Pyrimidine derivatives for labeled binding partners

• US 6,967,079 B2
• Filed: 11/14/2002
• Issued: 11/22/2005
• Est. Priority Date: 09/17/1993
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Jones, Robert J., Lin, Kuei-Ying, Matteucci, Mark
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/294,066
• Publication Number:
  US 20030207824A1
• Time in Patent Office: 1,104 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/26.6
• US Class Current: 435/6.11
• 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 primer for amplification or detection of a nucleic acid, the primer comprising a compound of the formula B_x-R¹, a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the formula:

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 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, CN or halo, or R⁶ taken together with an adjacent R⁶ to complete a ring containing 5 to 6 ring atoms, and tautomers, solvates and salts thereof; and

R³ is a protecting group or H;

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, taken together with A, is not unsubstituted phenyl.

2. The primer of claim 1, wherein R¹ is an oligomer.

3. The primer of claim 1, wherein R¹ is a polymer.

4. The primer of claim 1, wherein R¹ is an oligonucleotide.

5. The primer of claim 1, wherein Z, together with A, is of the subformula:

wherein A¹ is N or CR⁶; and

G is CH, S, O or NR⁴, wherein R⁴ H or C₁-C₆ alkyl.

6. A probe for amplification or detection of a nucleic acid, the probe comprising a compound having the formula B_x-R¹, a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the formula:

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 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, CN or halo, or R⁶ taken together with an adjacent R⁶ complete a ring containing 5 to 6 ring atoms, and tautomers, solvates and salts thereof; and

R³ is a protecting group or H;

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

in which D² 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, taken together with A, is not unsubstituted phenyl.

7. The probe of claim 6, wherein R¹ is an oligomer.

8. The probe of claim 6, wherein R¹ is a polymer.

9. The probe of claim 6, wherein R¹ is an oligonucleotide.

10. The probe of claim 6, wherein Z, together with A, is of the subformula:

wherein A¹ is N or CR⁶; and

G is CH, S, O or NR⁴, wherein R⁴ is H or C₁-C₆ alkyl.

11. A pair of primers for amplification or detection of a nucleic acid, at least one of said primers comprising a compound of the formula B_x-R¹, a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the formula:

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 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, CN or halo, or R⁶ is taken together with an adjacent R⁶ to complete a ring containing 5 to 6 ring atoms, and tautomers, solvates and salts thereof; and

R³ is a protecting group or H;

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, taken together with A, is not unsubstituted phenyl.

12. The pair of primers of claim 11, wherein R¹ is an oligomer.

13. The pair of primers of claim 11, wherein R¹ is a polymer.

14. The pair of primers of claim 11, wherein R¹ is an oligonucleotide.

15. The pair of primers of claim 11, wherein Z, together with A, is of the subformula:

wherein A¹ is N or CR⁶; and

G is CH, S, O or NR⁴, wherein R⁴ is H or C₁-C₆ alkyl.

16. A pair of primers for amplification or detection of nucleic acid, each of said pair of primers comprising a compound of formula B_x-R¹, a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the formula:

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

R³ is a protecting group or H;

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, taken together with A, is not unsubstituted phenyl.

17. The pair of primers of claim 16, wherein R¹ is an oligomer.

18. The pair of primers of claim 16, wherein R¹ is a polymer.

19. The pair of primers of claim 16, wherein R¹ is an oligonucleotide.

20. The pair of primers of claim 16, wherein Z, together with A, is of the subformula:

wherein A¹ is N or CR⁶; and

G is CH, S, O or NR⁴, wherein R⁴ is H or C₁-C₆ alkyl.

21. A method for amplification of nucleic acid, the method comprising:

selecting at least one primer for use in said method, the primer comprising a compound of the formula B_x-R¹, a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the formula;

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 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, CN or halo, or R⁶ is taken together with an adjacent R⁶ to complete a ring containing 5 to 6 ring atoms, and tautomers, solvates and salts thereof; and

R³ is a protecting group or H;

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, taken together with A, is not unsubstituted phenyl; and

performing at least one step for amplifying or detecting a nucleic acid sequence.

22. The method of claim 21, wherein R¹ is an oligomer.

23. The method of claim 21, wherein R¹ is a polymer.

24. The method of claim 21, wherein R¹ is an oligonucleotide.

25. The method of claim 21, wherein Z, together with A, is of the subformula:

wherein A¹ is N or CR⁶; and

G is CH, S, O or NR⁴, wherein R⁴ H or C₁-C₆ alkyl.

26. The method of claim 21, wherein the primer is a member of a set of primers.

27. The method of claim 21, wherein said primer is a member of a set of primers, and both members of the set of primers are independently of the formula:

wherein the variables are as defined in claim 21.