EP1198592A1 - Oligonucleotides comportant a la fois 2-aminoadenine et pyrimidines 5-substituees - Google Patents

Oligonucleotides comportant a la fois 2-aminoadenine et pyrimidines 5-substituees

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Publication number
EP1198592A1
EP1198592A1 EP00947043A EP00947043A EP1198592A1 EP 1198592 A1 EP1198592 A1 EP 1198592A1 EP 00947043 A EP00947043 A EP 00947043A EP 00947043 A EP00947043 A EP 00947043A EP 1198592 A1 EP1198592 A1 EP 1198592A1
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Prior art keywords
oligonucleotides
oligonucleotide
ethyl acetate
product
reaction
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EP00947043A
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German (de)
English (en)
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EP1198592A4 (fr
Inventor
Muthiah Manoharan
Phillip Dan Cook
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Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Publication of EP1198592A1 publication Critical patent/EP1198592A1/fr
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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
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/333—Modified A
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • This invention is related to oligonucleotides having non-standard bases.
  • this invention provides oligonucleotides having a combination of 2-aminoadenine and 5-substituted pyrimidines.
  • the 5-substituted pyrimidine is 5- methylcytosine.
  • Oligonucleotides and their analogs have been developed for various uses in molecular biology, including use as probes, primers, linkers, adapters, and gene fragments. In a number of these applications, the oligonucleotides specifically hybridize to a target nucleic acid sequence. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
  • One application utilizing an oligonucleotide's ability to hybridize to a complementary region in RNA or DNA is the diagnostic testing of materials including, for example, biological fluids, tissues, intact cells and isolated cellular components.
  • Oligonucleotides are also widely used as research reagents. They are particularly useful in studies exploring the function of biological molecules, as well as in the preparation of biological molecules. For example, the use of both natural and synthetic oligonucleotides as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology- trained personnel in applying PCR. Oligonucleotides are also used in other laboratory procedures.
  • oligonucleotides involving hybridization are the inhibition of specific gene expression, where the oligonucleotides are complementary to specific target messenger RNA (mRNA) or other sequences.
  • mRNA target messenger RNA
  • antisense This mode of action is commonly known as "antisense".
  • the specific binding of an antisense oligonucleotide to its target mRNA or other sequence can inhibit gene expression by at least two major mechanisms.
  • the binding of the oligonucleotide to its target may hinder protein binding for translation and/or regulation.
  • the oligonucleotides may act through RNase- mediated degradation of a DNA/RNA duplex.
  • Antisense technology is used in research applications to study the functions of certain genes.
  • Antisense oligonucleotides can also be therapeutic agents, with one antisense drug having been approved for use and several oligonucleotides currently undergoing clinical trials.
  • Other oligonucleotide based therapeutics include ribozymes and triplex forming oligonucleotides (TFOs) .
  • TFOs triplex forming oligonucleotides
  • ribozymes a region of the oligonucleotide hybridizes to a complementary region in a target RNA.
  • TFOs the oligonucleotide hybridizes to a complementary DNA strand, displacing the other DNA strand. Both of these therapeutic strategies could also benefit from increased hybridization to their targets.
  • oligonucleotides be able to be synthesized to have customized properties which are tailored for desired uses.
  • modifications include those designed to increase hybridization or binding to a target strand (i.e.
  • Tm melting temperatures
  • oligonucleotides generally fall into three classes, alternative intemucleoside linkages, modified sugars and non-standard bases.
  • Oligonucleotides containing both 2-aminoadenosine and 5-methylcytidine have been used in limited diagnostic applications.
  • Prosnyak, M.I., et al. (Genomics, 1994, 21, 490- 494) used a (CAC) 5 sequence where all the cytidines were 5-methylcytidines and all the adenosines were 2-aminoadenosine as a DNA fingerprinting probe.
  • PCR primers containing both these modifications were used by Lebedev, Y., et al. (Genet. Anal. Biomol Eng., 1996, 13, 15-21). Bailly, C, et al. (Mol.
  • oligonucleotides comprising at least one 2-aminoadenosine nucleoside, at least one 5-substituted pyrimidine nucleoside and a modified intemucleoside linkage. Additional oligonucleotides are provided comprising at least one 2-aminoadenosine nucleoside, at least one 5-substituted pyrimidine nucleoside, a modified intemucleoside linkage and a modified sugar residue.
