Arylstyrylimidazo[1,2-a]pyridine-based donor-acceptor acidochromic fluorophores: Synthesis, photophysical, thermal and biological properties

Article abstract of DOI:10.1016/j.jphotochem.2015.05.030

A series of new fluorescent arylstyrylimidazo[1,2-a]pyridines was synthesized and fully characterized. It is observed that the E isomers are stable in the solid state and in solutions. The absorption and emission properties of the compounds changed drasti

Full text of DOI:10.1016/j.jphotochem.2015.05.030

Accepted Manuscript
Title: Arylstyrylimidazo[1,2-a]pyridine-based donor-acceptor
acidochromic fluorophores: Synthesis, photophysical, thermal
and biological properties
Author: Burcu Aydıner Ergin Yalc¸ın Heiko Ihmels Leyla
˘
Arslan Leyla Ac¸ık Zeynel Seferoglu
PII:
S1010-6030(15)00195-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2015.05.030
JPC 9918
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Journal of Photochemistry and Photobiology A: Chemistry
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Accepted date:
14-4-2015
21-5-2015
23-5-2015
Please cite this article as: Burcu Aydiner, Ergin Yalc¸in, Heiko Ihmels, Leyla
˘
Arslan, Leyla Ac¸ik, Zeynel Seferoglu, Arylstyrylimidazo[1,2-a]pyridine-based
donor-acceptor acidochromic fluorophores: Synthesis, photophysical, thermal and
biological properties, Journal of Photochemistry and Photobiology A: Chemistry
http://dx.doi.org/10.1016/j.jphotochem.2015.05.030
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Arylstyrylimidazo[1,2-a]pyridine-based donor-acceptor acidochromic fluorophores:
Synthesis, photophysical, thermal and biological properties
Burcu Aydınera, Ergin Yalçına, Heiko Ihmelsb, Leyla Arslanc, Leyla Açıkc, Zeynel
Seferoğlua,b*znseferoglu@gazi.edu.tr
aGazi University, Department of Chemistry, 06500 Ankara, Turkey
bUniversity of Siegen, Organic Chemistry II, Adolf-Reichwein-Str. 2, D-57068 Siegen,
Germany
cGazi University, Department of Biology, 06500 Ankara, Turkey
* Corresponding author Tel.: +90 312 2021525; fax.: +90 312 2122279
1

Graphical abstract
Highlights
 A series of new fluorescent arylstyrylimidazo[1,2-a]pyridines was synthesized and
fully characterized.
 The absorption and emission properties of the compounds change drastically upon
acidification.
 The compounds show good thermal stability for practical applications.
 They show DNA cleavage activity.
2

Abstract
A series of new fluorescent arylstyrylimidazo[1,2-a]pyridines was synthesized and fully
characterized. It is observed that the E isomers are stable in the solid state and in solutions.
The absorption and emission properties of the compounds changed drastically upon
acidification. The compounds showed good thermal stability for practical applications. In
addition, antimicrobial, antioxidant, and DNA-cleavage activity of the compounds were
examined. The results indicate that the compounds did not show any antimicrobial and
antioxidant activity. However, they caused decreased mobility of form I DNA and also
showed DNA cleavage activity.
Keywords
Styryl dyes, heterocycles, imidazo[1,2-a]pyridines, pH sensitive dyes, fluorescent probes,
DNA cleavage activity.
3

1. Introduction
Functional fluorescent dyes bearing donor-acceptor groups are widely investigated as active
materials in dye chemistry. These type of dyes, with appropriate absorption and emission
color, are useful components for the development of optical chemosensors, photonic materials
or photosensitizers [1-4]. Styryl dyes were described as fluorescent compounds with high
quantum yield after interaction with biomolecules such as DNA, RNA etc. [5-8]. Also, the
dyes have been established as a promising class of compounds that exhibit a great potential to
be employed in advanced materials such as laser dyes, optical or electro-optical devices or
sensor materials due to their excellent optical-electronic properties [9-30]. Moreover, many
potential metal-free solar cell sensitizers are neutral styryl dyes [31]. However, some neutral
styryl dyes also show thermally induced or photo-induced E-Z isomerization and dimerization
especially in solution [32]. Overall, this class of styryl dyes exhibits a remarkable structural
diversity.
Molecules which contain more than one nitrogen atoms, are important class of heterocyclic
chemistry [33]. The imidazo[1,2-a]pyridines have been intensively investigated, well-known
and with an attractive core inside the imidazopyridine ring system. They have significant
importance in the pharmaceutical industry owing to their interesting biological activity
display over a broad range of therapeutic classes. These molecules exhibit antiviral, anti-
inflammatory, antitumor, analgesic, antipyretic, antiulcer and antibacterial properties [34-38].
In addition, drugs containing imidazo[1,2-a]pyridine ring such as zolpidem (hypnotic) are
currently available on the market. Therefore, we proposed that the integration of
imidazopyridine units into styryl-type structures leads to novel dye structures with promising
properties that may be considered for the development of functional dyes. In our previous
study we synthesized and investigated some photophysical properties of
4

