WO1997014709A1 - Antisense oligomers - Google Patents

Antisense oligomers Download PDF

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Publication number
WO1997014709A1
WO1997014709A1 PCT/EP1996/004347 EP9604347W WO9714709A1 WO 1997014709 A1 WO1997014709 A1 WO 1997014709A1 EP 9604347 W EP9604347 W EP 9604347W WO 9714709 A1 WO9714709 A1 WO 9714709A1
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hydrogen
compound
oligomer
formula
protecting group
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PCT/EP1996/004347
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French (fr)
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Robert Alan Goodnow
Steve Yik-Kai Tam
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F. Hoffmann-La Roche Ag
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Priority to AU72866/96A priority Critical patent/AU7286696A/en
Publication of WO1997014709A1 publication Critical patent/WO1997014709A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • 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
    • C12N2310/3513—Protein; Peptide

Definitions

  • Oligonucleotides are used as hybridization probes for cloning, diagnostic assays, and as indispensable primer reagents in the polymerase chain reaction (PCR) technology.
  • PCR polymerase chain reaction
  • the specificity of binding is governed by the Watson Crick base-pairing rule. This feature allows the design of an oligonucleotide reagent to be fairly straightforward, requiring only a knowledge of the target RNA or DNA sequence.
  • binding of an oligonucleotide with its complementary mRNA sequence has been studied as a new tool for inhibiting the translation process of a specific protein product.
  • Zamecnik and Stephenson demonstrated the usefulness of the approach in the inhibition of Rous Sarcoma Virus development in infected chicken fibroblasts (Zamecnik, P.C.; Stephenson, M.L. Proc. Natl Acad. Sci. U.S.A. 1978 75, 280).
  • This pioneering work has led to a blossoming of studies in the field and generated enormous interest in developing this concept for therapeutic applications. Since the oligonucleotide agent used is complementary (anti) to the sense of the genetic message contained in the mRNA target, this new approach was dubbed "antisense DNA" technology.
  • Phosphorothioates are widely known antisense compounds. These are backbone analogs in which one oxygen in the phosphodiester group is replaced by a sulfur atom. This conservative modification renders the molecules nuclease resistant and allows them to exhibit biological activities in cell cultures and in animal models. Phosphorothioate antisense drugs have entered clinical trials for evaluation as antiviral or anticancer agents.
  • oligonucleotides incorporating a few modified residues have been synthesized (Milligan, J. F. ; Matteucci, M.D. ; Martin, J. C. J. Med.
  • PNA Peptide Nucleic Acid
  • an antisense molecule with a backbone linkage having several characteristics, namely the linkage should be stable to enzymatic cleavage, should be neutral to avoid side effects associated with polyanionic structures, should have acceptable affinity and specificity in its binding with RNA, and should have a desirable physical chemical properties, for example, water solubility.
  • the antisense molecules of this invention meet these requirements.
  • antisense molecules of this invention are a new class of
  • Oligomers of formula I bind to DNA and RNA with strong affinity and base sequence
  • the water soluble oligomers of formula I are stable to extremes in acidity and basicity and to the enzymes found in blood serum.
  • the present invention is directed to a new type of oligomer which is composed of pyranosyl nucleoside monomer units connected by amide linkages. Synthesis of these compounds can be achieved by oligomerization of monomeric intermediates which are also part of this invention, using for example solid phase or solution coupling methodology. Due to the specific structures of the oligomers of this invention, the antisense molecules disclosed herein have the advantages of stability and water solubility, and avoid side effects and nonspecific protein binding associated with phosphorothioates.
  • the invention relates to an oligomer of formula
  • R 1 , R 2 and R 4 are independently hydrogen, lower alkyl or acyl
  • R 3 is hydrogen or lower alkyl
  • B is a nucleobase or a protected nucleobase
  • n 5 to 30;
  • X is NR 3 R 4 ;
  • Y is OR 3 , or NHR 3 ;
  • the invention is also directed to monomeric building blocks of formula
  • R 5 and R 6 are independently hydrogen or a hydroxy
  • protecting group or R 5 and R 6 taken together are a 1 ,2-dihydroxy protecting group, for example a ketal;
  • R 3 ' is hydrogen or lower alkyl
  • R 7 is hydrogen or an amino protecting group
  • R 8 is hydrogen or an acid protecting group
  • B is a nucleobase or a protected nucleobase.
  • the invention relates to pharmaceutical compositions which decrease the production of a target protein in a cell, which have as the active ingredient a compound of formula I in an amount effective to bind to the mRNA encoding the target protein and by this binding decrease production of the protein, and a pharmaceutically acceptable carrier.
  • oligomers of this invention are useful in any application involving antisense nucleotides.
  • the oligomer of formula I has enhanced enzymatic and chemical stability and water solubility over oligomers with modified linkages of other types while retaining binding sequence specificity to target mRNA.
  • the oligomers of this invention are useful as antisense therapeutics which bind to a target mRNA to block or decrease the production of a target protein.
  • Antisense therapeutics are used as described in Akhtar and Ivinson, Nature Genetics, 1993 4, 215.
  • a target protein may be selected which causes or contributes to an undesirable condition.
  • the nucleotide sequence of the gene or mRNA corresponding to the protein is then obtained by known methods, and oligomers complementary to part of this sequence may then be designed.
  • the nucleobase or the protected nucleobase is selected so that the antisense oligomer has a sequence of bases which are complementary to a selected target RNA.
  • antisense oligomers when made by the method of this invention as provided below, form stable antisense compounds which may then be administered to alleviate the condition associated with the presence of the target protein.
  • the oligomers of this invention are especially useful as antisense compounds for treatment of conditions related to the production of undesired or excessive proteins whether native or foreign, and are also useful to block the proliferation of viruses or cancer cells.
  • Antisense oligomers are designed to be complementary to the mRNA of a target gene, and bind to the RNA to prevent its translation, consequently reducing or preventing synthesis of the protein encoded by the target gene. Therefore, the ability to bind stably to nucleic acid under physiological conditions indicates antisense utility.
  • an antisense compound must have
  • antisense compounds must be sufficiently soluble to be physiologically effective and reach the target mRNA.
  • oligomers are also useful as PCR clamps for use in PCR technology, and have the advantage of stable and specific binding in PCR, and are also useful as probes in diagnostic tests employing nucleic acid hybridization. (See, for example, Nucleic Acids Research 23 :217, 1995).
  • the carboxy and amino terminal residues of the oligomers of this invention may be modified in any conventional way to make the compound more compatible with solid phase synthesis or to confer desirable physical chemical properties.
  • a specific application for the oligomers with linkages of this invention is in the antisense treatment of hyperpigmentation. Hyperpigmentation results from overproduction of the enzyme tyrosinase in melanocytes (pigment cells). Adding an oligomer of this invention which has the sequence corresponding to relevant portions of the mRNA encoding tyrosinase (Muller, G.; Ruppert, S.;
  • lower alkyl denotes an alkyl group containing 1 to 4 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, and the like, preferably methyl or ethyl.
  • acyl denotes an organic radical derived from an organic acid by removal of a hydroxy group.
  • An organic acid comprises, for example, an aliphatic acid such as acetic acid, propionic acid, isobutyric acid, stearic acid, oleic acid, palmitolic acid and the like; an aromatic acid such as benzoic acid or substituted benzoic acid; a heteroaromatic acid, preferably comprising a 5 to 6 membered ring containing at least one of the heteroatoms O, S or N, such as 2-furan-carboxylic acid or 2-pyridine-carboxylic acid.
  • Examples for hydroxy-lower alkyl are hydroxy ethyl or hydroxypropyl.
  • aryl denotes a group derived from an aromatic hydrocarbon which may be substituted or unsubstituted such as phenyl, para-nitrophenyl, para-bromophenyl, para-chlorophenyl, para-methylphenyl and para-methoxyphenyl and the like.
  • aralkyl is meant aryl substituted alkyl, such as benzyl and the like.
  • hydroxy protecting group means any conventional hydroxy protecting group known in the art. Exemplary of such hydroxy protecting groups are lower alkyl, acyl, lower alkanol, aryl, aralkyl, trimethylsilyl ether, triethylsilyl ether,
  • amino protecting group means any conventional amino protecting group such as, for example, 9-fluorenylmethoxycarbonyl (F-moc), tert-butyloxycarbonyl (t-Boc), benzyloxycarbonyl, allyloxy carbonyl, triphenylmethyl and 4,4'-dimethyloxytrityl (DMT), preferably Fmoc and t-Boc.
  • acid protecting group means any conventional acid protecting group known in the art. Exemplary of such acid protecting groups are lower alkyl, benzyl, phenyl, 2-trimethylsilyl-ethyl, preferably benzyl or tert-butyl.
  • the nucleobases may include any combination of the known natural or modified nucleobases.
  • Preferred nucleobases are the natural ones, such as, adenine, cytosine, guanine, thymine, and uracil.
  • Modified bases which are known in the art, such as, 5-( 1 -propynyl)uracil, 5-( 1 -propynyl)cytosine, inosine, 5-methyl-cytosine and 2,6-diaminopurine may be used.
  • the sequence of bases for an antisense oligomer is selected such that the oligomer presents a base sequence that is complementary to a selected mRNA.
  • the mRNA sequence may be determined on the basis that the mRNA encodes a protein whose translation is desired to be blocked in order to reduce or eliminate production of that protein to alleviate undesired effects caused in a subject by the presence of the protein.
  • mRNA sequences which are not already known may be determined by translating the sequence of the encoded protein using the genetic code. If the protein sequence is not known, then it may be determined by isolating and sequencing the protein by known techniques. Alternatively, the mRNA or DNA encoding the protein may be identified, isolated, and sequenced by known methods.
  • protected nucleobase means a base as defined above which is protected by a conventional nucleoside protecting group known in the art.
  • Examples of a protected nucleobase include N-benzoyl- or N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, or N-acetyl- or N-isobutyrylguanine.
  • the various substituents are illustrated as joined to the nucleus by one of the following notations: a wedged solid line indicating a substituent which is above the plane of the molecule ( ⁇ -orientation) and a wedged dotted line indicating a substituent which is below the plane of the molecule ( ⁇ -orientation) .
  • the present invention encompasses all four combinations of isomers at the C-3 and C-4 positions of
  • R 1 , R 2 and R 4 are independently hydrogen, methyl, ethyl or acetyl; R 3 is hydrogen or methyl; B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyryl-guanine; n is 5 to 20; X is NH 2 or NHAc wherein Ac is acetyl; Y is hydroxy, OCH 3 , NH 2 or NHCH 3 .
  • R 1 and R 2 are independently hydrogen, methyl, ethyl or acetyl; R 3 is hydrogen or methyl; B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine,
  • oligomers of this invention may be prepared accordingly using intermediates of this invention. These intermediates are prepared and then appropriately linked together by the method below, which is also part of this invention.
  • the RNA binding property of the oligomer may be determined by conventional thermal melting techniques.
  • the invention also relates to compounds of formula II which are monomeric building blocks in the synthesis of oligomers of formula I.
  • compounds of formula II which are monomeric building blocks in the synthesis of oligomers of formula I.
  • R 5 and R 6 are independently hydrogen, methyl, ethyl, acetyl or a trisubstituted silyl ether, such as triethylsilyl, isopropyldimethylsilyl or tert-butyldimethylsilyl.
  • R 3 ' is hydrogen or lower alkyl;
  • R 7 is
  • R 8 is hydrogen, tert-butyl or benzyl;
  • B is preferably thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetyl-cytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetyl-guanine or N-isobutyrylguanine.
  • R 5 and R 6 are independently trisubstituted silylether with tert-butyldimethylsilyl being particularly preferred;
  • R 3 ' is hydrogen;
  • R 7 is 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl;
  • R 8 is benzyl or hydrogen and B is thymine, uracil, N-benzoyl-cytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine.
  • the monomer of formula II has preferably a stereochemistry as shown in the compound of formula IIa
  • B' is a protected nucleobase
  • R 3 ' is hydrogen or lower alkyl
  • R 7 ' is an amine protecting group
  • Rs is an acid protecting group
  • R 9 is an alcohol protecting group which reacts
  • a compound of formula IV is de-acetylated to the
  • trifluoroacetamide of formula V is hydrolyzed by conventional methods such as, reaction with a base, preferably 30% aqueous ammonium hydroxide to give the corresponding compound of formula VI.
  • R 3 is hydrogen
  • a compound of formula VI is converted to the corresponding compound of formula VII by reaction with N-(9-fluorenylmethoxycarbonyloxy)succinimide in a solvent such as aqueous dimethylformamide in the presence of
  • cyanoborohydride in a polar solvent, preferably methanol, then followed by reaction with N-(9-fluorenylmethoxycarbonyloxy)-succinimide in a solvent such as aqueous dimethylformamide in the presence of NaHCO 3 .
  • the compound of formula VII is preferably purified by trituration with excess ether and water.
  • nucleobase B in the compound of formula VII is then protected by reaction with an activated acyl donating group such as benzoic anhydride or acetyl chloride in an anhydrous solvent such as pyridine, to give the corresponding compound of formula VIII.
  • an activated acyl donating group such as benzoic anhydride or acetyl chloride in an anhydrous solvent such as pyridine
  • the preferred method is by reaction of formula VIII with excess silylating agent, such as chlorotrialkylsilane or trialkylsilyl trifluoromethanesulfonate in the presence of a base, specifically, with an excess of both tert-butyldimethylsilyl trifluoromethanesulfonate and 2,6-lutidine or with an excess of both isopropyldimethylsilyl chloride and imidazole in an anhydrous solvent such as CH 2 Cl 2 .
  • silylating agent such as chlorotrialkylsilane or trialkylsilyl trifluoromethanesulfonate
  • the protecting group of the primary alcohol in a compound of formula IX is then selectively removed by conventional methods to give the corresponding compound of formula X.
  • silyl ether is the hydroxy protecting group
  • transformation can be carried out by reaction with camphor sulfonic acid in a solvent such as methanolic CH 2 Cl 2 .
  • the primary alcohol in a compound of formula X is then oxidized to a carboxylic acid to give the corresponding compound of formula Ile for example, by reaction with aqueous sodium hypochlorite (NaClO) solution in the presence of an aprotic aqueous-immiscible solvent such as aqueous CH 2 Cl 2 and 2,2,6,6-tetramethyl- 1 -piperidinyloxy free radical followed by reaction with sodium chlorite (NaClO 2 ) in a solvent such as aqueous tert-butanol.
  • NaClO sodium hypochlorite
  • protecting group such as, a benzyl group.
  • a compound of formula XII a known compound or compound prepared by known methods, is converted to a corresponding compound of formula XIII, for example, by reaction with phthalic anhydride in an anhydrous solvent such as pyridine, followed by reaction with excess tert-butyldimethylsilyl chloride and subsequently reaction with acetic anhydride .
  • a compound of formula XIII is selectively deprotected to the corresponding compound of formula XIV by reaction with acidic catalysis in a protic solvent, preferably acid resin in CH 3 OH.
  • the resultant primary alcohol XIV is oxidized, preferably with ruthenium chloride catalyzed sodium periodate in a mixed solvent system of H 2 O/CH 3 CN/CCl 4 .
  • the resulting carboxylic acid is protected with a conventional acid protecting group by conventional means, such as with CH 3 I in DMF in the presence of NaHCO 3 giving compound of Formula XVI.
  • a compound of formula XVI is converted to the corresponding compound of formula XVII by reaction with a Lewis acid catalyst in the presence of persilylated, protected nucleobase in an anhydrous, aprotic solvent, preferably with SnCl 4 or trimethylsilyl trifluoromethansulfonate in CH 3 CN or ClCH 2 CH 2 Cl at a temperature of 25-80°C.
  • a compound of formula XIX is converted to the corresponding compound of formula XX by reaction with 1 equivalent of an aldehyde and sodium cyanoborohydride in a polar solvent, preferably methanol, then followed by reaction with N-(9-fluorenylmethoxycarbonyloxy)-succinimide in a solvent such as aqueous dimethylformamide in the presence of NaHCO 3 .
  • nucleobase B in the compound of formula XX is converted to its protected form by reaction with excess activated acyl donating group such as benzoic anhydride or acetyl chloride in an anhydrous solvent such as 1 ,4-dioxane to give a compound of formula XXI in which B' is a protected nucleobase.
  • activated acyl donating group such as benzoic anhydride or acetyl chloride in an anhydrous solvent such as 1 ,4-dioxane
  • a compound of formula XXI is converted to the corresponding compound of formula IIe by reaction with a conventional hydroxy protecting group reagent under conditions known in the art to effect selective reaction at hydroxy groups.
  • the preferred method is by reaction with excess silylating agent, such as chlorotrialkylsilane in the presence of a base, specifically, with an excess of both triethylsilyl chloride and imidazole in a polar solvent such as dimethylformamide.
  • a compound of formula Ile is converted to the corresponding compound of formula IIf by conventional methods of adding an acid protecting group, preferably by reaction with benzyl bromide in presence of NaHCO 3 in dimethylformamide.
  • substitution of X is determined by the cleavage of the final amine protecting group after completion of the
  • the resultant product of formula I is isolated by conventional reversed phase and/or size exclusion chromatography.
  • R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 , B and X are as described above and R 8 ' is an acid protecting group.
  • a compound of formula IIf is deprotected at the amino terminus and the resulting compound is coupled to another moiety represented by formula II using a coupling agent, such as [O-(7-azabenzotriazol- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium hexafluoro-phosphate] in a polar solvent such as dimethylformamide to form an oligomer of formula XXII.
  • a coupling agent such as [O-(7-azabenzotriazol- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium hexafluoro-phosphate] in a polar solvent such as dimethylformamide to form an oligomer of formula XXII.