  • the 5-substituted pyrimidine is a 5-methylcytosine.
  • Preferred intemucleoside linkages are phosphorothioate linkages.
  • 5-substituted pyrimidine is defined as a pyrimidine base with a substitution as described in Luyten, I. and Herdewijn, P. (Eur. J. Med. Chem., 1998, 33, 515-576) at the 5 position. Included are 5-halo pyrimidines, e.g.
  • alkyl, alkenyl and alkynyl substituents are groups having from 1 to about 30 carbons, with 1 to about 10 carbons being particularly preferred.
  • the aryl groups have from 6 to about 14 carbons, and aralkyl groups have from 7 to about 30 carbons.
  • the substituent groups listed above can themselves bear substituent groups such as alkoxy, hydroxyl, amine, benzyl, phenyl, nitro, thiol, thioalkoxy, halo, or alkyl, aryl, alkenyl, or alkynyl. Particularly preferred are 5-methylcytosine and 5- fluoro cytosine. It will be appreciated by those skilled in the art that a 5-substituted pyrimidine will typically be attached to a sugar moiety.
  • 5-modified pyrimidine nucleosides can be incorporated into oligonucleotides in ways well known to persons of ordinary skill in the art, especially via automated methods. Other structures such as those employing modified sugars, modified nucleotide bond and otherwise are also useful herein.
  • 5-methyl-2'-deoxycytidine (5- me-C) containing oligonucleotides can be synthesized according to published methods (Sanghvi et al, Nucl. Acids Res., 1993 21, 3197).
  • 2-aminoadenine is defined to include 2-aminoadenine as well as N2 substituted 2-aminoadenine analogs such as those described in US Patent 5,459,255, commonly assigned and herein incorporated by reference. Also included are N6 substituted 2-aminoadenine analogs.
  • a number of substituent groups can be introduced into 2-aminoadenosine nucleoside in a protected (blocked) form or otherwise subsequently incorporated into an oligonucleotide and de-protected if necessary to form a final, desired compound.
  • Substituent groups include groups covalently attached to the purine ring.
  • Substituents at the amino functions of the adenine are represented as R R 2, R 3 and R 4 in formula (I) which include hydrocarbyl groups such as alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl, cycloalkenyl, cycloaralkyl, aryl, aralkyl or substituted aralkyl.
  • R, to R 4 are independently alkyl, alkenyl and alkynyl substituent , collectively hydrocarbyls, groups having from 1 to about 30 carbons, with 1 to about 10 carbons being particularly preferred.
  • the aryl groups have from 6 to about 14 carbons, and aralkyl groups have from 7 to about 30 carbons.
  • the substituent groups listed above can themselves bear substituent groups such as alkoxy, hydroxyl, amine, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, or alkyl, aryl, alkenyl, or alkynyl groups, and ether groups. Examples of alkyl substitutions are further disclosed by Manoharan, M., Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993.
  • a substituted aminoalkyl (especially -(CH 2 ) n -N[CH 2 ) n NH 2 ] ] ] . 2
  • n 0 to 30, e.g. dimethylaminoethyl, -(CH 2 )
  • R, and R 2 , or R 3 and R 4 may optionally form a 5 to 13 membered ring or ring system optionally incorporating additional heteroatoms selected from N, O and S.
  • Such rings or ring systems may include additional purine heterocycles.
  • Preferred are rings without additional heteroatoms and rings with a single N.
  • Preferred are rings containing 5 to 7 members. Most preferred are rings with 6 members.
  • the rings may also contain unsaturated bonds.
  • 2-aminoadenosine phosphoroamidites and phospho-triesters are prepared according to Chollet, A., et al. (Chemica Scripta 1986, 26, 37-40) by protecting the N2 position of adenine with isobutyryl and the N6 position with l-methyl-2,2-diethoxy pyrrolidine. They are also available through vendors such as TriLink Biotechnologies, Inc., San Diego, CA). The monomers are incorporated in an oligonucleotide chain using standard synthesis methods. Methods for synthesizing oligonucleotides include conversion to the phosphoramidite followed by solution phase or solid phase chemistries. Representative solution phase techniques are described in United States Patent No.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of a plurality of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence ofnucleases.
