Fluorescence properties of some 2-(4-amino-substituted-3-nitrophenyl)-3- hydroxyquinolin-4(1H)-ones

Article abstract of DOI:10.1016/j.tetlet.2010.12.013

2-(4-Amino-substituted-3-nitrophenyl)-3-hydroxyquinolin-4(1H)-ones have been studied to evaluate their fluorescence properties and possible use as molecular fluorescent probes. The amino group was substituted with various alkyl moieties possessing a suita

Full text of DOI:10.1016/j.tetlet.2010.12.013

Tetrahedron Letters 52 (2011) 715–717
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Tetrahedron Letters
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Fluorescence properties of some 2-(4-amino-substituted-3-nitrophenyl)-3-
hydroxyquinolin-4(1H)-ones
⇑
ˇ
ˇ
Kamil Motyka , Jan Hlavác, Miroslav Soural, Pavel Hradil, Petr Krejcí, Lubomír Kvapil, Miloš Weiss
ˇ
´
Department of Organic Chemistry, Faculty of Science, Institute of Molecular and Translational Medicine, Palacky University, Tr. 17. listopadu 12, 771 46 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
2-(4-Amino-substituted-3-nitrophenyl)-3-hydroxyquinolin-4(1H)-ones have been studied to evaluate
their fluorescence properties and possible use as molecular fluorescent probes. The amino group was
substituted with various alkyl moieties possessing a suitable terminal functional group (such as hydroxy
or amino group) that could serve to bind a 3-hydroxyquinolin-4(1H)-one (3HQ) fluorescence label to a
biomolecule. Besides simple hydrocarbon chains, ligands containing ethylenoxy units as optimal spacers
were also tested. The structure–fluorescence properties and theoretical applicability of the studied mol-
ecules are discussed.
Received 2 September 2010
Revised 25 November 2010
Accepted 3 December 2010
Available online 9 December 2010
Keywords:
3-Hydroxyquinolones
Fluorescence
Ó 2010 Elsevier Ltd. All rights reserved.
Dual fluorescence probes
Fluorescent probes allow researchers to detect particular com-
ponents of complex biomolecular assemblies with exquisite sensi-
tivity and selectivity, as well as to observe interesting properties of
the biosystem or biological processes. Nevertheless, fluorescence
techniques require a suitable fluorescent label with optimal prop-
erties. In the case of common single-band fluorescent labels the
fluorescence intensity depends on the label concentration which
can vary because of various biological processes in a sample.1 Dual
fluorescence labels that exhibit two well-separated emission
bands are not dependent on the concentration because the ratio
of the intensities of the two bands can be applied as a signal.1,2
This possibility is an advantage in complex biological systems such
as, cells or tissues where the local concentration of the label can-
not be controlled easily and generally, the label is not distributed
homogenously.
These are reasons behind the intensive development of new
dual fluorescence labels with improved fluorescence properties.
Recently, studies1–6 dealing with 2-aryl-3-hydroxyquinolin-
4(1H)-ones (3HQs) have been published. The dual fluorescence
spectrum of 3HQs is a result of an excited state intramolecular
proton transfer (ESIPT) reaction leading to the formation of two
excited state tautomeric forms of the probe.2,4 Different photo-
physical properties result in sufficiently separated emission
bands.2,3
A study of the fluorescent properties of a system composed of an
appropriately substituted 3HQ as the fluorescent label bearing a
suitable spacer for attachment of the label to the target biomole-
cule was described.6 The effect of spacer location at the carboxam-
ide group at positions 6–8 of the hydroxyquinolinone skeleton was
described in the Letter mentioned above.6 This study is focused on
evaluation of the fluorescence properties of 2-(4-amino-substi-
tuted-3-nitrophenyl)-3HQs (Scheme 1) with the aim to evaluate
the possibility of binding the 3HQ fluorescence label to a biomole-
cule via the phenyl ring at position 2. The studied derivatives were
chosen because of their simple preparation via nucleophilic substi-
tution of the chlorine with various amines7 which leads to a large
number of diverse derivatives which may be suitable for an attach-
ment to the target biomolecule (Scheme 1).
In this Letter we examined a set of derivatives 2–17 were
prepared using aliphatic diamines (3–5, 16) or amino alcohols
(6–14, 17). To investigate the influence of amino group substitu-
tion on the resulting fluorescent properties, the prepared ligands
contained alkyl chains of 2–5 carbons (to compare the effect of
chain length), and ligands with ethyleneoxy chains (used as hydro-
philic spacers to avoid convolution) were studied (Table 1). The
syntheses of these 3HQs have been described elsewhere7 and the
purity was >98% as verified by HPLC–MS and NMR analysis.8
The excitation spectra of the studied compounds showed two
maxima, around 305 nm and 350 nm (for compound 16 as an
example, see Fig. 1). From a comparison of the emission spectra
excited at the excitation wavelengths of both the maxima it is
apparent that the emission intensity of the first maximum at
419 nm remains the same, and the intensity of the second maxi-
mum at 518 nm is diminished when a shorter excitation wave-
length is used (Fig. 1). For this reason, as well as for improved
The essential part of the fluorescence label-biomolecule system
is the spacer between both these parts as it reduces potential
undesirable interactions between the fluorescence label and the
biomolecule, especially steric hindrance and spatial interference.
⇑
Corresponding author. Tel.: +420 585 634 436; fax: +420 585 634 465.
E-mail address: motyka@orgchem.upol.cz (K. Motyka).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.12.013

716
K. Motyka et al. / Tetrahedron Letters 52 (2011) 715–717
1.0
3
4
5
0.8
0.6
Scheme 1. General method for the preparation of the target compounds.
0.4
0.2
0.0
Table 1
List of investigated 3HQs
Compound
R1
R2
2
3
4
5
6
7
8
9
H
H
H
H
H
(CH2)2NH2
(CH2)3NH2
(CH2)4NH2
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)2OH
400
450
500
550
600
650
700
Wavelength (nm)
CH3
CH3CH2
CH3(CH2)2
(CH2)2OH
Figure 2. Normalized fluorescence emission spectra of compounds 3,4 and 5 in
methanol.
N
N
OH
10
O
of the same carbon chain length were compared. The quantum
yields of both hydroxyethyl and hydroxybutyl derivatives were
approximately 500 times higher compared to the analogous amino
derivatives (compare compounds 3, 5 and 11, 13) whereas the dif-
ference in the quantum yield of propylene derivatives was very
low (compare compounds 4 and 12). Compound 11 exhibited the
11
12
13
14
15
16
17
H
H
H
H
H
H
H
(CH2)2OH
(CH2)3OH
(CH2)4OH
(CH2)5OH
(CH2)3OCH2CH3
(CH2)2O(CH2)2NH2
(CH2)2O(CH2)2OH
highest quantum yield of those studied (
u = 10.21%). Compounds
10, 16 and 17 which contain ethyleneoxy units as spacers (polyeth-
yleneglycols are often used as spacers due to their water solubility,
biocompatibility and ready availability in a variety of lengths)
exhibited similar quantum yields that were not influenced by the
nature of the terminal substituent (hydroxy group vs amino group,
compare compounds 16 and 17).
However, due to the low quantum yields of compounds 10, 16
and 17 (0.01–0.05%, Table 2), they appear to be ineligible candi-
dates for further fluorescence label-biomolecule system develop-
ment. For compounds 3–5, an interesting dependency between
length of the alkyl chain and the spectral properties was observed.
With increasing chain length, the ratio of emission maxima I1/I2
increased which shows that the ratio of the excited state tauto-
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Table 2
Spectroscopic properties of the 3HQs in methanol
a
b
c
d
-0.02
300
Compound
kex
kem,1 (nm)
kem,2 (nm)
I1/I2
ue (%)
400
500
600
700
1
2
3
4
5
6
7
8
348
349
360
354
345
352
356
355
355
352
348
345
347
347
350
347
351
428
408
410
410
404
430
430
429
416
429
426
415
417
413
411
419
416
508
516
522
511
514
511
510
515
510
513
502
516
506
514
510
518
518
0.21
0.12
0.14
1.38
2.73
0.091
0.10
0.10
0.15
0.13
0.11
0.05
0.10
0.05
0.11
0.08
0.06
0.04
1.6 Â 10À3
0.02
Wavelength (nm)
Figure 1. Excitation and emission spectra of compound 16 in methanol. (—),
excitation spectrum; (– –), emission spectrum at kex = 303 nm; (ꢀꢀꢀ), emission
spectrum at kex = 347 nm.
0.36
5.7 Â 10À3
0.03
0.03
0.01
0.94
0.01
10.21
0.14
2.74
0.07
3.34
9
utility for biological applications, the higher excitation wavelength
was applied in all further measurements.
It was observed that the nature of the amino group influenced
significantly the quantum yields of the compounds. The quantum
yields depended on the chain length (Fig. 2) and terminal substit-
uents on the alkyl chain. When the amino group was unsubstituted
10
11
12
13
14
15
16
17
0.05
0.01
the quantum yield was very low (compound 2,
u
= 1.6 Â 10À3%). In
the case of aminoalkyl derivatives 3–5 the quantum yield was
influenced by the number of carbon atoms (but unfortunately,
did not exceed 0.36%).