  • the resultant coupling product of formula XXII can be isolated by conventional means such as chromatographic separations, preferably by silica gel flash column chromatography.
  • An oligomer of formula XXII is further elongated by repetition of the procedure described for the conversion of formula IIf to an oligomer of formula XXII.
  • the substitution of X is determined by the cleavage of the final amine protecting group after completion of the oligomerization process followed by modification of the free amine where necessary, specifically for R 3 is lower alkyl, for example, by reaction with 1 equivalent of a lower alkylaldehyde and sodium cyanoborohydride in a polar solvent such as
  • An oligomer of formula I is obtained by removal of the silyl or acyl hydroxy protecting groups R 5 and R 6 and by
  • a substituted or unsubstituted carboxamide may also be obtained by coupling the free acid oligomer (R 8 is H) with the appropriate amine under amide bond forming conditions.
  • the resultant product of formula I is isolated by conventional chromatographic means, preferably by HPLC.
  • acceptable salts can be used as a medicament, for example in the form of a pharmaceutical preparation.
  • the invention relates to a pharmaceutical preparation comprising an oligomer of formula I and a pharmaceutically inert carrier.
  • a preparation which decreases the production of a target protein in a cell which comprises any of the above-described compounds of formula I in an amount effective to bind to the nucleic acid (for example mRNA) sequence encoding the target protein in said cell and thereby decrease production of said protein, and a pharmaceutically acceptable carrier.
  • the dose ranges for the administration of the antisense oligomers may be determined by those of ordinary skill in the art without undue experimentation. In general, appropriate dosages are those which are large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of disease in the patient, counter-indications, if any, immune tolerance and other such variables, to be adjusted by the individual physician.
  • the antisense oligomers can be
  • parenterally by injection or by gradual infusion over time. They can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously or orally. Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers are propylene glycol,
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • carriers capable of transporting oligomers to cells and into cells may be used, for example liposomes, PEGylated
  • liposomes or cationic lipids.
  • Preservatives and other additives may also be present, such as anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See, generally,
  • the present invention also relates to a process for the manufacture of a medicament, especially for decreasing the production or a target protein in a cell, which process comprises mixing a compound of formula I or a pharmaceutically acceptable salt thereof with a therapeutically inert carrier material and bringing the mixture into a galenical administration form.
  • the reaction mixture was cooled and 0.756 g of NaHCO 3 followed by 5 mL of H 2 O was added. The reaction mixture was stirred for an additional hour. At this time, the suspension was diluted with addition of 50 mL each of CH 2 Cl 2 and H 2 O. The suspension was filtered and the solid was rinsed with CH 2 Cl 2 . The filtrate was extracted successively with saturated, aqueous NaHCO 3 and brine. The organic layer was dried over MgSO 4 , filtered, and the filtrate concentrated in vacuo resulting in 2.14 g of a beige foam.
  • the layers were separated and the organic layer was twice extracted with H 2 O .
  • the aqueous layers were back extracted once with EtOAc.
  • the organic layers were combined, washed with brine, dried with
  • the desired carboxylic acid product 1 ,2-dideoxy- 1 -[ 1 ,2-dihydro-5-methyl]-2,4-dioxo- 1 -pyrimidinyl)- [ [(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3 ,4-bis-O- [( 1 , 1 -dimethylethyl)-dimethylsilyl]- ⁇ -D-glucopyranuronic acid was obtained by chromatography of the residue from a silica gel flash column eluted with 4 to 10% CH 3 OH/CHCl 3 in 62% yield (0.51 g).
  • Steps a)-c) were performed as set forth in Example 1A.
  • reaction mixture was brought to room temperature after 5 minutes. After 90 minutes, another portion (0.93 mL, 5.9 mmol) of chloroisopropyl-dimethylsilane was added. After 30 minutes, TLC indicated the conversion of starting material to a single product. The reaction was terminated by dilution with 50 mL of
  • cytosine building block a To a suspension of N-benzoylcytosine (29.1 1 g, 135.40 mmol) in 250 mL of distilled ClCH 2 CH 2 Cl was added 66.80 ml (270.82 mmol) of BSA. This suspension was heated to a gentle reflux for 40 min during which time a clear solution was effected.
  • the suspension was filtered and washed through a pad of celite with CHCl 3 .
  • the filtrate was washed with saturated, aqueous NaHCO 3 and brine.
  • the organic layer was further treated with charcoal before filtration through a pad of celite.
  • the filtrate was dried over
  • the reaction was stirred at 0°C for 2 h before quenching the reaction with saturated aqueous NaHCO 3 and dilution of the mixture with CH 2 Cl 2 .
  • the organic layer was twice extracted with saturated, aqueous NaHCO 3 ; the combined aqueous layers are extracted once with CH 2 Cl 2 .
  • the combined organic layers are washed with brine, dried over
  • the reaction mixture was cooled to -4°C with stirring and 0.0006 g (0.0037 mmol) of TEMPO was added. To this mixture was added in 3 portions separated by 10 min a solution containing 0.48 mL of 1.24 M NaClO, 0.48 mL saturated NaHCO 3 , and 0.48 mL saturated NaCl. The temperature was raised to 0°C and the reaction was stirred for 1 h. At this point, the reaction was terminated by dilution with CH 2 Cl 2 (50 mL) and the organic solution was twice extracted with H 2 O. The aqueous layers were extracted once with CH 2 Cl 2 . The combined organic layers were washed with brine, dried with MgSO 4 , filtered and concentrated in vacuo.
  • the combined organic layers were combined, washed with brine, dried with MgSO 4 , filtered and concentrated in vacuo to a white foam residue.
  • the desired carboxylic acid product 1 ,2-dideoxy- 1 - [ 1 ,2-dihydro-2-oxo-4-[(phenylcarbonyl )amino]- 1 -pyrimidinyl] - [3 ,4-bis-O- [( 1 , 1 -dimethylethyl )dimethylsilyl ] -2 [ [(9 H- fluoren-9-ylmethoxy)-carbonyl]amino]- ⁇ -D-glucopyranuronic acid was obtained by chromatography of the residue from a silica gel flash column eluted with CHCl 3 and 1 to 10% CH 3 OH/CHCl 3 in 93% yield (0.29 g).
  • the reaction mixture was cooled to -4°C with stirring and 0.0008 g (0.0052 mmol) of TEMPO was added. To this mixture was added in 3 portions separated by 10 min a solution containing 0.47 mL of 1.24 M NaClO and 0.50 mL saturated NaHCO 3 . At this point, the temperature was raised to 0°C and the reaction was stirred for 2 h. At this point, the reaction was terminated by dilution with CHCl 3 (50 mL) and the organic solution was twice extracted with H 2 O. The organic layers were combined, washed with brine, dried with MgSO 4 , filtered and concentrated to dryness in vacuo.
  • the yellow solid was partitioned between a two phase solvent mixture composed of 200 mL of H 2 O and 500 mL of CHCl 3 /ethanol (3 to 2 vol/vol). The aqueous layer was extracted twice more with the solvent mixture of CHCl 3 / ethanol (3 to 2 vol/vol). The combined organic extracts were evaporated in vacuo and the residue was dissolved in a solvent mixture composed of 40 mL of acetonitrile, 40 mL of acetic acid, and 40 mL of H 2 O. After 15 minutes, the mixture was evaporated to dryness yielding a slightly yellow solid. This solid was chromatographed on a silica gel drip column eluted with a quaternary solvent system
  • the reaction mixture was cooled to 0°C with stirring and 0.0006 g of TEMPO was added. To this mixture was added in one portion a solution containing 1.09 mL of 1.04 M NaClO, 1.50 mL saturated NaHCO 3 , and 2.37 mL of H 2 O. The reaction was stirred for 1 hour at 0°C . At this point, the reaction was terminated by dilution with CH 2 Cl 2 (50 mL) and the aqueous layer was separated. The aqueous layer was extracted once with CH 2 Cl 2 . The combined organic layers were concentrated in vacuo.
  • Example 5b was added 0.50 mL of 20% piperidine in DMF. This solution was stirred at room temperature for 20 min before concentration of the reaction mixture to dryness in vacuo. The residue was redissolved in 1 mL of DMF and concentrated again to dryness in vacuo three times.
  • Fmoc-Lys(e-Boc)-Knorr-BHA-polystyrene resin was added to a freshly silylated solid phase peptide micro reactor 50 mg of 0.1 mmol/g (5.0 mmol) charged Fmoc-Lys(e-Boc)-Knorr-BHA-polystyrene resin was added. The Fmoc protecting group was removed by three successive treatments with 0.25 mL of 20% piperidine in DMF for one min, seven min and seven min
  • the final Fmoc group was removed in the same manner as described above.
  • the free amino resin was then treated with TFA/CH 2 Cl 2 ( 1 : 1 ) for 1 hour.
  • the resin was removed from the solvent by filtration through cotton and the filtrate was concentrated to dryness in vacuo.
  • the residue was suspended in Et 2 O and then centrifuged; the Et 2 O layer was discarded. This Et 2 O procedure was repeated twice further.
  • the residual solid was then treated for 12 h at room temperature for 0.3 mL of 30% (wt/vol) NH 4 OH. At this time, the reaction mixture was concentrated to dryness in vacuo. At this time, the reaction mixture was concentrated to dryness in vacuo.
  • the residue was treated with 1 mL of 1.0 molar
  • thermodynamic constants were obtained from fits of data to a two-state model with linear sloping baselines (Petersheim, M. ; Turner, D.H. Biochemistry 1983, 22, 256-263).
  • Total strand concentration was 3mM in a buffer of 100 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 7.0. "Up” indicates the melting temperature; “Down” indicates the annealing temperature .
  • oligonucleotide of formula I As shown in Table I, the binding of oligonucleotide of formula I, Example 6 (H 2 N-Lys-TCTCTCTCCTTCT-H) to RNA and DNA is compared to the binding of DNA to DNA and RNA to DNA. A higher melting temperature indicates a tighter association of the indicated duplex. From the data in Table I, it is clear that an oligonucleotide of formula I (Example 6) binds with greatest affinity to a complementary segment of RNA in an antiparallel fashion (carboxamide terminal of compound of Example 6, binding to the 5' end of an oligo). This strength in binding is similar to that of DNA binding activity.
  • the melting temperatures determined by heating and cooling (up and down, respectively) are similar. The similarity of a melting temperature despite the direction of the change in temperature during these measurement is indicative of an association between two oligomers. EXAMPLE 8
  • Tm's Melting Temperatures
  • selectivity As is seen from the data in Table II, the oligomer of Example 6 binds in a selective fashion in the Watson-Crick complementary base-pairing manner.
  • Table II Numbers are for melting temperature (T m 's) in degrees Celsius. Total strand concentration was 3 ⁇ M . Strands are bound in the antiparallel orientation. Numbers in parenthesis indicate the annealing temperature.
  • Antisense molecules have been demonstrated to reduce levels of the enzyme tyrosinase in melanocytes by the methods described below. These antisense molecules have potential utility for treating several diseases of hyperpigmentation
  • hypomelanosis including, but not limited to café au lait macules, nevus spilus, post inflammatory mealnosis (exanthems, drug eruptions) and scleroderma.
  • a commonly used line of mouse melanoma cells for example,
  • B-16 is raised in cell culture plates using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 50 ⁇ g/ml gentamicin.
  • Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 50 ⁇ g/ml gentamicin.
  • antisense and control oligonucleotides are added to low density (that is, subconfluent) cell cultures, then treatment is continued for several days. Cells are then rinsed with saline, collected by scraping, then cells are extracted and analyzed for levels of protein by the
  • tyrosinase levels levels of the target enzyme tyrosinase from a constant amount of extracted protein is measured essentially as described by a published procedure (Pomerantz, S. H. J. Biol. Chem. 1966, 241 : 161 ). Briefly, extracts are incubated with tyrosine and the cofactor DOPA (3,4-dihydroxy-phenylalanine. ) The tyrosine is tritium labeled at the 3 and 5 positions such that tyrosinase activity is measured by the amount of tritiated water that is produced. Tritiated water is quantitated by liquid scintillation counting. The purpose of the tyrosinase assay is to establish the potency of each antisense molecule by allowing us to calculate the concentration necessary to cause 50% inhibition of tyrosinase levels (IC 50 ) .
  • melanoma cells are seeded into 96-well plates at low density, then allowed to grow in the presence of antisense and control oligonucleotides for several days. Cell proliferation is assayed by the widely used tetrazolium dye procedure (Mosmann, T. J. Immunol. Meth.
  • each oligonucleotide producing 50% inhibition of growth rate is compared to the concentration of that oligonucleotide which produces 50% inhibition of tyrosinase level.
  • Antisense effects are indicated by the following conditions: a) active oligonucleotides are complementary in sequence to the target messenger RNA, b) sense and mismatched controls are less active than fully matched antisense oligonucleotides, c) tyrosinase activity is inhibited by concentrations of antisense oligonucleotides which do not suppress cell growth.
  • the selectivity of the antisense mechanism of these antisense molecules is determined by measuring the enzyme levels of two non-targetted cellular enzymes: alkaline phosphotase and tyrosine kinase.

Abstract

Antisense oligomers of formula (I) wherein R1, R2 and R4 are independently hydrogen, lower alkyl or acyl; R3 is hydrogen or lower alkyl; B is a nucleobase or a protected nucleobase; n is 5 to 30; X is NR3R4; Y is OR3, or NHR3; as well as, pharmaceutically acceptable salts thereof.

Description

ANTISENSE OLIGOMERS
The highly specific interaction between synthetic oligonucleotides and RNA or single-stranded DNA has led to many applications in molecular biology. Oligonucleotides are used as hybridization probes for cloning, diagnostic assays, and as indispensable primer reagents in the polymerase chain reaction (PCR) technology. The specificity of binding is governed by the Watson Crick base-pairing rule. This feature allows the design of an oligonucleotide reagent to be fairly straightforward, requiring only a knowledge of the target RNA or DNA sequence. As a further extension of this attractive principle, binding of an oligonucleotide with its complementary mRNA sequence has been studied as a new tool for inhibiting the translation process of a specific protein product. In 1978, Zamecnik and Stephenson demonstrated the usefulness of the approach in the inhibition of Rous Sarcoma Virus development in infected chicken fibroblasts (Zamecnik, P.C.; Stephenson, M.L. Proc. Natl Acad. Sci. U.S.A. 1978 75, 280). This pioneering work has led to a blossoming of studies in the field and generated enormous interest in developing this concept for therapeutic applications. Since the oligonucleotide agent used is complementary (anti) to the sense of the genetic message contained in the mRNA target, this new approach was dubbed "antisense DNA" technology.
Phosphorothioates are widely known antisense compounds. These are backbone analogs in which one oxygen in the phosphodiester group is replaced by a sulfur atom. This conservative modification renders the molecules nuclease resistant and allows them to exhibit biological activities in cell cultures and in animal models. Phosphorothioate antisense drugs have entered clinical trials for evaluation as antiviral or anticancer agents.
However, while the potential of phosphorothioate antisense drugs remains promising, there is a need for new types of antisense agents due to side effects. Specifically, non-antisense side activities are exhibited by the phosphorothioates because their polythioate backbones are ionic. These side activities affect protein binding (Stein, C.A.; Narayanan, R. Current Opinion in Oncology, Vol. 6, No. 6, pgs. 587-94 ( 1994) ), activation of immune cells and transcription factor(s) (Branda, R.F. ; Moore, A.L.;
Mathews, L.; McCormack, J.J.; Zon, G. Biochem. Pharmacol. 1993, 8, 33; and McIntyre, K.W. et al. Antisense Res. Devel, 1993, 3, 309), and RNase H cleavage of non-target sequences due to partial complementary binding (Giles, R.V.; Spiller, D.G. ; Tidd, D.M.
Anticancer Drug Design 1993, 8, 33).
To reduce non-antisense activities due to ionic backbones, oligonucleotides incorporating a few modified residues have been synthesized (Milligan, J. F. ; Matteucci, M.D. ; Martin, J. C. J. Med.
Chem. 1993, 36, 1923 and Carbohydrate Modifications in
Antisense Research: Sanghvi, Y.S., Cook, P. D. Eds. : ACS: Washington, D. C. ( 1994)). Specifically, residues in which the phosphodiester linkage has been replaced with an amide have been synthesized (Burgess, K.; Gibbs, R.A.; Metzker, M.L.; Raghavachari, R. J.. C.S.
Chem. Commun. 1994, 915; Chur, A.; Hoist, B.; Dahl, O.; Valentin-Hansen, P.; Pedersen, E.B. Nucleic Acid Res. 1993, 21 , 5179; Idziak, I.; Just, G.; Damha, M.; Giannaris, P. Tet. Lett. 1993, 34, 5417; De Mesmaeker,A.; Waldner, A.; Lebreton, J.; Hoffmann, P.; Fritsch, N.; Wolf, R.; Freier, S. Angew. Chem. Intl. Ed. Engl. 1994, 33, 226).
Incorporation of these modified linkages into a single stranded DΝA molecule usually results in a lowering of binding affinity with either a complementary RΝA or DΝA. However, no example of a total replacement of the phosphodiester groups by amide groups has been reported. Another type of analog with higher binding affinity is the Peptide Nucleic Acid (PNA) (Nielsen, P.; Egholm, M.; Berg, R.;
Buchardt, O. Science, 1991 , 254, 1497). These molecules contain the familiar nucleic acid bases - adenine, guanine, thymine and cytosine - linked to a peptide backbone rather than the sugar-phosphate backbone. Although this type of compound has high binding affinity with DNA and RNA, this type of compound also shows poor specificity by binding to antiparallel as well as to parallel complementary sequences of RNAs. Moreover, the poor water solubility of these molecules has become a drawback to their practical application.
Thus, there is a need for an antisense molecule with a backbone linkage having several characteristics, namely the linkage should be stable to enzymatic cleavage, should be neutral to avoid side effects associated with polyanionic structures, should have acceptable affinity and specificity in its binding with RNA, and should have a desirable physical chemical properties, for example, water solubility. The antisense molecules of this invention meet these requirements. In contrast to existing art, antisense molecules of this invention are a new class of
oligoribonucleotides comprising pyranosyl nucleoside building blocks connected by amide linkages. Oligomers of formula I bind to DNA and RNA with strong affinity and base sequence
selectivity. The water soluble oligomers of formula I are stable to extremes in acidity and basicity and to the enzymes found in blood serum. The present invention is directed to a new type of oligomer which is composed of pyranosyl nucleoside monomer units connected by amide linkages. Synthesis of these compounds can be achieved by oligomerization of monomeric intermediates which are also part of this invention, using for example solid phase or solution coupling methodology. Due to the specific structures of the oligomers of this invention, the antisense molecules disclosed herein have the advantages of stability and water solubility, and avoid side effects and nonspecific protein binding associated with phosphorothioates.
Accordingly, in a first aspect the invention relates to an oligomer of formula
Figure imgf000006_0001
wherein
R 1 , R 2 and R4 are independently hydrogen, lower alkyl or acyl;
R3 is hydrogen or lower alkyl;
B is a nucleobase or a protected nucleobase;
n is 5 to 30; X is NR3R4;
Y is OR3, or NHR3;
and pharmaceutically acceptable salts thereof.
In another aspect, the invention is also directed to monomeric building blocks of formula
Figure imgf000007_0001
wherein
R 5 and R6 are independently hydrogen or a hydroxy
protecting group or R5 and R6 taken together are a 1 ,2-dihydroxy protecting group, for example a ketal;
R3' is hydrogen or lower alkyl;
R7 is hydrogen or an amino protecting group;
R8 is hydrogen or an acid protecting group;
and B is a nucleobase or a protected nucleobase.
In yet another aspect, the invention relates to pharmaceutical compositions which decrease the production of a target protein in a cell, which have as the active ingredient a compound of formula I in an amount effective to bind to the mRNA encoding the target protein and by this binding decrease production of the protein, and a pharmaceutically acceptable carrier.
The oligomers of this invention are useful in any application involving antisense nucleotides. In particular, the oligomer of formula I has enhanced enzymatic and chemical stability and water solubility over oligomers with modified linkages of other types while retaining binding sequence specificity to target mRNA. In addition, it is possible by means of this invention to obtain oligomers all of whose component nucleosides are
connected by amide linkages. Because of their amide linkage, the present oligomers can be assembled into antisense compounds which are stable, water soluble, and in particular nuclease resistant. Thus, the oligomers of this invention are useful as antisense therapeutics which bind to a target mRNA to block or decrease the production of a target protein. Antisense therapeutics are used as described in Akhtar and Ivinson, Nature Genetics, 1993 4, 215. As an example of designing an antisense oligomer, a target protein may be selected which causes or contributes to an undesirable condition. The nucleotide sequence of the gene or mRNA corresponding to the protein is then obtained by known methods, and oligomers complementary to part of this sequence may then be designed. A preferred size for such oligomers is in the range of about 2 to about 28, especially about 7 to about 22 monomers (n = 5 to 20). Preferably, the nucleobase or the protected nucleobase is selected so that the antisense oligomer has a sequence of bases which are complementary to a selected target RNA.
These antisense oligomers, when made by the method of this invention as provided below, form stable antisense compounds which may then be administered to alleviate the condition associated with the presence of the target protein. The oligomers of this invention are especially useful as antisense compounds for treatment of conditions related to the production of undesired or excessive proteins whether native or foreign, and are also useful to block the proliferation of viruses or cancer cells. Antisense oligomers are designed to be complementary to the mRNA of a target gene, and bind to the RNA to prevent its translation, consequently reducing or preventing synthesis of the protein encoded by the target gene. Therefore, the ability to bind stably to nucleic acid under physiological conditions indicates antisense utility. In addition, an antisense compound must have
appropriate sequence binding specificity in order to bind to the specific mRNAs of a target gene. Finally, antisense compounds must be sufficiently soluble to be physiologically effective and reach the target mRNA. These oligomers are also useful as PCR clamps for use in PCR technology, and have the advantage of stable and specific binding in PCR, and are also useful as probes in diagnostic tests employing nucleic acid hybridization. (See, for example, Nucleic Acids Research 23 :217, 1995).
The carboxy and amino terminal residues of the oligomers of this invention may be modified in any conventional way to make the compound more compatible with solid phase synthesis or to confer desirable physical chemical properties. A specific application for the oligomers with linkages of this invention is in the antisense treatment of hyperpigmentation. Hyperpigmentation results from overproduction of the enzyme tyrosinase in melanocytes (pigment cells). Adding an oligomer of this invention which has the sequence corresponding to relevant portions of the mRNA encoding tyrosinase (Muller, G.; Ruppert, S.;
Schmid, E.; Schutz, G. EMBO 1988, 7, 2723-30) inhibits tyrosinase production, thus eliminating hyperpigmentation in affected cells (Ando, S.; Ando, O.; Suemoto, Y.; Mishima, Y. J. Invest. Dermatol. 1993, 100, 1505- 1555) (Kuzumaki, T.; Matsuda, A.; Wakamatsu, K.; Ito, S.; Ishikawa, K. Expt. Cell Res. 1993, 207, 33-40). The efficacy of an antisense oligomer for reducing hyperpigmentation may be determined using a cell-based antisense assay, for example the assay of Example 1. As used herein, the term "lower alkyl" denotes an alkyl group containing 1 to 4 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, and the like, preferably methyl or ethyl. The term "acyl" denotes an organic radical derived from an organic acid by removal of a hydroxy group. An organic acid comprises, for example, an aliphatic acid such as acetic acid, propionic acid, isobutyric acid, stearic acid, oleic acid, palmitolic acid and the like; an aromatic acid such as benzoic acid or substituted benzoic acid; a heteroaromatic acid, preferably comprising a 5 to 6 membered ring containing at least one of the heteroatoms O, S or N, such as 2-furan-carboxylic acid or 2-pyridine-carboxylic acid. Examples for hydroxy-lower alkyl are hydroxy ethyl or hydroxypropyl. The term "aryl" denotes a group derived from an aromatic hydrocarbon which may be substituted or unsubstituted such as phenyl, para-nitrophenyl, para-bromophenyl, para-chlorophenyl, para-methylphenyl and para-methoxyphenyl and the like. By aralkyl is meant aryl substituted alkyl, such as benzyl and the like.
The term "hydroxy protecting group" means any conventional hydroxy protecting group known in the art. Exemplary of such hydroxy protecting groups are lower alkyl, acyl, lower alkanol, aryl, aralkyl, trimethylsilyl ether, triethylsilyl ether,
isopropyldimethylsilyl ether, tert-butyldimethylsilyl ether and the like.
The term "amino protecting group" means any conventional amino protecting group such as, for example, 9-fluorenylmethoxycarbonyl (F-moc), tert-butyloxycarbonyl (t-Boc), benzyloxycarbonyl, allyloxy carbonyl, triphenylmethyl and 4,4'-dimethyloxytrityl (DMT), preferably Fmoc and t-Boc.
The term "acid protecting group" means any conventional acid protecting group known in the art. Exemplary of such acid protecting groups are lower alkyl, benzyl, phenyl, 2-trimethylsilyl-ethyl, preferably benzyl or tert-butyl.
The nucleobases may include any combination of the known natural or modified nucleobases. Preferred nucleobases are the natural ones, such as, adenine, cytosine, guanine, thymine, and uracil. Modified bases which are known in the art, such as, 5-( 1 -propynyl)uracil, 5-( 1 -propynyl)cytosine, inosine, 5-methyl-cytosine and 2,6-diaminopurine may be used. The sequence of bases for an antisense oligomer is selected such that the oligomer presents a base sequence that is complementary to a selected mRNA. As noted above, the mRNA sequence may be determined on the basis that the mRNA encodes a protein whose translation is desired to be blocked in order to reduce or eliminate production of that protein to alleviate undesired effects caused in a subject by the presence of the protein. mRNA sequences which are not already known may be determined by translating the sequence of the encoded protein using the genetic code. If the protein sequence is not known, then it may be determined by isolating and sequencing the protein by known techniques. Alternatively, the mRNA or DNA encoding the protein may be identified, isolated, and sequenced by known methods.
The term protected nucleobase means a base as defined above which is protected by a conventional nucleoside protecting group known in the art. Examples of a protected nucleobase include N-benzoyl- or N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, or N-acetyl- or N-isobutyrylguanine. In the formulas presented herein, the various substituents are illustrated as joined to the nucleus by one of the following notations: a wedged solid line
Figure imgf000011_0001
indicating a substituent which is above the plane of the molecule (β-orientation) and a wedged dotted line
Figure imgf000011_0002
indicating a substituent which is below the plane of the molecule (α-orientation) .
Accordingly, the present invention encompasses all four combinations of isomers at the C-3 and C-4 positions of
carbohydrates, as illustrated below in formulas Ia-Id and IIa-IId. The isomers of formulas Ia and IIa are preferred.
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000013_0001
Figure imgf000013_0002
In a preferred embodiment of the oligomer of formula I the substituents and number of monomers are as follows: R1 , R2 and R4 are independently hydrogen, methyl, ethyl or acetyl; R3 is hydrogen or methyl; B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyryl-guanine; n is 5 to 20; X is NH2 or NHAc wherein Ac is acetyl; Y is hydroxy, OCH3, NH2 or NHCH3. In a particularly preferred embodiment, R 1 and R2 are
independently hydrogen, or acetyl, X is NH2 ; Y is hydroxy or NH2 ; R3 is hydrogen or CH3 ; n is 5- 15 and B is thymine, cytosine, adenine, guanine or uracil. Once the target base sequence is known, the antisense
oligomers of this invention may be prepared accordingly using intermediates of this invention. These intermediates are prepared and then appropriately linked together by the method below, which is also part of this invention. The RNA binding property of the oligomer may be determined by conventional thermal melting techniques.
The invention also relates to compounds of formula II which are monomeric building blocks in the synthesis of oligomers of formula I. In a preferred embodiments of a compound of formula
II the substituents and independently as follows: R5 and R6 are independently hydrogen, methyl, ethyl, acetyl or a trisubstituted silyl ether, such as triethylsilyl, isopropyldimethylsilyl or tert-butyldimethylsilyl. R3' is hydrogen or lower alkyl; R7 is
preferably 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl;
R 8 is hydrogen, tert-butyl or benzyl; B is preferably thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetyl-cytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetyl-guanine or N-isobutyrylguanine. In a particularly preferred embodiment of the compound of formula II, R5 and R6 are independently trisubstituted silylether with tert-butyldimethylsilyl being particularly preferred; R3 ' is hydrogen; R7 is 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl; R8 is benzyl or hydrogen and B is thymine, uracil, N-benzoyl-cytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine.
As noted above, the monomer of formula II has preferably a stereochemistry as shown in the compound of formula IIa
Figure imgf000015_0001
The compounds of formula II and oligomer of formula I are prepared as hereafter described in Schemes I. II, III and the
Examples, Scheme I being the preferred method of making compounds of formula II.
SCHEME I
Figure imgf000016_0001
wherein B' is a protected nucleobase, R3 ' is hydrogen or lower alkyl, R7' is an amine protecting group, Rs is an acid protecting group, R9 is an alcohol protecting group which reacts
preferentially with primary hydroxy groups such as tert- butyldimethylsilyl or triphenylmethyl, Ac is acetyl, TFA is trifluoroacetyl, and B, R5 , R6 and R7 are as described above. In above Scheme I, the compound of formula III, a known compound or compound prepared by known methods (see
Synthetic Procedures in Nucleic Acid Chemistry, Volume I, Edited by W. Zorbach and R.S. Tipson, Interscience Publishers, John
Wiley & Sons, Inc.: New York, 1968, pp. 323-325), is converted to the compound of formula IV by reaction with a Lewis acid catalyst such as SnCl4 or trimethylsilyl trifluoromethanesulfonate in presence of a persilylated, protected base in an anhydrous, aprotic solvent such as CH3CN or ClCH2CH2Cl solvent at a
temperature in the range of 20- 100°C, preferably at 80- 100°C.
A compound of formula IV is de-acetylated to the
corresponding compound of formula V by conventional methods such as, base catalyzed hydrolysis in protic solvents, preferably a system composed of CH3OH / H2O / (CH3CH2)3N. The
trifluoroacetamide of formula V is hydrolyzed by conventional methods such as, reaction with a base, preferably 30% aqueous ammonium hydroxide to give the corresponding compound of formula VI.
When in formula VII R3 is hydrogen, a compound of formula VI is converted to the corresponding compound of formula VII by reaction with N-(9-fluorenylmethoxycarbonyloxy)succinimide in a solvent such as aqueous dimethylformamide in the presence of
NaHCO3.
When in formula VII R3 is lower alkyl, a compound of formula VI is converted to the corresponding compound of formula VII by reaction with 1 equivalent of an aldehyde and sodium
cyanoborohydride in a polar solvent, preferably methanol, then followed by reaction with N-(9-fluorenylmethoxycarbonyloxy)-succinimide in a solvent such as aqueous dimethylformamide in the presence of NaHCO3. The compound of formula VII is preferably purified by trituration with excess ether and water.
The nucleobase B in the compound of formula VII is then protected by reaction with an activated acyl donating group such as benzoic anhydride or acetyl chloride in an anhydrous solvent such as pyridine, to give the corresponding compound of formula VIII. A compound of formula VIII is converted to the
corresponding compound of formula IX by reaction with a primary hydroxy selective protecting group reagent, followed by reaction with a conventional hydroxy protecting group reagent, such as alkyl halide, acetic anhydride or trisubstituted silyl ether. The preferred method is by reaction of formula VIII with excess silylating agent, such as chlorotrialkylsilane or trialkylsilyl trifluoromethanesulfonate in the presence of a base, specifically, with an excess of both tert-butyldimethylsilyl trifluoromethanesulfonate and 2,6-lutidine or with an excess of both isopropyldimethylsilyl chloride and imidazole in an anhydrous solvent such as CH2Cl2.
The protecting group of the primary alcohol in a compound of formula IX is then selectively removed by conventional methods to give the corresponding compound of formula X. For example, when silyl ether is the hydroxy protecting group, this
transformation can be carried out by reaction with camphor sulfonic acid in a solvent such as methanolic CH2Cl2. The primary alcohol in a compound of formula X is then oxidized to a carboxylic acid to give the corresponding compound of formula Ile for example, by reaction with aqueous sodium hypochlorite (NaClO) solution in the presence of an aprotic aqueous-immiscible solvent such as aqueous CH2Cl2 and 2,2,6,6-tetramethyl- 1 -piperidinyloxy free radical followed by reaction with sodium chlorite (NaClO2) in a solvent such as aqueous tert-butanol. The synthesis of a compound of formula IIe by oxidation of a compound of formula X as described before is a preferred embodiment.
A carboxylic acid compound of formula Ile is converted to the corresponding compound of formula IIf by conditions known in the art to effect acid group protection by adding an acid
protecting group, such as, a benzyl group.
A compound of formula IIf can be deprotected to the
corresponding free amine wherein R7 is hydrogen by
conventional methods. It is also possible to synthesize the monomer building blocks of formula II according to Scheme II.
Figure imgf000020_0001
wherein Ac is acetyl, tBDMSO is tert-butyldimethylsilyl ether, Phth is phthalimide, R8 is an acid protecting group, B' is a protected nucleobase, and B, R3 ', R5, R6, and R7 are as described above .
As set forth in Scheme II, a compound of formula XII, a known compound or compound prepared by known methods, is converted to a corresponding compound of formula XIII, for example, by reaction with phthalic anhydride in an anhydrous solvent such as pyridine, followed by reaction with excess tert-butyldimethylsilyl chloride and subsequently reaction with acetic anhydride .
A compound of formula XIII is selectively deprotected to the corresponding compound of formula XIV by reaction with acidic catalysis in a protic solvent, preferably acid resin in CH3OH. The resultant primary alcohol XIV is oxidized, preferably with ruthenium chloride catalyzed sodium periodate in a mixed solvent system of H2O/CH3CN/CCl4. The resulting carboxylic acid is protected with a conventional acid protecting group by conventional means, such as with CH3 I in DMF in the presence of NaHCO3 giving compound of Formula XVI.
A compound of formula XVI is converted to the corresponding compound of formula XVII by reaction with a Lewis acid catalyst in the presence of persilylated, protected nucleobase in an anhydrous, aprotic solvent, preferably with SnCl4 or trimethylsilyl trifluoromethansulfonate in CH3CN or ClCH2CH2Cl at a temperature of 25-80°C.
A compound of formula XVII is converted to the
corresponding compound of formula XVIII by reaction with aqueous acid in a polar co-solvent, preferably HCl in tetrahydrofuran at a temperature range of 20-50°C. A compound of formula XVIII is converted to the
corresponding compound of formula XIX by reaction with a primary amine in a polar protic solvent, preferably methylamine or hydrazine in CH3OH at temperature range of 25-50°C.
When in formula XIX R3 is hydrogen, a compound of formula XIX is converted to the corresponding compound of formula XX by reaction with N-(9-fluorenylmethoxycarbonyloxy)succinimide in a solvent such as aqueous dimethylformamide in the presence of NaHCO3.
When in formula XIX R3 is lower alkyl, a compound of formula XIX is converted to the corresponding compound of formula XX by reaction with 1 equivalent of an aldehyde and sodium cyanoborohydride in a polar solvent, preferably methanol, then followed by reaction with N-(9-fluorenylmethoxycarbonyloxy)-succinimide in a solvent such as aqueous dimethylformamide in the presence of NaHCO3. At this point, the nucleobase B in the compound of formula XX is converted to its protected form by reaction with excess activated acyl donating group such as benzoic anhydride or acetyl chloride in an anhydrous solvent such as 1 ,4-dioxane to give a compound of formula XXI in which B' is a protected nucleobase.