  • modified oligonucleotides envisioned for this invention include those containing phosphorothioates, phosphotri esters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides with NR-C(*)-CH 2 -CH 2 , CH 2 -NR-C(*)- CH 2 , CH 2 -CH 2 -NR-C(*), C(*)-NR-CH 2 -CH 2 and CH 2 -C(*)-NR-CH 2 backbones, wherein "*” represents O or S (known as amide and thioamide backbones; DeMesmaeker et al, WO 92/20823, published November 26, 1992).
  • the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen etal, Science, 1991, 254, 1497; U.S. Patent No. 5,539,082).
  • modified oligonucleotides may contain one or more substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 2 CH 2 OCH 3 , OCH 2 CH 2 O(CH 2 ) n CH 3 , O(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 where n is from 1 to about 10; O-R or O-R-O-R where R is C, to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; O-substituted lower alkyl, Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;
  • substitutions at the 2' position include those disclosed in US Serial No. 09/016520, commonly assigned and herein incorporated by reference. Substitutions therein disclosed include aminooxy modifications, including O-aminooxyalkyl, O- alkylaminooxyalkyl and dialkylaminooxyethyl (e.g. dimethylaminooxyethyl) and are preferred. Another preferred modification includes 2'-O-methoxyethyl [which can be written as 2'-O-CH 2 CH 2 OCH 3 , and is also known in the art as 2'-O-(2-methoxyethyl) or 2'- methoxyethoxy] (Martin, et al, Helv. Chim. Ada 1995, 78, 486 ).
  • modifications include 2'-methoxy (2'-O-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro ( - F).
  • a further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE.
  • Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • oligonucleotides produced by this invention may additionally include other nucleobase modifications or substitutions.
  • "unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more lipophilic moieties which enhance the cellular uptake of the oligonucleotide.
  • lipophilic moieties may be linked to an oligonucleotide at several different positions on the oligonucleotide. Some preferred positions include the 3' position of the sugar of the 3' terminal nucleotide, the 5' position of the sugar of the 5' terminal nucleotide, and the 2' position of the sugar of any nucleotide.
  • the N 6 position of a purine heterocycle may also be utilized to link a lipophilic moiety to an oligonucleotide of the invention (Gebeyehu, G., et al, Nucleic Acids Res. 1987, 15, 4513).
  • lipophilic moieties include but are not limited to a cholesteryl moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett. 1995, 36, 3651; Shea et al, Nucl. Acids Res. 1990, 18, 3111), a polyamine or a polyethylene glycol chain (Manoharan et al.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA.DNA or RNA.RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA.DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • chimeric oligonucleotides include but are not limited to "gapmers," in which three distinct regions are present, normally with a central region flanked by two regions which may be chemically equivalent to each other but are distinct from the gap.
  • a preferred example of a gapmer is an oligonucleotide in which a central portion (the "gap") of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2'-deoxynucleotides, while the flanking portions (the 5' and 3' "wings") are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., 2'-fluoro- or 2'-O-methoxyethyl- substituted).
  • chimeras include "wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions.
  • the 5' portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2'-deoxynucleotides, whereas the 3' portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2'-fluoro- or 2'-O-methoxyethyl- substituted), or vice-versa.
  • the oligonucleotides of the present invention contain a 2'-O-methoxyethyl (2'-O-CH 2 CH 2 OCH 3 ) modification on the sugar moiety of at least one nucleotide. This modification has been shown to increase both affinity of the oligonucleotide for its target and nuclease resistance of the oligonucleotide.
  • one, a plurality, or all of the nucleotide subunits of the oligonucleotides of the invention may bear a 2'-O-methoxyethyl (-O-CH 2 CH 2 OCH 3 ) modification.
  • Oligonucleotides comprising a plurality of nucleotide subunits having a 2'-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and maybe chimeric oligonucleotides.
  • oligonucleo-tides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred. Oligonucleotides in accordance with this invention are from 5 to 50 nucleo tides in length. In the context of this invention it is understood that this encompasses non-naturally occurring oligomers as hereinbefore described, having from 5 to 50 monomers.