A similar dependence was observed with hydroxyalkyl deriva-
tives 11–14. On the other hand, an interesting fact was observed
when the corresponding hydroxyalkyl and aminoalkyl derivatives
a
b
c
kex, excitation wavelength.
kem,1, the fluorescence emission maximum at lower wavelengths.
kem,2, the fluorescence emission maximum at higher wavelengths.
I1/I2, the ratio of fluorescence maxima intensities.
d
e
u
, fluorescence quantum yield (determined with quinine sulfate in 0.5 M H2SO4
(
u
= 0.5779), taken as a reference fluorescence standard).

K. Motyka et al. / Tetrahedron Letters 52 (2011) 715–717
717
Table 3
Although compounds 11 and 13 can, in theory, be used for
attachment directly or via any other spacer to a biomolecule
through the terminal hydroxy group, elongation of the hydroxy-
ethyl ligand in compound 11 with an aminoethyl group (16) or
hydroxyethyl group (17) led to a crucial decrease of the fluores-
cence quantum yields. A relatively small change in the structure
of the studied molecules affected significantly the quantum yields
(up to five orders of magnitude) which indicates the possibility to
find improved, substituted 3HQs, as dual fluorescent labels bound
to a biomolecule via the phenyl ring at position 2 and having suit-
able spectral parameters. The value of the quantum yield after
binding of such systems to biomolecules is also an interesting
question, which could provide information on the actual use of
such derivatives as fluorescent probes.
Spectroscopic properties of compounds 11, 16 and 17 in various solvents
Compound
11
16
17
I1/I2
u
(%)
I1/I2
u
(%)
I1/I2
u (%)
MeCN
DMSO
EtOAc
MeOH
MeOH/H2O 1:1
Toluene
0.01
0.08
0.01
0.08
0.08
0.02
13.28
18.77
15.28
10.21
4.91
0.15
0.33
0.15
0.08
0.16
0.18
0.05
0.07
0.07
0.05
0.06
0.09
0.04
0.11
0.04
0.06
0.11
0.05
0.04
0.04
0.03
0.01
0.01
0.05
21.30
1.0
0.8
0.6
0.4
0.2
MeCN
DMSO
EtOAc
MeOH
Acknowledgements
MeOH/H2O
toluene
The authors are grateful to the Ministry of Education, Youth and
Sport of the Czech Republic, for grant MSM6198959216, and to
EEA/Norway, grant A/CZ0046/1/0022. The infrastructural part of
this project (Institute of Molecular and Translational Medicine)
was supported by the Operational Programme Research and Devel-
opment for Innovations (Project CZ.1.05/2.1.00/01.0030).
Supplementary data
Supplementary data (analytical data for compounds 2 and 4–
16) associated with this article can be found, in the online version,
at doi:10.1016/j.tetlet.2010.12.013.
0.0
400
450
500
550
600
650
700
Wavelength (nm)
Figure 3. Normalized fluorescence emission spectra of compound 16 in various
solvents.
References and notes
1. Yushchenko, D. A.; Bilokin’, M. D.; Pyvovarenko, O. V.; Duportail, G.; Mely, Y.;
Pivovarenko, V. G. Tetrahedron Lett. 2006, 47, 905–908.
2. Yushchenko, D. A.; Shvadchak, V. V.; Klymchenko, A. S.; Duportail, G.; Mely, Y.;
Pivovarenko, V. G. New J. Chem. 2006, 30, 774–778.
3. Yushchenko, D. A.; Shvadchak, V. V.; Klymchenko, A. S.; Duportail, G.;
Pivovarenko, V. G.; Mely, Y. J. Phys. Chem. A 2007, 111, 8986–8992.
4. Bilokin‘, M. D.; Shvadchak, V. V.; Yushchenko, D. A.; Klymchenko, A. S.;
Duportail, G.; Mely, Y.; Pivovarenko, V. G. Tetrahedron Lett. 2009, 50, 4714–4719.
5. Bilokin‘, M. D.; Shvadchak, V. V.; Yushchenko, D. A.; Duportail, G.; Mely, Y.;
Pivovarenko, V. G. J. Fluoresc. 2009, 19, 545–553.
meric forms2,4 grew to the benefit of those with fluorescence emis-
sion at lower wavelength. Surprisingly, a similar dependence be-
tween length of alkyl chain and the spectral properties was not
observed for compounds containing hydroxyalkyl chains (entries
11–14).
The effect of solvents (Table 3, Fig. 3) on the emission spectra of
compounds 11, 16 and 17 (representative compounds with higher
and lower quantum yields) was not significant and any obvious
relationship between polarity and the ratio of maxima intensities
was not found (see Fig. 3 with the emission spectra of compound
16 as an example). On the other hand, the quantum yields of these
representatives were meaningfully affected by solvents (Table 3).
Generally, the highest quantum yield was observed for toluene
and the lowest for methanol (16) and/or methanol/water 1:1 (11,
17). In the case of compound 11 the quantum yield of a toluene
solution (21.30%) was twice than that of a methanolic solution
(10.21%).
In conclusion, the fluorescence properties of 2-phenyl-substi-
tuted-3HQs were studied from the point of view of their applica-
tion as a part of a biomolecule-fluorescence probe system. It was
found that the 2-phenyl substituents of the 3HQs affected signifi-
cantly the fluorescence quantum yield, which varied from
1.6 Â 10À3 (2) to 10.21% (11). Moreover, in the case of compounds
3–5 the shape of the emission spectra was influenced by the car-
bon chain length. However, this was not the case for compounds
11–14 with hydroxyalkyl chains of different length.
ˇ
6. Motyka, K.; Hlavác, J.; Soural, M.; Funk, P. Tetrahedron Lett. 2010, 51, 5060–5063.
ˇ
ˇ
7. Krejcí, P.; Hradil, P.; Hlavác, J.; Hajdúch, M. Patent WO 2008028427, 2008; Chem.
Abstr. 2008, 148, 331,570. Compounds 1 and 2 were prepared by the following
procedure: 2-(4-chloro-3-nitrophenyl)-2-oxoethyl 2-aminobenzoate or 2-(4-
amino-3-nitrophenyl)-2-oxoethyl
2-aminobenzoate
(44.8 mmol)
was
suspended in polyphosphoric acid (167.3 g). The reaction mixture was heated
to 100 °C and stirred for 90 min. The mixture was then poured into H2O/crushed
ice (700 ml). The precipitated product was filtered, washed with H2O, dried and
recrystallized from 2-methoxyethanol. Compounds 3–17 were prepared by the
following general procedure: quinolinone 1 (200 mg, 0.63 mmol) was added to a
solution of amine (6.3 mmol) and N-methylpyrrolidone (1.0 ml) and the mixture
was stirred at 110 °C for 2 h. After cooling to room temperature, H2O (20 ml)
was added and the pH adjusted to 7 with dilute HCl (1:3). The precipitated solid
was collected by suction, washed thoroughly with H2O and dried at 80 °C. The
crude product was recrystallized from 2-methoxyethanol.
8. Yields and analytical data of selected compounds (the remainder are provided as
Supplementary data): 2-(4-Chloro-3-nitrophenyl)-3-hydroxyquinoline-4(1H)-one
(1): Yellow powder, 11.93 g (84%), mp 284–287 °C. 1H NMR (300 MHz, DMSO-
d6): d 7.29 (t, J = 7.9 Hz, 1H, ArH); 7.63 (t, J = 5.3 Hz, 1H, ArH); 7.69 (d, J = 8.4 Hz,
1H, ArH); 7.99 (d, J = 8.4 Hz, 1H, ArH); 8.14 (d, J = 2.4 Hz, 1H, ArH); 8.17 (d,
J = 2.3 Hz, 1H, ArH); 8.52 (s, 1H, ArH); 11.67 (br s, 1H, OH). MS: m/z 319.9
[M(37Cl)+H]+,
C15H9ClN2O4
calcd
316.70.
3-Hydroxy-2-(4-amino-3-
nitrophenyl)quinolin-4(1H)-one (3): Dark red powder, 170 mg (75%), mp 150–
156 °C. 1H NMR (300 MHz, DMSO-d6): d 2.88 (t, J = 5.85 Hz, 2H); 3.38–3.53 (t,
2H); 7.18–7.33 (m, 2H); 7.58 (t, J = 6.9 Hz, 1H); 7.72 (d, J = 7.8 Hz, 1H); 7.97–8.18
(m, 3H); 8.63 (br s, 2H). MS: m/z 341.1 [M+H]+, C17H16N4O4 calcd 340.33.
9. Melhuish, W. H. J. Phys. Chem. 1961, 65, 229–235.
The results show that introducing substituted alkyl groups
(with terminal amino or hydroxy groups) to compound 1 led
mostly to increased fluorescence quantum yields.