Process for the synthesis of modified P-chiral nucleotide...

Details Process for the synthesis of modified P-chiral nucleotide... Process for the synthesis of modified P-chiral nucleotide...

1999-10-14

2002-06-18

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025310, C536S025320, C536S025330, C536S025340, C536S025600, C536S026260, C536S026700, C536S026710, C536S026800

Reexamination Certificate

active

06407223

ABSTRACT:

An object of the invention is to provide a process for the synthesis of modified P-chiral nucleotide analogues of general formula 1, where R
1
stands for protecting group, preferably 4,4′-dimethoxytrityl (DMT), 9-phenylxanthene-9-ol (Px) or trialkylsilyl group, R
2
is a hydrogen atom, protected hydroxyl group, halogen, chloroalkyl, nitrile, azide, protected amine, perfluoroalkyl (containing up to four carbon atoms), perfluoroalkoxyl (containing up to four carbon atoms and up to nine fluorine or chlorine atoms), alkoxyalkyl, vinyl, ethynyl, OQ
1
, SQ
1
, NHQ
1
, where Q
1
stands for alkyl (C
1
-C
4
), aryl (C
6
-C
12
), alkenyl (C
3
-C
12
) or alkynyl (C
3
-C
12
), B stands for a purine or pyrimidine base (appropriately protected if necessary), Z is selected from Q
1
or vinyl, ethynyl, aminomethyl or aminoethyl substituents, X means oxygen, sulfur or selenium atom, R
x
is a protecting group, preferably aroyl, acyl, alkoxycarbonyl, benzenesulfonic, alkyl, trialkylsilyl group or the next unit of elongated oligonucleotide chain.
Bacterial or viral infection, as well as uncontrolled proliferation of cancer cells in a living organism, lead to a fully developed disease predominantly by synthesis of “unwanted”, harmful proteins. Viral diseases result from incorporation of viral genetic information into a host's genome followed by synthesis of viral proteins, which are damaging to the host organism.
Caused by different factors aberrations of protooncogenes and formation of oncogenes responsible for synthesis of “unwanted” proteins are recognized as important factors in cancer cells proliferation processes.
Recent achievements in molecular biology, including explanation of molecular bases of such diseases as AIDS, different viral and cancer diseases or blood circulation disesaes, resulted in intensive search for new selective treatments aimed at inhibition of the expression of genes which code “unwanted” proteins, or at tuning of the level of known regulatory proteins.
Two newly developed therapeutic approaches are ANTISENSE mRNA (C. A. Stein,
Cancer Res.,
1988, 48, 2659) and ANTIGENE (N. T. Thuong et al.,
Angew.Chem.Int.Ed.Engl.,
1993, 32) strategies, which stem from the knowledge on interactions between oligo(deoxyribonucleotide)s and DNA or RNA molecules. These conceptions are based on the assumption that short synthetic oligo(deoxyribonucleotide)s after being delivered inside a cell, form stable duplexes with complementary DNA or RNA molecules, and on this way slow down either transcription or translation process (E. Wickstrom, ed. Wiley-Liss, New York N.Y. 1993, “
Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS
”).
Nucleolytic enzymes present in cells and body fluids are able to hydrolyze exogenous DNA molecules very rapidly, thus stability of oligo(deoxyribonucleotide)s and their analogues against nucleases is a crucial factor in respect to their in vivo activity. Majority of modifications introduced to the oligo(deoxyribonucleotide)s with the aim of their enhanced nucleolytic stability, involved changes of ligands attached to the phosphorus atom of the internucleotide phosphodiester bond. Among them phosphorothioate, methanephosphonate, phosphoramidate and triester analogues to various extent fulfill the criterion of full or, at least, significantly enhanced stability. However, such modifications usually result in reduced hybridization properties towards complementary DNA and RNA strands (J. S. Cohen, ed.
Oligonucleotides: Antisense Inhibitors of Gene Expression,
CRC Press, Inc., Boca Raton, Fla., 1989).
Applicability of antisense oligonucleotides as potential therapeutics depends upon their ability to cross the cellular membranes to reach necessary therapeutic concentration at the site of target molecules inside the cell (e.g. mRNA in cytoplasm). The cellular membranes made of protein-lipid layers are permeable only for small non-ionic molecules and are not permeable for most of natural metabolites and many drugs.