  • Examples 1-13 are depicted in Reaction scheme 1.
  • Examples 14-20 are depicted in Reaction scheme 2.
  • the underlined numbers in parentheses following the Example's title compound correspond to the compound numbers on the respective reaction schemes.
  • a 2 L polyethylene bottle was equipped with a magnetic stirrer, thermometer, dry ice/acetone bath and a stream of argon gas.
  • Anhydrous pyridine 750 mL was added and the solution was cooled to -20°C.
  • 70% hydrogen fluoride in pyridine 500 mL.
  • 2,6-diaminopurine riboside (1, 2-aminoadenosine, 105 g, 0.372 mol, R.I. Chemical, Orange, CA, Reliable Biopharmaceuticals, St. Louis, MO) was suspended in the liquid.
  • the oil was partitioned between ethyl acetate (300 mL) and sat'd sodium bicarbonate solution (300 mL). The aqueous was extracted with more solvent ( 100 mL) and the combined organic layer was concentrated under reduced pressure to a small volume. A solid began to form. It was suspended in a mixture of hexanes-ethyl acetate (300 mL, 1 : 1) and then collected by filtration, washed with the same mix (3x150 mL) and dried (1 mm Hg, 25°C, 24 h) to 26.2 g of product as light yellow crystal, mp sh 139 °C, melts 146-148 °C.
  • the combined filtrate was concentrated and purified by column chromatography (silica, 200 g) using a gradient of ethyl acetate in hexanes (70%) to 80%) to all ethyl acetate to 5% methanol in ethyl acetate). The appropriate fractions were combined, evaporated and dried as above to give 5.9 g more product as a foam for a total of 32.1 g (75.4%).
  • the reaction was diluted with ethyl acetate (200 mL) and washed with sat'd sodium bicarbonate solution (300 mL). The aqueous layer was back-extracted with ethyl acetate (75 mL). The combined organic layer was washed with brine (200 mL), concentrated under reduced pressure to a thin oil and then directly applied to a silica gel column (100 g). The product was eluted with a mixture of hexanes-acetone-triethylamine (70:29:1).
  • the reaction was quenched by the addition of water (10 mL) and then concentrated under reduced pressure to an oil.
  • the oil was partitioned between ethyl acetate (200 mL) and sat'd sodium bicarbonate solution (200 mL).
  • the aqueous layer was back extracted with more ethyl acetate.
  • the combined organic phase was concentrated under reduced pressure and applied to a silica gel column (20 g).
  • the product was eluted with a mix of ethyl acetate-hexanes (1 :1).
  • the oil was partitioned between ethyl acetate (300 mL) and sat'd sodium bicarbonate solution (300 mL).
  • the aqueous was extracted with more solvent (300 mL) and the combined organic layer was concentrated under reduced pressure to a small volume and applied onto a silica gel column (700 g).
  • the product was eluted with ethyl acetate-hexanes (9:1) and then ethyl acetate to obtain all the product.
  • the appropriate pure fractions were combined, concentrated causing a precipitate to form.
  • the product was dried (1 mm Hg, 25°C, 24 h) to 49.4 g yellow crystalline solid, mp 88-96 °C.
  • Impure fractions were combined and recolumned as above to give an additional 4.8 g for a total of 54.2 g (82%) as a light yellow foam.
  • H-NMR (DMSO-d6) d 3.18 (s, 3, CH 3 OCH 2 ), 3.20-3.80 (m, 6, 5'and 5"-H and OCH 2 CH 2 O), 3.75 (s, 6, O-CH 3 ), 4.05 (m, 1, 4'-H), 4.40 (dd, 1, 3'-H), 4.59 (dd, 1, 2'-H), 5.22 (d, 1, 3'-OH), 5.95 (d, 1, l'-H), 6.83 (m, 4, arom.), 7.15-7.4 (m, 9, arom.), 7.93 (br s, 2, NH 2 ), 8.27 (s, 1, 8-H).
  • F-NMR (DMSO-d6) d -52.8 (s).
  • the reaction was washed with sat'd sodium bicarbonate solution.
  • the aqueous layer was back-extracted with dichloromethane ( 100 mL).
