WO1997027206A1 - Analogues d'oligonucleotides - Google Patents

Analogues d'oligonucleotides Download PDF

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Publication number
WO1997027206A1
WO1997027206A1 PCT/US1997/001236 US9701236W WO9727206A1 WO 1997027206 A1 WO1997027206 A1 WO 1997027206A1 US 9701236 W US9701236 W US 9701236W WO 9727206 A1 WO9727206 A1 WO 9727206A1
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Prior art keywords
substituted
compound
independently
group
protecting group
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PCT/US1997/001236
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English (en)
Inventor
Alexander A. Khorlin
Kyoichi A. Watanabe
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Codon Pharmaceuticals, Inc.
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Application filed by Codon Pharmaceuticals, Inc. filed Critical Codon Pharmaceuticals, Inc.
Priority to EP97903121A priority Critical patent/EP0885236A1/fr
Priority to AU17109/97A priority patent/AU1710997A/en
Priority to JP9527063A priority patent/JP2000505418A/ja
Publication of WO1997027206A1 publication Critical patent/WO1997027206A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a class of
  • oligonucleotides modified oligonucleotides, or
  • oligonucleosides i.e., phosphate-free oligonucleotides
  • linker of a non-nucleosidic nature containing at least one (aza) nitrogen as an achiral site for attachment of a DNA-binding group or a DNA-interacting group or carrier or targeting ligand.
  • Oligonucleotides can be of value as therapeutic agents for the treatment of a wide variety of diseases.
  • Classical therapeutics has generally focused on interactions with proteins, which either acting directly or through their enzymatic functions, contribute in major proportion to many disease states in animals and man.
  • oligonucleotides offer the potential for a highly efficient specificity due to their capability for base pairing with complementary nucleic acid strands in a Watson-Crick or Hoogsteen manner. In that sense
  • oligonucleotides provide a unique opportunity for gene therapy or the regulation of translation or transcription.
  • RNA messenger RNA
  • mRNA messenger RNA
  • tRNA transfer RNAs
  • Transcription initiation requires specific recognition of a promoter DNA sequence by the RNA-synthesizing enzyme, RNA polymerase. In many cases in procaryotic cells, probably in all cases in eucaryotic cells, this recognition is based on sequence-specific binding of protein
  • telomere binding factor RNA polymerase
  • repressors proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors.
  • Synthetic oligonucleotides could be used as
  • antisense probes involved in binding to transcellular RNA in a sequence-specific fashion such as Watson-Crick base pairing interactions.
  • synthetic DNA could suppress translation in vivo. It also may be possible to effect the genome by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents.
  • phosphoramidates bridged phosphorothioates, bridged methylenephosphonates, dephospho internucleotide analogues with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various "plastic" DNAs , alpha-anomeric bridges, and borane derivatives.
  • United States Patent 5,216,141 relates to DNA analogs containing sulfides, sulfoxides and sulfones as linking groups between subunits capable of forming bonds with natural oligonucleotides.
  • United States Patent 5,034,506 relates to polymeric compositions containing morpholino subunits linked together by achiral linkages. Each subunit is said to contain a purine or pyrimidine base pairing moiety.
  • United States Patents 5,405,938 and 5,166,315 relate to polymers containing an uncharged 5- or 6- membered cyclic backbone having selected bases attached to the backbone.
  • the polymer is said to be able to bind in sequence specific manner to a target sequence of a duplex polynucleotide.
  • PNAs peptide nucleic acids
  • phosphorodiester inter-sugar linkages are replaced with four atom linking groups.
  • the present invention relates to a macromolecule, at least a portion of which is of the structure :
  • each B is independently hydrogen, hydroxy, a
  • nucleobase naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, or an aromatic moiety;
  • each B 1 is independently hydrogen, hydroxy, amino, mercapto, a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, an aromatic moiety, a targeting group, a carrier, a reporter group, or a soluble or non-soluble polymer;
  • n is an integer from 1 to 50;
  • each X is independently a single bond, methylene, methylenecarbonyl, C 7 -C 12 aralkylene or substituted
  • aralkylene C 7 -C 12 aralkylenecarbonyl or substituted aralkylenecarbonyl, or a group of the formula: or
  • each Z 1 is independently O, S, NR 5 , methylene, or
  • each of p, q, r, and s is independently an integer from 0 to 20;
  • each of R 1 , R 2 , R 3 and R 4 is independently hydrogen; C 1 -C 8 alkyl, which may be hydroxy-, or alkoxy-, or alkylthio-substituted; hydroxy; alkoxy; alkylthio; amino or halogen;
  • each of R 5 and R 6 is independently hydrogen; C 1 - C 8 alkyl, which may be hydroxy-, or alkoxy-, or alkylthiosubstituted; hydroxy; alkoxy; alkylthio; or amino;
  • each of Q 1 or Q 2 independently comprises at least three atoms, at least one of which is carbon;
  • each V is independently oxygen, sulfur, NR 8 or
  • each J is independently hydrogen, azido, halogen, -OR 7 , -R 7 or -NR 7 R 8 , wherein each R 7 is independently
  • each of R 8 or R 9 is independently hydrogen, C 3 -C 10 branched alkyl or substituted alkyl, C 1 -C 10 unbranched alkyl or substituted alkyl, C 1 -C 10 unbranched oxaalkyl or substituted oxaalkyl, C 6 -C 10 aryl or substituted aryl, C 7 -C 12 aralkyl or substituted aralkyl, C 1 -C 10
  • the present invention relates to pharmaceutical compositions comprising an effective amount of a compound above, and a pharmaceutically suitable carrier.
