Phosphotriester oligonucleotides, amidities and method of preparation

• US 6,124,445 A
• Filed: 01/09/1998
• Issued: 09/26/2000
• Est. Priority Date: 11/23/1994
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Rayner, Bernard, Gosselin, Gilles, Manoharan, Muthiah, Imbach, Jean-Louis
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/004,940
• Time in Patent Office: 991 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34, 536/22.1, 536/23.1, 536/26.7, 536/26.71, 536/26.72, 536/26.74, 536/26.8
• US Class Current: 536/23.1
• CPC Class Codes: C07F 9/65616 containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Oligonucleotides having bioreversible phosphate blocking groups can be used as pro drugs for the oligonucleotides. The bio-reversible blocking groups can be removed by intercellular enzymes, particularly carboxyesterases, to provide the unprotected oligonucleotide. The oligonucleotides can be prepared via post synthesis alkylation reactions or utilizing amidite type chemistry wherein the bioreversible phosphorus protecting group is formed as an integral part of the amidite reagent.

Claims

1. A compound of the structure:

##STR6## wherein Pg is a nucleoside blocking group,

B is a heterocyclic base,

Y₁ is S and Y₂ is O or S or Y₁ is O and Y₂ is S,

Q is F or O-alkyl,

Z is hydrocarbyl,

X is O, S, or CH₂,

Y₇ is O or S, and

R₅, R₆, and R₇ are, independently, hydrocarbyl.

2. The compound of claim 1 wherein Z is C₁ -C₄ alkyl.

3. The compound of claim 1 wherein Z is t-butyl.

4. The compound of claim 1 wherein Z in methyl.

5. The compound of claim 1 wherein said hydrocarbyl group is substituted with at least one of a halogen, hydroxyl, thiol, keto, carboxyl, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, or nitro group.

6. The compound of claim 1 wherein R₅, R₆, and R₇ are, independently, C₁ -C₂₀ alkyl.

7. The compound of claim 1 wherein R₅, R₆, and R₇ are, independently, C₁ -C₄ alkyl.

8. The compound of claim 1 wherein R₅, R₆, and R₇ are methyl.

9. The compound of claim 1 wherein B is a purine or pyrimidine.

10. The compound of claim 9 wherein an exocyclic nitrogen atom of said purine or pyrimidine further includes a base-stable protecting group.

11. The compound of claim 10 wherein said purine or pyrimidine is adenine, guanine, or cytosine.

12. The compound of claim 10 wherein said protecting group is selected from the group consisting of pent-4-enoyl, 4-methylsulphinyl-benzyloxycarbonyl, 2-nitrophenylsulfenyl, and triphenyl-methanesulfenyl.

13. The compound of claim 1 wherein Pg is trityl, monomethoxytrityl, dimethoxytrityl or pixyl.

14. The compound of claim 1 wherein Pg is dimethoxytrityl.

15. The compound of claim 1 wherein Y₂ is S.

16. The compound of claim 1 wherein Q is O--C₁ --C₂₀ alkyl.

17. The compound of claim 16 wherein said alkyl group is substituted with at least one of a halogen, hydroxyl, thiol, keto, carboxyl, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, or nitro group.

18. The compound of claim 1 wherein Q is F.

19. A compound of the structure:

##STR7## wherein Pg is a nucleoside blocking group,

B is a heterocyclic base,

Y₁ is S and Y₂ is O or S or Y₁ is O and Y₂ is S,

Q is F or O-alkyl,

Z is hydrocarbyl,

X is O, S, or CH₂, and

Y₇ is O or S.

20. The compound of claim 19 wherein Z is C₁ -C₄ alkyl.

21. The compound of claim 19 wherein Z is t-butyl.

22. The compound of claim 19 wherein Z in methyl.

23. The compound of claim 19 wherein said hydrocarbyl group is substituted with at least one of a halogen, hydroxyl, thiol, keto, carboxyl, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, or nitro group.

24. The compound of claim 19 wherein B is a purine or pyrimidine.

25. The compound of claim 24 wherein an exocyclic nitrogen atom of said purine or pyrimidine further includes a base-stable protecting group.

