Novel oligonucleotides and related compounds

• US 20050159375A1
• Filed: 01/30/2004
• Published: 07/21/2005
• Est. Priority Date: 11/28/2003
• Status: Active Grant

• Current Assignee: Chemgenes Corporation
• Original Assignee: Chemgenes Corporation
• Inventors: Sit, Kwok-Hung, Srivastava, Suresh C., Bajpai, Satya K.
• Granted Patent: US 7,846,436 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present invention relates generally to oligonucleotides and more specifically to oligonucleotides which have a sequence including at least two CDG dinucleotides and a prodrug of an antimetabolite. The prodrug can be part of a CpG dinucleotide or may be attached elsewhere on the oligonucleotide.

Claims

19. The oligonucleotide of claim 19, wherein the alkyl is methyl.

23. An oligonucleotide for preferentially killing cancerous cells over non-cancerous cells comprising a motif represented by the formula:

5′

PGXCG3′

wherein P is a prodrug for an antimetabolite and X represents between 0 and 50 nucleotides.

1. An oligonucleotide for preferentially killing cancerous cells over non-cancerous cells comprising at least two CpG moieties and a prodrug for an antimetabolite covalently linked to the oligonucleotide.

2. The oligonucleotide of claim 1, wherein the antimetabolite is selected from the group consisting of 2′

-deoxy-3′

-thiacytidine, 3′

-azido-3′

-deoxythymidine, 2′

,3′

-dideoxycytidine, 2′

,3 ′

-didehydro-3′

-deoxythymidine, 2′

,3′

-dideoxyinosine, 5-fluoro-2′

-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine, 1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′

-deoxycytidine, 6-mercaptopurineriboside, 2-chlorodeoxyadenosine, and pentostatin.

3. The oligonucleotide of claim 1, wherein the pro drug is a prodrug for the antimetabolite 2′

-deoxy, 2′

,2′

difluorocytidine.

4. The oligonucleotide of claim 1, wherein two of the at least two CpG moieties are separated by a number of nucleotides selected from the numbers 2, 5, and 9.

5. The oligonucleotide of claim 1, wherein said prodrug is 5′

to the at least two CpG moieties.

6. The oligonucleotide of claim 1, wherein said prodrug is 3′

to the at least two CpG moieties.

7. The oligonucleotide of claim 1, wherein said prodrug is 3′

to at least one CpG moiety and 5′

to at least a second CpG moiety.

8. The oligonucleotide of claim 1, wherein said prodrug is linked to the oligonucleotide by a 3′

-3′

linkage.

9. The oligonucleotide of claim 1, wherein said prodrug is linked to the oligonucleotide by a 5′

-5′

linkage.

10. The oligonucleotide of claim 1, wherein said prodrug is linked to the oligonucleotide by a 3′

-5′

linkage.

11. The oligonucleotide of claim 1, wherein said prodrug is covalently linked to the oligonucleotide by a 5′

-3′

linkage.

12. The oligonucleotide of claim 1, wherein said prodrug is at a position that is selected from 10 nucleotides upstream from one of the at least two CpG moieties, 9 nucleotides upstream from the CpG moiety, 8 nucleotides upstream from the CpG moiety, 7 nucleotides upstream from the CpG moiety, 6 nucleotides upstream from the CpG moiety, 5 nucleotides upstream from the CpG moiety, 4 nucleotides upstream from the CpG moiety, 3 nucleotides upstream from the CpG moiety, 2 nucleotides upstream from the CpG moiety, 1 nucleotides upstream from the CpG moiety, 10 nucleotides downstream from a CpG moiety, 9 nucleotides downstream from the CpG moiety, 8 nucleotides downstream from the CpG moiety, 7 nucleotides downstream from the CpG moiety, 6 nucleotides downstream from the CpG moiety, 5 nucleotides downstream from the CpG moiety, 4 nucleotides downstream from the CpG moiety, 3 nucleotides downstream from the CpG moiety, 2 nucleotides downstream from the CpG moiety, and 1 nucleotides downstream from the CpG moiety.

13. The oligonucleotide of claim 1, wherein the prodrug is covalently linked to the oligonucleotide by a linker having the formula.

14. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one nucleotide having a ribose sugar moiety.

15. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one nucleotide having a 2′

-deoxyribose sugar moiety.

16. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one 2′

-halogen nucleotide.

17. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one 2′

-N-alkyl nucleotide wherein the alkyl has between about 1 and about 6 carbon atoms.

18. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one 2′

-O-alkyl nucleotide, one 2′

-N-Alkyl nucleotide, or one 2′

-O-halogen nucleotide, wherein the alkyl has between about 1 and about 6 carbon atoms

20. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a plurality of nucleotides connected by covalent internucleoside linkages, wherein each of the linkages are selected from the group consisting of a phosphodiester linkage, a C1-C6 alkoxy phosphotriester linkage, a phosphorothioate linkage and a phosphoramidate linkage.

21. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of any of claims 1-20.

22. The oligonucleotide of claim 21 wherein said pharmaceutically acceptable carrier is lipofectin.

24. The oligonucleotide of claim 23, wherein the antimetabolite is 2′

-deoxy, 2′

-,2′

-difluorocytidine.

25. The oligonucleotide of claim 23, wherein the metabolite is selected from the group consisting of 2′

-deoxy-3′

-thiacytidine, 3′

-azido-3′

-deoxythymidine, 2′

,3′

-dideoxycytidine, 2′

,3 ′

-didehydro-3 ′

-deoxythymidine, 2′

,3′

-dideoxyinosine, 5-fluoro-2′

-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine, 1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′

-deoxycytidine, 6-mercaptopurineriboside, 2-chlorodeoxyadenosine, and pentostatin.

26. The oligonucleotide of claim of 23, where X is selected from the group consisting of 2, 5, and 9.

27. The oligonucleotide of claim 23, wherein the oligonucleotide comprises multiple nucleotides and the prodrug is covalently linked to one of the nucleotides by a 3′

-3′

linkage.

28. The oligonucleotide of claim 23, wherein the oligonucleotide comprises multiple nucleotides and the prodrug is covalently linked to one of the nucleotides by a 5′

-5′

linkage.

29. The oligonucleotide of claim 23, wherein the oligonucleotide comprises multiple nucleotides and the prodrug is covalently linked to one of the nucleotides by a 3′

-5′

linkage.

30. The oligonucleotide of claim 23, wherein the oligonucleotide comprises multiple nucleotides and the prodrug is covalently linked to one of the nucleotides by a 5′

-3′

linkage.

31. The oligonucleotide of claim 23, wherein the oligonucleotide comprises at least one nucleotide having a ribose sugar moiety.

32. The oligonucleotide of claim 23, wherein the oligonucleotide comprises at least one nucleotide having a 2′

-deoxyribose sugar moiety.

33. The oligonucleotide of claim 23, wherein the oligonucleotide comprises at least one 2′

-O-Alkyl nucleotide, 2′

-N-Alkyl nucleotide, or 2′

-O-halogen nucleotide, wherein the alkyl has between about 1 and about 6 carbon atoms.

34. The oligonucleotide of claim 23, wherein the oligonucleotide comprises a plurality of nucleotides connected by covalent internucleoside linkages, wherein the linkages are selected from the group consisting of phosphodiester linkage, a C1-C6 alkoxy phosphotriester linkage, a phosphorothioate linkage and a phosphoramidate linkage.

35. The oligonucleotide of claim 23, wherein the oligonucleotide comprises multiple nucleotides and the prodrug is attached to at least one of the multiple nucleotides by a linker having the formula

36. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of any of claims 23-35.

37. The oligonucleotide of claim 36 wherein said pharmaceutically acceptable carrier is lipofectin.

39. The compound of claim 23, wherein the group that is attachable to a solid support has the formula O—

C(═

O)-M-C(═

O)—

NH-Spacer, where M is selected from the group consisting of succinyl, oxalyl, and hydroquinolynyl, and wherein the Spacer is selected from the

29-1. The oligonucleotide of claim 23, wherein the oligonucleotide comprises multiple nucleotides and the prodrug is covalently linked to one of the nucleotides by a 3′

-5′

linkage.

38. A compound having purity in excess of 98% by HPLC, having the formula:

40. group consisting of a C1-C6 alkyl, ethyloxyglycol, and a combination of alkyl and ethyleneglycoxy.

41. A compound having the formula:

42. A compound of the formula:

43. A compound having a purity in excess of 97% by HPLC, as shown by the formula:

44. A compound having purity in excess of 97% by HPLC, and having the formula: