5,5-disubstituted luciferins and their use in luciferase-based assays
Described are luciferin analogues, methods for making the analogues, kits including the analogues, and methods of using the compounds for the detection of luminescence in luciferase-based assays.
1. A method for detecting luminescence in a sample, the method comprising contacting the sample with a compound of formula (I′):
or a tautomer or a salt thereof, wherein
R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 1a , —NR 1b R 1c , or —NR 1x G 10 ;
R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 2a , —NR 2b R 2c , —SR 2d , —SO 2 R 2e , —S(O)R 2f , or —P(O)OR 2g R 2h ;
R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 3a , —NR 3b R 3c , —SR 3d , —SO 2 R 3e , —S(O)R 3f , or —P(O)OR 3g R 3h ;
R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 4a , —NR 4b R 4c , —SR 4d , —SO 2 R 4e , —S(O)R 4f , or —P(O)OR 4g R 4h ;
R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 5a , —NR 5b R 5c , —SR 5d , —SO 2 R 5e , —S(O)R 5f , or —P(O)OR 5g R 5h ;
or R 2 and R 3 together with the atoms to which they are attached, R 3 and R 4 together with the atoms to which they are attached, or R 4 and R 5 together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO and SO 2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylanino, aminoakyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl;
R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , R 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g , and R 5h , are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
R 1x , R 2x , R 3x , R 4x , and R 5x are each independently hydrogen or C 1 -C 12 alkyl;
—NR 1x G 10
wherein R AA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH 2 , —SH, —SCH 3 , phenyl, —COOH, —CO—NH 2 ,
and t is 1-10; and
W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl;
contacting the sample with a luciferin-utilizing luciferase, if it is not present in the sample; and
detecting luminescence.
2. The method of claim 1 , wherein the compound is a compound of formula (I),
or a tautomer or a salt thereof, wherein
R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 1a , or —NR 1b R 1c ;
R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 2a , —NR 2b R 2c , —SR 2d , —SO 2 R 2e , —S(O)R 2f , or —P(O)OR 2g R 2h ;
R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 3a , —NR 3b R 3c , —SR 3d , —SO 2 R 3e , —S(O)R 3f , or —P(O)OR 3g R 3h ;
R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 4a , —NR 4b R 4c , —SR 4d , —SO 2 R 4e , —S(O)R 4f , or —P(O)OR 4g R 4h ;
R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 5a , —NR 5b R 5c , —SR 5d , —SO 2 R 5e , —S(O)R 5f , or —P(O)OR 5g R 5h ;
or R 2 and R 3 together with the atoms to which they are attached, R 3 and R 4 together with the atoms to which they are attached, or R 4 and R 5 together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteratoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO and SO 2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl;
R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , R 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4g , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g and R 5h , are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; and
W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl.
3. The method of claim 1 , wherein R 1 is —OH.
4. The method of claim 2 , wherein R 2 , R 3 , R 4 , and R 5 , are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, C 1 -C 4 -alkyl, C 2 -C 4 -alkenyl, C 2 -C 4 -alkynyl, —OH, and —NH 2 .
5. The method of claim 2 , wherein R 4 is —OR 4a , or —NR 4b R 4c .
6. The method of claim 2 , wherein R 3 and R 4 together with the atoms to which they are attached form a 5- or 6-membered optionally substituted saturated, partially unsaturated or fully unsaturated ring.
7. The method of claim 2 , wherein R 4 and R 5 together with the atoms to which they are attached form a 5- or 6-membered optionally substituted saturated, partially unsaturated or fully unsaturated ring.
8. The method of claim 2 , wherein R 3 and R 4 together with the atoms to which they are attached form a 6-membered optionally substituted heterocycle, and R 4 and R 5 together with the atoms to which they are attached form a 6-membered optionally substituted heterocycle, wherein the two 6-membered rings are fused.
9. The method of claim 2 , wherein W 1 and W 2 are each alkyl.
10. The method of claim 2 , wherein the compound of formula (I) is a compound of formula (I-a),
or a tautomer or a salt thereof, wherein
A and B are each independently an optional 5- or 6-membered ring selected from the group consisting of aryl, heteroaryl, and heterocycle; wherein A, when present, is optionally substituted with one or more R A ; B, when present, is optionally substituted with one or more R B ; and each of R and R B , where present, is independently alkyl, halo, haloalkyl, hydroxyalkyl, —OH, —NH 2 , or alkyl —NH—;
Q is —OR Q1 or —NR Q1 R Q1 when both A and B are absent, wherein R Q1 at each occurrence is independently hydrogen, alkyl, or hydroxyalkyl; or Q is C, CR Q2 , CR Q2 R Q2 , N, NR Q2 , or O when at least one of A and B is present, wherein R 2 at each occurrence is independently hydrogen, alkyl, or hydroxyalkyl;
R is alkyl, alkoxy, halo, haloalkyl, hydroxyalkyl, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; and
p is 0, 1, 2, or 3.
11. The method of claim 10 , wherein the compound is a compound of formula (I-b) or (I-c):
wherein
in is 0, 1, 2, or 3; and
p is 0, 1, or 2.
12. The method of claim 10 , wherein the compound is a compound of formula (I-d):
wherein
n is 0, 1, 2, or 3; and
p is 0, 1, or 2.
13. The method of claim 10 , wherein the compound is a compound of formula (I-e):
wherein
m is 0, 1, 2, or 3;
n is 0, 1, 2, or 3; and
p is 0, or 1.