  • the present invention relates to methods for the treatment of diseases caused by pathogenic organisms, which comprises administering to a host organism in need of such treatment an effective amount of a compound or pharmaceutical composition described above.
  • the host organism may be any organism in need of such treatment, and includes mammals and humans.
  • the present invention relates to methods for the treatment of tumors, which comprises administering to an organism in need of such treatment an effective amount of a compound or pharmaceutical
  • the organism may be any organism in need of such treatment, and includes mammals and humans.
  • the present invention relates to a compound having the formula :
  • each B is independently a naturally occurring
  • nucleobase a non-naturally occurring nucleobase, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group;
  • each B 1 is independently hydrogen, hydroxy, amino, mercapto, a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group;
  • n is an integer from 1 to 50;
  • each X is independently an optionally protected group selected from a single bond, methylene group,
  • each Z 1 is independently O, S, Se, NR 5 , methylene, or C(CH 3 ) 2 ;
  • each of p, q, r, and s is independently an integer from 0 to 20;
  • each of R 1 , R 2 , R 3 and R 4 is independently hydrogen; C 1 -C 8 alkyl, which may be hydroxy-, or alkoxy-, or alkylthio-substituted; hydroxy; alkoxy; alkylthio; amino or halogen;
  • each of R 5 and R 6 is independently hydrogen; C 1 - C 8 alkyl, which may be hydroxy-, or alkoxy-, or alkylthiosubstituted; hydroxy; alkoxy; alkylthio; or amino;
  • each of Q 1 or Q 2 independently comprises at least three atoms, at least one of which is carbon;
  • each V is independently oxygen, sulfur, NR 8 or
  • R 8 is independently hydrogen, C 3 -C 10 branched alkyl or substituted alkyl, C 1 -C 10 unbranched alkyl or substituted alkyl, C 1 -C 10 unbranched oxaalkyl or
  • substituted oxaalkyl C 6 -C 10 aryl or substituted aryl, C 7 -C 12 aralkyl or substituted aralkyl, C 1 -C 10 unbranched aminoalkyl or substituted unbranched aminoalkyl; C 1 -C 10 unbranched aminooxaalkyl or substituted unbranched aminooxaalkyl, C 3 -C 10 and N 1 -N 4 branched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C 10 and N 1 -N 4 unbranched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C 10 and N 1 -N 4 unbranched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C
  • each J is independently, hydrogen, OR 7 , halogen, azide or R 7 , any of which is optionally protected, wherein each R 7 is independently -NR 8 R 9 or R 8 , wherein R 9 is independently hydrogen, C 3 -C 10 branched alkyl or substituted alkyl, C 1 -C 10 unbranched alkyl or substituted alkyl, C 1 -C 10 unbranched oxaalkyl or substituted oxaalkyl, C 6 -C 10 aryl or substituted aryl, C 7 -C 12 aralkyl or substituted aralkyl, C 1 - C 10 unbranched aminoalkyl or substituted unbranched
  • aminoalkyl C 1 -C 10 unbranched aminooxaalkyl or substituted unbranched aminooxaalkyl, C 3 -C 10 and N 1 -N 4 branched
  • polyamino- or polyaza-alkyl or substituted (polyamino- or polyaza-) alkyl C 1 -C 10 and N 1 -N 4 unbranched (polyamino- or polyaza-) alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C 10 and N 1 -N 4 unbranched (polyamino- or polyaza-)oxaalkyl or substituted unbranched (polyamino- or polyaza-)oxaalkyl, a natural or non-natural amino acid side chain radical, or a protecting group;
  • each Q 3 is independently -OX-, -SX- or -NR 8 X-, any of which is optionally protected;
  • each Q 4 is independently oxygen, sulfur or NR 8 , any of which is optionally protected;
  • Y is a protecting group
  • Y 1 is a spacer group linked to a solid support.
  • the present invention relates to a compound represented by the formula:
  • each B is independently a naturally occurring nucleobase, a non-naturally occurring nucleobase, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group;
  • each B 1 is independently hydrogen, hydroxy, amino, mercapto, a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group;
  • n is an integer from 1 to 50;
  • each X is independently one of the following optionally protected groups: a single bond, methylene, methylenecarbonyl, C 7 -C 12 aralkylene or substituted
  • aralkylene C 7 -C 12 aralkylenecarbonyl or substituted aralkylenecarbonyl or a group of formula: or
  • each Z 1 is independently O, S, Se, NR 5 , methylene, or C(CH 3 ) 2 ;
  • each of p, q, r and s is independently an integer from 0 to 20;
  • each of R 1 , R 2 , R 3 and R 4 is independently hydrogen; C 1 -C 8 alkyl, which may be hydroxy-, or alkoxy-, or alkylthio-substituted; hydroxy; alkoxy; alkylthio; amino or halogen;
  • each of R 5 and R 6 is independently hydrogen; C 1 - C 8 alkyl, which may be hydroxy-, or alkoxy-, or alkylthiosubstituted; hydroxy; alkoxy; alkylthio; or amino;
  • each of Q 1 or Q 2 comprises at least three atoms, at least one of which is carbon;
  • each V is independently oxygen, sulfur, NR 8 or methylene
  • each J is independently one of the following
  • optionally protected groups hydrogen, OR 7 , halogen, azide or R 7 , wherein each R 7 is independently -NR 8 R 9 or R 8 , wherein each of R 8 or R 9 is independently hydrogen, C 3 -C 10 branched alkyl or substituted alkyl, C 1 -C 10 unbranched alkyl or substituted alkyl, C 1 -C 10 unbranched oxaalkyl or
  • substituted oxaalkyl C 6 -C 10 aryl or substituted aryl, C 7 -C 12 aralkyl or substituted aralkyl, C 1 -C 10 unbranched aminoalkyl or substituted unbranched aminoalkyl; C 1 -C 10 unbranched aminooxaalkyl or substituted unbranched aminooxaalkyl, C 3 - C 10 and N 1 -N 4 branched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C 10 and N 1 -N 4 unbranched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C 10 and N 1 -N 4 unbranched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C 1 -C
  • each of Q 1 x and Q2x comprising at least one atom, is independently selected from optionally protected or activated fragments of Q 1 or Q 2 .
  • Figures 1-8 each depicts comparisons of prior art compounds (labeled (A) in each Figure) with various compounds of the present invention, labeled (B-1) through (B-24) and (C-1) through (C-6).
  • FIG. 9-11 each depicts compounds of the present invention.
  • FIGS 12-14 each depicts comparisons of prior art compounds (labeled (A) in each Figure) with various compounds of the present invention, labeled (E-1) through (E-13).
  • Figure 15 depicts compounds of the present invention.
  • the present invention is directed to macromolecules that are able to function like oligonucleotides and which also possess other useful properties.
  • the macromolecules are constructed from basic nucleoside units and specific linker units bearing nucleobase or other nucleic acid-binding elements.
  • the nucleoside units, joined by a nucleobase bearing linker of the present invention i.e., the Q 1 -N-Q 2 segment of Formula I), forms trimeric units.
  • the trimeric units can be further extended to pentameric, heptameric and other, higher order
  • the trimeric units can be connected via linkages other than those of the invention, as for example, via a normal phosphodiester linkage, a phosphothioate linkage, a phosphodithioate linkage, a phosphoroamidate linkage, a phosphotriester linkage, a methyl or other alkylphosphonate linkage or other linkage.
  • the linkage of the present invention contains two parts (or sublinkages, i . e. , Q 1 and Q 2 in Formula I) separated by an aza-nitrogen atom.
  • the aza-nitrogen atom is an achiral site of attachment of a nucleobase or any other nucleic acid binding moiety.
  • a single type of sublinkage is used to join nucleosides and aza-nitrogen atom(s).
  • two different sublinkages are used to form trimeric units, or two or more different sublinkages are used to form the higher order units.
  • sublinkages of different units may be the same or of different types as described in this specification
  • Some trimeric units carry naturally occurring or non-naturally occurring nucleic bases attached to an achiral aza-nitrogen atom within an internucleosidic linkage, other units carry a nucleobase-binding ligand other than a nucleobase, described below more fully.
  • the naturally occurring nucleobases which are referred to herein include the four main naturally occurring nucleobases
  • nucleobases i.e. thymine, cytosine, adenine or guanine, or other naturally occurring nucleobases, e. g. hypoxanthine, uracil, thiouracil, 5-methylcytosine, etc.
  • non-naturally occurring nucleobases which are referred to herein include, for example, fluorouracil, bromovinyluracil, triazolcarboxamide, benzimidazole, etc.
  • heterocyclic moieties which are referred to herein include any heterocycle containing at least one heteroatom fused or non-fused ring systems, for example, nitroindole derivatives, nitroimidazole derivatives, nitrotriazole derivatives, etc.
  • the DNA-binding groups which are referred to herein include: 1) covalent DNA-binding groups, which interact with DNA by means of chemical modification (for example, mustard gas derivatives, psoralen and its derivatives, mitomycin C, etc.); 2) non-covalent DNA-binding groups, which interact with DNA by means of hydrogen bond
  • Hydrogen bond formation between functional groups of DNA (or RNA) and functional groups of nucleic acid binding ligands plays an important role in specific recognition of nucleic acids (especially double stranded nucleic acids) by antibiotics (e.g., distamycin, netropsin, echinomycin, etc.), or proteins (e.g., repressors, restrictases, etc.), or oligonucleotides (e.g., DNA double strand formation, or DNA triple strand formation).
  • antibiotics e.g., distamycin, netropsin, echinomycin, etc.
  • proteins e.g., repressors, restrictases, etc.
  • oligonucleotides e.g., DNA double strand formation, or DNA triple strand formation.
  • intercalator associated with separation of two adjacent base pairs to allow insertion of a planar aromatic (hetero)-cyclic group, known as the intercalator.
  • the intercalators capable of binding to double stranded nucleic acids which are referred to herein include, for example, acridine or its
  • intercalators capable of binding to preferably triple stranded clusters of nucleic acids include, for example, coralyne (a member of the protoberberine family of alkaloids), propidium bromide, etc.
  • the carriers which are referred to herein include, for example, a polyamine group (e.g., polyethyleneimine, spermine, spermidine, poly-L-lysine, starburst dendrimers, etc.), or lipophilic groups (e.g., cholesterol, alkyl chain, etc.), or a soluble polymer (e.g., polyethylene glycols, polysaccharides, proteins, etc.), or by a non-soluble polymer (e. g. , dextranes, polyacrylamide
  • a targeting ligand e. g. , sugar or sugar phosphate residues which act as binding sites to receptors on the surface of target cell, antibodies, immunoglobulins, etc.
  • nucleoside refers to a unit composed of a heterocyclic base and a sugar, and includes the natural occurring nucleosides, including 2'-deoxy and 2'- hydroxyl forms, e. g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
  • the heterocyclic base typically is adenine, cytosine, guanine, thymine or uracil; the sugar is normally deoxyribose, i . e. , erythropentofuranosyl, or ribose, i.e., ribo-pentofuranosyl.
