Deoxynucleic alkyl thiourea compounds and uses thereof

Details Deoxynucleic alkyl thiourea compounds and uses thereof Deoxynucleic alkyl thiourea compounds and uses thereof

1999-09-28

2001-01-02

Schwartzman, Robert A. (Department: 1635)

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

Organic compounds

Carbohydrates or derivatives

C536S024330, C536S024310, C536S024300, C536S023100, C536S024500, C435S006120

Utility Patent

active

06169176

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to oligonucleotide compounds formed by replacing phosphodiester linkages in nucleic acids and more particularly to the synthesis and use of novel deoxynucleic alkyl thiourea (dNXt) compounds.
BACKGROUND OF THE INVENTION
The use of nucleic acid (DNA and RNA) as antisense or antigene drugs, to bind to DNA or RNA of disease-causing proteins to prevent their production, has been hampered by features of the DNA and RNA. For example, the negatively charged phosphodiester linkages of double- and triple-stranded DNA and RNA reside side by side, causing considerable charge—charge electrostatic repulsion, particularly at low (physiological) ionic strength. This feature, as well as the susceptibility of DNA and RNA to damage from nuclease activity, limits the usefulness of RNA and DNA as therapeutic agents. (See, Temsamami and Guinot,
Biotechnol. Appl. Biochem.
26:65 (1997); Uhlmann and Peyman,
Chem. Rev.
90:543 (1990); Crooke,
Anticancer Drug Des.
6:609 (1991); and Crooke,
Ann. Rev. Pharmacol. Toxicol.
32:329 (1992)).
Among other requirements, successful development of antisense therapeutics presupposes the oligonucleotides a) be stable in vivo, b) have improved permeability and cellular uptake and c) have greater binding affinity with high specificity. (Miller and Ts'o,
Anticancer Drug Des.
2:117 (1987); Milligan et al.,
J Med. Chem.
36:1923 (1993); and Mesmaeker et al.,
Curr. Opin. Struct. Biol.
343 (1995)).
Suggestions have been made to replace the phosphodiester linkages by other linkages that are either neutral or positively charged and resistant toward nuclease degradation in order to provide more effective antigene/antisense agents. (See, Crooke, (1992), supra, Cook,
In Antisense Research and Applications,
Lebleu (Ed.), CRC Press, Boca Raton, Fla., (1993), p. 149; and Morvan et al.,
J. Med. Chem.
36:280 (1993)). In addition, modification of oligonucleotides so as to enhance cellular uptake has been considered. (Cook et al., supra).
Antisense oligonucleotides having various backbone modifications have been prepared (see Bennett,
Biochemical Pharmacology
55:9 (1998); Alama et al.,
Pharmacological Research
36:171 (1997); Manoharan,
Designer Antisense oligonucleotides: conjugation chemistry and functionality placement,
CRC Press, Boca Raton, Fla. (1993); and Mesmaeker et al.,
Pure Appl. Chem.
69:43 7 (1997)).
The replacement of the phosphate linkages in DNA and RNA by achiral guanido groups providing a new class of guanidinium (g) linked nucleosides which are designated as DNG has been reported. (Dempcy et al.,
Proc. Nati. Acad. Sci. USA
91:7864 (1994); Dempcy et al.,
Proc. Nati. Acad. Sci. USA
92 (1995); Dempcy et al.,
J. Am. Chem. Soc.
11 7:6140 (1995); and Dempcy et al.,
Proc. Natl. A cad. Sci. USA
93:4326 (1996)).
Some examples of backbone-modified oligonucleotides having different electrostatic atttractions include: peptide (PNA-neutral) (Egholm et al.,
J. Am. Chem. Soc.
114:1895 (1992); and Nielsen and Haaima,
Chem. Soc. Rev.
73 (1997)); PHONA (Peyman et al.,
Angew. Chem., Int. Ed. Engl.
35:2636 (1996)); methyl phosphonate (DNAmp-neutral) (Stein and Cheng,
Science
261:1004 (1993); and Tseng and Ts'o,
Antisense Res. Dev.
5:251 (1995)); phosphorothioate (DNAs-anionic) (Cook, supra; Morvan et al., supra and Marshall and Caruthers,
Science
259:1564 (1993)); phosphoramidate (Gryaznov and Chen,
J. Am. Chem. Soc.
116:3143 (1994); amido (Mesmaeker et al.,
Angew, Chem. Int. Ed. Engl.
35:2790 (1996); MMI (Sanghvi et al.,
Nucleosides Nucleotides
16:907 (1997); boronated oligonucleotides (Sood et al.,
J. Am. Chem. Soc.
111:9234 (1989); Sood et al.,
J. Am. Chem. Soc.
223:9000 (1990) and Spielvogel et al.,
Pure Appl. Chem.
63:415 (1991)); ethylmorpholino and diniethylamino phosphoramidates (Jung et al.,
Nucleosides and Nucleotides
13:1597 (1994) and Letsinger et al.,
J. Am. Chem. Soc.
110:4470 (1988)); aminomethyl phosphonates (Huang et al.,
Bioconjugate Chem.
5:47 (1994); and guanido (Vasseur et al.,
J. Am. Chem. Soc.
114:4006 (1992); Blattler et al.,
J. Am. Chem. Soc.
120:2674 (1991); James et al.,
Nucleosides Nucleotides
16:1821 (1997); Jones et al.,
J. Org. Chem.
58:2983 (1993); Mesmaeker et al.,
Acc. Chem. Res.
28:366 (1995); Rao et al.,
Nucleosides Nucleotides
13:255 (1997); Stirchak and Summerton,
J. Org. Chem.
52:4202 (1987); and Thibon et al.,
J. Org. Chem.
62:4635 (1997)) (See FIG.
1
).
Small positively charged oligonucleotides (DNG) show unprecedented binding to nucleic acids with retention of specificity. (Dempcy et al.,
Proc. Natl. Acad. Sci. USA
92 (1995), supra and Dempcy et al.,
J. Am. Chem. Soc.
117:6140 (1995), supra; and Browne et al.,
Proc. Natl. Acad. Sci. USA
92:7051-7055 (1995)). The nonionic oligonucleotide DNAmp exhibits the ability to be transported into cells by passive diffusion/fluid phase endocytosis and is more resistant to degradation than DNA. (Cook et al., supra). Both DNAmp and DNAs, however have individual limited drawbacks of stereoisomeric complexity (Huang et al., supra), solubility (DNAmp) and toxicity (DNAs). (Morvan et al., supra, and Agrawal et al.,
Nucleosides Nucleotides
16:927-936 (1997)). These findings have led recently to the development of mixed backbone oligonucleotides (MBOs) where the phosphorothioates and methyl phosphonates have been alternated in an oligonucleotide backbone to produce improved antisense properties. (Morvan et al., supra, Agrawal et al., supra and Iyer et al.,
Tetrahedron
52:14419-14436 (1996)).
There remains a need for oligonucleotides that may perform better as antisense or antigene drugs, for example by having stronger affinity for DNA and RNA, as a result of changes in charge characteristics and resistance to nuclease degradation which can form stable constructs.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides novel oligonucleotide compounds having thiourea backbones for use as therapeutic antisense and/or antigene agents. Specifically in the compounds of the invention, negatively charged phosphodiester linkages {—O(PO
2
—
)O—} in nucleic acid (DNA or RNA) are replaced by a novel alkyl thiourea backbone having alkyl or alkoxy linkages to form novel compounds designated as dNXtsIn one embodiment, the phosphodiester linkages are replaced by a methylthiourea {—NHC(═SMe
+
)NH—} backbone to form dNmt. The backbone is positively charged, achiral, stable and can be readily synthesized.
The Thymidyl dNmt compounds of the invention are the first example of a positively charged polynucleotide backbone incorporating a methyl isothiouronium salt.
The dNXt compounds have higher affinity for DNA and RNA then, DNA has for RNA, or vice versa. Because of the alkyl group present in these compounds, properties such as hydrophobicity and cellular uptake of these compounds, may be readily modified to form improved compounds for use as therapeutic agents.


