Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-12-01
2002-06-11
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S046000, C514S047000, C514S048000, C514S049000, C514S050000, C514S051000, C536S026700, C536S026800
Reexamination Certificate
active
06403566
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is nucleoside and oligonucleotide analogues and methods for their preparation.
BACKGROUND
Nucleoside and nucleotide analogs have long been used as pharmaceutical ingredients against a variety of viruses and cancers. Currently, a number of nucleoside and nucleotide analogues are in clinical trials for several diseases.
In the cell, nucleosides and nucleotides are phosphorylated or further phosphorylated to the corresponding nucleoside triphosphates. Nucleoside triphosphates serve as inhibitors of DNA or RNA polymerases. Nucleoside triphosphates can also be incorporated into DNA or RNA, which interferes with the elongation of DNA or RNA.
Active nucleoside analogues are generally readily phosphorylated in the target cell. Corresponding nucleoside triphosphates have high affinity to catalytic sites of the polymerases and compete with the natural nucleoside triphosphates as the substrate of the polymerases.
Certain nucleoside analogues work at the nucleoside or the monophosphate level. One group of promising nucleoside analogues is the nucleosides with conformationally locked sugar moieties. It has been reported that certain conformationally locked carbocyclic nucleoside analogues demonstrated potent activity against HCMV, HSV, and EBV (Siddiqui et al.
Nucleosides Nucleotides
1996, 15, 235-250; Marquez et al.
J. Med. Chem
. 1996, 39, 3739-3747). A conformationally locked, carbocyclic AZT 5′-triphosphate has been reported to be an equipotent inhibitor of HIV reverse transcriptase (Marquez et al.
J. Am. Chem. Soc
. 1998, 120, 2780-2789). Other nucleosides with bicyclic sugar moieties were also prepared even though no activity was found or reported (Chao et al.
Tetrahedron
1997, 53, 1957-1970; Okabe et al.
Tetrahedron lett
. 1989, 30, 2203-2206, Hong, et al.
Tetrahedron Lett
. 1998, 39, 225-228).
Favorable, conformationally locked nucleosides are expected to have a positive impact on antisense oligonucleotides. Oligonucleotides, as potential antisense therapeutics, have been recognized and explored for two decades. Oligonucleotides are capable of forming double or triple helix with complementary DNA or RNA and have the ability to target the specific sequences in the viral and cancer genome. Specific binding of oligonucleotides to the DNA or RNA targets of interest would inactivate the function associated with the DNA or RNA such as replication, transcription, and translation. Therefore, viral cycles, or cancerous process can be interrupted while the normal cell cycles are not affected.
Since natural oligonucleotides are labile to the cellular and extracellular nucleases, a great deal of efforts has been made on the study of oligonucleotide modifications, especially those modifications aimed at improving nuclease resistance and binding affinity. Oligonucleotides containing certain bicyclic nucleosides have been shown to demonstrate improved nuclease stability (Leumann et al. Bioorg.
Med. Chem. Letts
. 1995, 5, 1231-4; Altmann et al.
Tetrahedron Lett
. 1994, 35, 2331-2334, 7625-7628). Recently, 2′-O,4′-C-methylene ribonucleosides, which have a locked 3′-endo sugar pucker, were synthesized and incorporated into oligonucleotides. Hybridization studies show that conformationally locked nucleosides can significantly enhance hybridization of modified oligonucleotides to the complementary RNA and DNA (Obika et al.
Tetrahedron Lett
. 1997, 38, 8735-8738; Koshkin et al.
Tetrahedron
1998, 54, 3607-3630).
There is a need for new, conformationally locked nucleosides with bicyclic sugar moieties. These novel nucleosides should be useful in antiviral, anti-cancer, and other therapies. In addition, oligonucleotides composed of these novel, modified nucleosides should have desired stability to cellular nucleases and strong binding affinity to nucleic acid targets. Therefore, these oligonucleotides should be potentially useful in therapeutics and diagnostics.
