Comprehensive exploration of the optical and biological properties of new quinoline based cellular probes

Article abstract of DOI:10.1016/j.dyepig.2017.05.029

A series of quinoline derivatives containing bithiophene and N-octyl-carbazole moieties and a variety of substituents in the quinoline core were synthesized and characterized. The influence of bithienyl and N-octyl-carbazyl substituents on the photophysical properties of novel quinolines were thoroughly investigated and supported by DFT calculations. The absorption and emission maxima of quinolines are located at 373-412 and 410–606?nm, respectively, which considerably red-shifts when an electron-withdrawing substituent (NO₂) is introduced to the quinoline ring. The quantum yield and lifetime for carbazyl-substituted quinolines are considerably higher (Φ_em?=?9–70%, τ?=?0.83–5.72?ns) compared to their bithiophene substituted counterparts (Φ_em?=?18–53%, τ?=?0.61–1.46?ns) resulting from greater planarity of carbazyl-substituted quinolines as proved by theoretical calculations. The carbazyl functional group in quinolines has immense impact on values of Δμ (13.83–15.50 D) indicating that the electron-donating ability of carbazole moiety relative to bithiophene motif (Δμ?=?9.16–13.83) in studied quinolines is more predisposed to intramolecular charge transfer (ICT). The capability of the novel compounds for cellular staining was investigated. All examined quinoline derivatives penetrate cell organelles quickly and efficiently with good overall fluorescence and signal to noise ratio. Relative lack of toxicity make them useful in such applications.

Full text of DOI:10.1016/j.dyepig.2017.05.029

Accepted Manuscript
Comprehensive exploration of the optical and biological properties of new quinolines
based cellular probes
Barbara Czaplinska, Anna Maron, Jan Grzegorz Malecki, Grazyna Szafraniec-Gorol,
Marek Matussek, Katarzyna Malarz, Anna Mrozek-Wilczkiewicz, Witold Danikiewicz,
Robert Musiol, Aneta Slodek
PII:
S0143-7208(17)30635-6
10.1016/j.dyepig.2017.05.029
DYPI 5991
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 29 March 2017
Revised Date: 11 May 2017
Accepted Date: 12 May 2017
Please cite this article as: Czaplinska B, Maron A, Malecki JG, Szafraniec-Gorol G, Matussek M, Malarz
K, Mrozek-Wilczkiewicz A, Danikiewicz W, Musiol R, Slodek A, Comprehensive exploration of the optical
and biological properties of new quinolines based cellular probes, Dyes and Pigments (2017), doi:
10.1016/j.dyepig.2017.05.029.
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Comprehensive exploration of the optical and biological
ACCEPTED MANUSCRIPT
properties of new quinolines based cellular probes
Barbara Czaplinska,[a] Anna Maron,[a] Jan Grzegorz Malecki,[a] Grazyna Szafraniec-Gorol,[a]
Marek Matussek,[a] Katarzyna Malarz,[a,b] Anna Mrozek-Wilczkiewicz,[b,c] Witold
Danikiewicz,[d] Robert Musiol,[a]* Aneta Slodek.[a]*
[a]
Faculty of Mathematics, Physics and Chemistry, Institute of Chemistry, University of
Silesia, Katowice, Szkolna 9, 40-007, Poland
Silesian Center for Education and Interdisciplinary Research, University of Silesia,
[b]
Chorzów, 75 Pułku Piechoty 1A, 41-500, Poland
A. Chełkowski Institute of Physics, University of Silesia, Katowice, Uniwersytecka 4,
[c]
40-007, Poland
[d]
Polish Academy of Science, Institute of Organic Chemistry,Warsaw, Kasprzaka 44/52,
PO Box 58, 01-224, Poland
*Corresponding authors:
E-mail addresses:
robert.musiol@us.edu.pl (R. Musiol)
aneta.slodek@us.edu.pl, a.slodek@wp.pl (A. Slodek)
Abstract
A series of quinoline derivatives containing bithiophene and N-octyl-carbazole moieties and a
variety of substituents in the quinoline core were synthesized and characterized. The influence
of bithienyl and N-octyl-carbazyl substituents on the photophysical properties of novel
quinolines were thoroughly investigated and supported by DFT calculations. The absorption
and emission maxima of quinolines are located at 373-412 and 410-606 nm, respectively,
which considerably red-shifts when an electron-withdrawing substituent (NO2) is introduced
to the quinoline ring. The quantum yield and lifetime for carbazyl-substituted quinolines are
considerably higher (Φem=9-70%, τ=0.83-5.72 ns) compared to their bithiophene substituted
counterparts (Φem=18-53%, τ=0.61-1.46 ns) resulting from greater planarity of carbazyl-
substituted quinolines as proved by theoretical calculations. The carbazyl functional group in
quinolines has immense impact on values of ∆µ (13.83-15.50 D) indicating that the electron-
donating ability of carbazole moiety relative to bithiophene motif (∆µ=9.16-13.83) in studied
quinolines is more predisposed to intramolecular charge transfer (ICT). The capability of the
novel compounds for cellular staining was investigated. All examined quinoline derivatives
penetrate cell organelles quickly and efficiently with good overall fluorescence and signal to
noise ratio. Relative lack of toxicity make them useful in such applications.

Keywords: quinoline; optical properties; solvatochromism; DFT study; intracellular probe
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Highlights
• Quinoline derivatives substituted by bithiophene and carbazole moieties were
examined.
• Photophysical properties of the obtained compounds were studied in details and
compared with DTF calculations.
• The effect of substituents in examined compounds on optical properties was discussed.
• Imaging of quinoline derivatives in cells was tested.
Graphical abstract
1.
Introduction
Nitrogen containing heterocyclic compounds mainly quinoline and carbazole derivatives have
strong position in drug and material chemistry. An enormous versatility of quinoline and
carbazole derivatives is also reflected the development in optoelectronics [1-4] and electronics
[5-8]. Quinolines as outstanding fluorescent compound with high quantum efficiency invite
much interest in fluorescence probes in chemosensors [9-13].
The highly luminescence quinoline derivatives were investigated as sensors for the detection
of bacteria using fluorescent imaging studies. They have shown sensitivity for both gram
positive and gram negative bacteria at a concentration in the micromolar range [14]. A series
of pyranoquinoline derivatives display staining property to the cultured HeLa cells after fixing
and give fluorescence in the sky blue region [15]. An 8-aminoquinoline-based sensor was
explored for Zn2+ detection with excellent selectivity and intracellular Zn2+ sensing in HeLa
cells and rat hippocampal slices [16]. The cellular imaging studies of novel coumarin-
quinoline compounds showed strong fluorescence under acidic conditions and low
cytotoxicity proved to be valuable probes to monitor pH changes from neutral to the acidic
conditions in living cells [17]. Another interesting applications arisen for carbazole based

imino-phenol that has been successfully used for detection and imaging of caffeine in living
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cells. For good fluorescence and solubility such a dye can be effective fluorescent probe for
xanthine alkaloids as caffeine in aqueous media like body fluids [16].
Much effort is made to synthesize chemosensors based on donor-acceptor (D-A) structure that
allow for effective intramolecular charge transfer (ITC) and thus constitute a fluorescent
probes with large Stokes shift. Recently D-A molecule based on carbazole and benzoindole
units has been evaluated to monitor pH fluctuations in live cells such as extremely acidic E.
coli [18]. Among the advantages of these compounds low cytotoxicity, good selectivity and
photostability should be mentioned.
Fluorescence probe based on carbazole moiety connected with cationic pyridinium moiety via
triple bond resulted in high selectivity, low detection limit, large Stokes shift and two-photon
absorption cross sections, being realized in bio-imaging experiments established its utility for
imaging Au3+ in mitochondria under two-photon excitation and good biocompatibility [19].
Besides of nitrogen-based aromatic compounds, molecules that contain sulfur such as
thiophene derivatives have been widely evaluated as fluorescent probes [20-23]. The
thiophene or bithiophene functionalized ligands as part of ruthenium complexes have been
recently explored in photodynamic therapy (PDT) [21]. The derivatives containing
bithiophene moieties have been tested as fluorescent imaging markers for β-amyloid plaques
(Aβ) [20,22], where they may serve as potential candidates for diagnosis of Alzheimer’s
disease. Cell imaging studies of fluorescent probe containing thiophene moiety for detection
of Cr3+ have shown high sensitivity of thiophene-based sensor on traces of Cr3+ in living cells
[23].
Those promising properties prompted us to involve both of these motifs in our search for
materials with interesting photophysical parameters. Recently we have reported work on
bithiophene-quinoline based compounds with their extensive optical and electrochemical
characteristics [24]. Further development of this valuable group of compounds is however
somewhat hampered by complex and inefficient synthesis. Thus we decided to explore the
field of synthetic availability as well as application as intracellular probes. Herein we present
synthesis of a series of novel compounds based on carbazyl- and bithienyl-substituted
quinoline of D-A type obtained via Ru-catalyzed reaction. The photophysical properties of
novel compounds have been thoroughly studied and confronted with theoretical calculations.
2.
Experimental
2.1. Materials

