5,5-disubstituted luciferins and their use in luciferase-based assays
View Patent ↗Described are luciferin analogs, methods for making the analogs, kits including the analogs, and methods of using the compounds for the detection of luminescence in luciferase-based assays.
1. 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 , —OG 1 , —NR 1x G 1 , 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 2c , —S(O)R 2f , —P(O)OR 2g R 2h , —OG 1 , or —NR 2x G 1 ;
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 , —OG 1 , or —NR 3x G 1 ;
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 , —P(O)OR 4g R 4h , —OG 1 , or —NR 4x G 1 ;
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 , —P(O)OR 5g R 5h , —OG 1 , or —NR 5x G 1 ;
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, or any combination thereof, 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, acyl, —OG 1 , —NHG 1 , and —N(C 1 -C 10 alkyl)G 1 ;
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;
G 1 comprises a substrate of a first enzyme, wherein biotransformation of the substrate by the first enzyme converts G 1 to H;
—NR 1x G 10 is a group that is cleavable by a second enzyme to convert the —NR 1x G 10 group to —OH; and
W 1 and W 2 together with the carbon to which they are attached form a cycloalkyl, cycloalkenyl, or heterocycle;
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.
2. The compound of claim 1 , which 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 4a , or —NR 4b 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 2 , —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 4 , —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, or any combination thereof, 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;
and
W 1 and W 2 together with the carbon to which they are attached form a cycloalkyl, cycloalkenyl, or heterocycle;
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 compound of claim 2 , or a tautomer or a salt thereof, wherein R 1 is —OH.
4. The compound of claim 2 , or a tautomer or a salt thereof, 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 compound of claim 2 , or a tautomer or a salt thereof, wherein R 4 is —OR 4a , or —NR 4b R 4c .
6. The compound of claim 2 , or a tautomer or a salt thereof, 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 compound of claim 2 , or a tautomer or a salt thereof, 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 compound of claim 2 , or a tautomer or a salt thereof, 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 compound of claim 2 , or a tautomer or a salt thereof, wherein W 1 and W 2 together with the carbon to which they are attached form a cycloalkyl.
10. The compound of claim 2 , which 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 A 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 Q2 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 compound of claim 10 , or a tautomer or a salt thereof, which is a compound of formula (I-b) or (I-c):
wherein
m is 0, 1, 2, or 3; and
p is 0, 1, or 2.
12. The compound of claim 10 , or a tautomer or a salt thereof, which is a compound of formula (I-d):
wherein
n is 0, 1, 2, or 3; and
p is 0, 1, or 2.
13. The compound of claim 10 , or a tautomer or a salt thereof, which 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 compound of claim 10 , or a tautomer or a salt thereof, which is a compound of formula (I-f):
wherein
n is 0, 1, 2, 3, 4, 5, 6, 7, or 8.
15. The compound of claim 10 , or a tautomer or a salt thereof, which is a compound of formula (I-g):
wherein
each of which is optionally substituted.
16. The compound of claim 2 , selected from the group consisting of:
5-(6-hydroxybenzo[d]thiazol-2-yl)-4-thia-6-azaspiro[2.4]hept-5-ene-7-carboxylic acid;
6-(6-hydroxybenzo[d]thiazol-2-yl)-5-thia-7-azaspiro[3.4]oct-6-ene-8-carboxylic acid;
2-(6-hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.4]non-2-ene-4-carboxylic acid;
2-(6-hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.5]dec-2-ene-4-carboxylic acid;
2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.5]dec-2-ene-4-carboxylic acid;
2-(6-hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.6]undec-2-ene-4-carboxylic acid;
2-(6-hydroxybenzo[d]thiazol-2-yl)-8-methyl-1-thia-3-azaspiro[4.5]dec-2-ene-4-carboxylic
2-(6-hydroxybenzo[d]thiazol-2-yl)-8-methyl-1-thia-3,8-diazaspiro[4.5]dec-2-ene-4-carboxylic acid;
2-(6-hydroxybenzo[d]thiazol-2-yl)-8-oxa-1-thia-3-azaspiro[4.5]dec-2-ene-4-carboxylic acid, or a tautomer or a salt thereof.
17. The compound of claim 1 , which is a compound of formula (II),
or a tautomer or a salt thereof, wherein R 4 is —OG 1 , —NHG 1 , or —N(C 1 -C 12 alkyl)G 1 .
18. The compound of claim 17 , which is a compound of formula (II-a),
or a tautomer or a salt thereof, wherein G 2 is a peptide.
19. The compound of claim 18 , or a tautomer or a salt thereof, wherein G 2 is a substrate for a non-luciferase enzyme.
20. A kit comprising a 5,5-disubstituted luciferin analogue, wherein the 5,5-disubstituted luciferin analogue is a compound according to claim 1 , or a tautomer or a salt thereof.
21. A kit comprising a 5,5-disubstituted luciferin analogue, wherein the 5,5-disubstituted luciferin analogue is a compound according to claim 2 , or a tautomer or a salt thereof.
22. The kit of claim 20 , further comprising a luciferase.
23. The kit of claim 20 , further comprising a buffer reagent.
24. A method for detecting luminescence in a sample, the method comprising
contacting a sample with a 5,5-disubstituted luciferin analogue, wherein the 5,5-disubstituted luciferin analogue is a compound according to claim 1 , or a tautomer or a salt thereof;
contacting the sample with a luciferin-utilizing luciferase, if it is not present in the sample; and
detecting luminescence.
25. A method for detecting luminescence in a sample, the method comprising
contacting a sample with a 5,5-disubstituted luciferin analogue, wherein the 5,5-disubstituted luciferin analogue is a compound according to claim 2 , or a tautomer or a salt thereof;
contacting the sample with a luciferin-utilizing luciferase, if it is not present in the sample; and
detecting luminescence.
26. The method of claim 24 , wherein the sample contains live cells.
27. The method of claim 24 , wherein the sample contains a luciferin-utilizing luciferase.
28. A method for detecting luminescence in a transgenic animal comprising
administering a 5,5-disubstituted luciferin analogue to a transgenic animal, wherein the 5,5-disubstituted luciferin analogue is a compound according to claim 1 , or a tautomer or a salt thereof; and
detecting luminescence;
wherein the transgenic animal expresses a luciferin-utilizing luciferase.
29. A method for detecting luminescence in a transgenic animal comprising
administering a 5,5-disubstituted luciferin analogue to a transgenic animal, wherein the 5,5-disubstituted luciferin analogue is a compound according to claim 2 , or a tautomer or a salt thereof; and
detecting luminescence;
wherein the transgenic animal expresses a luciferin-utilizing luciferase.
30. A method of detecting ATP in a sample comprising: (a) adding to the sample a reagent composition comprising a compound of claim 1 , or a tautomer or a salt thereof; and (b) detecting luminescence.