  • nucleosides in reference to nucleosides includes synthetic nucleosides having modified base moieties (for example, 5(6)-nitroindole, 4-nitrotriazole, 3(4)-nitrobenzimidazole, 2-aminopurine, benzimidazole, 5-fluorouracil, and the like) and/or modified sugar moieties (for example, arabino, xylo or lyxo pentafuranosyl sugars; or substituted arabino, erythro, ribo, xylo or lyxo pentafuranosyl sugars; or acyclic moieties mimicking sugar; or hexose sugars; etc.) e.g. described by Scheit, Nucleotide Analogues (John Wiley, New York, 1980). Such analogues include the natural and synthetic nucleosides with or without an appropriate protecting group for synthesis.
  • modified base moieties for example, 5(6)-nitroindole, 4-nitrotri
  • nucleotide refers to a nucleoside having a phosphate group esterified to at least one of the sugar hydroxyl groups.
  • oligonucleotide as used herein includes linear oligomers of natural or modified nucleosides, including deoxyribonucleosides, ribonucleosides,
  • alpha-anomeric forms thereof, and the like usually linked by phosphodiester bonds or analogues thereof ranging in size from a few monomeric units, e.g. 2-3, to several hundreds of monomeric units.
  • "Analogues" in reference to oligonucleotide refers to structures including modified portions such as modified sugar moieties, modified base moieties or modified sugar linking moieties.
  • oligonucleotides of the present invention are oligomers of the natural nucleosides having a length in the range of 2 to 50, and more
  • the compounds of the present invention are represented by an oligonucleotide, or modified oligonucleotide, or so-called “oligonucleoside” (i.e. phosphate-free oligonucleotide) optionally modified at the ends, at least a portion of which has the structure of formula la:
  • substituents W and W 1 represent the remainder of the macromolecule.
  • W and W 1 may be any substituents which do not detract from the utility of the present compounds.
  • each of W and W 1 are independently -H; -OH; optionally modified phosphate or phosphate analogs; nucleosides or analogs thereof;
  • nucleotides or analogs thereof oligonucleotides or analogs thereof; amino; mercapto; a DNA intercalator; a covalent or non-covalent DNA-binding group; a heterocyclic moiety; or an aromatic moiety, any of which optionally contains a protecting group; peptides or analogs thereof; chelating groups ( e. g. , EDTA) ; polymers ( e. g. polyamides,
  • polysaccharides or lipophilic groups.
  • each B independently comprises naturally occurring nucleobases, non-naturally occurring nucleobases, heterocyclic moieties, aromatic moieties, DNA
  • each B is a naturally occurring nucleobase or non-naturally occurring nucleobase.
  • the most preferred choice for each B is a naturally occurring nucleobase.
  • At least one of B 1 is a naturally occurring nucleobase; in other preferred embodiments at least one of B 1 is a non-naturally occurring nucleobase; in other preferred embodiments at least one of B 1 is a DNA intercalator (such as acridine derivatives, phenazine derivatives, etc.); in other preferred
  • At least one of B 1 is a covalent DNA-binding group (such as mustard gas derivatives, psoralen
  • At least one of B 1 is a DNA-binding antibiotic (such as daunomycin, actinomycin D or another representative of the actinomycin family, netropsin and its derivatives, distamycin and its derivatives, etc.); in other preferred embodiments at least one of B 1 is a reporter group (such as a fluorescent or chemiluminescent label, biotin, etc.); in other preferred embodiments at least one of B 1 is a targeting group for recognition of definite cells (such as an antibody or saccharide); in other preferred embodiments at least one of B 1 is a soluble or non-soluble polymer; in other preferred embodiments at least one of B 1 is a reactive functional group suitable for postsynthetic modification of an oligonucleotide (such as amino, mercapto, aldehyde, carboxyl, etc.).
  • a DNA-binding antibiotic such as daunomycin, actinomycin D or another representative of the actinomycin family, netropsin and its derivatives, distamycin
  • X comprises from 1 to 4 atoms; in other preferred embodiments, when B 1 is selected from DNA
  • X 1 comprises from 1 to 12 atoms; in a most preferred embodiment, when B 1 is a
  • X 1 is methylenecarbonyl
  • each of Q 1 and Q 2 is independently selected from two of Q 1 and Q 2
  • each of Z 4 or Z 5 is independently selected from the group consisting of a single bond, O, S, and NR 7 , wherein R 7 has been specified above;
  • each Z 3 is independently selected from the group consisting of hydrogen, R 8 , OR 7 , SR 7 , and NR 7 R 8 , wherein R 7 and R 8 have been specified above;
  • each Z 2 is independently selected from the group consisting of O, S, and NR 7 , wherein R 7 has been specified above.
  • each of Q 1 and Q 2 independently contains from 2 to 8 atoms; in more preferred embodiments Q 1 and Q 2 each independently contains from 3 to 6 atoms; most preferably 4 or 5 atoms. Most preferably, each Q 1 is independently selected from the following groups:
  • At least one of V is oxygen.
  • the most preferred choice for each V is oxygen.
  • At least one of J is independently selected from the group of hydrogen, fluorine or OCH 3 ; the most preferred choice for J is hydrogen.