arylstyrylimidazo[1,2-a]pyridine-based fluorescent dyes [39]. In our current study, we have
synthesized and evaluated phtophysical, biological and thermal properties of some
arylstyrylimidazo[1,2-a]pyridine dyes bearing chloro and bromo substituents. These
substituents can be beneficial in increasing the electronic delocalization along the skeleton of
the donor-acceptor structures using cross-coupling reactions [11b].
2. Experimental Section
2.1.
General Instrumentations and Materials
All the chemicals used were commercially available, of spectroscopic grade, and used without
further purification. The melting points were determined with a Büchi 510 K melting point
apparatus and were not corrected. Mass spectra (ESI in the positive-ion mode, source voltage
6 kV) were recorded with a Finnigan LCQ Deca instrument; only m/z values in the range of
100-2000 units were analyzed. NMR spectra were measured on Bruker Avance 400 (1H: 400
13
MHz, C: 100 MHz) spectrometers at 20 °C (293 K); chemical shifts are given in ppm (δ)
relative to TMS (δ = 0.00 ppm). Coupling constants (J) are given in hertz (Hz). Signals are
abbreviated as follows: singlet, s; doublet, d; doublet-doublet, dd; triplet, t; multiplet, m.
Elemental microanalysis of the compounds was performed with a HEKAtech EuroEA
combustion analyzer by Mr. H. Bodenstedt (Organische Chemie I, Universität Siegen). The
X-ray diffraction data collection was performed on a Rigaku R-AXIS RAPID-S
diffractometer equipped with Mo Kα (λ=0.71073 Å) radiation using w scans.
5

2.2.
Spectrophotometric measurements
Absorption spectra were recorded on a Varian Cary 100 double-beam spectrophotometer;
fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer.
All spectrophotometric measurements were performed in thermostated quartz sample cells at
20 °C, using spectral grade solvents (Fluka, Riedel-de Haën). The solution concentrations
were 10 μM for absorption spectroscopy and 1.0-5.0 μM for fluorescence spectroscopy.
Spectrophotometer slit widths were set to bandwidths of 5 nm for emission spectroscopy. The
relative fluorescence quantum yields, fl, were determined by standard methods with
Coumarin 1 (laser grade, Acros Organics, fl = 0.73 in ethanol) as a reference [40,41].
2.3.
X-ray crystallography
Single crystal X-ray diffraction data were collected on Rigaku R-AXIS RAPID-S
diffractometer equipped with a IP detector, using monochromatized Mo Kα (λ=0.71073 A°)
radiation. Integration of the intensities, correction for Lorentz and polarization effects and cell
refinements were performed using CrystalClear (Rigaku/MSC Inc., 2005) software [42]. The
structures were resolved by direct methods SHELXS97 [43] and refined on F2 by full-matrix
least-squares using the programs SHELXL97 [43]. All non-hydrogen atoms were refined
anisotropically. Positions of H atoms bonded to C atoms were calculated at ideal positions
and refined by the riding model with Uiso -1.5Ueq(C) for methyl, and Uiso -1.2Ueq(C) for
remaining H atoms. The final difference Fourier maps showed no peaks of chemical
significance.
6

2.4.
The synthesis of imidazo[1,2-a]pyridine derivatives
2-(4-Chlorophenyl)-7-methylimidazo[1,2-a]pyridine
(3a)
and
2-(4-bromophenyl)-7-
methylimidazo[1,2-a]pyridine (3b) were synthesized using literature methods. Compound 3b
was synthesized and characterized in our previous paper [39]. Compound 3a was synthesized
using literature method and characterization data for 2-(4-chlorophenyl)-7-
methylimidazo[1,2-a]pyridine are shown below [39].
2-(4-chlorophenyl)-7-methylimidazo[1,2-a]pyridine: The light colorless crystals were
obtained after recrystalization with ethanol at 79 % yield (1.91 g); mp. 197-198 °C (lit. mp.
1
196-197 ℃ [44]); H NMR (400 MHz, CDCl3) (δ, ppm): 7.98 (d, J = 6.8 Hz, 1H), 7.87 (d,
2H, J = 8.6 Hz), 7.72 (s, 1H), 7.31 (d, 2H, J = 8.6 Hz), 7.24 (s, 1H), 6.62 (d, J = 6.9 Hz, 1H),
2.40 (s, 3H); 13C NMR (100 MHz, CDCl3) (δ, ppm): 145.77, 143.79, 136.36, 133.56, 131.88,
128.78, 127.11, 124.76, 115.59, 115.42, 107.54, 21.33; MS (ESI+): m/z = 243.5 [M+H, 35Cl]+,
245.5 [M+H, 37Cl]+; Anal. Calcd for C14H11ClN2: C, 69.28; H, 4.57; N, 11.54. Found: C,
69.12; H, 4.48; N, 11.61.
2.5.
The synthesis of the imine derivatives 4a-f
All imine derivatives were synthesized and their structures were confirmed using literature
methods [39].
7

2.6.
General procedure for the synthesis of styrylimidazo[1,2-a]pyridine derivatives
A stirred mixture of 2-substituted-7-methylimidazo[1,2-a]pyridine (10 mmol) and Potassium
tert-butoxide (t-BuOK) (25 mmol) and the corresponding imine derivatives (10 mmol) in
DMF (20 mL) was heated at 90 °C for 4 h. The mixture was allowed to cool and
methanol/water mixture (10:1) was added. The precipitate was filtered and dried. The crude
product was recrystallized from EtOAc or EtOAc/MeOH to yield the pure compound as
crystalline solid or as fine powder.
7-[(E)-2-(phenyl)vinyl]-2-(4-chlorophenyl)imidazo[1,2-a]pyridine (5a): The solid formed
was collected by filtration and recrystallized using EtOAc/DME to obtain a brown solid
1
compound at 84 % yield (0.28 g); mp: 254-255 °C (lit. mp 252-253 °C [44]); H NMR
(CDCl3) (δ, ppm): 8.13 (d, 1H), 7.93 (m, 3H), 7.85 (s, 1H), 7.71 (s br 1H), 7.55 (d, 2H, J = 8.6
Hz), 7.38-7.44 (m, 3H), 7.33 (t, 1H), 7.19-7.23 (d, 1H, Jtrans = 16.3 Hz), 7.16 (d, 1H), 7.09-
13
7.15 (d, 1H, Jtrans = 16.3 Hz); C NMR (100 MHz, CDCl3) (δ, ppm): 159.6, 146.4, 144.5,
137.19, 132.03, 129.83, 129.66, 129.25, 128.11, 127.64, 127.19, 115.43, 115.66, 109.25; MS
(ESI+): m/z = 331.8 [M+H, 35Cl]+, 333.8 [M+H, 37Cl]+; Anal. Calcd for C21H15ClN2: C, 69.28;
H, 4.57; N, 11.54. Found: C, 69.12; H, 4.48; N, 11.61.
7-[(E)-2-(phenyl)vinyl]-2-(4-bromophenyl)imidazo[1,2-a]pyridine (5b): The solid formed
was collected by filtration and recrystallized using EtOAc to obtain a solid yellowish
compound at 78 % yield (0.48 g); mp: 262-263 °C; 1H NMR (CDCl3) (δ, ppm): 8.10 (d, 1H),
7.85 (m, 3H), 7.68 (s, 1H), 7.54-7.58 (m, 3H), 7.40 (m, 3H), 7.32 (m, 1H), 7.17-7.21 (d, 1H,
Jtrans = 16.3 Hz), 7.15 (d, 1H), 7.08-7.13 (d, 1H, Jtrans = 16.3 Hz); 13C NMR (100 MHz,
79
CDCl3) (δ, ppm): It was not obtained good spectrum; MS (ESI+): m/z = 377.6 [M+H Br]+,
8

378.7 [M+H 81Br]+; Anal. Calcd for C21H15BrN2: C, 67.21; H, 4.03; N, 7.47. Found: C, 66.98;
H, 3.89; N, 7.35.
7-[(E)-2-(4-Piperidinylaminophenyl)vinyl]-2-(4-chlorophenyl)phenylimidazo[1,2-
a]pyridine (5c): The solid formed was collected by filtration and recrystallized using
EtOAc/MeOH to obtain the compound as a light yellowish powder at 88 % yield (0.47 g);
1
mp: 308-309 °C; H NMR (CDCl3) (δ, ppm): 8.03 (d, 1H), 7.89 (d, 2H, J = 8.7 Hz), 7.78 (s,
1H), 7.52 (s br 1H), 7.43 (d, 2H, J = 8.6 Hz), 7.39 (d, 2H, J = 8.7 Hz), 7.07-7.10 (d, 1H, Jtrans
= 16.3 Hz), 7.33 (t, 1H), 6.91-6.95 (m, 3H), 3.26-3.29 (m, 4H), 1.69-1.73(m, 4H), 1.61-1.64
(m, 2H); 13C NMR (100 MHz, CDCl3) (δ, ppm): 160.4, 155.7, 144.6, 137.13, 133.13, 129.12,
128.13, 127.38, 127.32, 125.32, 122.19, 115.71, 113.68, 108.20, 49.80, 25.56, 24.3; MS
(ESI+): m/z = 414.9 [M+H, 35Cl]+, 416.8 [M+H, 37Cl]+; Anal. Calcd for C26H24ClN3: C,
75.44; H, 5.84; N, 10.15 Found: C, 75.11; H, 5.72; N, 10.01.
7-[(E)-2-(4-Piperidinylaminophenyl)vinyl]-2-(4-bromophenyl)imidazo[1,2-a]pyridine
(5d): The solid formed was collected by filtration and recrystallized using EtOAc to obtain
1
the compound as light yellowish solid at 74 % yield (0.48 g); mp: 272-273 °C; H NMR
(CDCl3) (δ, ppm): 8.04 (d, 1H), 7.95 (d, 2H, J = 8.8 Hz), 7.80 (m, 4H), 7.78 (s, 1H), 7.52 (s br
1H), 7.43 (d, 2H, J = 8.6 Hz), , 7.30 (m, 1H), 7.04-7.10 (m, 3H), 6.90-6.94 (m, 3H), 3.22-3.25
(m, 4H), 1.70-1.74 (m, 4H), 1.63-1.69 (m, 2H); 13C NMR (100 MHz, CDCl3) (δ, ppm): 159.6,
152.1, 144.6, 131.85, 128.78, 128.13, 127.88, 127.51, 126.04, 125.14, 123.03, 115.80, 110.56,
108.13, 49.87, 25.62, 24.32; MS (ESI+): m/z = 459.6 [M+H 79Br]+, 460.5 [M+H 81Br]+; Anal.
Calcd for C26H24BrN3: C, 68.12; H, 5.28; N, 9.17. Found: C, 68.28; H, 5.45; N, 9.57.
7-[(E)-2-(4-Diethylaminophenyl)vinyl]-2-(4-chlorophenyl)phenylimidazo[1,2-a]pyridine
(5e): The solid formed was collected by filtration and recrystallized using EtOAc/MeOH to
1
obtain the compound as yellow crystals at 81 % yield (0.47 g); mp: 224-225 °C; H NMR
(CDCl3) (δ, ppm): 8.01 (d, 1H), 7.87 (d, 2H, J = 8.9 Hz), 7.76 (s, 1H), 7.49 (s br 1H), 7.38-
9

