08 中国人民解放军军事医学科学院毒物药物研究所 Structure d'acide nucléique modifiée présentant une activité de fusion anti-vih-1
CN102766183B (zh) * 2011-05-05 2016-09-14 中国人民解放军军事医学科学院毒物药物研究所 具有抗hiv-1融合活性的修饰的核酸结构
WO2019222264A1 (fr) * 2018-05-15 2019-11-21 Illumina, Inc. Compositions, procédés de clivage chimique et procédés de déprotection d'oligonucléotides liés à une surface
AU2019271121B2 (en) * 2018-05-15 2021-05-20 Illumina Cambridge Limited Compositions and methods for chemical cleavage and deprotection of surface-bound oligonucleotides

Also Published As

Publication number Publication date
AU9460898A (en) 1999-05-03

Similar Documents

Publication Publication Date Title
JP6038860B2 (ja) リン原子修飾核酸の合成方法
JP6178744B2 (ja) モルホリノオリゴマーの合成方法
US8304532B2 (en) Method for preparing oligonucleotides
WO2002018388A1 (fr) Analogues de nucleosides et derives d'oligonucleotides renfermant ces analogues
JPH05504553A (ja) 新規の結合を有するオリゴヌクレオチドアナログ
JPH03501128A (ja) ヌクレオシドおよびポリヌクレオチドチオホスホラミダイトおよびホスホロジチオエイト化合物並びに方法
JPH0787982A (ja) 修飾オリゴデオキシリボヌクレオチド
WO1995024413A1 (fr) Compositions et procedes s'utilisant dans la synthese d'oligonucleotides
CA2159628A1 (fr) Nucleosides bifonctionnels et leurs oligomeres; methodes de preparation et d'utilisation
JP2000506849A (ja) オリゴヌクレオチド類似体
US5674856A (en) Modified oligodeoxyribonucleoditides
KR101437824B1 (ko) 올리고뉴클레오티드의 합성
WO1999019474A1 (fr) Oligodesoxyribonucleotides modifies comportant la sequence tggg
EP0739899B1 (fr) Dérivés d'oligoribonucléotide et leur application comme agents antiviraux
PL211566B1 (pl) Analog 2',5'-oligoadenylanowy, kompozycja farmaceutyczna zawierająca analog 2',5'-oligoadenylanowy oraz zastosowanie analogu 2',5'-oligoadenylanowego
JPH0892275A (ja) 修飾オリゴデオキシリボヌクレオチド
JP2000290185A (ja) Ｔｇｇｇ配列を持つ修飾オリゴデオキシリボヌクレオチドを含有する抗エイズ剤
WO1999021874A1 (fr) Oligodesoxyribonucleotides contenant un nucleoside modifie et autre
Ju et al. Synthesis of oligodeoxyribonucleoside phosphorothioates using Lawesson's Reagent for the Sulfur Transfer Step
JPH11246592A (ja) Ｔｇｇｇ配列を持つ修飾オリゴデオキシリボヌクレオチド
CA2096658A1 (fr) Derive d'acide nucleique
JP2000302684A (ja) 修飾ヌクレオシド等を含有するオリゴデオキシリボヌクレオチドを含有する抗エイズ剤
WO1990004603A1 (fr) Derives de nucleoside
JP3911703B2 (ja) アンチセンス核酸同族体
JPH07501542A (ja) アミノ炭化水素ホスホン酸塩成分を有するオリゴヌクレオチド

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA CN CZ HU ID IL KR MX NO NZ PL RU TR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: KR

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA