Coelenterazine derivatives and methods of using same

Patent metadata

Field	Value
Publication number	US-9487520-B2
Application number	US-201113287519-A
Country	US
Kind code	B2
Filing date	Nov 2, 2011
Priority date	Nov 2, 2010
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

The invention provides coelenterazine derivatives which are substrates for a non-luminescent enzyme and a pro-substrate for a luminescent enzyme. The invention also provides a method of using the derivatives. The derivatives are of Formula II:

First claim

The invention claimed is:

1. A compound of formula (II):

wherein R 2 is —(CH 2 ) n -T or C 1-5 alkyl;
R 6 is selected from the group consisting of —H, —OH, —NH 2 , —OC(O)R or —OCH 2 OC(O)R;
R 8 is selected from the group consisting of

 H or lower cycloalkyl;
R 11 is selected from the group consisting of a peptide containing from 2 to 35 amino acids, an amino acid, —O—R A , —OC(O)O—R A , —N(R B ) 2 , or —NHC(O)OR A ;
wherein R 3 and R 4 are both H or both C 1-2 alkyl;
R A is C 1-4 alkyl, substituted C 1-4 alkyl, —CH 2 —R c or —CH 2 —V—R c ;
each R B is independently —H or —R A ;
R c is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L′ is a direct bond or a linker, wherein the linker is selected from the group consisting of

 wherein each R E is independently H, halogen or NO 2 , each R D is independently H or Me, and each X is independently NH, NMe, O or S;
n is 0 to 3;
each R is independently a C 1-7 alkyl;
T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl; and
V is —S— or —O—.

2. A compound according to claim 1 , wherein R 2 is

or C 2-5
straight-chain alkyl;
each X is independently —S—, —O—or —NH—;
Z is —CH— or —N—;
Y is —H or —OH;
W is —NH 2 , halo, —OH, —NHC(O)R, —CO 2 R; and
R is C 1-7 alkyl.

3. A compound according to claim 1 , wherein R 2 is

and X is O or S.

4. A compound according to claim 1 , wherein R 2 is C 2-5 straight-chain alkyl.

5. A compound according to claim 1 , wherein R 8 is

lower cycloalkyl or H, wherein R 3 and R 4 are both H or C 1-2 alkyl.

6. A compound according to claim 1 , wherein R 8 is benzyl.

7. A compound according to claim 1 , wherein V is S.

8. A method of detecting the presence or amount of an enzyme comprising:
contacting a sample suspected of containing the enzyme with a compound according to claim 1 ; and
detecting luminescence of the sample.

9. The method of claim 8 , wherein the luminescence is quantified.

10. A method of detecting the presence of an enzyme in vivo comprising:
administering a compound according claim 1 to a transgenic animal; and
detecting luminescence.

11. A method of detecting the presence of an enzyme comprising:
administering a compound according to claim 1 to an animal;
obtaining a sample from the animal; and
detecting luminescence of the sample.

12. A method of detecting a second enzyme in a sample suspected of containing more than one enzyme comprising
contacting a sample suspected of containing more than one enzyme with a first compound according to claim 1 ;
contacting the sample with a second compound according to claim 1 ; and
detecting the luminescence of the sample; and
wherein the first compound contains a substrate for a first enzyme and the second compound contains a substrate for a second enzyme.

13. A compound selected from the group consisting of:

14. A compound of formula (II):

wherein R 2 is —(CH 2 ) n -T or C 1-5 alkyl;
R 6 is selected from the group consisting of —H, —OH, —NH 2 , —OC(O)R or —OCH 2 OC(O)R;
R 8 is

R 11 is selected from the group consisting of a peptide having from 2 to 35 amino acids, an amino acid, a saccharide, —O—R A , —OC(O)O—R A , —N(R B ) 2 , or —NHC(O)OR A ;
R A is C 1-4 alkyl, substituted C 1-4 alkyl, —CH 2 —R c or —CH 2 —V—R c ;
each R B is independently —H or —R A ;
R c is aryl, heteroaryl, substituted aryl or substituted heteroaryl;
L′ is a direct bond or a linker, wherein the linker is selected from the group consisting of

 wherein each R E is independently H, halogen or NO 2 , each R D is independently H or Me, and each X is independently NH, NMe, O or S;
n is 0 to 3;
each R is independently a C 1-7 alkyl;
T is heteroaryl, substituted heteroaryl or cycloalkyl; and
V is —S— or —O—.

15. A compound according to claim 14 , wherein R 2 is

or C 2-5 straight-chain alkyl;
each X is independently —S—, —O— or —NH—;
Z is —CH— or —N—; and
R is C 1-7 alkyl.

16. A compound according to claim 14 , wherein R 2 is

and
X is O or S.

17. A method of detecting the presence or amount of an enzyme comprising:
contacting a sample suspected of containing the enzyme with a compound according to claim 14 ; and
detecting luminescence of the sample.

18. The method of claim 17 , wherein the luminescence is quantified.

19. A method of detecting the presence of an enzyme in vivo comprising:
administering a compound according claim 14 to a transgenic animal; and
detecting luminescence.

20. A method of detecting the presence of an enzyme comprising:
administering a compound according to claim 14 to an animal;
obtaining a sample from the animal; and
detecting luminescence of the sample.

21. A method of detecting a second enzyme in a sample suspected of containing more than one enzyme comprising contacting a sample suspected of containing more than one enzyme with a first compound according to claim 14 ;
contacting the sample with a second compound according to claim 14 ; and
detecting the luminescence of the sample; and
wherein the first compound contains a substrate for a first enzyme and the second compound contains a substrate for a second enzyme.

Assignees

• Klaubert Dieter H
• Meisenheimer Poncho
• Unch James
• Zhou Wenhui
• Promega Corp

Inventors

• Klaubert Dieter H
• Meisenheimer Poncho
• Unch James
• Zhou Wenhui

Classifications

• C07D487/04 Primary

  Ortho-condensed systems · CPC title

• C07H17/02

  Heterocyclic radicals containing only nitrogen as ring hetero atoms · CPC title

• C12Q1/66

  involving luciferase · CPC title