All chemicals and starting materials were commercially available and were used without
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further purification. Solvents were distilled as per the standard methods and purged with
nitrogen before use. All reactions were carried out under argon atmosphere unless otherwise
indicated. Column chromatography was carried out on Merck silica gel. Thin layer
chromatography (TLC) was performed on silica gel (Merck TLC Silica Gel 60). The reactions
under microwave-assisted conditions were conducted on a CEM Discover instrument.
2.2. Synthesis and characterization
2.2.1. Representative procedure for quinoline derivatives of 6a-d
4-Methyl-2-(9-octylcarbaz-3-yl)-quinoline (6a). A mixture of 3-ethynyl-9-octylcarbazole
(4) (0.5 g, 1.6 mmol) and Ru3(CO)12 (16 mg, 0.02 mmol) in toluene (5 mL) was stirred under
argon for 15 minutes. Then 2-aminoacetophenone (5a) (0.675 g, 0.12 mL, 1 mmol), and 50%
aqueous HBF4 (10 µL, 0.08 mmol) were injected through the septum and the mixture was
heated at 110 °C for 3 hours. After being cooled to room temperature, the solvent was
removed under reduced pressure, and a dark brown residue was purified by column
1
chromatography on SiO2 (hexane/CH2Cl2, 3:1) to give 6a as yellow oil with 65% yield. H
NMR (400 MHz, CDCl3) δ 8.92 (d, J = 0.8 Hz, 1H), 8.35 (dd, J = 8.0, 1.6 Hz, 1H), 8.27 –
8.18 (m, 2H), 8.01 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.72 (dd, J = 8.0, 7.2 Hz, 1H), 7.51 (m,
3H), 7.44 (d, J = 8.0 Hz, 1H), 7.29 (dd, J = 7.6, 7.2 Hz, 1H), 4.34 (t, J = 7.2, 2H), 2.81 (s,
13
3H), 1.91 (m, 2H), 1.31 (m, 10H), 0.88 (m, 3H). C NMR (126 MHz, CDCl3) δ 157.88,
148.33, 144.54, 141.37, 141.02, 130.72, 130.06, 129.27, 127.03, 125.87, 125.53, 125.52,
123.67, 123.35, 123.32, 120.73, 119.86, 119.82, 119.18, 108.98, 108.96, 43.25, 31.85, 29.44,
29.23, 29.04, 27.35, 22.66, 19.10, 14.14. FTIR (KBr, cm-1): 3054, 3025 (CAr-H stretching),
2929 (C-H stretching -CH2-), 699 (CAr-H deformation). ESI-HRMS calcd for C30H33N2
421.2643 [M+H]+ found 421.2644.
2-(9-Octylcarbaz-3-yl)-4-phenylquinoline (6b). Compound 6b was prepared from 2-
aminobenzophenone (5b) (0.19 g, 1 mmol) according to the synthetic route described for 6a.
A crude product was purified by column chromatography (hexane/CH2Cl2, 2:1) to give yellow
oil of 6b. Yield: 70%. 1H NMR (400 MHz, CDCl3) δ 8.96 (d, J = 1.2 Hz, 1H), 8.40 (dd, J =
7.6, 1.2 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.92 (d, J =
8.0 Hz, 1H), 7.79 – 7.72 (m, 1H), 7.66 – 7.42 (m, 9H), 7.29 (dd, J = 8.0, 7.6 Hz, 1H), 4.35 (t,
13
J = 7.2 Hz, 2H), 1.92 (m, 2H), 1.31 (m, 10H), 0.88 (m, 3H). C NMR (126 MHz, CDCl3) δ
157.73, 149.14, 149.00, 141.52, 141.09, 138.80, 130.62, 130.04, 129.74, 129.74 129.53,
128.70, 128.44, 125.99, 125.90, 125.76, 125.62, 123.48, 123.38, 120.81, 119.96, 119.51,

119.32, 109.10, 109.06, 43.27, 31.93, 29.51, 29.31, 29.11, 27.41, 22.75, 14.24. FTIR (KBr,
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cm-1): 3059 (CAr-H stretching), 2922 (C-H stretching -CH2-), 696 (CAr-H stretching). ESI-
HRMS calcd for C35H35N2 [M+H]+ 483.2796 found 483.2800.
6-Chloro-2-(9-octylcarbaz-3-yl)-4-phenylquinoline (6c). Compound 6c was prepared from
2-amino-5-chlorobenzophenone (5c) (0.23 g, 1 mmol) according to the synthetic route
described for 6a. A crude product was purified by column chromatography (hexane/CH2Cl2,
1
2:1) to give yellow oil of 6c. Yield: 55%. H NMR (400 MHz, CDCl3) δ 8.99 (d, J = 1.2 Hz,
1H), 8.41 (dd, J = 7.6, 1.6 Hz, 1H), 8.28 – 8.23 (m, 2H), 8.03 (s, 1H), 7.92 (d, J = 2.2 Hz,
1H), 7.71 (dd, J = 8.8, 2.0 Hz, 1H), 7.66 – 7.57 (m, 5H), 7.54 (m, 2H), 7.47 (d, J = 7.6 Hz,
1H), 7.35 – 7.30 (dd, J = 7.2, 6.8 Hz, 1H), 4.35 (t, J = 7.2 Hz, 2H), 1.95 (m, 2H), 1.35 (m,
13
10H), 0.93 (m, 3H). C NMR (126 MHz, CDCl3) δ 157.77, 148.12, 147.52, 141.60, 141.10,
138.15, 131.63, 131.60, 130.31, 130.05, 129.64, 128.90, 128.71, 126.27, 126.08, 125.52,
124.58, 123.48, 123.36, 120.81, 120.04, 119.90, 119.42, 109.11, 109.07, 43.25, 31.97, 29.54,
29.36, 29.13, 27.43, 22.81, 14.31. FTIR (KBr, cm-1): 3061, 3054, 3028 (CAr-H stretching),
2921 (C-H stretching -CH2-), 703, 699 (CAr-H stretching). ESI-HRMS calcd for C35H34N2Cl
[M+H]+ 517.2407 found 517.2411.
6-Nitro-2-(9-octylcarbaz-3-yl)-4-phenylquinoline (6d). A mixture of 3-ethynyl-N-
octylcarbazole (4) (1 g, 3.2 mmol), 2-amino-5-nitrobenzophenone (5d) (0.5 g, 2 mmol),
Ru3(CO)12 (64 mg, 0.1 mmol), and 54% HBF4 in diethyl ether (40 µL, 0.3 mmol) in toluene
(5 mL) was stirred for 5 minutes under argon in a microwave reaction glass tube. Then the
glass tube was irradiated in the microwave reactor at 110 °C at a power of 50 W for 10
minutes. The crude product was purified by column chromatography on SiO2
(hexane/CH2Cl2, 2:1) to give yellow solid of 6d. Yield: 65%. m.p. = 152 °C. 1H NMR (400
MHz, CDCl3) δ 8.99 (d, J = 1.2 Hz, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.47 (dd, J = 9.2, 2.4 Hz,
1H), 8.43 (dd, J = 8.4, 1.6 Hz, 1H), 8.33 (d, J = 9.2 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.11 (s,
1H), 7.68 – 7.57 (m, 5H), 7.52 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.30 (dd, J = 8.0, 6.8 Hz,
13
1H), 4.35 (t, J = 7.2 Hz, 2H), 1.91 (m, 2H), 1.32 (m, 10H), 0.87 (m, 3H). C NMR (101
MHz, CDCl3) δ 160.35, 151.25, 150.55, 144.73, 141.95, 141.04, 137.21, 129.56, 129.19,
129.09, 129.00, 126.26, 125.66, 125.53, 124.27, 123.42, 123.14, 122.84, 122.81, 120.47,
120.30, 120.24, 120.20, 109.08, 109.01, 43.26, 31.87, 29.38, 29.23, 29.03, 27.35, 22.70,
14.17. FTIR (KBr, cm-1): 2916 (C-H stretching -CH2-), 702, 696 (CAr-H stretching). ESI-
HRMS calcd for C35H34N3O2 [M+H]+ 528.2649 found 528.2655.
2.2.2. Representative procedure for synthesis of compound 6e

