Oligonucleotide analogues

• US 6,562,960 B1
• Filed: 10/09/1998
• Issued: 05/13/2003
• Est. Priority Date: 03/05/1996
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Taylor, Roger John, Douglas, Mark Edward, Collingwood, Stephen Paul, Baxter, Anthony David
• Primary Examiner(s): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/142,212
• Time in Patent Office: 1,677 Days
• Field of Search: 536/24.5, 536/25.1, 536/25.34, 536/23.1, 536/25.3, 536/25.33, 525/54.11, 435/6, 435/91.1
• US Class Current: 536/25.3
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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

An oligonucleotide analogue having 10 to 200 natural and/or synthetic nucleoside units, linked by internucleoside linkages, at least one of the internucleoside linkages being of formula

where the indicated methylene group is attached to a 3′ carbon atom of a nucleoside, the indicated oxygen atom is attached to a 5′-carbon atom of an adjacent nucleside, R¹ is hydrogen, hydroxy, O—, thiol, S—, —NH₂ or a group of formula R_1a, —OR_1a, —SR_1a, —NHR_1b, or —NR_1bR_1c wherein R_1a is an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group; and R_1b and R_1c are each independently an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group or R_1b and R_1c together with the nitrogen atom to which they are attached denote a five- or six-membered heterocyclic ring, and X is oxygen or sulfur.

Claims

1. An oligonucleotide analogue having 10 to 200 natural and/or synthetic nucleoside units linked by internucleoside linkages, and a 3′

-terminal hydroxyl group, wherein at least one of the internucleoside linkages is of formula

where

the indicated methylene group is attached to a 3′

carbon atom of a nucleoside,

the indicated oxygen atom is attached to a 5′

-carbon atom of an adjacent nucleoside,

R¹ is hydrogen, a group of formula R¹_a, or —

NR¹_bR¹_c wherein

R¹_a is a C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group or a C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group bearing one or more terminal substituents; and

R¹_b and R¹_c are each independently an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group; and

x is oxygen or sulfur.

2. An oligonucleotide analogue according to claim 1, which has 15 to 40 nucleoside units.

3. An oligonucleotide analogue according to claim 1, which has 15 to 25 nucleoside units.

4. An oligonucleotide analogue according to claim 1 in which all the internucleoside linkages are of formula I.

5. An oligonucleotide analogue according to claim 1 in which up to 75% of the internucleoside linkages are of formula I.

6. An oligonucleotide analogue according to claim 5, in which up to 50% of the internucleoside linkages are of formula I.

7. An oligonucleotide analogue according to claim 6, in which up to 25% of the internucleoside linkages are of formula I.

8. An oligonucleotide analogue of claim 1, wherein at least two consecutive internucleoside linkages are of formula I.

9. An oligonucleotide analogue of claim 1, in which internucleoside linkages of formula I alternate with other internucleoside linkages.

10. An oligonucleotide analogue of claim 1, wherein the remaining internucleoside linkages are phosphodiester, phosphorothioate or phosphorodithioate linkages or a mixture of two or more thereof.

11. An oligonucleotide analogue according to claim 10, in which the remaining internucleoside linkages are phosphorothioate linkages.

12. An oligonucleotide analogue according to claim 10, further comprising a region having phosphodiester and/or phosphorothioate and/or phosphorodithioate internucleoside linkages between two regions having internucleoside linkages of formula I or a mixture thereof with phosphorothioate or phosphodiester linkages.

13. An oligonucleotide analogue according to claim 1, in which at least one nucleoside is modified at the 2′

position thereof.

14. An oligonucleotide analogue according to claim 13, in which at least one nucleoside has a halogen atom or a group of formula —

OR² at the 2′

position, where R² is a C₁ to C₁₀ aliphatic group.

15. An oligonucleotide analogue according to claim 14, in which at least one nucleoside has a group —

OR² at the 2′

position, where R² is an unsubstituted or substituted C₁ to C₁₀ alkyl group.

16. An oligonucleotide analogue according to claim 15, in which R² is C₁ to C₄ alkyl, C₁ to C₄ alkoxy-substituted C₁ to C₄ alkyl or a group of formula —

(CH₂CH₂O)—

_nR³ where R³ is methyl or ethyl and n is 2 to 4.

17. An oligonucleotide analogue according to claim 16, in which R² is methyl, ethyl, methoxyethyl, ethoxyethyl or a group of formula —

(CH₂CH₂O)—

₃CH₃.

18. An oligonucleotide analogue according to claim 13, in which at least two consecutive nucleosides are modified at the 2′

position and are linked by phosphodiester internucleoside linkages and/or there is a linkage of formula I between a nucleoside unmodified at the 2′

position and a 5′

carbon atom of a nucleoside modified at the 2′

position.

19. An oligonucleotide according to claim 1, in which R¹_a, R¹_b or R¹_c as alkyl, alkenyl, cycloalkyl, aryl, or aralkyl are unsubstituted or substituted by hydroxy, C₁ to C₄ alkoxy, halogen, cyano, tri(C₁-C₁₅ hydrocarbyl)silyl or primary, secondary or tertiary amino.

20. An oligonucleotide analogue according to claim 1 in which R¹ is hydrogen, hydroxy, O⁻

, SH, S⁻

, an unsubstituted or substituted C₁ to C₄ alkyl or phenyl group, a group of formula —

OR¹_a, where R¹_a is an unsubstituted or substituted C₁ to C₄ alkyl, C₃ to C₅ alkenyl, C₅ to C₈ cycloalkyl or C₇ to C₉ aralkyl group, or a group of formula —

SR¹_a where R¹_a is an unsubstituted or substituted C₁ to C₄ alkyl or phenyl group.

21. An oligonucleotide analogue according to claim 20, in which R¹ is hydrogen, hydroxy, O⁻

, SH, S⁻

, methoxy, ethoxy or 2-cyanoethoxy.

22. An oligonucleotide analogue according to claim 1 which is complementary to a region of mRNA for human c-raf kinase.

23. An oligonucleotide analogue according to claim 22 in which the nucleoside sequence is

5′ -TCC CGC CTG TGA CAT GCA TT-3′

(SEQ ID NO;

1).

24. An oligonucleotide analogue according to claim 1, which is complementary to a region of mRNA for human PKC-α

.

25. An oligonucleotide analogue according to claim 24, in which the nucleoside sequence is

5′

-GTT CTC GCT GGT GAG TTT CA-3′

(SEQ ID NO;

2).

26. A method of preparing an oligonucleotide analogue having at least one internucleoside linkage of formula I

where R¹ and X are as defined in claim 1, which comprises (i) carrying out a coupling reaction or successive coupling reactions between (A) a natural or synthetic nucleoside or oligonucleotide having a 5′

-hydroxyl group and (B) a natural or synthetic nucleoside or dinucleotide having at the 3′

-position thereof a group reactive with said 5′

-hydroxyl group until an oligonucleotide having the desired number of nucleosides is obtained, in at least one of said coupling reactions (B) being a nucleoside of formula

where B¹ is a nucleoside base radical, R⁴ is a hydroxy-protecting group, R⁵ is hydrogen, hydroxy or a 2′

modifying atom or group, M⁺ is a metal or unsubstituted or substituted ammonium ion or a cation of a heterocyclic base, and X is oxygen or sulphur, and being reacted with (A) in the presence of a sterically hindered organic acid halide or anhydride to form an oligonucleotide analogue having a phosphinate internucleoside linkage of formula

where X is oxygen or sulphur, and (ii)(a) oxidising the phosphinate linkage or (b) sulphurising the phosphinate linkage, or (c) reacting the phosphinate linkage with a compound of formula R¹_aY where R¹_a is as defined in claim 1 and Y is a leaving atom or group or (d) oxidising and reacting the phosphinate linkage with an alcohol of formula R¹_aOH or an amine of formula R¹_bNH₂ or R¹_bR¹_c NH where R¹_a, R¹_b and R¹_c are as defined in claim 1, or (e) silylating the phosphinate linkage and reacting the silylated linkage with a thioalkylating or thioarylating agent to give a phosphinate linkage of formula I where R¹ is —

SR¹_a where R¹_a is as defined in claim 1.

27. A method according to claim 26, in which the oligonucleotide analogue is further reacted to replace the protecting group R⁴ by hydrogen or, where R⁴ is on a terminal nucleoside in the oligonucleotide analogue, by a 5′

modifying group.

28. A method according to claim 26 which comprises the step of:

carrying out the successive coupling reactions (i) and step (ii) of claim 26 with the nucleoside or oligonucleotide (A) attached to the solid support;

(iii) detaching the oligonucleotide from the solid support and removing protecting groups to give an oligonucleotide having a terminal 5′

free hydroxyl group; and

(iv) optionally reacting the 5′

free hydroxyl group to introduce a modifying group at the terminal 5′

position.

29. A method according to claim 26 wherein, in formula II, B¹ is a pyrimidine base, R⁴ is a methoxytrityl, dimethoxytrityl or tris tert-butyltrityl group, R⁵ is hydrogen and M⁺ is an unsubstituted ammonium, mono-, di- or tri-C₁-C₁₀ alkyl- or hydroxyalkyl-ammonium ion or a cation of a heterocyclic base.

