5,5-disubstituted luciferins and their use in luciferase-based assays

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
• R1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR1a, —NR1bR1c, or —NR1xG10;
• R2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR2a, —NR2bR2c, —SR2d, —SO2R2e, —S(O)R2f, —P(O)OR2gR2h;
• R3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR3a, —NR3bR3c, —SR3d, —SO2R3e, —S(O)R3f, —P(O)OR3gR3h;
• R4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR4a, —NR4bR4c, —SR4d, —SO2R4e, —S(O)R4f, or —P(O)OR4gR4h;
• R5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR5a, —NR5bR5c, —SR5d, —SO2R5e, —S(O)R3f, —P(O)OR5gR5h;
• or R2 and R3 together with the atoms to which they are attached, R3 and R4 together with the atoms to which they are attached, or R4 and R5 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 SO2 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;
• R1a, R1b, R1c, R2a, R2b, R2c, R2d, R2e, R2f, R2g, R2h, R3a, R3b, R3c, R3d, R3e, R3f, R3g, R3h, 4a, R4b, R4c, R4d, R4e, R4f, R4g, R4h, R5a, R5b, R5c, R5d, R5e, R5f, R5g, and R5h, are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
• R1x, R2x, R3x, R4x, and R5x are each independently hydrogen or C1-C12alkyl;
• —NR1xG10 is
• wherein RAA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH2, —SH, —SCH3, phenyl, —COOH, —CO—NH2,
• and t is 1-10;
• and
• W1 and W2 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.

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
• R1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR1a, —NR1bR1c or —NR1x G10;
• R2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR2a, —NR2bR2c, —SR2d, —SO2R2e, —S(O)R2f, or —P(O)OR2gR2h;
• R3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR3a, —NR3bR3c, —SR3d, —SO2R3e, —S(O)R3f, or —P(O)OR3gR3h;
• R4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR4a, —NR4bR4c, —SR4d, —SO2R4e, —S(O)R4f, or —P(O)OR4gR4h;
• R5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR5a, —NR5bR5c, —SR5d, —SO2R5e, —S(O)R5f, or —P(O)OR5gR5h;
• or R2 and R3 together with the atoms to which they are attached, R3 and R4 together with the atoms to which they are attached, or R4 and R5 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 SO2 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;
• R1a, R1b, R1c, R2a, R2b, R2c, R2d, R2e, R2f, R2g, R2h, R3a, R3b, R3c, R3d, R3e, R3f, R3g, R3h, R4a, R4b, R4c, R4d, R4e, R4f, R4g, R4h, R5a, R5b, R5c, R5d, R5e, R5f, R5g, and R5h, are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
• R1x, R2x, R3x, R4x, and R5x are each independently hydrogen or C1-C12alkyl;
• —NR1xG10
• wherein RAA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH2, —SH, —SCH3, phenyl, —COOH, —CO—NH2,
• and t is 1-10;
• and
• W1 and W2 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.

A kit comprising a compound of formula (I′):

• or a tautomer or a salt thereof, wherein
• R1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR1a, —NR1bR1c, or —NR1xG10;
• R2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR2a, —NR2bR2c, —SR2d—, —SO2R2e, —S(O)R2f, or —P(O)OR2gR2h;
• R3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR3a, —NR3bR3c, —SR3d, —SO2R3e, —S(O)R3f, or —P(O)OR3gR3h;
• R4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR4a, —NR4bR4c, —SR4d, —S(O)2R4e, —S(O)R4f, or —P(O)OR4gR4h;
• R5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR5a, —NR5bR5c, —SR5d, —SO2R5e, —S(O)R5f, or —P(O)OR5gR5h;
• or R2 and R3 together with the atoms to which they are attached, R3 and R4 together with the atoms to which they are attached, or R4 and R5 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 SO2 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;
• R1a, R1b, R1c, R2a, R2b, R2c, R2d, R2e, R2f, R2g, R2h, R3a, R3b, R3c, R3d, R3e, R3f, R3g, R3h, R4a, R4b, R4c, R4d, R4e, R4f, R4g, R4h, R5a, R5b, R5c, R5d, R5e, R5f, R5g, and R5h, are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
• R1x, R2x, R3x, R4x, and R5x are each independently hydrogen or C1-C12alkyl;
• —NR1xG10
• wherein RAA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of —OH, —NH2, —SH, —SCH3, phenyl, —COOH, —CO—NH2,
• and t is 1-10;
• and
• W1 and W2 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.