Pro-substrates for live cell applications

1. A compound of formula (I), or a salt thereof,

wherein,

R A is selected from the group consisting of —(CR 1 R 2 ) m —N(R 3 )C(O)R 4 , —(CR 5 R 6 ) m —SO 3 R 7 , and —(CR 10 R 11 ) m —CO 2 R 12 , wherein

m at each occurrence is independently 2, 3, or 4;

R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 10 , R 11 , and R 12 at each occurrence are independently hydrogen or C 1 -C 4 alkyl;

R 4 is C 1 -C 4 alkyl, C 1 -C 4 alkoxy, carboxy-C 1 -C 4 alkoxy-C 1 -C 4 alkyl, phenyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of halogen, C 1 -C 4 alkoxy, and C 1 -C 4 haloakyl,

 wherein

R 41 , R 42 , and R 44 at each occurrence are each independently carboxy-C 1 -C 4 alkyl;

R 43 and R 45 are each independently selected from the group consisting of hydrogen, —C(O)alkyl, and

x and y are each independently an integer selected from 1 to 20;

R B is —(CR 46 R 47 ) t —NR 48 R 49 , wherein

t is 1, 2, 3, 4, 5, 6, 7, or 8

R 46 and R 47 at each occurrence are independently hydrogen or C 1 -C 4 alkyl;

R 48 and R 49 at each occurrence are independently hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aminoalkyl, alkylaminoalkyl, di(alkyl)aminoalkyl, cyanoalkyl, —(CR 50 R 51 ) z —OC(O)R 52 ; or R 48 and R 49 together with the nitrogen atom to which they are attached form a heterocyclyl, wherein said heterocyclyl is unsubstituted or substituted with 1, 2, 3, 4, 5 or 6 substituents independently selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 4 alkenyl, C 1 -C 4 alkynyl, —NO 2 , —CN, halogen, oxo, —OR 96 , —OC(O)R 97 , and —C(O)R 116 , wherein

R 50 and R 51 at each occurrence are independently hydrogen or C 1 -C 4 alkyl;

R 52 , R 96 , R 97 , and R 116 at each occurrence are independently hydrogen, C 1 -C 6 alkyl, or aryl;

z is 1, 2, 3, or 4;

R C is —(CH) 0-3 -T or C 1-5 alkyl; wherein T is aryl, heteroaryl, or cycloalkyl, wherein said aryl, heteroaryl, and cycloalkyl are each independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aminoalkyl, alkylaminoalkyl, di(alkylaminoalkyl), cyanoalkyl, alkoxy, haloalkoxy, cyano, hydroxy, amino, alkylamino, and di(alkyl)amino;

R D is hydrogen, C 1 -C 4 alkyl, benzyl, or C 3 -C 6 cycloalkyl;

R E is hydrogen, hydroxy, alkoxy, amino, alkylamino, di(alkyl)amino, —OC(O)alkyl, or —OCH 2 OC(O)alkyl;

wherein the dash bonds together indicate the presence of an optional 6-membered ring in the compound of formula (I), wherein the optional ring is saturated or unsaturated; and

wherein the optional 6-membered ring is absent.

2. The compound of claim 1 , or a salt thereof, wherein R A is —(CR 1 R 2 ) m —N(R 3 )C(O)R 4 , wherein

m, R 1 , R 2 , R 3 , and R 4 are as defined in claim 1 .

3. The compound of claim 1 , or a salt thereof, wherein R A is selected from the group consisting of:

4. The compound of claim 1 , or a salt thereof wherein R B is selected from the group consisting of:

5. The compound of claim 1 , or a salt thereof wherein R C is furylmethyl or benzyl, R D is benzyl, or R E is hydrogen.

6. The compound of claim 1 , having formula (I-iv), or a salt thereof,

wherein

R 200 is aryl or heteroaryl, each optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aminoalkyl, cyanoalkyl, alkoxy, haloalkoxy, cyano, hydroxy, and amino; and

R A , and R B are as defined in claim 1 .

7. The compound of claim 1 , having formula (I-x), or a salt thereof,

wherein

s is 1 or 2;

R 201 is aryl or heteroaryl, each optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aminoalkyl, cyanoalkyl, alkoxy, haloalkoxy, cyano, hydroxy, and amino; and

R A is as defined in claim 1 .

8. The compound of claim 2 , having formula (I-xvii), or a salt thereof,

wherein

q is 1 or 2;

s is 1 or 2.

9. The compound of claim 1 , or a salt thereof, selected from the group consisting of:

or a salt thereof.

10. The compound of claim 1 , or a salt thereof, wherein

R A is

wherein R 41 , R 42 , R 43 , R 44 , R 45 , x, and y are as defined in claim 1 ; and

R B is

11. A composition comprising a mixture of a compound of formula (I) according to claim 1 , or a salt thereof, and at least one compound of formula (II),

wherein R C , R D , and R E in formula (II) are as defined in claim 1 ; and wherein the dash bonds of formula (II) indicates an optional 6-membered ring, which is absent.

12. The composition according to claim 11 , wherein the compound of formula (II) is selected from the group consisting of (II-i), (II-ii), (II-iii), (II-iv), and (II-v), or a combination thereof,

13. A method to detect an interaction between a first protein and a second protein in a sample, the method comprising:

(a) contacting a sample with the compound of claim 1 , or a salt thereof, wherein the sample comprises:

(i) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first fragment of a luminescent enzyme and a first protein; and

(ii) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises:

a. a second fragment of the luminescent enzyme and a second protein; or

b. a fluorescent acceptor molecule and a second protein; and

(b) detecting luminescence in the sample if the second fusion protein comprises a second fragment of the luminescent enzyme and a second protein, wherein the detection of luminescence indicates an interaction between the first protein and the second protein, or detecting bioluminescence resonance energy transfer (BRET) in the sample if the second fusion protein comprises a fluorescent acceptor molecule and a second protein, indicating an interaction or close proximity of the bioluminescent donor and the fluorescence acceptor.

14. A bioluminescence resonance energy transfer (BRET) system comprising: a first fusion protein including a first target protein and a bioluminescence donor molecule, wherein the bioluminescence donor molecule is a luminescent enzyme; a second fusion protein including a second target protein and a fluorescent acceptor molecule; and the compound of claim 1 , or a salt thereof.

15. A kit comprising a compound of claim 1 , or a salt thereof.