6-Amino-2-(9-octylcarbaz-3-yl)-4-phenylquinoline (6e). A 6-nitro-2-(9-octylcarbaz-3-yl)-4-
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phenylquinoline (6d) (0.06 g, 0.15 mmol) was dissolved in 1,2-dimethoxyethane (DME) (3
mL) under stirring. The mixture was kept under reflux with activated charcoal (4 mg) within 1
hour and left overnight under stirring at room temperature. The reaction mixture was flushed
with argon and stirred for 30 min. Then Pd/C (2 mg) was added and the mixture was heated to
40 ºC. Hydrazine monohydrate (19 µL, 0.4 mmol) was added to the suspension. The obtained
mixture was refluxed for 3h, cooled, and filtered off. The filtrate was concentrated under
reduced pressure to afford crude product. The product was purified by column
chromatography on Al2O3 (hexane/AcOEt = 5:1 to 2:1) to give 6e with 83% yield. m.p. = 123
1
°C. H NMR (400 MHz, CDCl3) δ 8.87 (d, J = 1.6 Hz, 1H), 8.32 (dd, J = 8.4, 1.6 Hz, 1H),
8.20 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.86 (s, 1H), 7.63 – 7.45 (m, 7H), 7.43 (d, J
= 8.0 Hz, 1H), 7.24 (m, 1H), 7.19 (dd, J = 8.8, 1.6 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 4.34 (t, J
= 7.2 Hz, 2H), 3.87 (m, 2H), 1.90 (m, 2H), 1.29 (m, 10H), 0.86 (m, 3H). 13C NMR (101 MHz,
CDCl3) δ 154.40, 146.66, 144.33, 144.09, 141.11, 141.00, 139.29, 131.14, 131.00, 129.57,
128.60, 128.11, 126.89, 125.80, 125.24, 123.36, 123.33, 121.39, 120.72, 119.81, 119.36,
119.10, 108.97, 108.93, 106.00, 43.26, 31.85, 29.44, 29.23, 29.06, 27.36, 22.67, 14.14. FTIR
(KBr, cm-1): 3454, 3377 (-NH2 stretching ), 3064, 3047, 3028 (CAr-H stretching), 2922 (C-H
stretching -CH2-), 1622 (-NH2 deformation), 703, 700 (CAr-H stretching). ESI-HRMS calcd
for C35H36N3 [M+H]+ 498.2908 found 498.2916.
2.2.3. Representative procedure for synthesis of compound 7c
2-(2,2'-Bithiophen-5-yl)-6-chloro-4-phenylquinoline (7c). 2-Amino-5-chlorobenzophenone
(5c) (2 g, 8.6 mmol), 5-ethynyl-2,2'-bithiophene [25] (2.5 g, 9.5 mmol), Ru3(CO)12 (166 mg,
0.26 mmol), 50% aqueous HBF4 (48 µL, 0.78 mmol) in toluene (18 mL) was stirred at 140 °C
under argon for 6 hours. The solvent was removed under reduced pressure and the resulting
crude product was purified by column chromatography on SiO2 (hexane/CH2Cl2, 2:1) and
recrystallized from CHCl3/MeOH (10:1) to give light yellow crystals of 7c. Yield: 65%. m.p.
= 174 °C. 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 9.2 Hz, 1H), 7.77 (d, J = 2.4 Hz, 1H),
7.68 (s, 1H), 7.62 – 7.58 (dd, J = 9.2, 2.4 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.56 – 7.48 (m,
5H), 7.29 (dd, J = 3.6, 0.8 Hz, 1H), 7.27 – 7.23 (dd, J = 5.2, 1.2, 1H), 7.18 (d, J = 3.6 Hz,
1H), 7.04 (dd, J = 5.2, 3.6 Hz, 1H).13C NMR (101 MHz, CDCl3) δ 151.79, 148.18, 147.08,
143.39, 140.74, 137.49, 137.39, 131.97, 131.09, 130.61, 129.40, 128.85, 128.81, 128.06,
126.82, 126.56, 125.09, 124.60, 124.51, 124.37, 118.22. FTIR (KBr, cm-1): 3060, 3025 (CAr-
H stretching), 1069 (C-S stretching), 699 (CAr-H stretching). ESI-HRMS calcd for
C23H15NS2Cl [M+H]+ 404.0323 found 404.0334.

2.3. Measurements
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The NMR spectra were recorded on a Bruker Avance 500 MHz instrument by using CDCl3 as
solvent. High resolution mass spectra (HRMS) were recorded on a MALDI Synapt G2-S mass
spectrometer (Waters) using electrospray ionization (ESI). Samples were dissolved in
methanol and injected using an automatic sampler. The accuracy of the HRMS measurements
was better than 2 ppm. Melting point measurements were performed on Stuart automatic
melting point SMP40 apparatus.
The steady-state emission and excitation spectra were measured for dichloromethane (DCM)
solutions and on the FLS-980 spectrophotometer in ambient temperature using 450W Xe arc
lamp as a light source and PMT (Hamamatsu, R928P) in cooled housing as a detector. The
quantum yields of fluorescence were determined by absolute method in room temperature,
using the integrating sphere with solvent as a blank. Compounds are excited in the wavelength
corresponding to the absorption wavelength of the compounds. The time-resolved
measurement has been prepared at optically diluted solutions at room temperature using the
time correlated single photon counting methods on the FLS-980 spectrophotometer.
Excitation wavelengths were obtained using the picosecond pulsed diode EPLED−340 nm
and the picosecond pulsed diode EPL−375 nm with 100 ns pulse period as light sources,
depending on the obtained excitation maxima. PMT (Hamamatsu, R928P) in cooled housing
was used as a detector. The system was alignment at emission wavelengths. Additionally for
the analysis of a fluorescence decay, an instrument response function (IRF) needs to be
obtained. The IRF contains the information about the time response of the overall optical and
electronic system. The IRF was designate using ludox solution as a standard at 340 or 375
nm. The influences of the Raman scattering of solvent on emission of sample was avoided
using filter.
The crystals of compound 7a-d were mounted in turn on a Gemini A Ultra Oxford Diffraction
automatic diffractometer equipped with a CCD detector, and used for data collection. X-ray
intensity data were collected with graphite monochromated MoKα radiation (λ = 0.71073 A)
at a temperature of 295(2) K, with
ω scan mode. Ewald sphere reflections were collected up
to 2
θ
= 50.100. Details concerning crystal data and refinement are gathered in Table S1.
Lorentz, polarization and empirical absorption corrections using spherical harmonics
implemented in SCALE3 ABSPACK scaling algorithm [26] were applied. The structures
were solved by the direct method and subsequently completed by difference Fourier
recycling. All the non-hydrogen atoms were refined anisotropically using full-matrix, least-
squares techniques. All hydrogen atoms were positioned in geometrically idealized positions

and were allowed to ride on their parent atoms with Uiso(H) = 1.2 Ueq. The Olex2 [27] and
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SHELXS, SHELXL [28] programs were used for all the calculations. Atomic scattering
factors were incorporated in the computer programs.
2.4. DFT calculations
The quantum theoretical calculations using density functional theory (DFT), with an exchange
correlation hybrid functional B3LYP and the 6-31G++ basis for carbon, nitrogen, chloride,
oxygen and 6-31G for hydrogen atoms have been performed. The calculations were carried
out with use of Gaussian 09 program [29]. The geometries of these compounds were
optimized in dichloromethane solvent and no imaginary frequencies were found for any of
these species, computed at Ci symmetry, by vibrational analysis. Based on the optimized
geometries the energy and electronic distribution of molecular frontier orbitals were
calculated in dichloromethane. The solvent effect was introduced by PCM (with dielectric
constant ε=8.93) for an comparison with experimental electrochemical potentials.
2.5. Biological properties
2.5.1. Cell culture
Human colon adenocarcinoma cells (HCT116) and normal human fibroblast (NHDF) cells
were obtained from the American Type Culture Collection. Cells were grown as monolayer
cultures in 75 cm2 flasks (Nunc) in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-
12 (DMEM/F12). The medium was supplemented with 12% heat inactivated fetal bovine
serum (Sigma) and for NHDF with 15% fetal bovine serum (Gibco) and 100 µg/mL of
gentamycin (Gibco). The cell lines were maintained at 37°C in a 5% CO2 incubator and
passaged every 3-4 days as required.
2.5.2. Cytotoxicity assay
Exponentially growing cells were harvested by the trypsinization of sub-confluent cultures.
Cells were seeded into 96-well cell culture microtiter plates (Nunc) at concentrations of 5.0 x
103 cells per well and cultured for 24h. After this time, the growth medium was exchanged for
a medium containing the compounds in concentrations ranging from 0.5 µM- 25 µM. Stock
solutions of the compounds being investigated were prepared in sterile DMSO. The final
concentration of DMSO in the medium did not exceed 0.2%. After 72h incubations with the
compounds being investigated under standard cell culture conditions, the medium was
replaced with 100 µL of DMEM without phenol red. The metabolic activity of viable cells

was determined by adding 20 µL of CellTiter 96 AQueousOne Solutions – MTS (Promega)
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to each well followed by a 1 h incubation. The MTS assay is a colorimetric method for
determining the number of viable cells. A standard solution containing 100 µL of DMEM
without phenol red and 20 µL of MTS solution was used to determine ”blank” absorbance.
The absorbance was measured at 490 nm using a SynergyTM4 microplate reader (BioTek).
The inhibitory concentration (IC50) was defined as the compound concentration that was
necessary to reduce the cells proliferation to 50% of the untreated control cells and expressed
as means ± standard deviation (SD) in GraphPad Prism 5 software. Each individual
compound was tested in triplicate in a single experiment, with each experiment being repeated
at least 3 times.
2.6. Dying properties
2.6.1. The cellular staining
To visualize the accumulation of the compounds within the cells, 10×103 HCT116 cells in a
300 µL growth medium were plated into an 8-well LabTek chambered cover glass (Nunc) and
incubated under standard conditions at 37 °C in a humidified atmosphere at 5% CO2 for 24h.
After this, the cells were treated with the compounds being tested (6e, 7f) at concentration of
25 µM and incubated for a further 2h. After incubation the cells were washed three times with
PBS and then 300µL of PBS was added. The observation of the cells was carried out using an
inverted fluorescence microscope (IX81, Olympus) immediately after staining.
2.6.2. Subcellular localization
Compound localization in living cell cultures was studied using fluorescence microscopy.
Cells were seeded at a density of 20,000 cells/well on 8-well Lab-Tek chambered cover
glasses (Nunc). After 18h, a medium containing the compounds at a concentration that
inhibits 50% viability of cells was added for an additional 2 h (standard conditions). Then the
cells were rinsed with PBS (pH 7.2) and staining procedures were performed accordingly to
providers protocols. Briefly, a serum-free medium that contained MitoTracker® Orange
CMTMRos dye (100 nM, 30-min incubation, Molecular Probes), LysoTracker® Red DND-99
(500nM, 1h incubation, Molecular Probes) or CellMask Deep Red Plasma Membrane (8
µg/mL, 15 min, Molecular Probes) was added. After incubation, the cells were washed three
times with PBS and maintained in a phenol-free medium. Image observation was carried out
immediately after staining using an inverted fluorescence microscope (IX81, Olympus)
equipped with a CO2 incubator (temperature, humidity and gas flow under control). The

analysis and processing of images were performed using an ImageJ 1.41 (Wayne Rasband,
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National Institutes of Health, USA).
3.
Results and discussion
3.1. Synthesis and characterization
3.1.1. Synthesis of quinolines with bithiophene and carbazole motifs
Structures of the quinoline derivatives containing bithiophene and carbazole motifs are
depicted in Figure 1. The quinoline derivatives containing one and two bithiophene (bt)
moieties (7a-b and7d-f) have been recently synthesized and described in our previous papers
[24,30]. Additionally, the new 2-(2,2'-bithiophen-5-yl)-6-chloro-4-phenylquinoline (7c) was
synthesized in a similar manner as 7b from 5-ethynyl-2,2'-bithiophene (Scheme 1).
R1
N
R1
R2
R2
S
N
S
N
7a-f
6a-e
C8H17
a
Me
H
b
Ph
H
c
d
e
f
R1
R2
Ph
Cl
Ph
Ph
bt
H
NO2
NH2
Figure 1. Structure of quinoline derivatives 6a-e and 7a-f.
Compound 7c was obtained as light yellow crystals and the structure was determined by X-
ray analysis (Table S1 in Supporting Information). Furthermore, we present a series of novel
quinoline derivatives with a carbazole motif in the 2-position and various substituents in the
quinoline ring (Figure 1). The new carbazole-based quinoline derivatives (6a-d) were
synthesized from commercially available acyloanilines (5a-d) and 3-ethynyl-N-octylcarbazole
(4) in the presence of catalytic system ([Ru3(CO)12]/HBF4). The 3-ethynyl-N-octylcarbazole
(4) was prepared in four step synthesis according to the reference methods or by modification
of known procedures (Scheme 1). First, the N-alkylation of commercially available carbazole
was conducted with 1-octylbromide in the presence of a phase-transfer catalyst (TBAB) to

provide 2 with a quantitative yield [31]. Then, transformation of 2 into 3-iodo-N-
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octylcarbazol (3) was carried out by NIS at room temperature within 24 hours [32]. A Pd/Cu-
catalyzed Sonogashira coupling reaction of 3 with trimethylsilylacetylene (TMSA) in the
presence of triethylamine (TEA) afforded 3-(trimethylsilyl)ethynyl-N-octylcarbazole in 76 %
yield, and, subsequent deprotection gave the desired 4 in excellent 96% yield [33]. Compound
4 was used to obtain the main carbazole-based quinoline derivatives (6a-d). Preparation of
6a-c proceeded at 110 ºC with addition of 2mol% of [Ru3(CO)12] and 6 mol% of HBF4 within
3h giving the desired compounds 6a-c with excellent yield. An attempt to obtain compound
6d in a similar manner failed. Moreover, increasing either the duration of reaction and
temperature or concentration of [Ru3(CO)12] to 16 mol% or even a change of solvent did not
result in formation of 6d. Finally, the synthesis of 6d in the presence of [Ru3(CO)12] under
microwave irradiation at 100 ºC within 10 minutes succeeded to afford the desired compound
in satisfactory 75% yield (Scheme 1).
TMS
I
c)
d)
a)
b)
95%
76%
96%
96%
N
H
N
N
N
N
C8H17
C8H17
C8H17
C8H17
4
2
3
R1
5a-c
5d
O
O
R2
O2N
R2
O N
R1
2
Ph
e)
f)
NH
NH
2
2
N
N
N
75%
N
N
C8H17
6d
g)
6a-c
4
C8H17
C8H17
6d
NO2
a
c
b
R1
R2
83%
Me
Ph
Ph
Cl
NH2 6e
H
H
5c
O
Cl
Ph
NH
Cl
e)
S
2
S
N
65%
S
S
7c
Scheme 1. The pathway of the preparation of compounds 6a-e and 7c. Reagents and conditions: a)
BrC8H17, TBAB, 50% NaOH(aq), toluene, reflux, 24h; b) NIS, AcOH/CHCl3, rt, 24h;c)
[PdCl2(PPh3)2]/CuI, PPh3, TMSA, TEA, 70 °C, 24h; d) 50% NaOH, (THF/MeOH = 4:1; V/V), rt, 4h;
e) [Ru3(CO)12]/HBF4, toluene, Ar, 110 °C, 3h; f) MW, [Ru3(CO)12]/HBF4, toluene, Ar, 100 °C,
15min.; g) Cakt, DME, rfx, 2h, Pd/C, N2H4,3h.

To explore the influence of a strong electron donating substituent in the quinoline ring on the
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optical and biological properties, the amino group (NH2) was introduced into 6 position of
quinoline ring by reduction of nitro group (NO2) of compound 6d (Scheme 1).The reduction
was carried out in the same manner as for 7e [30] to afford the desired 6e with high yield of
83%.
3.2. Optical properties
The photophysical properties of compounds 6a-e and 7a-f were studied by UV-Vis and
fluorescence spectroscopy in dichloromethane (DCM) solution and are summarized in Table
1. The UV-Vis absorption and emission spectra of 6a-e and 7c are presented in Figure 2 and
Figure 3. The spectra of 7a-f except 7c are included in previous papers [24,30].
Table 1. Photophysical data for quinolines 6a-e and 7a-f.
Emission
τ[a]
kr 107[c]
[s-1]
knr 107[c]
[s-1]
Eg opt
[eV]
[d]
Absorption λ [nm]
(ε [M-1cm-1])
[b]
No
Φem
λ [nm] /Stokes
shift [cm-1]
410/5460
[ns]
339 (69868)
343 (84627)
369 (42284)
408 (41523)
377 (25944)
2.36
2.57
2.54
0.83
5.72
0.48
0.63
0.49
0.09
0.70
0.18
0.44
0.42
0.03
0.23
0.53
20.34
24.46
19.16
10.90
12.28
12.53
63.43
68.95
2.44
22.03
14.45
20.21
109.58
5.20
3.02
2.88
2.81
2.05
6a
6b
6c
431/6210
441/4352
606/8190
440/3588
6d
6e
2.82
2.80
55.96
81.49
94.98
68.31
76.67
35.77
373 (55014)
377 (67335)
386 (24643)
444/4287
1.46
7a
7b
7c
2.58
2.70
2.08
2.66
2.59
450/4303
460/4167
0.69
0.61
412 (68146)
400 (37143)
595/7465
466/3541
1.42
1.00
7d
7e
23.33
52.73
380 (80717)
479/5439
1.13
7f
[a] Estimated as average values of decay constants obtained after bi- or triexponential fitting of
measured decay curves. [b] Absolute quantum yields obtained using integrating sphere in optically
diluted dichloromethane solutions at 298K. [c] Radiative (kr) and non-radiative (knr) decay rates
assuming that emission excited states are produced with unit efficiency are estimated using following
equations: kr = Φem/τ; knr = (1
−Φem)/τ. [d] estimated from emission maximum at dichloromethane
solutions Egopt= 1241/λem.

The UV-Vis absorption spectra of 6a-e and 7c are presented in Figure 2. All quinoline
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derivatives with carbazole motif 6a-e display set of absorption bands in the range of 235 to
408 nm, which can be mainly attributed to π→π* transitions, however the shoulders visible in
the near UV-Vis region can be recognized as n→π* transitions. The structureless and broad
shape of spectra characteristic for studied compounds are typical for polyatomic moieties with
charge transfer (CT) character [34]. The lowest-energy absorption bands, which maxima were
served as excitation wavelengths in fluorescence measurements, are mainly situated in the UV
region – 6a (339 nm), 6b (343 nm), 6c (369 nm), 6d (408 nm), 6e (377 nm) (Table 1). The
longest excitation wavelength is observed for 6d, which can be easily explained by decreased
energy gap between HOMO and LUMO orbitals arising from the presence of strong electron-
withdrawing nitro group (NO2) in the quinoline ring. The absorption spectra of quinoline
derivatives with bithiophene motif (7a-f) are bathochromically shifted (5 to 30 nm) compared
to their counterparts containing carbazole motif (6a-e). This phenomenon is caused by
influence of stronger electron-donating nature of bithiophene moiety in 7a-f than electron-
donating character of carbazole unit in 6a-e.
Figure 2. Absorption spectra of 6a-e and 7c recorded in dichloromethane solution (2x10-5 mol/dm3).
The inserted plot: Comparison of the lower energy absorption bands of 6a-e and 7c.

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The dichloromethane solutions of compounds 6a-e and 7c revealed luminescence with
maxima in the range of 410-606 nm (Table 1). The emission spectra cover the visible region
and their maxima indicating violet fluorescence with the exception of 6d which exhibits
strongly bathochromically shifted orange fluorescence (Table 1 and Figure 3). The same
situation is observed for 7d where emission maximum is bathochromically shifted around 100
nm compared to its counterpart with a bithiophene substituent [24]. The quantum yields of
fluorescence for the studied quinoline derivatives with carbazole moiety are in the range of 9-
70%. The measured effective lifetime values are from 0.83 to 5.72 ns. The monoexponential
fit was estimated in the case of compound 6b-c and 6e, however the biexponential fit was
obtained in the case of 6a and 6d (Figure S1 in Supporting Information). The lowest excited
state lifetime is visible in the case of 6d with the nitro group as a substituent. Moreover,
compared to the 7a-f the significant increasing of lifetime values are visible for studied
compounds. The largest Stokes shift occurs for 6d due to an electron withdrawing -NO2 group
in the quinoline ring. While the smallest difference between absorption edge and emission
peak is observed in the case of 6e due to the electron donating – NH2 substituent in the
quinoline ring. The Stokes shift reflects the ability of the excited state to reorganize to a lower
energy state prior to photoemission [35]. Thus the very strong electron withdrawing nitro
group (NO2) in 6d facilitates the relaxation processes in the excited state by increasing
acceptor properties of the quinoline ring. While the amino group in 6e by increasing electron
density within the quinoline ring destabilizes the excited state, whereby the emission
maximum shifts towards higher energies. Moreover, the emission energy is decreased in the
case of 6b-e comparing to 6a due to electron-accepting properties of phenyl substituent
stabilizing the LUMO of compounds in the greater extent than methyl substituent and thus
decreasing the energy band gaps. The studied compounds 6a-e and 7a-f were additionally
tested for their solvatochromic behavior to attribute the excited state as locally excited (LE) or
charge transfer (CT) in character.

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Figure 3. Fluorescence spectra of 6a-e and 7c recorded in dichloromethane solution (2x10-5 mol/dm3).
The solvatochromic properties of novel quinoline derivatives with the carbazole motif 6a-e
and bithienyl substituted quinolines 7a-f were tested in diverse solvents including chloroform
(CHCl3), dichloromethane (DCM), dimethylsulfoxide (DMSO), dimethylformamide (DMF),
acetonitrile (ACN), toluene and methanol (MeOH). The obtained results are presented in
Figure 4 and Table S2 (Supporting Information). All of the tested compounds exhibit positive
solvatochromism (Figure 4).

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Figure 4. Fluorescence spectra of 6a-e and 7a-f recorded in solvents of different polarity. Red shifts of
emission wavelengths accompanying the increase of polarity (Burdick & Jackson polarity index)
indicate the positive solvatochromism. Spectra of compound 6d and 7d are not presented due to the
lack or very weak fluorescence.
The exceptions are the results obtained for methanolic solutions with respect to novel
quinoline derivatives 6a-e with the bathochromic shifts much higher than would result from
the polarity index. Methanol as a protic solvent introduces additional hydrogen bonding
effect, which is crucial in this type of investigations. Such significant effect of solvent polarity

on the emission properties of compounds may indicate the CT character of the excited state.
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To elucidate more precisely possibility of the intramolecular charge transfer (ICT) processes
in studied compounds, the difference between the excited and ground state dipole moments
(∆µ=µe–µg) was estimated using the Lippert–Mataga equation (eq (1)), where ∆Eexc-em is
Stokes shift (cm-1), h is Planck’s constant, c is the speed of light in vacuum, a is the Onsager
cavity radius and ∆f is the orientation polarizability of the solvent which measures both
electron mobility and dipole moment of the solvent molecule. ∆f was calculated with use of
equation (2) where ε is the dielectric constant of the solvent, n – the optical refractive
index of solvent. Onsager cavity radii (a) were calculated theoretically with use of the
Gaussian 09 [29] and collected in Table S2.
(1)
(2)
For compounds 6a-c, 6e, 7a-c, and 7e-f the Stokes shift ∆Eexc-em against ∆f is plotted in Figure
5 and Figure S2 (Supporting Information).
Figure 5. Lippert-Mataga plots for compounds 6a-c, 6e and 7a-f.
The calculated values of ∆µ are equal to 15.12 D, 14.47 D, 15.50 D, 13.83 D, respectively for
6a-c and 6e. The ∆µ above 14 D for 6a-c indicate CT character of excited state [36]. However
for compound 6e the significance of ICT processes should not be excluded, but the
assumption should consider the mixing excited state character (ICT/LE). The obtained results
are in excellent agreement with visualization of calculated molecular frontier orbitals involved
in photoexcitation (Figure S3 in Supporting Information). From these data one can see that

electron density is separated between HOMO and LUMO for 6a-c, but this evidence cannot
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be adduced in the case of 6e. The HOMO’s of 6a-e are mainly localized on the carbazole
moiety and in the LUMO’s the main contribution of quinoline π* orbitals is visible.
Additionally, for the compounds 7a-e comparing to 6a-e the ∆µ is lower. Such effect
indicates that the electron-donating ability of carbazole moiety relative to bithiophene motif in
studied quinoline derivatives in greater extent predisposed to ICT. For 7a-f, the clear ICT
character of excited state according to data in Table 2 have been detected in the case of 7f
compound. This example indicate the role of number of electron-donating substituents
connected to the quinoline core.
Table 2. Onsager cavity radius (a0), slope of Lippert-Mataga plots and ∆µ for compounds
6a-e and 7a-f.
a0 [Å]
6.03
6.20
6.17
6.44
6.92
5.43
5.60
5.56
5.78
5.74
5.92
Slope of Lippert-Mataga plot [cm-1]
∆µ [D]
15.12
14.47
15.50
—
10468.20
8820.61
10269.07
—
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
5791.19
4839.26
7126.52
5755.22
—
13.83
8.79
11.20
9.93
—
4455.78
13602.86
9.16
16.77
Due to changes of dipole moment in the excited state, solvent relaxation around the excited
molecule takes place to obtain a state of minimum energy. This is a reason of red shifted
emission in polar solvents and increasing Stokes shift as well. Another visible result of
fluctuations in “solvent shell” is inhomogeneous band broadening along with polarity of
solvent. Attention should be also paid to the differences in fluorescence intensity for each
compound in various solvents. Interestingly, the examined compounds exhibit the strongest
fluorescence in nonpolar solvents, like toluene. On the other hand, emission is weaker in more

polar solvents, while in protic solvents it is strongly diminished. This phenomenon is hard to
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explain without any deeper research due to the opportunity of possible competitive processes
responsible for these variations. Presumably hydrogen bonding enhances intersystem crossing
as in the case of 4-aminophthalimide (4-AP) – the compound whose similar property allow
for imaging of hydrophobic region in biological structures [37]. On the other hand, it is well
known that molecules undergoing CT processes display the increasing non-radiative decay
constants in more polar solvent [38] due to the stronger interaction between dipoles of solvent
with dipoles of excited compounds.
3.3. DFT calculations
To get an insight in the electronic structures of the compounds, the calculations by the DFT
were carried out. As the higher is the energy of the HOMO (less negative), more susceptible
is the molecule to donate electrons and, consequently, the easier the oxidation. A similar
argument can be used to interpret the tendency of a given molecule to suffer reduction, on the
basis of the energy of the LUMO. The contours of HOMOs and LUMOs are presented in
Figure S3 (Supporting Information) and the density of states diagrams (generated with use of
Gaussian program [39]) with the percentage constitutions of the HOMO and LUMO orbitals
are depicted on the Figure 6. As one can see the aliphatic –C8H17 chain does not participate in
the frontier occupied and virtual orbitals. HOMOs are mainly localized on the carbazole
except the 6e where quinoline moiety play significant role in the HOMO due to strong
electron donating amino (NH2) substituent in the quinoline ring. LUMOs are localized mainly
on the quinoline moieties whereas in the case of 6d LUMO the strong withdrawing -NO2
group plays a significant role (for this reason the energy of 6d LUMO is decreased).
6a
6b

ACCEPTED MANUSCRIPT
6c
6d
6e
Figure 6. Density of states diagrams for 6a-e.
The dichloromethane solutions of these compounds present absorption bands with
maxima above 400 nm (Figure 2) resulting from transitions between HOMO and LUMO.
Taking into account the electronic structures of these compounds it can be concluded that the
excitation occurs between carbazole (donor) and quinoline (acceptor) fragments. Thus for 6a-
d the excitation can be characterized as πcarbazole→π*quinoline. In the case of 6e in the electronic
absorption in the visible range of the spectrum both aromatic parts i.e. carbazole and
quinoline rings are involved in the transition (Figure 6).
3.4. Molecular structures
The quinoline compounds with a bithiophene moiety 7a-f crystallize in orthorhombic Pna21
(7a) and monoclinic P21/c and P21/n (7b-d) space groups respectively and details concerning
crystal structures determination and refinement are presented in Table S1. The quinoline
derivatives containing carbazole motif are oils so their structures cannot be determined by X-
ray. For this reason the geometry of the molecules have been calculated with use of DFT

method. In the solutions all of compounds reveal emission. Thus, considering that the
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planarity of the molecule influence the emission properties we briefly examine the geometries
of the molecules particularly in terms of the angles between quinoline and thiophene,
carbazole moieties and phenyl substituent. In the molecules 7a and 6a, angle between the
least squares planes of the rings containing quinoline and thiophene atoms is equal to 17.95°
and the thiophene ring in bithienyl substituent are twisted by 7.85°. In the 7b-d compounds in
which the methyl group in the position 4 of quinoline ring is replaced by –C6H5 (phenyl) the
angles between quinoline and phenyl are in the range of 44.01° to 49.29°. The introduction of
–NO2 (7d) or –Cl (7c) substituents increases the value of the angle between quinoline and
phenyl compared to unsubstituted quinoline at 6 position in compound 7b. The presence of
the phenyl substituent causes decreases the angle between the thiophene rings in the
bithiophene moiety from 26.02° in 7b to 19.50° and 19.02° in the case of 7d and 7c,
respectively. In the case of carbazole substituted quinoline derivatives with carbazole moiety
in 2 position (6a-e) the changes of the angles between quinoline and substituent planes i.e.
carbazole and phenyl are less dependent on the substituents on the quinoline ring. In 6a angle
between quinoline plane and carbazole is equal to 14.98° and the smallest value equal to
11.99° reaches for nitro substituted compound 6d. The highest value 15.65° is observed for
unsubstituted in position 6 compound 6b. Similarly the angles between quinoline and phenyl
substituent planes are very close to each other changing from 51.30° in 6b to 53.73° in the
case of 6e. These data suggest that quinoline with carbazole moiety should exhibit better
emission performance which is consistent with experimental data. The lifetimes and quantum
yields of quinoline with carbazole moiety are superior to their bithienyl substituted
counterparts due to the greater planarity of molecules.
3.5. Biological imaging properties
To assess the dying properties of the compounds that were obtained we first tested to
cytotoxicity as IC50. This parameter is crucial for staining the living cells without altering
their morphology or proliferation. It is important to determine safe level of the dye
concentration before further steps of investigation. This activity was determined by means of
tetrazolium bases assay MTS in vitro for all compounds that were synthesized. We have
chosen human colon adenocarcinoma and normal human fibroblasts cell lines as model for
these experiments. The rationale for this was the fact of changed biology of normal and
cancerous cells that may cause differences in reaction on the xenobiotic substances. In brief;
the cells were seeded in multiwell plates and incubated to maintain adhesion and exponential
growth phase. Then the cytotoxicity was determined in triplicates by incubation 96h with