7.42 (m, 4H), 7.04-7.09 (m, 2H), 6.84-6.88 (d, 1H, Jtrans = 16.3 Hz), 6.67 (d, 2H, J = 8.8 Hz),
3.37-3.44 (q, 6H), 1.17-1.21 (t, 4H); 13C NMR (100 MHz, CDCl3) (δ, ppm): 147.8, 146.1,
145.1, 135.8, 133.5, 132.5, 130.9, 128.8, 128.2, 127.1, 125.0, 123.7, 121.5, 113.7, 111.61,
35
37
110.36, 108.1, 44.4, 12.6; MS (ESI+): m/z = 402.6 [M+H, Cl]+, 404.6 [M+H, Cl]+; Anal.
Calcd for C25H24ClN3: C, 74.71; H, 6.02; N, 10.45 Found: C, 74.55; H, 5.98; N, 10.41.
7-[(E)-2-(4-Diethylaminophenyl)vinyl]-2-(4-bromophenyl)phenylimidazo[1,2-a]pyridine
(5f): The solid formed was collected by filtration and recrystallized using EtOAc/MeOH to
1
obtain the compound as bright yellowish crystals at 76 % yield (0.33 g); mp: 239-240 °C; H
NMR (CDCl3) (δ, ppm): 7.99 (d, 1H), 7.82 (d, 2H, J = 8.8 Hz), 7.75 (s, 1H), 7.55 (d, 2H, J =
8.8 Hz), 7.55 (s br 1H), 7.40 (d, 2H, J = 8.9 Hz), 7.03-7.08 (m, 2H), 6.83-6.87 (d, 1H, Jtrans
=
16.3 Hz), 6.66 (d, 2H, J = 8.8 Hz), 3.36-3.42 (q, 6H), 1.17-1.21 (t, 4H); 13C NMR (100 MHz,
CDCl3) (δ, ppm): 147.9, 145.4, 144.2, 136.2, 131.8, 131.2, 128.4, 128.3, 127.5, 127.1, 125.0,
123.6, 121.8, 121.3, 113.3, 111.6, 110.6, 108.1, 44.4, 12.6; MS (ESI+): m/z = 446.6 [M+H
79Br]+, 448.6 [M+H 81Br]+; Anal. Calcd for C25H24BrN3: C, 67.27; H, 5.42; N, 9.41 Found: C,
67.15; H, 5.31; N, 9.41.
7-[(E)-2-(4-Dimethylaminophenyl)vinyl]-2-(4-chlorophenyl)phenylimidazo[1,2-
a]pyridine (5g): The solid formed was collected by filtration and recrystallized using
EtOAc/MeOH to obtain the compound as bright yellowish powder at 84 % yield (0.31 g); mp:
1
308-309 °C; H NMR (CDCl3) (δ, ppm): 8.07 (d, 1H), 7.91-7.93 (d, 2H, J = 8.8 Hz), 7.78 (s,
1H), 7.73 (s br 1H), 7.40-7.44 (m, 4H), 7.14-7.18 (m, 2H), 6.85-6.89 (d, 1H, Jtrans = 16.3 Hz),
13
6.70-6.72 (d, 2H, J = 8.8 Hz), 3.01 (s, 6H); C NMR (100 MHz, CDCl3) (δ, ppm): 155.1,
151.5, 146.8, 145.1, 144.8, 134.7, 131.0, 128.0, 127.1, 125.0, 123.9, 122.1, 114.6, 113.0,
35
37
110.9, 107.6, 55.8; MS (ESI+): m/z = 374.7 [M+H, Cl]+, 376.7 [M+H, Cl]+; Anal. Calcd
for C23H20ClN3: C, 73.89; H, 5.39; N, 11.24 Found: C, 73.40; H, 5.54; N, 11.18.
10

7-[(E)-2-(4-Dimethylaminophenyl)vinyl]-2-(4-bromophenyl)phenylimidazo[1,2-
a]pyridine (5h): The solid formed was collected by filtration and recrystallized using
EtOAc/MeOH to obtain the compound as bright yellowish powder at 77 % yield (0.32 g); mp:
1
310-311 °C; H NMR (CDCl3) (δ, ppm): 8.09 (d, 1H), 7.86-7.88 (d, 2H, J = 8.7 Hz), 7.79 (s,
1H), 7.77 (s br 1H), 7.56-7.58 (d, 2H, J = 8.7 Hz), 7.42-7.44 (d, 2H, J = 8.9 Hz), 7.17-7.21 (d,
1H, Jtrans = 16.3 Hz), 7.22 (s, 1H), 6.85-6.89 (d, 1H, Jtrans = 16.3 Hz), 6.70-6.72 (d, 2H, J = 8.9
Hz), 3.01 (s, 6H); 13C NMR (100 MHz, CDCl3) (δ, ppm): It was not obtained good spectrum;
79
81
MS (ESI+): m/z = 418.8 [M+H Br]+, 420.7 [M+H Br]+; Anal. Calcd for C23H20BrN3: C,
66.04; H, 4.82; N, 10.04 Found: C, 65.99; H, 4.70; N, 9.92.
3. Results and Discussion
3.1. Preparation of arylstyrylimidazo[1,2-a]pyridine based fluorophores
The aryl-substituted methylimidazo[1,2-a]pyridine derivatives 3a,b were synthesized from 2-
bromoacetophenone derivatives 2a,b and 4-methyl-2-aminopyridine in good yields (Scheme
1). Light colorless crystal for 3a was obtained by slow evaporation using methanol and it was
also characterized using X-ray diffraction analysis (Figure 1). The imine derivatives 4a–d
were prepared by the reaction of aromatic aldehydes and the corresponding 4-chloroaniline
derivative [39]. The arylstyrylimidazo[1,2-a]pyridine derivatives 5a–h were obtained by the
base-catalyzed condensation of the imine derivatives 4a–d and the 2-aryl-substituted-7-
methylimidazo[1,2-a]pyridine derivatives 3a–c at 90 oC in DMF (Scheme 1). The
experimental protocol is straightforward and offers easy access to a wide variety of
arylstyrylimidazo[1,2-a]pyridines containing electron-donating groups in good yields. All
compounds 5a–h were characterized by NMR spectroscopy, mass spectrometry, and
11

elemental analysis. NMR spectroscopy proved to be very useful in assessing the configuration
of the vinylic double bond. Within the limit of NMR-spectroscopic analysis only the
formation of the E isomers could be detected. The assignment of the E-configuration of the
vinylic double-bond was based on the analysis of the vicinal coupling constants of the olefinic
protons, because the latter exhibit the characteristic coupling constant of J  16 Hz. This
coupling constant for the vinylic protons clearly support the selective formation of E-
configuration of the vinylic double-bond.
Figure 1 is here
Scheme 1 is here
3.2. Absorption and emission properties
The styryl derivatives 5a-h are colored in solid state and in solution. To assess the
solvatochromic behavior, the absorption and emission data of the styryl derivatives were
determined in DMSO, acetonitrile, THF, CH2Cl2 and toluene (Table 1).
Table 1 is here
The compounds 5a–h are fluorescent and show absorption wavelengths (λmax) in the UV or
visible region (356-392 nm). The most remarkable feature of the absorption and emission
spectra is the close similarity of the positions and intensities of absorption and emission bands
for all derivatives in the neutral styryl dyes. Although the compounds 5a,b exhibit two
absorption bands in all solvents used, the derivatives 5c–h showed just one absorption band.
In addition, the compounds 5e-h exhibit one shoulder at long wavelength in toulene used. The
dyes which have two absorption bands or one absorption band with shoulder, may exhibit E-Z
isomerization [27].
12

In addition these styryl derivatives are fluorescent. Generally, the dyes 5c–h show lower
fluorescence quantum yields than derivatives 5a,b with R2 = H. It may be proposed that the
relative low fluorescence quantum yields are as a result of a photo-induced intramolecular
electron transfer (PET) [46-48] reaction between the excited fluorophore (imidazopyridine
ring as an electron acceptor) and the electron donating dialkylamino groups. In general, larger
Stokes shifts are obtained for alkylamino derivatives 5c-h under investigation (e.g., 5c: 5801
cm–1; 5d: 5861 cm–1 in CH2Cl2).
With respect to absorption, the compounds 5a–h did not exhibit solvatochromism, absorption
maxima of compounds are almost same with increased solvent polarity. In contrast, all
compounds exhibit fluorosolvatochromism, i.e. a bathochromic shift of the emission band was
observed with increasing solvent polarity. This solvatochromic behavior, which results from
the stabilization of the highly polar emitting state by polar solvents, is typical for compounds
exhibiting an internal charge transfer upon excitation and has been fully documented with
donor-acceptor fluorophores [49].
The absorption maxima of derivatives with an electron-donating substituent, such as the
dialkylamino substituents showed significant bathochromic shifts as compared to the ones of
the reference compounds 5a,b. The same trend was observed in the emission spectra of
compounds 5c–h. The bathochromic shift is most likely the result of the more pronounced
donor-acceptor interplay between the donor functionalities and the eletron-poor
imidazopyridine ring. Hence, when the dialkylamino groups are attached at the para position
of phenyl ring, a more pronounced donor-acceptor system is established between
dialkylamino functionality and the imidazopyridine ring resulting in a larger red shift. These
results are compatible with our previous study [39].
3.3. Acidochromic properties
13

Application of fluorescent compounds in monitoring pH change has attracted considerable
attention. In our study, the presence of an alkylamino substituent and nitrogen atoms in
imidazopyridine ring prompted us to study the possibility of protonation of this compound in
CH2Cl2. Thus, the effect of protonation on the optical properties of 5a–h in CH2Cl2 solution
was also studied. The change in the UV-vis absorption spectra of 5e and 5g upon addition of
acid is illustrated in Figures 2 and 3. The general trend shall be illustrated exemplarily with
the representative derivatives 5e and 5g. Upon addition of trifluoroacetic acid (TFA) to a
solution of 5e and 5g in CH2Cl2, compounds 5e and 5g exhibited minor color changes from
yellow to deep-yellow with the growth of a new absorption band at 441 and 430 nm and a
well-defined isosbestic point at 415 and 406 nm respectively (Figures 2 and 3). This result
shows that a ground-state equilibrium in between neutral (5e and 5g) and monoprotonated
(5e+H+ and 5g+H+) species.
Table 2 is here
Figure 2 and 3 are here
The observed bathochromic shifts (51 nm for 5e and 50 nm for 5g) of the absorption
maximum may be explained by initial protonation of the imidazopyridine ring that leads to an
increased donor-acceptor interplay between the amino functionality and the imidazonium ion
(Scheme 2).
Scheme 2 is here
The addition of excess TFA to the solutions of 5e and 5g in CH2Cl2 led to a shift of the
absorption maximum from 441 nm to 348 nm, and from 430 nm to 342 nm respectively.
Hypsochromic shifts were observed at 93 nm and 88 nm with respect to the neutral forms 5e
and 5g. Apparently, with excess of TFA the compound is also protonated at the amino-
14

functionality which leads to a collapse of the donor-acceptor system and double protonated
species occur for both compounds (5e+2H+ and 5g+2H+) (Scheme 2, Figures 2 and 3). The
same hypsochromic shifts were also observed for dialkylamino-substituted
styrylimidazopyridines/styrylbenzimidazole and arylvinyldiazine derivatives [11,39,50].
Upon adition of acid the emission properties of the derivatives 5a–h also changed
significantly (Table 2). The emission intensity of 5c–h increased due to the presence of the
acid. For example, upon addition of acid to 5e and 5g in CH2Cl2, the fluorescence intensity
increased (Figures 2 and 3). In neutral form of dialkylamino-substituted imidazopyridines,
the PET process is active, but in double protonated species, PET is deactivated between the
amino functionality and the excited imidazopyridine unit. Therefore, the light-up effect of 5c–
h on acidification is most likely the result of a suppressed PET by protonation of the amino
functionality [11,39].
3.4.Thermal Properties
To investigate the thermal stability of the chromophores, thermogravimetric analysis (TGA)
was used. Thermal analyses were performed with a Shimadzu DTG-60H system, up to 500 °C
(10 °C min−1) under a dynamic nitrogen atmosphere (15 mL min−1). The change in weight of
the compounds was measured as a function of temperature. Thermal stability of the
compounds 5a-h is shown in Figure 4. The absence of weight loss up to 80 oC indicates that
the solids have no water molecule. The compounds, except 5b and 5e, have the same trend,
o
which showed weight loss at about 300 C and are decomposed in one step. The initial
decomposition temperatures (Td) are for 311 (5a), 158 (5b), 372 (5c), 355 (5d), 185 (5e), 351
o
(5f), 363 (5g) and 360 C (5h) respectively. Therefore, the chromophores exhibited good
thermal stabilities up to 300 °C except 5b and 5e, with thermal decomposition occuring
15

between 300 and 400 °C, which is high enough for applications in electro-optical device
preparation.
3.5.
Antimicrobial activity
The antimicrobial effects of the newly prepared styryl derivatives 5a-h were studied against
some bacteria and fungi to discover new antimicrobial agents. The agarwell-diffusion method
was used to test for the antimicrobial activities of the compounds against the reference
bacteria strains Staphylococcus aureus ATCC 25923 (G+), Bacillus subtilis ATCC 6633
(G+), Bacillus cereus NRRL-B-3711 (G+), Enterococcus faecalis ATCC 292112 (G+),
Pseudomonas aeruginosa ATCC 27853 (G-), Escherichia coli ATCC 25922 (G-),
Escherichia coli ATCC 35218 (G-), Klebsiella pneumonia ATCC 13883 (G-), Salmonella
typhimurium ATCC 14028 (G-), Proteus vulgaris RSKK 96029(G-) and Enterococcus hirae
ATCC 9790 (G+). In addition, Candida albicans ATCC 10231, Candida tropicalis NRRL-Y-
12968 and Candida krusei ATCC6258 were used for antifungal activities [51]. Gram positive
and gram negative bacteria were grown on Nutrient agar plates and incubated at 37 °C for 24
h. The yeast strains were grown in Sabourauddextrose agar medium and incubated at 30 °C
for 48 h. After incubation, microbial suspensions were adjusted to a turbidity of 0.5 Mc
Farland. Mueller Hinton agar (for bacterial strains) and sabourauddextrose agar (for fungal
strains) were mixed with 1% culture suspension and poured into plates. Upon examination of
bioactivity results of compounds, It has been noticed that none of the compounds was found
to be active against tested bacteria and fungi. Therefore, we can conclude that, the compounds
have no antimicrobial activity between concentration 50-250 µg/mL.
3.6.
Antioxidant activity
16

Antioxidant activity of the compounds was analysed by using DPPH (2,2-Diphenyl-1-
picrylhydrazyl) radical scavenging activity. DPPH method is applied for the estimation of
antioxidant activity of the compounds [52]. Briefly, to 1 µL of DPPH working solution, 50,
100, 150, 200, 250 µg/mL of the test compounds were added. The mixture was left in the dark
for 30 min before absorbance was read at 517 nm. DPPH radical scavenging activity was
calculated using the following formula: DPPH radical scavenging activity = (Acontrol
-
Asample)/Acontrol x 100. Antioxidant activity of the compounds was investigated by DPPH
scavenging activty. None of the compounds exhibited antioxidant activity at the tested
concentrations. DPPH scavenging activty is related to hydrogen atom transfer and number of
active group [52]. Many diseases such as cancer, dieabetes, Alzheimer are associated with
oxidative stress and reactive oxygen species. The studies revealed that antioxidant compounds
may reduce the risk of oxidative stress by their antioxidant activity. In order to determine the
antioxidant activity of the compounds, we tested DPPH scevanging activity.
3.7.
Interactions of DNA with the compounds
Interactions of styryl derivatives 5a-h with supercoiled pBR322 plasmid DNA were studied
using agarose gel electrophoresis [53]. The compounds were incubated over a range of
concentrations with supercoiled pBR322 plasmid DNA in the dark at 37°C for 24 h. The
compound/DNA mixtures were loaded onto the 1% agarose gel with a loading buffer (0.1 %
bromophenol blue, 0.1 % sucrose). Electrophoresis was carried out in 0.05 M Trisbase, 0.05
M glacial acetic acid and 1 mM EDTA (TAE buffer, pH=8.0) for 2 h at 70V [54]. The gel was
stained with ethidiumbromide (0.5 µg/mL), visualized under UV light using a transilluminator
17

