Preparation of deoxynucleosides

Details Preparation of deoxynucleosides Preparation of deoxynucleosides

2000-03-29

2004-11-23

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S027140, C536S028200

Reexamination Certificate

active

06822089

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to methods for forming deoxynucleosides from their corresponding ribonucleosides by first forming tert-butylphenoxythiocarbonyl derivatives and subsequently effecting a radical deoxygenation reaction at the carbon attached to the site of the tert-butylphenoxythiocarbonyl group.
BACKGROUND OF THE INVENTION
The potential therapeutic use of oligonucleotides represents a new paradigm for novel drug discovery. Over the last decade, oligonucleotide based antisense, triplex, ribozyme and aptamer techniques have emerged as powerful tools in the discovery of more specific and effective drugs (Sanghvi, Y. S.,
In Comprehensive Natural Product Chemistry
; Barton, D. H.; Nakanishi, K. (ed. in chief); vol. 7: DNA and Aspects of Molecular Biology; Kool. E. (ed.); Pergamon: New York, 1999, 285). Among these techniques, the antisense approach leads the trend with over a dozen oligonucleotides currently undergoing human clinical trials for the treatment of viral infections, cancers, and inflammatory disorders. For example, ISIS 2922 (formivirsen sodium) is a 21 mer antisense phosphorothioate that inhibits the replication of the human cytomegalovirun (HCMV). The recent success of antisense drugs in clinical trials is creating a growing demand for the manufacture of oligonucleotides.
Advances in automated synthesis on solid support and commercialization of synthetic nucleic acid building blocks now allows the generation and screening of an unprecedented number of synthetic oligonucleotides. Oligonucleotides are synthesized on automated DNA/RNA synthesizers with nucleoside phosphoramidites employed as the most commonly used monomers. Nucleoside phosphoramidites can be produced from the phosphitylation of 5′-dimethoxytrityl protected 2′-deoxynucleosides.
For the commercialization of antisense drugs, consumption of large amounts of 2′-deoxynucleosides are necessary. 2′-deoxynucleosides currently originate from natural sources, especially from salmon fish milt. The worldwide output of fish milt is about twenty thousand tons per year. From this, only about one hundred tons of DNA salt can be generated. The DNA salt is degraded to give approximately ten tons of 2′-deoxynucleosides in an even distribution of the four 2′-deoxynucleosides (dA, dC, dG, and T). A maximum of one ton of oligonucleotides can be produced from ten tons of 2′-deoxynucleosides, assuming that all ten tons would be available for oligonucleotide production.
According to our predictions, the market for the first three antisense drugs alone, not to mention the market for oligonucleotides used as other types of drugs and as diagnostic reagents, will require at least one ton of oligonucleotides, indicating that natural resources are insufficient to provide enough 2′-deoxynucleosides to meet future antisense drug demand. In addition, due to declining fish stock, fish milt may be an unreliable source of 2′-deoxynucleotides. Because the demand for 2′-deoxynucleosides exceeds the supply to such a great extent, a need exists for alternative sources of 2′-deoxynucleosides.
The supply of RNA and ribonucleosides is much greater than deoxynucleosides. RNA is derived from yeast and ribonucleosides can be produced in large amounts by fermentation processes. Due to their increased availability, ribonucleosides are much less expensive than 2′-deoxynucleosides. Methods exist for synthetically deriving deoxynucleosides from their ribonucleoside counterparts. Nevertheless, these methods are not economically feasible for the large scale production of 2′-deoxynucleosides. For example, ribonucleotides in their 5′-di or triphosphate form can be biosynthetically converted to their 2′-deoxy counterparts by ribonucleotide reductases. However, these processes are undesirable due to multiple inherent difficulties in the scaled-up production of 2′-deoxynucleosides catalyzed by these reductases.
Other possibilities exist for deriving deoxynucleosides from ribonucleosides. For instance, the chemical transformations used for converting alcohol groups to their corresponding deoxy derivatives are viable options. This chemistry involves radical chain reactions wherein thiocarbonyl derivatives of the alcohol groups are deoxygenated using free radical initiators and tributyltin hydride, as described by Barton and McCombie (Barton, D. H. R.; McCombie, S. J.,
J. Chem. Soc., Perkin Trans. I,
1975, 1574). These reactions are useful for the 2′-deoxygenation of ribonucleosides as well. (Robins, M. J.; Wilson, J. S.,
J. Am. Chem. Soc.,
1981, 103, 932, Robins, M. J.; Wilson, J. S.; Hansske, F.,
J. Am. Chem. Soc.,
1983, 105, 4059). Robins developed a thiocarbonyl reagent, phenyl chlorothionoformate (PhOCSCl, $44.75/5 g, Aldrich™ 1998-1999), that is introduced onto the 2′position of a ribonucleoside by a simple acylation. Chemical 2′-deoxygenation of the 2′-thiocarbonyl ribonucleoside is subsequently effected by a radical reaction. In addition to having a higher cost associated with the reagents these reactions use tin reagents for reductions which are toxic and difficult to dispose of.
The method developed by Robins was improved when the phenyl groups of the thiocarbonyl reagents were substituted with electron donating groups, such as halogens. (Barton, D. H.; Jaszberenyi, J. C.,
Tetrahedron Letters,
1989, 30, 2619, Barton, D. H. R.; Dorchak, J.; Jaszberenyi, J. C.,
Tetrahedron Letters,
1992, 36, 7435). Barton found that substituted phenyl chlorothionoformates, such as, 2,4,6-trichlorophenyl chlorothionoformate ($58.70/5 g Aldrich™ 1998-1999), or especially when pentafluorophenyl chlorothionoformate ($64.00/5 g Aldrich™ 1998-1999) is used to make the thiocarbonyl derivative, radical deoxygenation reaction rates with tributyltin hydride are considerably increased, occurring in minutes rather than hours. Additionally, the yields were found to be excellent. The electron withdrawing inductive effect of the substituents increases the radicophilicity of the thiocarbonyl group, thereby speeding up reaction rates. Although this method may be effective for the large scale production of 2′-deoxynucleosides from their corresponding ribonucleosides, the cost of the substituted phenylthiocarbonyl compounds is prohibitively high.
The use of a series of substituted 3′-phenyl thionocarbonates has been described wherein a free radical coupling using oximes mediated by bis(trimethylstannyl)-benzopinacolate. These reactions led to the formation of carbon-carbon bonds in the preparation of a series of dimeric nucleosides as mimics of nucleic acids (Bhat, B.; Swayze, E. E.; Wheeler, P.; Dimock, S.; Perbost, M.; Sanghvi, Y.,
J. Org. Chem.,
1996, 61, 8186, Dimock S.; Bhat, B.; Peoc'h, D.; Sanghvi, Y. S.; Swayze, E. E.,
Nucleosides
&
Nucleotides,
1997, 16(7-9) 1629).
The present invention addresses the need for cost-effective methods for the large-scale production of 2′-deoxynucleosides from their corresponding ribonucleosides.
SUMMARY OF THE INVENTION
The present invention provides processes for preparing a 2′-deoxynucleoside comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with at least one protecting agent for a time and under conditions effective to form a 3′-O,5′-O-bisprotected ribonucleoside;
contacting the 3′-O,5′-O-bisprotected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 2′-O-tert-butylphenoxythiocarbonyl derivatives of the bisprotected ribonucleoside; and
treating the derivatives with a reducing agent for a time and under conditions effective to give the 2′-deoxynucleoside.
Preferred protecting agents include 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane.

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