  • the combined organic layer was dried over sodium sulfate, concentrated to a thin oil and then directly applied to a silica gel column (200 g).
  • the product was eluted with ethyl ac ⁇ tate-hexanes-triethylamine (80:19:1).
  • the appropriate fractions were combined, concentrated under reduced pressure, coevaporated with anhydrous acetonitrile and dried (1 mm Hg, 25 °C, 24 h) to 9.3 g (70%) of product as a light yellow foam.
  • the oil was partitioned between ethyl acetate (75 mL) and 20% aqueous citric acid (75 mL).
  • the organic phase was washed with water (2x75 mL), dried over sodium sulfate, concentrated under reduced pressure, coevaporated with acetonitrile (100 mL) and dried (1 mm Hg, 25°C, 24 h) to 1.0 g (83%) of product as a white foam.
  • a 2 L polyethylene bottle was equipped with a magnetic stirrer, thermometer, dry ice/acetone bath and a stream of argon gas.
  • Anhydrous pyridine 200 mL was added and the solution was cooled to -20°C.
  • 70%> hydrogen fluoride in pyridine 100 mL
  • 2'-O-Methyl-2-aminoadenosine (2, 30 g, 0.101 mol, R.I. Chemical, Orange, CA, Reliable Bio-pharmaceuticals, St. Louis, MO) was dissolved in the solution.
  • Teit-butylnitrite (42 mL, 0.35 mol) was added in one portion and the reaction was stirred at 5-13 °C until the reaction was complete as judged by TLC (2 h, Rf 0.15, starting material; Rf 0.28, product, ethyl acetate-methanol 9:1).
  • Sodium bicarbonate 600 g was suspended with manual stirring in water (1 L) in a 20 L bucket. The reaction solution was slowly poured (to allow for evolution of carbon dioxide) into the aqueous layer with vigorous stirring. The resulting solution (pH 7-8) was extracted with ethyl acetate (5x400 mL).
  • the reaction was quenched by the addition of methanol (50 mL) and after 30 min, the reaction was concentrated under reduced pressure to an oil.
  • the oil was taken up in ethyl acetate (50 mL) and adsorbed onto silica gel (60 g).
  • the silica was dried under reduced pressure to a free flowing powder and placed on top of a silica gel column (200 g).
  • the product was eluted with a gradient of ethyl acetate in hexanes (50% to 100 % ethyl acetate). The appropriate fractions were combined, concentrated and dried (1 mm Hg, 25 °C, 24 h) to 19.1 g (58%>) of product as a yellow foam.
  • the product containing fractions were combined, concentrated under reduced pressure, coevaporated with acetonitrile (20 mL) and triethylamine (5 mL) and dried (1 mm Hg, 25°C, 24 h) to 2.1 g (90%) of the triethylamine salt of the product as a white foam.
  • the column was eluted with ethyl acetate until the product started to appear and then the polarity was increased by the addition of 5% methanol to drive off the balance of the product.
  • the appropriate fraction were combined, concentrated under reduced pressure and dried (1 mm Hg, 25 °C, 24 h) to a foam, 62 g which was still heavily contaminated with silyl reagent
  • the reaction was quenched by the addition of a sat'd sodium bicarbonate solution (450 mL). The layers were separated and the aqueous layer was extracted with more dichloro-methane (200 mL). The combined organic layers were concentrated under reduced pressure to an oil which in turn was redissolved in a minimum of ethyl acetate. A crystalline precipitate formed. This was collected, washed with ethyl acetate (2x100 mL) and dried (1 mm Hg, 25 °C, 24 h) to a white solid, 25.2 g, mp 226-228 °C.
  • TLC indicated a complete reaction (Rf 0.50, starting material; Rf 0.35, product; hexanes-ethyl acetate 1:1).
  • the reaction was allowed to cool and then it was diluted with methanol (100 mL).
  • the solution was concentrated under reduced pressure to an oil which in turn was applied onto a silica gel column (700 g) and eluted with hexanes and then a mixture of hexanes-ethyl acetate (7:3).
  • the reaction was concentrated under reduced pressure to an oily solid.
  • the residue was redissolved in a minimum of methanol and stirred with Dowex-50 beads (sulfonic acid resin, H+ form, 150 g of dry weight).