REFERENCES:
Branch, “A good antisenese is hard to find”TIBS23, pp.45-50 2/1998.
Vandendriessche et al. “Synthesis, Enzymatic Stability and Base-pairing Properties of Oligothymidylates Containing Thymidine Dimers with Different N-Substituted Guanidine Linkages”, J. Chem. Soc. Perkin Trans, pp.1567-1575 1993.
Browne et al. “Binding Studies of Cationic Thymidyl Deoxyribonucleic guanidine to RNA Homopolynucleotides”, Proc. Natl. Acad. Sci. vol 92, pp. 7051-7055 7/1995.
Crooke et al. Antisense Research and Application, pp.1-50 7/1998.
Agrawal, Sudhir et al., “Mixed-Backbone Oligonucleotides Containing Phophorothioate and Methylphosphonate Linkages as Second Generation Antisense Oligonucleotide,”Nucleosides and Nuclesides and Nucleotides,1997, 16(7-9):927-36. (Exhibit 1).
Alama, Angela et al., “Antisense Oligonucleotides as Therapeutic Agents,”Pharmacological Research,1997, 36(3):171-8. (Exhibit 2).
Blasko, Andrei et al., “Association of Short Strand DNA Oligomers with Guanidinium-Linked Nucleosides. A Kinetic and Thermodynamic Study,”Journal of the American Chemical Society,Aug. 28, 1996,

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