Pyrimidine derivatives for labeled binding partners

• US 20030207824A1
• Filed: 11/14/2002
• Published: 11/06/2003
• Est. Priority Date: 09/17/1993
• Status: Active Grant

• Current Assignee: Kuei-Ying Lin, Mark D. Matteucci, Robert J. Jones
• Original Assignee: Kuei-Ying Lin, Mark D. Matteucci, Robert J. Jones
• Inventors: Jones, Robert J., Lin, Kuei-Ying, Matteucci, Mark D.
• Granted Patent: US 6,967,079 B2
• Time in Patent Office: Days
• US Class Current: 514/43
• CPC Class Codes: C07D 239/47 One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
• CPC Class Codes: C07D 239/54 as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07F 7/2208 Compounds having tin linked only to carbon, hydrogen and/or halogen
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• 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: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Compounds having particular interest as labels and various novel uses in diagnostics and therapeutics are provided which have structure (1)

wherein R¹ is a binding partner, a linker or H;

a and b are 0 or 1, provided that the total of a and b is 0 or 1;

A is N or C;

X is S, O, —C(O)—, NH or NCH₂R⁶;

Y is —C(O)—;

Z is taken together with A to form an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a carbon atom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ring heteroatoms at least two of which are separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is substituted with R⁶ or ═O;

R⁶ is independently H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, NO₂, N(R³)₂, C≡N or halo, or an R⁶ is taken together with an adjacent R⁶ to complete a ring containing 5 or 6 ring atoms;

R³ is a protecting group or H; and tautomers, solvates and salts thereof; and provided that where a is 0, b is 1, and R¹ is

in which D² is independently hydroxyl, blocked hydroxyl, mono, di or triphosphate, or an oligodeoxyribonucleotide otherwise containing only the bases A, G, T and C; and

D³ is H or OH;

then Z is not unsubstituted phenyl.

Also provided are novel intermediates and methods for the preparation and use of the structure (1) compounds.

Claims

1. A compound having the structure:

2. The compound of claim 1 wherein the linker group comprises hydroxyl, phosphate or phosphonate.

3. The compound of claim 1 wherein R¹ is a linker group selected from the group consisting of:

E—

CHR⁷—

R¹¹—

(CH₂)_m1—

C(R⁸)(CH²)_m1(R⁹)—

(CH₂)_m1—

R¹⁰—

(CH₂)_m1,

E—

Q—

C₆H₄—

CH₂—

;

E—

CHR⁷—

O—

CHR⁷—

O—

CHR⁷—

,

E—

CHR⁷—

(CHR¹³)—

_m1—

CHR¹⁴—

R¹⁰—

;

H(CH₂)_m1CH(COOR²⁰)(CH₂)_m1—

D is an oligonucleotide coupling group;

D¹ is independently F, H, O-alkyl, S-alkyl or an oligonucleotide coupling group, but only one D¹ is an coupling group;

Q is independently —

C(R¹²)₂—

CH₂—

, C(R¹²)₂—

O—

, —

CR¹²═

CR¹²—

, or —

C≡

C—

;

R⁷ is independently H or C₁-C₄ alkyl;

R⁸ is H or C₁-C₄ alkyl, C₂-C₄ alkenyl or azidomethyl;

R⁹ is halo, H or OR²⁰

R¹⁰ is O, CH₂ or a covalent bond;

R¹¹ is O, S, CH₂, CHF or CF₂;

R¹² is independently H or halogen;

R¹³ is H, halogen, OR²⁰, CH₃, CH₂OR²⁰ or C₃-C₆ acyloxyalkyl;

R¹⁴ is H, halogen, OR₂₀, CH₃, CH₂OR²⁰, C₃-C₆ acyloxymethyl, or C₂-C₆ acyloxy,

R¹⁵ is CH₂, CHF or O;

R¹⁶ is CH or S, provided that when R¹⁹ is 0 or S, or R¹⁵ is CH, then R¹⁶ is not S;

R¹⁷ is H, OR²⁰, halogen, N₃, C₁-C₄ alkyl or C₁-C₄ alkoxy or is absent when R¹⁶ is S;

R¹⁸ is H, OR²⁰, halogen, N₃, C₁-C₄ alkyl or C₁-C₄ alkoxy;

R¹⁹ is O, S, CH₂, CHF or CF₂;

R²⁰ is H or a protecting group;

m1 is independently 0 or an integer from 1 to 4; and

E is OH, OR²⁰, —

PO₂ or —

OP(O)₂.

4. The compound of claim 1 wherein R¹ is H;

HOCH(CHR¹³)CH₂—

;

ECH₂OCH(CHR¹³)CH₂—

;

or

5. The compound of claim 1 wherein Z is

6. The compound of claim 5 wherein Z is

7. The compound of claim 5 wherein Z is

8. The compound of claim 5 wherein Z is

9. The compound of claim 5 wherein adjacent R⁶ are taken together to complete a phenyl, thiazole, imidazole, oxazole, pyridine or pyrimidine ring.

