3 modified oligonucleotide derivatives

Details 3 modified oligonucleotide derivatives 3 modified oligonucleotide derivatives

1997-07-17

2001-12-04

Houtteman, Scott W. (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C536S026600

Reexamination Certificate

active

06326487

ABSTRACT:

The present invention relates to novel oligonucleotide analogs with valuable physical, biological and pharmacological properties and to a process for the preparation thereof. Application thereof relates to the use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), as probes for detecting nucleic acids and as aids in molecular biology.
Oligonucleotides are increasingly being used as inhibitors of gene expression (J. F. Milligan, M. D. Matteucci and J. C. Martin, J. Med. Chem. 36 (1993) 1923; E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543).
Antisense oligonucleotides are nucleic acid fragments whose base sequence is complementary to a mRNA to be inhibited. This target mRNA may be of cellular, viral or other pathogenic origin. Examples of appropriate cellular target sequences are those of receptors, enzymes, growth factors, immunomodulators, ion channels or oncogenes. Inhibition of virus replication using antisense oligonucleotides has been described, for example, for RSV (Rous sarcoma virus), HSV-1 and -2 (herpes simplex virus type I and II), HIV (human immunodeficiency virus) and influenza viruses. This entails use of oligonucleotides which are complementary to the viral nucleic acid.
Sense oligonucleotides are, by contrast, designed in their sequence so that they bind (“trap”), for example, nucleic acid-binding proteins or nucleic acid-processing enzymes and thus inhibit the biological activity thereof (C. Hélène and J. J. Toulmé, Biochim. Biophys. Acta 1049 (1990) 99). Examples of viral targets which may be mentioned in this connection are reverse transcriptase, DNA polymerase and transactivator proteins. Triplex forming oligonucleotides generally have DNA as target and, after binding thereto, form a triple helix structure.
Whereas antisense oligonucleotides are used in general to inhibit the processing (splicing etc.) of the mRNA or the translation thereof into protein, triplex forming oligonucleotides inhibit the transcription or replication of DNA (N. T. Thuong and C. Hélène, Angew. Chem. 105 (1993) 697; Uhlmann and Peyman, Chemical Reviews 90 (1990) 543). However, it is also possible to bind single-stranded nucleic acids in a first hybridization with an antisense oligonucleotide to form a double strand which then, in a second hybridization with a triplex-forming oligonucleotide, forms a triplex structure. The antisense and triplex binding regions can moreover be located either in two separate oligonucleotides or else in one oligonucleotide.
A further application of synthetic oligonucleotides is in so-called ribozymes which destroy the target RNA as a consequence of their ribonuclease activity (D. Castanotto, J. J. Rossi, J. O. Deshler, Critical Rev. Eukar. Gene Expr. 2 (1992) 331).
Nucleic acid fragments with suitable labeling are used in DNA diagnosis as so-called DNA probes for specific hybridization onto a nucleic acid which is to be detected. The specific formation of the new double strand is in this case followed by means of the labeling, which is preferably not radioactive. It is possible in this way to detect genetic, malignant or viral diseases or diseases caused by other pathogens.
For most of the said applications, oligonucleotides in their naturally occurring form are of little suitability or completely unsuitable. They must be chemically modified so that they meet specific requirements. For oligonucleotides to be usable in biological systems, for example inhibiting virus replication, they must comply with the following conditions:
1. They must have a sufficiently high stability under in vivo conditions, that is to say both in serum and inside cells.
2. Their properties must be such that they can pass through the plasma membrane and nuclear membrane.
3. They must under physiological conditions bind in a base-specific manner to their target nucleic acid in order to display the inhibitory effect.
