Coelenterazine derivatives and methods of using same
View Patent ↗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:
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.