Modified oligonucleotides, their preparation and their use

• US 6,479,651 B1
• Filed: 08/22/2000
• Issued: 11/12/2002
• Est. Priority Date: 11/04/1994
• Status: Expired due to Term

• Current Assignee: Sanofi-Aventis Deutschland GmbH (Sanofi )
• Original Assignee: Aventis Pharma Deutschland GmbH (Sanofi )
• Inventors: Seela, Frank, Thomas, Horst
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/643,233
• Time in Patent Office: 812 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/24.3, 536/25.3, 514/44
• US Class Current: 536/22.1
• CPC Class Codes: C07H 19/044 Pyrrole radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

Modified oligonucleotides which possess at least one substituted 7-deazapurine base form more stable hybridization complexes with nucleic acids than unsubstituted analogs. They are useful as inhibitors of gene expression, as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents. Processes for preparing them are provided.

Claims

1. An oligonucleotide of the formula I

or a physiologically acceptable salt thereof, in which

B is, independently of each other, a base which is customary in nucleotide chemistry, and at least on B is a base of the formula II

 

in which

R¹⁵ and R¹⁶ are, independently of each other,

(1.) hydrogen,

(2.) halogen,

(3.) (C₁-C₁₀)-alkyl,

(4.) (C₁-C₁₀)-alkenyl,

(5.) (C₁-C₁₀)-alkynyl,

(6.) NO₂,

(7.) NH₂,

(8.) cyano,

(9.) —

S—

(C₁-C₅)-alkyl,

(10.) (C₁-C₅)-alkoxy,

(11.) (C₅-C₂₀)-aryloxy,

(12.) SiH₃,

(13.)

(14.) a radical as described under (3.), (4.), or (5.) which is substituted by one or more radicals selected from the group consisting of SH, —

S—

(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —

NR(c)R(d), —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(g), —

NR(e)R(f), and —

NR(e)R(g), or by a polyalkyleneglycol radical of the formula —

[O—

(CH₂)_r]_s,—

NR(c)R(d), where r and s are, independently of each other an integer between 1 to 18, wherein any of the foregoing OH, SH, —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(d), —

NR(e)R(f), and —

NR(e)R(g) or —

NR(c)R(g) groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target mucleic acid, attack the latter while binding, cross-linking or cleaving, or

(15.) are a radical as defined under (3.), (4.), or (5.) in which from one to all the H atoms are substituted by halogen;

R(a) is OH, (C₁-C₅)-alkoxy, (C₅-C₂₀)-aryloxy, NH₂ or NH—

T, where T is an alkylcarboxyl group or alkylamino group which is linked to one or more groups, where appropriate via a further linker, which intra-cellular uptake or serve for labeling a DNA or RNA probe or, when the oiigonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,

R(b) is hydroxyl, (C₁-C₆)-alkoxy or —

NR(c)R(d),

R(c) and R(d) are, independently of each other, H or (C₁-C₆)-alkyl which is unsubstituted or substituted by —

NR(e)R(f) or —

NR(e)R(g),

R(e) and R(f) are, independently of each other, H or (C₁-C₆)-alkyl,

R(g) is (C₁-C₆)-alkyl-COOH;

with the proviso that when R¹⁵ and R¹⁶ are identical, they cannot by hydrogen, NO₂, NH₂, cyano or SiH₃;

E and F are, independently of each other, H, OH, or NH₂,

R¹ is hydrogen, (C₁-C₁₈)alkyl, (C₂-C₁₉)-alkenyl, (C₂-C₁₈)-alkynyl, (C₂-C₁₈)-alkylcarbonyl, (C₃-C₁₉)-alkenylcarbonyl, (C₃-C₁₉)-alkynylcarbonyl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIIa

R^1a is hydrogen, C₁-C₁₈-alkyl, C₂-C₁₈-alkenyl, C₂-C₁₈-alkynyl, C₂-C₁₈-alkylcarbonyl, C₃-C₁₉alkenylcarbonyl, (C₃-C₁₉)-alkenylcarbonyl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, or a radical of the formula IIIb

is hydrogen, hydroxyl, C₁-C₁₈-alkoxy, C₁-C₆-alkenyloxy, halogen, azido or NH₂;

a is oxy, sulfanediyl or methylene;

is an integer ≧

1;

