Fluorescence Study of Imidazoquinoxalines

Article abstract of DOI:10.1007/s10895-017-2097-z

The fluorescence properties of eleven novel derivatives based on the imidazo[1,2-a]quinoxaline structures have been studied. The absorption and emission spectra of these compounds have been recorded in dimethylsulfoxide solution. The phenyl substituting group on position 1 gives them particular properties thanks to the diverse hydroxy or methoxy decorating moieties, especially when they are multiplied or mixed. The investigated fluorescence auto-quenching revealed that the decreasing fluorescence intensity correlated only with the chemical structures of the aromatic compounds.

Full text of DOI:10.1007/s10895-017-2097-z

J Fluoresc
DOI 10.1007/s10895-017-2097-z
ORIGINAL ARTICLE
Fluorescence Study of Imidazoquinoxalines
Cindy Patinote1,2 & Kamel Hadj-Kaddour1 & Marjorie Damian1
&
Carine Deleuze-Masquéfa1 & Pierre Cuq1 & Pierre-Antoine Bonnet1
Received: 19 January 2017 /Accepted: 4 April 2017
#
Springer Science+Business Media New York 2017
Abstract The fluorescence properties of eleven novel deriv-
atives based on the imidazo[1,2-a]quinoxaline structures have
been studied. The absorption and emission spectra of these
compounds have been recorded in dimethylsulfoxide solution.
The phenyl substituting group on position 1 gives them par-
ticular properties thanks to the diverse hydroxy or methoxy
decorating moieties, especially when they are multiplied or
mixed. The investigated fluorescence auto-quenching re-
vealed that the decreasing fluorescence intensity correlated
only with the chemical structures of the aromatic compounds.
example as probes in chemosensors. [8] Quinoxaline deriva-
tives are used for example as base for probes in the field of
bio-imaging. [9.]
Imiquimod, the international nonproprietary name of 1-
isobutyl-1H-imidazo[4,5-c]quinolin-4-amine (Scheme 1) is
the analogue of our entire synthetized molecules. [ 10–13]
This compound was already the subject of experimental and
theoretical fluorescent studies. [14, 15] Its relatively high fluo-
rescence intensity results from an extended π-conjugated
system.
In the course of our studies concerning the synthesis of
imidazo[1,2-a]quinoxaline derivatives as potential anti-
cancer compounds, we were confronted to the peculiar fluo-
rescence response of our compounds incubated with cancer-
ous cells. [16] In fact, a cytometric study using propidium
iodide revealed fluorescence more important than predicted
(data not shown). Thus, in order to determine whether the
signal increase is due to our organic molecules, we were con-
sequently interested by a fluorescence study of our com-
pounds. We present herein briefly the synthesis of our mole-
cules and their absorption and emission spectra.
.
.
.
Keywords Imidazoquinoxaline Imiquimod Fluorescence
Absorption
Introduction
Heterocyclic compounds are known to have fluorescent prop-
erties and are therefore of interest in many fields such as mo-
lecular probes for biochemical research [1, 2], fluorescent
whitening agents [3, 4], photoconducting material [5] and
electroluminescent devices [6]. Because of their high quantum
yield, quinolines are fluorescent compounds [7] used for
Experimental Part
Chemistry
Cindy Patinote and Kamel Hadj-Kaddour contribute equally to this work.
The synthetic pathway and the structures of the imidazo[1,2-
a]quinoxaline derivatives used in this study are given re-
spectively in Schemes 2 and 3. Compounds were synthe-
sized thanks to a route we previously described [11, 13,
16]. Briefly, the carbonylimidazole dimer 2 results from
the condensation of the 2-imidazole carboxylic acid 1 in
presence of thionyl chloride. Addition of the o-fluoroaniline
on the dimer 2 gives the intermediate 3. Cyclisation is
* Cindy Patinote
cindy.patinote@gmail.com
1
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247,
CNRS, Université de Montpellier, Faculté de Pharmacie, 15 avenue
Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France
2
Société d’Accélération du Transfert de Technologies (SATT AxLR),
CSU, 950 rue Saint Priest, 34090 Montpellier, France

J Fluoresc
Methods
Absorption spectra were recorded on a UV Visible spec-
trophotometer (Cary 400, Varian) from 250 to 400 nm.
Fluorescence spectra were recorded on a Fluorescence
spectrophotometer (Cary Eclipse, Varian) from 300 to
600 nm.
Solutions at 1.10−2 M of each compound were prepared in
DMSO. These working standard solutions were then diluted
with DMSO to obtain 1.10−4 M as final concentrations.
Results and Discussion
Scheme 1 Chemical structures of Imiquimod
In a first study, UV spectra of all substituted compounds at
1.10−4 M were recorded (Fig. 1). Higher concentrations
caused absorbance saturation for some molecules. Lower con-
centrations gave no analyzable results for some compounds.
Spectra show similar profiles with maxima around 270 nm
which is characteristic of the phenol moiety, more particularly
here of the catechol part, then a decrease until 300 nm.
Successive waves with secondary maxima could be observed
around 320 nm. It seems to be a characteristic of the
imidazo[1,2-a]quinoxaline, as depicted by compound EAPB
0003. No absorbance was detected after 360 nm in the visible
region.
allowed by using sodium hydroxide in dimethylacetamide.
Treatment of compound 4 with phosphorus oxychloride and
N,N-diethylaniline gives the chlorinated compound 5. Then,
an amino group substitutes the chlorine. To obtain the
NHCH3 residue 6, methylamine in ethanol is grafted under
microwave asssitance. To give the NH2 residue, ammonia is
used in acetonitrile also under microwave asssitance. The
bromination of the intermediate 6 by N-bromosuccinimide
led to compound 7. Appropriate aryl boronic acids are in-
troduced in position 1 via a Suzuki-Miyaura cross-coupling
reaction to furnish compounds 8. Some intermediates were
submitted to boron tribromide to give the hydroxylated de-
rivatives 9.
A slight tone could be done for compounds EAPB
02503 and EAPB 02902, which first maxima are closer
than 260 nm. Nevertheless, they also display second max-
ima at 270 nm.
Scheme 2 General synthetic
pathway