A compound of formula XXI is converted to the corresponding compound of formula IIe by reaction with a conventional hydroxy protecting group reagent under conditions known in the art to effect selective reaction at hydroxy groups. The preferred method is by reaction with excess silylating agent, such as chlorotrialkylsilane in the presence of a base, specifically, with an excess of both triethylsilyl chloride and imidazole in a polar solvent such as dimethylformamide. A compound of formula Ile is converted to the corresponding compound of formula IIf by conventional methods of adding an acid protecting group, preferably by reaction with benzyl bromide in presence of NaHCO3 in dimethylformamide.
A compound of formula IIf can be deprotected to the
corresponding free amine wherein R7 is hydrogen by
conventional methods. SYNTHESIS OF OLIGOMERS
Assembly of the suitably protected monomeric building blocks into oligomers can be carried out by solid phase methodology or by conventional solution phase coupling procedures. A detailed procedure for the solid phase synthesis is illustrated in Example 6. For reference, a number of reviews on the solid phase
procedure have been published including one that utilizes 9-fluorenylmethoxycarbonyl-protected amino acids (Fields, G. and Noble, R. Int. J. Peptide Protein Res. 1990, 35, 161 -214). By selecting the appropriate linkers known in the art for attachment of the first residue of formula II and by application of the appropriate cleavage conditions after completion of the
oligomerization process, it is possible to obtain the carboxy terminal such that Y is as described. It is also possible to chose a linker which, upon cleavage after completion of the
oligomerization process on the solid phase, generates a free acid which is then modified to the appropriate carboxy substitution by conventional means.
The substitution of X is determined by the cleavage of the final amine protecting group after completion of the
oligomerization process on the solid phase followed by
modification of the free amine where necessary, specifically by reaction with 1 equivalent of a lower alkylaldehyde and sodium cyanoborohydride in a polar solvent, such as, methanol and/or by reaction with an acyl anhydride in polar solvent. The oligomer is cleaved and deprotected from a solid phase by application of conditions known in the art.
Deprotection of the resultant oligomer is effected in 2 steps: treatment with NH3 saturated EtOH or with 30% (wt/vol) NH4O H
(aqueous) at 70°C for 12 hours. After removal of the solvent, the residue is treated with a desilylating agent, preferably
tetrabutylammonium fluoride in tetrahydrofuran at room
temperature for 12 hours followed by purification using a size exclusion column according to standard ribooligonucleotide purification protocols known in the literature.
The resultant product of formula I is isolated by conventional reversed phase and/or size exclusion chromatography.
Synthesis by solution phase procedure is illustrated in Scheme III and Example 5.
Figure imgf000025_0001
wherein R1 , R2, R3, R5, R6, R7, R8, B and X are as described above and R8' is an acid protecting group.
As set forth in Scheme III, a compound of formula IIf is deprotected at the amino terminus and the resulting compound is coupled to another moiety represented by formula II using a coupling agent, such as [O-(7-azabenzotriazol- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium hexafluoro-phosphate] in a polar solvent such as dimethylformamide to form an oligomer of formula XXII. The resultant coupling product of formula XXII can be isolated by conventional means such as chromatographic separations, preferably by silica gel flash column chromatography.
An oligomer of formula XXII is further elongated by repetition of the procedure described for the conversion of formula IIf to an oligomer of formula XXII. The substitution of X is determined by the cleavage of the final amine protecting group after completion of the oligomerization process followed by modification of the free amine where necessary, specifically for R3 is lower alkyl, for example, by reaction with 1 equivalent of a lower alkylaldehyde and sodium cyanoborohydride in a polar solvent such as
methanol and/or by reaction with an acyl anhydride in polar solvent. An oligomer of formula I is obtained by removal of the silyl or acyl hydroxy protecting groups R5 and R6 and by
applications of the appropriate conditions to transform the R8 ' ester into a free acid by hydrolysis, into another ester by
transesterification, or into substituted or unsubstituted amine by treatment with the appropriate amine under amidation
conditions. A substituted or unsubstituted carboxamide may also be obtained by coupling the free acid oligomer (R8 is H) with the appropriate amine under amide bond forming conditions.
In both solution and solid phase synthesis cases, a compound of formula XXII is converted to the corresponding compound of formula I with respect to transformation of R5 and R6 to R1 and R2 according to known conventional methods.
The resultant product of formula I is isolated by conventional chromatographic means, preferably by HPLC.
The oligomer of formula I and their pharmaceutically
acceptable salts can be used as a medicament, for example in the form of a pharmaceutical preparation.
In particular the invention relates to a pharmaceutical preparation comprising an oligomer of formula I and a pharmaceutically inert carrier. In particular it relates to a preparation which decreases the production of a target protein in a cell, which comprises any of the above-described compounds of formula I in an amount effective to bind to the nucleic acid (for example mRNA) sequence encoding the target protein in said cell and thereby decrease production of said protein, and a pharmaceutically acceptable carrier.
The dose ranges for the administration of the antisense oligomers may be determined by those of ordinary skill in the art without undue experimentation. In general, appropriate dosages are those which are large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of disease in the patient, counter-indications, if any, immune tolerance and other such variables, to be adjusted by the individual physician. The antisense oligomers can be
administered topically, for example, intradermally or as an ointment on the skin, parenterally by injection or by gradual infusion over time. They can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously or orally. Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. In
particular, carriers capable of transporting oligomers to cells and into cells may be used, for example liposomes, PEGylated
liposomes, or cationic lipids. Preservatives and other additives may also be present, such as anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See, generally,
Remington 's Pharmaceutical Science, 18th Ed., Mack Eds., 1990.
Moreover, the present invention also relates to a process for the manufacture of a medicament, especially for decreasing the production or a target protein in a cell, which process comprises mixing a compound of formula I or a pharmaceutically acceptable salt thereof with a therapeutically inert carrier material and bringing the mixture into a galenical administration form.
The following Examples are provided to further describe the invention and are not intended to limit it in any way.
All reactions were performed under dry nitrogen atmosphere at ambient temperature except where noted. Reagents were used without further purification except where noted. Dichloromethane (CH2Cl2), dichloroethane (ClCH2CH2Cl), and 2,6-lutidine were distilled over CaH2 under dry nitrogen atmosphere. Other solvents were taken from freshly open bottle purchased from Fisher Scientific. Silica gel flash columns were run with EM Science (230-400 Mesh). Ion exchange columns were run with Biorad Ag 2X8 resin. HPLC was performed with a dual pump TSP HPLC system and integrator. The following abbreviations are used in the description of experimental procedures: eq for equivalent; TMSOTf for trimethylsilyl trifluoro-methanesulfonate; NH4OH for ammonium hydroxide; Fmoc-O-succinimide for N-(9-fluorenylmethoxycarbonyloxy)succinimide, DMF for dimethyl-formamide, NaHCO3 for sodium bicarbonate, TEMPO for 2,2,6,6-tetramethyl- 1 -piperidinyloxy free radical, NaClO for sodium hypochlorite, NaClO2 for sodium chlorite, tBDMSOTf for tert-butyldimethylsilyl trifluoromethane-sulfonate, HATU [O-(7-azabenzotriazol- 1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate] , DIPEA for diisopropyl-ethylamine, Knorr for p-(R, S)-α- [ 1 -(9H-fluoren-9-yl)-methoxy-formamido]-2 ,4-dimethoxybenzyl ] -phenoxyacetic acid, CH3CN for acetonitrile, CH2Cl2 for dichloromethane, CH3O H for methyl alcohol, ClCH2CH2Cl for dichloroethane. EXAMPLE 1 A
Synthesis of thymine building block a ) To a suspension of 1.14 g (9.05 mmol) of thymine in 30 mL of dry CH3CN was added BSA (N,O-bis(trimethylsilyl)acetamide) (4.46 mL, 18.04 mmol) and this mixture was heated to 80°C for
15 minutes resulting in a clear solution. After the solution was cooled to room temperature, tetraacetate, 2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranose- 1 ,3 ,4,6-tetraacetate, (2.00 g, 4.51 mmol) was added followed by 1.74 mL (9.00 mmol) of TMSOTf (trimethylsilyltrifluoromethanesulfonate). The resulting clear solution was then stirred at room temperature for 15 minutes before heating to 85°C for 12 h. At this time, TLC indicated the consumption of the tetraacetate. The reaction mixture was cooled and 0.756 g of NaHCO3 followed by 5 mL of H2O was added. The reaction mixture was stirred for an additional hour. At this time, the suspension was diluted with addition of 50 mL each of CH2Cl2 and H2O. The suspension was filtered and the solid was rinsed with CH2Cl2. The filtrate was extracted successively with saturated, aqueous NaHCO3 and brine. The organic layer was dried over MgSO4, filtered, and the filtrate concentrated in vacuo resulting in 2.14 g of a beige foam. This residue was recrystallized from CH3OH/H2O yielding 1.76 g of the desired product, 1 -[3,4,6-tri-O-acetyl-2-(trifluoroacetylamino)-2-deoxy-β-D-gluco-pyranosyl]thymine; the mother liquor yielded a further 0.058 g of this product. The total yield was thus 1.82 g
(79%). 1 H NMR: (DMSO-d6) δ anomeric H- 1 5.88 (d, 1H), thymine -CH3 1.769 (s, 3H). Microanalysis: calculated for C19H22F3N3O 10: C 44.80, H 4.35, N 8.25; found: C 44.82, H 4.23, N 8.16. FAB-MS: m/z 510 for (M+1 )+. b ) To 1 1 g (21.61 mmol) of 1 -[3,4,6-tri-O-acetyl-2-(trifluoroacetylamino)-2-deoxy-β-D-glucopyranosyl]thymine, the thymine building block was added a solution containing 22 mL of triethylamine (Et3N), 1 10 mL of CH3OH, and 88 mL of H2O forming a clear and colorless solution. After 4 hours, when TLC indicated the completion of the reaction, the solution was concentrated in vacuo. Additional CH3OH was added and the solution was evaporated again to a beige foam. The residue was recrystallized from H2O yielding 5.97 g (72%) of the expected triol. 1 H NMR: (DMSO-d6): δ anomeric H- 1 5.57 (d, I H), thymine -CH3 1.78 (s,
3H). FAB-MS: m/z 384 for (M+1 )+.
To 4.37 g ( 1 1.44 mmol) of the triol obtained as described above was added 80 mL of a solution containing 20 mL of 30% (wt/vol) NH4OH and 60 mL of H2O. The resulting solution was stirred for 12 h at room before thorough evaporation to a light brown solid: 1 -(2-amino-2-deoxy-β-D-glucopyranosyl)thymine , 4.84 g (crude yield > 100%). 1 H NMR: (D2O): δ anomeric H-1 5.69 (d, 1H), thymine -CH3 1.91 (s, 3H). FAB-MS: m/z 288 for (M+1 )+. c) To 4.87 g (16.97 mmol) of 1-(2-amino-2-deoxy-β-D-glucopyranosyl)thymine, amino sugar was added 48 mL of H2O and 127 mL of 1 ,4-dioxane. To this solution was added N-(9-fluorenylmethoxy-carbonyloxy)succinimide ( 10.22 g, 30.91 mmol) as a solution in 60 mL of 1 ,4-dioxane. This was followed by addition of 1.15 g (13.67 mmol) of NaHCO3. This turbid solution was stirred for 12 h at room temperature. At this time when TLC indicated the consumption of the amino sugar, the reaction mixture was concentrated to dryness in vacuo. To the residue was added 200 mL of H2O and 700 mL of ethyl ether
(Et2O). This 2 phase suspension was stirred for 24 h at room temperature before filtration and rinsing of the resultant solid with H2O and Et2O. The solid was dried under vacuum, then suspended in Et2O again. Filtration and drying of the solid resulted in 3.84 g (66%) of the N-Fmoc protected amino sugar, 1 - [2-deoxy-2- [[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-glucopyranosyl]-thymine. 1H NMR: (DMSO-d6): δ anomeric H-1 5.49 (d, 1H), Fmoc methine 4.59 (m, 1H), thymine -CH3 1.75 (s, 3H). FAB-MS: m/z 510 for (M+1 )+. d ) To a 20 mL dry CH2Cl2 suspension of 2.0 g (3.94 mmol) of 1-[2-deoxy-2- [[9H-fluoren-9-ylmethoxy)carbonyl] amino] - β- D-glucopyranosyl]thymine was added 8.25 mL (70.42 mmol) of 2,6-lutidine. The resulting solution was cooled to ≈ 3°C with stirring under nitrogen atmosphere before addition of 4.07 mL ( 17.7 mmol) of tert-butyldimethylsilyl trifluoromethanesulfonate.
After 30 minutes, another 4.07 mL of tert- butyldimethylsilyl trifluoromethanesulfonate was added. The reaction mixture was brought to room temperature after 30 minutes and the reaction was stirred in this manner overnight. At this time, the reaction was worked up by dilution with 100 mL of CH2Cl2 and this solution was extracted once with 50 mL of 1 N HCl aq., followed by extraction twice with H2O. The combined aqueous layers were extracted once with CH2Cl2. The combined CH2Cl2 layers were washed twice with saturated, aqueous NaHCO3, once with brine, dried with MgSO4, filtered, and concentrated in vacuo. The residue was chromatographed on silica eluted sequentially with CHCl3 and 1% CH3OH/CHCl3. This yielded the desired 1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4,5-tris-O-[(1,1-dimethyl- ethyl)dimethylsilyl]-β-D-glucopyranosyl]thymine as a white foam (2.56 g, 76%).1H NMR: (DMSO-d 6): δ thymine H-6 7.55 (s, 1H), anomeric H-1 5.64 (d, 1H), Fmoc methine 4.37 (m, 1H), thymine -CH3 1.74 (s, 3H). FAB-MS: m/z 852 for (M+1)+. e) To a 3°C solution of 5.6 mL of distilled CH2Cl2 and 5.6 mL of CH3OH containing 1.14 g (1.34 mmol) 1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4,6-tris-O-[(1,1-dimethylethyl)-dimethyl-silyl]-β-D-glucopyranosyl]thymine was added 0.53 g (2.28 mmol) of CSA (dl-10-camphorsulfonic acid). The resulting solution was stirred under nitrogen atmosphere and the
temperature was maintained at approximately 3°C for 120 minutes. The reaction was terminated by addition of saturated, aqueous NaHCO3, followed by dilution with 50 mL of CH2Cl2. The resulting solution was extracted twice with saturated, aqueous
NaHCO3 and once with H2O. The combined aqueous layers were extracted once with CH2Cl2. The combined CH2Cl2 layers were washed once with brine, dried with MgSO4, filtered, and
concentrated in vacuo. The residue was chromatographed on silica eluted sequentially with 1% to 2% CH3OH/CHCl3. This yielded,
1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4-bis-O-[(1,1-dimethyl-ethyl) dimethylsilyl]- β-D-glucopyranosyl]-thymine as a white foam (0.84 g, 85%). Microanalysis: calculated for C38H55N3O8Si2-H2O C 61.84, H 7.51, N 5.69; found: C 61.56, H 7.28, N 5.48. f) To 0.82 g (0.1.11 mmol) of 1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4-bis-O-[(1,1-dimethylethyl)-dimethylsilyl]- β-D-glucopyranosyl]thymine in 33 mL of distilled CH2Cl2 was added 0.039 g (0.33 mmol) of KBr and 0.019 g (0.056 mmol) of (n-Bu)4NHSO4. This suspension was cooled to 3°C with stirring under nitrogen atmosphere. To the cooled suspension was added 0.0088 g (0.056 mmol) of TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy free radical). To this mixture was added a solution containing 1.33 mL of 1.0 M (aq) NaClO, 1.33 mL of saturated NaHCO3, and 1.33 mL of saturated NaCl. The reaction was stirred for 30 minutes. At this time, another solution containing 1.33 mL of 1.0 M (aq) NaClO, 1.33 mL of saturated NaHCO3, and 1.33 mL of saturated NaCl was added. This caused the formation of a bright yellow color. At this point, the reaction was terminated by dilution with CH2Cl2 (50 mL) and the aqueous layer was separated. The organic layer was concentrated in vacuo. The resulting opaque residue was dissolved in 10 mL of tert-butyl alcohol and 10 mL of H2O. To this stirred solution was added 1.53 g ( 1 1.10 mmol) of NaH2PO4 and 4.70 mL (44.40 mmol) of 2-methyl-2-butene. To this solution was added 0.80 g (8.88 mmol) of NaClO2. This mixture was stirred at room temperature for 30 minutes. At this time, the reaction mixture was poured into a mixture of EtOAc ( 100 mL) and H2O ( 10 mL) acidified to pH=2 with 10% (wt/vol) aqueous citric acid. The layers were separated and the organic layer was twice extracted with H2O . The aqueous layers were back extracted once with EtOAc. The organic layers were combined, washed with brine, dried with
M gS O4, filtered and concentrated in vacuo to a white foam
residue. The desired carboxylic acid product, 1 ,2-dideoxy- 1 -[ 1 ,2-dihydro-5-methyl]-2,4-dioxo- 1 -pyrimidinyl)- [ [(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3 ,4-bis-O- [( 1 , 1 -dimethylethyl)-dimethylsilyl]- β-D-glucopyranuronic acid was obtained by chromatography of the residue from a silica gel flash column eluted with 4 to 10% CH3OH/CHCl3 in 62% yield (0.51 g). 1H NMR: (DMSO-d6): δ anomeric H-1 5.92 (d, 1 H), Fmoc methine 4.28 (m, 1 H), thymine -CH3 1.77 (s, 3H), 0.91 tert-butyl (s, 9H), 0.77 ppm tert-butyl (s, 9H). FAB-HRMS: calculated for (M+1 )+: 752.3398; found for (M+1)+: 752.3397.
