Pro-substrates for live cell applications

Patent metadata

Field	Value
Publication number	US-9927430-B2
Application number	US-201514608910-A
Country	US
Kind code	B2
Filing date	Jan 29, 2015
Priority date	Jan 29, 2014
Publication date	Mar 27, 2018
Grant date	Mar 27, 2018

Abstract

Provided are pro-substrates useful in assays of living cells. The pro-substrates can be used to detect the presence or absence of enzymes, such as luciferase, in living cells. The pro-substrates can be coelenterazine derivatives or analogs.

First claim

What is claimed is:

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 fusio

Assignees

• Promega Corp

Inventors

• Zhou Wenhui
• Binkowski Brock
• Wang Hui
• Butler Braeden
• Machleidt Thomas
• Wood Keith

Classifications

• C07D487/04

  Ortho-condensed systems · CPC title

• C07K5/0819

  with the first amino acid being acidic · CPC title

• G01N33/542 Primary

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C12Q1/66

  involving luciferase · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title