# US 9,518,981 B2 · Modified nucleic acid binding cyanine dyes for the detection of reactive oxygen species

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- Number: US 9,518,981 B2
- Document ID: US9518981B2
- Status: Granted
- Kind: B2
- Application number: 14/384,843
- Filed: 2013-02-26
- Published: 2015-03-19
- Granted: 2016-12-13
- Earliest claimed: 2012-03-30
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Yi-Zhen Hu; Aimei Chen; Hee Chol Kang; Kyle Gee; Bhaskar Mandavilli
- Web page: https://patentgrep.com/patent/US9518981B2

## CPC

- G01N 33/84: involving inorganic compounds or pH
- C07D 417/06: linked by a carbon chain containing only aliphatic carbon atoms
- C09B 23/02: the polymethine chain containing an odd number of >CH- {or >C[alkyl]-} groups
- C09B 23/04: one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
- C09B 23/06: three >CH- groups, e.g. carbocyanines
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- 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)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

Disclosed herein are compounds, compositions, methods, and kits for detecting reactive oxygen species (ROS) by conventional fluorescence microscopy, fluorescence spectroscopy, flow cytometry, and/or high content imaging. The compounds disclosed herein are novel reduced nucleic acid binding cyanine dyes, which dyes are probes for detecting ROS and measuring oxidative stress in cells either in vitro and/or in vivo. Also described herein are processes for preparing novel reduced dyes, i.e., ROS probes, for use in the disclosed compositions, methods and kits.

## Claims

### Claim 1

A reduced dye compound having structural formula (II):
wherein
each R1 is H;
t is 4;
R2 is a substituted or unsubstituted C1-C6 alkyl;
R5 is selected from the group consisting of C1-C6 alkyl, C1-C6 alkenyl, C1-C6 polyalkenyl, C1-C6 alkynyl, C1-C6 polyalkynyl and an OMEGA;
R3, R4, R11, R12, R13 and R14 are each independently selected from the group consisting of H, halogen, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 polyalkenyl, C1-C6 alkynyl, C1-C6 polyalkynyl, —OR8, —SR8, —(NR8R9), —OSO2R19, and an OMEGA, wherein R8 and R9, which may be the same or different, are independently selected from the group consisting of H and C1-C6 alkyl;
R19 is a C1-C6 alkyl, C1-C6 perfluoroalkyl, or aryl;
OMEGA is a substituted or unsubstituted phenyl,
wherein the OMEGA is attached at R3, R4, R5, R11, R12, R13, or R14 by a single bond,
wherein when more than one of R3, R4, R5, R11, R12, R13, and R14 is an OMEGA, each OMEGA is optionally the same or different, and
wherein one or two of R3, R4, R5, R6, R7, R11, R12, R13 and R14 is an OMEGA;
X is O, S, Se, NR15 or CR16R17;
R16 and R17, which may be the same or different, are independently selected from the group consisting of H and C1-C6 alkyl;
R15 is H or C1-C6 alkyl;
R20, R21 and R22 are independently H, C1-C6 alkyl, C3-C10 cycloalkyl, aryl or heteroaryl;
W is H or D in either the R or S configuration; and
n is 0, 1, 2, or 3.

### Claim 2 (depends on 1)

The compound according to claim 1, wherein:
R2 is methyl or ethyl;
R4 is C1-C6 alkyl;
R5 is phenyl;
X is S; and
n is 0.

### Claim 3

A reduced dye compound selected from the group consisting of:
(Z)-2-((2-butyl-1-phenylquinolin-4(1H)-ylidene)methyl)-3-methyl-2,3-dihydrobenzo[d]thiazole;
(Z)-2-((2-butyl-1-phenylquinolin-4(1H)-ylidene)methyl)-3-methyl-2-dueterio-3-hydrobenzo[d]thiazole;
(Z)-3-methyl-2-((1-propylquinolin-4(1H)-ylidene)methyl)-2,3-dihydrobenzo[d]thiazole;
(Z)-2-((2-methoxy-1-phenylquinolin-4(1H)-ylidene)methyl)-3-methyl-2,3-dihydrobenzo[d]thiazole;
(Z)—N1,N1-dimethyl-N3-(4-((3-methyl-2,3-dihydrobenzo[d]thiazol-2-yl)methylene)-1-phenyl-1,4-dihydroquinolin-2-yl)-N3-propylpropane-1,3-diamine;
N-ethyl-N-(4-((Z)-4-((E)-3-(2-methyl-2,3-dihydrobenzo[d]thiazol-2-yl)allylidene)-1-phenyl-1,4-dihydroquinolin-2-yl)benzyl)ethanamine; and
N-ethyl-N-(4-((Z)-1-methyl-4-((E)-3-(3-methyl-2,3-dihydrobenzo[d]thiazol-2-yl)allylidene)-1,4-dihydroquinolin-2-yl)benzyl)ethanamine.

### Claim 4 (depends on 1)

A composition for the detection of reactive oxygen species (ROS), the composition comprising:
a) one or more reduced dyes; and
b) a carrier,
wherein said reduced dyes are compounds according to claim 1 and are present in an amount effective to detect the presence of ROS upon reaction with ROS.

### Claim 5 (depends on 4)

The composition according to claim 4, wherein the composition is suitable for in vitro applications.

### Claim 6 (depends on 4)

The composition according to claim 4, wherein the composition is suitable for in vivo applications.

### Claim 7 (depends on 1)

A method of detecting reactive oxygen species (ROS) in a sample, the method comprising the steps of:
a) contacting the sample with an effective amount of one or more of the reduced dye compounds according to claim 1; and
b) determining if the reduced dye compound has been oxidized.

### Claim 8 (depends on 4)

A method of detecting reactive oxygen species (ROS) in a sample, the method comprising the steps of:
a) contacting the sample with an effective amount of the composition according to claim 4; and
b) determining if the reduced dye compound has been oxidized.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the sample comprises cells, tissues, biological fluids, or combinations thereof.

### Claim 10 (depends on 7)

The method according to claim 7, wherein oxidation of the reduced dye compound is detected by fluorescence spectroscopy.

### Claim 11 (depends on 7)

The method according to claim 7, wherein oxidation of the reduced dye compound is detected by fluorescence microscopy.

### Claim 12 (depends on 7)

The method according to claim 7, wherein oxidation of the reduced dye compound is detected by confocal laser scanning fluorescence microscopy.

### Claim 13 (depends on 7)

The method according to claim 7, wherein oxidation of the reduced dye compound is detected by total internal reflection fluorescence microscopy.

### Claim 14 (depends on 7)

The method according to claim 7, wherein the detection of the reactive oxygen species (ROS) is used to diagnose a disease or disorder selected from the group consisting of carotid artery injuries, atherosclerosis, hypertension, cancers, diseases and disorders characterized by inflammation, radiation-induced late normal tissue damage; tissue damages due to chemotherapy, reperfusion after ischemia, or transplantation; diabetes, such as type 1 diabetes (T1D), neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease; cerebrovascular disease, cystic fibrosis, chronic kidney disease, cardiovascular disease, preeclampsia, ophthalamic diseases, and combinations thereof.

### Claim 15 (depends on 1)

A kit for detecting reactive oxygen species (ROS) in a sample, the kit comprising:
a) one or more reduced dye compounds according to claim 1; and
b) one or more containers.

### Claim 16 (depends on 4)

A kit for detecting reactive oxygen species (ROS) in a sample, the kit comprising:
a) the composition according to claim 4; and
b) one or more containers.

### Claim 17 (depends on 15)

The kit according to claim 15, wherein the kit further comprises instructions for performing an assay for detecting one or more reactive oxygen species (ROS) comprising the steps of:
a) contacting the sample with an effective amount of one or more of the reduced dye compounds;
b) measuring fluorescence intensity of the one or more dye compounds; and
c) determining if the reduced dye compound has been oxidized;
wherein an increase in fluorescence intensity is indicative of the presence of ROS in the sample.

### Claim 18 (depends on 17)

The kit according to claim 17, wherein the assay is performed in vivo or in vitro.

### Claim 19 (depends on 1, 2)

A process for preparing a reduced dye compound according to claim 1, the process comprising:
a) reacting a cyanine compound having structural formula (IV)
with a reducing agent, wherein:
X, R1, R2, R3, R4, R5, R11, R12, R13, R14, R20, R21, R22, n, and t are as defined in claim 2.