EXAMPLE 1 B
Synthesis of thymine building block
Steps a)-c) were performed as set forth in Example 1A. d) To a 5 mL dry DMF solution of 0.751 g ( 1.50 mmol) of 1-[2-deoxy-2- [[9H-fluoren-9-ylmethoxy)carbonyl] amino]-β-D-glucopyranosyl]thymine was added 1.60 g (23.6 mmol) of imidazole and a catalytic quantity (≈ 1 mg) of DMAP (para-N,N-dimethylaminopyridine). The resulting solution was cooled to = 2°C with stirring under nitrogen atmosphere before addition of 0.93 mL (5.9 mmol) of chloroisopropyl-dimethylsilane. The reaction mixture was brought to room temperature after 5 minutes. After 90 minutes, another portion (0.93 mL, 5.9 mmol) of chloroisopropyl-dimethylsilane was added. After 30 minutes, TLC indicated the conversion of starting material to a single product. The reaction was terminated by dilution with 50 mL of
EtOAc (ethyl acetate) and this solution was. extracted twice with H2O, followed by extraction with 10% (wt/vol) aqueous citric acid. The organic layer was extracted once again with H2O. The combined aqueous layers were extracted once with EtOAc. The combined EtOAc layers were washed once with brine, dried with
M g S O4, filtered, and concentrated in vacuo. The residue was chromatographed on silica eluted sequentially with CHCl3 and 1 % CH3OH/CHCl3. This yielded the desired 1-[2-deoxy-2-[[9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3 ,4,5-tris-O-[( 1 -methylethyl)dimethylsilyl]-β-D-glucopyranosyl]thymine as a white foam ( 1.01 g, 83%). 1H NMR: (CDCl3): δ anomeric H- 1 5.62 (d, 1H), Fmoc methine 4.29 (m, 1H), thymine -CH3 1.91 (s, 3H). FAB-MS: m/z 810 for (M+1)+. Microanalysis: calculated for
C41H63N3O8Si3: C 60.78, H 7.84, N 5.19; found: C 60.75, H 7.82, N 5.27. e ) To a -30°C solution of 5 mL of distilled CH2Cl2 and 1.2 mL of CH3OH containing 0.25 g (0.31 mmol) of N-Fmoc protected amino sugar 1 -[2-deoxy-2-[[9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3 ,4,6-tris-O-[( 1 -methylethyl)dimethylsilyl]-β-D-glucopyranosyl] -thymine was added 0.014 g (0.062 mmol) of CSA (dl- 10-camphorsulfonic acid). The resulting solution was stirred under nitrogen atmosphere and the temperature was maintained at approximately -30°C for 105 minutes. The reaction was
terminated by addition of saturated NaHCO3, followed by dilution with 50 mL of EtOAc. The resulting solution was extracted twice with saturated, aqueous NaHCO3 and once with H2O. The
combined aqueous layers were extracted once with EtOAc. The combined EtOAc layers were washed once with brine, dried with M g S O4, filtered, and concentrated in vacuo. The residue was chromatographed on silica eluted sequentially with CHCl3 and then 1 % to 2% CH3OH/CHCl3. This yielded 1 -[2-deoxy-2-[[9H-fluoren-9-ylmethoxy)carbonyl]amino]- [3 ,4-bi s-O- [( 1 -methylethyl)dimethylsilyl]-β-D-glucopyranosyl]thymine as a white foam (0.196 g, 89%). 1H NMR: (DMSO-d6): δ anomeric H- 1
5.62 (d, 1H), Fmoc methine 4.36 (m. 1H), thymine -CH3 1.76 (s, 3H). 1.75- 1.96 silylisopropyl (m, 14H), -0.03 0.00 ppm
silylmethyl (4 singlets, 12H). FAB-MS: (m/z 910 for (M+1 )+. f) To 0.18 g (0.25 mmol) of 1 -[2-deoxy-2-[[9H-fluoren-9-ylmethoxy) carbonyl]amino]-[3,4-bis-O-[( 1 -methylethyl)-dimethylsilyl]-β-D-glucopyranosyl]thymine in 10 mL of distilled CH2Cl2 was added 0.0030 g (0.025 mmol) of KBr and 0.0043 g (0.013 mmol) of (n-Bu)4NHSO4. This suspension was cooled to -6°C with stirring under nitrogen atmosphere. To the cooled suspension was added 0.0004 g (0.0025 mmol) of TEMPO (2,2,6,6-tetramethyl- 1 -piperidinyloxy free radical). To this mixture was added in 3 portions separated by 10 minutes a solution
containing 0.33 mL of 1.24 M NaClO, 0.33 mL saturated NaHCO3, and 0.33 mL saturated NaCl. The temperature was raised to 0°C and the reaction was stirred for 30 minutes. At this point, the reaction was terminated by dilution with EtOAc (50 mL) and the organic solution was twice extracted with H2O. The aqueous layers were extracted once with EtOAc. The organic layers were combined, washed with brine, dried with MgSO4, filtered and concentrated in vacuo.
The resulting opaque residue was dissolved in 3 mL of tert-butyl alcohol and 3 mL of H2O. To this stirred solution was added 0.28 g (2.03 mmol) of NaH2PO4 and 1.08 mL ( 10.16 mmol) of 2-methyl-2-butene. To this solution was added 0.23 g (2.54 mmol) of NaClO2. This mixture was stirred at room temperature for 30 min. At this time, the reaction mixture was poured into a mixture of EtOAc (50 mL) and H2O acidified to pH=2 with 10% (wt/vol) aqueous citric acid. The layers were separated and the organic layer was twice extracted with H2O. The organic layers were combined, washed with brine, dried with MgSO4, filtered and concentrated in vacuo to a white foam residue. The desired carboxylic acid product, 1 ,2-dideoxy- 1 - [ 1 ,2-dihydro-5-methyl]-2 ,4-dioxo- 1 -pyrimidinyl)- [ [(9H-fluoren-9-ylmethoxy)carbonyl]-amino]- [3 ,4-bis-O-[( 1 -methylethyl) dimethylsilyl]]-β-D-glucopyranuronic acid was obtained by chromatography of the residue from a silica gel flash column eluted with 5 to 20%
CH3OH/CHCl3 in 74% yield (0.136 g). 1 H NMR: (DMSO-d6): δ anomeric H- 1 5.55 (d, 1 H), Fmoc methine 4.36 (m, 1H), thymine
-CH3 1.75 (s, 3H), 1.75- 1.96 silylisopropyl (m, 14H), -0.03 to 0.00 ppm silylmethyl (m, 12H). FAB-MS : 724 for (M+1)+.
Microanalysis: calculated for C36H49N3O9Si2-H2O C 58.99, H 6.88, N 5.73; found: C 58.82, H 6.54, N 5.86.
EXAMPLE 2
Synthesis of cytosine building block a ) To a suspension of N-benzoylcytosine (29.1 1 g, 135.40 mmol) in 250 mL of distilled ClCH2CH2Cl was added 66.80 ml (270.82 mmol) of BSA. This suspension was heated to a gentle reflux for 40 min during which time a clear solution was effected. At this time, the solution was cooled to room temperature and 20 g (45.15 mmol) of 2-deoxy-2-[(trifluoro-acetyl)amino]-β-D-glucopyranose- 1 ,3,4,6-tetraacetate was added, followed by a solution of 100 mL of ClCH2CH2Cl containing freshly distilled SnCl4 ( 15.17 mL, 135.42 mmol). This resulting slightly yellow solution was refluxed for 2 h. At this time, the reaction was cooled to room temperature and 57 g of NaHCO3 was added and stirred before the addition of 22 mL of H2O. The mixture was stirred for an additional 20 min at room temperature. The suspension was filtered and washed through a pad of celite with CHCl3. The filtrate was washed with saturated, aqueous NaHCO3 and brine. The organic layer was further treated with charcoal before filtration through a pad of celite. The filtrate was dried over
Na2S O4, filtered and concentrated in vacuo to a yellowish foam. This residue was chromatographed on silica gel eluted with CH2Cl2 and 1 to 3.5% CH3OH/CH2Cl2. The product was further purified by recrystallization from CH3OH/H2O. As a single crop of white crystals the desired glycosylation product, N- [ 1 ,2-dihydro-2-oxo- 1 - [3,4,6-tri-O-acetyl-2-deoxy-2- [( trifluoroacetyl)amino] -β-D-glucopyranosyl]]-4-pyrimidinyl]benzamide was obtained (9.22 g, 34%). 1 H NMR: (CDCl3): δ anomeric H- 1 6.21 (d, 1 H), acetyl ester 2.06 (s, 3H), acetyl ester 1.82 (s, 3H). FAB-MS: m/z 599 for (M + 1 )+, 1 197 for (2M+1 )+. Microanalysis: calculated for
C25H25N4O 10 C 50.17, H 4.21, N 9.36; found: C 50.01 , H 4.00, N 9.16. b ) 19 g (31.77 mmol) of N-[ 1 ,2-dihydro-2-oxo- 1 -[3,4,6-tri-O-acetyl-2-deoxy-2- [(trifluoroacetyl)amino] -β-D-glucopyranosyl] ] - 4-pyrimidinyl]-benzamide was treated with a solution of 57 mL of Et3N, 285 mL of CH3OH, and 228 mL of H2O. This solution was stirred at room temperature for 4.5 h. At this time, the solution was concentrated to dryness in vacuo. The residue was treated with 60 mL of 30% (wt/vol) NH4OH diluted with 140 mL of H2O for 12 h at room temperature. The reaction mixture was then concentrated to dryness in vacuo. The residue was dissolved in 50 mL of H2O and loaded to an ion exchange column containing 400 mL of Ag 2X8, HO- form and eluted with H2O. The
appropriate fractions according to TLC were combined, frozen, and lyophilized to a white powder. The free amino sugar, 1 -(2-amino-2-deoxy-β-D-glucopyranosyl)cytosine was obtained in 96% yield (8.30 g). 1 H NMR: (D2O): δ cytosine-H6 7.71 (d, 1H), cytosine-H5 6.10 (d, 1H), anomeric H- 1 5.64 (d, 1H). CSI-MS: m/z 273 for (M+1 )+, 545 for (2M+ 1 )+. Microanalysis: calculated for
C 10H 16N4O5 C 44.12, H 5.92, N 20.81 ; found: C 44.02, H 5.94, N 20.8 1 . c ) To 0.80 g (2.94 mmol) of 1 -(2'-amino-2'-deoxy-β-D- glucopyranosyl)cytosine was added 4 mL of H2O. To this solution was added a 20 mL solution of 1 ,4-dioxane containing 1.19 g (3.53 mmol) N-(9-fluorenylmethoxycarbonyloxy)succinimide and an additional volume of 1 ,4-dioxane ( 12 mL) was added. Sodium bicarbonate (0.15 g, 1.79 mmol) was added as an 8 mL H2O solution to the reaction mixture. This mixture was stirred at room temperature for 12 h. At this time TLC indicated the consumption of starting material and the reaction solvents were removed in vacuo leaving a white gum. This residue was treated with 20 mL of H2O and excess ether (200 mL). The suspension was stirred for several hours, filtered and resuspended in CH2Cl2,
Et2O and H2O for 12 h. The suspension was filtered and the solid dried under vacuum yielding 1.00 g (57%) of 1 -[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D -glucopyranosyl]cytosine. 1 H NMR: (DMSO-d6): δ cytosine-H6 7.50 (d, 1H), cytosine-H5 6.67 (d, 1H), anomeric H- 1 5.66 (d, 1H). CSI- MS: m/z 495 for (M+1)+. Microanalysis: calculated for C25H26N4O7 C 60.98, H 5.82, N 10.61 ; found: C 61.10, H 5.63, N 10.50. d ) To 15. 50 g (31.38 mmol) of 1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-glucopyranosyl]cytosine was added 1.2 liters of 200 proof ethanol and this solution was heated to reflux. At reflux, 7.09 g (31.37 mmol) of benzoic anhydride was added. Every hour for 4 hours, an additional portion of 7.09 g of benzoic anhydride was added to the refluxing solution. After a total of 5 hours reaction time, TLC indicated the consumption of the starting material. At this time the reaction mixture was concentrated to near dryness in vacuo and 500 mL of Et2O was added; this suspension was filtered and rinsed with Et2O. The resulting solid was recrystallized from CHCl3, giving 7.48 g (40%) of N- [ 1 - [2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-gluco-pyranosyl]- 1 ,2-dihydro-2-oxo-4-pyrimidinyl] -benzamide. 1 H NMR: (DMSO-d6): δ anomeric H- 1 5.75 (m, 1 H). CSI-MS: m/z 599 for (M+1)+ and 621 for (M+Na)+. e ) To 0.250 g (0.42 mmol) of N-[ 1 ,2-dihydro-2-oxo- 1 -(2-deoxy-2- [[(9H-fluoren-9-ylmethoxy)carbonyl]amino] -β-D-glucopyranosyl)-4-pyrimidinyl]benzamide was added 3 mL of distilled CH2Cl2 and 0.876 mL (7.52 mmol) of distilled 2,6-lutidine. To this white suspension cooled to 0°C was added 0.43 mL ( 1.88 mmol) of tBDMSOTf (tert-butyldimethylsilyl trifluoromethanesulfonate) . Stirring was continued at 0°C for 5 minutes before the cold bath was removed and the reaction was allowed to stir at room temperature. After 90 minutes, an additional portion of
tBDMSOTf (0.43 mL) was added. The reaction was stirred at room temperature for 4 h. At this time, the reaction mixture was poured into EtOAc and twice extracted with H2O, once with 5% (wt/vol) citric acid, once with saturated, aqueous NaHCO3 , and finally once with brine. The organic layer was dried with MgSO4, filtered, and concentrated in vacuo to a slight white solid. This solid was loaded to a silica gel flash column and the desired silylated product, N-[ 1 -[2-deoxy-2- [[(9H-fluoren-9-ylmethoxy)-carbonyl]amino]- [3,4,6-tris-O- [( 1 , 1 -dimethylethyl)dimethylsilyl]-β-D-glucopyranosyl]- 1 ,2-dihydro-2-oxo-4-pyrimidinyl] benzamide was obtained by elution of the column with CHCl3 and 1%
CH3OH/CHCl3: 0276 g (70%). 1H NMR: (CDCl3): δ cytosine-H6, H5 6.89 (br, 2H), anomeric H-1 6.12 (d, 1H). FAB-MS: m/z 941 for (M+1)+. FT-IR (KBr pellet): Si-CH3837 cm-1. f) To 0.46 g (0.50 mmol) of N-[1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4,6-tris-O-[(1,1-dimethylethyl)dimethylsilyl]-β-D-glucopyranosyl]-1,2-dihydro-2-oxo-4-pyrimidinyl] benzamide was added 2.0 mL of distilled CH2Cl2 and 2.0 mL of CH3OH. This clear solution was cooled to 0°C before the addition of 0.20 g (0.84 mmol) of CSA. The reaction was stirred at 0°C for 2 h before quenching the reaction with saturated aqueous NaHCO3 and dilution of the mixture with CH2Cl2. The organic layer was twice extracted with saturated, aqueous NaHCO3; the combined aqueous layers are extracted once with CH2Cl2. The combined organic layers are washed with brine, dried over
MgSO4, filtered and the filtrate was concentrated in vacuo to a white foam. The desired primary alcohol, N-[1-[2-deoxy-2-[(trifluoroacetyl)amino]-[3,4-bis-O-[(1,1-dimethylethyl)dimethylsilyl]-[β-D-glucopyranosyl]-1,2-dihydro-2-oxo-4-pyrimidinyl]-benzamide was obtained by elution of the crude foam from a silica gel flash column eluted with CHCl3 and 1 to 2%