various concentrations of tested compounds followed by spectrophotometric count of cells
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viability. In general all tested compounds from groups 6a-e and 7a-f can be regarded as
biologically inert in concentrations up to 50 µM. The exception is 6e with IC50>12.5 µM.
From the point of view microbiological applications this lack of toxicity is crucial for
observations of living cells during prolonged incubation times. On the other hand,
pharmacokinetic parameters of the tested compounds allow to penetrate cell walls effectively
in relatively short time. This prompted us to test those compounds as cellular dyes. According
to our preliminary experiments full fluorescence can be observed after 1 hour of incubation
with 25µM (Figure 7). During this short period of time the safe (inactive) concentration of
compounds that were tested is consequently higher. However most of the compounds show
interesting spectroscopic properties they are not equally effective as cellular dyes. The overall
strength of fluorescence and quality of the image is a result of yield of fluorescence,
absorption and emission range as well as pharmacokinetic properties as protein bonding,
metabolism and photobleaching [40]. With this in mind we have selected 6e and 7f as the
most effective in staining of the cellular structures (Figure 7), while other exemplary
micrographs for other compounds from this group can be found in Supporting Information.
Figure 7. Micrographs of colon cancer cells stained with 6e and 7f. Scale bars = 50 µm.
To explore further the nature of dye accumulation within cell organelles we performed the co-
localization experiments with commercial dyes for specific cellular substructures. On the
basis of preliminary observations of dyes fluorescence we have chosen plasma membrane
lizosomes and mitochondria as potential sites of the dye accumulation. As presented on
Figure 8, the compound 7f selected for this experiment penetrate the cell body evenly and
accumulate in lipophilic parts as cell membrane and lysosomes. However some concentration
of the dye can be also noticed within mitochondria. These observations are explained on the
basis of the structural and physicochemical properties of the molecules that were tested.
Relatively high lipophilicity (for 7f CLogP=7.73) and basic properties are prerequisites of
lysosome accumulation. On the other hand the sulfur atoms present in bithiophene moiety
may further facilitate accumulation in membranes [41,42].

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Figure 8. Colocalization experiments with organelle staining. Scale bars = 50 µm.
4. Conclusion
The novel quinoline derivatives (6a-e) containing carbazole motif in the 2-position and
different substituents in 4 and 6 positions were synthesized and thoroughly studied. In
addition, a series of known quinolines derivatives (7a-b and 7d-e) containing bithiophene
fragment, among them one new compound 2-(2,2'-bithiophen-5-yl)-6-chloro-4-
phenylquinoline (7c) were systematically studied and compared to their carbazole substituted
counterparts. The bithienyl-substituted quinolines (7a-f) show red-shifted absorption and
emission bands with respect to carbazyl-substituted quinolines (6a-e). The 6a-e possess the
absorption maxima at 235 to 408 nm, significant emission from blue to orange region (410-
606 nm), high quantum yield (9-70%), and lifetimes ranging from 0.83 to 5.72 ns. The
introduction of the carbazole moiety allowed to considerably improved the properties of 6a-e
series. Higher quantum yield of fluorescence and lifetime was the effect of higher electron
density separation between substituent and core substructure in this series comparing to 7a-f.
This in turn emerged from slightly electron-donating effect of carbazole moiety on the whole
molecule as proved by DFT calculations. Stronger electron density separation in 6a-e render
them also more susceptible for intermolecular charge transfer in protic polar solvents.
Accordingly it may be concluded that introduction of carbazole substituent in the 2-position

of quinoline results in a desirable improvement of photophysical properties. Finally the ability
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of the novel compounds for cellular staining was investigated. According to structural
properties the quinoline derivatives penetrate cell organelles quickly and efficiently with good
overall fluorescence and signal to noise ratio. Relative lack of toxicity make them useful in
such applications.
Acknowledgments
This study is supported by the NCN grants 2014/13/D/NZ7/00322 (A.M.W.),
2016/21/B/ST5/00805, 2013/09/B/NZ7/00423 (R.M.), and 2015/17/N/ST5/02838 (G.S.G).
The Gaussian 09 calculations were carried out in the Wroclaw Centre for Networking and
Supercomputing, WCSS, Wrocław, Poland; http://www.wcss.wroc.pl, under calculational
Grant No. 18
Appendix A. Supplementary Data
CCDC 947485, 945129, 945128 and 1059636 contains the supplementary crystallographic
data for compounds 7a-e, respectively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk
References
[1]
Ates M, Karazehir T, Sarac AS. Conducting Polymers and their Applications. Current
Physical Chemistry 2012;2:224-40. DOI: 10.2174/1877946811202030224
[2]
Ho CL, Li H, Wong WY. Red to near-infrared organometallic phosphorescent dyes for
OLED
applications.
J
Organomet
Chem
2014;751:261-85.
DOI:
10.1016/j.jorganchem.2013.09.035
[3]
[4]
Young PJ, Kyu SK, Kyeong KL, In KM, Dong CC, Jun YL, Tae WK. Blue
phosphorescent organic light-emitting devices based on carbazole/thioxanthene-S,S-
dioxide with a high glass transition temperature. J Mater Chem C 2015;3:6192-99.
DOI: 10.1039/C5TC00279F
Slodek A, Filapek M, Schab-Balcerzak E, Grucela M, Kotowicz S, Janeczek H,
Smolarek K, Mackowski S, Malecki JG, Jedrzejowska A, Szafraniec-Gorol G,
Chrobok A, Marcol B, Krompiec S, Matussek M. Highly Luminescence Anthracene

Derivatives as Promising Materials for OLED Applications. Eur J Org Chem
ACCEPTED MANUSCRIPT
2016;23:4020-31. DOI: 10.1002/ejoc.201600532
[5]
[6]
Lai H, Hong P, Liu P, Yuan C, Li Y, Fang Q. Multi-carbazole derivatives: new dyes
for highly efficient dye-sensitized solar cells. RSC Adv 2012;2:2427-32. DOI:
10.1039/C2RA01002J
Slodek A, Matussek M, Filapek M, Szafraniec-Gorol G, Szlapa A, Grudzka-Flak I,
Szczurek M, Malecki JG, Maron A, Schab-Balcerzak E, Nowak EM, Sanetra J,
Olejnik M, Danikiewicz W, Krompiec S. Small Donor–Acceptor Molecules Based on
a Quinoline–Fluorene System with Promising Photovoltaic Properties. Eur J Org
Chem 2016;14: 2500-08. DOI: 10.1002/ejoc.201600318
[7]
[8]
Burland DM, Miller RD, Walsh CA. Second-order nonlinearity in poled-polymer
systems. Chem Rev 1994;94:31-37. DOI: 10.1021/cr00025a002
Szafraniec-Gorol G, Slodek A, Filapek M, Boharewicz B, Iwan A, Jaworska M, Żur L,
Sołtys M, Pisarska J, Grudzka-Flak I, Czajkowska S, Sojka M, Danikiewicz W,
Krompiec S. Novel iridium(III) complexes based on 2-(2,2'-bithien-5-yl)-quinoline.
Synthesis, photophysical, photochemical and DFT studies. Mat Chem Phys
2015;162:498-508. DOI: 10.1016/j.matchemphys.2015.06.020
[9]
Perin N, Hranjec M, Pavlovi G, Karminski-Zamola G. Novel aminated
benzimidazo[1,2-a]quinolines as potential fluorescent probes for DNA detection:
Microwave-assisted synthesis, spectroscopic characterization and crystal structure
determination. Dyes and Pigments 2011;91:79-88. DOI: 10.1016/j.dyepig.2011.02.003
[10] Zhang XH, Liu Q, Shi HJ, Wang LY, Fu LY, Wei XC, Yang LF. Synthesis, spectral
properties of cell-permeant dimethine cyanine dyes and their application as fluorescent
probes in living cell imaging and flow cytometry. Dyes and Pigments 2014;100:232-
40. DOI: 10.1016/j.dyepig.2013.09.011
[11] Tseng CH, Chen YR, Tzeng CC, Liu W, Chou CK, Chiu CC, Chen YL. Discovery of
indeno[1,2-b]quinoxaline derivatives as potential anticancer agents. Eur J Med Chem
2016;108:258-73. DOI: 10.1016/j.ejmech.2015.11.031
[12] Slavik R, Herde AM, Bieri D, Weber M, Schibli R, Kraemer SD, Ametamey SM, Mu
L. Synthesis, radiolabeling and evaluation of novel 4-oxo-quinoline derivatives as PET
tracers for imaging cannabinoid type 2 receptor. Eur J Med Chem 2015;92:554-64.
DOI: 10.1016/j.ejmech.2015.01.028
[13] Kim H, You GR, Park GJ, Choi JY, Noh I, Kim Y, Kim SJ, Kim C, Harrison RG.
Selective zinc sensor based on pyrazoles and quinoline used to image cells. Dyes and
Pigments 2015;113:723-29. DOI: 10.1016/j.dyepig.2014.10.006

[14] Dhanapal R, Perumal PT, Damodiran M, Ramprasath C, Mathivanan N. Synthesis of
ACCEPTED MANUSCRIPT
quinoline derivatives for fluorescent imaging certain bacteria. Bioorg Med Chem Lett
2012;22:6494-97. DOI: 10.1016/j.bmcl.2012.08.039
[15] Majumdar KC, Ponra S, Ghosh T, Sadhukhan R, Ghosh U. Synthesis of novel
pyrano[3,2-f]quinoline, phenanthroline derivatives and studies of their interactions
with proteins: An application in mammalian cell imaging. Eur J Med Chem
2014;71:306-315. DOI: 10.1016/j.ejmech.2013.10.067
[16] Pradhan AB, Mandal SK, Banerjee S, Mukherjee A, Das S, Bukhsh ARK, Saha A. A
highly selective fluorescent sensor for zinc ion based on quinoline platform with
potential applications for cell imaging studies. Polyhedron 2015;94:75-82. DOI:
10.1016/j.poly.2015.04.005
[17] Zhu S, Lin W, Yuan L. Development of a ratiometric fluorescent pH probe for cell
imaging based on a coumarin–quinoline platform. Dyes and Pigments 2013;99:465-71.
DOI: 10.1016/j.dyepig.2013.05.010
[18] Fan L, Gao SQ, Li ZB, Niu WF, Zhang WJ, Shuang SM, Dong C. An indole-
carbazole-based ratiometric emission pH fluorescent probe for imaging extreme
acidity. Sensor Actuat B 2015;221:1069-76. DOI: 10.1016/j.snb.2015.07.076
[19] Feng Y, Li D, Wang Q, Wang S, Meng X, Shao Z, Zhu M, Wang X. A carbazole-
based mitochondria-targeted two-photon fluorescent probe for gold ions and its
application in living cell imaging. Sensor Actuat B 2016;225:572-78. DOI:
10.1016/j.snb.2015.11.081
[20] Cui M, Li Z, Tang R, Jia H, Liu B. Novel (E)-5-styryl-2,2′-bithiophene derivatives as
ligands for β-amyloid plaques. Eur J Med Chem 2011;46:2908-16. DOI:
10.1016/j.ejmech.2011.04.015
[21] Shi G, Monro S, Hennigar R, Colpitts J, Fong J, Kasimova K, Yin H, DeCoste R,
Spencer C, Chamberlain L, Mandel A, Lilge L, McFarland SA. Coord Chem Rev
2015;282-283:127-38. DOI: 10.1016/j.ccr.2014.04.012
[22] Nesterov EE, Skoch J, Hyman BT, Klunk WE, Bacskai BJ, Swager TM. In Vivo
Optical Imaging of Amyloid Aggregates in Brain: Design of Fluorescent Markers.
Angew Chem Int Ed Engl 2015;44:5452-56. DOI: 10.1002/anie.200500845
[23] Guha S, Lohar S, Banerjee A, Sahana A, Hauli I, Mukherjee SK, Matalobos JS, Das
D. Thiophene anchored coumarin derivative as a turn-on fluorescent probe for Cr3+:
Cell imaging and speciation studies. Talanta 2012;91:18-25. DOI:
10.1016/j.talanta.2011.12.014

[24] Slodek A, Filapek M, Szafraniec G, Grudzka I, Pisarski WA, Malecki JG, Zur L, Grela
ACCEPTED MANUSCRIPT
M, Danikiewicz W, Krompiec S. Synthesis, Electrochemistry, Crystal Structures, and
Optical Properties of Quinoline Derivatives with a 2,2' Bithiophene Motif. Eur J Org
Chem 2014:5256-64. DOI: 10.1002/ejoc.201402241
[25] Krompiec S, Filapek M, Grudzka I, Kula S, Slodek A, Skorka L, Danikiewicz W,
Ledwon P, Lapkowski M. An ambipolar behavior of novel ethynyl-bridged
polythiophenes-A comprehensive study. Synth Met 2013;165:7-16. DOI:
10.1016/j.synthmet.2012.12.030
[26] CrysAlis RED, Oxford Diffraction Ltd., Version 1.171.37.35g
[27] Dolomanov OV, Bourhis LJ, Gildea RJ, Howard JAK, Puschmann H. OLEX2: a
complete structure solution, refinement and analysis program. J Appl Cryst
2009;42:339.
[28] Sheldrick GM. A short history of SHELX. Acta Cryst A 2008;64:112.
[29] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR,
Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X,
Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M,
Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H,
Vreven T, Montgomery, Jr. JA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers
E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A,
Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE,
Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O,
Austin AJ, Cammi R, Pomelli C, Ochterski J. W, Martin RL, Morokuma K,
Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD,
Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian, Inc.,
Gaussian 09, Revision A.1, Wallingford CT
[30] Kotowicz S, Siwy M, Filapek M, Malecki JG, Smolarek K, Mackowski S, Slodek A,
Schab-Balcerzak E. New donor-acceptor-donor molecules based on quinoline acceptor
unit with Schiff base bridge: synthesis and characterization. J Lumin 2017;183:458-69.
DOI: 10.1016/j.jlumin.2016.11.058
[31] Ren MG, Guo HJ, Qi F, Song QH. Fluorescent pyrene-centered starburst
oligocarbazoles with excellent thermal and electrochemical stabilities. Org Biomol
Chem 2011;9:6913-16. DOI: 10.1039/C1OB05857F
[32] Finke AD, Gross DE, Han A, Moore JS. Engineering Solid-State Morphologies in
Carbazole–Ethynylene Macrocycles. J Am Chem Soc 2011;133:14063-70. DOI:
10.1021/ja204795q

[33] Wu W, Ji S, Guo H, Zhao J. Tuning the emission property of carbazole-caped
ACCEPTED MANUSCRIPT
cyclometalated platinum(II) complexes and its application for enhanced luminescent
oxygen
sensing.
J
Organomet
Chem
2011;696:2388-98.
DOI:
10.1016/j.jorganchem.2011.03.002
[34] Resch-Genger U, Hoffmann K, Pa J. Comprehensive Biomedical Physics. In:
Reference Module in Biomedical Sciences 2014;4:15-26.
[35] Jones BA, Facchetti A, Wasielewski MR, Marks TJ. Tuning Orbital Energetics in
Arylene Diimide Semiconductors. Materials Design for Ambient Stability of n-Type
Charge Transport. J Am Chem Soc 2007;129:15259-78. DOI: 10.1021/ja075242e
[36] Mutai T, Cheon JD, Arita S, Araki K. Phenyl-substituted 2,2′:6′,2″-terpyridine as a
new series of fluorescent compounds—their photophysical properties and fluorescence
tuning. J Chem Soc Perkin Trans 2001;2:1045-50. DOI: 10.1039/B102685M
[37] Zhang S, Yang H, Singh L. Increased information leakage from text. CEUR Workshop
Proc. 2014;1225:41–42
[38] Bogado AL, Carlos RM, Daolio C, Ferreira AG, Neumann MG, Rominger F,
Machado SP, Da Silva JP, De Araujo MP, Batista AA. Observation of vinylidene
emission in mixed phosphine/diimine complexes of Ru(II) at room temperature in
solution.
J
Organomet
Chem
2012;696:4184-90.
DOI:
10.1016/j.jorganchem.2011.09.017
[39] O’Boyle NM, Tenderholt AL, Langer KM. cclib: A library for package-independent
computational chemistry algorithms. J Comp Chem 2008;29:839-45. DOI:
10.1002/jcc.20823
[40] Ettinger A, Wittmann T. Chapter 5 – Fluorescence live cell imaging. Methods Cell
Biol 2014;123:77-94. DOI: 10.1016/B978-0-12-420138-5.00005-7
[41] Campbell ID, Humphries MJ. Integrin Structure, Activation, and Interactions. Cold
Spring
Harb
Perspect
Biol
2011;3:
a004994–a004994.
DOI:
10.1101/cshperspect.a004994
[42] Sevier CS, Kaiser CA. Formation and transfer of disulphide bonds in living cells. Nat
Rev Mol Cell Biol 2002;3:836-47. DOI: 10.1038/nrm954