Fluorinated resorufin compounds and their application

• US 7,432,372 B2
• Filed: 11/01/2004
• Issued: 10/07/2008
• Est. Priority Date: 10/31/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Patch, Brian, Leung, Wai-Yee, Liu, Jixiang, Steinberg, Thomas Harry, Ge, Yue, Batchelor, Robert Hardy, Smalley, Peter Aaron Emrys, Johnson, Iain D.
• Primary Examiner(s): HABTE, KAHSAY
• Application Number:
  US10/980,139
• Publication Number:
  US 20050096315A1
• Time in Patent Office: 1,436 Days
• Field of Search: 544/99, 544/101
• US Class Current: 544/99
• CPC Class Codes: C07D 265/38 [b, e]-condensed with two six-membered rings
• CPC Class Codes: C07D 413/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C09B 19/00 Oxazine dyes
• CPC Class Codes: C12Q 1/28 involving peroxidase

Abstract

The invention provides novel fluorinated resorufin compounds that are of use in a variety of assay formats. Also provided are methods of using the compounds and kits that include a compound of the invention and instructions detailing the use of the compound in one or more assay formats.

Claims

1. A compound having the formula:

2. The compound according to claim 1, wherein at least one of R³, R⁴, R⁵, and R⁶ is fluorine.

3. The compound according to claim 1, wherein A is OR⁸.

4. The compound according to claim 3, wherein R⁸ is hydrogen.

5. The compound according to claim 1, wherein E is OR⁸.

6. The compound according to claim 5, wherein R⁸ is hydrogen.

7. The compound according to claim 1, wherein R³ and R⁶ are each fluorine.

8. The compound according to claim 1, wherein R⁴ and R⁵ are each fluorine.

9. The compound according to claim 1, wherein R³, R⁴, R⁵ and R⁶ are each fluorine.

10. The compound according to claim 1, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.

11. The compound according to claim 1, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are each hydrogen.

12. The compound according to claim 1, wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.

13. The compound according to claim 1, wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are each hydrogen.

14. The compound according to claim 1, wherein A is NR⁹R¹⁰.

15. The compound according to claim 1, wherein A is NR⁹R¹⁰ and R⁹ and R¹⁰ are independently hydrogen, substituted alkyl or unsubstituted alkyl.

16. The compound according to claim 1, wherein E is NR⁹R¹⁰.

17. The compound according to claim 1, wherein E is NR⁹R¹⁰ and R⁹ and R¹⁰ are independently hydrogen, substituted alkyl or unsubstituted alkyl.

18. The compound according to claim 1, wherein R¹ is hydrogen, substituted alkyl or unsubstituted alkyl.

19. The compound according to claim 1, wherein R¹ is methyl.

20. The compound according to claim 1, wherein X¹ is oxygen.

21. The compound according to claim 1, having the formula:

22. The compound according to claim 1, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide or a thiol.

23. The compound according to claim 1, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

24. An oxazine dye compound having the formula:

25. The compound according to claim 24, wherein A is OR⁸.

26. The compound according to claim 25, wherein R⁸ is hydrogen.

27. The compound according to claim 24, wherein Z is O.

28. The compound according to claim 24, wherein at least one of R³, R⁴, R⁵, and R⁶ is fluorine.

29. The compound according to claim 24, wherein R³ and R⁶ are each fluorine.

30. The compound according to claim 24, wherein R⁴ and R⁵ are each fluorine.

31. The compound according to claim 24, wherein R³, R⁴, R⁵ and R⁶ are each fluorine.

32. The compound according to claim 24, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, or a reactive group.

33. The compound according to claim 24, wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are independently hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, or a reactive group.

34. The compound according to claim 24, wherein A is NR⁹R¹⁰.

35. The compound according to claim 24, wherein A is NR⁹R¹⁰ and R⁹ and R¹⁰ are independently hydrogen, substituted alkyl or unsubstituted alkyl.

36. The compound according to claim 24, wherein Z is N+R⁹R¹⁰.

37. The compound according to claim 24, wherein Z is N+R⁹R¹⁰ and R⁹ and R¹⁰ are independently hydrogen, substituted alkyl or unsubstituted alkyl.

38. The compound according to claim 24, wherein at least one of the R², R³, R⁴, R⁵, R⁶ and R⁷ is a reactive group.

39. The compound according to claim 24, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide or a thiol.

40. The compound according to claim 24, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

41. A compound having formula:

42. The compound according to claim 41, wherein at least one of R³, R⁴, R⁵, and R⁶ is fluorine.

43. The compound according to claim 41, wherein A is OR⁸.

44. The compound according to claim 43, wherein R⁸ is hydrogen.

45. The compound according to claim 41, wherein Z is O.

46. The compound according to claim 41, wherein R³ and R⁶ are each fluorine.

47. The compound according to claim 41, wherein R⁴ and R⁵ are each fluorine.

48. The compound according to claim 41, wherein R³, R⁴, R⁵ and R⁶ are each fluorine.

49. The compound according to claim 41, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.

50. The compound according to claim 41, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are each hydrogen.

51. The compound according to claim 41, wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.

52. The compound according to claim 41, wherein wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are each hydrogen.

53. The compound according to claim 41, wherein X¹ is oxygen.

54. A compound having the formula: