Pyrimidine nucleosides

Details Pyrimidine nucleosides Pyrimidine nucleosides

1999-08-27

2002-04-09

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S050000, C514S051000

Reexamination Certificate

active

06369040

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the design and synthesis of certain 2- and 4-substituted pyrimidine nucleosides that are useful for incorporation into oligonucleotides. The oligonucleotides, in turn, are useful as diagnostic reagents, research reagents and in therapeutics.
BACKGROUND OF THE INVENTION
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect might be obtained with minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides. Oligonucleotides complementary to a specific target messenger RNA (mRNA) sequence are used. Several oligonucleotides are currently undergoing clinical trials for such use.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate the action of transcription factors. Several recent reports describe such interactions (see, Bielinska, et. al.,
Science
1990, 250, 997-1000; and Wu, et al.,
Gene
1990, 89, 203-209.)
Oligonucleotides also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with the above gene expression inhibition, diagnostic use can take advantage of an oligonucleotides ability to hybridize with a complementary strand of nucleic acid. 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.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of such other biological molecules. One particular use, the use of oligonucleotides as primers in the reactions associated with polymerase chain reaction (PCR), has been the cornerstone for the establishment of an ever expanding commercial business. The use of such PCR reactions has seemingly “exploded” as more and more use of this very important biological tool are practiced. The uses of PCR have extended into many areas in addition to those contemplated by its nobel laureate inventor. Examples of such new areas include forensics, paleontology, evolutionary studies and genetic counseling to name just a few. Primers are needed for each of these uses. Oligonucleotides, both natural and synthetic, serve as the primers.
Oligonucleotides also are used in other laboratory procedures. A number of these uses are described in common laboratory manuals such as
Molecular Cloning, A Laboratory Manual
, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and
Current Protocols In Molecular Biology
, F. M. Ausubel, et. al., Eds., Current Publications, 1993. Such uses include Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA from Book 2 of
Molecular Cloning, A Laboratory Manual
, ibid. and DNA-Protein Interactions and The Polymerase Chain Reaction from Vol. 2 of
Current Protocols In Molecular Biology
, ibid.
To supply the users of oligonucleotides, many scientific journals now contain advertisements for either oligonucleotide precursors or for custom-synthesized oligonucleotides. This has become an important commercial use of oligonucleotides. Other oligonucleotides can be synthesized to have properties that are tailored for the desired use. Thus, a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents, and as therapeutic entities. These modifications are designed to increase binding to a target strand, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to provide stability against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonuclectides, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, or to improve the pharmacokinetic properties of the oligonucleotides.
To serve some of the above-noted uses, reactive functionalities can be tethered to an oligonucleotide and directed at phosphodiester and heterocyclic centers in target compounds. The positioning of these functionalities may require the synthesis of C-2 and C-4 substituted pyrimidine nucleosides with the following general features: (a) preservation of Watson-Crick hybridization characteristics; (b) stability during automated DNA synthesis; and (c) the conformational rigidity of a carbon tether to accurately place functionalities near their target. Criteria (a) and (b) are not met by readily available 2-O-, 2-S-, or 2-N-alkyl pyrimidine nucleosides since these groups are sensitive to nucleophilic displacement. For example, deprotecting DNA oligomers containing S-alkylated moieties with concentrated ammonia likely would yield displacement products at C-2 and C-4 pyrimidine positions. In addition, 2-O-, 2-S-, and 2-N-alkyl groups would preclude the availability of an imino proton at N-3, which is necessary for hydrogen bonding. Likewise, nucleosides having 2-N-alkyl groups at these positions would fail to meet criteria (b) and (c) due to their chemical instability and the spatial instability inherent in an inversion of configuration of a tertiary nitrogen. Accordingly, there remains a need in the art for novel 2- and 4-substituted pyrimidines that meet the design criteria set forth above.
OBJECTS OF THE INVENTION
It is one object of this invention to provide oligonucleotides for use in diagnostics, therapeutics and as research reagents.
It is another object of this invention to provide oligonucleotides that effectively modulate the activity of RNA or DNA or proteins.
It is a further object to provide novel 2-substituted pyrimidines.
These and other objects will become apparent to persons of ordinary skill in the art from a review of the present specification and appended claims.
SUMMARY OF THE INVENTION
In accordance with these and other objects, the present invention provides novel C-2 and C-4 modified pyrimidine nucleosides and oligonucleosides. In certain embodiments, the compounds of the invention have structure 1 or structure 2:
wherein:
J is N or CH;
R
5
is H or CH
3
;
one of R
2
and R
4
is ═O, ═NH, or ═NH
2
+
; and the other of R
2
and R
4
is Q, ═C(R
A
)—Q, C(R
A
) (R
B
)—C(R
C
) (R
D
)—Q, C(R
A
)═C(R
C
)—Q or C≡C—Q;
R
A
, R
B
, R
C
and R
D
independently are H, SH, OH, NH
2
, or C
1
-C
20
alkyl, or one of (R
A
) (R
B
) or (R
C
) (R
D
) is ═O;
Q is halogen, hydrogen, C
1
-C
20
alkyl, C
1
-C
20
alkylamine, C
1
-C
20
alkyl-N-phthalimide, C
1
-C
20
alkylimidazole, C
1
-C
20
alkylbis-imidazole, imidazole, bis-imidazole, amine, N-phthalimide, C
2
-C
20
alkenyl, C
2
-C
20
alkynyl, hydroxyl, thiol, keto, carboxyl, nitrates, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, O-(

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