Fluorogenic pH-sensitive dyes and their methods of use (II)

Patent metadata

Field	Value
Publication number	US-9599622-B2
Application number	US-201314402358-A
Country	US
Kind code	B2
Filing date	Mar 14, 2013
Priority date	May 30, 2012
Publication date	Mar 21, 2017
Grant date	Mar 21, 2017

Abstract

Disclosed herein are compounds, compositions, methods and kits for detecting pH in samples using pH-sensitive fluorescent dyes. The compounds disclosed herein are novel xanthene-derivative dyes comprising an aniline moiety with one or more electron donating groups, which dyes are for detecting pH in samples either in vitro or in vivo. Also described herein are processes for preparing said dyes for use in the disclosed compositions, methods and kits.

First claim

What is claimed is:

1. A pH-sensitive fluorescent dye compound of structural formula (I):

wherein
R 1 is alkoxy or thioalkyl;
R 2 and R 6 , which may be the same or different, are each independently H, halogen, —OR a , —SR a , —NR a R b , or an electron donating group;
R 3 is —NR′R″, wherein R′ and R″, which may be the same or different, are each independently alkyl or substituted alkyl;
R 4 is selected from the group consisting of alkyl and substituted alkyl;
R 5 is selected from the group consisting of alkyl; substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl; substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl; (CH 2 ) n CO(O)R; (CH 2 ) n C(O)R; (CH 2 ) n C(O)NHR; (CH 2 ) n C(O)NRR c , wherein n is an integer from 1 to 6, and R and R c , which may be the same or different, are each independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted amino, alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic group, or R d , wherein R d is (CH 2 ) n C(O)NHR x , wherein n is an integer from 1 to 6, and R x is a reactive group; -L-R x ; and -L-S c , wherein L is a linker, R x is a reactive group, and S c is a conjugated substance;
R a is H, alkyl, or substituted alkyl;
R b is alkyl or substituted alkyl; and
R 7 , R 8 , R 9 , and R 10 , which may be the same or different, are each independently alkenyl.

2. The compound according to claim 1 , wherein
R 1 is alkoxy or thioalkyl;
R 2 and R 6 , which may be the same or different, are each independently H or halogen;
R 3 is —NR′R″, wherein R′ and R″, which may be the same or different, are each independently alkyl;
R 4 is alkyl;
R 5 is selected from the group consisting of alkyl; substituted alkyl; alkenyl; substituted alkenyl;
acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl; substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl; (CH 2 ) n CO(O)R; (CH 2 ) n C(O)R; (CH 2 ) n C(O)NHR; (CH 2 ) n C(O)NRR c , wherein n is an integer from 1 to 6, and R and R c , which may be the same or different, are each independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted amino, alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic group, or R d , wherein R d is (CH 2 ) n C(O)NHR x , wherein n is an integer from 1 to 6, and R x is a reactive group; -L-R x ; and -L-S c ; and
R 7 , R 8 , R 9 , and R 10 , which may be the same or different, are each independently alkenyl.

3. The compound according to claim 1 , wherein
R 1 is alkoxy or thioalkyl;
R 2 and R 6 , which may be the same or different, are each independently H, Cl or F;
R 3 is —NR′R″, wherein R′ and R″, which may be the same or different, are each independently alkyl;
R 4 is alkyl;
R 5 is selected from the group consisting of alkyl; substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl; substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl; (CH 2 ) n CO(O)R; (CH 2 ) n C(O)R; (CH 2 ) n C(O)NHR; (CH 2 ) n C(O)NRR c , wherein n is an integer from 1 to 6, and R and R c , which may be the same or different, are each independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted amino, alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic group, or R d , wherein R d is (CH 2 ) n C(O)NHR x , wherein n is an integer from 1 to 6, and R x is a reactive group; -L-R x ; and -L-S c ; and
R 7 , R 8 , R 9 , and R 10 , which may be the same or different, are each independently alkenyl.

4. The compound according to claim 1 , wherein
R 1 is alkoxy or thioalkyl;
R 2 and R 6 are each H;
R 3 is —NR′R″, wherein R′ and R″, which may be the same or different, are each independently alkyl;
R 4 is alkyl;
R 5 is selected from the group consisting of alkyl; substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl; substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl; (CH 2 ) n CO(O)R; (CH 2 ) n C(O)R; (CH 2 ) n C(O)NHR; (CH 2 ) n C(O)NRR c , wherein n is an integer from 1 to 6, and R and R c , which may be the same or different, are each independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted amino, alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic group, or R d , wherein R d is (CH 2 ) n C(O)NHR x , wherein n is an integer from 1 to 6, and R x is a reactive group; -L-R x ; and -L-S c ; and
R 7 , R 8 , R 9 , and R 10 , which may be the same or different, are each independently alkenyl.

5. The compound according to claim 1 , wherein
R 1 is methoxy;
R 2 and R 6 are each H;
R 3 is —NR′R″, wherein R′ and R″, which may be the same or different, are each independently methyl or ethyl;
R 4 is methyl or ethyl;
R 5 is methyl; ethyl; carboxyalkyl; (CH 2 ) n CO(O)R; (CH 2 ) n C(O)R; (CH 2 ) n C(O)NHR; (CH 2 ) n C(O)NRR c ; wherein n is an integer from 1 to 6, and R and R c , which may be the same or different, are each independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted amino, alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic group, or R d , wherein R d is (CH 2 ) n C(O)NHR x , wherein n is an integer from 1 to 6, and R x is a reactive group selected from carboxyl, carboxylester, amide, maleimide, succinimidyl ester (SE), sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP) ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester, nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R x ; or -L-S c ; and
R 7 , R 8 , R 9 , and R 10 , which may be the same or different, are each independently propenyl.

6. A pH-sensitive fluorescent dye compound of structural formula (II):

wherein
R 1 is alkoxy or thioalkyl;
R 2 and R 6 , which may be the same or different, are each independently H, halogen, —OR a , —SR a , —NR a R b , or an electron donating group;
R 3 is —NR′R″, wherein R′ and R″, which may be the same or different, are each independently alkyl or substituted alkyl;
R 4 is selected from the group consisting of alkyl and substituted alkyl;
R 5 is selected from the group consisting of alkyl; substituted alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl; substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl; (CH 2 ) n CO(O)R; (CH 2 ) n C(O)R; (CH 2 ) n C(O)NHR; (CH 2 ) n C(O)NRR c , wherein n is an integer from 1 to 6, and R and R c , which may be the same or different, are each independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted amino, alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic group, or R d , wherein R d is (CH 2 ) n C(O)NHR x , wherein n is an integer from 1 to 6, and R x is a reactive group; -L-R x ;

Assignees

• Life Technologies Corp

Inventors

• Gee Kyle
• Singh Upinder
• Rukavishnikov Aleksey
• Beacham Daniel
• Huang Shih-Jung
• Janes Michael
• Zhou Wenjun

Classifications

• G01N33/582 Primary

  with fluorescent label · CPC title

• C09B11/24 Primary

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C07D311/90

  with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9 · CPC title

• A61K49/0041

  Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal (in vivo G01N) · CPC title

• G01N33/84

  involving inorganic compounds or pH · CPC title