CH3OH/CHCl3: 0.31 g (74%). 1H NMR: (CDCl3): δ anomeric H-16.12 (d, 1H), NH 5.30 (br d, 1H), t-butyl Si 0.96 (s.9H), t-butyl Si 0.91 (s, 9H), (CH3)2Si 0.19 (s, 6H), (CH3)2Si 0.16 (s, 6H). FAB-MS: m/z 827 for (M+1)+. FT-IR (KBr pellet): Si-CH3839 cm-1. g) To 0.31 g (0.371 mmol) of N-[1-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4-bis-O-[(1,1-dimethylethyl)-dimethylsilyl]-β-D-glucopyranosyl]-1,2-dihydro-2-oxo-4-pyrimidinyl]benzamide was added 0.0052 g (0.037 mmol) of KBr, 0.0063 g (0.019 mmol) of (n-Bu)4NHSO4, and 10 mL of distilled CH2Cl2. The reaction mixture was cooled to -4°C with stirring and 0.0006 g (0.0037 mmol) of TEMPO was added. To this mixture was added in 3 portions separated by 10 min a solution containing 0.48 mL of 1.24 M NaClO, 0.48 mL saturated NaHCO3, and 0.48 mL saturated NaCl. The temperature was raised to 0°C and the reaction was stirred for 1 h. At this point, the reaction was terminated by dilution with CH2Cl2 (50 mL) and the organic solution was twice extracted with H2O. The aqueous layers were extracted once with CH2Cl2. The combined organic layers were washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The resulting opaque residue was dissolved in 4 mL of tert-butyl alcohol and 4 mL of H2O. To this stirred solution was added 0.41 g (3.00 mmol) of NaH2PO4 and 1.57 mL ( 14.84 mmol) of 2-methyl-2-butene. To this solution was added 0.34 g (3.71 mmol) of NaClO2. This mixture was stirred at room temperature for 30 min. At this time, the reaction mixture was poured into a mixture of EtOAc (50 mL) and H2O acidified to pH=2 with 10% (wt/vol) aqueous citric acid. The layers were separated and the organic layer was twice extracted with H2O. The combined organic layers were combined, washed with brine, dried with MgSO4, filtered and concentrated in vacuo to a white foam residue. The desired carboxylic acid product, 1 ,2-dideoxy- 1 - [ 1 ,2-dihydro-2-oxo-4-[(phenylcarbonyl )amino]- 1 -pyrimidinyl] - [3 ,4-bis-O- [( 1 , 1 -dimethylethyl )dimethylsilyl ] -2 [ [(9 H- fluoren-9-ylmethoxy)-carbonyl]amino]-β-D-glucopyranuronic acid was obtained by chromatography of the residue from a silica gel flash column eluted with CHCl3 and 1 to 10% CH3OH/CHCl3 in 93% yield (0.29 g). 1 H NMR: (CDCl3): anomeric H- 1 6.75 (br, 1 H), t-butyl Si 0.98 (s, 9H), t-butyl Si 0.87 (s, 9H), (CH3)Si 0.22 (s, 3H), (CH3)Si 0.19 (s, 3H), (CH3)Si 0.10 (s, 3H), (CH3)Si 0.08 (s, 3H). FAB-MS: m/z 841 for (M+1 )+. Microanalysis: calculated for C44H56N4O9Si2-H2O C
62.17, H 6.76, N 6.59; found: C 62.33, H 5.83, N 6.18. EXAMPLE 3
Synthesis of adenine building block a ) In 25 mL of dry CH3CN was suspended 0.54 g (2.26 mmol) 6-N-benzoyladenine and to this suspension was added 0.50 mL
(4.51 mmol) of freshly distilled SnCl4. An immediate solution was achieved at room temperature. To this solution was added 0.50 g ( 1.13 mmol) of 2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranose- 1 ,3,4,6-tetraacetate and the reaction mixture was heated for 12 h at 40°C at which time TLC indicated the complete consumption of starting material. The reaction was cooled to room temperature and 1.89 g (22.5 mmol) of NaHCO3 was added followed by the addition of 0.71 mL (39.44 mmol) of H2O. This mixture was stirred at room temperature for 30 min and then filtered and washed through a pad of celite with CH3CN. The filtrate was concentrated to dryness in vacuo and the residue was dissolved in EtOAC. This solution was washed with saturated, aqueous NaHCO3 and brine, dried over NaSO4, and filtered. The filtrate was concentrated in vacuo leaving a white foam. This foam was chromatographed on silica with 5 to 10% CH3OH/CH2Cl2 yielding 0.46 g (66%) of the desired product, N-[9-[3,4,6-tri-O-acetyl-2-deoxy-2- [(trifluoroacetyl )amino )- β-D -glucopyranosyl ) -9H-purin-6-yl]-benzamide. 1 H NMR: (CDCl3 ): adenine aromatic 8.38 (s, 1 H), adenine aromatic 8.25 (s, 1 H), anomeric H- 1 6.25 (d, 1H). FAB-MS : m/z 623 for (M+ 1 )+. Microanalysis: calculated for
C26H25N6O9-H2O C 48.75, H 4.24, N 13.12; found: C 48.84, H 4.13, N 12.88. b ) 6.59 g ( 10.50 mmol) of N-[9-[3,4,6-tri-O-acetyl-2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranosyl ] -9H-purin-6-yl]benzamide was treated with a solution of 20 mL of Et3N, 100 mL of CH3OH, and 80 mL of H2O. This solution was stirred at room temperature for 12 h. At this time, the solution was concentrated to dryness in vacuo. The residue was treated with 160 mL of 30% (wt/vol) NH4OH for 72 h at room temperature. The reaction mixture was then concentrated to dryness in vacuo. The residue was dissolved in 50 mL of H2O and loaded to an ion exchange column containing 265 mL of Ag 2X8, HO- form and eluted with H2O. The appropriate fractions according to TLC were combined, frozen, and lyophilized to a white powder. The amino sugar 9-(2-amino-2-deoxy-β-D-glucopyranosyl)adenine was obtained in 63% yield ( 1.98 g). 1H NMR: (D2O): δ adenine aromatic 8.34 (s, 1H), adenine aromatic 8.21 (s, 1H), anomeric H- 1 5.60 (d, 1H). FAB-MS: m/z 297 for (M+1 )+. c) To 0.69 g (2.33 mmol) of 9-(2-amino-2-deoxy-β-D-glucopyranosyl)adenine was added 3.45 mL of H2O. To this solution was added a 28 mL 1 ,4-dioxane solution of 0.94 g (2.80 mmol) of N-(9-fluorenylmethoxycarbonyloxy)succinimide. This resulted in a cloudy mixture. To this system was added 0.13 g ( 1.50 mmol) of NaHCO3. This mixture was stirred for 12 h at room
temperature. At this time, TLC indicated the consumption of starting material and the mixture was concentrated in vacuo to a dry residue. This residue was stirred for 18 h in 250 mL of Et2O and 250 ml of H2O. The suspension was then filtered and the solid dried in a vacuum oven for 2 days. Thus was obtained 1.0 g (82%) of the desired 9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)-carbonyl]-amino]-β-D-glucopyranosyl]adenine 1 H NMR: (DMSO-d6): adenine aromatic 8.19 (s, 1 H), adenine aromatic 8.17 (s, 1 H), anomeric H- 1 6.60 (d, 1 H), Fmoc-methine 4. 17 (m, 1H). FAB-MS : m/z 519 for (M+1 )+. d ) Azeotropic evaporation of 0.50 g (0.97 mmol) of 9-[2-deoxy-2- [ [(9H-fluoren-9-ylmethoxy)carbonyl] amino]-β-D-glucopyranosyl]adenine in dry pyridine was repeated three times before dissolving the residue in 20 mL of dry pyridine. To this solution was added 0.74 mL (5.79 mmol) of chlorotrimethylsilane. This reaction mixture was stirred at room temperature for 30 min. At this time, 0.56 mL (4.82 mmol) of benzoyl chloride was added and the resultant solution was stirred at room temperature for 2.5 h at which time TLC indicated the complete conversion of the starting material into product. The reaction mixture was quenched by addition of 10 mL of H2O, stirred for 1.5 h, and then 40 mL of cold saturated NaHCO3 was added. This 2 phase system was stirred for an additional 3 h at which time the mixture was extracted twice with EtOAc. The EtOAc layer was washed with brine and dried over MgSO4, filtered and concentrated to dryness in vacuo. The residue was dissolved in EtOAc and this solution was washed thrice with 2 N HCl, thrice with saturated, aqueous NaHCO3, once with brine and dried with MgSO4, and filtered. The filtrate was evaporated to dryness. The residue was
recrystallized from CHCl3 yielding 0.48 g (64%) of the desired N-[2-deoxy-2- [[(9H-fluoren-9-ylmethoxy)carbonyl] amino]- β-D-glucopyranosyl]-9H-purin-6-yl]-dibenzamide. 1 H NMR: (DMSO-d6): adenine aromatic 8.76 (s, 1 H), adenine aromatic 8.67 (s, 1 H), anomeric H- 1 6.70 (d, 1H), Fmoc-methine 4.07 (m, 1H). FAB-MS: m/z 727 for (M+ 1)+, 749 for (M+Na)+. FT-IR (KBr): amide resonance 1700 cm- 1 . e ) To 0.050 g (0.069 mmol) of N-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino] - β-D-glucopyranosyl]-9H-purin-6-yl] -dibenzamide was added 2 mL of distilled CH2Cl2. To this
suspension was added 0.16 mL ( 1.38 mmol) of 2,6-lutidine followed by 0.16 mL (0.69 mmol) of tBDMSOTf. This solution was stirred for 72 h. At this time, the reaction mixture was diluted with CHCl3 and washed once with H2O, twice with 1 N HCl, twice with saturated NaHCO3 and once with brine. The organic layers was dried with Na2S O4 and filtered, and the filtrate was
concentrated to dryness in vacuo. The desired silylated product N- [2-deoxy- [3,4,6-tris-O- [( 1 , 1 -dimethylethyl)dimethylsilyl]-2- [ [(9H-fluoren-9-ylmethoxy)carbonyl] amino]-β-D-glucopyranosyl]-9H-purin-6-yl]-dibenzamide was isolated by silica gel
chromatography using CH2Cl2 and 0.5% to 1 % CH3OH/CH2Cl2. The appropriate fractions were collected, evaporated to dryness, and lyophilized from 1 ,4-dioxane. This yielded 0.059 g of product (81%). 1H NMR: (CDCl3): adenine aromatic 8.46 (s, 1H), adenine aromatic 8.33 (s, 1H), anomeric H-l 5.97 (d, 1H). FAB-MS: m/z 1069 for (M+1)+. Microanalysis: calculated for C58H75N6O9Si3-(two molecules of 1,4-dioxane, C4H8O2) C 64.77, H 7.16, N 7.55; found: C 64.37, H 7.34, N 7.63. f) To 2.32 g (2.17 mmol) of N-[2-deoxy-[3,4,6-tris-O-[(1,1-dimethylethyl)dimethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)-carbonyl]-amino]-β-D-glucopyranosyl]-9H-purin-6-yl]dibenzamide was added 9 mL of distilled CH2Cl2 and 9 mL of
CH3OH. The resultant solution was cooled to -5°C and 0.76 g (3.25 mmol) of CSA was added. The reaction mixture was stirred for 2 h before dilution with CHCl3 and extraction with H2O twice, with saturated, aqueous NaHCO3 once, with H2O once again, and finally with brine. The organic layer was dried over Na2SO4 and filtered.
The filtrate was concentrated in vacuo to a white foam. This foam was purified by elution from a silica gel flash column eluted with CHCl3 and 1 to 2% CH3OH/CHCl3. Thus was obtained 1.68g (81%) of the desired primary alcohol, N-[2-deoxy-[3,4-bis-O-[(1,1-dimethylethyl)dimethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)-carbonyl]amino]-β-D-glucopyranosyl]-9H-purin-6-yl]dibenzamide. 1H NMR: (CDCl3): adenine aromatic 8.51 (s, 1H), adenine aromatic 8.33 (s, 1H), anomeric H-1 5.93 (d, 1H). FAB-MS: m/z 955 for (M+1)+. g) To 0.50 g (0.524 mmol) of N-[2-deoxy-[3,4-bis-O-[(1,1-dimethylethyl)dimethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)-carbonyl]-amino]-β-D-glucopyranosyl]-9H-purin-6-yl]dibenzamide was added 0.006 g (0.050 mmol) of KBr, 0.009 g (0.027 mmol) of (n-Bu)4NHSO4, and 20 mL of distilled CH2Cl2. The reaction mixture was cooled to -4°C with stirring and 0.0008 g (0.0052 mmol) of TEMPO was added. To this mixture was added in 3 portions separated by 10 min a solution containing 0.47 mL of 1.24 M NaClO and 0.50 mL saturated NaHCO3. At this point, the temperature was raised to 0°C and the reaction was stirred for 2 h. At this point, the reaction was terminated by dilution with CHCl3 (50 mL) and the organic solution was twice extracted with H2O. The organic layers were combined, washed with brine, dried with MgSO4, filtered and concentrated to dryness in vacuo.
The resulting opaque residue was dissolved in 4.6 mL of tert-butyl alcohol and 4.6 mL of H2O. To this stirred solution was added 0.62 g (4.46 mmol) of NaH2PO4 and 2.36 mL (22.27 mmol) of 2-methyl-2-butene. To this solution was added 0.50 g (5.53 mmol) of NaClO2. This mixture was stirred at room temperature for 30 min. At this time, the reaction mixture was poured into a mixture of EtOAc (50 mL) and H2O acidified to pH=2 with 10% (wt/vol) aqueous citric acid. The layers were separated and the organic layer was twice extracted with H2O. The organic layers were combined, washed with brine, dried with MgSO4, filtered and concentrated in vacuo to a white foam residue. This white foam was purified by silica gel flash column chromatography eluted with CHCl3 and 1 to 4% CH3OH/CHCl3 . The combined fractions were collected, evaporated, and lyophilized from dioxane and the desired adenine building block, 1 -[6-[bis(phenylcarbonyl)amino] -9H-purin-9-yl] - 1 , 2-dideox y-3 ,4-bis-O- [( 1 , 1 -dimethylethyl)dimethylsi lyl ] -2- [ [ ( 9H-fluoren -9-ylmethoxy )-carbonyl] amino] - β-D-glucopyranuronic acid was obtained in 74% yield (0.38 g). 1 H NMR: (CD3OD): adenine aromatic 9.06 (s, 1H), adenine aromatic 8.47 (s, I H), anomeric H- 1 6.20 (d, 1 H), t-butyl Si 0.99 (s, 9H), t-butyl Si 0.85 (s, 9H), (CH3)Si 0.22 (s, 3H), (CH3)Si 0.21 (s, 3H), (CH3)Si 0.1 1 (s, 3H), (CH3)Si -0.01 (s, 3H). FAB-MS: m/z 969 for (M+1 )+. Microanalysis: calculated for
C44H56N4O9Si2-(2 molecules of 1 ,4-dioxane, C4H8O2) C 62.91 , H 6.68, N 7.34; found: C 63.09, H 5.86, N 7.10. EXAMPLE 4A
Synthesis of guanine building block a ) To 7.68 g of 2-acetylamino-6-hydroxypurine suspended in 400 mL of dry acetonitrile was added 20 mL of bis-silylacetamide
(BSA); the mixture was heated to reflux under inert and dry atmosphere for 30 minutes. At this time, to the cooled mixture was added 8.86 g of 2-deoxy-2-[(trifluoroacetyl)amino]- β-D-glucopyranose- 1 ,3,4,6-terraacetate and 7.72 mL of trimethylsilyl trifluoromethanesulfonate. The mixture was heated to reflux under inert atmosphere for 12 hours. At this time, the clear solution was concentrated to 100 mL volume, before the addition of 200 mL 5% (wt.vol) NaHCO3 and 400 mL of EtOAc. The resulting suspension was filtered and washed with EtOAc through celite. The aqueous layer was separated and the organic layer was extracted twice with saturated NaHCO3, brine, and dried with M g S O4. Upon filtration and evaporation in vacuo, the resulting residue was chromatographed on silica gel eluted with 5/95 CH3OH/CHCl3. The product, N-[7-[3,4,6-tris-O-acetyl-2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranosyl] -6,9-dihydro-6-oxo- 1 H-purin-2-yl]-acetamide, eluted first (3.1 1 g, 27%): 1 H NMR (DMSO-d6) δ H-8 8.39 (br s (broad), 1 H), H- 1 ' 6.09 (br, 1 H); 13C NMR (DMSO-d6) δ 158.0, 151.8, 147.6, 144.1 , 1 10.8. This was followed by the coupling product, N- [9-[3,4,6-tris-O-acetyl-2-deoxy-2-[(trifluoroacetyl) amino- β-D-glucopyranosyl]-6,9-dihydro-6-oxo- 1 H-purin-2-yl]-acetamide (4.97 g, 43%). 1H NMR (DMSO-d6) δ H-8 8.19 (br, 1H), H-1 ' 5.93 (s 1H); 13C NMR (DMSO-d6) δ 154.6, 148.6, 148.1 , 138.5, 120.3. b ) To 14.56 g (25.35 mmol) of N-[9-[3,4,6-tris-O-acetyl-2-deoxy- 2- [(trifluoroacetyl)amino]-β-D-glucopyranosyl] -6,9-dihydro-6-oxo- 1 H-purin-2-yl]-acetamide was added a solution composed of 45 mL of Et3N, 225 mL of CH3OH, and 180 mL of H2O. This solution was stirred at room temperature for 12 hours. At this time, the solution was concentrated to dryness in vacuo. The residue was recrystallized from H2O yielding a white powder (7.2 g, 69%).
