Serum stable pro-coelenterazine analogues

Patent metadata

Field	Value
Publication number	US-10836767-B2
Application number	US-201816023950-A
Country	US
Kind code	B2
Filing date	Jun 29, 2018
Priority date	Jul 6, 2017
Publication date	Nov 17, 2020
Grant date	Nov 17, 2020

Abstract

Described are pro-coelenterazine analogues, methods for making the analogues, kits comprising the analogues, and methods of using the compounds for the detection of luminescence in luciferase-based assays or fragment complementary luciferase. The disclosed pro-coelenterazine analogues provide increased serum stability for live cell assays, and are capable of tuning the brightness and assay windows as needed for the applications.

First claim

What is claimed is:

1. A compound of formula (I)

or a tautomer, or a salt thereof, wherein:
Ar 1 is phenyl, Ar 2 is furyl, and Ar 3 is phenyl;
R A is selected from the group consisting of C 2 -C 10 linear or branched alkyl, alkoxy, alkoxyalkyl, amido, acetoxy, methyl ether polyethylene glycoxy, methyl ether polyethylene glycoxyalkyl, haloalkyl, haloalkoxy, aryl, arylalkyl, cycloalkyl, hydroxyl alkyl, hydroxyl polyethylene glycoxyl, carboxyalkyl, heteroaryl, heteroarylalkyl, heterocycle, heterocyclic alkyl, R B CH 2 —, R C O—, R C C(O)NH—, and R C C(O)O—, wherein R B is selected from the group consisting of C 1 -C 9 , linear or branched alkyl, alkoxy, alkoxyalkyl, amido, acetoxy, methyl ether polyethylene glycoxy, methyl ether polyethylene glycoxyalkyl, haloalkyl, haloalkoxy, aryl, arylalkyl, cycloalkyl, hydroxyl alkyl, hydroxyl polyethylene glycoxyl, carboxyalkyl, heteroaryl, heteroarylalkyl, heterocycle, and heterocyclic alkyl; and R C is selected from the group consisting of C 1 -C 2 linear or branched alkyl, alkoxyalkyl, methyl ether poly ethylene glycoxy alkyl, haloalkyl, aryl, arylalkyl, cycloalkyl, hydroxyl alkyl, hydroxyl polyethylene glycoxy alkyl, carboxyalkyl, heteroaryl, heteroarylalkyl, heterocycle, and heterocyclic alkyl;
R x1 , R x2 , at each occurrence, are each independently selected from the group consisting of C 1 -C 6 linear or branched alkyl, optionally substituted by one or more substituents selected from the group consisting of alkoxy, aryl, cycloalkyl, heteroaryl, and heterocycle.

2. The compound of claim 1 , wherein R x1 and R x2 are methyl.

3. The compound of claim 1 , wherein: R A is R B CH 2 —, wherein R B is CH 3 (OCH 2 CH 2 ) n O—, wherein n is any number from 0-10; or wherein R A is R C O—, wherein R C is linear or branched C 1 —C 5 —alkyl, or CH 3 (OCH 2 CH 2 ) n —, wherein n is any number from 0-10; or wherein R A is R C C(O)NH—, wherein R C is linear or branched C 1 —C 5 —alkyl; or wherein R A is R C C(O)O—, wherein R C is linear or branched C 1 —C 5 —alkyl.

4. The compound of claim 1 , wherein R A —C(R x1 R x2 )— is formula:

5. The compound of claim 1 , wherein the compound of formula (I) has formula (I-d):

6. The compound of claim 1 , wherein the compound of formula (I) has formula (I-e):

7. The compound of claim 1 , wherein R A —C(R x1 R x2 )— is formula:

8. The compound of claim 1 , wherein the compound of formula (I) has formula (I-f):

9. The compound of claim 1 , wherein the compound of formula (I) has formula (I-g):

10. The compound of claim 1 , wherein the compound of formula (I) has formula (I-h):

11. The compound of claim 1 , wherein the compound of formula (I) has formula (I-j):

12. The compound of claim 1 , wherein the compound of formula (I) has formula (I-k):

13. A compound of formula (II)

or a tautomer, or a salt thereof, wherein, Ar 1 , Ar 2 , and Ar 3 are each independently selected from the group consisting of aryl and heteroaryl, wherein Ar 1 , Ar 2 , and Ar 3 are each optionally substituted; Ar 4 is aryl, furan or thiophene, optionally substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, aryl, cycloalkyl, heteroaryl, and heterocycle.

14. The compound of claim 13 , wherein Ar 1 is phenyl, Ar 2 is furyl, and Ar 3 is phenyl.

15. The compound of claim 13 , Ar 4 is phenyl, furan, thiophene, optionally substituted by one or more alkoxy.

16. The compound of claim 13 , wherein the compound of formula (II) has formula (II-a), (II-b), or (II-c)

wherein Ar 1 is selected from the group consisting of aryl and heteroaryl; Ar 2 is selected from the group consisting of aryl and heteroaryl; and Ar 4 is phenyl, furan, thiophene, optionally substituted by one or more alkoxy.

17. A compound of formula (III)

or a tautomer, or a salt thereof, wherein, Ar 1 , Ar 2 , and Ar 3 are each independently selected from the group consisting of aryl and heteroaryl, wherein Ar 1 , Ar 2 , and Ar 3 are each optionally substituted; R C is selected from the group consisting of C 1 -C 9 , linear or branched alkyl, alkoxyalkyl, methyl ether poly ethylene glycoxy alkyl, haloalkyl, aryl, arylalkyl, cycloalkyl, hydroxyl alkyl, hydroxyl polyethylene glycoxy alkyl, carboxyalkyl, heteroaryl, heteroarylalkyl, heterocycle, and heterocyclic alkyl.

18. The compound of claim 17 , wherein Ar 1 is phenyl, Ar 2 is furyl, and Ar 3 is phenyl.

19. The compound of claim 17 , wherein R C is selected from the group consisting of C 1 -C 9 , linear or branched alkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, hydroxyl alkyl, heteroaryl, heteroarylalkyl, heterocycle, and heterocyclic alkyl.

20. The compound of claim 17 , wherein the compound of formula (III) has formula (III-a), (III-b), or (III-c)

wherein R C is selected from the group consisting of C 1 -C 9 linear or branched alkyl, alkoxyalkyl, aryl, arylalkyl, cycloalkyl, hydroxyl alkyl, heteroaryl, heteroarylalkyl, heterocycle, and heterocyclic alkyl.

21. The compound of claim 1 , selected from the group consisting of:
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 3-methoxy-2,2-dimethylpropanoate;
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 3-(2-(2-methoxyethoxy)ethoxy)-2,2-dimethylpropanoate;
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 3-(2-methoxyethoxy)-2,2-dimethylpropanoate;
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 13,13-dimethyl-2,5,8,11-tetraoxatetradecan-14-oate;
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 16,16-dimethyl-2,5,8,11,14-pentaoxaheptadecan-17-oate;
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 2-methoxy-2-methylpropanoate; and
((8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3-yl)oxy)methyl 2-acetamido-2-methylpropanoate.

22. The compound of claim 13 , selected from the group consisting of:
((8-benzyl-2-(furan-2-ylmethyl)-6-phen

Assignees

• Promega Corp

Inventors

• Zhou Wenhui
• Binkowski Brock
• Meisenheimer Poncho
• Niles Andrew L
• Kupcho Kevin
• Unch James

Classifications

• C12Q1/66

  involving luciferase · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N2333/916

  acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4) · CPC title

• C07D487/04 Primary

  Ortho-condensed systems · CPC title

• G01N2333/90241

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title