J Fluoresc
Scheme 3 Compounds
structures
Maxima at 270 nm for compounds EAPB 02203 and EAPB
02303 should be nuanced. Even if they absorb in this region, the
maxima are less marked than the other compounds and corre-
spond to a steady state. The decrease is evident after 290 nm.
The substitute-devoid compound EAPB 0403 absorbs
slightly less than the mono-hydroxylated compound EAPB
0603. Absorbance of the mono-methoxy derivative EAPB
0503 is quite divided by two between 260 nm and 330 nm.
Whereas absorbance for compounds EAPB 02403 and EAPB
02503 are equivalent, it is not the case for the others. Compounds
with two methoxy groups, EAPB 01803 and EAPB 02203, ab-
sorb more than their respective di-hydroxy derivatives EAPB
01903 and EAPB 02303. Despite being less intense, compound
EAPB 2902 shows a similar profile than compound EAPB 2503.
According to the UV spectra results, fluorescence spectra
were recorded by excitation at 270 nm (Fig. 2) for all com-
pounds at 1.10−4 M. As already observed for the absorbance
study, compounds EAPB 0403, EAPB 01813 and EAPB 0603
show higher fluorescence intensities than compounds EAPB
0503, EAPB 02403 and EAPB 01903. They display the same
maximum at around 395 nm. Fluorescence spectra of com-
pounds EAPB 02203 and 02303 are shifted to the higher wave-
lengths. The maximum is around 420 nm for EAPB 02203 and
around 445 nm for EAPB 02303. The fluorescence intensity is
divided by three between those two compounds. In a general
way, the di-hydroxy compounds show lower fluorescence in-
tensities than their respective di-methoxy derivatives.
Compounds EAPB 02503 and EAPB 02902 show a very
low fluorescence intensity when excited at 270 nm or 260 nm
(first maxima in their absorbance spectra).
The fluorescence intensities are clearly different for these
compounds of the same chemical family tested at the same
concentration. Those results suggest a kind of auto-quenching
phenomenon for EAPB 02303, EAPB 02902 and EAPB
3
2,5
2
Fig. 1 UV spectra
EAPB 0003 + catechol
EAPB 0403
EAPB 0503
EAPB 0603
1,5
1
EAPB 02203
EAPB 02303
EAPB 01803
EAPB 01903
EAPB 02403
EAPB 02503
0,5
0
EAPB 02902
catechol
254
274
294
314
334
354
374
-0,5
-1
EAPB 0003
Waveleng th (nm)

J Fluoresc
700
600
500
400
300
200
100
0
Fig. 2 Fluorescence spectra
EAPB 0403
EAPB 0503
EAPB 0603
EAPB 02203
EAPB 02303
EAPB 01803
EAPB 01903
EAPB 02403
EAPB 02503
EAPB 02902
300
350
400
450
500
550
600
Wavelength (nm)
40
Fig. 3 Fluorescence Spectra
of EAPB 02902
35
30
25
20
15
10
5
1.10-6
5.10-6
1.10-5
5.10-5
1.10-4
M
M
M
M
M
0
300
350
400
450
500
550
600
Wavelength (nm)
40
Fig. 4 Fluorescence Spectra
of EAPB 02503
35
30
25
20
15
10
5
1.10-4
5.10-5
1.10-5
5.10-6
1.10-7
M
M
M
M
M
0
300
350
400
450
500
550
600
Wavelength (nm)

J Fluoresc
450
400
350
300
250
200
150
100
50
Fig. 5 Fluorescence Spectra of
EAPB 02303
1.10-5
5.10-5
1.10-4
5.10-4
1.10-3
1.10-2
M
M
M
M
M
M
0
300
350
400
450
500
550
600
Wavelength (nm)
02503. Catechols are known to behave as quencher, thanks to
their ability to absorb photon energy, resulting in their oxida-
tion [17–19]. Fluorescence quenching is easily characterized
by a decrease of fluorescence with increase substrate concen-
tration. To explore the quenching properties of some of our
compounds, concentrations range in dimethylsulfoxide have
been prepared for EAPB 02902, EAPB 02503 and EAPB
02303, ranging respectively from 1.10−6 M to 1.10−4 M,
1.10−7 M to 1.10−4 M and 1.10−5 M to 1.10−2 M. Their fluo-
rescence spectra are recorded and depicted respectively on
Fig. 3, Fig. 4 and Fig. 5. The limit of detection for EAPB
02902 and EAPB 02503 correspond to 1.10−7 M. We observe
on these spectra that fluorescence intensities increase with the
concentration. So, there is no quenching of our compounds in
the concentrations used for biological experiments.
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UV and fluorescence spectra of eleven compounds based on
the imidazo[1,2-a]quinoxaline structure are presented here.
Even if all of these compounds refer to the same chemical
family, differences were observed in their absorbance and
emission responses, particularly the intensities. In our condi-
tions, no auto-quenching fluorescence was observed for these
compounds. The observed decreasing fluorescence intensity
is only correlated to the particular structures and polyhydroxy
substitutions of the aromatic studied compounds.
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Acknowledgements We would like to thank the Société d’Accélération
du Transfert de Technologies (SATT AxLR) for financial support for Dr.
Cindy Patinote. We also thank the team Cellular Pharmacology from the
IBMM directed by Dr. Jean-Louis Banères for technical support with UV
and fluorescence spectra recording.