Quinolinium- and pyridinium-based fluorescent dye compounds

Details Quinolinium- and pyridinium-based fluorescent dye compounds Quinolinium- and pyridinium-based fluorescent dye compounds

1999-10-05

2002-01-01

Huang, Evelyn Mei (Department: 1625)

Organic compounds -- part of the class 532-570 series

Organic compounds

Nitrogen attached directly or indirectly to the purine ring...

C544S363000, C544S124000, C544S128000, C546S172000, C546S330000, C546S334000, C546S194000, C546S276400

Reexamination Certificate

active

06335446

ABSTRACT:

1. FIELD OF THE INVENTION
The present invention relates to quinolinium- and pyridinium-based fluorescent dyes, and to their use in the staining of proteins in solution, in gels and on solid supports.
2. BACKGROUND OF THE INVENTION
Structures of formula II are disclosed in WO 96/36882, which discloses unsubstituted compounds (R1=R3=H) and the corresponding 2- and 2,6-substituted anilines, and those where the olefinic bond is optionally substituted (R4, R5=H, alkyl or phenyl), having fluorescence properties. A specific example of such a compound is shown in formula III.
Structures of formula IV are also disclosed in WO 96/36882, which discloses unsubstituted compounds (R1=R3=H) and the 2- and 2,6-substituted anilines, having fluorescence properties. A specific example of such a compound is shown in formula V.
Citation of a reference herein shall not be construed as indicating that such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to compounds of formula I
wherein
R
1
is (C
1
-C
6
) straight or branched chain alkyl, halogen or —CF
3
;
either R
2a
and R
2b
are independently a lipophilic group or H, R
2a
and R
2b
not simultaneously being H, or R
2a
and R
2b
are taken together and form a morpholinyl, piperidinyl or pyrrolidinyl ring;
R
3
is H or (C
1
-C
6
) straight or branched chain alkyl; and
either R
4
and R
5
are both H, or R
4
and R
5
taken together are —CH═CH—CH═CH—,
the aromatic rings A and B, the —(CH
2
)
4-5
— group, and the —C(H)═C(R
3
)— group being optionally substituted with one or more —OH, (C
1
-C
6
) straight or branched chain alkoxyl, halogen, (C
1
-C
6
) straight or branched chain haloalkyl, pyridyl, thiophenyl, furyl, and phenyl, the phenyl being optionally substituted with one or more —OH, (C
1
-C
6
) straight or branched chain alkyl or (C
1
-C
6
) straight or branched chain alkoxyl.
In a second aspect, the present invention relates to compounds of formula I wherein
R
1
is H, (C
1
-C
6
) straight or branched chain alkyl, halogen or —CF
3
;
R
2a
and R
2b
are taken together to form —(CH
2
)
2
—NR
6
—(CH
2
)
2
—, wherein R
6
is (C
1
-C
12
) straight or branched chain alkyl, (C
1
-C
12
) straight or branched chain alkylcarbonyl or (C
1
-C
12
) straight or branched chain alkylsulphonyl;
R
3
is H; and
either R
4
and R
5
are both H, or R
4
and R
5
are taken together and form —CH═CH—CH═CH—,
the aromatic rings A and B, the —(CH
2
)
4-5
— group, and the —C(H)═C(R
3
)— group being optionally substituted with one or more —OH, (C
1
-C
6
) straight or branched chain alkoxyl, halogen, (C
1
-C
6
) straight or branched chain haloalkyl, pyridyl, thiophenyl, furyl, and phenyl, the phenyl being optionally substituted with one or more —OH, (C
1
-C
6
) straight or branched chain alkyl or (C
1
-C
6
) straight or branched chain alkoxyl.
In a third aspect, the present invention relates to compounds of formula I wherein
R
1
is H, (C
1
-C
6
) straight or branched chain alkyl, halogen, or —CF
3
;
either R
2a
and R
2b
are independently a lipophilic group or H, R
2a
and R
2b
not simultaneously being H, or R
2a
and R
2b
are taken together and form a morpholinyl, piperidinyl, pyrrolidinyl, or piperazinyl ring, wherein the piperazinyl ring is optionally substituted with (C
1
-C
12
) straight or branched chain alkyl, (C
1
-C
12
) straight or branched chain alkylcarbonyl or (C
1
-C
12
) straight or branched chain alkylsulphonyl;
R
3
is —CN, CONH
2
, —COOH, or —COOR, wherein R is (C
1
-C
6
) straight or branched chain alkyl or (C
1
-C
10
) straight or branched chain aralkyl, the —CONH
2
group being optionally substituted with one or two (C
1
-C
6
) alkyl groups or one or two (C
1
-C
10
) aralkyl groups; and
either R
4
and R
5
are both H, or R
4
and R
5
are taken together and form —CH═CH—CH═CH—,
the aromatic rings A and B, the —(CH
2
)
4-5
— group, and the —C(H)═C(R
3
)— group being optionally substituted with one or more —OH, (C
1
-C
6
) straight or branched chain alkoxyl, halogen, (C
1
-C
6
) straight or branched chain haloalkyl, pyridyl, thiophenyl, furyl, and phenyl, the phenyl being optionally substituted with one or more —OH, (C
1
-C
6
) straight or branched chain alkyl or (C
1
-C
6
) straight or branched chain alkoxyl.
The present invention may be understood more fully by reference to the detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.
4. DETAILED DESCRIPTION OF THE INVENTION
4.1 Definitions
As used herein, halogen refers to F, Cl, Br or I.
As used herein, (C
1
-C
6
) straight or branched alkyl includes but is not limited to methyl, ethyl, n-propyl, propan-1-yl, propan-2-yl, cyclopropan-1-yl, n-butyl, butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, 3-methyl-n-butyl, n-pentyl, pentan-1-yl, pentan-2-yl, cyclopentan-1-yl, n-hexyl, hexan-1-yl, hexan-2-yl, and cyclohexan-1-yl.
As used herein (C
1
-C
6
) straight or branched chain alkoxy includes but is not limited to 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl, and 6-hydroxyhexyl.
As used herein, a lipophilic group refers to (C
1
-C
20
) straight or branched chain alkyl or (C
1
-C
20
) straight or branched chain aralkyl.
As used herein, (C
1
-C
20
) straight or branched chain alkyl includes but is not limited to methyl, ethyl, n-propyl, propan-1-yl, propan-2-yl, cyclopropan-1-yl, n-butyl, butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, 3-methyl-n-butyl, n-pentyl, pentan-1-yl, pentan-2-yl, cyclopentan-1-yl, n-hexyl, hexan-1-yl, hexan-2-yl, and cyclohexan-1-yl, n-octyl, n-decyl, n-hexadecyl, n-octadecyl, and n-eicosyl.
As used herein, aryl refers to phenyl, naphthyl, or anthracenyl.
As used herein, (C
1
-C
6
) straight or branched chain haloalkyl refers to an alkyl moiety, such as one of those listed above, having at least one halogen attached, including, but not limited to, —CF
3
, —CH
2
F, —CHF
2
, —CH
2
I, —CH
2
Br, —CH
2
Cl, —CHCl
2
, —CCl
3
, —CH
2
CHClCH
3
, —CH
2
C(CH
3
)(CH
2
Br), —CH
2
CH
2
CH
2
CH
2
I, —C(CH
3
)(CH
3
)(CH
2
F), and —CH
2
CHClCH
2
CHClCH
2
CH
3
. Preferably, the haloalkyl group is —CF
3
.
As used herein, (C
1
-C
12
) straight or branched chain alkylcarbonyl refers to an alkyl moiety, such as those listed above, having one or more C═O groups attached.
As used herein, (C
1
-C
12
) straight or branched chain alkylsulphonyl refers to an alkyl moiety, such as those listed above, having one or more —SO
2
— groups attached.
One skilled in the art will readily appreciate that when R
4
and R
5
taken together are —CH═CH—CH═CH—, aromatic ring B and —CH═CH—CH═CH— generate a quinoline system.
4.2 Compounds
The compounds of formula I have been found to display fluorescence properties superior to those of the prior art. Thus, in particular, certain compounds of the invention display much increased brightness over the compounds of formulae III and V when they are bound to protein (e.g. bovine serum albumin) and when sodium dodecyl sulphate is present in an amount below its critical micelle concentration. Furthermore, certain compounds of this invention display greater differences in the fluorescence intensities of their protein-bound and unbound states when compared to the compounds of formulae III and V.
Without being bound by any particular theory, Applicants believe that the compounds of formula I have properties superior to those described in WO 96/36882. In general terms, other substituents (both those shown in formula I, and also those not shown) may be less critical; thus, each of the aromatic rings, the —(CH
2
)
4-5
— group and the conjugating C═C link may carry other/further substituents that do not substantially affect the fluorescence intensity of the compounds (e.g. compounds have at least 50%, and preferably at least 75%, of the intensity of any compound illustrated herein under the same conditions). Examples of substituents

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