  • the compounds of the invention are synthesized by adaptation of standard oligonucleotide synthesis
  • the compounds of the present invention may be prepared by incorporation of fragments of formula I onto the 5'-end of a growing oligonucleotide or modified oligonucleotide chain, represented by the formula II:
  • each of B, B 1 , Q 1 , Q 2 , V and J are as described above, any of which is optionally blocked with a protecting group if appropriate (e. g. , acetyl, isobutyryl,
  • each Q 3 independently comprises a single bond, oxygen, sulfur, -NR 8 -, -OX-, -SX-, -X-, or -NR 8 X-, wherein X and R 8 have the meanings specified above in connection with formula I; more preferably each Q 3 independently comprises a single bond, -OCH 2 -, -SCH 2 -, -CH 2 -, -NR 8 CH 2 -, -CH 2 CH 2 -, and -OCH 2 CH 2 -; the most preferred choice for Q 3 is -OCH 2 -;
  • each Q 4 independently comprises a single bond, oxygen, sulfur and NR 8 , wherein R 8 has the meanings specified above for formula I;
  • Y is a protecting group such as triphenylmethyl, p- anisyldiphenylmethyl, di-p-anisylphenylmethyl, pixyl, trialkylsilyl having from 3 to 14 carbon atoms, 9- fluorenylmethyl carbamate, trifluoroacetyl, or the like; more preferably Y is p-anisyldiphenylmethyl, di-p- anisylphenylmethyl, or pixyl;
  • Y 1 is a spacer group linked to a solid support, which spacer comprises carbonyl, ester, carbamate, urethane, hydrazide, C 1 -C 14 alkylene or modified alkylene, C 6 -C 14 aralkylene or modified aralkylene, C 6 -C 14 alkylarene or modified alkylarene, C 1 -C 100 oxaalkylene or thiaalkylene or azaalkylene each containing from one to fifty different heteroatoms or hetroatoms of the same type, where aza groups are, optionally, protected by amino protecting groups, C 1 -C 14 alkylenecarbonyl or alkylenethiocarbonyl or alkylenesulfone or alkylenesulfoxide, C 1 -C 100
  • azaalkylenecarbonyl (or their thiocarbonyl or sulfone or sulfoxide analogues) each containing from one to fifty different heteroatoms or hetroatoms of the same type, where aza groups are, optionally, protected by amino protecting groups; or a group of the formula (III):
  • each of R 10 or R 11 independently comprises C 3 -C 10 branched alkyl, C 1 -C 10 unbranched alkyl or oxaalkyl, C 6 -C 10 aryl, C 7 -C 12 aralkyl; a more preferred choice for each of R 10 or R 11 is C 3 -C 5 branched alkyl or C 1 -C 4 unbranched alkyl; the most preferred choice for each of R 10 or R 11 is isopropyl;
  • R 12 is C 2 -C 8 alkylene, C 2 -C 8 alkenylene or -C 2 -C 8 oxaalkylene, comprising one or two heteroatoms; most preferably R 12 is a morpholino group;
  • Z 5 is any phosphate protecting group; preferably, 4-Cl-C 6 H 4 -O-, 2-Cl-C 6 H 4 -O-, 4-NO 2 -C 6 H 4 CH 2 CH 2 -O-,
  • NCCH 2 CH 2 -O- NCCH 2 C(CH 3 ) 2 -O-, CH 3 O-, (Z) 3 CCH 2 -O-, R 10 S-,
  • oligonucleotide chain represented by the group of formula IV:
  • each of Q 1 x and Q 2 ⁇ comprises optionally protected or activated fragments of Q 1 or Q 2 ; more preferably each of Q 1 x and Q 2 ⁇ contains from one to three atoms, any of which optionally contains a protecting group; in most preffered embodiments each Q 1 x independently comprises Y 2 -NH-CH 2 -,
  • R 13 is selected from halogen, hydroxyl, pentafluorophenoxy, tetrafluorophenoxy, p-nitrophenoxy, or N-succinimidoxy.
  • the compounds of the present invention may be divided into five groups:
  • oligonucleotide analogues wherein the non-nucleosidic linkage bears a nucleic base
  • oligonucleotide analogues wherein the non-nucleosidic linkage bears a covalent DNA-binding group, or a non-covalent DNA-binding group, or a DNA-intercalator, or a DNA-binding antibiotic;
  • oligonucleotide analogues wherein the non-nucleosidic, preferrably non-phosphate containing linkage, is used as a new type of linker connecting clusters of oligonucleotides or modified oligonucleotides;
  • oligonucleotide analogues wherein the non-nucleosidic linkage bears a carrier, or polymer, or
  • oligonucleotide analogues wherein the non-nucleosidic linkage bears a reporter group, such as biotin.
  • the oligonucleotide analogues (1)-(5) are able to recognize both single stranded and double stranded nucleic acids.
  • the oligonucleotide analogues (1) with fragments of formula I placed on the 3'- and/or 5'-end or distributed along the oligonucleotide chain demonstrate the ability to bind DNA fragments and at the same time possess increased enzymatic stability.
  • the oligonucleotide analogues (2) with fragments of formula I placed in definite positions along the oligonucleotide chain bind efficiently to double stranded DNA or RNA, and attachment of a covalent DNA-binding group, or a non-covalent DNA-binding group, or a DNA-intercalator, or a DNA-binding antibiotic, provides another opportunity to overcome the problem of recognition of polypyrimidine tracts by triplex forming oligonucleotides (TFO).
  • TFO triplex forming oligonucleotides
  • the oligonucleotide analogues (3) can be used as a new type of TFO.
  • the oligonucleotide analogues (4) and (5) are useful in the synthesis of oligonucleotide conjugates.
  • the improved enzymatic stability and binding ability and cell membrane penetration of the compounds of the invention render them efficient as antisense (binding to RNA) or antigene (binding to DNA) agents.
  • the invention provides reagents and methods for inhibiting transcription and/or replication of particular genes or for degradation of particular regions of double stranded DNA in cells of an organism by administering to said organism a compound of invention as defined above.
  • the invention provides reagents and methods for killing or mutating cells (such as tumor cells) or pathogenic organisms (such as viruses, bacteria, fungi, etc.) by contacting said cells or organisms with compounds or compositions of the present invention which have specificity for such cells or organisms.
  • cells such as tumor cells
  • pathogenic organisms such as viruses, bacteria, fungi, etc.
  • Viruses susceptable to treatment according to the present invention would be readily determined by one of ordinary skill, and could include herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), etc.
  • HSV herpes simplex virus
  • HPV human papillomavirus
  • HAV human immunodeficiency virus
  • the compounds of the present invention may be formulated in a pharmaceutical composition, which may include, in addition to an effective amount of active ingredient,
  • compositions may also include one or more other active ingredients such as antimicrobial agents, antiinflammatory agents, and the like.
  • Administration may be done topically, orally, by inhalation, or parenterally. for example.
  • Topical formulations may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Oral formulations include powders, granules, suspensions or solutions in water or non-aqueous media, capsules or tablets, for example. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be used as needed.
  • Parenteral formulations may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • the dose regimen will depend on a number of factors which may readily be determined, such as severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with a course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved.
  • One of ordinary skill may readily determine optimum dosages, dosing methodologies and repetition rates.
  • unit dosage form compositions according to the present invention will contain from about 0.01 mg to about 100 mg of active ingredient, preferably about 0.1 mg to about 10 mg of active ingredient.
  • Topical formulations (such as creams, lotions, solutions, etc.) may have a concentration of active ingredient of from about 0.01% to about 50%,
  • Figures I-VI represent some trimeric fragments of the present invention with the variations in structures of linking moieties d and Q 2 (see Formula I of present
  • All depicted structures from B-1 (Fig. 1) through B-24 (Fig. 6) represent some oligonucleoside trimeric fragments of the invention with variations in the linking moieties, wherein B n is preferably a nucleic base.
  • the structures C-1 (Fig. 7) through C-9 (Fig. 9) represent examples wherein the aza nitrogen atom in Q 1 -N-Q 2 serves as a site of attachment for an intercalating moiety.
  • Formula C-1 (Fig. 7) illustrates the case where an acridine residue is attached through an acyl-type spacer; in formula C-2 the acridine residue is attached through an alkyl spacer.
  • conjugated to a triple helix forming oligonucleotide can be favorable for self-stabilization of the formed triplex.
  • Figures 10 and 11 represent situations where a DNA covalent binding moiety (i.e., psoralen) is conjugated to a internucleoside linker of the present invention.
  • psoralen binds preferentially to double-stranded DNA molecules and attachment of psoralen to an abasic site within the triplex forming oligonucleotide can considerably increase accuracy of site modification of the DNA duplex.
  • Figures 12-14 demonstrate examples of trimeric units of the present invention bearing a functional group
  • oligonucleotides with DNA-active substances such as intercalators, alkylators, DNA-binding antibiotics, or any other nucleic acid binding group as described above.
  • Scheme I illustrates the synthesis of trinucleoside units containing fragments of formula B-1 (Fig. 1) with -OCH 2 CH 2 CH 2 N[X-B 1 ]-CH 2 CH 2 CH 2 OCH 2 - (3' ⁇ 4') intersugar linkage.
  • Nucleoside with a protected base and partially protected sugar moiety (compound 1) is cyanoethylated (as in Example 2 , infra) at the 3'-position, reduced to the aminopropyl derivative (compound 5, Example 5) and then trifluoroacetylated (compound 6, Example 6).
  • the other chain of transformation is allylation of the 5'-position of the nucleoside followed by conversion to the 3-bromopropyl derivative (compound 9).
  • incorporation of this trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having an amino modified (abasic) site (motif E-2, Fig. 12) in its structure, wherein the aliphatic amino-group serves as a site of attachment for various DNA-active groups.
  • oligonucleotide analogs include compounds having formulas B-2, B-3, or B-4 (Fig. 1).
  • Compound 6 (see Scheme I and Example 6) is a starting substance for the synthesis of such intermediates.
  • Compound 6 is alkylated with the tert-butyl ester of bromoacetic acid in DMF in the presence of sodium hydride to give compound 19 (Example
  • the oligonucleosides include compounds having formulas B-5, B-6, or B-7 (Fig. 2).
  • Compound 7 (see Scheme I, Example 7) is a starting material for the synthesis of those intermediates.
  • Compound 7 is
  • Example 35 cyanoethylated to give compound 25 (Example 35), that after reduction is trifluoroacetylated to compound 26 (Example 36).
  • Compound 26 is alkylated with the tert-butyl ester of bromoacetic acid in DMF in the presence of sodium hydride to give compound 27 (Example 37), which after acidic deprotection is converted into a carboxylic acid derivative 28 (Example 38).
  • Compound 28 is treated with a saturated solution of ammonia in ethanol and acylated with an activated carboxymethyl derivative of a nucleic base to give compound 29 (Examples 39, 40 and 41).
  • Compound 29 is dimethoxytritylated to protect the 3'-hydroxyl group and to give compound 30 (Example 42), which can be used for condensation directly, or transformed, for example, into activated ester 31 (Example 43).
  • Scheme IV illustrates examples of synthesis of trinucleosides 33 containing motifs of formulas B-2, B-3, or B-4 (Fig. 1) starting from compounds of general formula 24. Also, Scheme IV shows a route of conversion of partially protected trinucleoside 32 into phoshoramidite 34 and CPG derivative 35.
  • Scheme V illustrates examples of synthesis of trinucleosides 37 containing motifs of formulas B-5, B-6, or B-7 (Fig. 2) starting from compounds of general formula 31. Also Scheme V demonstrates the conversion of partially protected trinucleoside 36 into phoshoramidite 38 and CPG derivative 39.
  • Scheme VI illustrates synthesis of peptide-like oligonucleosides 54 containing
  • Example 50 which after hydrgenolysis and ammonia treatment is converted to compound 48.
  • Compound 48 is acylated with an activated carboxymethyl derivative of a nucleic base to give modified dinucleoside 49 (Examples 52 and 53).
  • Acid labile protecting groups (Boc- and tert-butyl protecting groups) are removed with 50% TFA/DCM and the 6'-amino function is blocked with a monomethoxytrytil protecting group to give compound 50 (Examples 54 and 55).
  • Compound 50 can be used as a building block for solid-phase peptide-like synthesis of oligonucleosides with regular repeating elements depicted in square brackets in formula of compound 54.
  • Compound 50 also can be used as a building block for the synthesis of peptide-like
  • the methodology of synthesis is similar to described in Scheme VI .
  • Scheme IX illustrates the synthesis of 5'-monomethoxytrytil protected trinucleoside (compound 71) with an abasic site containing an aminoalkyl linker, and conversion of that compound into phoshoramidite 72.
  • Scheme X illustrates the synthesis of 5'-monomethoxytrytil protected trinucleoside (compound 74) with an abasic site containing an aliphatic mercapto group, and conversion of this compound into phoshoramidite 75.
  • Scheme XI illustrates the synthesis of trinucleoside phoshoramidite 75 containing a Fmoc-protected hydrazido modified abasic site (motif E-5, Fig. 13).
  • trinucleoside phoshoramidite 81 with a 2,3-di-O-Ac-1-O-carboxymethyl glycerol modified abasic site (motif E-4, Fig. 12). Incorporation of that trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having a cis-diol modified abasic site (motif E-4, Fig. 12) in its structure, wherein a cis-diol group, after periodate oxidation, provides an aldehyde group as a site of attachment for various DNA-active groups.
  • Scheme XIV illustrates the synthesis of an activated ester of a carboxyl derivative of coralyne 88.
  • Compound 88 is used for postsynthetic modification of
  • oligonucleotides containing an aminoalkyl linker with a coralyne moiety oligonucleotides containing an aminoalkyl linker with a coralyne moiety.
  • Scheme XV illustrates the synthesis of an activated ester of a carboxyl derivative of psoralen 93.
  • Compound 93 is used for postsynthetic modification of
  • oligonucleotides containing an aminoalkyl linker with a psoralen moiety oligonucleotides containing an aminoalkyl linker with a psoralen moiety.
  • phoshoramidite 101 with several abasic sites containing trifluoroacetyl protected aza-nitrogens in an
  • oligonucleoside backbone (motif E-9, Fig. 14).
  • Thy 158.8 (C-4, Phe-OMe, MMT); 150.9 (C-2, Thy); 143.7 (C-1, Phe, MMT); 137.9 (C-1, Phe, BOM); 134.6 (C-1, Phe-OMe, MMT); 134.1 (C-6, Thy); 130.3-127.3 (gs, C-2, C-3, C-4, Phe, BOM&MMT); 117.4 (-CN); 113.3 (C-3, Phe-OMe, MMT); 110.5 (C-5, Thy); 87.2 (-C(Phe) 2 Phe-OMe); 85.3 (C-1', dT);
  • the residue is coevaporated with toluene (2 ⁇ 300), dissolved in 3% TFA/DCE (700 ml) and kept at ambient temperature for 15 minutes.
  • the reaction mixture is washed successively with 5% NaHCO 3 (3 ⁇ 250 ml) and water (2 ⁇ 250 ml), dried (MgSO 4 ) and evaporated in vacuo to dryness.
  • the residue is
  • Thy 150.9 (C-2, Thy); 137.8 (C-1, Phe, BOM); 137.4 (C-1, Phe, Bzl), 135.6 (C-6, Thy); 128.5&128.2&127.9&127.6 (C-2, C-3, C-4, Phe, BOM&Bzl); 110.2 (C-5, Thy); 87.6 (C-1', dT); 85.0 (C-4', dT); 78.6 (C-3', dT); 72.1 (OCH 2 , BOM); 71.5
  • CH 2 CH-CH 2 -); 4.69 (s, 2H, OCH 2 , BOM); 4.57&4.48 (d&d, 2H,
  • the flask is charged with a solution of compound 8 (4.5 g, 9.1 mmole) in anhydrous THF (10 ml) and cooled to 0°C.
  • a 1M solution of BH 3 /THF (3.1 ml, 9.3 mmole) is added dropwise over a period of 5 minutes. The resulting mixture is stirred for 30 min at 0°C and 30 min at ambient
  • Thy 2 6.92 (m, 2H, Phe, MMT); 6.29 (gm, 2H, H-1', dT 2 &dT 3 );
  • Trinucleoside 16 is converted to the succinimide ester and treated with long chain alkylamine CPG (500 A, Sigma).
  • the resulting CPG 17 with a loading of 15-20 ⁇ mole/g is used for solid phase oligonucleotide synthesis according standard protocols.
  • Chloro(diisopropylamino)-ß-cyanoethoxyphosphine (0.75 mmol) is added to the reaction mixture and the reaction mixture is allowed to warm to 20°C and stirred for 4 hours.