14. The method of claim 10 , wherein the compound is a compound of formula (I-f):
wherein n is 0, 1, 2, 3, 4, 5, 6, 7, or 8.
15. The method of claim 10 , wherein the compound is a compound of formula (I-g):
wherein
which is optionally substituted.
16. The method of claim 2 , wherein the compound is selected from the group consisting of:
5,5-diethyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid;
(S)-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid;
(R)-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid;
(S)-2-(7-aminonaphtho[2,1-d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid;
(S)-5,5-dimethyl-2-(6-(pyrrolidin-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid;
(S)-2-(6,7-dihydro-5H-thiazolo[4,5-f]indol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid;
(S)-2-(6-((3-hydroxypropyl)amino)benzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid;
5,5-dibenzyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid;
5,5-diethyl-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; and
2-(6-hydroxybenzo[d]thiazol-2-yl)-5,5-dipropyl-4,5-dihydrothiazole-4-carboxylic acid;
or a tautomer or a salt thereof.
17. The method of claim 2 , wherein the sample contains live cells.
18. The method of claim 2 , wherein the sample contains a luciferin-utilizing luciferase.
19. A method for detecting luminescence in a transgenic animal comprising
administering to a transgenic animal a compound of formula (I′):
or a tautomer or a salt thereof, wherein
R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 1a , —NR 1b R 1c , or —NR 1x G 10 ;
R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 2a , —NR 2b R 2c , —SR 2d , —SO 2 R 2e , —S(O)R 2f , —P(O)OR 2g R 2h ;
R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 3a , —NR 3b R 3c , —SR 3d , —SO 2 R 3e , —S(O)R 3f , —P(O)OR 3g R 3h ;
R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 4a , —NR 4b R 4c , —SR 4d , —SO 2 R 4e , —S(O)R 4f , or —P(O)OR 4g R 4h ;
R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 5a , —NR 5b R 5c , —SR 5d , —SO 2 R 5e , —S(O)R 3f , —P(O)OR 5g R 5h ;
or R 2 and R 3 together with the atoms to which they are attached, R 3 and R 4 together with the atoms to which they are attached, or R 4 and R 5 together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of, N, S, NO, SO and SO 2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, or acyl;
R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4g , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g , and R 5h , are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
R 1x , R 2x , R 3x , R 4x , and R 5x are each independently hydrogen or C 1 -C 12 alkyl;
—NR 1x G 10 is
wherein R AA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH 2 , —SH, —SCH 3 , phenyl, —COOH, —CO—NH 2 ,
and t is 1-10; and
W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl; and
detecting luminescence;
wherein the transgenic animal expresses a luciferin-utilizing luciferase.
20. A method of detecting ATP in a sample comprising:
(a) adding to the sample a reagent composition comprising a compound of formula (I′):
or a tautomer or a salt thereof, wherein
R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 1a , —NR 1b R 1c or —NR 1x G 10 ;
R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 2a , —NR 2b R 2c , —SR 2d , —SO 2 R 2e , —S(O)R 2f , or —P(O)OR 2g R 2h ;
R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 3a , —NR 3b R 3c , —SR 3d , —SO 2 R 3e , —S(O)R 3f , or —P(O)OR 3g R 3h ;
R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 4a , —NR 4b R 4c , —SR 4d , —SO 2 R 4e , —S(O)R 4f , or —P(O)OR 4g R 4h ;
R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 5a , —NR 5b R 5c , —SR 5d , —SO 2 R 5e , —S(O)R 5f , or —P(O)OR 5g R 5h ;
or R 2 and R 3 together with the atoms to which they are attached, R 3 and R 4 together with the atoms to which they are attached, or R 4 and R 5 together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO and SO 2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl;
R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , R 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4g , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g , and R 5h , are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
R 1x , R 2x , R 3x , R 4x , and R 5x are each independently hydrogen or C 1 -C 12 alkyl;
—NR 1x G 10
wherein R AA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH 2 , —SH, —SCH 3 , phenyl, —COOH, —CO—NH 2 ,
and t is 1-10; and
W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl; and
(b) detecting luminescence.
21. A kit comprising a compound of formula (I′):
or a tautomer or a salt thereof, wherein
R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 1a , —NR 1b R 1c , or —NR 1x G 10 ;
R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 2a , —NR 2b R 2c , —SR 2d —, —SO 2 R 2e , —S(O)R 2f , or —P(O)OR 2g R 2h ;
R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 3a , —NR 3b R 3c , —SR 3d , —SO 2 R 3e , —S(O)R 3f , or —P(O)OR 3g R 3h ;
R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 4a , —NR 4b R 4c , —SR 4d , —S(O) 2 R 4e , —S(O)R 4f , or —P(O)OR 4g R 4h ;
R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR 5a , —NR 5b R 5c , —SR 5d , —SO 2 R 5e , —S(O)R 5f , or —P(O)OR 5g R 5h ;
or R 2 and R 3 together with the atoms to which they are attached, R 3 and R 4 together with the atoms to which they are attached, or R 4 and R 5 together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO and SO 2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl;
R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , R 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4g , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g , and R 5h , are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
R 1x , R 2x , R 3x , R 4x , and R 5x are each independently hydrogen or C 1 -C 12 alkyl;
—NR 1x G 10
wherein R AA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH 2 , —SH, —SCH 3 , phenyl, —COOH, —CO—NH 2 ,
and t is 1-10; and
W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl.
22. The kit of claim 21 , further comprising a luciferase.
23. The kit of claim 21 , further comprising a buffer reagent.