Natural and modified oligonucleotides complementary to fragments of viral DNA (RNA) are reported to show antiviral and anticancer properties in cell lines (in vivo), thus they are able to permeate through cell membranes and hybridize to the target DNA or RNA molecules. Several nucleolytically stable DNA analogues, as alkyl triesters (P. S. Miller,
Biochemistry,
1977, 16, 1988), and methanephosphonates (C. H. Marcus-Sekura et al.,
Nucleic Acids Res.,
1987, 15, 5749; P. S. Miller et al.,
Biochemistry,
1986, 25, 5092; S. K. Loke et al.,
Top. Microbiol. Immunol.,
1988, 141, 282; A. M. Tari et al.,
J.Biol.Med.,
1996, 74, 623; S. Agrawal et al.,
Clin.Pharmacokinet.,
1995, 28, 7) were used for the research in different cell lines including human HL60, Syrian hamster fibroblasts, U 937, L 929, CV-1 and ATH 8. For modified oligonucleotides the cellular uptake is usually rather low, what results in reduced in vivo activity compared to that expected from in vitro studies.
So far, DNA analogues have worse hybridization properties than natural DNA, thus the inhibition of transcription or translation, and, consequently, inhibition of protein biosynthesis are less effective than expected. There are several reasons for this phenomenon, such as complicated third-order structure of RNA, limited accessibility of its particular segments, or DNA/RNA interactions with proteins.
In order to overcome these obstacles several DNA analogues possessing internucleotide linkages without phosphorus atom, like methylene group (M. Matteuci,
Tetrahedron Lett.,
1990, 31, 2385) dialkylsilyl groups (R. Stirczak,
J.Org.Chem.,
1987, 52, 202) or sulfonyl group (S. Benner,
J.Org.Chem.,
1995, 61, 7620) have been synthesized. Research on their application as therapeutics is in an initial phase, mostly because of unfavorable physicochemical properties, as poor solubility and hybridization properties, and low chemical stability. Triester analogues are degradable by esterases, what renders them unusable in the antisense strategy (Goodrick et al.,
Bioconj.Chem.,
1990, 1, 165).
In the case of phosphorothioate and methanephosphonate analogues of DNA, which possess chiral center at the phosphorus atom, an additional problem is encountered, since the synthesis of oligomers with n internucleotide bonds results in formation of 2
n
diastereoisomers, unless the method of synthesis is stereospecific.
It was found, that for oligo(nucleoside-3′,5′-methanephosphonate)s of R
P
-, S
P
- or random configuration at each phosphorus atom, their hybridization properties towards complementary DNA or RNA depend on the configuration of the phosphorus centers (P. S. Miller et al.,
J.Biol.Chem.,
1980, 255, 9659;
Biochemistry,
1982, 21, 2507). For phosphorothioate DNA analogues the stereodifferentiation of hybridization properties is accompanied by their stereoselective susceptibility to enzymatic hydrolysis by certain nucleases (Potter et al.,
Biochemistry,
1983, 22, 1369; Bryant et al.,
Biochemistry,
1979, 18, 2825).
Leśnikowski et al.(
Nucleic Acids Res.,
1990, 18, 2109) found that stereospecifically synthesized octamer possessing six out of seven internucleotide methanephosphonate bonds of R
P
configuration has much stronger affinity towards pentadeoxyadenylic template than its counterpart possessing these bonds of S
P
configuration, or the oligomer obtained by non stereoselective method. The stereoregular oligomers were obtained by non stereoselective condensation of corresponding two stereoregular tetramers synthesized in solution starting from diastereomerically pure 5′-O-MMT-thymidine-3′-O-(O-p-nitrophenylmethanephosphonate)s and 3′-O-acetylthymidine with Grignard reagent used as an activator (Leśnikowski et al.,
Nucleic Acids Res.,
1990, 18, 2109; ibid, 1988, 16, 11675; Leśnikowski et al.,
Nucleosides
&
Nucleotides,
1991, 10, 773).
Other examples of synthesis of diastereomerically pure (or, at least, significantly enriched with an R
P
diastereoisomer) methanephosphonate analogues of DNA involve reaction

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