Pyrimidine derivatives for labeled binding partners

• US 6,800,743 B2
• Filed: 05/21/2002
• Issued: 10/05/2004
• Est. Priority Date: 11/07/1997
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Lin, Kuei-Ying, Matteucci, Mark D.
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Lewis, Patrick T.
• Application Number:
  US10/152,123
• Publication Number:
  US 20030072712A1
• Time in Patent Office: 868 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.31, 536/25.32, 536/25.34, 536/25.4, 536/25.33, 514/256, 514/267, 435/6, 435/87, 435/90, 544/242, 544/245, 544/249
• US Class Current: 536/22.1
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• 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 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Compounds having structure (1)

wherein R¹ is —H a protecting group, a linker or a binding partner; and R² and R³⁴ are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.

Claims

1. A compound having the formula:

wherein;

D is hydroxyl, protected hydroxyl, an oligonucleotide coupling group or a solid support;

D¹ is an oligonucleotide coupling group, hydroxyl, protected hydroxyl or a solid support, wherein D¹ is bonded to one 2′

or 3′

position in the oligonucleotide and another 2′

or 3′

position is substituted with R²¹, provided that D and D¹ are not both an oligonucleotide coupling group and they are not both a solid support;

each R⁴ is, independently, a phosphodiester linkage or a phosphodiester substitute linkage;

each R²¹ is, independently, hydroxyl, protected hydroxyl, halogen or a moiety that enhances nuclease resistance;

n is an integer from 0 to 98;

each B, independently, is a naturally occurring or non-naturally occurring base, or an analog or a protected derivative thereof, provided that at least one B is a base of structure II;

 

wherein R³⁴ is independently —

O—

, —

S—

or —

N(CH₃)—

,

R² is —

R⁶—

(CH₂)_tN(R⁵)C(NR⁵)N(R³)₂, —

R⁶—

CH₂—

CHR³¹—

N(R³)₂, —

R⁶—

(R⁷)_v—

N(R³)₂, —

R⁶—

(CH₂)_t—

N(R³)₂, —

(CH₂)_1-2—

O(CH₂)_t—

N(R³)₂, or A(Z)_x1;

A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R² is not amine, protected amine, nitro or cyano;

x1 is 1, 2, or 3;

R³ is —

H, —

CH₃, —

C₂H₅ or a protecting group;

R⁵ is independently —

H or a protecting group;

R⁶ is independently —

S—

, —

N(R⁵)—

, —

O—

or —

CH₂—

;

R⁷ is independently linear alkyl having 1 to 4 carbon atoms optionally including one —

CH═

CH—

, —

C═

C—

or —

CH₂ —

O—

CH₂—

moiety, or R⁷ is cyclic alkyl having 3, 4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms is optionally substituted with a single —

CH₃, —

CN, ═

O, —

OH or protected hydroxyl, provided that the carbon atoms in any —

CH═

CH—

or —

CH₂—

O—

CH₂—

moiety are not substituted with ═

O, —

OH or protected hydroxyl;

R³¹ is the side chain of an amino acid;

t is 1, 2, 3, or 4, but when R⁶ is —

O—

, —

S—

, or —

N(R⁵)—

, t is not 1; and

v is independently 0, 1, or 2.

2. The compound of claim 1 wherein R² is —

O(CH₂)₂—

NH₂, —

O(CH₂)₃—

NH₂, —

CH₂—

(CH₂)_tN(R³)₂, —

N(R⁵)—

(CH₂)_tN(R³)₂, —

S—

(CH₂)_tN(R³)₂, —

O(CH₂)_tN(R³)₂, —

O(CH₂)₂—

N(CH₃)₂, —

O(CH₂)₃—

N(CH₃)₂, —

O(CH₂)₂—

NHCH₃, —

O(CH₂)₃—

NHCH₃, —

O—

CH₂—

CH(CH₃)—

NH₂, —

CH₂—

O(CH₂)₂—

NH₂, —

CH₂—

O(CH₂)₃—

NH₂ or —

(CH₂)₂—

O(CH₂)₂—

NH₂.