30. A method according to claim 26 wherein the nucleoside of formula II is a stereoisomer having the formula:

31. A method according to claim 26 wherein the coupling reaction of the nucleoside of formula II with (A) in the presence of a sterically hindered organic acid halide is carried out in the presence of a heterocyclic base having a tertiary nitrogen atom in the ring or an oxide thereof.

32. A method according to claim 26 wherein oxidation (ii)(a) is effected by treatment with iodine and water, or with tert-butyl hydroperoxide.

33. A method according to claim 26 wherein sulphurisation (ii)(b) is effected by treatment with sulphur in the presence of a tertiary amine in an organic solvent.

34. A method according to claim 26 wherein the reaction (ii)(c) of the phosphinate internucleoside linkage of formula 1A with a compound of formula R¹_aY where R¹_a is alkyl, cycloalkyl or aralkyl as defined in claim 1 and Y is halogen is carried out in the presence of a strong base.

35. A method according to claim 26 wherein the reaction (ii)(c) of the linkage of formula IA with a compound of formula R¹_aY, where R¹_aY an alkenyl or aryl halide or triflate, is carried out in the presence of a palladium catalyst.

36. A method according to claim 26 wherein the oxidative reaction (ii)(d) of the phosphinate internucleoside linkage of formula IA with an alcohol of formula R¹_aOH is carried out by reaction with an oxidant in the presence of the alcohol R¹_aOH and a base.

37. A method according to claim 36, in which the oxidant is iodine, carbon tetrachloride or bromotrichloromethane, and the base is pyridine.

38. A method according to claim 26 wherein the oxidative reaction (ii)(d) of the phosphinate internucleoside linkage of formula IA is effected with an amine of formula R¹_bNH₂ or R¹_bR¹_cNH where R¹_b and R¹_c are as defined in claim 1 and carbon tetrachloride or bromotrichloromethane or iodine to give an oligonucleotide analogue of the invention in which R¹ is —

NHR¹_b or —

NR¹_bR¹_c respectively.

39. A method according to claim 26 wherein the reaction (ii)(e) of the phosphinate linkage of formula IA is carried out by silylating the linkage using a trialkylsilyl halide and a base, and reacting the silylated linkage with a thioalkylating or thioarylating agent.

40. A method according to claim 39, in which the thioalkylating or thioarylating agent is a thiosulphonate of formula ArSO₂SR¹_a where R¹_a is as defined in claim 1 and Ar is an aromatic group.

41. A method according to claim 26 wherein when an oligonucleotide analogue having the desired number of nucleosides has been synthesised on a solid support, it is detached from the solid support, by treatment with concentrated aqueous ammonia, before or after treatment to remove hydroxy-protecting groups.

42. A method according to claim 26 wherein hydroxy-protecting groups are removed by treatment with an aqueous organic acid.

43. A method according to claim 26 wherein the nucleoside of formula II is replaced by a dinucleotide having the formula:

where B¹ is a nucleoside base radical, R¹ is hydrogen, hydroxy, O⁻

, thiol, S⁻

, —

NH₂ or a group of formula R¹_a, —

OR¹_a, —

SR¹_a, —

NHR¹_b or —

NR¹_bR¹_c where R¹_a is an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group, R⁴ is a hydroxy-protecting group and R⁵ is hydrogen, hydroxy or a 2′

modifying atom or group, B² is a nucleoside base radical, R⁶ is hydrogen, hydroxy or a 2′

modifying atom or group and R⁷ is a group reactive with, or activatable to be reactive with, a 5′

hydroxyl group in a nucleoside.

44. A method according to claim 43, in which R⁷O in formula VIII is a H-phosphonate group, a phosphoramidite group or a phosphodiester group.

45. A dinucleotide of formula

where B¹ is a nucleoside base radical, B² is a nucleoside base radical, R¹ is hydrogen, hydroxy, O⁻

, thiol, S⁻

, —

NH₂ or a group of formula R¹_a, —

OR¹_a, —

SR¹_a, —

NHR¹_b or —

NR¹_bR¹_c where R¹_a is an unsubstituted or substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₃ to C₈ cycloalkyl, C₆ to C₁₀ aryl or C₇ to C₁₃ aralkyl group, R⁴ is a hydroxy-protecting group, R⁵ is hydrogen, hydroxy or a 2′

modifying atom or group, and R⁶ is hydrogen, hydroxy or a 2′

modifying atom or group except that where R¹ is other than hydrogen at least one of R⁵ and R⁶ is a 2′

modifying atom or group and R⁹ is a group reactive with, or activatable to be reactive with, a 5′

hydroxyl group in a nucleoside or a hydroxy-protecting group.