Pyrimidine derivatives for labeled binding partners

• US 6,617,437 B1
• Filed: 12/21/1999
• Issued: 09/09/2003
• Est. Priority Date: 09/17/1993
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Matteucci, Mark, Jones, Robert J., Lin, Kuei-Ying
• Primary Examiner(s): Carr, Deborah D.
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US09/468,454
• Time in Patent Office: 1,358 Days
• Field of Search: 435/91.2, 514/44, 536/28-- 5, 536/23.1, 536/26.6, 544/242
• US Class Current: 536/23.1
• 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 alkeny, 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:

wherein

R¹ is a binding partner, a linker or H;

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

A is independently N or C;

X is independently S, O, —

C(O)—

, NH or NCH₂R⁶;

Y is —

C(O)—

;

Z is taken together with A to form a 5- or 6-membered aryl or heteroaryl ring structure wherein the heteroaryl ring has 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, a single S and a single N ring heteroatom separated by a carbon, 2N 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 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 provided that where a is 0, b is 1 and R¹ is

 

in which

D² is independently hydroxyl, protected 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 taken together with the A and carbon to which it is bound does not form an unsubstituted phenylene;

and tautomers, solvates and salts thereof.

2. The compound of claim 1 wherein R¹ is a linker and said linker has a substituent group which is hydroxyl, O—

P(═

O)(O⁻

)₂ or —

P(═

O)(O⁻

)₂.

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

E—

CHR⁷—

R¹¹—

(CH₂)_m1—

C(R⁸)((CH₂)_m1(R⁹))—

(CH₂)_m1—

(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 a 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 O or S, or R¹⁵ is CH₂, 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, O—

P(═

O)(O⁻

)₂, and —

P(═

O)(O⁻

)₂.

4. The compound of claim 1 wherein

R¹ is H;

HOCH(CHR¹³)CH₂—

;

ECH₂OCH(CHR¹³)CH₂—

;

or

E is OH, OR²⁰, O—

P(═

O)(O⁻

)₂, and —

P(═

O)(O⁻

)₂;

D² is independently F, H, O-alkyl, S-alkyl or an oligonucleotide coupling group;

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

R²⁰ is H or a protecting group.

5. The compound of claim 1 wherein Z is

A¹ is N or CR⁶;

and

G is CH, S, O or NR⁴; and

R⁴ is H or C₁-C₆ alkyl.

6. The compound of claim 5 wherein Z is

R⁴ is H or C₁-C₆ alkyl;

and

R⁵ is H, NO₂ or C₁-C₆ alkyl.

7. The compound of claim 5 wherein Z is

R² is C₁-C₆ alkyl and R⁶ is H.

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 A and the carbon to which Z is bound 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 the A and carbon to which Z is bound completes a heteroaryl residue which has 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;

or 3 ring N atoms at least two of which are separated by at least 1 carbon atom.

23. The compound of claim 1 wherein Z together with the A and carbon to which Z is bound completes a heteroaryl ring consisting of 5 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 A and the carbon to which Z is bound completes an aryl ring consisting of 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 A and the carbon to which Z is bound 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. An oligomer having the structure

wherein

D is OH or blocked OH;

D¹ is an oligonucleotide coupling group or OH;

X¹ is independently a phosphodiester linkage or a phosphodiester substitute linkage bonded to the 2′

or 3′

position of a furanose ring, and the remaining 2′

or 3′

position is substituted with R²¹;

R²¹ is H, OH, F, —

O-alkyl (C₁-C₁₂), —

S-alkyl (C₁-C₁₂), OC₃H₅, or SC₃H₅;

n is an integer from 0 to 98; and

B is a purine or pyrimidine base provided that at least one B has the structure

 

wherein

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 together with a and the carbon which Z is bound to form 5- or 6-membered aryl or heteroaryl ring structure wherein the heteroaryl ring has 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, a single S and a single N ring heteroatom separated by a carbon, 2N 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 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

provided that where a is 0, b is 1 and R¹ is

 

in which

D² is independently hydroxyl, protected 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 phenylene;

and tautomers, solvates and salts thereof.

31. The oligonucleotide of claim 30 wherein X¹ is —

P(S)(O⁻

)—

, —

P(O)(O⁻

)—

, —

P(ME)(O)—

, or —

P(ME)(S)—

.

32. The oligomer of claim 30 wherein Z together with A and the carbon to which Z is bound is

A¹ is N or CR⁶;

G is CH, S, O or NR⁴; and

R⁴is H or C₁-C₆ alkyl;

and the tautomers, solvates and salts thereof.

33. The oligomer of claim 30 wherein Z together with A and the carbon to which Z is bound is

R⁴ is H or C₁-C₆ alkyl;

and

R⁵ is H, NO₂ or C₁-C₆alkyl.

34. The oligomer of claim 30 wherein Z together with A and the carbon to which Z is bound is

R² is C₁-C₆ alkyl and R⁶ is H.

35. The oligomer of claim 30 wherein Z together with A and the carbon to which Z is bound is

36. The oligomer of claim 30 wherein adjacent R⁶ are taken together to form phenyl, thiazole, imidazole, oxazole, pyridine or pyrimidine radicals.

37. The oligomer of claim 30 wherein Z together with A and the carbon to which Z is bound is

38. The oligomer of claim 30 wherein Z together with A and the carbon to which Z is bound is

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

40. The oligomer of claim 30 wherein R²¹ is H, OH, —

O-allyl, —

O-methyl, —

O-ethyl or —

O-propyl.

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

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

43. The oligomer of claim 30 wherein n is 0 to 20.

44. The oligomer of claim 30 wherein D¹ is an H-phosphonyl group, a methylphosphonamidityl group, a β

-cyanoethylphosphoramidityl group or an alkylphosphoramidityl group.

45. The oligomer of claim 30 wherein D is OH, —

PO₃, dimethoxytrityl-O or monomethoxytrityl-O.

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

or 3′

end of the oligomer.

47. The oligomer of claim 30 wherein at least one B is 5-(1-propynyl) cytosinyl or uracilyl.