Oligonucleolotides having site specific chiral phosphorothioate internucleoside linkages

• US RE39,464 E1
• Filed: 08/24/2004
• Issued: 01/09/2007
• Est. Priority Date: 07/14/1998
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Cook, Phillip Dan
• Primary Examiner(s): Wilson; James O.
• Assistant Examiner(s): Owens, Jr.; Howard V.
• Application Number:
  US10/925,348
• Time in Patent Office: 868 Days
• Field of Search: 514/43, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 435/7.2
• US Class Current: 514/43
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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

Novel chiral compounds that mimic and/or modulate the activity of wild-type nucleic acids are disclosed. In general, the compounds are phosphorothioate oligonucleotides wherein the 5′, and the 3′-terminal internucleoside linkages are chirally Sp and internal internucleoside linkages are chirally Rp.

Claims

1. An oligomeric compound comprising a plurality of covalently-bound nucleosides;

said compound having the formula;

5′

-T₁—

(Nu—

Sp)_n—

(Nu—

Lp)_m—

(NU—

Sp)_p—

Nu—

T₂-3′

wherein;

T₁ and T₂ are each, independently, hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleoside, a nucleotide, an oligonucleotide, a conjugate group or a 5′

or 3′

substituent group;

each Sp is a chiral Sp phosphorothioate internucleoside linkage;

each Lp is, independently, a chiral Rp phosphorothioate internucleoside linkage, a racemic phosphorothioate internucleoside linkage or an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage;

each n and m is, independently, from 1 to 100;

each p is from 0 to 100;

where the sum of n, m and p is from 3 to about 200;

each Nu independently, has the formula;

 

wherein;

Bx is a heterocyclic base moiety; and

R₁ is H, hydroxyl, a protected hydroxyl, a 2′

-substituent group or a protected 2′

-substituent group.

2. The oligomeric compound of claim 1 wherein each R, is H or hydroxyl.

3. The oligomeric compound of claim 1 wherein R₁ is C₁-C₁₀ O-alkyl or C₁-C₁₀ substituted O-alkyl.

4. The oligomeric compound of claim 3 wherein R₁ is 2′

-O-methoxyethyl or 2′

-O-methyl.

5. The oligomeric compound of claim 1 wherein each Nu is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.

6. The oligomeric compound of claim 1 wherein the sum of n, m, and p is from 5 to about 50.

7. The oligomeric compound of claim 1 wherein the sum of n, m, and p is from 8 to about 30.

8. The oligomeric compound of claim 1 wherein the sum of n, m, and p is from 10 to about 25.

9. The oligomeric compound of claim 1 wherein p is 1 or 2.

10. The oligomeric compound of claim 1 wherein n and p are each 1 and m is from 3 to about 20.

11. The oligomeric compound of claim 1 wherein T₁ and T₂ are, independently, hydroxyl or a protected hydroxyl.

12. The oligomeric compound of claim 1 wherein each Lp is an Rp phosphorothioate internucleoside linkage.

13. The oligomeric Compound of claim 1 wherein at least one Lp is a racemic phosphorothioate internucleoside linkage.

14. The oligomeric Compound of claim 1 wherein at least one Lp is an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage.

15. The oligomeric Compound of claim 1 wherein R₁ is a 2′

-substituent group or a protected 2′

-substituent group.

16. A pharmaceutical composition comprising a compound of claim 1 and an acceptable pharmaceutical carrier.

46. A method of modulating the production or activity of a protein in an organism, comprising contacting said organism with a compound of claim 1, wherein said protein is protein kinase C, ICAM-1, VCAM-1, PECAM-1, ELAM-1, H-ras, K-ras, AP-1, a Jun N-terminal kinase, or a matrix metalloproteinase.

47. A method of treating an organism having a disease characterized by the undesired production of a protein, comprising contacting said organism with a compound of claim 1, wherein said disease is psoriasis, an inflammatory disorder of the skin, an infectious disease of the skin, or skin cancer.

48. The oligomeric compound of claim 47 wherein R₁ is 2′

-O-methoxyethyl or 2′

-O-methyl.

17. A nucleoside having the formula:

wherein;

Bx is a heterocyclic base moiety;

R₄ is a hydroxyl protecting group;

R₁ is H, hydroxyl, a protected hydroxyl, a 2′

-substituent group or a protected 2′

-substituent group; and

R₂ is an Sp chiral auxiliary group.

18. The nucleoside of claim 17 wherein said chiral auxiliary group has one of formulas I, II, III, IV, V or VI:

19. The nucleoside of claim 17 wherein Bx is adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.

20. The nucleoside of claim 17 wherein each R₁ is H or hydroxyl.

21. The nucleoside of claim 17 wherein R₁ is C₁-C₁₀ alkyl or C₁-C₁₀ substituted alkyl.

22. The nucleoside of claim 21 wherein R₁ is 2′

-O-methoxyethyl or 2′

-O-methyl.

23. The nucleoside of claim 17 wherein at least one R₁ is 2′

-O-methoxyethyl or 2′

-O-methyl.

24. The nucleoside of claim 17 wherein R₁ is a 2′

-substituent group or a protected 2′

-substituent group.

25. A method of preparing an oligomeric compound of formula:

5′

-T₁—

(Nu—

Sp)_n—

(NU—

Lp)_m—

(NU—

Sp)_p—

Nu—

T₂-3′

wherein;

each T₁ and T₂ is, independently, hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleotide, a nucleotide or an oligonucleotide, a conjugate group or a 5′

or 3′

substituent group;

each Sp is an Sp phosphorothioate internucleoside linkage;

each Lp is, independently, an Rp phosphorothioate internucleoside linkage, a racemic phosphorothioate internucleoside linkage or an internucleoside linkage other than a chiral Rp phosphorothioate internucleoside linkage;

each n and m is, independently, from 1 to 100;

each p is from 0 to 100 where the sum of n, m and p is from 3 to about 200;

each Nu, independently, has the formula;

 

wherein

Bx is a heterocyclic base moiety; and

R₁ is H, hydroxy, a protected hydroxyl, a 2′

-substituent group or a protected 2′

-substituent group;

comprising the steps of;

(a) providing a compound of formula;

wherein;

R₄ is a labile hydroxyl protecting group;

R₃ is a covalent attachment to a solid support;

(b) deblocking said labile hydroxyl protecting group to form a deblocked hydroxyl group;

(c) optionally treating said deblocked hydroxyl group with a further compound having the formula;

wherein;

R₂ is an Sp chiral auxiliary group;

and a condensing reagent to form an extended compound;

(d) optionally repeating steps (b) and (c);

(e) treating said deblocked hydroxyl group with a compound having the formula;

wherein;

R₅ is an Rp chiral auxiliary group or an activated phosphorus group;

and a condensing reagent to form a further extended compound;

(f) optionally repeating steps (e) and (f) to add further nucleosides;

(g) deblocking said labile hydroxyl protecting group to form a deblocked hydroxyl group;

(h) treating said deblocked hydroxyl group with a further compound having the formula;

wherein;

R₁ is an Sp chiral auxiliary group;

and a condensing reagent to form a protected oligomeric compound; and

(i) optionally repeating steps (h) and (i) to add at additional nucleosides thereby forming a further protected oligomeric compound.

26. The method of claim 25 further comprising the step of deblocking the product of step (i).

27. The method of claim 25 wherein said Sp chiral auxiliary group has one of formulas I, II or III:

28. The method of claim 25 wherein said Rp chiral auxiliary group has one of formulas IV, V or VI:

29. The method of claim 25 further comprising one or more capping steps after steps c, d, e, f, h, or i;

said capping steps comprising treatment with a capping agent.

30. The method of claim 25 further comprising one or more oxidation steps;

said oxidation steps comprising treatment with an oxidizing agent.

31. The method of claim 25 wherein said labile hydroxyl protecting group is dimethoxytrityl, monomethoxy trityl, trityl or 9-phenyl-xanthene.

32. The method of claim 25 wherein said heterocyclic base moiety is a purine or a pyrimidine.

33. The method of claim 32 wherein said purine or pyrimidine is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.

34. The method of claim 25 wherein the sum of n, m, and p is from 5 to about 50.

35. The method of claim 25 wherein the sum of n, m, and p is from 8 to about 30.

36. The method of claim 25 wherein the sum of n, m, and p is from 10 to about 25.

37. The method of claim 25 wherein T₁ and T₂ are, independently hydroxyl or a protected hydroxyl.

38. The method of claim 25 wherein each Lp is an Rp phosphorothioate internucleoside linkage.

39. The method of claim 25 wherein at least one Lp is a racemic phosphorothioate internucleoside linkage.

40. The method of claim 25 wherein n and p are each 1 and m is from 3 to about 20.

41. The method of claim 25 wherein n and p are each 2 and m is from 3 to about 20.

42. The method of claim 25 wherein p is 0.

43. The method of claim 25 wherein at least one R₁ is a 2′

-substituent group or a protected 2′

-substituent group.

44. The method of claim 25 wherein said activated phosphorus group is a phosphoramidite, an H-phosphonate or a phosphate triester.

45. The method of claim 25 wherein said covalent attachment to a solid support is a sarcosinyl-succinonyl linker.

49. A compound of formula:

wherein;

R₆₂ is H or a hydroxyl protecting group;

R₁ is H, hydroxyl, a protected hydroxyl, a 2′

-substituent group or a protected 2′

-substituent group;

B is a heterocyclic base moiety; and

R₆₁ is a chiral auxiliary selected from formulas I-VI;

50. The oligomeric compound of claim 49 wherein each R₁ is H or hydroxyl.

51. The oligomeric compound of claim 49 wherein R₁ is C₁-C₁₀ O-alkyl or C₁-C₁₀ substituted O-alkyl.

52. The oligomeric compound of claim 51 wherein R₁ is 2′

-O-methoxyethyl or 2′

-O-methyl.

53. The oligomeric compound of claim 49 wherein each Nu is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine.

54. A compound having the formula:

wherein;

B is a heterocyclic base moiety;

q is 0 to about 50;

R₆₂ is H or a hydroxyl protecting group;

R₁ is H, hydroxyl, a protected hydroxyl, a 2′

-substituent group or a protected) 2′

-substituent group;

R₆₄ is H, a hydroxyl protecting group, or a linker to a solid support;

R₆₃ is a radical selected from the group consisting of

55. The oligomeric compound of claim 54 wherein each R₁ is H or hydroxyl.

56. The oligomeric compound of claim 54 wherein R₁ is C₁-C₁₀ O-alkyl or C₁-C₁₀ substituted O-alkyl.

57. The oligomeric compound of claim 54 wherein R₁ is 2′

-O-methoxyethyl or 2′

-O-methyl.

58. The oligomeric compound of claim 54 wherein each Nu is, independently, adenosine, guanosine, uridine. 5-methyluridine, cytidine 5-methylcytidine or thymine.

59. The oligomeric compound of claim 54 wherein q is 5 to about 50.

60. The oligomeric compound of claim 54 wherein q is from 8 to about 30.

61. The oligomeric compound of claim 54 wherein q is from 10 to about 25.

62. The oligomeric compound of claim 54 wherein q is 1.

63. The oligomeric compound of claim 54 wherein q is 0.

64. A compound having the formula: