Cell impermeable coelenterazine analogues

1. A compound of formula (I)

or a tautomer, or a pharmaceutically acceptable salt thereof, wherein

X and Y are independently absent, amino, COOR 2 , —SO 2 —OR 3 , —PO(OR 4 )(OR 5 ), or —O—(CR 1a R 1b ) m —Z;

R 1 is aryl, heteroaryl, heterocycle, or cycloalkyl, wherein the aryl, heteroaryl, heterocycle, or cycloalkyl are each substituted with Q-L-Z and are each optionally further substituted;

Z at each occurrence is independently —COOR 2 , —SO 2 —OR 3 , —PO(OR 4 ) (OR 5 ), halogen, —NR 6 R 7 , or —NR 8 —CO—R 9 ;

Q is —O—, —NR Q —, —NR Q —CO—, —CO—NR Q —, —O—CO—NR Q —, or —NR Q —CO—O—;

L is —(CR 1a R 1b ) m — or —(CR 1x R 1y —CR 1x R 1y —O) t1 —(CR 1x R 1y ) t2 -Q 1 -, wherein Q 1 is absent, —O—, or —NR Q1 —;

R 2 , R 3 , R 4 , R 5 , R 8 , and R 9 at each occurrence are independently hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycle;

R 6 and R 7 at each occurrence are independently hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycle; or R 6 and R 7 , together with the nitrogen atom to which they are attached, together form an optionally substituted ring;

R 1a , R 1b , R Q , R Q1 , R 1x , and R 1y at each occurrence are independently hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl;

q is 0, 1, or 2;

m at each occurrence is independently 1-12;

t1 is 1-10; and

t2 is 0-5.

2. The compound of claim 1 , wherein

Q is —O—, —NH—, —NH—CO—, —CO—NH—, —O—CO—NH—, or —NH—CO—O—; and

L is —(CR 1a R 1b ) m —.

3. The compound of claim 1 , wherein

Q is —O—; and

L is —(CR 1a R 1b ) m —.

4. The compound of claim 1 , wherein

L is —(CR 1x R 1y —CR 1x R 1y —O) t1 —(CR 1x R 1y ) t2 —Q 1 —.

5. The compound of claim 1 , wherein R 1 is phenyl or furyl optionally further substituted with 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arylsulfonyl, aminosulfonyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl.

6. The compound of claim 1 , wherein R 1 is phenyl or furyl substituted with -Q-L-Z, in which Q is —O— and L is —(CR 1a R 1b ) m —.

7. The compound of claim 1 , wherein q is 1.

8. The compound of claim 1 , having formula (I-a):

or a tautomer, or a pharmaceutically acceptable salt thereof, wherein

R 1c is selected from the group consisting of alkyl, halogen, cyano, nitro, haloalkyl, hydroxy, hydroxyalkyl, amino, and —COOH;

n is 0, 1, 2, 3, or 4; and

X, Y, R 1a , R 1b , m, and Z are as defined in claim 1 .

9. The compound of claim 8 , wherein at least one of X and Y is absent.

10. The compound of claim 8 , wherein R k is halogen.

11. The compound of claim 8 , wherein m is 3, 4, 5, 6, 7, 8, 9, or 10.

12. The compound of claim 8 , wherein Z is —COOR 2 , —SO 2 —OR 3 , or —PO (OR 4 )(OR 5 ), or pharmaceutically acceptable salt thereof.

13. The compound of claim 8 , wherein Z is —NR 6 R 7 or —NR 8 —CO—R 9 , or pharmaceutically acceptable salt thereof, and wherein

R 9 is —(CR 9a R 9b —NH—CO) u —R 10 ;

R 9a and R 9b at each occurrence are independently hydrogen or C 1 -C 4 alkyl optionally substituted with —COOR 9c ;

R 9c at each occurrence is independently hydrogen or C 1 -C 4 alkyl;

R 10 is optionally substituted C 1 -C 8 alkyl; and

u is 0-10.

14. The compound of claim 13 , wherein Z is —NR 8 —CO—R 9 , or pharmaceutically acceptable salt thereof, and wherein

R 8 is hydrogen; and

R 9 is

15. A compound having formula (I-b):

or a tautomer, or a pharmaceutically acceptable salt thereof, wherein

R 1d is selected from the group consisting of alkyl, halogen, cyano, haloalkyl, hydroxyalkyl, and —COOH;

v is 0, 1, 2, or 3;

X and Y are each independently absent, —COOR 2 , —SO 2 —OR 3 , —PO(OR 4 )(OR 5 ), or —O—(CR 1a R 1b ) m —Z;

at least one of X and Y is present;

Z at each occurrence is independently —COOR 2 , —SO 2 —OR 3 , —PO(OR 4 ) (OR 5 ), halogen, —NR 6 R 7 , or —NR 8 —CO—R 9 ;

R 2 , R 3 , R 4 , R 5 , R 8 , and R 9 at each occurrence are independently hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycle;

R 6 and R 7 at each occurrence are independently hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycle; or R 6 and R 7 , together with the nitrogen atom to which they are attached, together form an optionally substituted ring;

R 1a and R 1b at each occurrence are independently hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl; and

m at each occurrence is independently 1-12.

16. The compound of claim 15 , wherein

X is —COOR 2 , —SO 2 —OR 3 , —PO(OR 4 ) (OR 5 ), or —O—(CR 1a R 1b ) m —Z; and

Y is absent.

17. The compound of claim 15 , wherein

X is absent; and

Y is —COOR 2 , —SO 2 —OR 3 , or —PO(OR 4 ) (OR 5 ), or —O—(CR 1a R 1b ) m —Z.

18. A compound selected from the group consisting of:

2,2,2-trichloroethyl 6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)hexane-1-sulfonate;

tert-butyl 6-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)hexanoate;

6-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)hexanoic acid;

8-benzyl-2-(3-((6-bromohexyl)oxy)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3 (7H)-one;

sodium 6-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)hexane-1-sulfonate;

tert-butyl 4-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)butanoate;

sodium 3-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy) propane-1-sulfonate;

4-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)butanoic acid;

tert-butyl 8-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)octanoate;

8-benzyl-2-(4-((6-bromohexyl)oxy)-3-chlorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;

8-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)octanoic acid;

tert-butyl 6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)hexanoate;

6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)hexanoic acid;

sodium 6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)hexane-1-sulfonate;

(S)-3-acetamido-4-(((S)-1-(((S)-1-((6-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenoxy)hexyl)amino)-3-carboxy-1-oxopropan-2-yl)amino)-3-carboxy-1-oxopropan-2-yl)amino)-4-oxobutanoic acid;

sodium 8-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)octane-1-sulfonate;

sodium 10-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)decane-1-sulfonate;

sodium 6-(5-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)hexane-1-sulfonate;

sodium 6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-fluorophenoxy)hexane-1-sulfonate;

sodium 6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2,6-difluorophenoxy)hexane-1-sulfonate;

2-(4-((6-aminohexyl)oxy)-3-fluorobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;

6-(4-((8-benzyl-6-(3-hydroxyphenyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-chlorophenoxy)hexane-1-sulfonic acid;

(S)-3-acetamido-4-(((S)-1-(((S)-1-((6-(4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-2-fluorophenoxy) hexyl)amino)-3-carboxy-1-oxopropan-2-yl)amino)-3-carboxy-1-oxopropan-2-yl)amino)-4-oxobutanoic acid;

tert-butyl 3-(8-benzyl-2-(furan-2-ylmethyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-6-yl)benzoate;

3-(8-benzyl-2-(furan-2-ylmethyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-6-yl)benzoic acid;

4-(8-benzyl-2-(furan-2-ylmethyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-6-yl)benzoic acid; and

6-(4-((6-aminohexyl)oxy)phenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one.

19. A kit comprising a compound of claim 1 .

20. A bioluminescence resonance energy transfer (BRET) system comprising a compound according to claim 1 .

21. A method of detecting an enzyme in a sample, the method comprising,

(a) contacting the sample with a compound according to claim 1 ; and

(b) detecting luminescence in the sample.

22. A method for detecting luminescence in a sample, the method comprising,

(a) contacting a sample with a compound of claim 1 ;

(b) contacting the sample with a coelenterazine-utilizing luciferase, if it is not present in the sample; and

(c) detecting luminescence in the sample.

23. A method for detecting cell death in a sample, the method comprising:

(a) contacting a sample with a compound that induces cell death;

(b) contacting the sample with a compound according to claim 1 ; and

(c) detecting luminescence in the sample, wherein the sample comprises cells expressing a coelenterazine-utilizing luciferase.

24. A method for detecting secretion of a bioluminescent enzyme in a sample, the method comprising

(a) contacting the sample with a compound of claim 1 ; and

(b) detecting luminescence in the sample, wherein the sample comprises cells expressing a secretable bioluminescent enzyme.

25. A method for detecting luminescence in a transgenic animal comprising

(a) administering a compound of claim 1 to a transgenic animal; and

(b) detecting luminescence;

wherein the transgenic animal expresses a coelenterazine-utilizing luciferase.