(Biodoc Analyzer, Biometra), photographed with a video-camera and save as a TIFF file. The
experiments were repeated three times.
BamHI and HindIII restriction enzyme digestion of compounds-pBR322 plasmid DNA. The
compound/DNA mixtures were incubated for 24 h, and then restricted with the enzymes
BamHI and HindIII for 1 h at 37 °C. The restricted DNA was run in 1 % agarose gel
electrophoresis for 2 h at 70 V in TAE buffer.
The interaction of DNA with the compounds could convert covalently supercoiled form I
DNA into singly nicked circular form II, and then into a linear form III DNA [54]. This
cleavage reaction could be monitored by gel electrophoresis. The DNA cleavage by these
compounds has been examined by observing the conversion of supercoiled DNA to the open
circular form II and linear form III DNA. Restriction enzymes cut DNA near specific
recognition nucleotide sequences known as restriction sites [55]. BamHI and HindIII are
known to recognize the sequences G/GATCC and A/AGCTT respectively [56]. pBR322
plasmid DNA contain a single restriction site for each enzyme which convert supercoiled
form I DNA and singly nicked circular form II DNA to linear form III DNA. Figure 5 shows
the electrophoretograms to grams for the interaction of pBR322 plasmid DNA with 5a-h at
concentrations of compounds ranging from 100 to 62.5 µM. Lane P applies to the untreated
pBR322 plasmid DNA (control DNA), showing the supercoiled (Form I) and open nicked
(Form II) forms. Lanes 1–5 apply to pBR322 plasmid DNA incubated with the compounds
with concentrations ranging from 1000 to 62.5 µM. The mobility of form I DNA was
decreased for all the compounds. There are smaller bands than form I in each case indicating
cleavage of DNA.
In case of 5a-c, 5e, 5g and 5h there is also linear band (Form III) between form I and form II
DNA indicating double strand breaks by the compounds (Figure 5). Figures 6 and 7 show the
electrophoretograms applied incubated mixtures of pBR322 plasmid DNA compounds
18

followed by HindIII and BamHI digestion. After digestion of plasmid DNA HindIII enzyme
the linear form III band is observed for all compounds indicating that the compounds DNA
mixtures are restricted by the enzyme. However, BamHI digestion single form III band is not
observed indicating the compounds DNA mixtures are not fully restricted by the enzyme. The
results suggest that compounds bind to G/G nucleotides of DNA.
4. Conclusion
In this paper, a new series of arylstyrylimidazo[1,2-a]pyridine-based donor-acceptor
1
fluorophores were synthesized and their structures were confirmed by H NMR, 13C NMR,
Elemental analysis and Mass spectra. All the fluorophores have an E-configuration of the
1
vinyl double bond as unequivocally shown by H NMR spectroscopy. The optical properties
of these dyes were studied in solvents of different polarities. The absorption of the
compounds 5a–h did not exhibit solvatochromism, absorption maxima of compounds are
almost same with increased solvent polarity. However, they exhibited fluorosolvatochromism,
for instance the emission band is red shifted with increasing solvent polarity. The probes had
high fluorescent in acidic media but low fluorescent in neutral environments. After double
protonation of compounds 5c-h, light up effect was observed due to inhibition of PET
process. This phenomenon should enable the development of fluorometric pH sensors after
suitable design of the molecules. The TGA results show that the compounds are having good
thermal stability (except 5b and 5e).
DNA is the molecular target of the anticancer drugs. Small molecules which are bind to DNA,
inhibit enzymatic DNA substrate such as DNA polymerase and topoisomerase activity [57].
In our study, we determined DNA-Ligand interaction using gel electrophoresis method. This
mehod is useful for determining DNA-Ligand interactions, but additional experiments should
be done for more detailed evaluation of interactions. The compounds 5a-c, 5e, 5g and 5h have
19

promising DNA activities by causing double strand break and bind to G/G nucleotides of
DNA.
In conclusion, these compounds can be used as promising candidate for the development of
light up probe for pH differences in biological and enviromental media and may have various
applications such as nonlinear optics (NLO).
Crystallographic data
Crystallographic data that were deposited in CSD under CCDC-1059253 registration number
contain the supplementary crystallographic data for this manuscript. These data can be
obtained free of charge from the Cambridge Crystallographic Data Centre (CCDC) via
www.ccdc.cam.ac.uk/data_request/cif and are available free of charge upon request to CCDC,
12 Union Road, Cambridge, UK (fax: +441223 336033, e-mail: deposit@ccdc.cam.ac.uk).
Acknowledgements
This investigation was supported by The Scientific and Technological Research Council of
Turkey (TUBITAK) Grant no:1059B191100170.
20

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27

Scheme Captions
Scheme 1. Synthesis of arylstyrylimidazo[1,2-a]pyridine derivatives 5a-h.
Scheme 2. Prototropic equilibria of 5g.
Figure Captions
Figure 1. Mercury drawing of the molecule 3a. Thermal ellipsoids are drawn at the 50%
probability level.
Figure 2. (A) The absorption spectra of 5e (above, c = 10 μM) upon addition of TFA (100
mM) in CH2Cl2; Pictures of absorption (above) and emission (below, under UV light (365
nm)) color of 5e in CH2Cl2 solutions (c = 1 mM; λex = 365 nm). (B) Spectrofluorimetric
titration of 5e (below, c = 1 μM; λex = 360 and 450 nm) in CH2Cl2, arrow indicate the
increase/decrease of the fluorescence intensity upon addition 100 mM TFA in CH2Cl2.
Figure 3. (A) The absorption spectra of 5g (above, c = 10 μM) upon addition of TFA (100
mM) in CH2Cl2; Pictures of absorption (above) and emission (below, under UV light (365
nm)) color of 5g in CH2Cl2 solutions (c = 1 mM; λex = 365 nm). (B) Spectrofluorimetric
titration of 5g (below, c = 1 μM; λex = 350 and 440 nm) in CH2Cl2, arrow indicate the
increase/decrease of the fluorescence intensity upon addition 100 mM TFA in CH2Cl2.
Figure 4. TGA curves of chromophores 5a-h.
Figure 5. Electrophoretograms applied to the incubated mixtures of Plasmid DNA (P) and
varying concentrations of the compounds 5a-h (lane 1: 1000 µM, lane 2: 500 µM, lane 3: 250
µM, lane 4: 125 µM, line 5: 62.5 µM).
Figure 6. Electrophoretograms for the HindIII digested mixtures of pBR322 plasmid DNA
after their treatment with various concentrations of compounds, followed by their digestion
with HindIII (untreatedplasmid DNA pBR322 was applied to lane 1, lane 2 applies pBR322
DNA digested with HindIII. Lanes 3-10 apply to pBR322 plasmid DNA interacted with the
compounds 5a-h followed by their digestion with HindIII, respectively).
Figure 7. Electrophoretograms for the BamHI digested mixtures of pBR322 plasmid DNA
after their treatment with various concentrations of compounds, followed by their digestion
with BamHI (untreated plasmid DNA pBR322 was applied to lane 1, lane 2 applies pBR322
DNA digested with BamHI. Lanes 3-10 apply to pBR322plasmid DNA interacted with the
compounds 5a-h followed by their digestion with BamHI, respectively).
Table Captions
Table 1. Photophysical data of arylstyrylimidazo[1,2-a]pyridine based dyes 5a-h
Table 2. pH-Dependent Absorption and Emission Properties of arylstyrylimidazo[1,2-
a]pyridine based dyes 5a–h.
28

Tables
Table 1. Photophysical data of arylstyrylimidazo[1,2-a]pyridine based dyes 5a-h
DMSO
Acetonitrile
THF
CH2Cl2
cλfl
Toluene
Stok
es
Stok
es
Stok
es
aλabs
aλabs
(nm)
(log
εb)
aλabs
(nm)
(log
εb)
aλabs
(nm)
(log
εb)
aλabs
(nm)
(log
εb)
cλfl
(n
Stokes
Shift,
cm-1
Stokes
Shift,
cm-1
(nm) cλfl
(log (nm)
εb)
cλfl
(nm)
cλfl
(nm)
Dye
5a
dφfl Shift
dφfl Shift
dφfl Shift
dφfl
dφfl
(nm)
, cm-
m)
, cm-
, cm-
1
1
1
362( 424 0.4 2730 356(4. 41 0.36 2757 359(4. 416 0.34 2416 358(
416
0.26 2486 362(4. 396,
0.31
2323
4.56
),
1
50),
374(4.
44)
7
52),
378(4.
48)
4.58)
,
48),
381(4.
42)
418
380(
4.53
)
377(
4.53)
423 0.2 2606 357(4. 41 0.19 2699 360(4. 416 0.18 2346 359( 395,41 0.13 2429 362(4. 396,
0.36
2323
5b
362(
4.52
),
381(
4.46
)
1
52),
374(4.
46)
6
50),
379(4.
46)
4.47)
,
5
47),
381(4.
42)
418
377(
4.41)
383( 497 0.2 5988 371(4. 48 0.09 6293 376(4. 460 0.13 4856 373(
476
479
475
0.03 5801 380(4. 459
53)
0.07
0.20
0.13
4529
4809
3939
5c
5d
5e
4.47
)
8
29)
4
50)
4.34)
382( 497 0.2 6057
373
49 0.11 6401 374(4. 467 0.14 5324 374(
0.09 5861 376(4. 459,
4.34
)
4
(4.17)
0
17)
4.47)
43)
439(
sh.)
391( 494 0.1 5332 383(4. 49 0.07 5743
475 0.09 4854 389(
4.36)
0.07 4854 388(40 436,
4.42
)
8
33)
1
386(4.
47)
8
458
sh.)
(4.43)
392 495 0.1 5308
383
(4.53)
48 0.06 5617
8
384
(4.61) 445
417, 0.08 3570 386(
475
469
471
0.06 4854 388(40 434,
9 sh.) 459
0.13
0.18
0.24
3986
4063
5921
5f
5g
5h
(4.4
7)
5
4.47)
(4.46)
389( 494 0.2 5464
48 0.09 5907 380(4. 464 0.12 4764 381(
49) 4.45)
0.06 4787 384(40 455,
3 sh.) 434(
(4.49) sh.)
0.08 5153 384(40 434,
2 sh.) 497
4.52
)
2
377(4.
42)
5
384( 492 0.2 5716 384(4. 48 0.13 5465 382(4. 461 0.05 4486 379(
4.25
)
3
58)
6
62)
4.43)
(4.33)
aLong wavelength absorption maximum, in nm; c= 10 μ M; bε = molar absorption coefficient, cm-1M-1; cFluorescence
maximum, in nm;c= 1-5 μ M; dFluorescence quantum yield relative to Coumarin 1 (0.78) in ethanol [40,41].
Table 2. pH-Dependent Absorption and Emission Properties of arylstyrylimidazo[1,2-
a]pyridine based dyes 5a–h.
Dyes
UV/vis (TFA 100 mM in
FL (TFA 100 mM in
CH2Cl2) λmax (nm)
Stokes shift
CH2Cl2) λmax (nm)
cm-1
355
355
349
350
348
350
342
346
413
416
407
408a
410
426a
430
415
3955
4130
4083
4061
4345
5097
5983
4805
5a
5b
5c
5d
5e
5f
5g
5h
aLow fluorescence intensity
29