Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-11-06
2002-08-27
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S027110, C536S027300, C536S027800, C536S027810, C536S028100, C536S028500
Reexamination Certificate
active
06441161
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of modified nucleosides, and in particular, in the field of modified nucleosides for use in diagnostic applications and as therapeutic agents.
BACKGROUND OF THE INVENTION
There has been considerable interest in developing modified nucleosides as therapeutic agents, diagnostic agents, and for incorporation into oligonucleotides. For example, modified nucleosides such as AZT, ddI, d4T, and others have been used to treat AIDS. 5-trifuoromethyl-2′-deoxyuridine is active against herpetic keratitis and 5-iodo-1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)cytosine has activity against CMV, VZV, HSV-1, HSV-2 and EBV (A Textbook of Drug Design and Development, Povl Krogsgaard-Larsen and Hans Bundgaard, Eds., Harwood Academic Publishers, 1991, Ch. 15).
Modified nucleosides have shown utility in diagnostic applications. In these applications, the nucleosides are incorporated into DNA in determinable locations, and various diagnostic methods are used to determine the location of the modified nucleosides. These methods include radiolabeling, fluorescent labeling, biotinylation, and strand cleavage. An example of strand cleavage involves reacting the nucleoside with hydrazine to yield urea nucleosides, then reacting the urea nucleoside with piperidine to cause strand cleavage (the Maxam-Gilbert method).
Modified nucleosides have also been incorporated into oligonucleotides. There are several ways in which oligonucleotides may be useful as therapeutics. Antisense oligonucleotides can bind certain genetic coding regions in an organism to prevent the expression of proteins or to block various cell functions. Further, a process known as the SELEX process, or
S
ystematic
E
volution of
L
igands for
EX
ponential Enrichment, allows one to identify and produce oligonucleotides that selectively bind target molecules. The SELEX process is described in U.S. Pat. No. 5,270,163 issued Dec. 14, 1993, entitled “Methods for Identifying Nucleic Acid Ligands,”. the contents of which are hereby incorporated by reference.
The SELEX method involves the selection of oligonucleotides from a mixture of candidates to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a random mixture of oligonucleotides, the method involves contacting the mixture with a target under conditions favorable for binding (or interacting), partitioning unbound oligonucleotides from oligonucleotides which have bound to (or interacted with) target molecules, dissociating the oligonucleotide-target pairs, amplifying the oligonucleotides dissociated from the oligonucleotide-target pairs to yield a ligand-enriched mixture of oligonucleotides, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired.
Modified nucleosides can be incorporated into antisense oligonucleotides, ribozymes, and oligonucleotides used in or identified by the SELEX process. These nucleosides can impart in vivo and in vitro stability of the oligonucleotides to endo and exonucleases, alter the charge, hydrophilicity or lipophilicity of the molecule, and/or provide differences in three dimensional structure.
Modifications of nucleosides that have been previously described include 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, and methylations. Modifications have also included 3′ and 5′ modifications such as capping. PCT WO 91/14696, incorporated herein by reference, describes a method for chemically modifying antisense oligonucleotides to enhance entry into a cell.
U.S. Pat. No. 5,428,149 to Eaton, which is incorporated herein in its entirety, describes modifying pyrimidine nucleosides via a palladium coupling reaction in which a nucleophile and carbon monoxide are coupled to pyrimidine nucleosides containing a leaving group on the 5-position of the pyrimidine ring, preferably forming ester and amide derivatives.
A variety of methods have been used to render oligonucleotides resistant to degradation by exonucleases. PCT WO 90/15065 describes a method for making exonuclease-resistant oligonucleotides by incorporating two or more phosphoramidite, phosphoromonothionate and/or phosphorodithionate linkages at the 5′ and/or 3′ ends of the oligonucleotide. PCT WO 91/06629 discloses oligonucleotides with one or more phosphodiester linkages between adjacent nucleosides replaced by forming an acetal/ketal type linkage which is capable of binding RNA or DNA.
RNA has been stabilized against endonucleolytic cleavage by modifying the 2′-position of the ribonucleosides. One approach to stabilization against base-specific endonucleolytic cleavage rests on the interference with base recognition by enzymes. Several strategies for this modification are known, including modification with 2′-amino and 2′-fluoro (Hobbs et al.,
Biochemistry
, 12:5138 (1973), Guschlbauer et al.,
Nucleic Acid Res
. 4:1933 (1977)), and 2′-OCH
3
(Shibahara et al., 15:4403 (1987); Sproat et al.,
Nucleic Acids Res
., 17:3373 (1989), each of which is hereby incorporated by reference). PCT WO 91/06556, also incorporated by reference, describes nuclease-resistant oligomers with substituents at the 2′-position. PCT WO 91/10671 describes antisense oligonucleotides chemically modified at the 2′-position and containing a reactive portion capable of catalyzing, alkylating, or otherwise affecting the cleavage of RNA, a targeting portion, and a tether portion for connecting the targeting and reactive portions.
It would be advantageous to provide new nucleosides for therapeutic and diagnostic applications and for inclusion in oligonucleotides. When incorporated in oligonucleotides, it would be advantageous to provide new oligonucleotides that exhibit different high affinity binding to target molecules, and/or show increased resistance to exo and endonucleases than oligonucleotides prepared from naturally occurring nucleosides. It would also be useful to provide nucleotides with modifications that impart a biological activity other than, or in addition to, endo and exonuclease resistance.
It is therefore an object of the present invention to provide modified nucleosides that are useful for therapeutic administration and/or diagnostic applications.
It is a further object of the present invention to provide modified nucleosides that are useful for incorporation into oligonucleotides to allow for binding to or otherwise interacting with target molecules that may be different than would be obtained if naturally occurring nucleosides were used.
It is still a further object of the present invention to provide nucleosides with modifications that may impart a biological activity other than, or in addition to, endo and exonuclease resistance. It is yet a further object of the present invention to provide novel methods for preparing modified nucleosides.
SUMMARY OF THE INVENTION
Novel compounds which are useful as diagnostic agents and as antitumor, antiviral and/or antibiotic therapeutic agents, and methods of preparation and use thereof, are disclosed. The compounds are modified nucleosides, specifically pyrimidine and purines, where the modifications are at the 5- or 6-position of a pyrimidine ring or at the 2-, 6- or 8-positions of the purine ring. Most preferably, the modifications are at the 5-position of the pyrimidine ring and at the 8-position of the purine ring. Although naturally occurring purine nucleosides contain an amine (or carbonyl) group at the 6-position, the amine group can be diazotized and replaced with a halogen using routine chemistry.
The modified purine compounds of the invention are structurally illustrated by formulae (I) and (II) and the modified pyrimidine compounds are structurally illustrated by formulae (III) and (IV), below. The modified urea nucleosides of the present invention are illustrated by fo
Eaton Bruce
Kirschenheuter Gary P.
Gilead Sciences, Inc.
Swanson & Bratschun L.L.C.
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