SUMMARY OF THE INVENTION
Conformationally locked bicyclic-sugar nucleosides, which have a common geometrical shape, and methods for producing conformationally locked bicyclic-sugar nucleosides are described. Nucleosides are provided having bicyclic sugar moieties and oligonucleotides comprising the following formula:
Wherein X, Y and Z are independently selected from a group of O, S, CH
2
, NR, C═O, C═CH
2
or nothing, where R is selected from a group of hydrogen, alkyl, alkenyl, alkynyl, acyl; R
1
is selected from a group of adenine, cytosine, guanine, hypoxanthine, uracil, thymine, heterocycles, H, OCH
3
, OAc, halogen, sulfonate; R
2
, R
3
are independently selected from a group of H, OH, DMTO, TBDMSO, BnO, THPO, AcO, BzO, OP(NiPr
2
)O(CH
2
)
2
CN, OPO
3
H, PO
3
H, diphosphate, triphosphate; R
2
and R
3
together can be PhCHO
2
, TIPDSO
2
or DTBSO
2
.
The novel nucleosides described herein are anticipated to be useful in antiviral, anti-cancer, and other therapies. Oligonucleotides composed of these modified nucleosides have desired physiological stability and binding affinity that enable them to be useful in therapeutics and diagnostics.
DETAILED DESCRIPTION
Conformationally locked nucleosides which have a 3′-endo sugar pucker, and methods of their preparation are provided. Processes for preparation of previously reported bicyclic nucleoside analogues cannot be applied to the novel nucleoside analogues described herein. The analogues described resulted from the successful linking between C2′ and C4′ positions of ribose in the nucleoside analogues.
As used herein, the abbreviation “Ac” refers to acetyl; the abbreviation “Bn” refers to benzyl; the abbreviation “Bz” refers to benzoyl; the abbreviation “DMT” refers to dimethoxytrityl; the abbreviation “THP” refers to tetrahydropyranyl; the abbreviation “TBDMS” refers to t-butyldimethylsilyl; the abbreviation “TIPDS” refers to tetraisopropyldisilyl; and the abbreviation “DTBS” refers to di(t-butyl)silyl.
Synthesis of 2,4-Bridged Ribofuranose Derivatives
1-&agr;-Methylarabinose 1, prepared according to a published procedure (Tejima et al.
J. Org. Chem
. 1963, 28. 2999-3003), was protected with 1,1,3,3-tetraisopropyldisiloxanyl (TIPS) at O3 and O5 to give 2, which was converted to the ketone 3 by treatment with DMSO/DCC/TFA. The subsequent Wittig reaction and removal of TIPS afforded the alkene 4 in very good yield. Compound 4 was protected with t-butyidimethylsilyl (TBS) at O5 and with benzyl (Bn) at O3 to give 5. Hydroboration of 5 was conducted with 9-BBN to give exclusively the 2-deoxy-2-hydroxymethyl derivative 6 in excellent yield. 2-deoxy-2-hydroxymethyl derivative 6 was subjected to tritylation with 4,4′-O-dimethoxytrityl (DMT) chloride and removal of TBS with tetrabutylammonium fluoride (TBAF) to yield 7.
Compound 7 was oxidized to give the aldehyde 8, which was treated with formaldehyde and sodium hydroxide to yield the 4-hydroxymethyl derivative 9 in excellent yield. The mesylation of 9 and the subsequent removal of DMT afforded 10. The cyclization effected with NaH in THF and the subsequent removal of the mesyl afforded the bicyclic sugar 11. Treatment of compound 11 with acetic anhydride in the presence of DMAP yields 12, whereas treatment with acetic anhydride/acetic acid in the presence of sulfuric acid yields 13, in which the acetoxy at C1 has an inverted orientation (1-&bgr;), as compared to the methoxy of 11.
Synthesis of 2′,4′-Bridged Bicyclonucleosides
The bicyclonucleosides having the 2′,4′-bridged sugar moiety were synthesized from condensations of silylated nucleoside bases and the bicyclic sugars as shown below. The condensation of 13 with bis(trimethylsilyl)thymine yielded the product 14, the &agr;-anomer, in excellent yield. Treatment of 14 with BCl
3
removed acetyl and benzyl simultaneously to yield the bicyclic &agr;-thymidine 15.
The condensation of 13 with 6-chloro-9-trimethylsilylpurine gave a mixture of the &agr;- and &bgr;-purine nucleosides, 16 and 17 (ratio of &agr;: &bgr;, 1:1 to 2:3), which could be separated b
Fish Robert D.
ICN Pharmaceuticals, Inc.
Rutan & Tucker LLP
Wilson James O.
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