W is oxo, thioxo or selenoxo;

V is oxy, sulfanediyl or imino;

Y is oxy, sulfanediyl, imino or methylene;

Y′

is oxy, sulfanediyl, imino, (CH₂)_m or V(CH₂)_m, in which

m is an integer from 1 to 18;

X is hydroxyl or mercapto;

U is hydroxyl, mercapto, SeH, C₁-C₁₈-alkoxy, C₁-C₁₈-alkyl, C₆-C₂₀-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, NHR³, NR³R⁴ or a radical of the formula IV

2. An oligonucleotide as claimed in claim 1, wherein R¹⁵ is

(1.) NO₂,

(2.) NH₂,

(3.) —

S—

(C₁-C₆)-alkyl,

(4.) (C₁-C₆)-alkoxy,

(5.) (C₆-C₂₀)-aryloxy,

(6.) SiH₃,

(7.)

(8.) (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl which is substituted by one or more radicals from the group SH, —

S—

(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —

NR(c)R(d), —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(g), —

NR(e)R(f), or —

NR(e)R(g), or by a polyalkylene glycol radical of the formula —

[O—

(CH₂)_r]_s—

NR(c)R(d), where r and s are, independently of each other an integer between 1 to 18, with it being possible for functional groups selected from the group consisting of OH, SH, —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(d), —

NR(e)R(f), and —

NR(e)R(g) and —

NR(c)R(g) additionally to be linked to one or more groups, where appropriate via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, crosslinking or cleaving, or

(9.) (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl in which from one to all the H atoms are substituted by halogen; and

R¹⁶ is hydrogen.

3. An oligonucleotide as claimed in claim 2, wherein R¹⁵ is (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl in which from one to all the H atoms are substituted by halogen, and said halogen is fluorine.

4. An oligonucleotide as claimed in claim 1, wherein the base B is located in the β

position, the nucleotides are present in the D configuration, R² is located in the 2′

position, a is oxy and n is an integer from 2 to 99.

5. An oligonucleotide as claimed in claim 1, wherein

R¹ is hydrogen, C₁-C₆-alkyl, or a radical of the formula IIIa;

R^1a is hydrogen, C₁-C₆-alkyl, or a radical of the formula IIIb;

R² is hydrogen, C₁-C₆-alkoxy, C₁-C₆-alkenyloxy, or hydroxyl;

n is an integer from 4 to 39;

m is an integer from 1 to 6;

U is hydroxyl, mercapto, C₁-C₆-alkoxy, C₁-C₆-alkyl, NR³R⁴ or NHR³, or C₁-C₆-alkyl, in which

R³ is C₁-C₈-alkyl, or methoxyethyl,

and B, W, V, Y, Y′

, X and Z are as defined in claim 1.

6. An oligonucleotide as claimed in claim 5, wherein R¹ and R^1a are methyl;

R² is hydrogen;

n is an integer from 5 to 29;

m is 1;

U is hydroxyl; and

R³ is C₁-C₄-alkyl.

7. An oligonucleotide as claimed in claim 1, wherein V, Y and Y′

are oxy, sulfanediyl or imino, and W is oxo or thioxo.

8. An oligonucleotide as claimed in claim 1, wherein U is hydroxyl, methyl or mercapto.

9. An oligonucleotide as claimed in claim 1, wherein at least one of R¹ and R^1a is hydrogen.

10. An oligonucleotide as claimed in claim 1, wherein one or both of R¹⁵ and R¹⁶ is/are —

[O—

(CH₂)_r]_s—

NR(c)R(d); and

r and s are, independently of each other, an integer from 1 to 6.

11. An oligonucleotide as claimed in claim 1, wherein one or both of R¹⁵ and R¹⁶ is/are a radical as defined under (3.), (4.), or (5.) in which from one to all the H atoms are substituted by halogen.

12. A diagnostic reagent, comprising at least one oligonucleotide as claimed in claim 1, together with one or more of a solid support to which it is bound, a solvent or diluent, and a diagnostically relevant auxiliary reagent.

13. A pharmaceutical composition, comprising at least one oligonucleotide as claimed in claim 1, together with a physiologically acceptable excipient.

14. An oligonucleotide as claimed in claim 1, wherein R² is allyloxy.