# US 10,428,075 B2 · Coelenterazine analogues

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- Number: US 10,428,075 B2
- Document ID: US10428075B2
- Status: Granted
- Kind: B2
- Application number: 15/982,514
- Filed: 2018-05-17
- Published: 2018-11-22
- Granted: 2019-10-01
- Earliest claimed: 2016-07-28
- Assignees: Promega Corporation
- Applicants: Promega Corporation
- Inventors: Mary Hall; Thomas Kirkland; Thomas Machleidt; Anton Shakhmin; Joel R. Walker
- Web page: https://patentgrep.com/patent/US10428075B2

## CPC

- C07D 487/04: Ortho-condensed systems
- A61K 49/0021: {the fluorescent group being a small organic molecule}
- C07D 519/00: Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
- C09K 11/06: containing organic luminescent materials
- C12Q 1/66: involving luciferase
- C12Y 113/12005: Renilla-luciferin 2-monooxygenase (1.13.12.5), i.e. renilla-luciferase
- C12Y 113/12013: Oplophorus-luciferin 2-monooxygenase (1.13.12.13)
- C09K 2211/1011: Condensed systems
- C09K 2211/1018: Heterocyclic compounds
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

Described are 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.

## Claims

### Claim 1

A method for in vivo bioluminescent imaging, the method comprising:
contacting a live cell with a compound of Formula I or Formula II:
wherein R5 and R5a are hydrogen;
R6 and R6a are aryl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, acyl amino, aminoalkyl, alkyl, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, alkoxy, sulfonylamino, sulfonyl, alkylsulfonyl, amide, carbamate, silyl, substituted silyl, acyl, or aminosulfonyl;
R8 and R8a are optionally substituted aryl, heteroaryl, alkyl substituted with aryl, or alkyl substituted with heteroaryl;
Rx is halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, or heteroalkyl; and
p is 0, 1, 2, 3, 4, or 5;
provided that the following compounds are excluded from formula (I): 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; and
8-benzyl-2-(furan-2-ylmethyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one; and
Rx is not 4-hydroxy when p is 1, R5a is not hydrogen, or R8a is not benzyl, or any combination thereof;
and
detecting luminescence; wherein the live cell expresses a coelenterazine-utilizing luciferase.

### Claim 2 (depends on 1)

The method of claim 1, wherein:
R6 and R6a are phenyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, acyl amino, aminoalkyl, alkyl, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, alkoxy, sulfonylamino, sulfonyl, alkylsulfonyl, amide, carbamate, silyl, substituted silyl, acyl, or aminosulfonyl.

### Claim 3 (depends on 1)

The method of claim 1, wherein
R6 and R6a are
X1-X5 are each independently CR11; and
R11, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, nitro, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C6-alkoxy, C1-C6-haloalkyl, C1-C6-haloalkoxy, C1-C6-alkylamino, C1-C6-dialkylamino, and C1-C6-heteroalkyl.

### Claim 4 (depends on 3)

The method of claim 3, wherein R8 and R8a are selected from the group consisting of:

### Claim 5 (depends on 3)

The method of claim 3, wherein the compound is of formula (I); R11 at X1, X3, X4, and X5 is hydrogen; and the R11 at X2 is hydrogen, hydroxy, amino, C1-C6-alkoxy, C1-C6-haloalkoxy, C1-C6-alkylamino, C1-C6-dialkylamino, or C1-C6-heteroalkyl.

### Claim 6 (depends on 3)

The method of claim 3, wherein the compound is of formula (I); and R6 is selected from the group consisting of:

### Claim 7 (depends on 5)

The method of claim 5, wherein R8 is selected from the group consisting of:

### Claim 8 (depends on 5)

The method of claim 5, wherein R8 is

### Claim 9 (depends on 7)

The method of claim 7, wherein the compound is

### Claim 10 (depends on 1)

The method of claim 1, wherein the live cell is in an animal.

### Claim 11 (depends on 1)

The method of claim 1, wherein the coelenterazine-utilizing luciferase is selected from the group of a Oplophorus luciferase or variant thereof and a Renilla luciferase or a variant thereof.

### Claim 12 (depends on 1)

The method of claim 1, wherein the coelenterazine-utilizing luciferase is expressed as a fusion protein.

### Claim 13 (depends on 12)

The method of claim 12, wherein the fusion protein comprises the coelenterazine-utilizing luciferase and a protein of interest.

### Claim 14 (depends on 13)

The method of claim 13, wherein the coelenterazine-utilizing luciferase is selected from the group of a Oplophorus luciferase or variant thereof and a Renilla luciferase or a variant therof.