  • the solution was filtered, concentrated to an oil and applied to a silica gel column (400 g) .
  • the product was eluted with a gradient of methanol in ethyl acetate (0-20%) .
  • the appropriate fractions were combined, concentrated under reduced pressure and dried (1 mm Hg, 25 °C, 24 h) to a solid, 12.1 g (98%), mp darkens above 178°C but melts above 250 °C.
  • the reaction was concentrated under reduced pressure to a thin oil and then directly applied to a silica gel column (200 g).
  • the product was eluted with ethyl acetate- triethylamine (99: 1).
  • the appropriate fractions were combined, concentrated under reduced pressure, coevaporated with anhydrous acetonitrile and dried (1 mm Hg, 25°C, 24 h) to 9.8 g (73%) of white foam.
  • Example 20 Preparation of 5'-0-(4,4'-Dimethoxytrityl)-3'-0-succinyl-2'-deoxy-2- fluoroadenosine (23) 5'-0-(4,4'-Dimethoxytrityl)-2'-deoxy-2-fluoroadenosine (21, 2.3 g, 4.03 mmol), dimethylaminopyridine (0.16 g, 1.3 mmol) and succinic anhydride (1.7 g, 17 mmol) were dissolved in anhydrous pyridine (20 mL) and stirred at ambient temperature under an argon atmosphere until the reaction was complete (6 h) by TLC (Rf 0.80, starting material; Rf 0.20, product, ethyl acetate-methanol 4:1).
  • the reaction was quenched by the addition of water (20 mL) and then concentrated under reduced pressure to an oil.
  • the oil was partitioned between ethyl acetate ( 100 mL) and 20%> aqueous citric acid ( 100 mL) .
  • the organic phase was washed with water (2x 100 mL), dried over sodium sulfate, concentrated under reduced pressure to an oil.
  • the oil was redissolved in a minimum of dichloromethane (10 mL) and added with vigorous stirring to hexanes (100 mL) to give a precipitate.
  • Oligonucleotides were synthesized on a Perseptive Biosystems Expedite 8901
  • the 1 ⁇ mole syntheses were deprotected and cleaved from the CPG in 1-2 mL 28.0- 30%) ammonium hydroxide (NH 4 OH) for approximately 16 hours.
  • the lO ⁇ mole synthesis CPG's was split into 2 vials for cleavage as described above. 5mg NH 4 OH was added to each vial.
  • the samples were filtered from CPG using Gelman 0.45 um nylon acrodisc syringe filters. Excess NH 4 OH was evaporated away in a Savant AS 160 automatic speed vac. The cmde yield was measured on a Hewlett Packard 8452 A Diode Array Spectrophotometer at 260 ran. Cmde samples were then analyzed by mass spectrometry (MS) on a Hewlett Packard electrospray mass spectrometer. Trityl-on oligos were purified by reverse phase preparative high performance liquid chromatography (HPLC).
  • MS mass spectrometry
  • HPLC reverse phase preparative high performance liquid chromatography
  • HPLC conditions were as follows: Waters 600E with 991 detector; Waters Delta Pak C4 column (7.8X300mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH 7.0; B: 100%) acetonitrile; 2.5 mL/min flow rate; the gradient: 5% B for first five minutes with linear increase in B to 80%> during the next 55 minutes. Fractions containing the desired product were collected and the solvent was dried off in the speed vac. Oligos were detritylated in 80%> acetic acid for approximately 60 minutes and lyophilized again.
  • Free trityl and excess salt were removed by passing detritylated oligos through Sephadex G-25 (size exclusion chromatography) and collecting appropriate samples through a Pharmacia fraction collector. The solvent was again evaporated away in a speed vac. Purified oligos were then analyzed for purity by CGE, HPLC (flow rate: 1.5 mL/min; Waters Delta Pak C4 column, 3.9X300mm), and MS. The final yield was determined by spectrophotometer at 260 nm.
  • 0.5 OD sample was first desalted by osmosis on a Millipore filter (filter type VS with 0.025 mM pore size) for one hour. (Water with 10 drops of 28.0-30% NH 4 OH were added to a petri dish. The 0.5 OD oligo sample was placed on the filter, floating on the water, for approximately one hour.) Purified yields were quantitated using a Hewlett Packard 8452A Diode Array Spectrophotometer at 260 nm.