3. The compound of claim 1 wherein R²¹ is selected from the group consisting of —

CH₃, —

O—

alkyl, —

NH-alkyl, —

S-alkyl, —

OR⁵, —

NHR⁵, —

SR⁵, -halogen, —

O(CH₂)_2-8—

O-alkyl, —

O—

(CH₂)_2-8—

CH₂CO₂H, —

O(CH₂)_2-8—

CH₂N(R⁵)₂, —

O—

(CH₂)_2-4—

O—

(CH₂)_2-4—

O(CH₂)_2-4—

R⁶⁵, —

O(CH₂CH₂O)_rCH₂—

R⁶⁵, —

O(CH₂)₂—

R⁶⁵, —

O(CH₂)₂—

O(CH₂)₂—

R⁶⁵ and —

OCH₂CF₂CF₃;

R⁶⁵ is —

H, halogen, —

OR⁵, —

OCH₃, —

NHR⁵, —

SR⁵, substituted alkyl, —

NHOR⁵⁵, —

N₃, —

CN, —

CH₂CN, —

CHCl₂, —

CFH₂, —

CF₂H, —

CH₂CH═

CH₂, —

OC(S)OC₆H₅, t-butyldimethylsilyl ether, triisopropylsilyl ether, an N-phthaloyl protected amino group or a fluorescent label; and

each R⁵⁵ is, independently, R⁵, C₁-C₁₀ alkyl or substituted C₁-C₁₀ alkyl.

4. The compound of claim 3 wherein said halogen is fluorine.

5. The compound of claim 3 wherein said substituted alkyl is selected from the group consisting of —

O—

alkylamino, —

S—

alkylamino, —

NH-alkylamino, —

O—

methyl, —

O—

ethyl, —

O-n-propyl, —

O—

allyl, —

O(CH₂)_2-6OH, —

O(CH₂)₂F, —

OCH₂CHF₂, —

OCH₂CF₃, —

O(CH₂)_2-6OCH₃, —

O(CH₂)_2-OCH₂CH₃, —

O(CH₂)₂OCH₂CH₂OH, —

O(CH₂)₂—

OCH₂CH₂F, —

O(CH₂)₂NHR⁵, —

O(CH₂)₃NHR⁵, —

O(CH₂)₄NHR⁵, —

O(CH₂)₂F, —

O—

(CH₂)₃F, —

O—

(CH₂)₄F, —

OCH₂—

CF₂CF₃, —

O(CH₂)_sR⁶⁵, —

O(CH₂)₂—

[O(CH₂)₂]_rR⁶⁵, —

NH-methyl, —

NH-ethyl, —

NH-n-propyl, —

NH(CH₂)₂OH, —

NH(CH₂)₃OH, —

NH(CH₂)₂F, —

NHCH₂CF₂CF₃, —

NH(CH₂)_sR⁶⁵, —

S—

methyl, —

S-ethyl, —

S-n-propyl, —

S-allyl, —

S(CH₂)_sOH, —

S(CH₂)₃OH, —

S(CH₂)₂F and —

S(CH₂)_s[O(CH₂)₂]_rR⁶⁵, wherein each s is an integer from 2 to 8.

6. The compound of claim 3 wherein said —

O(CH₂)_2-8—

O-alkyl is —

O(CH₂)₂—

O—

CH₃.

7. The compound of claim 2 wherein each B not having structure II is selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine, 5-(1-butynyl)uracil and 5-(1-butynyl)cytosine).

8. The compound of claim 1 wherein R² is —

R⁶(CH₂)_tN(R⁵)C(NR⁵)N(R³)₂, —

O(CH₂)₂NH₂, —

O(CH₂)₃NH₂, or —

R⁶(R⁷)_vN(R³)₂.

9. The compound of claim 8 wherein R₂ is —

R⁶(CH₂)_tN(R⁵)C(NR⁵)N(R³)₂.

10. The compound of claim 8 wherein R² is —

R⁶(R⁷)_vN(R³)₂.

11. The compound of claim 1 wherein R⁴ is P(O)ON(R⁴⁰)₂

each R⁴⁰ is, independently, hydrogen, C_1-8 alkyl, a protecting group or both R⁴⁰ together with the nitrogen atom to which they are attached form a moiety having one of the formulas:

 

or both R⁴⁰ together are a protecting group, or each R⁴⁰ is, independently, branched or unbranched C_1-12 alkyl, substituted branched or unbranched C_1-12 alkyl, with substituents including one or more —

O—

, —

C(O)—

, —

OC(O)—

, —

C(O)O—

, —

OR⁴², —

SR⁴³, —

C(O)NR³⁹—

, —

C(O)N(R⁴¹)₂, —

NR⁴¹—

, —

N(R⁴¹)₂, halogen, —

CN or —

NO₂;

each R⁴¹ is, independently, hydrogen, a protecting group or both R⁴¹ together are a protecting group or C_1-4 alkyl;

R⁴² is hydrogen or a protecting group; and

R⁴³ is —

H, a counter ion or a moiety having the formula

R⁴⁶ is C_1-8 alkyl.