Compounds and methods for assaying redox state of metabolically active cells and methods for measuring NAD(P)/NAD(P)H

1. A method for detecting cellular metabolites in a sample comprising,

a. contacting the sample with a compound of formula (II), (III), or (IV), a dehydrogenase amplification enzyme system, NAD or NADP, a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate, and a luciferase reaction mixture; and

b. detecting bioluminescence;

wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;

the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;

the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;

wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;

R 9 and R 10 are independently selected from C 1-4 alkyl;

R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;

X is O, NH or a direct bond;

L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;

wherein at least one of X or L is not a direct bond when the compound has formula (II);

R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;

R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;

n is an integer from 0-2;

Y is O or NR 15 ; and

R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

2. The method of claim 1 , wherein the compound, dehydrogenase amplification enzyme, diaphorase, NAD or NADP and luciferase reaction mixture are in a single composition.

3. A method of detecting NADH or NADPH in a sample comprising:

a. contacting the sample with a compound of formula (II), (III), or (IV), a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate and a luciferase reaction mixture; and

b. detecting bioluminescence;

wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;

the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;

the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;

wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;

R 9 and R 10 are independently selected from C 1-4 alkyl;

R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;

X is O, NH or a direct bond;

L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;

wherein at least one of X or L is not a direct bond when the compound has formula (II);

R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;

R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;

n is an integer from 0-2;

Y is O or NR 15 ; and

R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

4. The method of claim 3 , wherein the sample is further contacted with a diaphorase inhibitor.

5. The method of claim 3 , wherein the compound, diaphorase and luciferase reaction mixture are in a single composition.

6. A method of determining total NAD(P)/NAD(P)H in a sample comprising:

a. contacting the sample with a dehydrogenase amplification enzyme system, a compound of formula (II), (III) or (IV), a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate, and a luciferase reaction mixture; and

b. detecting bioluminescence;

wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;

the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;

the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;

wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;

R 9 and R 10 are independently selected from C 1-4 alkyl;

R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;

X is O, NH or a direct bond;

L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;

wherein at least one of X or L is not a direct bond when the compound has formula (II);

R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;

R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;

n is an integer from 0-2;

Y is O or NR 15 ; and

R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

7. The method of claim 6 , wherein the dehydrogenase amplification enzyme system comprises a dehydrogenase amplification enzyme and a substrate for the dehydrogenase amplification enzyme.

8. The method of claim 6 , wherein the dehydrogenase amplification enzyme system, diaphorase, the compound, and luciferase reaction mixture are in a single composition.

9. A method of determining total NAD(P)/NAD(P)H in a sample comprising:

a. contacting the sample with a dehydrogenase amplification enzyme system;

b. adding to the reaction of a) a compound of formula (II), (III) or (IV), and a diaphorase that utilizes NADH or NADPH as a cofactor, wherein the compound of formula (II), (III), or (IV) as a diaphorase substrate, and a luciferase reaction mixture; and

c. detecting bioluminescence;

wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;

the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;

the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;

wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;

R 9 and R 10 are independently selected from C 1-4 alkyl;

R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;

X is O, NH or a direct bond;

L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;

wherein at least one of X or L is not a direct bond when the compound has formula (II);

R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;

R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;

n is an integer from 0-2;

Y is O or NR 15 ; and

R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

10. The method of claim 9 , wherein the dehydrogenase amplification enzyme system comprises a dehydrogenase amplification enzyme and a substrate for the dehydrogenase amplification enzyme.

11. A method for detecting the presence or absence of or determining the amount of NAD or NADH or NADP or NADPH or any combination thereof in cells, comprising:

a. contacting the cells with a cell lysis reagent and luciferase reaction mixture, wherein the luciferase reaction mixture also contains a dehydrogenase amplification enzyme system, a diaphorase and a compound of formula (II), (III), or (IV); and

b. detecting bioluminescence thereby detecting the presence or absence of or determining the amount of NAD or NADH or NADP or NADPH or any combination thereof;

wherein the compound of formula (II) is

wherein R 1 is H, C 1-4 alkyl, C 1-4 hydroxylalkyl, C 3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH 2 ) n′ —P(Ph) 3 ; R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n′ is an integer from 2-7;

the compound of formula (III) is

wherein R 4 and R 5 are independently selected from H, halogen, methyl, and trifluoromethyl;

the compound of formula (IV) is

wherein R 2 is —CH 2 -aryl or —CH 2 -heteroaryl; R 5 is —CH 2 -aryl or —CH 2 -heteroaryl; and R 7 is aryl or heteroaryl;

wherein R 14 is H, C 1-4 alkyl, C 1-4 alkoxyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide;

R 9 and R 10 are independently selected from C 1-4 alkyl;

R 11 , R 12 and R 13 are independently selected from H, C 1-4 alkyl, C 1-4 alkoxyl, bromo, chloro or amino, or R 11 and R 12 can form a fused phenyl ring;

X is O, NH or a direct bond;

L is a direct bond or —C 6 (R 16 ) 4 CH 2 — or —(CH 2 ) m C(R 17 ) 2 (CH 2 ) n —Y—C(O)—;

wherein at least one of X or L is not a direct bond when the compound has formula (II);

R 16 is independently H, halogen, CH 3 , OCH 3 , or NO 2 ;

R 17 is independently H, C 1-4 alkyl or both R 17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;

n is an integer from 0-2;

Y is O or NR 15 ; and

R 15 is H, C 1-4 alkyl, C 2-4 hydroxylalkyl, C 2-4 alkoxyl, C 2-4 carboxylic acid, or C 2-4 amide.

12. The method of claim 11 , wherein the cell lysis reagent contains a detergent.

13. The method of claim 11 , wherein the dehydrogenase amplification enzyme system comprises a dehydrogenase amplification enzyme and a substrate for the dehydrogenase amplification enzyme.