  • NCH 2 COOBu t 4.64&4.59 (m&m, 1H, H-1, THP); 4.18 (m, *, H- 4', dT); 4.15&4.08 (m&m, *, H-3', dT); 4.10-3.40 (gm, 10H with *, H-5', dT; H-5, THP; OCH 2 CH 2 CH 2 N(COCF 3 )-); 2.44 (m,
  • H-1', dT 4.64&4.59 (m&m, 1H, H-1, THP); 4.30-4.00 (gm, 4H, H-4', dT;H-3', dT; -NHCH 2 COOBu t ) ; 4.00-3.40 (gm, 6H, H- 5', dT; H-5, THP; OCH 2 CH 2 CH 2 NH-); 2.75 (m, 2H, OCH 2 CH 2 CH 2 NH);
  • Thy 137.8&137.3 (C-1, Phe, BOM; C-1, Bzl); 134.1 (C-6, Thy); 128.3-127.4 (gs, C-2, C-3, C-4, Phe, BOM&Bzl); 117.4 (-CN); 110.1 (C-5, Thy); 85.4 (C-1', dT); 83.1 (C-4', dT); 78.4 (C-3', dT); 71.9 (OCH 2 , BOM); 71.4 (OCH 2 , Bzl); 70.7
  • Compound 43 is trifluoroacetylated and purified as per the procedure of Example 6 to give compound 44.
  • Compound 44 is alkylated with tert-butyl ester of bromoacetic acid and purified as per the procedure of
  • a solution of compound 52 (1 mmole) in 80% AcOH 100 ml is kept overnight at ambient temperature.
  • the reaction mixture is evaporated in vacuo to dryness and the residue is coevaporated with Py (3 ⁇ 50 ml) and anhydrous DMF (3 ⁇ 25 ml).
  • the residue is dissolved in anhydrous DMF (5 ml) and solution of compound 51 (1.2 mmole) and DIPEA (1.1 mmole) in anhydrous DMF (5 ml) is added.
  • the reaction mixture is kept at ambient temperature for 2h.
  • phosphoramidites of modified nucleosides and oligonucleoside synthetic blocks have been prepared, they can be incorporated into oligonucleotide analogues, which are synthesized by a stantard solid phase approach, using automated nucleic acid synthesizer such as Applied
  • the macromolecules of the invention can be compared in their ability to bind to complementary nucleic acids by determining the melting temperature of a particular double- stranded (ds) or triple-stranded (ts) complex. Upon formation of dsDNA from two single strands, due to base stacking the extinction coefficient decreases
  • Duplexes are formed from single-stranded
  • oligodeoxyribonucleotides and the macromolecules of the present invention are synthesized according the description or examples presented herein. Oligodeoxyribonucleotides are synthesized on solid phase with an Applied Biosystems, Inc. 392 DNA/RNA Synthesizer. The oligonucleotide species is purified as their dimethoxytrityl derivatives by reverse-phase chromatography using HPLC (Gilson). Typically 0.3-0.5 OD 260 oligonucleotide analog is hybridized with 1 equivalent of the other strand and the absorbance (260 nm) hyperchromicity upon duplex to random coil transition is monitored using a Gilford Response II spectrophotometer.
  • the buffers used are 10 mM in phosphate, 0.1 mM in EDTA and either 0.1 M or 1 M in NaCl.
  • the following extinction coefficients are used dA: 15.4 ml/ ⁇ mol ⁇ cm; dT 8.8; dG: 11.7 and dC: 7.3 for both regular oligonucleotides and
  • the melting curves are recorded in steps of 0.5 °C/min.
  • the T m is determined from the maximum of the 1st derivative of the plot A 260 vs
  • oligonucleotide analogues of the invention were evaluated for their stability in media containing various concentrations of fetal calf serum or adult human serum. Oligonucleotide analogs are incubated for various times, treated with protease K and then analyzed by reverse-phase or ion-exchange HPLC or by gel-electrophoresis on 20% polyacrylamide-urea denaturating gels and subsequent autoradiography. Based on the location of the modified linkage and the known length of the oligonucleotide it is possible to determine the effect on nuclease degradation depending on the particular modification of the linkage. For the cytoplasmic nucleases, an HL 60 cell line can be used.
  • oligonucleotide analogs are assessed for degradation as mentioned above for serum nucleolytic degradation. Autoradiography. results are quantitated to compare the regular oligonucleotides and macromolecules of invention.
  • endonucleases 3', 5'-exo-, and 5', 3'-exonucleases
  • the oligonucleotide analogs are incubated in defined reaction buffers specific for various selected nucleases, treated with proteinase K and then analyzed by reverse-phase or ion-exchange HPLC, or by gel-electrophoresis on 20% polyacrylamide-urea denaturating gels and subsequent autoradiography.

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Abstract

La présente invention concerne des macromolécules contenant la structure représentée par la formule générale (1) où la définition des substituants est conforme à celle donnée dans les spécifications. L'invention concerne également des composés convenant à la synthèse de ces composés, ainsi que des compositions pharmaceutiques contenant l'un de ces composés comme principe actif.
PCT/US1997/001236 1996-01-26 1997-01-24 Analogues d'oligonucleotides WO1997027206A1 (fr)

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US6919441B2 (en) 1994-03-14 2005-07-19 Aventis Pharma Deutschland Gmbh Polyamide-oligonucleotide derivatives, their preparation and use
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections

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US5386023A (en) * 1990-07-27 1995-01-31 Isis Pharmaceuticals Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling
US5393877A (en) * 1992-04-03 1995-02-28 Zeneca Limited Linkers for the synthesis of multiple oligonucleotides in seriatim from a single support attachment
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6919441B2 (en) 1994-03-14 2005-07-19 Aventis Pharma Deutschland Gmbh Polyamide-oligonucleotide derivatives, their preparation and use
US7485421B2 (en) 1994-03-14 2009-02-03 Hoechst Gmbh Polyamide-oligonucleotide derivatives, their preparation and use
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections

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