Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-11-19
2004-06-29
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S026700, C536S026740, C536S026800, C536S026900
Reexamination Certificate
active
06756496
ABSTRACT:
The present invention is directed to novel hydrogen phosphonodithioates, and in particular to nucleosides, deoxynucleosides, nucleotides and deoxynucleotides containing novel hydrogen phosphonodithioate groups.
BACKGROUND OF INVENTION
Internucleotide phosphate analogs have recently gained interest as having uses in and of themselves, other than for making recombinant DNA for application in genetic engineering techniques. For example, methanephosphonate linked deoxyoligonucleotides inhibit viral replication in mammalian cells (Smith, C. C., et al.,
Proc. Natl. Acad. Sci. USA
83, 2787-2791 (1986)) and some phosphorothioates have been shown to have anti-HIV activity (Matsukura, et al.,
Proc. Natl. Acad. Sci. USA
84, 7706-7710 (1987)). Internucleotide phosphate analog linkages, such as the phosphoramidate linkages, are useful as an attachment for reporter groups, intercalating agents and cross-linking agents. Such linkages also can be resistant to certain nucleases which would otherwise act upon naturally-occurring phosphate internucleotide linkages. Moreover, the modification of the phosphorus-containing internucleotide linkages has been shown to affect gene expression (Marcus-Sekura, et al.,
Nucl. Acids Res.
15, 5749-5763 (1987)). Therefore, there is a continuing interest in phosphate analogs for all of these utilities, as well as for use in synthesizing sequence-defined oligonucleotides and analogs thereof.
SUMMARY OF THE INVENTION
The present invention provides novel and useful hydrogen phosphonodithioates of the formula I:
in general wherein each R is independently a substituted or unsubstituted hydrocarbyl group, or substituted or unsubstituted hydrocarbyl group containing one or more heteroatoms; Y is O, S, —NH— or —NR—; and A
+
is a cation. By the term hydrocarbyl it is meant any group which contains at least the elements hydrogen and carbon. This term is not intended to be limited to groups which contain only. hydrogen and carbon.
Particularly preferred compounds are those wherein the R group is a nucleoside, nucleotide, deoxynucleoside,.deoxynucleotide or any protected or modified derivative of any of those groups. The most preferred class comprises those compounds wherein Y is oxygen. The nucleosides, nucleotides, deoxynucleosides or deoxynucleotides may be protected at any functional site on the molecule.
It will be appreciated that as used herein the terms “nucleosides” and “nucleotides” will include those moieties which contain not only the known purine and pyrimidine bases, but also bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, and the like.
Modified nucleosides or nucleotides will also include modifications on the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with halogen, or functionalized as ethers, etc.
Examples of modified nucleosides or nucleotides which may comprise R in formula I include, but are not limited to:
2-aminoadenosine
deoxy-2-aminoadenosine
5-bromouridine
deoxy-5-bromouridine
5-chlorouridine
deoxy-5-chlorouridine
5-fluorouridine
deoxy-5-fluorouridine
5-iodouridine
deoxy-5-iodouridine
5-methyluridine
(deoxy-5-methyluridine is the same as thymidine)
inosine
deoxy-inosine
xanthosine
deoxy-xanthosine
In general, the compounds according to the present invention are useful in oligonucleotide synthesis to generate analogous internucleotide linkages. The nucleotide (or nucleoside) monomers and oligomers may also themselves have antiviral activity, anticancer utility, or other therapeutic utility, and may also have diagnostic, pesticidal, agricultural, animal health care, or plant health care uses. In particular, the compounds of the invention are useful research reagents for forming internucleotide linkages. A particularly preferred use of the compounds of the present invention is to form internucleotide hydrogen phosphonothioate linkages, which can, in turn, be converted to other phosphothioate linkages.
DESCRIPTION OF PREFERRED EMBODIMENT
In general, and as defined in more detail hereinbelow, the compounds according to the present invention include those in which R may be alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, substituted aryl, or substituted aralkyl, including a protected or unprotected nucleoside, nucleotide, deoxynucleoside or deoxynucleotide.
As described in more detail hereinbelow, the compounds according to the present invention may be prepared by treatment of an acidic azole with phosphorus trihalide to form a reactive intermediate which can be treated directly with the compound ROH, RSH, RNH
2
or RRNH, wherein R is as described herein. Subsequent treatment with hydrogen sulfide, followed by a base, results in compounds of the present invention. It will be appreciated that 1,2,4-triazole, tetrazoles, or other reactants may be utilized in the synthesis provided they form an intermediate upon reaction with phosphorus trihalide which, in turn, undergoes the desired addition of the ROH, RSH, RNH
2
or RRNH utilized in the synthesis.
In a preferred embodiment of the present invention, the compounds of the invention may be made into activated intermediates by conversion of one of the sulfur atoms to a suitable leaving group, under appropriate conditions, such as a thioalkanoate group,
This activated intermediate is useful for conversion to other useful compounds, such as by reaction with a nucleoside or nucleotide to form hydrogen phosphonothioate ester-linked nucleosides or nucleotides.
The compounds according to the present invention may be made in general in situ by treatment of one equivalent of a phosphorus trihalide, preferably phosphorus trichloride, in a suitable solvent with an excess of a reactive acidic azole, such as 1,1,4-triazole, in 3 to 4 equivalent excess. Then an equivalent amount or less, of a suitable reagent, ROH, RSH, RNH
2
or RRNH, wherein R is as defined herein, can be added in the presence of a base. Then treatment with hydrogen sulfide followed by neutralization, such as with trialkyl ammonium bicarbonate, results in compounds according to the present invention, which can be isolated by chromatography, crystallization, distillation, and the like, by conventional procedures.
While generally the synthesis has been described utilizing 1,2,4-triazole, any other suitable acidic azole may be utilized in the synthesis, since the triazole does not appear in the final compound. Other azoles include, but are not limited to, for example, tetrazoles, and the like. Suitable solvents for the in situ reaction will depend, of course, on the solubility of the particular azole utilized in the initial reaction with the phosphorus trihalide, and upon the solubility of the reagent ROH, RSH, RNH
2
or RRNH.
Since R may contain one or more functional groups, these groups will be protected during the synthesis of the compounds according to the present invention, particularly if such functional groups are reactive with electrophilic phosphorus species or hydrogen sulfide under the reaction conditions of the synthesis. Conventional blocking groups may be utilized, such as those used for blocking hydroxyl, sulfhydryl, carboxyl and amino groups in chemical synthesis, particularly those groups used for blocking functionalities in peptide synthesis or oligonucleotide synthesis.
In a preferred embodiment of the present invention, R will be a nucleoside, nucleotide, deoxynucleoside or deoxynucleotide, which contains hydroxyl functionalities as well as amine functionalities in the basic side chains. Under the conditions of reactions for the synthesis of compounds according to the present invention described above, typically the 5′ or 3′ hydroxyl group of the ribonucleoside will be protected by a hydroxyl protecting group, preferably the dimethoxytrityl group. The reactive amine groups on the basic side chains may be protected by an amine protecting group, such as an acyl group or amidine. It will be recognize
Buhr Chris A.
Froehler Brian C.
Crane L. Eric
ISIS Pharmaceuticals Inc.
Wilson James O.
Woodcock & Washburn LLP
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