26. The compound of claim 25 wherein said purine or pyrimidine is adenine, guanine, or cytosine.

27. The compound of claim 25 wherein said protecting group is selected from the group consisting of pent-4-enoyl, 4-methylsulphinyl-benzyloxycarbonyl, 2-nitrophenylsulfenyl, and triphenyl-methanesulfenyl.

28. The compound of claim 19 wherein Pg is trityl, monomethoxytrityl, dimethoxytrityl or pixyl.

29. The compound of claim 19 wherein Pg is dimethoxytrityl.

30. The compound of claim 19 wherein Y₂ is S.

31. The compound of claim 19 wherein Q is O--C₁ -C₂₀ alkyl.

32. The compound of claim 31 wherein said alkyl group is substituted with at least one of a halogen, hydroxyl, thiol, keto, carboxyl, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, or nitro group.

33. The compound of claim 19 wherein Q is F.

34. A compound of the structure ##STR8## wherein:

Pg is a nucleoside blocking group,

B is a heterocyclic base,

Y₁ is S and Y₂ is O or S or Y₁ is O and Y₂ is S,

R₃ and R₄, independently, are hydrocarbyl or together R₃ and R₄ are part of a nitrogen heterocyclic ring,

Q is F or O-alkyl,

n is 1 to 6,

said Y₂ is covalently bound to S-Z, C(═

Y₇)--Z, or C(═

Y₇)--Z--N⁺ (R₅) (R₆) (R₇),

Z is hydrocarbyl,

X is O, S, or CH₂,

Y₇ is O or S, and

R₅, R₆, and R₇ are, independently, hydrocarbyl.

35. The compound of claim 34 wherein Y₂ is covalently bound to --C(═

Y₇)--Z.

36. The compound of claim 34 wherein Y₂ is covalently bound to --S--Z.

37. The oligonucleotide of claim 34 wherein Y₂ is covalently bound to --C(═

Y₇)--Z--N(R₅) (R₆) (R₇).

38. The compound of claim 34 wherein n is 2.

39. The compound of claim 34 wherein Z is C₁ -C₂₀ alkyl.

40. The compound of claim 35 wherein Z is C₁ -C₂₀ alkyl.

41. The compound of claim 34 wherein Z is C₁ -C₄ alkyl.

42. The compound of claim 35 wherein Z is C₁ -C₄ alkyl.

43. The compound of claim 35 wherein Z is i-propyl or t-butyl.

44. The compound of claim 35 wherein Z in methyl.

45. The compound of claim 34 wherein said hydrocarbyl group is substituted with at least one of a halogen, hydroxyl, thiol, keto, carboxyl, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, or nitro group.

46. The compound of claim 37 wherein R₅, R₆, and R₇ are, independently, C₁ -C₂₀ alkyl.

47. The compound of claim 37 wherein R₅, R₆, and R₇ are, independently, C₁ -C₄ alkyl.

48. The compound of claim 37 wherein R₅, R₆, and R₇ are methyl.

49. The compound of claim 34 wherein B is a purine or pyrimidine.

50. The compound of claim 49 wherein said purine or pyrimidine includes an exocyclic amine bearing a base-stable protecting group.

51. The compound of claim 50 wherein said purine or pyrimidine is adenine, guanine, or cytosine.

52. The compound of claim 50 wherein said protecting group is selected from the group consisting of pent-4-enoyl, 4-methylsulphinyl-benzyloxycarbonyl, 2-nitrophenylsulfenyl, and triphenyl-methanesulfenyl.

53. The compound of claim 34 wherein Pg is trityl, monomethoxytrityl, dimethoxytrityl or pixyl.

54. The compound of claim 53 wherein Pg is dimethoxytrityl.

55. The compound of claim 34 wherein each of R₃ and R₄ are C₁ -C₁₀ alkyl.

56. The compound of claim 34 wherein each of R₃ and R₄ are iso-propyl.

57. The compound of claim 34 wherein Y₁ is O and Y₂ is S.

58. The compound of claim 34 wherein Y₁ is S and Y₂ is O.

59. The compound of claim 34 wherein Y₁ is S and Y₂ is S.

60. The compound of claim 34 wherein Q is O--C₁ -C₂₀ alkyl.

61. The compound of claim 60 wherein said alkyl group is substituted with at least one of a halogen, hydroxyl, thiol, keto, carboxyl, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, or nitro group.

62. The compound of claim 34 wherein Q is F.