Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-02-18
2002-11-26
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S025340, C536S025600
Reexamination Certificate
active
06486313
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel methods for the preparation of alkylphosphonate nucleoside dimers, oligomeric compounds and methods of using such oligomeric compounds. In accordance with preferred embodiments of the invention, the present methods provide for the preparation of substantially diastereomerically pure (or enriched) alkylphosphonate compounds from substantially diastereomerically pure (or enriched) starting materials.
BACKGROUND OF THE INVENTION
Modified oligonucleotides are of great value in molecular biological research and in applications such as anti-viral therapy. Modified oligonucleotides which can block RNA translation, and are nuclease resistant, are useful as antisense reagents. One such modification is the alkylphosphonate internucleotide linkage.
There are a number of properties associated with oligonucleotides having alkylphosphonate internucleoside linkages that are superior to native phosphodiester internucleotide linkages. The majority of research in this area has been with methylphosphonates (Miller et al.,
Antisense Research and Applications,
1993, pp. 189-203, Crooke, S. T. and Lebleu, B. (eds) CRC Press, Boca Raton). Oligonucleotides having methylphosphonate internucleotide linkages are isosteric to natural phosphate groups, have non-ionic backbones, are resistant to nucleases, have weak interactions with plasma proteins and can enter cells by passive diffusion or through absorptive endocytosis. Oligonucleotides having methylphosphonate internucleotide linkages are not substrates for RNase H when duplexed with RNA. When a chimeric oligonucleotide is prepared having a 2′-deoxy region flanked on either side by methylphosphonate regions, the resulting oligonucleotide is able to withstand intracellular nuclease degradation until bound to a target RNA. Upon formation of an oligonucleotide/target RNA duplex the 2′-deoxy region becomes a substrate for RNase H and thus can give rise to a terminating event.
In addition to chemical modifications of the internucleoside linkage, the chirality of each phosphorus containing internucleoside linkage also effects the ability of a modified oligonucleotide to bind to RNA. The standard oxidation of a phosphite to a phosphate internucleotide linkages results in attack on either side of the phosphorous atom resulting in Rp and Sp diastereomers. Modified phosphorus oligonucleotides are racemic mixtures having 2
n
isomers, with n equal to the number of intersugar linkages in the oligonucleotide. Thus, a 15-mer modified oligonucleotide, containing 14 asymmetric centers has 2
14
or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to specifically hybridize to a target mRNA or DNA with optimal affinity.
Backbone chirality may also affect the susceptibility of an oligonucleotide-RNA heteroduplex to RNase H activity. The ability to serve as a template for RNase H has significant therapeutic implications since it has been suggested that RNase H causes cleavage of the RNA component in an RNA-DNA oligonucleotide heteroduplex. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that contain the correct chirality for maximum hybridization efficiency and termination of translation. Thus, increasing the percentage of internucleotide linkages that are diastereomerically enriched that can serve as substrates for RNase H in a heteroduplex will likely lead to a more efficacious compound for antisense and other oligonucleotide therapies.
Oligonucleotides having pure Rp or Sp methylphosphonate internucleotide linkages have been prepared by a number of routes. The separation of a mixture of H-phosphonate diastereomers into pure Rp and Sp stereoisomers followed by alkylation with retention of configuration has given the corresponding pure Sp or Rp methylphosphonate (Seela, F., Kretschmer, U.,
J. Org. Chem.,
1991, 56, 3861-3869). Another method requires the separation of a mixture of Rp and Sp 5′-O-protected-2′-O-deoxynucleosyl-3′-O-methylphosphinate (International Patent Application PCT/US93/06251, entitled “Trivalent Synthesis of Oligonucleotides Containing Stereospecific Alkylphosphonates and Arylphosphonates”, filed Jun. 30, 1993), 3′-O-methylphosphonate (Jaworskamaslanka et al.,
Antisense
&
Nucleic Acid Drug Development,
1997, 7, 23-30), or -3′-O-methylphosphonothioate (International Patent Application PCT/US93/06277, entitled “Pentavalent synthesis of Oligonucleotides Containing Stereospecific Alkylphosphonates and Arylphosphonates”, filed Jun. 30, 1993) before their stereoselective incorporation in the oligonucleotide.
The incorporation of alkylphosphonate internucleoside linkages into modified oligonucleotide has been limited to only a few groups. The primary group that has been used is the methylphosphonate group. The use of benzylphosphonate internucleotide linkages is another group that has been synthesized (Eisenhardt et al.,
Nucleosides
&
Nucleotides,
1997, 16, 1669-1672).
The synthesis of a T-T dimer having a phosphorothioate internucleoside linkage has been previously reported using chiral auxiliary indole-oxazaphosphorine with 3′-OTBDPS-thymidine in the presence of several equivalents of 1,8-diazabicyclo[5.40]undec-7-ene (DBU) (Wang, J. C., Just, G.,
Tetrahedron Lett.,
1997, 38, 3797-3800). After removal of the chiral auxiliary and sulfurization, the T-T phosphorothioate dimer is isolated in very high diastereomeric excess.
There remains a need for improved methods for preparing alkylphosphonates. There is further need for methods for preparing alkylphosphonates that are substantially diastereomerically pure or enriched. The present invention is directed to these, as well as other, important ends.
SUMMARY OF THE INVENTION
The present invention provides processes for preparing alkylphosphonate compounds having the structure:
wherein each M
1
and M
2
independently, are hydrogen or a hydroxyl protecting group or one of M
1
and M
2
is hydrogen or a hydroxyl protecting group and the other of M
1
and M
2
is a covalent attachment to a solid support. In the foregoing structure, each J is, independently, hydrogen, a 2′-substituent group, or a protected 2′-substituent group and R
1
is an electron withdrawing group. In such compounds, X
2
is hydrogen or —(CH
2
)
m
—E
2
; E
2
is hydrogen, alkyl, aryl, aralkyl, substituted aryl, a heterocycle, mixed heterocycle or heteroaryl and m is 0, 1, 2 or 3. Each Bx is a heterocyclic base moiety, preferably of the type associated with nucleobases, such as the preferred purines and pyrimidines. The methods comprise the steps of selecting a dimeric trialkylphosphite compound of the structure:
and treating said dimeric trialkylphosphite compound, in a solvent, with a base under conditions of time temperature and pressure to form said alkylphosphonate compound.
It is preferred that one of M
1
and M
2
be a hydroxyl protecting group and the other of M
1
and M
2
be a covalent attachment to a solid support. In other preferred embodiments, both of M
1
and M
2
are hydroxyl protecting groups. It is preferred for some embodiments, that the electron withdrawing group be CN, COOR
5
wherein R
5
is C
1
-C
8
alkyl, NO
2
, CF
3
, aryl, halogen or substituted aryl wherein said substituents are halogen, cyano, C
1
-C
6
alkyl, perhalo C
1
-C
3
, alkyl, alkoxy, nitro, nitroso or carboxylate, with CN or NO
2
being more preferred. In some preferred embodiments, X
2
is H and R
1
is CN.
In the processes of the invention, the alkylphosphonate compound is prepared as substantially a single diastereomer. It is also preferred that the dimeric trialkylphosphite compound be substantially diastereomerically pure as well.
In accordance with some embodiments, it has been found useful for the base to be
Guzaev Andrei
Jung Michael E.
Just George
Manoharan Muthiah
Roland Arlène
ISIS Pharmaceuticals Inc.
Owens Howard V.
Wilson James O.
Woodcock & Washburn LLP
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