These conditions are not indispensable for DNA probes; however, these oligonucleotides must be derivatized in such a way that detection, for example, by fluorescence, chemiluminescence, colorimetry or specific staining, is possible (Beck and Köster, Anal. Chem. 62 (1990) 2258).
Chemical modification of oligonucleotides usually takes place by appropriate modification of the phosphate backbone, ribose unit or the nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543). Another frequently used method is to prepare oligonucleotide 5′-conjugates by reacting the 5′-hydroxyl group with appropriate phosphorylation reagents. Oligonucleotides modified only at the 5′ end have the disadvantage that they are broken down in serum. If, on the other hand, all the internucleotide phosphate residues are modified there are often drastic alterations in the properties of the oligonucleotides. For example, the solubility of methylphosphonate oligonucleotides in aqueous medium is diminished and the hybridization capacity is reduced. Phosphorothioate oligonucleotides have non-specific effects so that, for example, even homooligomers (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543) are active against viruses.
The breakdown of oligonucleotides by 3′-nucleolytic activity is generally regarded as the predominant breakdown by nucleases in serum. The object therefore is to provide 3′-derivatized oligonucleotide analogs with specific activity, increased serum stability and good solubility.
This invention therefore relates to compounds of the formula I and formula II
and the physiologically tolerated salts thereof, in which
a is a number from zero to 20, preferably from zero to 10, particularly preferably from zero to 6, very particularly preferably from zero to 4;
b is a number from zero to 20, preferably from zero to 10, particularly preferably from zero to 4, very particularly preferably of zero;
R
1
is hydrogen, C
1
-C
18
-alkyl, preferably C
1
-C
6
-alkyl, in particular methyl, C
2
-C
18
-alkenyl, C
3
-C
18
-alkynyl, C
1
-C
18
-alkylcarbonyl, C
2
-C
19
-alkenylcarbonyl, C
3
-C
19
-alkynyl-carbonyl, C
6
-C
20
-aryl, C
6
-C
14
-aryl-C
1
-C
8
-alkyl, or a radical of the formula III
 preferably hydrogen or a radical of the formula III, very particularly preferably hydrogen;
R
2
is hydrogen, hydroxyl, C
1
-C
18
-alkoxy, halogen, azido or NH
2
, preferably hydrogen, hydroxyl, C
1
-C
4
-alkoxy, fluorine or NH
2
, particularly preferably hydrogen or hydroxyl, very particularly preferably hydrogen;
D is hydroxyl, O—PO
3
2−
, very particularly preferably hydroxyl;
B is a base customary in nucleotide chemistry, for example natural bases such as adenine, cytosine, guanine, uracil and thymine or unnatural bases such as, for example, purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N
4
,N
4
-ethanocytosine, N
6
,N
6
-ethano-2,6-diaminopurine, pseudoisocytosine, 5-propinuracil, 5-propincytosine, 5-fluorocytosine, 5-fluorouracil, 5-hydroxymethyluracil and 5-bromocytosine and very particularly preferably adenine, cytosine, guanine, uracil, thymine, 5-propinuracil and 5-propincytosine;
n is an integer from 1 to 100, preferably 5 to 40, particularly preferably 6 to 30, very particularly preferably 7 to 25;
n′ is an integer from zero to 50, preferably zero to 40, particularly preferably zero to 30, very particularly preferably zero to 25;
m is an integer from zero to 5, very particularly preferably zero;
m′ in formula I is an integer from zero to 5, very particularly preferably zero or 1;
m′ in formula II is an integer from 1 to 5, very particularly preferably 1;
A is oxy, thioxy or methylene, preferably oxy;
W is oxo, thioxo or selenoxo, preferably oxo or thioxo, particularly preferably oxo;
V is oxy or thio, very particularly preferably oxy;
T is oxy, thio or imino, very particularly preferably oxy;
Y is oxy, thio, imino or methylene, very particularly preferably oxy;
X is hydroxyl or mercapto;
U is hydroxyl, mercapto, BH
3
, SeH, C
1
-C
18
-alkoxy, preferably C
1
-C
6
-alkoxy, C
1
-C
18
-alkyl, preferably C
1
-C
6
-alkyl, C
6
-C
20

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