10. The compound of claim 5 wherein G is S, O or NR⁴.

11. The compound of claim 5 wherein Z is

12. The compound of claim 5 wherein Z is

13. The compound of claim 1 wherein b is 0.

14. The compound of claim 1 wherein a is 1 and X is O.

15. The compound of claim 1 wherein b is 1 and a is 0.

16. The compound of claim 1 wherein A is C.

17. The compound of claim 1 wherein A is N.

18. The compound of claim 5 wherein A is N.

19. The compound of claim 5 wherein A is CH.

20. The compound of claim 1 wherein Z together with CH—

A completes a aryl ring containing 6 ring atoms.

21. The compound of claim 20 wherein the aryl ring contains a nonbridging ring carbon atom which is singly substituted with R⁶.

22. The compound of claim 1 wherein Z together with CH—

A completes a heteroaryl residue containing 1 ring N atom, 2 ring N atoms, 1 ring oxygen atom 1 ring nitrogen and 1 ring sulfur atom separated by at least 1 carbon atom, and 3 ring N atoms, two of which are separated by at least 1 carbon atom.

23. The compound of claim 1 wherein Z together with CH—

A completes a heteroaryl ring containing 5 ring atoms, one of which is N.

24. The compound of claim 1 wherein A is C, b is 0, a is 1, and X is O, C(O) or S.

25. The compound of claim 1 wherein a and b are both 0.

26. The compound of claim 1 wherein A is N, b is 0, a is 1 and X is O.

27. The compound of claim 24 wherein Z together with CH—

A completes an unsubstituted aryl ring or an aryl ring substituted with R⁶.

28. The compound of claim 25 wherein A is C and Z together with CH—

A completes an unsubstituted pyridine ring or is a pyridine ring substituted with R⁶.

29. The compound of claim 1 wherein a is 0, b is 1 and Y is C(O).

30. A compound having the structure

31. The compound of claim 30 wherein R¹ is H;

HOCH(CHR¹³)CH₂—

;

ECH₂OCH(CHR¹³)CH₂—

;

or

32. A compound having the structure

33. The compound of claim 32 wherein R¹ is H;

HOCH(CHR¹³)CH₂—

;

ECH₂OCH(CHR¹³)CH₂—

;

or

35. A compound having the structure

34. The compound of claim 35 wherein J′

is unsubstituted with other that H or is substituted with C₁-C₃ alkyl.

36. The compound of claim 35 wherein R¹ is H;

HOCH(CHR₁₃)CH₂—

;

ECH₂OCH(CHR¹³)CH₂—

;

or

37. The compound of claim 35 wherein R⁶ is hydrogen or C₁-C₃ alkyl.

38. A compound having the structure

39. The compound of claim 38 wherein R¹ is H;

HOCH(CHR¹³)CH₂—

;

ECH₂OCH(CHR¹³)CH₂—

;

or

40. A compound having the structure

41. A method for the preparation of a phenoxazine, the method comprising contacting KF with a compound having the structure

42. An oligomer having the structure

43. The oligonucleotide of claim 42 wherein X¹ is —

P(S)(O)—

, —

P(O)(O)—

, —

P(Me)(O)—

, or —

P(Me)(S)—

.

44. The oligomer of claim 42 wherein Z is

45. The oligomer of claim 42 wherein Z is

46. The oligomer of claim 42 wherein Z is

47. The oligomer of claim 42 wherein Z is

48. The oligomer of claim 42 wherein adjacent R⁶ are taken together to form phenyl, thiazole, imidazole, oxazole, pyridine or pyrimidine radicals.

49. The oligomer of claim 42 wherein Z is

50. The oligomer of claim 42 where Z is

51. The oligomer of claim 42 containing from 2 to about 30 nucleotide units.

52. The oligomer of claim 42 wherein R²¹ is H, OH, —

O-alkyl, O-methyl, —

O-ethyl or —

O-propyl.

53. The oligomer of claim 42 wherein adjacent B groups have substructure (30).

54. The oligomer of claim 42 wherein bases other than substructure (30) consist of naturally-occurring nucleotide bases.

55. The oligomer of claim 42 wherein n is 0 to 20.

56. The oligomer of claim 42 wherein D¹ is H-phosphonate, methylphosphonamidite, β

-cyanoethylphosphoramidite or alkylphosphoramidite.

57. The oligomer of claim 42 wherein D is OH, —

PO₃, DMTO or MMTO.

58. The oligomer of claim 42 wherein a single B group having substructure (30) is located at the 5′

or 3′

end of the oligomer.

59. The oligomer of claim 42 further comprising 5-(1-propynyl) cytosine or uridine.

60. A method comprising contacting a nucleic acid with an oligomer of claim 42.

61. The method of claim 60 further comprising determining whether the oligomer has formed a duplex with the nucleic acid.

62. The method of claim 60 wherein the nucleic acid is present within a cell and the sequence of bases in the oligomer are selected such as to be complementary to a predetermined sequence within the nucleic acid, wherein B groups of substructure (30) are employed as cytosine surrogates.

63. The method of claim 60 wherein the nucleic acid is RNA or single stranded DNA.

64. A method comprising contacting a target nucleic acid sequence with an oligonucleotide substantially complementary to the target sequence, the oligonucleotide comprising at least one base analogue capable of increasing the Tm of a duplex of the oligonucleotide and the target to about 85 to 95°

C., heating the duplex to about from 85 to 95°

C., and contacting the duplex with a DNA polymerase.