  • Example 22 Oligonucleotide Sequences Oligonucleotides synthesized with both 2-aminoadenosines and 5- methylcytidines are shown in Table 1. Several oligonucleotides were synthesized as gapmers havings a central region of 2'-deoxy residues flanked by regions of 2'-methoxyethoxy residues.
  • oligonucleotides were synthesized as having all 2'-deoxy residues. All linkages in both sets of oligonucleotides were phosphorothioate linkages. The oligonucleotides were characterized by mass spectrometry and HPLC analysis to verify the correct chemical structure. Results are shown in Table 1. All oligonucleotides synthesized gave the expected molecular weights.
  • oligonucleotides are synthesized as described in Example 21.
  • the structures of the oligonucleotides, and their physical characteristics, are shown in Table 2.
  • Emboldened residues are 2'-methoxyethoxy residues (others are 2'-deoxy-).
  • Underlined cytidines are 5-methylcytidines; underlined adenos are 2-aminoadenosines; all linkages are phosphorothioate linkages.
  • All emboldened nucleosides are 2'-0 -methoxyethyl (2'-O-CH 2 -CH 2 -O-CH 3 ), all C's are 5-methylcytosine residues, all A's are 2-aminoadeno residues
  • Oligonucleotides 26481 (SEQ ID NO.2), 26482 (SEQ ID NO.3), 26483 (SEQ ID NO.4), and 26484 (SEQ ID NO.5), were synthesized in three additional variants: with no base modifications (parent), with all 5-methylcytosines (no 2-aminoadenosines), and with all 2 - am i n o a d e n o s i n e s ( n o 5 - m e thy l c y t o s i n e s ) .
  • HUVEC cells (Clonetics, San Diego, CA) were washed three times with OPTIMEMTM (Life Technologies, Inc., Rockville, MD) and prewarmed to 37°C. Oligonucleotides were premixed with 10 mg/ml LIPOFECTIN® (Life Technologies, Inc.) in OPTIMEMTM, serially diluted to the desired concentrations, and applied to washed cells. Basal and untreated (no oligonucleotide) control cells were also treated with LIPOFECTIN®. Cells were incubated for 4 h at 37°C, at which time the medium was removed and replaced with standard growth medium with or without 5 mg/ml TNF- ⁇ (R&D Systems, Minneapolis, MN). Incubation at 37 °C was continued until the indicated times.
  • nucleosides are 2'-0 -methoxyethyl (2'-O-CH 2 -CH 2 -O-CH 3 ), underlined C's are 5-methylcytosine residues, underlined A's are 2-aminoadenosine residues
  • H-ras targeted antisense oligonucleotides were tested for the ability to specifically reduce H-ras mRNA in T-24 cells (ATCC, Manassas, Va.).
  • T-24 cells were routinely maintained in complete growth media, DMEM supplemented with 10%> fetal calf semm and 100 units per milliliter penicillin and 100 micrograms per milliliter streptomycin (Life Technologies, Grand Island, NY) in a humidified incubator at 37°C.
  • T-24 cells were plated in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) at a density of 2x10 5 cells per well in complete growth medium and incubated as above.
  • oligonucleotide Twenty-four hours after plating the growth media was aspirated and the monolayer was washed once with semm free media (OPTIMEMTM, Life Technologies, Grand Island, NY). Oligonucleotides were formulated in semm free OPTIMEMTM and LIPOFECTIN® (Life Technologies, Grand Island, NY) at a constant ratio of 3 micrograms per milliliter LIPOFECTIN® per 100 nanomolar oligonucleotide. For oligonucleotide treatment two milliliters of formulated oligonucleotide was added to each well and the cells were incubated for four hours at 37°C. Following incubation the formulated oligonucleotide was aspirated from the monolayer, replaced with growth media, and incubated overnight.
  • RNA was prepared using RNAzol (TEL-TEST, Inc., Friendswood, TX.) following the manufacturer's protocol. RNA was fractionated through 1.2% agarose- formaldehyde gels and transferred to nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ) following standard protocols (Sambrook et al, Molecular Cloning a Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989).
  • Nylon membranes were probed for H-ras (Oncogene Research Products, Cambridge, MA) using standard 32 P random priming labeling and hybridization protocols (Sambrook et al, Molecular Cloning a Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989). Following hybridization, membranes were imaged using a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA) and the images quantified using Image Quant 5.0 software (Molecular Dynamics, Sunnyvale, C A). Following image analysis, membranes were striped of H-ras probe and reprobed for G3PDH (Clontech, Palo Alto, CA) and analyzed as above. H-ras signal was normalized to G3PDH. The mean normalized percent control of triplicates and standard deviation for H-ras signal was calculated.
  • oligonucleotides are shown in Table 6. Results are shown in Table 7.
  • the oligonucleotide with 2-aminoadenosines and 5-methylcytosines was about three-fold more effective than a gapmer having a central region of 2'-deoxynucleotides flanked by regions of 2'-methoxyethoxy nucleotides.
  • Emboldened residues are 2'-methoxyethoxy residues (others are 2'-deoxy-).
  • Underlined cytidines are 5-methylcytidines; underlined adenosines are 2-aminoadenosines; all linkages are phosphorothioate linkages.

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Abstract

La présente invention concerne des oligonucléotides comportant à la fois 2-aminoadénine et pyrimidines 5-substituées avec une liaison d"internucléoside modifiée. Dans certains modes préférés de réalisation, la pyrimidine 5-substituée représente 5-méthylcytosine. Les oligonucléotides obtenus présentent une plus forte hybridation avec leurs séquences cibles. En outre, ces oligonucléotides peuvent être des composé utiles, notamment dans des applications diagnostiques et thérapeutiques.
EP00947043A 1999-07-06 2000-07-05 Oligonucleotides comportant a la fois 2-aminoadenine et pyrimidines 5-substituees Withdrawn EP1198592A4 (fr)

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US6822089B1 (en) 2000-03-29 2004-11-23 Isis Pharmaceuticals, Inc. Preparation of deoxynucleosides
LT2578685T (lt) 2005-08-23 2019-06-10 The Trustees Of The University Of Pennsylvania Rnr, apimančios modifikuotus nukleozidus ir jų panaudojimo būdai
CN108586550A (zh) * 2018-06-14 2018-09-28 慎终（上海）生物科技有限公司 2-氟腺苷的合成工艺

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EP0431523A2 (fr) * 1989-12-04 1991-06-12 Enzo Biochem, Inc. Composés de nucléotides modifiés
WO1994002501A1 (fr) * 1992-07-23 1994-02-03 Isis Pharmaceuticals, Inc. Nouveaux 2'-o-alkyle nucleosides et phosporamidites, leurs procedes de preparation et d'utilisation
WO1998039353A1 (fr) * 1997-03-03 1998-09-11 The Perkin-Elmer Corporation Vecteurs oligonucleotidiques chimeres ameliores

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US5883082A (en) * 1990-08-14 1999-03-16 Isis Pharmaceuticals, Inc. Compositions and methods for preventing and treating allograft rejection
US5582986A (en) * 1991-06-14 1996-12-10 Isis Pharmaceuticals, Inc. Antisense oligonucleotide inhibition of the ras gene
EP0672180B1 (fr) * 1992-03-16 2002-01-16 Isis Pharmaceuticals, Inc. Modulation par oligonucleotide de la proteine kinase c
US5801154A (en) * 1993-10-18 1998-09-01 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of multidrug resistance-associated protein
US5744362A (en) * 1994-05-31 1998-04-28 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of raf gene expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0431523A2 (fr) * 1989-12-04 1991-06-12 Enzo Biochem, Inc. Composés de nucléotides modifiés
WO1994002501A1 (fr) * 1992-07-23 1994-02-03 Isis Pharmaceuticals, Inc. Nouveaux 2'-o-alkyle nucleosides et phosporamidites, leurs procedes de preparation et d'utilisation
WO1998039353A1 (fr) * 1997-03-03 1998-09-11 The Perkin-Elmer Corporation Vecteurs oligonucleotidiques chimeres ameliores

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Title
See also references of WO0102608A1 *

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