# US 10,308,975 B2 · Substituted imidazo[1,2-a]pyrazines as luciferase substrates

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- Number: US 10,308,975 B2
- Document ID: US10308975B2
- Status: Granted
- Kind: B2
- Application number: 15/887,735
- Filed: 2018-02-02
- Published: 2018-08-09
- Granted: 2019-06-04
- Earliest claimed: 2016-02-15
- Assignees: Promega Corporation
- Applicants: Promega Corporation
- Inventors: Anton Shakhmin; Thomas Kirkland; Joel Walker; Thomas Machleidt; Mary Hall; Keith V. Wood
- Web page: https://patentgrep.com/patent/US10308975B2

## CPC

- C12Q 1/66: involving luciferase
- A61K 49/0013: {Luminescence}
- C07D 487/04: Ortho-condensed systems
- 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
- G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)}
- H04N 1/6041: {for controlling uniformity of color across image area}
- G01N 2333/90241: acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
- 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
- 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)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- H04N: PICTORIAL COMMUNICATION, e.g. TELEVISION

## Abstract

Described are substituted imidazo[1,2-a]pyrazine compounds, which are coelenterazine analogs, kits comprising the compounds, and methods of using the compounds for the detection of luminescence in luciferase-based assays. Also described are methods for making the compounds, such as a method using aminopyrazine acetophosphonates as synthesis intermediates.

## Claims

### Claim 1

A compound of formula (I):
or a tautomer or pharmaceutically acceptable salt thereof,
wherein:
R1 is phenyl; and
q is 1;
wherein said phenyl is substituted with 1, 2, 3, 4, or 5 functional groups independently selected from the group consisting of cyano, carbamate, nitro, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, aryloxy, benzyloxy, amino, aminoalkyl, sulfonylamino, sulfinylamino, sulfonyl, sulfinyl, —COOH, amide, carbamate, silyl, silyloxy, sulfanyl, and acyl;
provided that R1 is not
wherein Rc is OH, OC(O)C1-C7-alkyl, or OCH2OC(O)C1-C7-alkyl.

### Claim 2 (depends on 1)

The compound of claim 1, or a tautomer or pharmaceutically acceptable salt thereof, wherein said phenyl is substituted with 1 or 2 functional groups independently selected from the group consisting of cyano, carbamate, alkyl, haloalkyl, heteroalkyl, aryl, hydroxy, alkoxy, allyloxy, amino, aminoalkyl, sulfonylamino, —COOH, and alkylsulfanyl.

### Claim 3 (depends on 1)

The compound of claim 1, wherein the compound is selected from the group consisting of:
8-benzyl-2-(3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
tert-butyl (3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl) carbamate;
2-(3-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-(tert-butyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
2-([1,1′-biphenyl]-4-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
tert-butyl (4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate;
2-(4-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
2-(4-(allyloxy)-3-methoxybenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-hydroxy-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic acid;
8-benzyl-2-(3,5-bis (trifluoromethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic acid;
2-(3-(aminomethyl)benzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
N-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl) methanesulfonamide;
8-benzyl-2-(4-methoxy-3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-(dimethylamino)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-(hydroxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3,4-dimethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-(methylthio)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-((dimethylamino)methyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-ethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-(2-methoxyethoxy)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; and
3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzonitrile;
or a tautomer or pharmaceutically acceptable salt thereof.

### Claim 4 (depends on 1)

A kit comprising a compound of claim 1, or a tautomer or pharmaceutically acceptable salt thereof.

### Claim 5 (depends on 4)

The kit of claim 4, wherein the kit further comprises a luciferase.

### Claim 6 (depends on 4)

The kit of claim 4, wherein the kit further comprises a buffer reagent.

### Claim 7 (depends on 1)

A method for detecting luminescence in a sample, the method comprising:
contacting a sample with a compound of claim 1, or a tautomer or pharmaceutically acceptable salt thereof;
contacting the sample with a coelenterazine-utilizing luciferase, if it is not present in the sample; and
detecting luminescence.

### Claim 8 (depends on 7)

The method of claim 7, wherein the sample contains live cells.

### Claim 9 (depends on 7)

The method of claim 7, wherein the sample contains a coelenterazine-utilizing luciferase.

### Claim 10 (depends on 1)

A method for detecting luminescence in a transgenic animal comprising:
administering a compound of claim 1, or a tautomer or pharmaceutically acceptable salt thereof to a transgenic animal; and
detecting luminescence;
wherein the transgenic animal expresses a coelenterazine-utilizing luciferase.

### Claim 11

A compound of formula (I):
or a tautomer or pharmaceutically acceptable salt thereof,
wherein:
R1 is phenyl; and
q is 1;
wherein said phenyl is substituted with 2, 3, 4, or 5 functional groups independently selected from the group consisting of halogen, cyano, carbamate, nitro, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, aryloxy, benzyloxy, amino, aminoalkyl, sulfonylamino, sulfinylamino, sulfonyl, sulfinyl, —COOH, amide, carbamate, silyl, silyloxy, sulfanyl, and acyl.

### Claim 12 (depends on 11)

The compound of claim 11, or a tautomer or pharmaceutically acceptable salt thereof, wherein said phenyl is substituted with 2, 3, 4, or 5 functional groups independently selected from the group consisting of halogen, haloalkyl, heteroalkyl, hydroxy, alkoxy, and allyloxy.

### Claim 13 (depends on 11)

The compound of claim 11, wherein the compound is selected from the group consisting of:
8-benzyl-2-(2,4-difluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-fluoro-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-fluoro-3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3-fluoro-4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-fluoro-2-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
2-(4-(allyloxy)-3-methoxybenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-hydroxy-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-((perfluorophenyl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3,5-bis (trifluoromethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(4-methoxy-3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one;
8-benzyl-2-(3,4-dimethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; and
8-benzyl-6-phenyl-2-(2,4,6-trifluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one,
or a tautomer or pharmaceutically acceptable salt thereof.

### Claim 14 (depends on 11)

A kit comprising a compound of claim 11, or a tautomer or pharmaceutically acceptable salt thereof.

### Claim 15 (depends on 14)

The kit of claim 14, wherein the kit further comprises a luciferase.

### Claim 16 (depends on 14)

The kit of claim 14, wherein the kit further comprises a buffer reagent.