To 7.08 g of this white powder was added 140 mL of 30% (wt/vol) NH4OH, and the reaction mixture was stirred for 15 hours at room temperature. The reaction mixture was then concentrated to dryness in vacuo. To the residue was added H2O and the mixture was frozen and lyophilized to a white powder yielding 9-(2-amino-2-deoxy-β-D-glucopyranosyl)guanine quantitatively. FAB-MS: m/z 313 for (M+ 1 )+. c) To 3.31 g ( 10.61 mmol) of 9-(2-amino-2-deoxy-β-D-glucopyranosyl) guanine was added 72 mL of H2O. To this solution was added a 100 mL 1 ,4-dioxane solution containing 4.65 g
( 13.79 mmol) of N-(9-fluorenylmethoxycarbonyloxy)succinimide. This resulted in a cloudy mixture. To this system was added 0.58 g (6.90 mmol) of NaHCO3. This mixture was stirred for 12 hours at room temperature. At this time, TLC indicated the consumption of starting material and the mixture was concentrated in vacuo to a dry residue. This residue was stirred for 3 hours in 310 mL of Et2O and 145 ml of H2O. The suspension was then filtered and the solid dried in a vacuum oven for 12 hours at 40°C. Thus was obtained 4.28 g (76%) of 9-(2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino]- β-D-glucopyranosyl)guanine. 1H
NMR: (DMSO-δ6): guanine H-8 7.72 (s, 1H), anomeric H- 1 5.37 (d, 1H), Fmoc-methine 4.05 (m, 1H). FAB-MS : m/z 535 for (M+1 )+. d ) To 2.85 g (5.34 mmol) of 9-(2-deoxy-2-[[(9H-fluoren-9-ylmethoxy) carbonyl]amino]- β-D-glucopyranosyl)guanine was added dry pyridine and the resulting solution was evaporated. This procedure was repeated twice. To the residue was then added 100 mL of pyridine. To this solution was added 6.8 mL of chlorotrimethylsilane. This reaction mixture was stirred at room temperature for 10 minutes. At this time, additional chlorotrimethylsilane (1.44 mL, 64.8 mmol total) was added. The reaction mixture was stirred at room temperature for an
additional 90 minutes. At this time, 2.69 mL ( 16.22 mmol) of isobutyric anhydride was added and the resultant solution was stirred at room temperature for 1.5 hours at which time TLC indicated the complete conversion of the starting material into product. The reaction mixture was quenched by cooling to 0°C before addition of 60 mL of cold saturated NaHCO3. This two phase system was stirred for an additional 30 minutes at which time the mixture was extracted twice with EtOAc. The EtOAc layer was concentrated to an oil in vacuo. The residue was dissolved acetonitrile and toluene and concentrated in vacuo; this procedure was repeated once more resulting in a yellow solid. The yellow solid was partitioned between a two phase solvent mixture composed of 200 mL of H2O and 500 mL of CHCl3 /ethanol (3 to 2 vol/vol). The aqueous layer was extracted twice more with the solvent mixture of CHCl3 / ethanol (3 to 2 vol/vol). The combined organic extracts were evaporated in vacuo and the residue was dissolved in a solvent mixture composed of 40 mL of acetonitrile, 40 mL of acetic acid, and 40 mL of H2O. After 15 minutes, the mixture was evaporated to dryness yielding a slightly yellow solid. This solid was chromatographed on a silica gel drip column eluted with a quaternary solvent system
composed of EtOAc / CH3CN / CH3OH / H2O (70/10/5/5). In this manner it was possible to isolate 2.44 g (75%) of N-[9-[2-deoxy- 2- [[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo- 1 H-purin-2-yl] 2-methylpropanamide. 1H NMR: (CD3OD):guanine H-8 8.19 (s, 1H), anomeric H- 1 5.85 (d, 1H), Fmoc-methine 4.10 (m, 1H), N-isobutyryl methyl 1.10 (d, 3H), 1.00 (d, 3H). FAB-MS: m/z 605 for (M+l )+. e ) 4.28 grams (7.08 mmol) of N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino] -β-D-glucopyranosyl]-6,9-dihydro-6-oxo- 1 H-purin-2-yl]acetamide was reacted in dry DMF with 8.0 eq (56.64 mmol) of imidazole and 4.0 eq (4.27 mmol) tert-butyldimethyldilyl chloride. After 2 hours reaction at room temperature, the solvent was evaporated in vacuo. The residue was partitioned between EtOAc and H2O, acidified to pH 2 by addition of 10% (wt/vol) aqueous citric acid. The EtOAc layer was twice extracted with H2O. The combined aqueous layers were back-extracted once with EtOAc. The combined EtOAc layers were then washed once each with saturated aqueous NaHCO3 and brine. The solution was dried over MgSO4, filtered and the filtrate concentrated in vacuo. The residue was dissolved in a minimum of CHCl3 and applied to a pad of silica gel (30 mL). The
impregnated gel was eluted with 100 mL of CHCl3 The bis-silyl product was eluted with 7.5% CH3OH/CHCl 3. The appropriate fractions were concentrated to a clear oil and the oil was dried under vacuum in the presence of P2O 5 for 12 hours.
To a 0.2 Molar CH2Cl2 solution of 2.96 g (3.56 mmol) of the bis-silyl product at 3°C stirred under a nitrogen atmosphere was added 4.14 mL (35.60 mmol) of 2,6-lutidine and 2.05 mL (8.90 mmol) of tert-butyldimethylsilyl trifluoromethanesulfonate.
Stirring was continued at 3°C for 20 minutes before an additional portion of tBDMSOTf (2.05 mL) was added. The reaction was stirred at room temperature for 5 hours. At this time, the reaction mixture was diluted with CH2Cl2 and extracted once with H2O acidified with 10% (wt/vol) aqueous citric acid, twice
extracted with H2O. The aqueous layers were back extracted once with CH2Cl2. The combined organic layers were washed once with saturated, aqueous NaHCO3 and finally once with brine. The organic layer was dried with MgSO4, filtered, and concentrated in vacuo to a white solid. This solid was loaded to a silica gel flash column and the desired silylated product, N- [9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino] - [3 ,4,6-tris-O- [( 1 , 1 -dimethylethyl) dimethylsilyl]-β-D-glucopyranosyl]-6 ,9-dihydro-6-oxo- 1 H-purin-2-yl]-2-methylpropanamide was obtained by elution of the column with 1 % CH3OH/CHCl3: 2.54 g (75%). 1H NMR: (CDCl3): δ H- 8 8.05 (s, 1H), H-1' 5.91 (5, 1H); calculated for
C48H74N6O8Si3 C 60.85, H 7.87, N 8.87; found: C 61.01, H 8.03, N 8.69; FAB-MS: m/z 947 for (M+1 )+. f) 5.66 g (6.00 mmol) of N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy) carbonyl]amino]-[3,4,6-tris-O-[( 1 , 1 -dimethylethyl)dimethylsilyl]]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo- 1 H-purin-2-yl]-2-methylpropanamide was treated with 1.70 equivalents of camphor sulfonic acid (CSA) (2.36 g) in 25 mL of distilled CH2Cl2 and 25 mL of CH3OH at - 10°C. The reaction was stirred at 0°C for
2 hours before quenching the reaction with saturated aqueous NaHCO3 and dilution of the mixture with CH2Cl2. The organic layer was twice extracted wtih saturated, aqueous NaHCO3; the combined aqueous layers were extracted once with CH2Cl2. The combined organic layers were washed with brine, dried over
M g S O4, filtered and the filtrate was concentrated in vacuo to a white foam. The desired primary alcohol, N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]- [3 ,4,-bis-O- [( 1 , 1 -dimethylethyl)dimethylsilyl]]-β-D-glucopyranosyl] -6,9-dihydro-6-oxo- 1 H-purin-2-yl]2-methylpropanamide was sufficiently pure to carry onto the next step for oxidation: 4.70 g (94%). 1H NMR: (DMSO-δ6) : δ anomeric H- 1 5.60 (d, 1H), t-butyl Si 0.92 (s, 9H), t-butyl Si 0.85 (s, 9H), t-butyl Si 0.79 (s, 9H). FAB-MS: m/z 833 for (M+1 ). g) To 0.79 g (0.95 mmoL) of N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino]- [3,4,-bis-O- [( 1 , 1 -dimethylethyl)dimethylsilyl]]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo- 1 H-purin-2-yl]2-methylpropanamide was added 0.0034 g of KBr, 0.0016 g of (n-Bu)4NHSO4, and 32 mL of distilled CH2Cl2. The reaction mixture was cooled to 0°C with stirring and 0.0006 g of TEMPO was added. To this mixture was added in one portion a solution containing 1.09 mL of 1.04 M NaClO, 1.50 mL saturated NaHCO3, and 2.37 mL of H2O. The reaction was stirred for 1 hour at 0°C . At this point, the reaction was terminated by dilution with CH2Cl2 (50 mL) and the aqueous layer was separated. The aqueous layer was extracted once with CH2Cl2. The combined organic layers were concentrated in vacuo.
The resulting opaque residue was dissolved in 10 mL of tert-butyl alcohol and 10 mL of H2O. To this stirred solution was added 1.05 g of NaH2PO4 and 4.00 mL of 2-methyl-2-butene. To this solution was added 0.86 g of NaClO2. This mixture was stirred at room temperature for 30 minutes. At this time, the reaction mixture was poured into a mixture of EtOAc and H2O acidified to pH=2 with 10% (wt/vol) aqueous citric acid. The layers were separated and the organic layer was twice extracted with H2O. The combined organic layers were combined, washed with brine, dried with MgSO4, filtered and concentrated in vacuo to a white foam residue. The desired product, 1-[2-(2-methylpropanoylamino)- 1 ,6-dihydro-6-oxo-9H-purin-9-yl ] - 1 ,2-dideoxy-2]-[[(9H- fluoren-9-ylmethoxy)carbonyl] amino]-[3,4-bis-O-[( 1 , 1 -dimethylethyl)dimethyl-silyl] -β-D-gluco-pyranuronic acid was obtained by chromatography of the residue from a silica gel drip column eluted with a quaternary solvent system
composed of EtOAc / CH3CN / CH3OH / H2O (70/10/7.55/7.5) in
75% yield (0.60 g). 1H NMR: (DMSO-δ6): anomeric H- 1 5.72 (d, 1H), t-butylSi 0.91 (s, 9H), t-butyl Si 0.18 (s, 6H), (CH3)2Si 0.17 (s, 3H), (CH3)2Si 0.10 (s, 3H). FAB-MS: m/z 869 for (M+Na)+. Alternatively, the guanine building block of formula II can be prepared as set forth in Example 4B.
EXAMPLE 4B
Synthesis of guanine building block a) To 5 g ( 17.65 mmol) of N-[6-(phenylmethoxy)- 1 H-purin-2-yl]-acetamide was added 150 mL of dry CH3CN and 9.60 mL (38.34 mmol) of BSA and this mixture was stirred at 80°C for 3 h. At this time, 15.65 g (33.80 mmol) of 2-deoxy-2- [(trifluoroacetyl)amino]-β-D-glucopyranose- 1 ,3 ,4,6-tetraacetate was added followed by 6.80 mL (35.30 mmol) of TMSOTf. This mixture was heated with stirring at 80°C for 12 hours. At this time, the reaction mixture was poured into 200 mL of saturated, aqueous NaHCO3 and this mixture was extracted twice with EtOAc. The combined organic layers were washed once with H2O and brine before drying over MgSO4. The crude product was obtained by filtration and concentration of the filtrate in vacuo to a brownish foam. The glycosylation product was obtained by silica gel column chromatography eluted with 50% to 75% EtOAc /hexanes. Appropriate fractions were collected and further purified by normal phase preparative HPLC eluting prepacked silica cartridges with 50% EtOAc/hexanes. In this manner, 1.75 g (15% yield) of the desired product, N-[6-(phenylmethoxy)-9-[3,4,6-tris-O-acetyl-2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranosyl]-1H-purin-2-yl]acetamide, was isolated. 1H NMR:
(CDCl3): guanine H-88.42 (s, 1H), anomeric H-1 6.19 (d, 1H), benzyl methylene 5.62 (m, 2H), N-acetamide methyl 2.30 (s, 3H). FAB-MS: m/z 667 for (M+1)+. b) To a CH3OH solution of N-[6-(phenylmethoxy)-9-[3,4,6-tris-O-acetyl-2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranosyl]-1H-purin-2-yl]-acetamide is added a catalytic quantity of 10% (wt/wt) Pd/carbon. This mixture is purged several times with hydrogen gas and then stirred at room temperature under 1 atmosphere pressure of hydrogen gas. When TLC indicates the consumption of the starting material, the mixture is filtered and washed through celite; the filtrate is then concentrated to yield N-[9-[3,4,6-tris-O-acetyl-2-deoxy-2-[(trifluoroacetyl)amino]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo-1H-purin-2-yl]-acetamide. c) To N-[9-[3,4,6-tris-O-acetyl-2-deoxy-2-[(trifluoroacetyl)-amino]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo-1H-purin-2-yl]acetamide is added a solution composed of a solution of Et3N, CH3OH, and H2O (1 / 5 / 4 volume ratio). This solution is stirred at room temperature for 5 h. At this time, the solution is concentrated to dryness in vacuo. The residue is treated with 30% (wt/vol) NH4OH for 72 h at room temperature. The reaction mixture is then concentrated to dryness in vacuo. The residue is dissolved in H2O and loaded to a C1 8 reversed phase gel column and eluted with H2O followed by CH3OH / H2O ( 1/1 ). Appropriate fractions are collected and evaporated to dryness in vacuo to result in 9-(2-amino-2-deoxy-β-D-glucopyranosyl)guanine . d ) To a H2O solution of 9-(2-amino-2-deoxy-β-D-glucopyranosyl)guanine is added a 1 ,4-dioxane solution
containing 1.2 eq of N-(9-fluorenylmethoxy-carbonyloxy)-succinimide. This results in a cloudy mixture. To this system is added 0.65 eq NaHCO3. This mixture is stirred for 12 h at room temperature. When TLC indicates the consumption of starting material, the mixture is concentrated in vacuo to a dry residue.
This residue is stirred for 18 h in excess Et2O and H2O. The suspension is then filtered and the solid dried in a vacuum oven for 12 h. The desired N-Fmoc protected amino sugar, 9-(2-deoxy-2- [[9H-fluoren-9-ylmethoxy)carbonyl]amino]- β-D-glucopyranosyl)guanine will be obtained. e ) To 9-(2-deoxy-2- [[(9H-fluoren-9-yl methoxy)carbonyl] amino] -β-D-glucopyranosyl)guanine is added dry pyridine and the resulting solution is evaporated. This procedure is repeated twice. The residue is then dissolved in pyridine. To this solution is added 6 eq of chlorotrimethylsilane. This reaction mixture is stirred at room temperature for 40 min. At this time, 5 eq of acetyl chloride is added and the resultant solution is stirred at room temperature until TLC indicates the complete conversion of the starting material into product. The reaction mixture is quenched by cooling to 0°C before addition of 10 mL of H2O and stirring for 1.5 h, followed by addition of 40 mL of cold saturated NaHCO3. This 2 phase system is stirred for an additional 3 h at which time the mixture is extracted twice with EtOAc. The EtOAc layer is washed with brine and dried over MgSO4, filtered and concentrated to dryness in vacuo. The residue is dissolved in EtOAc and this solution is washed thrice with 2 N HCl, thrice with saturated, aqueous NaHCO3, once with brine and dried with
MgSO4, and filtered. The filtrate is evaporated to dryness. The residue is purified by elution from a silica gel chromatography column eluted with 15% CH3OH / CHCl3 to yield the desired N-Fmoc protected amino sugar N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo-1H-purin-2-yl]acetamide. f) The tris-triisopropylsilyl ether of N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-glucopyranosyl]-6,9-dihydro-6-oxol-1H-purin-2-yl]-acetamide is formed by reaction of this material in dry DMF with excess imidazole and excess chlorodimethylisopropylsilane in presence of a catalytic quantity of DMAP. The product is isolated by evaporation of solvent, EtOAc and H2O by standard extraction procedures, concentration of the organic layer and silica gel column chromatography of the residue. This will result in good yields of tris-silyl ether, N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-3,4,6-tris- O-[(1-methylethyl)dimethylsilyl]]-β-D-glucopyranosyl]-6,9-dihydro-6-oxo-1H-purin-2-yl]acetamide. g) N-[9-[2-deoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4,6-tris-O-[(1-methylethyl)dimethylsilyl]]-β-D-glucopyranosyl]- 6,9-dihydro-6-oxo-1H-purin-2-yl]acetamide is transformed to the guanine building block 1-[2-(acetylamino)-1,6-dihydro-6-oxo-9H-purin-9-yl]-1,2-dideoxy-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-[3,4-bis-O-[(1-methylethyl)-dimethylsilyl]]-β-D-glucopyranuronic acid according to the procedures previously described. EXAMPLE 5
Synthesis of dimer and trimer of formula I a) To 0.13 g (0.20 mmol) of cytosine building block 1,2-dideoxy-1-[1,2-dihydro-2-oxo-4-[(phenylcarbonyl)amino]-1-pyrimidinyl]- [3,4-bis-O-[triethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]-β-D-glucopyranuronic acid was added 2 mL of DMF. To this solution was added 0.037 mL (0.31 mmol) of benzylbromide and 0.060 g (0.72 mmol) of NaHCO3. This mixture was stirred for 3.5 h. At this time the solvent was removed in vacuo and the residue was dissolved in EtOAc. The EtOAc solution was extracted twice with H2O, followed by washing with brine, drying with MgSO4 and filtration. The filtrate was concentrated to dryness and the residue was chromatographed on silica gel eluted with 2% CH3OH/CHCl3 by which was obtained in 75% yield (0.108 g) of the desired benzyl 1,2-dideoxy-1-[1,2-dihydro-2-oxo-4-[(phenylcarbonyl)amino]-1-pyrimidinyl]-[3,4-bis-O-triethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)-carbonyl]amino]-β-D-glucopyranuronate. 1H NMR: (CDCl3): cytosine-H-68.21 (d, 1H), anomeric H-1 5.97 (d, 1H), benzyl CH24.59 (s, 2H). FAB-MS: m/z 703 for (M+1)+. b) To 0.030 g (0.043 mmol) of benzyl 1,2-dideoxy-1-[1,2-dihydro-2-oxo-4-[(phenylcarbonyl)amino]-1-pyrimidinyl]-[3,4-bis-O-[triethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]-β-D-gluco-pyranuronate was added 0.50 mL of 20% piperidine in DMF. This solution was stirred at room temperature for 20 min before concentration of the reaction mixture to dryness in vacuo. The residue was redissolved in 1 mL of DMF and concentrated again to dryness in vacuo three times. In a separate ependorf vial was added 1,2-dideoxy-1-[1,2-dihydro-2-oxo-4-[(phenylcarbonyl)amino]-1-pyrimidinyl]-[3,4-bis-O-[triethylsilyl]-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-β-D-glucopyranuronic acid (0.046 g (0.055 mmol), 0.30 mL DMF, 0.019 mL (0.11 mmol) of DIPEA (diisopropylethylamine), and lastly 0.019 (0.050 mmol) of HATU ([O-(7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate]) . The components in the ependorf were mixed thoroughly and transferred after 1 min to the flask containing the first residue; the ependorf vial was rinsed with 0.3 mL of fresh DMF and that DMF was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. At this time, the reaction mixture was concentrated in vacuo and the residue was loaded to a silica gel flash column eluted with 2 to 4% CH3OH/CHCl3 by which was obtained 0.042 g of the desired cytosine-cytosine dimer (76%). 1 H NMR: (CD3OD): 1 -cytosine-H-6 8.16 (d, 1 H), 2-cytosine-H-6 8.4
(d, 1H), 1 st cytosine anomeric H- 1 5.91 (d, I H), 2nd cytosine anomeric H-1 5.90 (d, 1H), benzyl CH2 5.23 (s, 2H). FAB-MS: m/z 1303 for (M+1 )+. c) To 0.043 g (0.033 mmol) of the cytosine-cytosine dimer (of
Example 5b) was added 0.50 mL of 20% piperidine in DMF. This solution was stirred at room temperature for 20 min before concentration of the reaction mixture to dryness in vacuo. The residue was redissolved in 1 mL of DMF and concentrated again to dryness in vacuo three times. In a separate ependorf vial was added 1 ,2-dideoxy- 1 - [ 1 ,2-dihydro-5-methyl]-2 ,4-dioxo- 1 -pyrimidinyl)-2- [ [(9H-fluoren-9-ylmethox y )c arbonyl]amino] - [3 ,4-bis-O- [triethylsilyl]-β-D-glucopyranuronic acid (0.049 g (0.066 mmol), 0.30 mL DMF, 0.023 mL (0.132 mmol) of DIPEA, and lastly 0.024 g (0.063 mmol) of HATU. The components in the ependorf were mixed thoroughly and transferred after 1 min to the flask containing the first residue; the ependorf vial was rinsed with 0.3 mL of fresh DMF and the rinse was added to the reaction mixture. The reaction mixture was stirred at room temperature for 70 min. At this time, the reaction mixture was concentrated in vacuo and the residue was loaded to a silica gel flash column eluted with 2 to 3.5% CH3OH/CHCl3 by which was obtained 0.045 g of the desired cytosine-cytosine-thymine trimer (75%). 1 H NMR:
(CD3OD): 1-cytosine-H-6 8.40 (d, 1H), 2-cytosine-H-6 8.27 (d, 1H), 1 st cytosine anomeric H-1 6.25 (d, 1H), 2nd cytosine anomeric H- 1 6.13 (d, 1H), thymine anomeric H- 1 5.82 (d, 1H), benzyl CH2 5.23 (s, 2H). FAB-MS: m/z 1814 for (M)+.
EXAMPLE 6
Synthesis of oligomer: H2N-Lys-TCTCTCTCCTTCT-H
To a freshly silylated solid phase peptide micro reactor 50 mg of 0.1 mmol/g (5.0 mmol) charged Fmoc-Lys(e-Boc)-Knorr-BHA-polystyrene resin was added. The Fmoc protecting group was removed by three successive treatments with 0.25 mL of 20% piperidine in DMF for one min, seven min and seven min
durations. After each treatment, the resin was purged of the solution; after the third treatment, the resin was thrice washed with DMF and thrice with CH2Cl2. To the micro reactor was added 0.025 mmol of the thymine building block, followed by 0.35 mL of dry DMF and 0.0086 mL (0.05 mmol) of DIPEA and finally 8.6 mg (0.022 mmol) of HATU in such a fashion that this solution did not come in contact with the resin-bound free amine until one minute after addition of the HATU reagent. The resin was then gently shaken for 20 min before purging the reactor of solvent; the resin was thrice washed with DMF and thrice with CH2C I2. Subsequent couplings were performed by repetition of the same Fmoc-deprotection and coupling procedures. Optional capping was performed by treatment with 5% (vol/vol ) acetic anhydride in DMF for 5 min followed by washing the resin with DMF and CH2Cl2, three times each.
After completion of the coupling sequences, the final Fmoc group was removed in the same manner as described above. The free amino resin was then treated with TFA/CH2Cl2 ( 1 : 1 ) for 1 hour. The resin was removed from the solvent by filtration through cotton and the filtrate was concentrated to dryness in vacuo. The residue was suspended in Et2O and then centrifuged; the Et2O layer was discarded. This Et2O procedure was repeated twice further. The residual solid was then treated for 12 h at room temperature for 0.3 mL of 30% (wt/vol) NH4OH. At this time, the reaction mixture was concentrated to dryness in vacuo. At this time, the reaction mixture was concentrated to dryness in vacuo. The residue was treated with 1 mL of 1.0 molar
tetrabutylammonium fluoride in tetrahydrofuran for 12 hours at room temperature. At this time, 0.3 mL of aqueous 0.5 molar NH4OAc (ammonium acetate) was added and the mixture was concentrated in vacuo. The residue was diluted to a volume of 2.5 mL by addition of H2O and this solution was loaded to a washed NAP-25 column (Pharmacia). After the penetration of the solution into the solid phase of the column, elution of the desalted and crude product was effected with 3.5 mL of H2O. This was collected and lyophilized. The residue was dissolved in H2O and purified by reversed phase HPLC: (VYDAC-C4 10 μ 22 × 250 mm preparative column, 265 nm detection, 1.5 mL/min flow rate, elution with
20% CH3CN/H2O, 0.2% HFBA (heptafluorobutyric acid) for 0- 15 min, then 15-20 min a linear gradient to 20% CH3CN/H2O , 0.1 % TFA). The appropriate fractions were combined and lyophilized. The purified oligomer was homogenous as measured by analytical HPLC using another elution conditions: Waters μ Bondpak C1 8 3.9
× 300 mm eluted with a 20 min linear gradient from 20%
CH3CN/H2O to 20% CH3CN/H2O, 0.1 % TFA at 1.5 mL/min flow rate. The identity of the purified oligomer was confirmed by MALDI-TOF mass spectrometry as 3737 for (M+ 1 )+.
EXAMPLE 7
Hybridization Properties of Oligomer
Thermal Melting Studies Absorbance versus temperature curves were measured at 260 nm using a Cary 3 spectrophotometer equipped with an
electrothermal temperature controller and interfaced with an IBM PS2/50Z computer. Oligonucleotide concentration was 1.4 μM and the buffer contained 100 mM NaCl 10 mM sodium phosphate, and 0.1 mM EDTA, pH 7. Tm values were determined from the maxim of first derivative plots using the Reducep program
(Koerber, S.C.; Fink, A.L., Analytical Biochemistry 1987, 165, 75- 87) which utilizes the Savitzky-Golay algorithm (Savitzky, A.; Golay, M.J.E. Analytical Chemistry 1964, 36, 1627-39)
thermodynamic constants were obtained from fits of data to a two-state model with linear sloping baselines (Petersheim, M. ; Turner, D.H. Biochemistry 1983, 22, 256-263).
Figure imgf000060_0001
Table I Numbers are for melting temperature (Tm 's) in degrees
Celsius. Total strand concentration was 3mM in a buffer of 100 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA, pH 7.0. "Up" indicates the melting temperature; "Down" indicates the annealing temperature .
As shown in Table I, the binding of oligonucleotide of formula I, Example 6 (H2N-Lys-TCTCTCTCCTTCT-H) to RNA and DNA is compared to the binding of DNA to DNA and RNA to DNA. A higher melting temperature indicates a tighter association of the indicated duplex. From the data in Table I, it is clear that an oligonucleotide of formula I (Example 6) binds with greatest affinity to a complementary segment of RNA in an antiparallel fashion (carboxamide terminal of compound of Example 6, binding to the 5' end of an oligo). This strength in binding is similar to that of DNA binding activity. The melting temperatures determined by heating and cooling (up and down, respectively) are similar. The similarity of a melting temperature despite the direction of the change in temperature during these measurement is indicative of an association between two oligomers. EXAMPLE 8
Base-Pairing Specificity of Oligomer
Thermal Melting Studies Conditions: 100 mM NaCl, 10 mM sodium phosphate, 0.1 mM
EDTA, pH 7.
Melting Temperatures (Tm's) in °C In order to test the selectivity of binding, the binding of the same oligonucleotide of Example 6 was investigated for RNA targets sequences containing a single base that is not
complementary to the oligonucleotide of formula I base sequence. The results of these investigations are shown in Table II. A decrease in the melting temperature indicates that the oligomer of formula I binds a complementary sequence with greater affinity than to a sequence that is not perfectly complementary. A decrease in binding affinity due to the presence of base
mismatches is termed selectivity. As is seen from the data in Table II, the oligomer of Example 6 binds in a selective fashion in the Watson-Crick complementary base-pairing manner.
Figure imgf000062_0001
Table II Numbers are for melting temperature (Tm 's) in degrees Celsius. Total strand concentration was 3 μM . Strands are bound in the antiparallel orientation. Numbers in parenthesis indicate the annealing temperature.
EXAMPLE 9
Cell-based Antisense assay
Antisense molecules have been demonstrated to reduce levels of the enzyme tyrosinase in melanocytes by the methods described below. These antisense molecules have potential utility for treating several diseases of hyperpigmentation
(hypermelanosis) including, but not limited to café au lait macules, nevus spilus, post inflammatory mealnosis (exanthems, drug eruptions) and scleroderma. A commonly used line of mouse melanoma cells (for example,
B-16) is raised in cell culture plates using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin. To initiate an experiment, antisense and control oligonucleotides are added to low density (that is, subconfluent) cell cultures, then treatment is continued for several days. Cells are then rinsed with saline, collected by scraping, then cells are extracted and analyzed for levels of protein by the
Bradford method (Bradford, M.M. Anal. Biochem. 1976, 72 : 248).
Levels of the target enzyme tyrosinase from a constant amount of extracted protein is measured essentially as described by a published procedure (Pomerantz, S. H. J. Biol. Chem. 1966, 241 : 161 ). Briefly, extracts are incubated with tyrosine and the cofactor DOPA (3,4-dihydroxy-phenylalanine. ) The tyrosine is tritium labeled at the 3 and 5 positions such that tyrosinase activity is measured by the amount of tritiated water that is produced. Tritiated water is quantitated by liquid scintillation counting. The purpose of the tyrosinase assay is to establish the potency of each antisense molecule by allowing us to calculate the concentration necessary to cause 50% inhibition of tyrosinase levels (IC50) .
To control for effects due to nonspecific toxicity, melanoma cells are seeded into 96-well plates at low density, then allowed to grow in the presence of antisense and control oligonucleotides for several days. Cell proliferation is assayed by the widely used tetrazolium dye procedure (Mosmann, T. J. Immunol. Meth.
1983 , 65, 55). The concentration of each oligonucleotide producing 50% inhibition of growth rate is compared to the concentration of that oligonucleotide which produces 50% inhibition of tyrosinase level. Antisense effects are indicated by the following conditions: a) active oligonucleotides are complementary in sequence to the target messenger RNA, b) sense and mismatched controls are less active than fully matched antisense oligonucleotides, c) tyrosinase activity is inhibited by concentrations of antisense oligonucleotides which do not suppress cell growth. The selectivity of the antisense mechanism of these antisense molecules is determined by measuring the enzyme levels of two non-targetted cellular enzymes: alkaline phosphotase and tyrosine kinase.
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001

Claims

Claims 1. An oligomer of formula
Figure imgf000068_0001
w herei n
R 1 , R 2 and R4 are independently hydrogen, lower alkyl or acyl;
R3 is hydrogen or lower alkyl;
B is a nucleobase or a protected nucleobase;
n is 5 to 30;
X is NR3R4; and
Y is OR3, or NHR3;
and pharmaceutically acceptable salts thereof.
2. The oligomer of Claim 1 , wherein R1 , R2 and R4 are independently hydrogen, methyl, ethyl or acetyl.
3. The oligomer of Claim 2, wherein R1 , R2 and R4 are independently hydrogen or acetyl.
4. The oligomer of Claim 1 , wherein R3 is hydrogen or methyl .
5. The oligomer of Claim 4, wherein R3 is hydrogen.
6. The oligomer of Claim 1 , wherein B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine.
7. The oligomer of Claim 1 , wherein n is 5 to 20.
8. The oligomer of Claim 1 , wherein X is NH2 or NHAc wherein Ac is acetyl.
9. The oligomer of Claim 8. wherein X is NH2.
10. The oligomer of Claim 1 , wherein Y is hydroxy, OCH3, NHCH3, or NH2.
1 1 . The oligomer of Claim 10, wherein Y is hydroxy or NH2.
12. The oligomer of Claim 6, wherein B is thymine, cytosine, adenine, guanine or uracil.
1 3. The oligomer of Claim 1 , wherein R1 and R2 are independently hydrogen, methyl, ethyl or acetyl, R3 is hydrogen or methyl, B is thymine, cytosine, adenine, guanine, uracil, N- benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine; n is to 20; X is NH2 or NHAc wherein Ac is acetyl and Y is hydroxy, OCH3, NH2 or NHCH3.
14. The oligomer of Claim 13, wherein R1 and R2 are independently hydrogen or acetyl, R3 is hydrogen, X is NH2, Y is hydroxy or NH2, n is 5-15 and B is thymine, cytosine, adenine, guanine or uracil.
15. The oligomer of any one of claims 1- 14 having the formula
Figure imgf000070_0001
16. A compound of the formula
Figure imgf000071_0001
wherein
R3' is hydrogen or lower alkyl,
R5 and R6 are independently hydrogen or a hydroxy
protecting group or R5 and R6 taken together are a 1 ,2- dihydroxy protecting group,
R7 is hydrogen or an amino protecting group,
R8 is hydrogen or an acid protecting group, and
B is a nucleobase or a protected nucleobase.
17. The compound of Claim 16, wherein R5 and R6 taken together are a ketal.
1 8. The compound of Claim 16, wherein R5 and R6 are independently hydrogen, methyl, ethyl, acetyl or trisubstituted-silyl ether.
19. The compound of Claim 18, wherein the trisubstituted silyl ether is triethylsilyl, isopropyldimethylsilyl or tert-butyldimethyl silyl.
20. The compound of any one of Claims 16- 18, wherein
R7 is hydrogen, 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl.
21 . The compound of any one of Claims 16-18, wherein R 8 is hydrogen, tert-butyl or benzyl.
22. The compound of any one of Claims 16- 18, wherein B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine.
23. The compound of any one of Claims 16-18, wherein R5 and R6 are independently hydrogen, methyl, ethyl, acetyl or trisubstituted silyl ether; R3 ' is hydrogen or methyl; R7 is hydrogen, 9-fluorenylmethoxy-carbonyl or tert-butyloxycarbonyl; R8 is hydrogen, tert-butyl or benzyl, and B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine.
24. The compound of Claim 23, wherein R3' is hydrogen, R5 and R6 are independently trisubstituted silyl ether, R8 is benzyl or hydrogen, R7 is 9-fluorenylmethoxycarbonyl or tert-butoxycarbonyl and B is thymine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine.
25. The compound of Claim 23, wherein R5 and R6 are independently tert-butyldimethylsilyl.
26. A compound of formula
Figure imgf000073_0001
wherein R3 ' is hydrogen or lower alkyl, R5 and R6 are independently hydrogen or a hydroxy protecting group or R5 and R6 taken together are a 1,2-dihydroxy protecting group, R7 is hydrogen, or an amino protecting group, and B is a nucleobase or protected nucleobase.
27. The compound of Claim 26, wherein R3' is hydrogen, R5 and R6 are independently hydrogen, methyl, ethyl, acetyl or trisubstituted silyl ether; R7 is hydrogen, 9-fluorenylmethoxycarbonyl or tert-butyloxy-carbonyl; B is thymine, cytosine, adenine, guanine, uracil, N-benzoylcytosine, N-acetylcytosine, N-benzoyladenine, N,N-dibenzoyladenine, N-acetylguanine or N-isobutyrylguanine .
28. A compound according to any one of claims 16-25 having the formula
Figure imgf000073_0002
29. The compound, N-[9-[3,4,6-tris-O-acetyl-2-deoxy-2- [(trifluoro-acetyl)amino-β-D-glucopyranosyl] -6,9-dihydro-6-oxo-1 H-purin-2-yl] -acetamide .
30. A process for the preparation of a compound of formula
Figure imgf000074_0001
wherein R3 ' is hydrogen or lower alkyl, R5 and R6 are independently hydrogen or a hydroxy protecting group or R5 and R6 taken together are a 1 ,2-dihydroxy protecting group, R7 is hydrogen, or an amino protecting group, and B is a nucleobase or protected nucleobase, which comprises reacting a compound of formula
Figure imgf000074_0002
wherein R3 ' is hydrogen or lower alkyl, R5 and R6 are independently hydrogen or a hydroxy protecting group or R5 and R6 taken together are a 1 ,2-dihydroxy protecting group, R7 is hydrogen, or an amino protecting group, R8 is hydrogen or an acid protecting group, and B is a nucleobase or protected
nucleobase with an aqueous sodium hypochlorite solution in the presence of an aprotic aqueos-immiscible solvent and 2,2,6,6-tetramethyl- 1-piperidinyloxy free radical followed by reaction with sodium chlorite.
3 1 . A medicament comprising a compound according to any one of claims 1- 15, and a pharmaceutically inert carrier.
32. A medicament which decreases the production of a target protein in a cell, which medicament comprises a compound according to any one of claims 1- 15 in an amount effective to bind to the mRNA sequence encoding the target protein an said cell and thereby decrease production of said protein, and a pharmaceutically inert carrier.
33. A process for the manufacture of a medicament, especially for decreasing the production of a target protein in a cell, which process comprises mixing a compound of formula I or a pharmaceutically acceptable salt thereof with a therapeutically inert carrier material and bringing the mixture into a galenical administration form.
34. An oligomer according to any one of claims 1 - 15 for use as a therapeutically active substance, especially for
decreasing the production of a target protein in a cell.
35. The use of an oligomer according to any one of claims 1- 15 for decreasing the production of a target protein in a cell.
36. The invention as hereinbefore described, especially with reference to the oligomers, intermediates, processes and compositions as hereinbefore described.
***
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